.::

 

 

lll‘llllllllllllll *

 

This is to certify that the
dissertation entitled

Molecular analysis of the photosystem I reaction center
in the cyanobacterium synecbocysak sp. PCC 6803

presented by
Lawrence B. Smart
has been accepted towards fulfillment

of the requirements for

PhD. degree in Genetics

 

 

  
     

 
 

LIBRARY
6 Michigan State '
' University

‘PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

 

DATE DUE DATE DUE DATE DUE

 

 

 

wWWe-i

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution
c:\circ\datedue.pm3—p.1

TA

llllllllitiltlltl‘

 

This is to certify that the
dissertation entitled
Molecular analysis of the photosystem I reaction center
in the cyanobacterium synecbocysnis' sp. PCC 6803
presented by

Lawrence B. Smart

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Genetics

 

 

  
     

  

LIBRARY
Michigan State
University

[PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

 

DATE DUE DATE DUE DATE DUE

 

 

”4.»
K7? 1

l res ‘3 C’

 

7): W? F It;

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*7
MSU Is An Affirmative Action/Equal Opportunity Institution
c:\clrc\datedue.pm3-p.1

 

 

MOLECULAR ANALYSIS OF THE PHOTOSYSTEM I REACTION CENTER
IN THE CYANOBACI‘ERIUM SYNECHOCYSHS SP. PCC 6803

By

Iawrence B. Smart

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Genetics Graduate Program

1992

 

ABSTRACT

MOLECULAR ANALYSIS OF THE PHOTOSYSTEM I REACTION CENTER
IN THE CYANOBACTERIUM SHVECHOCYSIIS SP. PCC 6803

By
Lawrence B. Smart

The focus of this study is the investigation of the biogenesis, structure,
and function of the photosystem (PS) I reaction center in the unicellular
cyanobacterium Smecbocystis sp. PCC 6803 using molecular genetic techniques.
The PSI reaction center is comprised of a heterodimer of homologous 82 - 83-
Kda polypeptides, PsaA and PsaB, which bind all but two of the electron
transfer components of PSI and are encoded by the genes psaA and p.933. The
structure-function elements studied in this project were the [4Fe-4S] electron
transfer component Fx, which is coordinated by four cysteine residues, two from
PsaA and two from PsaB, and a leucine zipper motif in PsaA and PsaB,
proposed to act in reaction center dimerization. Synecbocystzls 6803 is an
excellent organism for the study of PSI, because it is readily transformable, has
an active homologous recombination system, has a photosynthetic apparatus
much like that of plants, and may be grown heterotrophically. The psaA-psaB
operon was isolated and its DNA sequence determined. Transcripts from
psaA-psaB accumulate in dark-grown cells to a level similar to that in light-
grown cells. Transcripts from psbA also accumulate in dark-grown cells, but to

levels three- to four-fold lower than in light-grown cells. Both the psaA and

psaB genes were inactivated by interposon mutagenesis and selection under
light-activated heterotrophic growth (LAHG) conditions, indicating that the PSI
reaction center is dispensable for heterotrophic growth of Smecbocystzs 6803.
Genetic inactivation of PSI was performed in strains to be used for P811
mutagenesis in order to facilitate the purification and analysis of PSII. Site-
directed amino acid substitutions were incorporated into PsaB, changing a
proposed cysteine ligand of Fx and changing leucines in a leucine zipper motif.
Analysis of these mutants indicates that Fx is an important structural as well as
functional component of PSI, but that the leucine zipper may not play a major

role in reaction center dimerization.

 

 

 

ACKNOWLEDGEMENTS

I wish to express my deepest gratitude to those people who have shared
their precious time, knowledge, experience, and enthusiasm with me in the
process of completing this project. I am pleased to have had the opportunity to
exchange ideas, techniques, and home-brews with my collaborators at the
University of Nebraska, Patrick Warren and Dr. John Golbeck. I am indebted

to Dr. Neil Bowlby for teaching me much of what I know about photosynthesis,

 

EPR spectroscopy, and ball bearings. I wish to thank Kurt Stepnitz for his

 

excellent and extensive photographic work on my behalf. I have been very
fortunate to have had the enthusiastic support and encouragement of my
advisory committee, Dr. Barbara Sears, Dr. Michael Thomashow, and Dr. C.
Peter Wolk. Equally supportive have been my co-workers in the McIntosh lab,
especially Dr. John Fitchen, Dr. Carrie Hiser, Dr. Idah Sithole, Dr. Shawn
Anderson, and Dr. David Rhoads. I must give a great deal of credit for the
success of this project and for the good start of my scientific career to Dr. Lee
McIntosh, who has given unending support and guidance. Finally, I would like
to thank my wife, the future Dr. Christine Durbahn Smart and my parents,
Robert and Marjorie Smart, for their loving understanding, tolerance, and

encouragement.

 

 

TABLE OF CONTENTS

Page
LIST OF TABLES ......................................... viii
LIST OF FIGURES ........................................ ix
LIST OF ABBREVIATIONS ................................. xi
CHAPTER 1. INTRODUCTION .............................. 1
PHOTOSYNTHESIS
PHOTOSYSTEM I

 

PSI Reaction Center
Peripheral PSI polypeptides
PSI Gene Organization
GENETIC MANIPULATION OF PSI
Procaryotic organisms
Eucaryotic organisms
FOCUS OF TI-HS STUDY
REFERENCES

CHAPTER 2. Expression of photosynthesis genes in the cyanobacterium
Synechocystis sp. PCC 6803: psaA—psaB and psbA transcripts accumulate in
dark-grown cells ............................................ 38
INTRODUCTION
MATERIALS AND METHODS
Materials
Strain and growth conditions
Cloning
Sequencing and sequence analysis
RNA isolation and northern blots
RESULTS
Cloning
DNA sequence
psaA—psaB deduced amino acid sequence
mRNA accumulation in cells grown with glucose
mRNA accumulation in cells grown without glucose

 

DISCUSSION

ACKNOWLEDGEMENTS
REFERENCES

CHAPTER 3. Targeted genetic inactivation of the photosystem I reaction
center in the cyanobacterium Smecbocysns sp. PCC 6803 . ............ 71
INTRODUCTION
RESULTS
Construct interrupting psaA
Transformation of Synechocystis 6803 and selection conditions
Genetic mapping and segregation analysis
Chlorophyll determination and oxygen evolution
Growth characteristics
EPR spectra
Detection of proteins by antibodies
DISCUSSION
Synecbocystis 6803 as a genetic system
Response of wild-type cells to LAHG conditions
Inactivation of psaA
Conclusions
MATERIALS AND METHODS
Materials
Strain and growth conditions
Nucleic acid manipulations
Oxygen evolution assays
Thylakoid membrane isolation
EPR spectra
SDS-PAGE and immunoblotting
ACKNOWLEDGEMENTS
REFERENCES

CHAPTER 4. Genetic inactivation of the [2538 gene in Synechocystis sp.
PCC 6803 disrupts assembly of photosystem I ...................... 102
INTRODUCTION
RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES

CHAPTER 5. Genetic manipulation of Synechocystis sp. PCC 6803:
development of strains lacking PSI for the study of PSII ............... 114
INTRODUCTION
MATERIALS AND METHODS
Materials

vi

 

 

 

 

CH.z
the I
sp. P

CHA

APP]

sitec

Strains and growth conditions
Nucleic acids
Chl and protein analysis
EPR spectroscopy
RESULTS
Plasmid constructs and transformations
Characterization of D1'/PSI' and D2'/PSI'
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES

CHAPTER 6. Mutational analysis of the structure and biogenesis of
the photosystem I reaction center in the cyanobacterium Synechocystis
sp. PCC 6803 .............................................. 138
INTRODUCTION
MATERIALS AND METHODS
Materials
Strains and growth conditions
DNA Manipulations
Membrane isolation and analysis
ESR spectroscopy
RESULTS
Recipient strain
Site-directed mutagenesis
Characterization
DISCUSSION
Site-directed mutagenesis of PSI
Conclusions
ACKNOWLEDGEMENTS
REFERENCES

CHAPTER 7. DISCUSSION AND CONCLUDING REMARKS ....... 173
Coordinate regulation of thylakoid membrane complexes
Light sensitivity of PSI mutants
Comparison of PSI to other photosynthetic reaction centers
Potential future experiments
References

APPENDIX. ESR and optical spectroscopy of wild type and

site-directed mutants ........................................ 183
Acknowledgements

Vii

LIST OF TABLES

Table Page

2.1 DNA probes used for screening, Southern, and northern hybridizations 46

3.1 Chlorophyll determination and photosynthesis assays 81
4.1 Chlorophyll determinations and 0; evolution assays 107
5.1 Chlorophyll content of whole cells and thylakoid membranes 125

6.1 Characterization of p533 mutants 153

viii

 

 

3.2 f

3.3 :4

3.4 E

3.5 11

3.6 p

4.111

LIST OF FIGURES

Figure

2.1 Restriction map and sequencing strategy of the
Synechocystis 6803 psaA and psaB operon

2.2 Nucleotide and deduced amino acid sequences of
the Synechocysti's 6803 psaA and p533 operon

2.3 Autoradiographs of a single northern blot,
stripped and reprobed

2.4 Relative levels of mRNA accumulation in
Synechocystis 6803 cells

2.5 Autoradiographs of a single lane from a northern blot
(48 h dark without glucose), stripped and reprobed

3.1 Restriction map of the insert region from plasmid pLSl8K
and of the psaA-psaB operon in Synechocystis 6803

3.2 Autoradiographs of Southern and northern blots of
wild type and ADK9

3.3 Autoradiographs of Southern blots of wild-type,
ADK9, and ALT6 DNA

3.4 EPR spectra of thylakoid membranes from WT-MIXO,
WT-LAHG, and ADK9

3.5 Immunoblots of thylakoid proteins from WT-MIXO, WT-LAHG,
and ADK9

3.6 Petri dishes with WT-MIXO, WT-LAHG, and ADK9

4.1 Partial restriction map of the psaA-psaB operon from
Synechocystis 6803 and of plasmid pLS1315K

Page

48

50

56

58

60

76

77

79

83

85

89

104

 

5.3 11

5.4 R

6.1C

6.2 P

6.3 I

6.4 i

6.5 I

A2

A3

A4

4.2 Autoradiographs of Southern and northern blots of
wild type and BDK8

4.3 Room temperature EPR spectra of BDK8 membranes

4.4 Immunoblots of WT-MIXO, WT-LAHG, and BDK8
membrane proteins

5.1 Autoradiographs of Southern blots of genomic DNA from
wild-type and mutant cells

5.2 Partial restriction maps of plasmids used for mutagenesis
and of portions of the Smechocystis 6803 chromosome

5.3 Immunoblots of membrane proteins isolated from wild type,
D1‘/PSI' and D2'/PSI'

5.4 Room temperature EPR spectra of thylakoids from
D1'/PSI' and D2'/PSI'

6.1 Cartoon of Fx and portions of PsaA and PsaB

6.2 Partial restriction maps of psaA-psaB and of plasmids
for mutagenesis

6.3 Immunoblots of membrane proteins probed with antibodies
recognizing PsaA/B, PsaC, or PsaD

6.4 Room temperature ESR spectra taken during
illumination of thylakoid membranes

6.5 Low temperature ESR spectra of PSI complex isolated
from the C565S mutant

A.1 Low temperature ESR spectra of wild-type PSI complex

A.2 Low temperature ESR spectra of membranes isolated from
wild-type and mutant cells

A.3 Low temperature ESR spectra of membranes isolated
from C5658

A.4 Optical spectroscopy of PSI complex isolated from
C5658 and wild type

106

108

109

122

123

127

128

141

150

156

158

160

185

186

187

188

LIST OF ABBREVIATIONS

American Type Culture Collection
chlorophyll a/b binding

chlorophyll
2,6-dichloro-p-benzoquinone
Nl-(dichlorophenyl)-N3-dimethylurea
electron-nuclear double resonance
electron spin echo envelope modulation
electron spin resonance

electron paramagnetic resonance
electron X-ray absorption fine structure
[4Fe-4S] center A

[4Fe-4S] center B

[4Fe-4S] center X

gentamycin

kilobases or kilobase pairs

kiloDalton

kanamycin

light-activated heterotrophic growth
light harvesting complex

milliTesla

nicotinamide adenine dinucleotide phosphate
polyacrylamide gel electrophoresis
Pasteur Culture Collection

polymerase chain reaction

polyethylene glycol

photosystem I

photosystem II

sodium dodecyl sulfate

spectinomycin

streptomycin
N-tris-(hydroxymethyl)-methyl-Z-aminoethanesulfonic acid
tris(hydroxymethyl)aminomethane
mixotrophically-grown wild type
LAHG-grown wild type

 

 

 

 

 

 

PHO'.

conve
annos
polyp
C02 i
win
mech

then}

zflgae
light.
ofca
Cyclic
elect]

not t

 

CHAPTERl

INTRODUCTION

PHOTOSYNTHESIS

Photosynthesis, the process used by plants, algae, and some bacteria to
convert light energy into chemical energy, dramatically affects the Earth and its
atmosphere. This series of chemical reactions, catalyzed by dozens of
polypeptides, is responsible for generating the 02 that we breathe and for fixing
C02 into organic compounds. Thus, to ensure our survival in an ever-changing
environment, it is vital for us to gain a complete understanding of the
mechanisms and regulation of these reactions and the proteins that catalyze
them.

Photosynthesis can be classified into oxygenic and non-oxygenic. Plants,
algae, and cyanobacteria perform oxygenic photosynthesis, evolving 02 from the
light-driven oxidation of water and transferring electrons through a linear series
of carriers, eventually yielding reducing equivalents, in addition to performing
‘ cyclic photosynthesis. Non-oxygenic photosynthesis is characterized by cyclic
electron flow through a single photo-reaction center, driving ATP synthesis, but

not the generation of reducing equivalents. Non-oxygenic photosynthesis is

 

 

 

 

 

perfor
bacter
ct a],
protei
to the

photo

flow C
phosp
many
cyano
comp
photc
gradi<
by th<
factol
fixatii
anabt

anYr

chiOI‘i

are: ]

Cificj.

2

performed by species of bacteria and has been best characterized in the purple
bacteria Rhodobactcr sphaeroides and Rhodopseudomonas viridis (Deisenhofer

et a1., 1985; Allen et a1., 1987). Further discussion of the photosynthetic

 

proteins in the purple and green bacteria and the heliobacteria will be limited
to their use as models for some of the proteins involved in oxygenic

photosynthesis and the evolutionary significance of that similarity.

 

As alluded to earlier, oxygenic photosynthesis involves the light-driven
flow of electrons from water to reduce nicotinamide adenine dinucleotide
phosphate (NADP+) to NADPH, which may be used as reducing power for
many anabolic reactions. This process is accomplished in plants, algae, and
cyanobacteria by three thylakoid membrane-bound, multi—subunit enzyme
complexes: photosystem II (PSII), the cytochrome b/f complex, and
photosystem I (PSI). As electrons are passed through certain carriers, a proton
gradient is generated across the thylakoid membrane. This gradient is released

by the flow of protons through the ATP synthetase (also called the coupling

 

factor), driving ATP synthesis. ATP and NADPH act as substrates for carbon
fixation and sugar synthesis, amino acid synthesis, fatty acid synthesis, or other
anabolic reactions. In plants and algae, the thylakoids and many of the
enzymes involved in biosynthetic processes are compartmentalized to the
chloroplasts.

The basic requirements for the light-dependent photosynthetic reactions
are: light capture, charge separation, and charge stabilization. To accomplish

efficient energy use, the charge separation created at the initial photochemical

   

 

Oxyge
equiv

relea:

mech
uncle:
elect]
Pilot
The I
elect;

I'Cpla

 

3

event must be quickly stabilized by a series of oxidation-reduction reactions
between spatially separate electron carriers. The initial step is the capture of

energy from light by antennae pigments, primarily chlorophylls. This energy is

 

then transferred to special, reaction center chlorophyll, which in P811 is termed
P680 (Rutherford, 1988). The energy captured by P680 generates an excited

electron, which is then passed to the primary acceptor, a phaeophytin, then to a

 

bound plastoquinone, QA (Rutherford, 1988). Finally, electrons are passed to a
second plastoquinone called QB, which when doubly reduced to plastoquinol,
exchanges with a quinone from a pool in the membrane (Rutherford, 1988).
P630+ is a strong oxidant and is reduced by electrons donated by tyrosine
radicals in the D1 and D2 polypeptides, termed Yz and YD (Debus et 31.,
1988a; Debus et 31., 1988b). YZ+ and YD+ are reduced by Mn atoms in the
oxygen evolving complex. The tetrad of Mn atoms accumulates oxidizing
equivalents in a linear series of oxidation or S-states, finally oxidizing water and

releasing oxygen in a single event (Kok ct a1., 1970; Forbush et a1., 1971).

 

The plastoquinol form of Q, reduces the cytochrome bé/f complex. The
mechanism of electron transport through this multisubunit complex is poorly
understood, but it catalyzes the net import of two protons for every two
electrons it transports (O’Keefe, 1988). The cyt bé/f complex is shared between
photosynthesis and respiration in the cyanobacteria (Sandmann et at, 1984).

9 The cytochrome b6/f complex also accomplishes the transition from the two
electron carrier QB to the single electron carrier plastocyanin, which may be

replaced by cytochrome 0553 in cyanobacteria (Sandmann, 1986). Plastocyanin,

 

 

er C
theth

donat

bkflog
energ
toA1
donat
fenec
Ferre
iCIiCI
natur
Capal
qnoc
not g

gradi

PHK]

PSI]

mem

 

4

like QB, is a diffusible carrier that can bridge the distinct protein complexes in
the thylakoid. Plastocyanin (or sometimes cytochrome 0553 in cyanobacteria)
donates electrons to the reaction center chlorophyll pair in PSI, P700.

PSI captures the second photon of light needed to complete reduction of
NADP+ and transfers the energy to P700, creating the strongest reductant in a
biological system (es-1.2 V) (Vos and van Gorkom, 1988). PM passes an
energized electron to the primary electron acceptor A0, which donates electrons
to A1, which then reduces Fx. Finally, Fx reduces FA and/or FB, which then
donate electrons to ferredoxin. In cyanobacteria, flavodoxin can substitute for
ferredoxin under conditions of iron-limitation (Ho and Krogmann, 1982).
Ferredoxin then reduces NADP+ to NADPH; a reaction catalyzed by the
ferredoxinzNADP+ oxidoreductase (Ho and Krogmann, 1982). The detailed
nature of the electron carriers in PSI will be discussed below. PSI is also
capable of catalyzing cyclic electron flow, in which electrons are returned to the
cytochrome complex to cycle back to P700 (O’Keefe, 1988). This process does
not generate reducing power (NADPH), but does cause formation of a proton

gradient for ATP synthesis.

PHOTOSYSTEM I

PSI reaction center
The PS1 complex is a conglomeration of many protein subunits, both

membrane-bound and extrinsic, which coordinate several different cofactors

 

 

 

lllVOlV
funcfi
valua‘
(iohx
liuthi
the g
systei
pane
enfin
tugsi
PSID
gene.
pron
bChN
(hhb:
nanu
of so
subu

m0it

83-k:
1987

Phi

5

involved in electron transport. Recently, detailed reviews of PSI structure,
function, and gene organization have been published and provide the reader a
valuable compilation of the current state of understanding of PSI (Bryant, 1987;
Golbeck, 1989; Golbeck, 1992; Golbeck and Bryant, 1991; Mathis and
Rutherford, 1987). The nomenclature used to describe the PSI subunits and
the genes that encode them is as follows: genes are named as in other bacterial
systems, with three lower case letters describing the function of the encoded
protein, then an upper case letter to describe a particular gene in a group; the
entire name is italicized (Hallick, 1989). In the case of PSI, the first two letters,
ps, signify photosynthesis, the next, a, signifies PSI (as opposed to b, signifying
P811), and the final letter currently ranges from A to L, designating a particular
gene. However, to avoid the confusion that has arisen from the naming of the
protein subunits by their order of electrophoretic migration, which varies
between organisms (Golbeck and Bryant, 1991), protein nomenclature will
differ from that of Hallick (1989). The protein name is the same as the gene
name, but is not italicized, and the first and last letters are capitalized. The list
of subunits presented here represents a complete list as we know it, but more
subunits may be discovered in the future as techniques to detect them become
more refined.

The reaction center of PS1 consists of a heterodimer of homologous 82 -
83-kDa (kiloDalton) polypeptides, named PsaA and PsaB (Cantrell and Bryant,
1987; Fish et 31., 1985). The amino acid sequences deduced from the psaA and

psaB genes are predicted to form 11 membrane-spanning a-helices (Cantrell

 

 

 

 

and i
ICSldl
chloru
(Goll
also :
harve
the c

prote

photu
peak
and I
chlox
bactt
(Dei
a diI
mole

abso

Spec
milli

plas

6
and Bryant, 1987; Fish et at, 1985). There are approximately 80 histidine

residues in PsaA and PsaB combined, which may bind many of the #100
chlorophyll molecules associated with the reaction center, including P700
(Golbeck and Bryant, 1991). In plants and algae, antennae chlorophst are
also associated with chlorophyll a/b-binding (cab) proteins in the light-
harvesting complex (LHC) (Golbeck, 1992). However, in cyanobacteria all of
the chlorophyll associated with PSI appears to be bound to the reaction center
proteins, as there are no LHC proteins (Ho and Krogmann, 1982).

The PsaA and PsaB proteins bind the electron transfer components P700,
A0, A], and FX. The reaction center chlorophyll, P700, is the site of the primary
photochemical charge separation in PSI. It is named by the wavelength at the
peak of the oxidized-minus-reduced difference spectrum (Kok, 1956). Optical
and electron paramagnetic resonance (EPR) spectroscopy indicate that P700 is a
chlorophyll a-dimer (Kok, 1957; Norris et a1., 1974), analogous to the
bacteriochlorophyll special pair P870 in the bacterial reaction center
(Deisenhofer et 31., 1985). Recent data suggest that the ground state of P700 is
a dimer, but that the P700”r cation is localized to one of the two chlorophyll
molecules (Golbeck and Bryant, 1991; Ikegami and Itoh, 1988). Upon
absorbing a photon of light, P700 is raised to an excited state, Pm', then passes
an electron to A0 and forms the cation PM)”. P700+ is detectable by EPR
spectroscopy, giving a signal centered at g=2.0025, with a linewidth of 0.8 - 1.0
milliTesla (mT), termed signal I (Norris et 31., 1971). P700+ is reduced by

plastocyanin or in cyanobacteria, by cytochrome c553 (Sandmann, 1986).

 

 

 

 

resoi
Pine
back
(hiat
indh:
(nib
conn
sate

rano

been
Spec
(fifie
char
Inok
Cent:
COnc
fenn

Inca

iion

sngg

cOnt

7

The primary electron acceptor, A0, has been best characterized by time-
resolved optical spectroscopy, in which the flash-induced absorption changes of
P700 are measured under highly reducing conditions. Using this technique, the
back reaction from A0' to Pm,” has been determined to be approximately 35 ns
(Mathis et a1., 1988). The flash-induced difference spectrum of Ao' - A0
indicates that this component is a chlorophyll a molecule (Mathis et a1., 1988;
Golbeck and Bryant, 1991). Photo-accumulation experiments, in which
continuous illumination is used under highly reducing conditions to obtain a
state with P700 A0“, also suggests that A.J is a chlorophyll a monomer present in a
ratio of 0.94 A0 /P—,00 (Mansfield and Evans, 1985).

The identity of the second electron acceptor, A1, is controversial. A1 has
been characterized by photoaccumulation studies, using optical and EPR
spectroscopy, and also by time-resolved optical spectroscopy. The absorption
difference spectra and EPR spectra obtained in photoaccumulation studies are
characteristic of some quinone species (Mansfield and Evans, 1985). Also, two
molecules of phylloquinone (vitamin K1) can be extracted from the PSI reaction
center (Schoeder and Lockau, 1986). However, conflicting data exist
concerning the role of phylloquinone in electron transport. Ether extraction to
remove phylloquinone also blocks electron flow past A, at room temperature as
measured by time-resolved optical spectrosc0py (Mansfield et at, 1987).
However, under cryogenic temperatures, electron transfer to FA is possible,
suggesting that a low-temperature bypass of A, occurs (Setif et a1., 1987). In

contrast, inactivation of phylloquinone by ultraviolet (UV) radiation does not

 

 

 

ehnn
ehct
speci
afiec
ontl
anah
extra

ehct

pron
[HEP
1978
K)P-
char
lDek
then
and
renu
Inni

thej

cent

anal

8

eliminate the A1 EPR signal (Ziegler et a1., 1987) or affect room temperature
electron flow to the terminal electron acceptors (Palace et a1., 1987). Likewise,
specific deuteration of quinone in a methionine auxotroph of Anabaena did not
affect the photoaccumulated A1 EPR signal (Barry et 31., 1988), casting doubt
on the source of this EPR signal. The presence of phylloquinone in PS1, the
analogy with the quinones in P811, and data from optical spectrosc0py after
extraction of quinones are strong evidence for a role of phylloquinone in PSI
electron transport.

The final electron transfer component bound to the reaction center
proteins is Fx. This component was first detected by EPR spectroscopy of PSI
preparations at low temperature (Evans et at, 1976; Malkin and Bearden,
1978). After extraction with mild detergent, the 30 ms backreaction of F A/FB'
to P700+ is replaced by a 1.2 ms backreaction with a difference spectrum
characteristic of an iron-sulfur center (Golbeck and Cornelius, 1986).
Determination of acid-labile sulfur content of the PSI complex indicated. that
there were 12 mol of sulfur per mol of P700 (Golbeck et a1., 1988). Since FA
and FE were known to be [4Fe-4S] centers (Wynn and Malkin, 1988a), the
remaining iron and sulfur form either a [4Fe-4S] center or 2 [2Fe-28] centers.
Initial attempts to characterize Fx were complicated by interfering signals from
the iron-sulfur centers F A and FE. Once a particle was resolved lacking FA and
F3 , but with Fx intact (Parrett et at, 1989), confirmation of Fx as a [4Fe-4S]
center was accomplished by extended X—ray absorption fine structure (EXAFS)

analysis (McDermott et a1., 1989) and by Mossbauer spectroscopy (Petrouleas

 

 

 

 

ct a].
from
The

g val
are 5
are 5
sulfu
31., l
Corr
PsalA
(001
both
pohq
1989
(Btu
inter

diffit

PsaE

00m

Gly.

pro;

9

etal., 1989). Also the acid-labile sulfur content of this preparation is reduced
from 12 mol / mol P700 to approximately 4 mol / mol P700 (Parrett et at, 1989).
The EPR spectrum of reduced Fx is only detected at low temperature and has
gvalues of 2.08, 1.88, and 1.78 (Warden and Golbeck, 1986). These resonances
are shifted slightly from those expected for a [4Fe-4S] center and the features
are somewhat broader than expected (Golbeck and Bryant, 1991). An iron-
sulfur center with the especially low redox potential of Fx (-670 mV) (Parrett et
a1., 1989) is assumed to be coordinated by four cysteine residues (Golbeck and
Cornelius, 1986). Analysis of conserved cysteines in the protein sequences of
PsaA and PsaB shows that there are only three in PsaA and two in PsaB
(Golbeck and Bryant, 1991), which indicates that Fx may be coordinated by
both the PsaA and PsaB proteins and that the stoichiometry is of one PsaA
polypeptide and one PsaB polypeptide per P700 (Golbeck, 1987b; Scheller et 31.,
1989a). This stoichiometry has been confirmed by radiolabelling experiments
(Bruce and Malkin, 1988b; Bruce and Malkin, 1988a). The role of Fx as an
intermediate between A1 and FA/FB is still not fully resolved, mainly due to the
difficulty associated with its detection (Golbeck and Bryant, 1991).

A comparison of the five conserved cysteine residues found in PsaA and
PsaB reveals that four of them lie in a motif conserved in both PsaA and PsaB
containing two of the cysteines each (Golbeck and Bryant, 1991; Cantrell and
Bryant, 1987). This 12 residue sequence is: Phe-Pro-Cys-Asp-Gly-Pro-Gly—Arg-
Gly-Gly-Thr-Cys. These four cysteines, two in PsaA and two in PsaB have been

proposed to act as the ligands to the [4Fe-4S] Fx (Golbeck and Cornelius,

 

 

 

 

1986
repe;
et al
leuci
were
funcr
at, 1
OH
prot<
anot
er a)
term
not
for c‘
but 1
a], 1
beer
Coile
puta
Proti

lr0n.

10

1986). Immediately adjacent to this motif in both PsaA and PsaB are heptad
repeats of conserved leucine residues that are predicted to lie in a-helices (Fish
et 31., 1985 ; Cantrell and Bryant, 1987) and have been proposed to form a
leucine zipper (Webber and Malkin, 1990; Kossel et 31., 1990). Leucine zippers
were discovered initially in some DNA-binding regulatory proteins, which
function as dimers (Turner and Tjian, 1989; Landschulz et 31., 1989; Gentz et
31., 1989). It was shown with the proteins F05 and Jun and with the protein
C/EBP that the leucine zippers were responsible for dimerization of the
proteins, which positions basic regions in each protein in proximity to one
another, forming a DNA-binding domain (Landschulz et 31., 1988; Landschulz
et 31., 1989; Gentz et 31., 1989). Proteins with these characteristics have been
termed bZIP proteins (Pu and Struhl, 1991). It has been shown by site-directed
mutagenesis that it is the specific interaction of the leucines that is responsible
for dimerization (Hu 61:31., 1990; Landschulz et 31., 1989; Gentz et 31., 1989),
but that other residues in the helices also play a role in specificity (O’Shea et
31., 1992). Synthetic peptides forming the GCN4 leucine zipper region have
been crystallized and the structure resolved, revealing that the helices form a
coiled-coil structure (O’Shea et 31., 1991). It has been proposed that the
putative leucine zipper in PSI acts in the dimerization of the reaction center
proteins, thus putting the cysteine ligands to Fx in position to coordinate the
iron-sulfur center, analogous to the DNA-binding domain of bZIP proteins

(Webber and Malkin, 1990; Kossel et 31., 1990).

 

 

 

Pcfil

Fa

roon
(Par
iEPR
eta}
extn
char
vahu
are]

then

Valur

vart
Chh
2941
be 1i
reac
inao
pron

1991

1 1
Peripheral PSI polypeptides

The 9-kDa PsaC protein carries the terminal electron acceptors FA and
FE. In time-resolved optical studies, FA/FB' backreact with Pm“ in 30 ms at
room temperature, but this charge separation is irreversible at low temperature
(Parrett et 31., 1989). FA and F3 have been shown to be [4Fe-4S] centers by
EPR spectroscopy (Cammack and Evans, 1975), EXAFS studies (McDermott
et 31., 1988), Mossbauer spectroscopy (Evans et 31., 1979), and 19F-NMR core
extrusion studies (Golbeck et 31., 1987a). The EPR spectrum of F A' has
characteristic gvalues of 2.05, 1.94, and 1.86, while the spectrum of F3' has g
values of 2.07, 1.92, and 1.89 (Malkin and Bearden, 1978). When both centers
are reduced either chemically with dithionite or by illumination during freezing,
their high-field and low-field resonances merge, yielding a spectrum with g
values of 2.05, 1.94, 1.92, and 1.89 (Evans et 31., 1974).

The psaC gene has been inactivated in a cyanobacterium, Anabaena
variabilis sp. ATCC 29413 (Mannan et 31., 1991), and in the alga,
Chlamydomonas reinhardtij (Takahashi et 31., 1991). In Anabaena variabilis
29413, the loss of PsaC prevented autotrophic growth and caused the cells to
be light-sensitive, but did not significantly alter stable assembly of the PSI
reaction center (Mannan ct 31., 1991). However, in Chlamydomonas,
inactivation of psaC prevented stable accumulation of the PSI reaction center

proteins and of seven additional peripheral PSI polypeptides (Takahashi et 31.,

1991).

The sequence of the PsaC protein includes an amino acid motif

 

 

knov
four
the (
Fukr
puril
Past:
Esct
Sync
(Zhe

pron

to as
Geo:
the 1
fune
II we

term

 

12

characteristic of bacterial-type ferredoxins, which also bind 2 [4Fe-4S] centers
(Haj et 31., 1987; Oh-oka et 31., 1988). This motif, Cys-X-X—Cys-X-X-Cys-X-X—
X-Cys-Pro, is duplicated in the protein sequence and includes the eight cysteine
ligands to the two iron-sulfur centers (Haj et 31., 1987; Oh-oka et 31., 1988).
The three-dimensional structures of several bacterial-type ferredoxins are
known, revealing that the protein backbone folds back on itself and that the

fourth cysteine in each motif coordinates the opposite iron-sulfur center than

 

the other three cysteines in the motif (Adman et 31., 1973; Stout et 31., 1988;
Fukuyama et 31., 1988). An in Vitro reconstitution system was developed using

purified reaction center protein from Synechococcus sp. PCC 6301 (PCC,

 

Pasteur Culture Collection) and PsaC and PsaD purified from strains of
Escherichia c011 that were expressing, respectively, the psaC gene from
Synechococcus sp. PCC 7002 and the psaD gene from Nostoc sp. PCC 8009
(Zhao et 31., 1990). This system has been used to study B. calf-expressed PsaC
protein with site-directed changes of some of the proposed cysteine ligands to
FA and F3 (Zhao et 3]., 1992). By changing the second cysteine in each motif
to aspartate, as can occur naturally in some ferredoxins (Conover et 31., 1990;
George et 31., 1989), an inactive [3Fe-4S] center was formed, without affecting
the formation of the other [4Fe-4S] center (Zhao et 31., 1992). The remaining
functional [4Fe-4S] center in the mutant proteins behaved similarly to wild type.
It was thus deduced that the amino-terminal motif binds F3 and the carboxyl—
terminal motif binds FA (Zhao et 31., 1992).

The PsaD polypeptide has a mass of 15 - 16 kDa in cyanobacteria and

 

 

of 1:
has :
ferrc
Ano
desc
ICSlt
com
of F
pr 0i
inae
unde
phoi
and

SUpF

fem

on t]

13
of 18 kDa in plants (Chitnis et 31., 1989a; Mfinch et 31., 1988). Cross-linking

has shown that it plays a role in the docking of the diffusible [2Fe-ZS]
ferredoxin to the PSI core (Zilber and Malkin, 1988; Zanetti and Merati, 1987).
Another role for PsaD was discovered using the in vitro reconstitution system
described earlier. Although PsaC was able to bind to the reaction center and
restore electron transfer to FA and FE, the addition of PsaD made the resulting
complex more resistant to detergent and allowed wild-type levels of reduction
of FA and F3 (Li et 31., 1991). Thus PsaD also appears to play a role in the
proper binding of PsaC to the reaction center. The psaD gene has been
inactivated in Smechocysns sp. PCC 6803, resulting in reduced growth rate
under autotrophic conditions, but wild-type grth rate under
photoheterotrophic conditions ( in the presence of glucose as a carbon source
and M-(dichlorophenyl)-N3-dimethylurea [DCMU]) (Chitnis et 31., 19893). This
supports the idea that PsaD plays a role in facilitating electron transfer to
ferredoxin, while not affecting cyclic photosynthesis.

The PsaE protein has a mass of 8 - 10-kDa and appears to be located
on the stromal side of PS1 (Franzen et 31., 1989b). This position was implied
by cross-linking of PsaE to PsaD, which also cross-links to ferredoxin (Oh-oka
et 31., 1989). This association with PsaD suggests that PsaE may play a role in
the association of PsaC and PsaD to the reaction center. The psaE gene has
been inactivated in Synecbocystis 6803 and in Synechococcus sp. PCC 7002, but
the phenotype was essentially wild-type, indicating that PsaE is dispensable for

PS1 function (Chitnis et 31., 1989b; Bryant et 31., 1990). The PsaF protein has a

 

 

 

mas:
whic
char
3]., i
plas
whei

auto

PSI

appr
men
amir
fron
CI 31
dllpl
(on
kDa.
at EU
Cyan
tOge-
“188'
PSI I

discc

14

mass of approximately 17 kDa and has several positively charged domains
which may serve in its proposed role of facilitating binding of the negatively
charged plastocyanin and/or cytochrome cm to the reaction center (Steppuhn et
31., 1988; Franzen et 31., 1989b). The PsaF protein has been cross-linked to
plastocyanin in spinach preparations (Wynn and Malkin, 1988b). However,
when the psaF gene was inactivated in Synechocystzs 6803, the cells grew
autotrophically at wild-type rates, indicating that this protein is dispensable for
PS1 function (Chitnis et 31., 1991). The PsaG protein has a mass of
approximately 10 kDa and, like PsaD, PsaB, and PsaF, does not appear to be
membrane bound (Steppuhn et 31., 1988; Franzen et 31., 19893). The deduced
amino acid sequence of the psaG gene from barley shows high identity to that
from spinach and is weakly homologous to PsaK from Chlamydomonas (Okkels
et 31., 1992). The authors suggest that either psaG or psaK arose by a
duplication of the other, and that they occupy symmetrical positions in PSI
(Okkels et 31., 1992). The PsaH protein has a mass of approximately 10 - 11
kDa, is polar, and appears to be located on the stromal side of PS1 (Steppuhn
et 31., 1989; Franzen et 31., 19893). PsaH does not appear to exist in
cyanobacteria (Koike et 31., 1989; Rhiel and Bryant, 1988). This information
together with a similar pattern of psaH gene regulation as the cab genes,
suggests that PsaH may play a role in facilitating the association of LHC I with
PSI (Steppuhn et 31., 1989).

Improved techniques of gel electrophoresis have allowed the recent

discovery of three very small polypeptides associated with PSI (Koike et 31.,

 

 

1989
kl)a
199C

Psbl

anyi
hydr
(Fra
far

front
fern]

not}

(()ki

bout
lbad
PsaH
been
PsaH
KONG

gene

 

 

15
1989). The P331 and PsaJ proteins both have masses of approximately 4 - 5

kDa, are quite hydrophobic, and are predicted to form a a helix (Moller et 31.,
1990; Ikeuchi et 31., 1991). The PsaI sequence has some identity to helix E of
Psz, inspiring a proposal that it is involved in quinone binding (Moller et 31.,
1990), but there is no direct evidence that either P531 or P831 is associated with
any cofactors. The PsaK protein has a mass of approximately 8 kDa, is quite
hydrophobic, and is predicted to form a single membrane-spanning helix
(Franzen et 31., 19893). No role for PsaK has been definitively assigned thus
far. The most recent PSI protein to be discovered is the 18-kDa PsaL protein
from barley (Okkels et 31., 1991). PsaL is very hydrophobic and is predicted to
form two membrane-spanning a helices (Okkels et 31., 1991). This protein may
not have been discovered in other organisms, because it comigrates with PsaE

(Okkels et 31., 1991).

PSI gene organization

In eucaryotes, the genes that encode the PSI polypeptides are located in
both the nucleus and the plastid. The genes encoded in the plastome are:
psaA, psaB, psaC, p531, and p33], the remaining genes psaD, psaE, psaF, ps3 G,
psaH, psaK, and psaL are encoded in the nucleus. Many of these genes have
been characterized in cyanobacteria, as well, with the notable exception of
psaI-I, which appears to be absent from cyanobacteria (Rhiel and Bryant, 1988;
Koike et 31., 1989). The following is a brief description of the structure of each

gene and a summary of the organisms in which particular genes have been

 

 

char

toba

spin

Chis

In p
Oper
intrc
et al
(Cus
0PP(
1987
psaE
0f 81
is 2113
(G01:

gene:

 

 

 

 

16

characterized.

The psaA and p533 genes have been cloned and sequenced from
tobacco (Shinozaki et 31., 1986), liverwort (Ohyama et 31., 1986), rice
(Hiratsuka et 31., 1989), maize (Fish et 31., 1985), pea (Lehmbeck et 31., 1986),
spinach (Kirsch et 31., 1986), Buglena gracr’lrs (Cushman et 31., 1987),
Chlamydomonas reinhardtii (Kiick et 31., 1987), Synechococcus sp. PCC 7002
(Cantrell and Bryant, 1987), and Synechococcus vulcanus (Shimizu et 31., 1992).
In plants and cyanobacteria, the psaA and p533 genes are arranged in an
operon, with psaA transcribed first. In the green algae, the psaA gene has
introns; in Chlamydomonas reinbardtii psaA is organized in three exons (Kiick
et 31., 1987), while in Euglena graciIis, the psaA gene is split into four exons
(Cushman et 31., 1987). In Chlamydomonas these exons are encoded on
opposite strands, and the transcripts are joined by trans-splicing (Kiick et 31.,
1987; Choquet et 31., 1988; Goldschmidt-Clermont et 31., 1990). The psaA and
psaB genes characterized so far are all highly similar to each other, in the range
of 80% and greater similarity (Golbeck and Bryant, 1991). The psaA sequence
is also homologous to psaB, exhibiting sequence identity of approximately 40%
(Golbeck and Bryant, 1991). This suggests that one of the two reaction center
genes arose by a duplication of the other.

The psaC gene has been cloned and sequenced from tobacco (Shinozaki
et 31., 1986; Hayashida et 31., 1987), rice (Hiratsuka et 31., 1989), liverwort
(Ohyama et 31., 1986), maize (Schantz and Bogorad, 1988), pea (Dunn and

Gray, 1988), wheat (Dunn and Gray, 1988), spinach (Steppuhn et 31., 1989),

 

 

hon:

gene
1988
char

Brya

anot
((iol

5595

6ta]

6! a]

17
Chlamydomonas remlrardtr'i (Takahashi et 31., 1991), Anabaena sp. PCC 7120

(Mulligan and Jackman, 1992), Anabaena variabilis sp. ATCC 29413 (ATCC,
American Type Culture Collection) (Mannan and Pakrasi, 1992),
Synechococcus vulcanus (Shimizu et 31., 1990), Nostoc sp. PCC 8009 (Bryant et
31., 1990), Synechococcus sp. PCC 7002 (Bryant et 31., 1990), and two copies
from Synechocystis sp. PCC 6803 (Anderson and McIntosh, 1991b; Steinmiiller,
1992). In chloroplasts and some cyanobacteria, the psaC gene is flanked by the
genes 11th and ndhE, and is cotranscribed with nth (Schantz and Bogorad,

1988; Anderson and McIntosh, 1991b; Hayashida et 31., 1987). The psaC genes

 

characterized thus far are all highly similar to one another (Golbeck and
Bryant, 1991).

The psaD gene has been cloned and sequenced from the following
cyanobacteria: Nostoc sp. PCC 8009 (Bryant et 31., 1990), Synechococcus sp.
PCC 6301 (Wynn et 31., 1989), and Synecbocystrs sp. PCC 6803 (Chitnis et 31.,
19893). cDNA clones of the psaD gene have been isolated and sequenced
from spinach (Munch et 31., 1988), tomato (Hoffman et 31., 1988), and tobacco
(Yamamoto et 31., 1991). The plant sequences are over 80% identical to one
another, while the cyanobacterial sequences are over 60% similar to each other
(Golbeck and Bryant, 1991). The cyanobacterial sequences are approximately
55% similar to those from plants (Golbeck and Bryant, 1991).

Clones of p535 have been isolated and sequenced from spinach (Miinch
et 31., 1988), barley (Okkels et 31., 1988), Chlamydomonas reinhardtr'i (Franzen

et 31., 1989b), Nostoc sp. PCC 8009 (Bryant et 31., 1990), Synechococcus sp.

 

 

PCC
1990
cyan
appr
high

prev

Ct 31
Sync
sequ
iden
C1115
clon.
1989
rein.
3qu
liver
a]. j
Sasa
3qu
and
seqt

has

 

18
PCC 6301 (Bryant et 31., 1990), Synecbococcus sp. PCC 7002 (Bryant et 31.,

1990), and Synechocystis sp. PCC 6803 (Chitnis et 31., 1989b). The
cyanobacterial genes are much shorter than those from plants and are
approximately 73% identical to one another (Golbeck and Bryant, 1991). The
high degree of similarity suggests some structural or functional constraints that
prevent divergence.

The psaF gene has been cloned and sequenced from spinach (Steppuhn
et 31., 1988), Chlamydomonas reinhardtii (Franzen et 31., 1989b), and
Sjmechocystis sp. PCC 6803 (Chitnis et 31., 1991). The deduced amino acid
sequence from the Synecbocystis sp. PCC 6803 gene is approximately 50%

identical to the deduced amino acid sequences from spinach or

 

 

Chlamydomonas (Chitnis et 31., 1991). The p336 and psaH genes have been
cloned and sequenced from spinach (Steppuhn et 31., 1988; Steppuhn et 31.,
1989), barley (Okkels et 31., 1992; Okkels et 31., 1989) and ChIamydomonas
reinbardtii (Franzen et 31., 1989a). The psaI gene has been cloned and
sequenced from the chloroplast genomes of tobacco (Shinozaki et 31., 1986),
liverwort (Ohyama et 31., 1986), rice (Hiratsuka et 31., 1989), barley (Scheller et
31., 1989b), pea (Nagano et 31., 1991) and wheat (Ogihara, Terachi, and
Sasahuma, unpublished data). The p53] gene has only been cloned and
sequenced from tobacco (Shinozaki et 31., 1986), rice (Hiratsuka et 31., 1989),
and liverwort (Ohyama et 31., 1986). The psaK gene has been cloned and
sequenced from Chlamydomonas reinhardtii (F ranzén et 31., 1989a), and psaL

has been cloned and sequenced from barley (Okkels et 31., 1991).

 

It

 

GE

a bi(
syste
of II
for I
stud;

toda

phor
to b
AT(
Sym
6802
reac

com

moc

(Wi'

19
GENETIC MANIPULATION OF PSI

Procaryotic organisms

Molecular biology offers the researcher the ability to alter an element of
a biological system in order to learn more about that element in the natural
system. This approach is ideal for studying the roles of particular components
of the photosynthetic apparatus. Certain organisms are especially well-suited
for these types of experiments and have been used to great advantage in the
study of photosynthesis. The following is a summary of the organisms used
today for molecular genetic studies of oxygenic photosynthesis.

The cyanobacteria represent excellent organisms for the study of
photosynthesis. Two strains in particular have been used most frequently and
to best advantage, Synechocystis sp. PCC 6803 and Anabaena variabfli's sp.
ATCC 29413. Occasionally, other strains have been used, such as
Synechococcus sp. PCC 7002 or Synechococcus sp. PCC 7942. Synechocystis
6803 is a unicellular cyanobacterium that is naturally competent, and thus is
readily transformable (Grigorieva and Shestakov, 1982). It has a genome size
comparable to E. 0011' and expresses bacterial drug resistance genes effectively
(Williams, 1988). This strain has an active homologous recombination system,
making gene replacement by double recombination a straightforward technique
(Williams, 1988). There does not appear to be an active restriction or
modification system in Synechocystis 6803 that would digest transforming DNA

(Williams, 1988). Of great importance is that this strain may be grown

 

 

 

 

phou

heun

Ines
cond
wfld-
Sync
(VVfl
cruc
Sin:
1991
adv;
Ban
'Ver
Nfls

1989

oil

Cyai

1hr
and

set

20
photoheterotrophically (with DCMU and glucose) (Williams, 1988) or

heterotrophically, under light-activated heterotrophic growth conditions
(LAHG), complete darkness except 5 min of light every 24 h and in the
presence of glucose (Anderson and McIntosh, 1991a). Growth under these
conditions eliminates the selective advantage conferred by the expression of
wild-type photosynthesis genes, allowing for complete segregation of mutations.
Synecbocystis 6803 has approximately 8 - 10 copies of its chromosome per cell
(Williams, 1988); thus in order to obtain a homozygous strain, segregation is
crucial. Techniques for the purification of both PSII and PS1 from
Smechocystrs 6803 have been developed (Rogner et 31., 1990; Noren et 31.,
1991; Burnap et 31., 1989). Synechocystis 6803 has been used to great
advantage in the study of PSII structure and function relationships (Noren and
Barry, 1992; Debus et 31., 1988b; Debus et 31., 1988a; Nixon and Diner, 1992;
Vermaas et 31., 1988; Pakrasi et 31., 1988), PSII biogenesis (Jansson et 31., 1987;
Nilsson et 31., 1990), and PS1 biogenesis (Chitnis et 31., 1989b; Chitnis et 31.,
19893; Chitnis et 31., 1991).

Another cyanobacterium that has been used for the genetic manipulation
of PSI is Anabaena van'abr'II's sp. ATCC 29413. This filamentous
cyanobacterium can grow heterotrophically, using fructose as a carbon source
(Wolk and Shaffer, 1976). Plasmids for the conjugal transfer of DNA into
Anabaena variabilzs 29413 have been engineered (Elhai and Wolk, 1988; Murry
and Wolk, 1991) and a method using a conditionally-lethal gene for the

selection of double recombinants was developed (Cai and Wolk, 1990). The

 

 

 

 

 

prir
whi-
exp
inte
sue]

gen

Eur

phr
ass<
alg:
has
Ph(
higi
rel:
CXti
(Cl
Chi
tee
ant
ger

get

21

primary integration event that occurs after conjugation is single recombination,
which is undesirable for site-directed mutagenesis or gene replacement
experiments (Elhai and Wolk, 1988). By using the 5308 gene, cells that have
integrated conjugated DNA by single recombination are killed when grown on
sucrose (Cai and Wolk, 1990). This strain has been used to inactivate the PSI

genes psaC (Mannan et 31., 1991) and psaB (Toelge ct 31., 1991).

Eucaryotic organisms

Although cyanobacteria are excellent organisms for studying oxygenic
photosynthesis, they lack the additional elements of regulation and complexity
associated with the interaction between the nucleus and plastids in plants and
algae. The development of a technique for the transformation of chloroplasts
has prompted some researchers to use molecular genetics to study
photosynthesis in a species of green algae, Chlamydomonas reinhardtii, and in a
higher plant, tobacco. Chlamydomonas is a unicellular alga with a single,
relatively large chloroplast. The photosynthetic apparatus has been studied
extensively in this organism, both at the protein level and the genetic level
(Chua et 31., 1975; Rochaix and Erickson, 1988). The Chlamydomonas
chloroplast may be transformed using the biolistic particle-bombardment
technique (Boynton et 31., 1988; Newman et 31., 1991; Boynton et 31., 1990),
and cells may be grown heterotrophically on acetate (Harris, 1989). Using a
gene conferring resistance to spectinomycin/streptomycin for selection, the psaC

gene has been inactivated in Chlamydomonas (Takahashi et 31., 1991). In

 

 

 

 

ad

tra

po
chi
11a
Inu
res
rna

(361'

FI)

and
for-
thru
in 0
asse
proj
Sine
accu
the 5

(ilSpc

 

22

addition, a psaB mutant has been restored to photosynthetic competence, by
transforming with a wild-type psaB gene (Bingham et 31., 1991).

Genetic manipulation of photosynthesis in higher plants is also now
possible, using the biolistic technique to transform tobacco (Nicotiana tabacum)
chloroplasts (Svab et 31., 1990; Staub and Maliga, 1992). In tissue culture, these
plants may be grown on glucose, allowing complete segregation of inactivation
mutations of psbA and rbcl, using mutations in the 16S rRNA to confer
resistance to spectinomycin (Svab and Maliga, 1991). Although the genetic
manipulation of PSI genes in tobacco has not yet been attempted, the potential

certainly exists for successful utilization of this organism in the study of PSI.

FOCUS OF THIS STUDY

The body of the work presented in this thesis addresses the development
and utilization of the cyanobacterium Synechocysn's sp. PCC 6803 as a system
for the study of PSI structure/function relationships and biogenesis. The main
thrust of the project was genetic manipulation of the PSI reaction center genes,
in order to gain insight into the structural framework needed for proper
assembly and function of the PSI reaction center. The initial stage of the
project was the isolation and characterization of the psaA-psaB operon from
Syneclrocysn's sp. PCC 6803. This included analysis of psaA-p.933 mRNA
accumulation in cells grown under LAHG conditions, which were to be used for
the selection of mutants in PSI. To ensure that the psaA and psaB genes are

dispensable for the growth of Syneclrocystr's 6803 under LAHG conditions and

 

 

t0

Sin
psa
p52
of I
sim
mu
ten
of 5
mm
liga
zipr
of t
was

rear

 

 

—— c“ -=*=*‘1"-~’-*-c-”"’- —

23

to study PSI biogenesis, both genes were inactivated by interposon mutagenesis.
Since PSII assemblednormally in cells with mutations that inactivated psaA or
psaB, strains were engineered which have genetic inactivation mutations in
psaA and could be used as recipients for PSII site-directed mutations. The lack
of PSI in strains with directed mutations in either psbA or psz allows for
simple and effective purification of PSII in order to analyze the effects of those
mutations. The final stage of the study addressed the role of the [4Fe-4S]
center Fx and of the array of leucines in the reaction center using the technique
of site-directed mutagenesis. A system for the rapid recovery of site-directed
mutants in the psaA gene was developed and utilized to change a potential
ligand to the iron-sulfur cluster Fx and to introduce mutations into the leucine
zipper motif. Analysis of these mutants gives us insight into the role of Fx and
of the leucine zipper in the biogenesis of the PSI reaction center. In addition I
was able to study the participation of FX in electron transport through the

reaction center.

 

 

Adr
ferr

Alle
the
Nat

the
p101

ndh

Syn.
and

Bar.
(192
radi

Bing
With
C6111
Bey
Ran

(195
min

BOy
J0hr
Ch];
Aca

Brill
Dun

Bru<
Phor

thOS

 

 

24
REFERENCES

Adman ET, Sieker LC and Jensen LH (1973) The structure of a bacterial
ferredoxin. J. Biol. Chem. 248:3987-3996.

Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of
the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc.
N3 t1. Acad. Sci. USA 84:5730-5 734.

Anderson SL and McIntosh L (19913) Light-activated heterotrophic growth of
the cyanobacterium Sjmechocystis sp. strain PCC 6803: a blue-light-requiring
process. J. Bacteriol. 173:2761-2767.

Anderson SL and McIntosh L (1991b) Partial conservation of the 5’

ndhE-psa C—nth 3’ gene arrangement of chloroplasts in the cyanobacterium
Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria
and chloroplasts. Plant M01. Biol. 16:487-499.

Barry BA, Bender CJ, McIntosh L, Ferguson-Miller S and Babcock GT
(1988) Photoaccumulation in photosystem I does not produce a phylloquinone
radical. Isr. J. Chem. 28:129-132.

Bingham SE, Xu R and Webber AN (1991) Transformation of chloroplasts
with the psaB gene encoding a polypeptide of the photosystem I reaction
center. FEBS Lett. 292: 137-140.

Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR,
Randolph-Anderson BL, Robertson D, Klein TM, Shark KB and Sanford JC
(1988) Chloroplast transformation in Chlamydomonas with high velocity
microprojectiles. Science 240.1534—1538.

Boynton JE, Gillham NW, Harris EH, Newman SM, Randolph-Anderson BL,
Johnson AM and Jones AR (1990) Manipulating the chloroplast genome of
Chlamydomonas—Molecular genetics and transformation. In: Current Research
in Photosynthesis, Vol. III. Baltscheffsky M, ed. Dordrecht, Netherlands: Kluwer
Academic Publishers, pp. 509-516.

Bruce BD and Malkin R (19883) Structural aspects of photosystem I from
DunalieIIa saIina. Plant Physiol. 88: 1201—1206.

Bruce BD and Malkin R (1988b) Subunit stoichiometry of the chloroplast
photosystem I complex. J. Biol. Chem. 263:7302-7308.

Bryant DA (1987) The cyanobacterial photosynthetic apparatus: comparison of
those of higher plants and photosynthetic bacteria. In: Photosynthetic

 

am
for

Ch
lac

 

 

25

Picoplankton, Canadian Bulletin of Fisheries and Aquatic Sciences, vol. 214.
Platt T and Li WKW, eds. Ottowa: Dept. Fisheries and Oceans, pp. 423-500.

Bryant DA, Rhiel E, De Lorimier R, Zhou J, Stirewalt VL, Gasparich GE,
Dubbs J M and Snyder W (1990) Analysis of phycobilisome and photosystem I
complexes of cyanobacteria. In: Current Research in Photosynthesis, vol. II.
Baltscheffsky M, ed. Dordrecht, Netherlands: Kluwer Academic Publishers, pp.
1-9.

Burnap R, Koike H, Sotiropoulou G, Sherman LA and Inoue Y (1989)
Oxygen evolving membranes and particles from the transformable
cyanobacterium Synechocystis sp. PCC6803. Photosynth. Res. 22:123-130.

Cai Y and Wolk CP (1990) Use of a conditionally lethal gene in Anabaena sp.
strain PCC 7120 to select for double recombinants and to entrap insertion
sequences. J. Bacteriol. 172:3138-3145.

Cammack R and Evans MCW (1975) EPR spectra of iron-sulfur proteins in
dimethylsulphoxide solutions: Evidence that chloroplast photosystem I particles
contain 4Fe-4S centres. Biochem. Biophys. Res. Comm. 67:544-549.

Cantrell A and Bryant DA (1987) Molecular cloning and nucleotide sequence

of the psaA and p833 genes of the cyanobacterium Synechococcus sp. PCC
7002. Plant M01. Biol. 9.45 3-468.

Chitnis PR, Reilly PA and Nelson N (19893) Insertional inactivation of the
gene encoding subunit II of photosystem I from the cyanobacterium
Synechocystis sp. PCC 6803. J. Biol. Chem. 264:18381-18385.

Chitnis PR, Reilly PA, Miedel MC and Nelson N (1989b) Structure and
targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I
from the cyanobacterium Synechocysns sp. PCC 6803. J. Biol. Chem.
264:18374-18380.

Chitnis PR, Purvis D and Nelson N (1991) Molecular cloning and targeted
mutagenesis of the gene psaF encoding subunit III of photosystem I from the
cyanobacterium Synechocysn's sp. PCC 6803. J. Biol. Chem. 266:20146-20151.

Choquet Y, Goldschmidt-Clermont M, Girard-Bascou J, Kiick U, Bennoun P
and Rochaix J-D (1988) Mutant phenotypes support a trans-splicing mechanism

for the expression of the tripartite psaA gene in the C reinhardtii chloroplast.
Cell 52:903-913.

Chua NH, Matlin K and Bennoun P (1975) A chlorophyll-protein complex
lacking in photosystem I mutants of Chlamydomonas reinhardtii J. Cell Biol.

 

 

 

 

67:3

Johl
clus

Cus
P70
In: 1
Net

Del
H1111

OXYI
Del

tyrc

Dei
the
Rb.

Du
gen

Elh
Me

Ev:
ele.
frit

Ev:
of ‘
Bir

EV;
Oxi
ele

Fis
lig]

 

26
67:361-377.

Conover RC, Kowal AT, Fu W, Park J-B, Aono S, Adams MW and
Johnson MK (1990) Spectroscopic characterization of the novel iron-sulfur
cluster in Pyrococcus fun'osus ferredoxin. J. Biol. Chem. 265:8533-8541.

Cushman J C, Hallick RB and Price CA (1987) The gene for the
P700-chlorophyll a protein in Euglena gracihs is interrupted by three introns.

In: Progress in Photosynthesis Research, Vol. IV. Biggens J, ed. Dordrecht,
Netherlands: Martinus Nijhoff Publishers, pp. 667-670.

Debus RJ, Barry BA, Babcock GT and McIntosh L (1988a) Site-directed
mutagenesis identifies a tyrosine radical involved in the photosynthetic
oxygen-evolving system. Proc. Natl. Acad. Sci. USA 85:427-430.

Debus RJ, Barry BA, Sithole I, Babcock GT and McIntosh L (1988b)
Directed mutagenesis indicates that the donor to P280 in photosystem II is
tyrosine-161 of the D1 polypeptide. Biochemistry 27:9071-9074.

Deisenhofer J, Epp 0, Miki K, Huber R and Michel H (1985) Structure of
the protein subunits in the photosynthetic reaction centre of
Rhodopseudomonas Viridls at 3 A resolution. Nature 318:618-624.

Dunn PPJ and Gray JC (1988) Localization and nucleotide sequence of the

gene for the 8 kDa subunit of photosystem I in pea and wheat chloroplast
DNA Plant Mol. Biol. 11:311-319.

Elhai J and Wolk CP (1988) Conjugal transfer of DNA to cyanobacteria.
Methods Enzymol. 167:747-754.

Evans EH, Cammack R and Evans MCW (1976) Properties of the primary
electron acceptor complex of photosystem I in the blue-green alga Chlorogloea
fn’tschii. Biochem. Biophys. Res. Comm. 68: 1212-1218.

Evans EH, Rush JD, Johnson CE and Evans MCW (1979) Mossbauer spectra
of photosystem-I reaction centres from the blue-green alga Chlorogloea fritschii.
Biochem. J. 182: 861-865.

Evans MCW, Reeves SG and Cammack R (1974) Determination of the
oxidation-reduction potential of the bound iron-sulphur proteins of the primary

electron acceptor complex of photosystem I in spinach chloroplasts. FEBS Lett.
49:111-114.

Fish LE, Kiick U and Bogorad L (1985) Two partially homologous adjacent
light-inducible maize chloroplast genes encoding polypeptides of the P700

 

 

 

 

ch10

Fort
ph0‘
Pho

Frat
chat

m0]
Ge:

chat

 

 

27
chlorophyll a-protein complex of photosystem I. J. Biol. Chem. 260:1413-1421.

Forbush B, Kok B and McGloin M (1971) Cooperation of charges in
photosynthetic 02 evolution - II. Damping of flash yield oscillation, deactivation.
Photochem. Photobiol. 14:307-321.

Franzén L-G, Frank G, Zuber H and Rochaix J-D (1989a) Isolation and
characterization of cDNA clones encoding Photosystem I subunits with

molecular masses 11.0, 10.0 and 8.4 kDa from Chlamydomonas reinhardtii Mol.
Gen. Genet. 219:137-144.

Franzen L—G, Frank G, Zuber H and Rochaix J-D (1989b) Isolation and

characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of
photosystem I from Chlamydomonas reinhardtii. Plant M01. Biol. 12:463-474.

Fukuyama K, Nagahara Y, Tsukihara T, Katsube Y, Hase T and Matsubara
H (1988) Tertiary structure of Bacillus thermoproteolyticus [4Fe- 4S]
ferredoxin. J. Mol. Biol. 199183-193.

Gentz R, Rauscher FJ III, Abate C and Curran T (1989) Parallel association
of F os and Jun leucine zippers juxtaposes DNA binding domains. Science
243:1695-1699.

George SJ, Armstrong FA, Hatchikian EC and Thomson AJ (1989)
Electrochemical and spectroscopic characterization of the conversion of the 7Fe
into the 8Fe form of ferredoxin 111 from Desulfovzbn’o africanus. Identification
of a [4Fe- 4S] cluster with one non-cysteine ligand. Biochem. J. 264:275-284.

Golbeck JH, McDermott AE, Jones WK and Kurtz DM (1987a) Evidence for
the existence of [2Fe-2S] as well as [4Fe-4S] clusters among F A, FE and Fx.
Implications for the structure of the Photosystem I reaction center. Biochim.

Biophys. Acta 891: 94-98.

Golbeck JH (1987b) Light-induced charge separation across the photosynthetic
membrane: a proposed structure for the photosystem I reaction center. J.
Membrane Sci. 33: 151-168.

Golbeck JH, Parrett KG, Mehari T, Jones KL and Brand JJ (1988) Isolation
of the intact photosystem I reaction center core containing P700 and iron-sulfur
center Fx. FEBS Lett. 228:268-272.

Golbeck JH (1989) Structure, function, and organization of the photosystem I
reaction center complex. Biochim. BI'Ophys. Acta 895:167-204.

Golbeck JH and Cornelius JM (1986) Photosystem I charge separation in the

 

 

 

28

absence of centers A and B. 1. Optical characterization of center ‘A2’ and
evidence for its association with a 64-kDa peptide. Biochim. Biophys. Acta
849: 16-24.

Golbeck JH (1992) Structure and function of photosystem 1. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 43:293-324.

Golbeck JH and Bryant DA (1991) Photosystem 1. Curr. Topics Bioenerg.
16: 83-177.

Goldschmidt-Clermont M, Girard-Bascou J, Choquet Y and Rochaix J-D
(1990) Trans-splicing mutants of Chlamydomonas reinhardtii. M01. Gen. Gene t.
223:417-425.

Grigorieva G and Shestakov S (1982) Transformation in the cyanobacterium
Synechocystis sp. 6803. FEMS M'crobiol. Lett. 13:367-370.

Hallick RB (1989) Proposals for the naming of chloroplast genes. II. Update to
the nomenclature of genes for thylakoid membrane polypeptides. Plant Mol.
Biol. Reporter 7: 266-275.

Harris EH (1989) The Chlamydomonas sourcebook. A comprehensive guide to
biology and laboratory use. San Diego, CA: Academic Press.

Hayashida N, Matsubayashi T, Shinozaki K, Sugiura M, Inoue K and
Hiyama T (1987) The gene for the 9 kd polypeptide, a possible apoprotein for
the iron-sulfur centers A and B of the photosystem I complex, in tobacco
chloroplast DNA. Curr. Genet. 12:247-250.

Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M,
Kondo C, Honji Y, Sun C-R, Meng B-Y, Li Y-Q, Kanno A, Nishizawa Y,
Hirai A, Shinozaki K and Sugiura M (1989) The complete sequence of the rice
(Ozyza sativa) chloroplast genome: intermolecular recombination between
distinct tRNA genes accounts for a major plastid DNA inversion during the
evolution of the cereals. Mol. Gen. Genet. 217: 185-194.

Ho K and Krogmann DW (1982) Photosynthesis. In: The Biology of
Cyanobacteria. Carr NG and Whitton BA, eds. Los Angeles: University of
California Press, pp. 191-214.

Hoffman NE, Pichersky E, Malik VS, Ko K and Cashmore AR (1988)

Isolation and sequence of a tomato clone encoding subunit II of the
photosystem I reaction center. Plant Mol. Biol. 10:435-445.

H¢j PB, Svendsen I, Scheller HV and Moller BL (1987) Identification of a

 

 

ch10
A at

29

chloroplast-encoded 9-kDa polypeptide as a 2[4Fe-4S] protein carrying centers
A and B of photosystem I. J. Biol. Chem. 262:12676-12684.

Hu J C, O’Shea EK, Kim PS and Sauer RT (1990) Sequence requirements for
coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions.
Science 250:1400-1403.

Ikegami I and Itoh S (1988) Absorption spectroscopy of P-700-enriched
particles isolated from spinach. Is P-700 a dimer or a monomer?. Biochim.
Biophys. Acta 934:39—46.

Ikeuchi M, Nyhus KJ, Inoue Y and Pakrasi HB (1991) Identities of four
low-molecular-mass subunits of the photosystem I complex from Anabaena
van'abilis ATCC 29413. Evidence for the presence of the ps3] gene product in
a cyanobacterial complex. FEBS Lett. 287:5 -9.

Jansson C, Debus RJ, Osiewacz HD, Gurevitz M and McIntosh L (1987)
Construction of an obligate photoheterotrophic mutant of the cyanobacterium
Synechocystis 6803. Plant Physiol. 85:1021-1025.

Kirsch W, Seyer P and Herrmann RG (1986) Nucleotide sequence of the
clustered genes for two P700 chlorophyll a apoproteins of the photosystem I
reaction center and the ribosomal protein 814 of the spinach plastid

chromosome. Curr. Genet. 10843-855.

Koike H, Ikeuchi M, Hiyama T and Inoue Y (1989) Identification of
photosystem I components from the cyanobacterium, Synechococcus vulcanus
by N-termina] sequencing. FEBS Lett. 253:257-263.

Kok B (1956) On the reversible absorption change at 705 mu in photosynthetic
organisms. Biochim. Biophys. Acta 22:399-401.

Kok B (1957) Absorption changes induced by the photochemical reaction of
photosynthesis. Nature 179:583-584.

Kok B, Forbush B and McGloin M (1970) Cooperation of charges in
photosynthetic 0; evolution - 1. A linear four step mechanism. Photochem.
Photobiol. 11:457-475.

Kfick U, Choquet Y, Schneider M, Dron M and Bennoun P (1987) Structural
and transcriptional analysis of two homologous genes for the P700 chlorophyll

a-apoproteins in Chlamydomonas reinhardii: evidence for in vivo trans-splicing.
EMBO J. 6:2185-2195.

Kossel H, Dory I, Igloi G and Maier R (1990) A leucine-zipper motif in

 

 

abst
Res

Mal
and
Else

30
photosystem 1. Plant Mol. Biol. 15:497-499.

Landschulz WH, Johnson PF and McKnight SL (1988) The leucine zipper: A
hypothetical structure common to a new class of DNA binding proteins. Science
240:1759-1764.

Landschulz WH, Johnson PF and McKnight SL (1989) The DNA binding
domain of the rat liver nuclear protein C/EBP is bipartite. Science
243:1681-1688.

Lehmbeck J, Rasmussen OF, Bookjans GB, Jepsen BR, Stummann BM and
Henningsen KW (1986) Sequence of two genes in pea chloroplast DNA coding

for 84 and 82 kDa polypeptides of the photosystem I complex. Plant Mol. Biol.
723-10.

Li N, Zhao J, Warren PV, Warden JT, Bryant DA and Golbeck JH (1991)
PsaD is required for the stable binding of PsaC to the photosystem I core
protein of Synechococcus sp. PCC 6301. Biochemistry 307863-7872.

Malkin R and Bearden AJ (1978) Membrane-bound iron-sulfur centers in
photosynthetic systems. Biochim. Biophys. Acta 505:147-181.

Mannan RM and Pakrasi HB (1992) Molecular analysis of the psaC gene
encoding the F A/FB apoprotein of photosystem I in the filamentous
cyanobacterium Anabaena variabihs ATCC 29413. Plant Physiol. 98: 798-800.

Mannan RM, Whitmarsh J, Nyman P and Pakrasi HB (1991) Directed
mutagenesis of an iron-sulfur protein of the photosystem I complex in the
filamentous cyanobacterium Anabaena variabzhs ATCC 29413. Proc. Natl.
Acad. Sci. USA 88:10168-10172.

Mansfield RW and Evans MCW (1985) Optical difference spectrum of the
electron acceptor A0 in photosystem 1. FEBS Lett. 190:237-241.

Mansfield RW, Hubbard JAM, Nugent JHA and Evans MCW (1987)
Extraction of electron acceptor A, from pea photosystem I. FEBS Lett.
220:74-78.

Mathis P, Ikegami I and Setif P (1988) Nanosecond flash studies of the
absorption spectrum of the Photosystem I primary acceptor A0. Photosynth.
Res. 16:203-210.

Mathis P and Rutherford AW (1987) The primary reactions of photosystem I
and II of algae and higher plants. In: Photosynthesis. Amesz J, ed. London:
Elsevier Science Publishers, pp. 63-96.

 

 

MCI
Sane
I: at

MCI
Fe-S

Mo]
Hall

poly
Balt

Mu]
the

cyar

Mm
of t]
rep]

M131

enc<
pho

Nag
anai
Cur

Nev
Tar
Gel

Nils
a C(
pelt

Nix.
cen
ma]

N01
OXY.

 

31

McDermott AE, Yachandra VK, Guiles RD, Britt RD, Dexheimer SL,
Sauer K and Klein MP (1988) Low-potential iron-sulfur centers in photosystem
I: an X-ray absorption spectroscopy study. Biochemistry 27:4013-4020.

McDermott AE, Yachandra VK, Guiles RD, Sauer K, Klein MP, Parrett
KG and Golbeck JH (1989) EXAFS structural study of Fx, the low-potential
Fe-S center in photosystem I. Biochemistry 28:8056-8059.

Moller BL, Scheller HV, Okkels JS, Koch B, Andersen B, Nielsen HL, Olsen 1,
Halkier BA and Hoj PB (1990) Chloroplast encoded photosystem I
polypeptides of barley. In: Current Research in Photosynthesis, Vol II.
Baltscheffsky M, ed. Netherlands: Kluwer Academic Publishers, pp. 523-530.

Mulligan ME and J ackman DM (1992) Nucleotide sequence and expression of
the gene for the 9 kDa F A/FB component of Photosystem I from the
cyanobacterium Anabaena sp. PCC7120. Plant Mol. Biol. 18:803-808.

 

Murry MA and Wolk CP (1991) Identification and initial utilization of a portion
of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of
replication in Anabaena sp. strain M-131. Mol. Gen. Genet. 227:113-119.

Miinch S, Ljungberg U, Steppuhn J, Schneiderbauer A, Nechushtai R,
Beyreuther K and Herrmann RG (1988) Nucleotide sequences of cDNAs
encoding the entire precursor polypeptides for subunits II and III of the
photosystem I reaction center from spinach. Curr. Genet. 14:511-518.

Nagano Y, Matsuno R and Sasaki Y (1991) Sequence and transcriptional
analysis of the gene cluster trnQ—zpr-psaI-ORF231-petA in pea chloroplasts.
Curr. Genet. 20:431-436.

Newman SM, Gillham NW, Harris EH, Johnson AM and Boynton JE (1991)
Targeted disruption of chloroplast genes in Chlamydomonas rernhardtii. Mol.
Gen. Genet. 230265-74.

 

Nilsson F, Andersson B and Jansson C (1990) Photosystem II characteristics of

a constructed Synechocystis 6803 mutant lacking synthesis of the D1
polypeptide. Plant Mo]. Biol. 14:1051-1054.

Nixon PJ and Diner BA (1992) Aspartate 170 of the photosystem II reaction
center polypeptide D1 is involved in the assembly of the oxygen-evolving
manganese cluster. Biochemistry 31:942-948.

Noren GH, Boerner RJ and Barry BA (1991) EPR characterization of an
oxygen-evolving photosystem II preparation from the transformable

   

 

cyant

Nore
exhil
Bloc,

Norr
r6501
Acat

Non
doul
chlm

 

32
cyanobacterium Synechocystrls 6803. Biochemistry 303943-3950.

Noren GH and Barry BA (1992) The YF161D1 mutant of Synechocystrls 6803
exhibits an EPR signal from a light-induced photosystem II radical.
Biochemistry 31:3335-3342.

Norris JR, Uphaus RA, Crespi HL and Katz JJ (1971) Electron spin
resonance of chlorophyll and the origin of signal I in photosynthesis. Proc. Natl.
Acad. Sci. USA 68:625-628.

Norris JR, Scheer H, Druyan ME and Katz JJ (1974) An electron-nuclear
double resonance (Endor) study of the special pair model for photo-reactive
chlorophyll in photosynthesis. Proc. Nat]. Acad. Sci USA 71:4897-4900.

O’Keefe DP (1988) Structure and function of the chloroplast cytochrome bf
complex. Photosyrrth. Res. 17:189-216.

O’Shea EK, Klemm JD, Kim PS and Alber T (1991) X-ray structure of the
GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254:539-544.

O’Shea EK, Rutkowski R and Kim PS (1992) Mechanism of specificity in the
F os-Jun oncoprotein heterodimer. Cell 68:699-708.

Oh-oka H, Takahashi Y, Kuriyama K, Saeki K and Matsubara H (1988) The
protein responsible for center A/B in spinach photosystem I: isolation with
iron-sulfur c1uster(s) and complete sequence analysis. J. Biochem. 103:962-968.

Oh-oka H, Takahashi Y and Matsubara H (1989) Topological considerations
of the 9-kDa polypeptide which contains centers A and B, associated with the

14— and 19- kDa polypeptides in the photosystem I complex of spinach. Plant
Cell Physiol. 30: 869-875.

Ohyama K, F ukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono
K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H and Ozeki H (1986)
Chloroplast gene organization deduced from complete sequence of liverwort
Marchantia polymorpha chloroplast DNA Nature 322:5 72-5 74.

Okkels JS, Jepsen LB, Honberg LS, Lehmbeck J, Scheller HV, Brandt P,
H¢yer-Hansen G, Stummann B, Henningsen KW, Von Wettstein D and
Meller BL (1988) A cDNA clone encoding a 10.8 kDa photosystem I
polypeptide of barley. FEBS Lett. 237:108-112.

Okkels JS, Scheller HV, Jepsen LB and Moller BL (1989) A cDNA clone
encoding the precursor for a 10.2 kDa photosystem I polypeptide of barley.
FEBS Lett. 250:575-579.

 

 

 

33

Okkels JS, Scheller HV, Svendsen I and Meller BL (1991) Isolation and
characterization of a cDNA clone encoding an 18-kDa hydrophobic

photosystem I subunit (PSI-L) from barley (Hordeum vulgare L.). J. Biol.
Chem. 266:6767-6773.

Okkels JS, Nielsen VS, Scheller HV and Moller BL (1992) A cDNA clone
from barley encoding the precursor from the photosystem I polypeptide PSI-G:
Sequence similarity to PSI-K. Plant Mol. Biol. 18:989-994.

Pakrasi HB, Williams JGK and Arntzen CJ (1988) Targeted mutagenesis of
the pst and pst genes blocks photosynthetic electron transport: evidence for
a functional role of cytochrome b559 in photosystem II. BABO J. 7:325-332.

Palace GP, Franke JE and Warden JT (1987) Is phylloquinone an obligate
electron carrier in photosystem 1?. FEBS Lett. 215:58-62.

Parrett KG, Mehari T, Warren PG and Golbeck JH (1989) Purification and
properties of the intact P-700 and Fx-containing Photosystem I core protein.
Biochim. Biophys Acta 973:324-332.

Petrouleas V, Brand JJ, Parrett KG and Golbeck JH (1989) A Mossbauer
analysis of the low-potential iron-sulfur center in photosystem I: spectroscopic
evidence that Fx is a [4Fe-4S] cluster. Biochemistry 28:8980-8983.

Pu WT and Struhl K (1991) The leucine zipper symmetrically positions the
adjacent basic regions for specific DNA binding. Proc. Natl. Acad. Sci. USA
88:6901—6905.

Rhiel E and Bryant DA (1988) Preliminary results concerning the psaC, psaD,
psaE, and psaF genes and their products in the cyanobacteria Synechococcus
sp. PCC 7002 and Nostoc sp. PCC 8009. In: Light-Energy Transduction in
Photosynthesis: Higher Plant and Bacterial Models. Stevens SEJr and Bryant
DA, eds. Rockville,MD: American Society of Plant Physiologists, pp. 320-323.

Rochaix JD and Erickson J (1988) Function and assembly of photosystem II:
genetic and molecular analysis. T IBS 13:56-59.

Rutherford AW (1988) Photosystem II, the oxygen evolving photosystem. In:
Light-Energy Transduction in Photosynthesis: Higher Plant and Bacterial
Models. Stevens SEJr and Bryant DA, eds. Rockville, MD: American Society of
Plant Physiologists, pp. 163-177.

Rogner M, Nixon PJ and Diner BA (1990) Purification and characterization of
photosystem I and photosystem II core complexes from wild-type and

 

 

phyc
Biol.

Sam
inter
algal
Rob

Sam
diffe

Schz
psat

Scht
pho
Blur

Sch:
(19%
by t

SCht
sub]

34

phycocyanin-deficient strains of the cyanobacterium Synechocystis PCC 6803. J.
Biol. Chem. 265:6189—6196.

Sandmann G, Scherer S and Edger P (1984) General aspects of location and
interaction of respiratory and photosynthetic electron transport in blue-green
algae. In: Compartments in Algal Cells and Their Interactions. Wiessner W,
Robinson D and Starr RC, eds. Berlin: Springer-Verlag, pp. 207-217.

 

Sandmann G (1986) Formation of plastocyanin and cytochrome c-553 in
difierent species of blue-green algae. Arch. Microbiol. 145:76-79.

Schantz R and Bogorad L (1988) Maize chloroplast genes nth, ndhE, and
psaC. Sequences, transcripts and transcript pools. Plant Mol. Biol. 11:239-247.

Scheller HV, Svendsen I and Moller BL (1989a) Subunit composition of
photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster. J.
Biol. Chem. 264:6929—6934.

Scheller HV, Okkels JS, Haj PB, Svendsen I, Roepstorff P and Moller BL
(1989b) The primary structure of a 4.0-kDa photosystem I polypeptide encoded
by the chloroplast psaI gene. J. Biol. Chem. 264:18402-18406.

Schoeder H-U and Lockau W (1986) Phylloquinone copurifies with the large
subunit of photosystem 1. FEBS Lett. 199:23-27.

Setif P, Ikegami I and Biggins J (1987) Light-induced charge separation in
Photosystem I at low temperature is not influenced by vitamin K-1. Biochim.
Biophys. Acta 894:146-15 6.

Shimizu T, Hiyama T, Koike H and Inoue Y ( 1990) Nucleotide sequence of
the psaC gene of the cyanobacterium Synechococcus vulcanus. Nucl. Acids Res.
18:3644.

 

Shimizu T, Hiyama T, Ikeuchi M and Inoue Y (1992) Nucleotide sequences of
the psaA and psaB genes encoding the Photosystem I core proteins from the
thermophilic cyanobacterium Synechococcus vulcanus. Plant Mol. Biol.

18:785-791.

Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi
T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C,
Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K,
Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H and Sugiura M (1986)
The complete nucleotide sequence of the tobacco chloroplast genome: its gene
organization and expression. EMBO J. 5:2043-2049.

   

 

Stat
inco
4:39

Stei
cyar

Stet
Lot
enc:

35

Staub JM and Maliga P (1992) Long regions of homologous DNA are
incorporated into the tobacco plastid genome by transformation. Plant Cell
4:39-45.

Steinmiiller K (1992) Identification of a second psaC gene in the
cyanobacterium Sjmechocystrls sp. PCC6803. Plant Mol. Biol. (in press)

Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thummler F,
Lottspeich F and Herrmann RG (1988) Nucleotide sequence of cDNA clones
encoding the entire precursor polypeptides for subunits IV and V of the
photosystem I reaction center from spinach. FEBS Lett. 237:218-224.

Steppuhn J, Hermans J, Nechushtai R, Herrmann GS and Herrmann RG
(1989) Nucleotide sequences of cDNA clones encoding the entire precursor
polypeptide for subunit VI and of the plastome-encoded gene for subunit VII
of the photosystem I reaction center from spinach. Curr. Genet. 16:99-108.

Stout GH, T urley LC, Sieker LC and Jensen LH (1988) Structure of
ferredoxin I from Azotobacter vinelandri. Proc. Na tl. Acad. Sci. USA
85: 1020-1022.

Svab Z, Hajdukiewicz P and Maliga P (1990) Stable transformation of plastids
in higher plants. Proc. Natl. Acad. Sci. USA 87:85 26-8530.

Svab Z and Maliga P (1991) Mutation proximal to the tRNA binding region of
the Nicotiana plastid 16S rRNA confers resistance to spectinomycin. Mol. Gen.
Genet. 228:316-319.

Takahashi Y, Goldschmidt-Clermont M, Soen S-Y, Franzen L-G and Rochaix
J -D (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii:

insertional inactivation of the psaC gene encoding the iron sulfur protein
destabilizes photosystem I. EMBO J. 10:2033-2040.

Toelge M, Ziegler K, Maldener I and Lockau W (1991) Directed mutagenesis
of the gene psaB of Photosystem I of the cyanobacterium Anabaena variabilis
ATCC 29413. Biochim. Biophys. Acta 1060233-236.

Turner R and Tjian R (1989) Leucine repeats and an adjacent DNA binding
domain mediate the formation of functional cFos-cJun heterodimers. Science
243: 1689-1694.

Vermaas WFJ, Rutherford AW and Hansson O (1988) Site-directed
mutagenesis in photosystem II of the cyanobacterium Synechocystrs sp. PCC
6803: donor D is a tyrosine residue in the D2 protein. Proc. Natl. Acad. Sci.
USA 85:8477—8481.

 

 

 

 

 

V05
Phc
Bio
Wa

abs
con

We

Wil
phc
680

 

36

Vos MH and van Gorkom HJ (1988) Thermodynamics of electron transport in
Photosystem I studied by electric field-stimulated charge recombination.
Biochim. Biophys. Acta 934: 293-3 02.

Warden JT and Golbeck JH (1986) Photosystem I charge separation in the
absence of centers A and B. 11. ESR spectral characterization of center ‘X’ and
correlation with optical signal ‘A2’. Biochim. Biophys. Acta 849.25-31.

Webber AN and Malkin R (1990) Photosystem I reaction-centre proteins
contain leucine zipper motifs. A proposed role in dimer formation. FEBS Lett.
264:1-4.

Williams JGK (1988) Construction of specific mutations in photosystem II
photosynthetic reaction center by genetic engineering methods in Synechocystis
6803. Methods Enzymol. 167: 766-778.

Wolk CP and Shaffer PW (1976) Heterotrophic micro- and macrocultures of a
nitrogen-fixing cyanobacterium. Arch. Microbiol. 110:145-147.

 

Wynn RM and Malkin R (19883) Characterization of an isolated chloroplast
membrane Fe-S protein and its identification as the photosystem I Fe-SA/Fe-SB
binding protein. FEBS Lett. 229.293-297.

 

Wynn RM and Malkin R (1988b) Interaction of plastocyanin with photosystem
I: a chemical cross-linking study of the polypeptide that binds plastocyanin.
Biochemistry 27:5863-5 869.

Wynn RM, Omaha J and Malkin R (1989) Structural and functional properties
of the cyanobacterial photosystem I complex. Biochemistry 28:5554-5560.

Yamamoto Y, Tsuji H, Hayashida N, Inoue K and Obokata J (1991)
Nucleotide sequence of cDNA clones encoding PSI-D2 protein of photosystem
I in Nicotiana sylvestrrls. Plant Mol. Biol. 17:125 1-125 4.

 

Zanetti G and Merati G (1987) Interaction between photosystem I and

ferredoxin: identification by chemical cross-linking of the polypeptide which
binds ferredoxin. Eur. J. Biochem. 169:143-146.

 

Zhao J, Warren PV, Li N, Bryant DA and Golbeck JH (1990) Reconstitution
of electron transport in photosystem I with PsaC and PsaD proteins expressed
in Escherichia coli. FEBS Lett. 276:175-180.

Zhao J, Li N, Warren PV, Golbeck JH and Bryant DA (1992) Site-directed
conversion of a cysteine to aspartate leads to the assembly of a [3Fe-4S] cluster

 

 

in P
BIO:
2le
pho

Zill
phc

 

 

37

in PsaC of photosystem I. The photoreduction of FA is independent of F3.
Biochemistry 31:5093-5099.

Ziegler K, Lockau W and Nitschke W (1987) Bound electron acceptors of
photosystem 1. Evidence against the identity of redox center A, with
phylloquinone. FEBS Lett. 217: 16-20.

Zilber AL and Malkin R (1988) Ferredoxin cross-links to a 22 kD subunit of
photosystem 1. Plant Physiol. 88:810-814.

 

 

 

 

 

JIM.
P

 

959

the

fro

pri

all

ICE

ex}

ph

Mr

 

CHAPTER2

Expression of photosynthesis genes in the cyanobacterium Synechocystis sp.
PCC 6803: psaA-psaB and psbA transcripts accumulate in dark-grown cells

Published as Smart, LB. and McIntosh, L. Plant Molecular Biology vol. 17, pp.

959-971 (1991).

 

INTRODUCTION

 

The study of oxygenic photosynthesis has been expedited by the use of
the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system.
The photosynthetic apparatus in cyanobacteria is highly homologous to that
from higher plants, including subunit composition, functional components, and
primary sequence (Ho and Krogmann, 1982). Synechocystis 6803 is very

amenable to molecular genetic manipulation. It is naturally competent, is

 

readily transformable, has an active homologous recombination mechanism,

expresses bacterial drug resistance genes, and can be grown

 

photoheterotrophically (Williams, 1988) or heterotrophically (Anderson and
McIntosh, 1991). Considerable progress has been made in the study of PSH in

Synechocystis 6803, including cloning and mutagenesis of the genes for the D1

38

 

 

 

"K

he
sel

an:

ext
Ct
3pc
poi
sul
an:
19:
Cat

[4i

 

 

39
and D2 core polypeptides, psbA (present in three copies) and psz (present in

two copies) (Debus et a1., 1988; Jansson et al., 1987). In addition, mutagenesis
of two peripheral proteins of PSI has been accomplished in Synechocystis 6803

(Chitnis et a1., 1989a; Chitnis et 31., 1989b). The recent discovery of

 

heterotrophic growth conditions for Synechocystis 6803, which negate the
selective advantage of wild-type PSI, make it an excellent system for molecular
analysis of the core of PS1 (Anderson and McIntosh, 1991). Synechocystis 6803

grows in the dark if supplied with glucose and 5 min of light every 24 h

 

(Anderson and McIntosh, 1991). Anderson and McIntosh have termed this
light-activated heterotrophic growth (LAHG) (Anderson and McIntosh, 1991).
The biochemistry of PSI from plants and cyanobacteria has been
extensively characterized (Bengis and Nelson, 1977; Lundell et a1., 1985; Wynn

et al., 1989). Electron transfer components have been defined by various
spectroscopic techniques and have been shown to be associated with three
polypeptides, the two P700-apoproteins (PsaA and PsaB) and the 9 kDa iron-

sulfur (Fe-S) protein (PsaC) (Golbeck, 1989). PsaA and PsaB bind P700, A0, A1,

 

and Fx, while F A and F3 are bound to PsaC (Golbeck et al., 1988; Hoj et 31.,

1987). PsaA and PsaB also bind approximately 100 chlorophyll molecules for

 

each P700 (Golbeck, 1989). The electron transfer component Fx is most likely a
[4Fe-4S] center bound by four cysteine residues, two in PsaA and two in PsaB
(Golbeck and Bryant, 1991). Immediately adjacent to those conserved cysteines
in both PsaA and PsaB are series of conserved leucines, spaced seven residues

apart, in regions predicted to form a-helices. It has been proposed that these

 

 

he

M:

dif

19:

Et
a].
p12

g6

19
an

a].

ps.

an

dil

TC!

 

40

helices may interact to form a leucine zipper (Kossel et a1., 1990; Webber and
Malkin, 1990).
The psaA and psaB genes have been cloned and sequenced from many

different organisms, including: maize (Fish et a1., 1985), spinach (Kirsch et a1.,

 

1986), pea (Lehmbeck et al., 1986), tobacco (Shinozaki et a1., 1986), rice
(Hiratsuka et a1., 1989), Marchantia polymorpha (Ohyama et a1., 1986),

Euglena gracilrs (Cushman et a1., 1987), Chlamydomonas rer'nhardtii (Kiick et

a1., 1987), and Synechococcus Sp. PCC 7002 (Cantrell and Bryant, 1987). In

 

plants and algae, these genes are located in the plastid genome. The psaA
genes cloned from algae contain introns, and in C. reinhardtii the exons are
joined by trans-splicing (Choquet et a1., 1988; Goldschmidt-Clermont et a1.,
1990). In plants and Synechococcus 7002, the genes are arranged in tandem
and have been shown to be co-transcribed (Cantrell and Bryant, 1987; Chen et
al., 1990; Meng et a1., 1988; Rodermel and Bogorad, 1985). All the psaA and
psaB genes sequenced so far show a very high degree of conservation (Cantrell
and Bryant, 1987).

The expression of photosynthesis genes in the process of plastid

 

 

differentiation and chloroplast maturation has been the subject of intense
research and has revealed the significant role of post-transcriptional regulation
(for a recent review, see (Mullet, 1988)). The expression of photosynthesis
genes has also been studied in cyanobacteria. Lonneborg et a]. (Lonneborg et
a1., 1988) showed there is greater accumulation of psbA transcript under

high-light conditions relative to low-light conditions in Synechococcus sp. PCC

 

   

cc
79
of

CI]

Sc

gel
Phl

CxI

Wili

 

41
6301. Greater accumulation of psbA and psz mRNA in high-light relative to

low-light was also seen in Synechocystis 6803 (Mohamed and J ansson, 1989).
However, no hybridization to mRNA from psbA, psz, or rbcL (encoding the
large subunit of ribulose-bisphosphate carboxylase/oxygenase [Rubisco]) could
be detected from Sjmechocystis 6803 grown in the dark without glucose
(Mohamed and Jansson, 1989). In Synechocystis 6803, copies 2 and 3 of psbA
are nearly identical, while copy 1 is more divergent (Metz et a1., 1990; Osiewacz
and McIntosh, 1987). By mutagenesis of two of the three copies of psbA,
Mohamed and J ansson (1989) demonstrated that copy 1 is not expressed, while
copies 2 and 3 are expressed in Synechocystis 6803. Synechococcus sp. PCC

7942 has three copies of psbA as well. However, copy 1, which encodes form I

 

 

of the D1 protein, is expressed at much higher levels than copies 2 or 3, which
encode form II of D1 (Bustos et a1., 1990; Schaefer and Golden, 1989b;
Schaefer and Golden, 1989a). In Synechococcus 7942, the accumulation of
mRNA from copies 2 and 3 decreases with decreasing light intensity, while the
accumulation of mRNA from copy 1 increases with decreasing light intensity
(Bustos et a1., 1990; Schaefer and Golden, 1989b).

This paper describes the cloning and sequencing of the psaA and psaB
genes from Synechocystis sp. PCC 6803, and their use, together with other
photosynthesis genes, as probes in the analysis of photosynthesis gene

expression. Quantification of transcript accumulation for psaA-psaB and psbA

 

 

in light-grown cells, cells put in total darkness with glucose, and LAHG cells

will be presented.

 

 

8.

al-

 

 

42
MATERIAIS AND METHODS

Materials

All chemicals and reagents used were of high quality and were obtained
from Sigma Chemical Co. (St.Louis, MO), Boehringer Mannheim Biochemicals
(Indianapolis, IN), or Research Organics (Cleveland, OH). Restriction and
modification enzymes were purchased from New England Biolabs (Beverly,
MA) or Gibco BRL (Gaithersburg, MD). Nitrocellulose was obtained from
Schleicher and Schuell (Keene, NH). [a-32P]dATP was obtained from

Amersham (Arlington Heights, IL).

Strain and growth conditions

A glucose-tolerant (Williams, 1988), dark-growth-adapted (Anderson and
McIntosh, 1991) strain of Synechocystis sp. PCC 6803 was grown in BG-11
medium supplemented with 5 mM TES (N-tris[hydroxymethyl]methyl-2-
aminoethanesulfonic acid) (pH 8.0) as previously described (Jansson et al.,
1987). Glucose was added to a final concentration of 5 mM. Cell grth was
measured by absorbance at 730 nm (OD730). Light-grown cells received
20 umol'm'zs1 of continuous white light. LAHG conditions were: total
darkness except for 5 min of white light (40 umol-m‘zs'l) every 24 h as
previously described (Anderson and McIntosh, 1991). For dark incubations,
cells were grown to early exponential phase (OD730=0.2-0.5), then wrapped in

aluminum foil and put in a light-tight box in a darkroom accessed by a

 

 

 

 

St

S)

W:

SC

 

 

 

43

darkened hallway for 24, 30, or 48 h. Cells grown without glucose were
incubated for 48 h only in complete darkness as described (Mohamed and

J ansson, 1989).

Cloning

All nucleic acid manipulations were performed using standard techniques
(Sambrook et al., 1989), except where otherwise noted. All DNA fragments
used as hybridization probes (see Table 2.1) were purified by agarose gel
electrophoresis and were random primer-labelled with 32P (Feinberg and
Vogelstein, 1983). Genomic DNA isolated from Synechocystis 6803 was
purified on cesium chloride gradients as described (Williams, 1988).
Conditions for hybridization with the Synechococcus 7002 probes were: 1X
Denhardt’s solution (0.2 mg/ml Ficoll, 0.2 mg/ml polyvinylpyrrolidone, 0.2 mg/ml
bovine serum albumin), 3X SSC (a solution of 3 M NaCl and 0.3 M sodium
citrate constitutes 20X SSC), 35% v/v formamide, 0.1% w/v sodium dodecyl
sulfate (SDS), 40 mg/l salmon sperm DNA, 37°C. Hybridization conditions for
Synechocystis 6803 probes were the same except 50% v/v formamide was used.
Blots were washed with 6X SSC; two times at 25 °C, then once at 37°C. A
mini-library was constructed by ligating size-fractionated Synechocystis 6803
Kpn I fragments purified from an agarose gel into pUC119 (Vieira and
Messing, 1987). Escherichia coIiHB101 (Boyer and Roulland-Dussoix, 1969)
was transformed with the ligation mixture and ampicillin resistant colonies were

screened by colony hybridization (Grunstein and Hogness, 1975). A AEMBL3

 

 

 

lit

19

44
library of Synechocystis 6803 DNA was previously described (Jansson et a1.,

1987)

Sequencing and sequence analysis

DNA sequence was determined from either single-stranded or double-
stranded templates generated from pUC118 or pUC119 (Vieira and Messing,
1987) by the chain-termination method (Sanger et a1., 1977) using Sequenase
version 2 (US. Biochemical, Cleveland, OH) as described by the manufacturer.
Nested deletions were generated using exonuclease III and mung bean nuclease
(Henikoff, 1984). Oligonucleotides for use as sequencing primers were
synthesized using an Applied Biosystems DNA synthesizer. These primers were
used to sequence those regions not recovered in the series of exonuclease III
deletions. The polymerase chain reaction (PCR) was performed using genomic
Synechocystis 6803 DNA, two of the aforementioned Oligonucleotides, and
AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk, CT), as described by the
manufacturer. The amplified fragment was purified, digested, and subcloned
into pUC118 for sequencing. DNA sequence analysis was performed using
Editbase (courtesy of N. Neilson, Purdue University), MCF and Amphi (A. R.
Crofts, Univ. of Illinois) and the programs of the University of Wisconsin

Genetics Computer Group.

 

 

 

 

 

45
RNA isolation and northern blots

RNA was isolated from cells in late exponential phase (OD730=0.7-0.9)
using a previously described technique (Golden et al., 1987), except that
immediately after harvest, the cells were frozen at -70°C in BG-11, then
thawed. Great care was taken to perform manipulations in complete darkness
or very dim light until the cells were exposed to chloroform. RNA was
denatured, fractionated on agarose/formaldehyde gels, and transferred to
nitrocellulose. Molecular sizes were estimated by comparison to an ethidium
bromide-stained lane of a 9.5-0.24 kb RNA ladder (Gibco-BRL). Northern
blots were hybridized (1X Denhardt’s solution, 3X SSC, 30 mM Tris pH 8.0, 0.1
mM EDTA, 50% v/v formamide, 0.1% w/v SDS, 40 mg/l salmon sperm DNA,
37°C) with 32F random primer-labelled probes (see Table 2.1), washed with 6X
SSC, then quantified using a Betascope machine (Betagen, Waltham, MA)
and/or exposed to x-ray fihn. The blots were then stripped using boiling water
with 0.2% w/v SDS (three times, 10 min each), verified to be non-radioactive,

and reprobed.

RESULTS

Cloning
Southern blots of genomic Synechocystis 6803 DNA digested with Kpn I
were probed with the plasmid pAQPR80 (the kind gift of DA Bryant), which

contains the psaA and psaB genes from Synechococcus sp. PCC 7002 (Cantrell

 

 

 

 

46

Table 2.1. DNA probes used for screening, Southern, and northern

 

 

 

hybridizations.
Sourcea Genes encoded Fragment
7002 psaApsaB pAQPR80 7.8 kb EcoR I-Bgl II-pUC9b
7002 85% of psaA 1.9 kb Hind III (from pAQPR80)
7002 60% of psaB 1.4 kb Hind III (from pAQPR80)
7002 5’ end of psaA 0.5 kb EcoR I-Hind III (from pAQPR80)
6803 80% of psaA 1.8 kb Kpn I (from pLSl8)
6803 half of psaB 0.9 kb Kpn I (from pLS19)
6803 3’ half of psaB 1.2 kb Hind III (from pLSlS)
6803 3’ half of psbAZ 0.5 kb Kpn I-Hinc II (from pKW1266°)
6803 3’ half of pszZ 0.6 kb Kpn I-Sma I (from pRD655")
6803 3’ half of rbcL 0.7 kb BamH I (from pSF2.6‘=)
6803 psaD 0.4 kb BstE II-BgII (from pPR8‘)
6803 3’ flanking psaB 1.0 kb EcoR I-Kpn I (from pLS31)
7942 16S rRNA 2.0 kb Xba I (from pAN48)

 

" Organisms are: 7002, Synechococcus sp. PCC 7002; 6803, Synechocystis sp.
PCC 6803; 7942, Synechococcus sp. PCC 7942

b (Cantrell and Bryant, 1987) kind gift of DA. Bryant, Dept. of Molecular and
Cell Biology, Pennsylvania State University, University Park, PA

° (Jansson et a1., 1987)

d (Debus et a1., 1988)

° T. Reich and L. McIntosh (unpublished)

f (Reilly et a1., 1988) kind gift of N. Nelson, Roche Institute, Nutley, NJ

3 (Tomioka et a1., 1981) kind gift of SS. Golden, Dept. of Biology, Texas
A & M University, College Station, TX

LE

 

 

 

47
and Bryant, 1987). Four hybridizing fragments of approximately 1.8, 1.7, 1.4,

and 0.9 kilobase pairs (kb) were detected (data not shown). The sum length of
these fragments corresponds to the expected length of the psaA-psaB operon
(Cantrell and Bryant, 1987). Kpn I-digested genomic Synechocystis 6803 DNA
was fractionated on an agarose gel, and the fragments corresponding in size to
the hybridizing bands were purified and ligated into pUC119. This mini-library
was screened by colony hybridization using two Hind III fragments from
pAQPR80 (Table 2.1); a 1.9 kb (containing most of psaA) and a 1.4 kb

Hind III fragment (containing the 3’ half of psaB). Two hybridizing clones
were isolated: one contained a 1.8 kb Kpn I fragment (pLS18) and the other
contained a 0.9 kb Kpn I fragment (pLSl9) (Figure 2.1). Plasmids pLSl8 and
pLSl9 were partially sequenced and, by comparison to the Synechococcus 7002
sequence, were verified to encode portions of psaA and psaB. Inserts from
pLSl8 and pLS19 and a 0.5 kb EcoR I-Hind III fragment from pAQPR80
(encoding the 5’ end of psaA) (Table 2.1) were then used as probes in
screening a AEMBL3 library of a partial Sau 3A digest of Synechocystis 6803
DNA to recover full-length clones. Several hybridizing A clones were isolated
and their DNA purified, mapped, and subcloned into pUC118 or pUC119. The
maps of two of the 1 clones, 311 and 121, and their subclones are shown in
Figure 2.1. Southern analysis of digested genomic Synechocystis 6803 DNA,
probed with the inserts from pLS18 and pLS19, revealed only one large or a
few small hybridizing fragments, as with pAQPR80, indicating psaA and psaB

are present in single copy in the Synechocystis 6803 genome (data not shown).

 

 

 

48

psaA psaB

V////////////J rf////////////I

 

 

 

 

 

 

 

 

 

 

5.x 8 K is W is E.H is,
311 ---------- -t r- ——————————————————— 121
pLS18 pLS19
—
_ I L ............ 1 r ...........
F I ------------- l J —
[u] - [ ....... l __j
4 >4 >4 4
1kb
L_...__._l

Figure 2.1. Restriction map and sequencing strategy of the Synechocystis 6803
psaA and psaB operon. Hatched boxes represent the protein coding regions of
psaA and psaB. A partial restriction map is shown on the solid line: X, Xba I;
B, Bgl 11; K, Kpn I; H, Hind III; E, EcoR I. Dashed lines represent the insert
regions from two of the 1 clones (311 and 121). Boxes below the dashed lines
represent clones from the mini-library or sub-clones of the 1 clones: black
regions were sequenced from both strands, stippled regions were sequenced
from one strand. Inserts from the plasmids pLS18 and pLS19 are labelled
above the boxes. Triangles represent Oligonucleotides used as sequencing
primers and in PCR.

 

 

 

 

 

49
DNA sequence

The sequencing strategy is shown in Figure 2.1. Sequencing revealed a
single base-pair deletion in one of the original clones from the mini-library
(pL818), evidenced by a disruption of the reading frame in a highly conserved
region of psaA. Unfortunately, this region was not overlapped by any of the 1
clones. Therefore, the PCR technique was utilized to amplify the region
between two of the sequencing primers from genomic Synechocystis 6803 DNA.
The PCR product was purified, subcloned into pUC118, and sequenced,
verifying the true sequence and our hypothesis of a deletion in our cloned
fragment in pLS18.

The DNA and deduced amino acid sequence of the Synechocystis sp.
PCC 6803 psaA and psaB genes and flanking regions is shown in Figure 2.2.
The genes are arranged in tandem with psaA upstream of psaB. The psaA
gene consists of 2256 nucleotides, which would encode a protein 751 amino
acids long with a predicted molecular mass of 82.9 kDa. The psaB gene is 2196
nucleotides long and would encode a protein with 731 amino acids and a
predicted molecular mass of 81.3 kDa. The region between the genes is 245
nucleotides long, with no apparent open reading frame or inverted repeat
sequences. The Synechocystis 6803 psaA gene shows 78.0% DNA sequence
identity to the Synechococcus 7002 psaA gene (Cantrell and Bryant, 1987) and
69.4% DNA identity to the spinach psaA gene (Kirsch et a1., 1986). The
Synechocystis 6803 psaB gene has 81.1% DNA sequence identity to the

Synechococcus 7002 psaB gene (Cantrell and Bryant, 1987) and 69.4% DNA

 

 

 

 

 

50

Figure 2.2. Nucleotide and deduced amino acid sequences of the Synechocystis
6803 psaA and psaB operon. The DNA sequence is numbered on the left; the
deduced amino acid sequence is numbered on the right. Sequences showing
similarity to E. coli promoters and ribosome binding sites are underlined. The
psaA gene extends from nucleotide 1 to 2256. The psaB gene extends from
nucleotide 2502 to 4697. The amino acid sequence FPCDGPGRGGTC,
conserved in PsaA and PsaB and including cysteines thought to bind Fx, as well
as the conserved leucines which may form a leucine zipper, upstream of those
cysteines, are enclosed in boxes. The DNA sequence data reported appear in
the EMBL/GenBank Database under the accession number X58825.

 

 

 

-213

-113

88

188

288

388

588

688

888

988

1088

1188

1288

1388

1488

1588

1688

1788

1888

1988

2088

2188

51

- (-1o
TGGGGCAATTTTTAATTATTGTTACGCAGGTCTTGCCTAGGGGGGGGGAGGCCGTATTATCTTCTAGTGATGTTTGCTGAAAACGCCTATCTGTGCAAGG

TTTAACATCGTTATTATGAAGCGAAAACTAATTCCCTTTTTTACGCTTCCTCTATTACACTATTCTGCATAGGAAACCCTTAATAGTTCATTGTCGAGCG

(S-D) H T I S P P E R E A X A K V 8 V D N N P V P T 8 F E K W G
AGGAGAACCCTGCATGACAATTAGTCCACCCGAAAGAGAGGCTAAGGCCAAAGTCTCGGTTGATAACAACCCGGTACCAACTTCCTTCGAGAAGTGGGGC

K P G H F D R T L A R G P K T T T W I W N L H A N A H D F D S Q T
AAGCCGGGTCACTTCGACCGGACTTTABCTAGAGGTCCCAAAACCACCACTTGGATTTGGAATCTCCATGCCAATGCCCATGATTTTGATAGTCAGACCA

S D L E D V B R K I F 8 A H F G H L A V V F V W L 8 G H Y F H G A K
GCGATTTAGAAGATGTTTCGCGCAAAATCTTCAGTGCTCACTTTGGGCACCTCGCTGTGGTCTTTGTTTGGTTAAGTGGAATGTACTTCCACGGCGCGAA

F S N Y E G W L A D P T H I K P B A Q V V W P I V G Q G I L N G D
ATTTTCCAATTACGAAGGTTGGTTAGCCGACCCTACCCACATTAAACCCAGTGCCCAAGTGGTTTGGCCCATTGTCGGTCAAGGCATCTTGAACGGGGAT

V G G G F H G I Q I T S G L F Y L W R A S G F T D S Y Q L Y C T A
GTGGGCGGTGGCTTCCACGGTATTCAGATTACGTCTGGCCTGTTTTATCTCTGGCGGGCCTCCGGTTTCACCGACAGCTATCAGCTCTACTGCACCGCCA

I G G L V M A A L H L F A G W F H Y H V R A P K L E W F Q N V E S H
TTGGCGGTTTGGTTATGGCTGCCCTGATGCTGTTCGCTGGCTGGTTCCACTACCACGTCAAAGCTCCCAAATTGGAATGGTTCCAAAATGTGGAGTCGAT

K N H H L A G L L G L G S L G W A G H O I H V S H P I N K L L D A
GATGAACCATCATTTGGCTGGTTTGTTGGGCTTAGGCTCCTTGGGATGGGCGGGTCACCAGATCCACGTCTCCATGCCCATTAATAAACTTTTGGATGCT

G V A P K D I P L P B E F I L E P S K H A E L Y P S F A Q G L T P
GGGGTTGCTCCTAAGGACATTCCCCTTCCCCACGAGTTTATTTTGGAACCGAGCAAGATGGCGGAACTCTACCCCAGCTTTGCCCAAGGTTTGACACCGT

F F T L N W G V Y S D F L T F R G G L N P V T G G L H L S D T A H H
TCTTTACCCTCAACTGGGGAGTCTACTCAGACTTCCTCACCTTTAAAGGGGGATTGAATCCAGTCACCGGCGGCCTCTGGTTGTCTGATACCGCTCACCA

H L A I A V L F I I A G H H Y R T N V G I G K S M X E I L E A H K
CCATTTGGCGATCGCCGTCCTGTTCATCATTGCCGGTCATATGTACCGCACGAACTGGGGCATCGGCCATAGCATGAAAGAGATCCTCGAAGCCCACAAG

G P F T G E G E K G L Y E I L T T S W H A Q L A I N L A L L G S L
GGCCCCTTCACTGGGGAAGGTCACAAAGGACTTTATGAAATCCTGACTACTTCTTGGCACGCTCAATTAGCCATTAACCTCGCCCTGTTAGGTTCTTTGA

T I I V A Q B H Y A H P P Y P Y Q A I D Y A T Q L S L F T H H M W I
CGATCATCGTTGCACAACACATGTATGCGATGCCGCCCTATCCCTACCAGGCGATCGATTACGCTACGCAGTTATCCCTGTTCACCCACCACATGTGGAT

G G F L I V G A G A H G A I F M V R D Y D P A K N V N N L L D R M
TGGCGGCTTCCTCATCGTAGGGGCCGGTGCCCATGGCGCCATCTTCATGGTGCGTGATTATGATCCCGCCAAGAACGTTAATAACTTGCTGGATCGGATG

L R B R D A I I S H L N W V C I F L G F H 8 F G L Y I B N D T H R
CTGCGCCACCGTGACGCCATCATTTCCCATCTCAACTGGGTATGTATTTTCCTCGGCTTCCATAGCTTCGGTCTCTACATCCACAACGACACCATGCGGG

A L G R P Q D H F S D T A I Q L Q P I F A Q N V O H L H T L A P G A
CTTTAGGCCGTCCCCAGGACATGTTCTCCGACACGGCCATCCAACTGCAACCGATCTTTGCCCAATGGGTACAGCACCTGCACACCTTGGCCCCCGGAGC

T A P N A L A T A S Y A F G G E T I A V A G K V A H M P I T L G T
CACCGCTCCCAATGCCTTGGCCACTGCCAGCTATGCCTTTGGCGGAGAAACCATTGCCGTTGCCGGTAAAGTCGCCATGATGCCCATCACCCTGGGTACT

A D F H V H B I H A F T I B V T A I L L K G V Y A R S 5 R L
GCCGACTTCATGGTTCACCACATCCACGCTTTTACCATCCACGTAACGGCC ATCCTCCTCAAAGGGGTTCTATATGCCCGTAGCTCTCGCCTTGTCC

p n x A A:J;;2 G F a.|r p c n G p G a G G r c] Q v s G n D a v r L G L F
crearaanscc GGTTTCCGTTTCCCCTGTSATGGCCCCGGCCGCGGCGGCACCTGCCAAGTATCTGGTTGGGACCACGTTTTCCTCGGTCTGTT

 

W M Y N S L S I V I F B F 8 V K M Q S D V W G T V S P D G S V T B
CTGGATGTACAACTCCCTTTCCATCGTCATCTTCCACTTCAGTTGGAAAATGCAATCCGATGTTTGGGGTACCGTTTCTCCCGATGGCAGTGTCACCCAT

V T L G N F A Q 8 A I T I N G W L R D F L W A Q A A N V I N S Y G
GTGACCCTAGGTAACTTTGCCCAGAGTGCCATCACCATCAATGGCTGGTTGCGGGACTTCCTCTGGGCCCAAGCCGCCAACGTCATCAACTCCTACGGTT

S A L S A Y G I M F L A G R F V F A F S L H F L F 8 G R G Y W Q E L
CCGCCCTGTCGGCCTATGGCATCATGTTCCTCGCCGGACACTTTGTCTTTGCCTTCAGCCTCATGTTCCTGTTCAGTGGACGGGGATATTGGCAAGAGTT

I E S I V W A H N R L N V A P A I Q P R A L S I I Q G R A V G V A
GATCGAGTCCATTGTCTGGGCTCACAACAAACTGAATGTGGCTCCGGCCATTCAACCCCGCGCTTTGAGCATCATTCAAGGTCGTGCGGTCGGTGTAGCG

H Y L L G G I V T T W A F F L A R S L S I G *
CACTATCTCCTCGGAGGTATTGTTACCACCTGGGCGTTCTTCCTCGCCCGCAGTCTTTCCATTGGCTAGACTTTGAGCTGAAGTTGGGTTTTCCGGGATA

 

 

29

62

96

129

162

196

229

262

296

329

362

396

429

462

496

529

562

596

629

662

696

729

751

 

2255

2355

2555

2555

2655

2755

2555

2955

3055

3255

3355

3455

3555

3655

3733

3855

3958

4056

4158

4258

5358

5458

tsee

tsee

AACCAGG

CTASAIT

G T
GGCACCI

F G
TTGGCG
GACCAC

P S
CCTAGC

E V
ACGTTG

CCACA

2288

2388

2488

2588

2688

2788

2888

2988

3088

3188

3288

3388

3488

3588

3688

3788

3888

3988

4088

4188

4288

4388

4488

4588

4688

52

AACCAGGGGGACTGGCTGAGGTTTTTACTATCCTTAATCCTCCCCCTGCCCTCCACCCTCCCCAGCATTTAATAGAAGCGGTTTTAATTTCGCTTTTACC

( 843)
CTATATTCACCCCTATAACCCCTTGTTATAGTGGCTTTAACAATCCGGAAATGAGCAGCGCTTCTGCATTAGCCTCATTTTGCTTGCTTAACGAGGAGAA

N A T R F P K F S Q D L A Q D P T T R R I W Y G I A T A R
TTTCCGAAAAAGCTATGGCAACTAAATTTCCTAAATTTAGCCAGGATCTCGCCCAAGACCCGACTACACGGCGTATTTGGTACGGGATTGCTACGGCCCA

D F E T H D G H T E E N L Y Q R I F A B H F G H I A I I F L W T 8
CGACTTTGAAACCCACGATGGGATGACTGAGGAGAATCTTTACCAAAAGATTTTTGCCTCCCACTTTGGACACATCGCCATCATTTTCCTGTGGACGTCT

G T L F B V A W Q G N F E Q W I K D P L N I R P I A H A I W D P H
GGCACCCTTTTCCACGTTGCGTGGCAAGGTAATTTTGAACAATGGATTAAAGATCCTTTAAACATTCGCCCGATCGCCCATGCGATTTGGGAGCCCCACT

F G E G A V N A F T Q A G A S N P V N I A Y S G V Y H W F Y T I G H
TTGGCGAGGGAGCTGTCAATGCTTTCACCCAAGCTGGGGCTTCTAACCCGGTTAACATTGCTTATTCCGGGGTTTACCACTGGTTCTACACCATTGGTAT

T T N Q E L Y S G A V F L L V L A S L F L F A G W L H L Q P K F R
GACCACCAACCAAGAGCTCTATTCTGGTGCGGTCTTCCTATTAGTGTTAGCTTCCCTGTTTTTATTTGCAGGCTGGTTACACCTCCAACCGAAGTTCCGT

P S L A W F X N A E S R L N B B L A G L F G V S 8 L A W A G H L V
CCTAGCTTAGCCTGGTTCAAAAATGCCGAATCCCGCTTAAATCACCACCTGGCTGGTTTGTTCGGGGTTAGCTCCTTGGCTTGGGCTGGTCACTTGGTCC

B V A I P E A R G Q B V G W D N F L 8 T P P H P A G L M P F F T G N
ACGTTGCGATTCCCGAAGCCCGGGGTCAACACGTTGGTTGGGATAACTTCCTGTCTACTCCTCCCCACCCCGCTGGTTTGATGCCTTTCTTCACCGGCAA

W G V Y A A D P D T A G H I F G T B E G A G T A I L T F L G G F H
CTGGGGCGTGTATGCGGCGGATCCCGACACTGCTGGCCACATTTTTGGTACTTCTGAAGGTGCTGGTACCGCAATCCTGACCTTCCTGGGTGGTTTCCAT

P Q T E 8 L W L T D I A R B H L A I A V I F I I A G H N Y R T N W
CCCCAAACGGAATCTCTCTGGTTGACGGACATTGCCCACCACCATTTGGCGATCGCCGTGATCTTCATCATTGCTGGTCACATGTATCGCACCAACTGGG

G I G H B I K E I L N A H K G P L T G A G R T N L Y D T I N N 5 L H
GCATTGGCCACAGCATCAAAGAAATTCTTAATGCCCATAAAGGTCCCCTAACCGGCGCAGGCCATACCAACCTGTACGACACCATCAATAACTCCCTCCA

F Q L G L A L A S L G V I T S L V A Q B H Y S L P S Y A F I A Q D
CTTCCAACTCGGCTTAGCCTTGGCAAGCTTAGGGGTTATTACTTCCCTGGTGGCGCAGCACATGTACTCCCTGCCCTCCTACGCCTTTATTGCCCAGGAC

H T T Q A A L Y T H B Q Y I A G F L M V G A F A H G A I F F V R D
CACACCACCCAGGCAGCCCTCTACACCCATCACCAGTACATTGCTGGATTCTTGATGGTTGGTGCTTTTGCCCACGGTGCCATCTTCTTTGTCCGGGATT

Y D P V A N K D N V L A R H L E H R E A L I S H L 8 W V S L F L G F
ACGATCCCGTGGCCAATAAAGATAACGTGCTGGCCCGCATGCTTGAACACAAAGAGGCTCTGATTTCCCACTTAAGCTGGGTGTCCCTCTTCTTGGGCTT

H T L G L Y V H N D V V V A F G T P E R Q I L I E P V F A Q W I Q
CCACACCCTTGGTCTTTATGTCCATAACGATGTGGTGGTGGCCTTCGGTACTCCCGAAAAACAAATTCTGATCGAGCCCGTTTTTGCCCAATGGATTCAA

A T S G K A L Y G F D V L L S N P D S I A S T T G A A W L P G W L
GCAACTTCCGGTAAAGCTCTCTATGGCTTTGATGTTTTGCTCTCCAATCCTGACAGCATTGCTTCCACCACTGGAGCCGCTTGGTTACCCGGCTGGTTGG

D A I N s G T N s L r L r I G p G D r'ti] v a a A I A [Z] G L a T r A [a]

ATGCTATCAACAGCGGCACCAACTCTCTGTTCTTGACCATTGGCCCTGGCGACTTCTTGGTTCACCACGCGATCGCCCTAGGGTTGCACACCACTGCCCT

 

I L r x G A,E:J D A R G s x M p D x x D r G Y s r p c D G p G a G
GATTCTAATCAAAGGTGCTTTGGATGCCCGTGGTTCTAAGTTGATGCCGGACAAAAAGGACTTCGGTTACTCCTTCCCCTGTGATGGCCCCGGCCGTGGC

G T CI D I 8 A W D A F Y L A H F W M L N T L G W L T F Y W H W K H

GGTACCTGCGACATCTCTGCTTGGGATGCCTTCTACCTAGCCATGTTCTGGATGCTGAACACCTTGGGTTGGTTGACCTTCTACTGGCACTGGAAACACC

L G V W 8 G N V A Q F N E N S T Y L M G W F R D Y L W A N S A Q L I
TCGGTGTTTGGAGCGGTAACGTTGCTCAGTTCAACGAAAACTCCACCTACCTGATGGGTTGGTTCCGGGATTACCTCTGGGCGAACTCTGCTCAGTTAAT

N G Y N P Y G V N N L S V W A W M F L F G H L V W A T G F H F L I
CAATGGTTACAACCCCTACGGTGTCAACAATCTGTCAGTTTGGGCTTGGATGTTCCTTTTCGGACACCTGGTCTGGGCTACTGGCTTCATGTTCTTGATC

8 W R G Y W Q E L I E T I V W A H E R T P L A N L V R W R D K P V
TCTTGGCGGGGTTACTGGCAAGAGTTGATTGAAACCATCGTTTGGGCCCACGAGCGCACTCCTTTGGCGAACTTGGTTCGTTGGAAAGATAAGCCCGTTG

A L S I V Q A R L V G L A H F T V G Y V L T Y A A F L I A S T A G K
CGTTGTCCATTGTTCAAGCCCGTTTGGTTGGTTTAGCCCACTTCACCGTTGGTTATGTGCTCACCTATGCGGCATTCCTAATTGCTTCCACAGCCGGTAA

F G *
GTTCGGTTAACGAATTCCTCTGTTAGGTAATTAAGCTT

Figure 2.2 (cont)

 

29

62

95

129

162

195

229

262

295

329

362

395

429

462

495

595

629

662

695

729

731

 

identity to
upstream i
start of p5
Shine-Dal,
and Dalgz
-10 seque
gene, whi
(TTGAC
(Hawley
region 3’
stem-loo
involved
The part
IPSI4 in
7002 (C;

in those

PsaA-pa

S
in the 5
Prfidicte
the Syn

t0 the I

53

identity to the spinach psaB gene (Kirsch et a1., 1986). Seven nucleotides
upstream from the start of psaA and fifteen nucleotides upstream from the
start of psaB is the sequence AGGAGAA, which has similarity to the
Shine-Dalgarno sequence and may constitute a ribosome binding site (Shine
and Dalgarno, 1974). A sequence (TA'I'I‘AT) similar to the E. coli consensus
-10 sequence (TATAAT) lies 154 nucleotides upstream of the start of psaA
gene, while a sequence ('ITGCCI‘) similar to the E. coli -35 sequence
(TTGACA) lies 17 nucleotides upstream from the potential -10 sequence
(Hawley and McClure, 1983). Partial nucleotide sequence obtained for the
region 3’ to the psaB gene includes G-C-rich inverted repeats that may form a
stem-loop structure followed by a series of thymines. This type of structure is
involved in transcription termination in E. coli (Rosenberg and Court, 1979).
The partial sequence data from the region 3’ to psaB show no homology to
rpsl4 from spinach (Kirsch et al., 1986) or to ORF128 from Synechococcus
7002 (Cantrell and Bryant, 1987), which have been found downstream of psaB

in those organisms.

psaA-psaB deduced amino acid sequence

Specific residues as well as predicted secondary structures are conserved
in the Synechocystis 6803 psaA-psaB deduced protein sequences. The
predicted amino acid sequence of the psaA gene (PsaA) is 88.4% identical to
the Synechococcus 7002 PsaA (Cantrell and Bryant, 1987) and 80.9% identical

to the spinach PsaA (Kirsch et a1., 1986). The Synechocystis 6803 psaB

 

 

 

deduced a]
7002 PsaB
PsaB (Kir:
for both P
sequences
Lehmbecl
the seque
PsaB), w]
all of the
terminal

residues

acids ap;
0f leucin

leucine :

mRNA
1‘
With prr
as well
from ce
PUt in t
cOnditit

hybridi:

54

deduced amino acid sequence (PsaB) is 92.8% identical to the Synechococcus
7002 PsaB (Cantrell and Bryant, 1987) and 79.6 % identical to the spinach
PsaB (Kirsch et 31., 1986). Eleven membrane-spanning a-helices are predicted
for both PsaA and PsaB, as has been predicted for previously characterized
sequences (Cantrell and Bryant, 1987; Fish et a1., 1985; Kirsch et 3]., 1986;
Lehmbeck et a1., 1986). Also conserved in both deduced protein sequences is
the sequence FPCDGPGRGGTC (residues 572 to 583 in PsaA, 554 to 565 in
PsaB), which is absolutely conserved in the deduced amino acid sequences of
all of the psaA and psaB genes sequenced to date. On the immediate amino-
terminal side of this sequence in PsaA and PsaB are conserved series of leucine
residues (four leucines in PsaA, five in PsaB), each leucine spaced seven amino
acids apart. These regions are predicted to form a-helices, each with a string
of leucines, that would have the potential to interact with each other, forming a

leucine zipper (Kossel et a1., 1990; Webber and Malkin, 1990).

mRN A accumulation in cells grown with glucose

Northern blots of total RNA from Synechocystis 6803 were hybridized
with probes encoding psaA, psaB, psbAZ, 16S rRNA, psaD, psz2, and rbcL,
as well as a probe encoding 1.0 kb 3’ to psaB (Table 2.1). RNA was isolated
from cells grown in continuous light; cells grown in the light with glucose, then
put in total darkness for 24, 30, or 48 h; and from cells grown under LAHG
conditions (Anderson and McIntosh, 1991). Autoradiographs from

hybridizations to a representative northern blot with samples from each of the

 

 

above eon
hybridized
probe alsr
to a 2.1-Id
5.0 kb. T
be a tram
operon fr
transcript
PSI, hybr
strongly t
2.5-kb trz
from Syn
3 Smear t
full-lengtj
Subunits
In
PsaA-psa
directly fj
“Ormalizi
Probe. (
regimts c

24‘kb p5

55

above conditions are shown in Figure 2.3. Both the psaA and psaB probes
hybridized to a 5.0-kb transcript, indicating they are co-transcribed. The psaA
probe also hybridized to a 2.4-kb message, while the psaB probe also hybridized

to a 2.1-kb message. Both probes hybridized to a smear of bands smaller than

 

5.0 kb. The fragment encoding psbAZ hybridized to a 1.2-kb message that may
be a transcript from one or both of psbAZ or psbA3. A fragment of the rrn

operon from Synechococcus 7942 specific for 16S rRNA hybridized to a 1.5-kb

 

transcript. The psaD probe from Synechocystis 6803, encoding subunit II of
PS1, hybridized to a single message of 0.5 kb. The pszZ probe hybridized
strongly to the 1.2-kb transcript from pszZ, but also hybridized weakly to the
2.5-kb transcript from the overlapping pszI-pst genes. The probe for rbcL
from Synechocystis 6803 hybridized to a 2.6-kb transcript, a 2.0-kb message, and
a smear of transcripts smaller than 2.6 kb. The 2.6-kb transcript represents the
full-length message [from the rbcLS operon, which encodes the large and small
subunits of Rubisco.

In order to quantify the steady-state levels of mRNA accumulation for
psaA-psaB and psbA, radioactivity in the region of the bands was measured
directly from the blots using a Betascope machine. These values were then
normalized based on the quantification of hybridization of the 16S rRNA
probe. Quantification of hybridization of the psaA probe was measured in the
regions of the 5.0-kb full-length psaA-psaB message and in the region of the

2.4-kb psaA-specific message. Values for at least one quantification from at

 

Figure 2.
Probes u
The Upp
are not I
c0mpat‘is
light-gun
dark Witl
each autr

56

psaA psaB psbA2

12345 12345 12345

2.4 -

 

v a...

168 rRNA pszz psaD rbcL
12345 12345 12345 12345
kb

2.6- —
1.5- ‘2'”. 2.0-
05-“...

Figure 2.3. Autoradiographs of a single northern blot, stripped and reprobed.
Probes used are indicated above each panel and are described in Table 2.1.

The upper three panels are aligned in the same scale. The lower four panels
are not in alignment. Sizes of bands are in kb and were determined by
comparison to an ethidium bromide-stained RNA ladder. Lanes are: 1,
light-grown control; 2, 24 h dark with glucose; 3, 30 h dark with glucose; 4, 48 h
dark with glucose; 5, LAHG conditions. Exposure times were different for
each autoradiograph.

 

least two
percentag
Accumule
the light 2
h, while t
the accur
the light ;
steady-Ste
accumula
incubatio
steady-st;
1ight-grov
quantifie.

dark witl

mRNA 8

R'
t0 early t
darkness
incubatic
Significar
formalde

57

least two preparations for each condition were averaged and are expressed as a
percentage of the accumulation in the light-grown samples (Figure 2.4).
Accumulation of the 5.0-kb psaA-psaB transcript drops to 87% of the level in
the light after 24 h in the dark, 47% after 30 h, then back up to 82% after 48
h, while the steady-state level in LAHG cells is 80% of light-grown. However,
the accumulation of the 2.4-kb psaA message increases to 152% of the level in
the light after 24 h, but drops to 97% after 30 h, 107% after 48 h, while the
steady-state level in LAHG cells is 85% of the level in light-grown cells. The
accumulation of the 1.2-kb psbA message steadily decreases through the dark
incubation: 68% after 24 h, 50% after 30 h, and 41% after 48 h, while the
steady-state level of this psbA message in LAHG cells is 28% of that in
light-grown cells. Although the hybridization of the other probes used was not
quantified, none showed a significant and repeatable change upon growth in the

dark with glucose.

mRNA accumulation in cells grown without glucose

RNA was isolated from duplicate cultures of Synechocystis 6803 grown
to early exponential phase in the light without glucose, then put in complete
darkness for 48 h. The final OD730 of the culture was lower than when the dark
incubation started, and the yield of RNA from these preparations was
significantly lower than for cells grown with glucose. When the RNA in the
formaldehyde gel was stained with ethidium bromide and visualized under UV

light, the samples from cells grown without glucose were indistinguishable from

 

 

 

8‘ ‘8‘

§

8

'on (96)
' e Accumulatl
Relatrv 8

a

9%

Figure 2..
Values 31
two prep
ljght‘grov
thel6S II
transcripr
transcripr

HIS are:
dark witl
heteiotrc

58

 

60—

Relative Accumulation (%)
8
fi

20—

  

psaA psaA psbA
(5.0 kb) (2.4 kb)
E Light 24h 30h 48h LAHG

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.4. Relative levels of mRNA accumulation in Synechocystis 6803 cells.
Values are averages of at least one quantification of hybridization to at least
two preparations from each condition and are expressed as a percentage of the
light-grown control. Values were normalized based on the hybridization of
the16S rRNA probe. The left group is a quantification of the 5.0-kb psaA-psaB
transcript. The middle group is a quantification of the 2.4-kb psaA-specific
transcript. The right group is a quantification of the 1.2-kb psbA transcript.
Bars are: Light, light-grown control; 24 h, 24 h dark with glucose; 30 h, 30 h
dark with glucose; 48 h, 48 h dark with glucose; LAHG, light-activated
heterotrophic growth conditions.

 

 

 

those frorr
representa
psbA2, ps
inspection
cells lacki:
mRNA in
while the
equal. Tl
smaller, v
the expec
hybridize.
prDI-ps
transcript

hybridize

DIScus

T]
of Oxygen
JElnsson .
DIOgeneS
haVe c101
e"liressir

whether

59

those from cells grown with glucose (data not shown). Autoradiographs of a
representative lane from a northern blot with this RNA hybridized to the psaA,
psbA2, pszZ, rbcl, and 16S rRNA probes are shown in Figure 2.5. By
inspection of autoradiographs, the level of hybridization to the mRNA from
cells lacking glucose was significantly lower than the level of hybridization to
mRNA from either the 48 h dark with glucose sample or the LAHG sample,
while the hybridization of the 16S rRNA probe appeared to be approximately
equal. The psaA probe hybridized to a smear of transcripts of 3.0 kb and
smaller, with bands of 2.4, 1.6, 1.1, and 0.7 kb. The psbA2 probe hybridized to
the expected 1.2-kb message and a 0.6-kb transcript. The pszZ probe
hybridized strongly to the 1.2-kb pszZ transcript and very weakly to the 2.5-kb
pszI-pst message. Hybridization of the rbcL probe to a smear of
transcripts 2.5 kb and smaller was nearly undetectable. The 16S rRNA probe

hybridized to the expected 1.5-kb transcript.

DISCUSSION

The use of Synechocystis sp. PCC 6803 as a model system for the study
of oxygenic photosynthesis has been well documented (Debus et a1., 1988;
J ansson et a1., 1987; Williams, 1988). In order to broaden the analysis of the
biogenesis and structure/function relationships of PSI in Synechocystis 6803, we
have cloned the psaA and psaB genes for use as probes to study gene
expression and for targeted mutagenesis. As a part of a study to determine

whether the photosystems assemble in the dark, a quantitative analysis of the

 

 

 

Flame 2
Without
ethldluH
rRNA- .
limes WI

60

48 h Dark (-glucose)

12345

kb
7.46 -

4.40 -

2.37 -

1.35-

 

0.24 -

Figure 2.5. Autoradiographs of a single lane from a northern blot (48 h dark
without glucose), stripped and reprobed. Size labels (in kb) are from an
ethidium bromide-stained RNA ladder. Probes used: 1, psaA; 2, psbA2; 3, 16S
rRNA; 4, psz2; 5, rbcL Probes used are described in Table 2.1. Exposure
times were different for each autoradiograph.

 

expression
psaA and
as well as
growth co
makes rm
Th
homologt
homologt
gene is h
acid cons
This sugg
the origir
structure
plant sec
in Synec
bp. The
SYRCCho
7002. A
(07314) :
A fragm
Probe 0
Neither

dOWnstr

 

61

expression of photosynthesis genes in the dark was performed. Although the
psaA and psaB genes have been cloned and sequenced from a cyanobacterium
as well as multiple plant and algal species, the recent discovery of heterotrophic
grth conditions for Synechocystis 6803 (Anderson and McIntosh, 1991)
makes mutagenesis of the PSI core tractable in this organism.

The psaA and psaB genes from Synechocystis 6803 are highly
homologous to the genes from Synechococcus 7002 and only slightly less
homologous to the genes cloned from higher-plant chlorOplasts. The psaA
gene is homologous to the psaB gene, with long stretches of absolute amino
acid conservation and 56.9% DNA sequence identity in Synechocystis 6803.
This suggests one of these genes arose from a duplication of the other prior to
the origin of chloroplasts. Fundamental differences in the psaA-psaB operon
structure from cyanobacteria and higher plant chloroplasts do exist. In the
plant sequences, there are only 25 bp between the psaA and psaB genes, while
in Synechococcus 7002 this region is 173 bp, and in Synechocystis 6803 it is 245
bp. There is no apparent open reading frame in the intergenic region of
Synechocystis 6803, while there is a short open reading frame in Synechococcus
7002. Also, in tobacco, rice, and spinach, the gene for ribosomal protein 814
(rps14) is found downstream of psaB and is co-transcribed with psaA and psaB.
A fragment encoding 1.0 kb immediately downstream of psaB was used as a
probe of northern blots, but no hybridization was detected (data not shown).
Neither Synechocystis 6803 nor Synechococcus 7002 have rps14 immediately

downstream of psaB, and in Synechocystis 6803, this region may not be

 

 

 

 

 

transcribe
Th
Synechoq
and highs
PsaA and
PsaB is t]
apart. It
zipper, p‘
Mafia]
regions c
(Golbeck
leucines
role in tl
one or n
assess th-
heterodi]
tranSpon
H
genes an
HOWever
productS
enCorie j

Intergen.

 

62

transcribed or if so, the message accumulates to undetectable levels.

The predicted amino acid sequences of psaA and psaB from
Synechocystis 6803 are highly homologous to those from Synechococcus 7002
and higher plants. Eleven membrane-spanning a-helices are predicted for both
PsaA and PsaB. An interesting feature of the eighth helix in both PsaA and
PsaB is the presence of conserved leucine residues, spaced seven amino acids
apart. It has been proposed that these helices may interact to form a leucine
zipper, pulling PsaA and PsaB together (Kossel et al., 1990; Webber and
Malkin, 1990). These helices are immediately adjacent to highly conserved
regions containing the four cysteines thought to bind the 4Fe-4S center Fx
(Golbeck and Bryant, 1991). We plan to use site-directed mutagenesis of the
leucines described above to address the leucine zipper hypothesis and assess its
role in the assembly of Fx and of the PSI core. In addition, we plan to mutate
one or more of the conserved cysteines to confirm their role as ligands to Fx, to
assess the importance of Fx in maintaining the integrity of the core
heterodimer, and to answer questions about the role of Fx in electron
transport.

Hybridization patterns of psaA and psaB to northern blots indicate these
genes are co-transcribed, as both genes hybridize to a 5.0 kb transcript.
However both genes also hybridize to smaller transcripts that may represent the
products of processing in the intergenic region, yielding transcripts that only
encode psaA or psaB. The lack of an open reading frame in the relatively long

intergenic region may leave the transcript susceptible to processing, as this

 

 

 

region a}
psaB hyt
seen in h
general c
this mess
maize (R
half-life (
chloropla
Tl
PSI and I
darkness
quantifier
Preparati
about 12
Condition.
tTansferre
ElPproach
Status, A
leiding,
tI‘mvgh n
light.gmw
transcript

aCcumular

 

 

63

region apparently would not be bound to ribosomes. Probes for psaA and
psaB hybridize to a smear of transcripts smaller than 5.0 kb. Smearing was not
seen in hybridizations with other probes, suggesting this is not the result of
general degradation of the RNA sample, but perhaps due to rapid turnover of
this message. A similar complex pattern of hybridization has been seen in
maize (Rodermel and Bogorad, 1985) and in tobacco (Meng et a1., 1988). The
half-life of the psaA-psaB transcript has not been measured in cyanobacteria or
chloroplasts.

The accumulation of transcripts from genes encoding core proteins of
PSI and PSII was quantified for three time points of incubation in total
darkness with glucose, for LAHG conditions, and for light-grown cells. These
quantifications were averaged from multiple trials from at least two
preparations of each type. The doubling time for cells in continuous light is
about 12 h (Williams, 1988), while the doubling time for cells in LAHG
conditions is 36 h (Anderson and McIntosh, 1991). When cells were
transferred from the light to total darkness, their growth rate slowed to
approach that for LAHG conditions, presumably due to a change in energy
status. After approximately 48 h without a light pulse, the cells would stop
dividing. The accumulation of the psaA-psaB transcript drOps only slightly
through the dark incubation, with a steady-state level of 80% of that of
light-grown cells in the LAHG sample. The accumulation of the psaA-specific
transcript generally shows the same pattern as the full-length transcript. The

accumulation of psbA transcript shows a consistent decline through the dark

incubatio
changes i
but rathe
accumula
regulatio:
are grow
assayed i

M
of an ex;
of psbA,
Our resu
these ger
glucose, '
actually i
lower th:
and MCI}
dark witl
cells gror
the Stud)

Tf
iota] dar
the trans

aVailabIe

 

 

64

incubation to a level in LAHG cells 28% of that in light-grown cells. These
changes in message accumulation may not be due to transcriptional regulation,
but rather to changes in mRNA stability. Also, any changes in message
accumulation seen under these conditions cannot be attributed solely to light
regulation, but may be the result of the difference in energy status when cells
are grown in the dark. It is clear that transcripts of the photosynthesis genes
assayed in this experiment do accumulate in cells grown in the dark.

Mohamed and Jansson (Mohamed and Jansson, 1989) published results
of an experiment using Symechocystis 6803 in which they saw no accumulation
of psbA, psz, or rbcL mRNA in cells put in the dark for 48 h without glucose.
Our results from a duplication of this experiment indicate that transcripts from
these genes and psaA-psaB do accumulate in cells grown in the dark without
glucose, but the mRNA is degraded (Figure 2.5). The OD730 of the cultures
actually decreased after the 48 h incubation, and the yield of RNA was much
lower than from cultures grown in complete darkness with glucose. Anderson
and McIntosh (1991) showed that Synechocystis 6803 does not divide in the
dark without supplemental glucose. We would conclude that the mRNA of
cells grown under these conditions has been degraded and is not satisfactory for
the study of gene expression.

The presence of transcripts from photosynthesis genes in cells grown in
total darkness indicates that either transcription continues in the dark or that
the transcripts are stable for as long as 48 h. In either case, the mRNA is

available for the synthesis and assembly of photosynthesis proteins. The

 

 

pattern 0:
over, thus
light-grow
estimated
similar in
after the
Changes
6803 (Mt
1988) are
of the w:

available

ACKNO
I
Clones. j
State Ur
William
of their j
given by
DE-FGc
Ln).

 

 

65

pattern of hybridization to psaA-psaB suggests this message is rapidly turned
over, thus transcription of this operon probably continues in the dark at near
light-grown rates. The half-life of psbA in Synechococcus 6301 has been
estimated to be approximately 120 min (Liinneborg et a1., 1988). If this is
similar in Synechocystis 6803, the rate of transcription of psbA may drop shortly
after the shift to the dark, but the psbA transcript pool decreases gradually.
Changes in the psbA transcript pool have been observed when Synechocystis
6803 (Mohamed and Jansson, 1989) or Synechococcus 6301 (Lonneborg et al.,
1988) are grown in high light. Transcriptional regulation of psbA may be one
of the ways this organism alters its photosynthetic apparatus to adjust to

available light conditions.

ACKNOWLEDGEMENTS

I thank Donald Bryant, Susan Golden, and Nathan Nelson for providing
clones. I am grateful to Chris Somerville (D.O.E.-Plant Research Lab, Mich.
State Univ.) for synthesizing Oligonucleotides. I thank James Tiedje and
William Holben (Dept. of Crop and Soil Science, Mich. State Univ.) for the use
of their Betascope. I deeply appreciate the advice and assistance generously
given by Shawn Anderson. This work was supported in part by DOE. grant

DE-FG02-90ER 20021 (to L.M.) and MSU. Research Excellence Funds (to

L.M.).

 

 

REFERE

Anderson
the cyano
process. .1

Bengis C
reaction t

Boyer H‘
restrictior
41:459-47

Bustos S.
of four q
1721998]

Cantrell .
of the ps
7002. Fla

Chen S-(
rice ch10:
heteroge

Chitnis l
targeted
fT0111 the
264:1837

Chitnis I
gene enc
Synecho.

ChOquet
and Roc
IOI the 8
Cell 52$

66
REFERENCES

Anderson SL and McIntosh L (1991) Light-activated heterotrophic growth of
the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring
process. J. Bacteriol. 173:2761-2767.

Bengis C and Nelson N (1977) Subunit structure of chloroplast photosystem I
reaction center. J. Biol. Chem. 252:4564-45 69.

Boyer HW and Roulland-Dussoix D (1969) A complementation analysis of the
restriction and modification of DNA in Escherichia coli. J. Mol. Biol.
41:459-472.

Bustos SA, Schaefer MR and Golden SS (1990) Different and rapid responses
of four cyanobacterial psbA transcripts to changes in light intensity. J. Bacteriol.
172: 1998-2004.

Cantrell A and Bryant DA (1987) Molecular cloning and nucleotide sequence
of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC
7002. Plant Mol. Bio]. 9.453-468.

 

Chen S-CG, Cheng M-C, Chen J and Hwang L-Y (1990) Organization of the

rice chloroplast psaA-psaB—rpsM gene and the presence of sequence
heterogeneity in this gene cluster. Plant Sci. 68:213-221.

Chitnis PR, Reilly PA, Miedel MC and Nelson N (1989a) Structure and
targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I
from the cyanobacterium Synechocystis sp. PCC 6803. J. Biol. Chem.
264:18374-18380.

Chitnis PR, Reilly PA and Nelson N (1989b) Insertional inactivation of the
gene encoding subunit II of photosystem I from the cyanobacterium
Symechocystis sp. PCC 6803. J. Biol. Chem. 264:18381—18385.

Choquet Y, Goldschmidt-Clermont M, Girard-Bascou J, Kiick U, Bennoun P
and Rochaix J-D (1988) Mutant phenotypes support a trans-splicing mechanism

for the expression of the tripartite psaA gene in the C. rer'nhardtii chloroplast.
Cell 52:903-913.

Cushman J C, Hallick RB and Price CA (1987) The gene for the
P700-chlorophyll a protein in Euglena gracilis is interrupted by three introns.
In: Progress in Photosynthesis Research, Vol. IV. Biggens J, ed. Dordrecht,
Netherlands: Martinus Nijhoff Publishers, pp. 667-670.

Debus RJ, Barry BA, Babcock GT and McIntosh L (1988) Site-directed

 

67

mutagenesis identifies a tyrosine radical involved in the photosynthetic
oxygen-evolving system. Proc. Natl. Acad. Sci. USA 85:427-430.

Feinberg AP and Vogelstein B (1983) A technique for radiolabelling DNA
restriction endonuclease fragments to high specific activity. Anal. Biochem.
132: 6-13.

Fish LE, Kiick U and Bogorad L (1985) Two partially homologous adjacent
light-inducible maize chloroplast genes encoding polypeptides of the P700
chlorophyll a-protein complex of photosystem I. J. Biol. Chem. 260:1413-1421.

Golbeck JH, Parrett KG, Mehari T, Jones KL and Brand JJ (1988) Isolation
of the intact photosystem I reaction center core containing P700 and iron-sulfur
center Fx. FEBS Lett. 228:268-272.

Golbeck JH (1989) Structure, function, and organization of the photosystem I
reaction center complex. Biochim. Biophys. Acta 895:167-204.

Golbeck JH and Bryant DA (1991) Photosystem I. Curr. Topics Bioenerg.
16:83-177.

Golden SS, Brusslan J and Haselkorn R (1987) Genetic engineering of the
cyanobacterial chromosome. Methods Enzynrol. 153:215-231.

Goldschmidt-Clermont M, Girard-Bascou J, Choquet Y and Rochaix J-D
( 1990) T rans-splicing mutants of Chlamydomonas reinhardtii. Mol. Gen. Genet.
223:417-425.

Grunstein M and Hogness DS (1975) Colony hybridization: A method for the
isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci.
USA 72:3961-3965.

Hawley DK and McClure WR (1983) Compilation and analysis of Escherichia
coli promoter DNA sequences. Nucl. Acids Res. 11:2237-2255.

Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted
breakpoints for DNA sequencing. Gene 28:351-359.

Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M,
Kondo C, Honji Y, Sun C-R, Meng B-Y, Li Y-Q, Kanno A, Nishizawa Y,
Hirai A, Shinozaki K and Sugiura M (1989) The complete sequence of the rice
(Oryza sa tiva) chloroplast genome: intermolecular recombination between
distinct tRNA genes accounts for a major plastid DNA inversion during the
evolution of the cereals. Mol. Gen. Genet. 217:185-194.

 

 

 

68

Ho K and Krogmann DW (1982) Photosynthesis. In: The Biology of
Cyanobacteria. Carr NG and Whitton BA, eds. Los Angeles: University of
California Press, pp. 191-214.

 

Hoj PB, Svendsen I, Scheller HV and Muller BL (1987) Identification of a
chloroplast-encoded 9-kDa polypeptide as a 2[4Fe-4S] protein carrying centers
A and B of photosystem I. J. Biol. Chem. 262: 12676-12684.

J ansson C, Debus RJ, Osiewacz HD, Gurevitz M and McIntosh L (1987)
Construction of an obligate photoheterotrophic mutant of the cyanobacterium
Synechocystis 6803. Plant Physiol. 85:1021-1025.

Kirsch W, Seyer P and Herrmann RG (1986) Nucleotide sequence of the
clustered genes for two P700 chlorophyll a apoproteins of the photosystem I
reaction center and the ribosomal protein 814 of the spinach plastid
chromosome. Curr. Genet. 10: 843-855.

Kiick U, Choquet Y, Schneider M, Dron M and Bennoun P (1987) Structural
and transcriptional analysis of two homologous genes for the P700 chlorophyll
a-apoproteins in Chlamydomonas reinhardr'i: evidence for in vivo trans-splicing.
EMBO J. 6:2185-2195.

 

Kossel H, Dory I, Igloi G and Maier R (1990) A leucine-zipper motif in
photosystem 1. Plant Mol. Biol. 15:497-499.

Lehmbeck J, Rasmussen OF, Bookjans GB, Jepsen BR, Stummann BM and
Henningsen KW (1986) Sequence of two genes in pea chloroplast DNA coding
for 84 and 82 kDa polypeptides of the photosystem I complex. Plant Mol. Biol.
7:3-10.

Lundell DJ, Glazer AN, Melis A and Malkin R (1985) Characterization of a
cyanobacterial photosystem I complex. J. Biol. Chem. 260:646-654.

Lonneborg A, Kalla SR, Samuelsson G and Oquist G (1988) Light-regulated
expression of the psbA transcript in the cyanobacterium Anacystis nidulans.
FEBS. Lett. 240:110-114.

Meng BY, Tanaka M, Wakasugi T, Ohme M, Shinozaki K and Sugiura M
(1988) Cotranscription of the genes encoding two P700 chlorophyll a
apoproteins with the gene for ribosomal protein C814: determination of the
transcriptional initiation site by in vitro capping. Curr Genet 14:395-400.

Metz J, Nixon P and Diner B (1990) Nucleotide sequence of the psbA3 gene
from the cyanobacterium Synechocystis PCC 6803. Nucl. Acids Res.
18:6715-6715.

 

69

Mohamed A and Jansson C (1989) Influence of light on accumulation of
photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803.
Plant Mol. Biol. 13:693-700.

Mullet JE (1988) Chloroplast development and gene expression. Annu. Rev.
Plant Physiol. Plant Mol. Bio]. 39: 475 -5 02.

 

Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono
K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H and Ozeki H (1986)

Chloroplast gene organization deduced from complete sequence of liverwort
Marchantia polymorpha chloroplast DNA. Nature 322:572-5 74.

Osiewacz HD and McIntosh L (1987) Nucleotide sequence of a member of the

psbA multigene family from the unicellular cyanobacterium Synechocystis 6803.
Nucl. Acids Res. 15:10585-10585.

Reilly P, Huhnes JD, Pan YCE and Nelson N (1988) Molecular cloning and
sequencing of the psaD gene encoding subunit II of Photosystem I from the
cyanobacterium, Synechocystis sp. PCC 6803. J. Biol. Chem. 263:17658-17662.

 

Rodermel SR and Bogorad L (1985) Maize plastid photogenes: mapping and
photoregulation of transcript levels during light-induced development. J. Cell
Biol. 100. 463-476.

Rosenberg M and Court D (1979) Regulatory sequences involved in the
promotion and termination of RNA transcription. Annu. Rev. Genet.
13:319-353.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory Press.

Sanger F, Nicklen S and Coulson AR (1977) DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

Schaefer MR and Golden SS (1989a) Differential expression of members of a
cyanobacterial psbA gene family in response to light. J. Bacteriol.
171:3973-3981.

Schaefer MR and Golden SS (1989b) Light availability influences the ratio of
two forms of D1 in cyanobacterial thylakoids. J. Biol. Chem. 264:7412-7417.

Shine J and Dalgarno L (1974) The 3’-terminal sequence of Escherichia coli
16S ribosomal RNA: complementarity to nonsense triplets and ribosome
binding sites. Proc. Natl. Acad. Sci. USA 71:1342-1346.

 

70

Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi
T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C,
Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K,
Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H and Sugiura M (1986)
The complete nucleotide sequence of the tobacco chloroplast genome: its gene
organization and expression. EMBO J. 5:2043-2049.

Tomioka N, Shinozaki K and Sugiura M (1981) Molecular cloning and
characterization of ribosomal RNA genes from a blue-green alga, Anacystis
nidulans. Mo]. Gen. Gene t. 184: 35 9-363.

Vieira J and Messing J (1987) Production of single-stranded plasmid DNA.
Methods Enzymol. 153:3—11.

Webber AN and Malkin R (1990) Photosystem I reaction-centre proteins

contain leucine zipper motifs. A proposed role in dimer formation. FEBS Lett.
264:1-4.

Williams J GK (1988) Construction of specific mutations in photosystem II
photosynthetic reaction center by genetic engineering methods in Synechocystis
6803. Methods Enzymol. 167:766-778.

Wynn RM, Omaha J and Malkin R (1989) Structural and functional properties
of the cyanobacterial photosystem I complex. Biochemistry 285554-5560.

 

 

CHAPTER3

Targeted genetic inactivation of the photosystem I reaction center in the

cyanobacterium synechocysns sp. PCC 6803

Published as Smart, L.B., Anderson, SL, and McIntosh, L. The EMBO Journal
vol. 10, pp. 3289-3296 (1991). Reprinted with permission of Oxford University

Press, Oxford, UK

INTRODUCTION
’ Photosystem (PS) I is a multi—subunit, membrane-bound protein complex
that functions as a light-driven, plastocyanin:ferredoxin oxidoreductase. PSI
operates in both cyclic electron flow, driving ATP synthesis, and in linear
electron flow, which drives ATP synthesis and the generation of reducing power
as NADPH. Light energy is captured by the PSI antenna and is funneled to a
chlorophyll (chl) a special-pair, P700, which passes an energized electron to A0 (a
specialized chl a). Pmt is then reduced by plastocyanin, which may be replaced
by cytochrome cm in cyanobacteria (Sandmann, 1986). Electron transfer
proceeds from A0 to A1 (most likely a phylloquinone), and then to Fx (a

[4Fe-4S] center), which donates electrons to FA and/or FB (both [4Fe-4S]

71

 

72

centers), the terminal acceptors of PSI. F A and/or FB reduce the soluble
electron carrier ferredoxin, which reduces NADP+ to NADPH, catalyzed by the
ferredoxin:NADP+ oxidoreductase (for a recent review of PSI, see (Golbeck
and Bryant, 1991)). The electron transfer components P700, A0, A1, and Fx are
bound to a heterodimer of homologous 82—83 kDa polypeptides, PsaA and
PsaB (Golbeck et a]., 1988; Golbeck and Bryant, 1991), encoded by the psaA
and psaB genes, which have been mapped to the plastome of plants and algae
(Westhoff et a1., 1983; Girard-Bascou, 1987b). Centers FA and FE are bound to
a 9 kDa polypeptide, PsaC, also encoded in the plastome by psaC (Hoj et a1.,
1987). Functions have been proposed for two of the eight additional known
PSI proteins: PsaD is thought to be involved in ferredoxin binding (Zilber and
Malkin, 1988), while PsaF may be involved in plastocyanin binding (Wynn and
Malkin, 1988).

Mutants with aberrant PSI have been isolated in Chlamydomonas
reinhardtii (Chua et a1., 1975; Girard et a1., 1980; Girard-Bascou, 1987b), maize
(Cook and Miles, 1990; Miles et a1., 1979), barley (Moller et a1., 1980),
Antirrhinum majus (Herrmann, 1971), Scenedesmus (Gregory et a1., 1971), and
Oenothera (Fork and Heber, 1968). Two nuclear mutants of C. reinhardtii
described by (Chua et a1., 1975) lack P700 activity and the P700- apoproteins, yet
still maintain wild-type levels of PSII activity and PSII core proteins. Further
analysis of those mutants and 23 other nuclear mutants of C. reinhardtii
deficient in the PSI reaction center proteins revealed the loss of a group of

small polypeptides that are enriched in PSI preparations from wild-type cells

 

 

73

(Girard et 31., 1980). Some of those nuclear mutations have been shown to
block the trans-splicing of the psaA message in the chloroplast, thus abolishing
PSI assembly (Choquet et a1., 1988; Goldschmidt-Clermont et a1., 1990). In
addition, mutations that mapped to the psaB gene in the plastome of C.
reinhardtii resulted in the loss of the PSI reaction center, as well as the same
group of small polypeptides deficient in the nuclear mutants (Girard-Bascou et
a1., 1987a). A nuclear mutant of barley, deficient in the PSI reaction center,
also lacks at least two small proteins that are enriched in wild—type PSI
particles, but maintains wild-type levels of PSII activity (Moller et a1., 1980).
Similar plastome mutants of Oenothera (Fork and Heber, 1968) and
Antirrhinum (Herrmann, 1971) and nuclear mutants of maize (Miles et 31.,
1979) that are deficient in the PSI reaction center also retain PSII activity.
Targeted mutagenesis of PS1 genes has not been accomplished in plants
or algae; however, inactivation of genes for two peripheral PSI proteins has
been achieved in the unicellular cyanobacterium Synechocystis sp. PCC 6803
(Chitnis et al., 1989b; Chitnis et al., 1989a). Inactivation of the psaD gene
resulted in reduced growth rate under photoautotr0phic conditions, but wild-
type growth rate under photoheterotrophic conditions (with DCMU [N1-
(dichlorOphenyl)-N3-dimethyl urea] and glucose in the light) (Chitnis et al.,
1989a). When the psaE gene, encoding an 8-kDa protein with unknown
function, was inactivated, only minor changes in PSI activity and
photoautotrOphic growth were observed (Chitnis et al., 1989b). Similar results

were obtained when psaB was inactivated in the cyanobacterium Synechococcus

 

 

 

 

 

 

74
sp. PCC 7002 (Bryant et a1., 1990).

Attempts at targeted mutagenesis of the psaA-psaB genes in
Synechococcus sp. PCC 7002 have resulted in only partial segregation of the
mutation (Zhao et a1., 1990), perhaps because selection was performed in the
light, which gave a selective advantage to cells maintaining wild-type PSI. This
problem is avoided by performing selection and segregation under
heterotrOphic growth conditions, which have recently been defined for
Synechocystis 6803 (Anderson and McIntosh, 1991). Termed light-activated
heterotrophic growth (LAHG), cells are grown with 5 mM glucose in complete
darkness except for 5 min of light (40 umolm'Z-S'l) every 24 h (Anderson and
McIntosh, 1991). Here we describe the first complete segregation of a directed

inactivation of psaA in Synechocystis 6803 by selection under LAHG conditions.

RESULTS

Construct interrupting psaA

A 1.8 kilobase pair (kb) Kpn I fragment, encoding most of the psaA
gene from Synechocystis 6803 (Smart and McIntosh, 1991, see Chapter 2), in
pUC119 (Vieira and Messing, 1987) (plasmid pLSl8), was digested with Nco I
and the resulting single-stranded DNA overhangs were digested briefly with
mung bean nuclease. A 1.25-kb Hinc II fragment, encoding an aminoglycoside
3’-phosphotransferase from Tn903 conferring resistance to kanamycin (Km)

(Oka et al., 1981), was purified from pUC4K (Vieira and Messing, 1982; Taylor

 

75
and Rose, 1988) and ligated into the blunted Nco I site of pL818. This was

transformed into Escherichia coli DH5a (Gibco-BRL) and Km-resistant (KmR)
colonies were recovered. The resulting plasmid, pLSl8K, had lost the Nco I
site and the Hinc II sites flanking the KmR cassette. A restriction map of the

construct from pLSl8K is shown in Figure 3.1.

Transformation of synechocystrs 6803 and selection conditions

Synechocystis 6803 that had been maintained under LAHG conditions
for two subcultures was transformed with the plasmid pLS18K. Selection for
KmR colonies was performed under both mixotrophic (continuous light and 5
mM glucose) and LAHG conditions. Single KmR colonies were streaked to at
least five serial plates to obtain full segregation of the mutation. One KmR
colony recovered under LAHG conditions (ADK9) and one recovered under

mixotrophic conditions (ALT6) were analyzed further.

Genetic mapping and segregation analysis

Southern blots of genomic digests from ADK9 were probed with the 1.8
kb Kpn I insert from pLS18 (Figure 3.2A). A restriction map of the psaA-psaB
region is shown in Figure 3.1. In all the digests of ADK9 DNA, no wild-type
copies of the psaA gene were detected (Figure 3.2A), even after prolonged

exposure (not shown). Hybridization of psaA to a Hind III digest of ADK9

 

76

psaA psaB

l///////////77] l/////////////1

 

 

 

5’N C K N K CCKCH KC CH CNNK 3’
X X
k 0'“) k pLS18K
R A
1 kb Km —
u___r (C) H (C)

Figure 3.1. Restriction map of the insert region from plasmid pLS18K and of
the psaA-psaB operon in Synechocystis 6803. Protein coding regions for psaA
and psaB are shown as hatched boxes. Cloned Synechocystis 6803 DNA in
pLSl8K is indicated as a line, while the KmR gene from pUC4K is indlcated as
a box. Potential regions of cross-over integration into the Synechocystis 6803
chromosome are shown as crosses. Restriction sites are: N, Nco I; C, Hinc 11;

K, Kpn I; H, Hind 111. Sites in parentheses were destroyed in the construction
of pLSlSK.

 

77

 

 

A E _ B
E 5 § 3 ‘é
a a a a - 9
E 9 E 9 E 9 E 9 E 9
kbp kb ‘
23.1 - 23.1 - 4.40 -
9.4— p ' 94
3: '. o 6"" ' 2-37-

MN

on)
MN
on:

0.24 - "

0.6 -
0.6 -

Figure 3.2. Autoradiographs of Southern and northern blots of wild type and
ADK9. A. Autoradiographs of Southern blots of wild-type and ADK9 DNA
probed with the insert from pL318. Enzymes used for digestion are indicated.
7.5 ug of DNA were loaded per lane. The Hind III, Kpn I, and Hinc II digests
were run on the same gel; the N00 I digest was run on a separate gel. Size
markers were from i digested with Hind III and are indicated in kilobase pairs
(kbp). B. Autoradiographs of northern blots of WT-LAHG RNA (15 pig) and
ADK9 RNA (15 pg) probed with the insert from pLSlS. Size markers are
from an RNA ladder (Gibco BRL) stained with ethidium bromide and are
indicated in kb.

78

DNA revealed a new Hind III site (within the KmR cassette), creating
fragments of 6.1 and 2.9 kb, rather than the wild-type 7.7 kb fragment. The
psaA probe hybridized to an 8.7 kb Nco I fragment of ADK9 DNA, rather than
to the 4.3 and 3.2 kb wild-type fragments, confirming the loss of this site by the
insertion of the KmR-cassette. Hybridization to Hinc II and Kpn I digests of
ADK9 DNA showed an increase in the length of the hybridizing fragments due
to the insertion of the KmR gene. The wild-type psaA and psaB genes are
arranged in tandem and are co-transcribed (Smart and McIntosh, 1991, see
Chapter 2). Insertion of the KmR cassette caused premature termination of
transcription, yielding a 1.2 kb message, rather than the wild-type 5.0 kb
message (Figure 3.2B).

DNA was isolated from ALT 6 at three stages of segregation: after five
serial platings under mixotrophic conditions (5 x MIXO), after five platings
under mixotrophic and one plating under LAHG conditions (1 x LAHG), and
after five platings under mixotrophic and five platings under LAHG conditions
(5 x LAHG). Km selection was maintained for all subcultures. These DNA
samples were digested with Hind III, separated by electrophoresis, blotted, and
hybridized with the psaA probe described above to determine the degree of
segregation of the psaA mutation at each stage (Figure 3.3). In addition, the
hybridization to fragments of 7.7, 6.1, and 2.9 kb was quantified using a
Betascope to determine the percentage of hybridization to wild-type or mutant
fragments (data not shown). The DNA from ALT6(5 x MIXO) has a high

percentage of wild-type copies of psaA (Figure 3.3); quantification showed the

 

 

 

 

7

\O

568
EII
31:125.
£551
O’cococom
SESSSEE
<<<<<

kbp

(75317 3 '0'“; 4

2.9 - ---.-

Figure 3.3. Autoradiographs of Southern blots of wild-type, ADK9, and ALT6
DNA. DNA was digested with Hind III and probed with the insert from
pLS18. 7.5 pg of DNA were loaded per lane. The four lanes on the left were
run on the same gel; the three lanes on the right were run on a separate gel.
DNA was isolated from ALT6 after 5 serial platings in mixotrophic conditions
(5xMIXO), after 5 platings in mixotrophic and 1 plating in LAHG conditions
(1xLAHG), and after 5 platings in mixotrophic and 5 platings in LAHG
(5xLAHG). Sizes were estimated from A digested with Hind III and are
indicated in kilobase pairs (kbp).

 

 

 

 

 

80

ratio of wild-type to mutant copies to be 3:1. After only one plate grown under
LAHG conditions, the ratio of wild-type to mutant copies shifts dramatically to
1:3. Five serial platings under LAHG conditions is sufficient for segregation of

the psaA mutation (Figure 3.3, 5 x LAHG).

Chlorophyll determination and oxygen evolution

The amount of chlorophyll in whole cells harvested at late log phase and
in isolated membranes is presented in Table 3.1 for WT-MIXO, WT-LAHG,
and ADK9 cells. The amount of chlorophyll per wild-type cell drops slightly
more than four-fold when grown under LAHG conditions. The ADK9 mutant
has less than half the chlorophyll per cell, when compared with WT-LAHG.
The relative amounts of chlorophyll per mg protein in membranes from the
three cell types approximately fits the same ratios as chlor0phyll per cell (Table
3.1). The oxygen evolution rates of all three cell types, assayed in the presence
of DCBQ, which accepts electrons from PSII in a DCMU-sensitive reaction
(Rutherford, 1988), are approximately equal, when normalized to equal cell
number (Table 3.1). The whole chain photosynthesis rates, measured with no
added acceptor, are only slightly lower for WT-LAHG compared with WT-
MIXO, on a per cell basis (Table 3.1). However, the oxygen evolution rate
with no artificial acceptor for ADK9 is z4—fold lower than for WT-LAHG,
when normalized to equal cell number (Table 3.1). Absorbance at 730 nm
(ODm) is a measure of the light scattering of a cell suspension and does not

assay absorption of any particular pigment. The number of cells per ml,

 

 

 

 

 

 

81

Table 3.1. Chlorophyll determination and photosynthesis assays.

 

 

 

 

 

 

WT-MIXO WT-LAHG ADK9
Chlorophyll
determination
Whole Cells'
ug chl/(mlO.D.-,3o) 3.661063 0.81 :031 0.37:0.09
Membranes”
ug chl/(mg protein) 43.1:72 99:04 52:14
Oxygen evolution
1 mM DCBQc
umol Oz/(mg chl°h) 206116 832: 194 16901306
umol Oz/(IO.D.730'h) 753 674 625
No added acceptord
umol 02/(mg chl-h) 148:30 537:79 287 :79
umol O,/(lO.D.730-h) 541 435 106

 

aAverages of at least five samples of each type.

t’Averages of two samples of each type.

cAverages of at least 14 trials for each type.

“Averages of at least six trials for each type.

 

 

 

82

relative to OD730, was approximately equal for all three cell type (data not

shown), indicating that OD730 is a good estimate of cell number.

Growth characteristics

The growth of ADK9 was indistinguishable from that of wild-type cells
under LAHG conditions (data not shown). However, after several attempts,
ADK9 would not divide under continuous illumination of 20 umolm'2s1, our

standard conditions for growth of wild-type Synechocystis 6803 (Jansson et a].,

1987)

EPR spectra

Spectra from membranes isolated from WT-MIXO, WT-LAHG and
ADK9 cells are shown in Figure 3.4. The spectra from illuminated wild-type
membranes show a prominent feature with a linewidth of 0.8-1.0 milliTesla
(mT) centered at g=2.0026, characteristic of signal I from Pm,” (panel A)
(Norris et a1., 1971). However, the signal from illuminated ADK9 membranes
had a 2.0 mT linewidth centered at g=2.0040, typical of signal 11 from YD+
(panels A and E) (Barry and Babcock, 1987). Signal 11 was detected from
membranes of all three cell types after dark adaptation (panel B). In ADK9,

there is no detectable signal I (panels A and E).

 

 

83

 

 

 

 

 

 

 

 

 

 

A Illuminated
d—x'l
d_)(" d“
dH
g
dH
d_X"
dH g-
dH

 

 

 

 

 

330 332 334 338 338 340
H (mT)

 

 

 

330 332 334 336 338 340

Figure 3.4. EPR spectra of thylakoid membranes from WT-MIXO, WT-LAHG,
and ADK9. Illumination was provided by a high-intensity microscope lamp;
dark-adaptation was for at least 10 min. Panel A contains spectra from all
three cell types under illumination, panel B shows spectra from all three cell -
types after dark-adaptation. Panels C, D, and E contain the same spectra as in
A and B presented as illuminated and dark-adapted for each cell type. Spectra

are standardized to equal gain and to 1 mg chlml'l.

 

84
Detection of proteins by antibodies

Thylakoid proteins from WT-MIXO, WT-LAHG, and ADK9 were
separated by SDS-PAGE, transferred to nitrocellulose, and probed with
antibodies raised to PS1 proteins and to the D1 protein of PSII (Figure 3.5).
Approximately equal protein was loaded in each lane, 120 ug protein
representing 5 ug chl for WT-MIXO, 150 ug protein for WT-LAHG (1.5 ug
chl), and 145 ug protein for ADK9 (0.75 ug chl). Antibodies raised to PsaA
and PsaB from spinach cross-reacted to a diffuse band with apparent molecular
mass of 60 - 65 kDa in both wild-type samples, but not in ADK9 (panel A).
This anomalous behavior of the P700-apoproteins in SDS-PAGE, migration at a
lower molecular weight than the predicted 83 kDa, has been observed
previously and is probably due to the highly hydrophobic nature of these
proteins (Fish et 31., 1985). Antibodies raised to PsaD cross-reacted to protein
of z15 kDa in both wild-type samples, both not in ADK9 (panel B). Similarly,
antibodies raised to PsaC cross-reacted to a protein of z9 kDa in the wild-type
samples (panel C), including a purified PSI preparation, but not in the ADK9
sample. The PsaC antibody cross-reacted to high molecular weight background
bands in wild-type and ADK9 thylakoids, but not in a PSI preparation. With all
three antibodies, development was allowed to continue until background bands
appeared in the ADK9 lane, to ensure there was no signal from a specific
cross-reaction in that lane. The intensity of signal from WT-MIXO was greater
than from WT-LAHG for all three PSI antibodies. Antibodies raised to PsbA

cross-reacted to a diffuse band of ”32 kDa in all three samples with

 

85

123 123 1234 123
kDa 'kDa kDa kDa

Figure 3.5. Immunoblots of thylakoid proteins from WT-MIXO, WT-LAHG,
and ADK9. Lane 1 is WT-MIXO (5 ug chl); lane 2 is WT-LAHG (1.5 pg chl);
lane 3 is ADK9 (0.75 ug chl); lane 4 (panel C) is purified PSI complex from
wild-type Synechocystis 6803 (5 ug chl). Immunoblots were probed with
PsaA/B antibody (panel A), PsaD antibody (panel B), PsaC antibody (panel C),
and PsbA antibody (panel D). Relative molecular weights were estimated by
comparison with prestained molecular size standards (Diversified Biotech) and
are expressed in kDa.

 

 

86

approximately equal intensity.

DISCUSSION

mechocysnls 6803 as a genetic system

Synechocystis 6803 serves as an excellent system for the genetic
alteration of photosynthetic proteins. Its ease of transformation, active
homologous recombination, and ability to express bacterial drug resistance
genes simplify targeted mutagenesis, providing that proper conditions for
selection are met. Since Synechocystis 6803 maintains multiple copies of its

genome per cell, complete segregation of a mutation may only be accomplished

 

if the presence of wild-type copies provides no selective advantage to the cell.
In order to mutagenize the core polypeptides of PSI, heterotrophic growth
conditions (LAHG) for Synechocystis 6803 were developed by Anderson and
McIntosh (Anderson and McIntosh, 1991). Procedures for isolation of PSI and
PSII complexes (Régner et a1., 1990) and well-characterized PSI and PSII EPR
signals (Barry and Babcock, 1987; Golbeck and Bryant, 1991) make analysis of

mutants relatively straightforward.

Response of wild-type cells to LAHG conditions ,
Wild-type Synechocystis 6803 grown under LAHG conditions has
dramatically less chlorophyll per cell, when compared to WT-MIXO. When

PSII activity and EPR signal 11 intensity are expressed per cell, the amount of

87
active PSII in WT-LAHG was only slightly less than in WT-MIXO. The signal

intensity from PsbA antibody was approximately equal in WT-MIXO and WT-
LAHG, while the signals from PS1 antibodies were lower in WT-LAHG
compared with WT-MIXO. The ratio of PSI to PSII, reflected by the relative
intensity of EPR signal I to signal II, was greater for WT-MIXO than for WT-
LAHG; on the order of 8 - 10:1 for WT-MIXO and 2 - 3:1 for WT-LAHG
(Figure 3.4, panels C and D). A similar ratio of PSI to PSII for light-grown
cells (9:1) was determined by (Rdgner et a1., 1990). Thus, the reduction in
chlorophyll in WT-LAHG cells was due primarily to a reduction in the amount
of PS1, while the amount of PSII remained approximately the same. However,
the pool of psaA-psaB transcript drops only 20% when Synechocystis 6803 is
grown under LAHG conditions, while the pool of psbA message drops >70%
(Smart and McIntosh, 1991, see Chapter 2). This indicates that there is some
form of post-transcriptional regulation of PSI and PSII accumulation in cells

grown under LAHG conditions.

Inactivation of psaA

We have used LAHG conditions for the selection of a psaA-inactivation
mutant of Synechocystis 6803. This mutant, ADK9, contains no detectable
functional PSI. Signal 1, from P700+, was not present in the EPR spectrum of
ADK9. Antibodies raised to the P700-apoproteins from spinach, which cross-
reacted to protein from wild-type Synechocystis 6803, did not detect the Pm- ’

apoproteins in ADK9 thylakoids. Upon probing a northern blot of ADK9

 

 

88

RNA with the psaA gene, only a truncated psaA-psaB transcript, z1.2 kb in
length was detected (5.0 kb in wild-type, Figure 3.2B). This is too short to
encode the full length psaA or psaB. ADK9 displayed greatly reduced rates of
oxygen evolution with no artificial acceptor. The low rate of oxygen evolution
that was observed in whole ADK9 cells may simply represent saturation of the
quinone pool, or it may indicate there was an undefined electron acceptor from
PSII. This activity was fully inhibited by DCMU, an inhibitor of PSII (data not
shown). Three possible electron acceptors for PSII in ADK9 are: ferredoxin,
which reduces NADP”, as proposed by (Arnon et a1., 1981); cytochrome
oxidase via the cytochrome b6/f complex, which is shared between
photosynthesis and respiration in cyanobacteria (Sandmann et al., 1984); or a
hydrogenase, which would reduce H+ to H2 gas and has been purified from
cyanobacteria (Ewert and Smith, 1989). The amount of chlorOphyll per cell
was greatly reduced in ADK9, allowing the turquoise-blue color of the
phycobilisomes to predominate. This would be expected with the loss of the
PSI core polypeptides, which bind over 100 chlorophyll molecules per P700
(Lundell et a1., 1985). By inspection of cells on plates illuminated with long-
wave UV light (366 nm), fluorescence yield from ADK9 was greater than from
WT-LAHG or WT-MIXO, a classic phenotype of mutants impaired in
photosynthesis (Figure 3.6) (Girard et a1., 1980; Miles et a1., 1979).

Antibodies raised to PsaC and to PsaD, membrane-extrinsic proteins of

 

 

 

89

BE? 3 223583:
We some 5% Eu

A8: 38 Em: 86.53:: BEE some“ 203 39 80309 of E Saga Jam:
5%: Es as, as as a: a seam 3&5 8:2 as 6253 0:593 .93 0&2;
m 28 Lo :33 88» mamauwouofi 35¢. Ea 653-; 652-; £5 856 Sam .3 pamfi

 

 

90

PSI, which cross-reacted to protein in wild-type thylakoids failed to detect those
proteins in ADK9 thylakoids. This suggests that PsaC and PsaD do not
accumulate in the absence of P700-apoproteins. Despite the absence of the PSI
core in ADK9, PSII assembled and formed functional complexes. The EPR
spectrum from ADK9 included signal II, from YD“, a tyrosine radical in the D2
polypeptide. Adding DCBQ, an acceptor of PSII, stimulated oxygen evolution
to near wild-type rates when expressed on a per cell basis. The D1 polypeptide
accumulated in ADK9, since antibodies raised to D1 from Amaranthus hybridus
cross-reacted to protein of the expected molecular mass (32 kDa) in ADK9
thylakoids. The loss of small PSI proteins concomitant with the inactivation of
the PSI core, with the continued assembly of PSII, corresponds to previously
characterized nuclear (Girard et 31., 1980; Chua et 31., 1975) and plastome
(Girard-Bascou, 1987b) mutants of C. reinhardtii and a nuclear mutant of
barley (Moller et 31., 1980). Genetic inactivation of PSI in strains with site-
directed mutations in PSII may facilitate their analysis by removing
contaminating signals from PSI.

Although ADK9 grew at wild-type rates under LAHG conditions, it did
not divide under continuous illumination of 20 umol'm'zs'l. The loss of PSI
and the chlorophyll bound to it probably made ADK9 extremely sensitive to
photoinhibition. We have not determined the maximum irradiance under which
ADK9 will grow. Since ADK9 did evolve oxygen, although at a much lower
rate, these cells may be able to grow autotrophically (in the light without

glucose) by oxidizing water and passing those electrons to an as yet undefined

 

 

 

91

acceptor.

Conclusions

The use of LAHG conditions is sufficient for complete segregation of
mutations in the PSI core in Smechocystils 6803. These conditions eliminate
the selective advantage conferred by the presence of wild-type PSI proteins and
avert the lethal effect of continuous light. The success of this inactivation of
psaA is encouraging for planned site-directed mutagenesis in the study of

structure/function relationships in PSI.

MATERIALS AND METHODS

Materials

All chemicals and reagents were of high purity and were obtained from
Sigma Chemical Co. (St. Louis, MO) or Research Organics (Cleveland, OH).
Enzymes were obtained from Gibco-BRL (Gaithersburg, MD) or New England
Biolabs (Beverly, MA). Nitrocellulose was obtained from Schleicher and
Schuell (Keene, NH). Radioactive nucleotide ([a-32P]dATP) was obtained from

Amersham (Arlington Heights, IL).

Strain and growth conditions
A glucose-tolerant (Williams, 1988), dark-growth-adapted (Anderson and

McIntosh, 1991) strain of Synechocystls sp. PCC 6803 was grown in BG-11

 

 

 

 

 

92

medium supplemented with 5 mM N-tris[hydroxymethy1]-2-aminoethanesulfonic
acid (TES) pH 8.0 and 5 mM glucose, as previously described (Jansson et 31.,
1987). Mixotrophic cultures (WT-MIXO) were grown in continuous light (20
umol'm'zsl), while heterotrophic cultures (WT-LAHG and ADK9) were
grown under LAHG conditions, complete darkness, except 5 min light

(40 umol-m'Z-S‘l) every 24 h in the presence of 5 mM glucose (Anderson and
McIntosh, 1991). For grth on solid medium, the above medium was

supplemented with 1.5% w/v agar and 0.3% sodium thiosulfate as described

 

(Anderson and McIntosh, 1991). When appropriate, media were supplemented
with 5 ug/ml kanamycin sulfate. Cells used for membrane isolation were grown
in 10 - 15 l of medium and were harvested using a Sorvall continuous flow
rotor. Cell growth was measured by absorbance at 730 nm (OD730) in a Gilford
Response spectrOphotometer using a glass, 10 mm pathlength cuvette. Cells
were counted in a hemacytometer. Chlorophyll was extracted with methanol
and quantified using described extinction coefficients (Lichtenthaler, 1987).
Fluorescence emission was assessed qualitatively from cells on solid medium

under illumination of long-wave UV light (366 nm).

Nucleic acid manipulations

All nucleic acid manipulations were performed using standard techniques
(Sambrook et a1., 1989), except where otherwise noted. Genomic DNA was
isolated from Synechocystzs 6803 essentially as described (Williams, 1988),

fractionated by agarose gel electrophoresis, and transferred to nitrocellulose

 

93

(Southern, 1975). RNA isolation and conditions for hybridizations were
previously described (Smart and McIntosh, 1991, see Chapter 2). A 1.8 kb Kpn
I fragment encoding most of psaA from Smechocystis 6803 (pLSlS) (Smart and
McIntosh, 1991, see Chapter 2) was random primer-labelled (Feinberg and
Vogelstein, 1983) and used as a probe. Quantification of hybridization to
Southern blots was performed using a Betascope machine (Betagen, Waltham,
MA). Transformation of Synechocystis 6803 was performed essentially as
described (Williams, 1988), except that cells were transferred to selective

medium 30 and 60 h after initial plating on non-selective medium.

Oxygen evolution assays

Rates of oxygen evolution were measured using whole cells as previously
described (Debus et a1., 1988). For wild-type cells, 10 pg chl‘ml'1 was used; for
ADK9 cells, 5 pg chl'rnl'1 was used. Light (from projector lamps) passed
through a copper sulfate solution and filtered with red cellophane provided
saturating illumination. DCBQ (1 mM) was added when appropriate. Cells for
oxygen evolution assays were grown to late exponential phase (OD-m = 0.6-

1.0), harvested, and washed twice with BG-11.

Thylakoid membrane isolation
Cells for thylakoid membrane isolation were grown to late exponential
phase, harvested by centrifugation, washed with BG-11, and resuspended in

break buffer (0.8 M sucrose, 20 mM MES [2-[N—morpholino]-ethansulfonic

 

 

 

 

 

94
acid] pH 6.0, 1 mM EDT A [ethylenediaminetetraacetic acid}, 2 mg/l DNase I, 3

mg/l PMSF [phenylmethylsulfonyl fluoride], 0.6 mg/l pepstatin a, 1.8 mg/l TPCK
[N-tosyl-L-phenylalanine chloromethyl ketone]). The cells were broken in a
Bead Beater (Biospec Products, Bartlesville, OK) essentially as described by
Rogner et a1. (Rogner et 31., 1990). Unbroken cells and cell debris were
pelleted at 6000g for 15 min (Sorvall GSA rotor). The membranes were then
precipitated with 25 mM calcium chloride and 5% w/v PEG-8000 (polyethylene

glycol MW 8,000) and pelleted at 27,000g for 15 min (Sorvall GSA rotor). The

 

membranes were resuspended in freeze buffer (25% v/v glycerol, 20 mM
calcium chloride, 20 mM MES pH 6.0), pelleted at 48,000g for 30 min (Sorvall
SS-34 rotor), then resuspended in freeze buffer and stored at -70°C. Purified
PSI complex from wild-type Synechocystis 6803 was provided by J. Golbeck
(Department of Biochemistry, University of Nebraska, Lincoln, NE). Protein

concentration was determined using the assay of Lowry (Lowry et a1., 1951).

EPR spectra

Isolated thylakoid membranes were pelleted and resuspended to a final

 

concentration of 0.28 - 1.7 mg chl'ml'1 in BG-11 / 16 ,uM DCMU / 0.25 mM

EDTA. EPR spectra were obtained as previously described (Barry and

 

Babcock, 1987). Illumination was provided by a high-intensity microscope light.
Dark-adaptation was for at least 10 min. Conditions were as follows: power, 2
mW; field modulation, 0.4 mT; time constant, 200 msec; sweep time, 100 sec.

Spectra were averaged from at least 5 scans. The gain used for dark-adapted

 

 

 

 

 

95

samples and for illuminated ADK9 was 8 x 106. The gain used for illuminated

WT-MIXO was 1 x 106 and for illuminated WT-LAHG was 2 x 10“.

SDS-PAGE and immunoblotting

Protein samples were separated by electrophoresis on a 10-17.5%
gradient acrylamide resolving gel with a 5% stacking gel (Laemmli, 1970)
(Laemmli, 1970). Samples were prepared as described Wynn et 31.,
(1989)(Wynn et 31., 1989). Gels were run at 25 mA constant current for 7-8 h.
Protein was visualized using Coomassie stain or transferred to nitrocellulose
overnight at 1 mA with cooling (Towbin et 31., 1979)(Towbin et 3]., 1979).
Relative molecular weights were estimated by comparison to prestained
molecular size standards (Diversified Biotech, Newton Centre, MA).
Immunoblots were incubated at least 1 h in blocking solution (10 mM Tris, pH
7.4; 0.9% sodium chloride (NaCl); 1% bovine serum albumin (BSA); 0.02%
sodium azide (NaNg); 2% dry milk), then incubated with the primary antibody
(1:500 dilution) for 1-1.5 h at room temperature. After washing off the primary
antibody (3 times for 15 min with 10 mM Tris, pH 7.4; 0.9% NaCl; 0.02%
NaN3; 0.1% Triton X-100; 0.05% SDS; 0.1% BSA) the blots were incubated
with goat anti-rabbit IgG conjugated to alkaline phosphatase (Kirkegaard and
Perry, Gaithersburg,MD) (1:3000 dilution of a 0.5 mg/ml solution in block
buffer). The detection reaction was performed in development buffer (100 mM
Tris, pH 8.8; 100 mM NaCl; 5 mM magnesium chloride) using BCIP

(5-bromo-4-chloro-3-indolyl phosphate p—toluidine salt) and NET (nitro blue

   

 

96

tetrazolium) as substrates (Knecht and Dimond, 1984). Antibodies raised in
rabbit against the P700-apoproteins (PsaA and PsaB) from spinach were the kind
gift of Nathan Nelson (Roche Institute, Nutley, NJ). Antibodies raised to the
PsaC protein from Synechococcus (Henry et a1., 1990) were the gift of James
Guikema (Div. of Biology, Kansas State Univ., Manhattan, KS). Antibodies
raised to the 32 kDa-D1 polypeptide from A. hybridus were previously
described (Ohad et 31., 1985). PsaD antibodies, raised in rabbit to spinach
protein, were the gift of Richard Malkin (Div. of Molecular Plant Biology, Univ.

of California, Berkeley, CA).

ACKNOWLEDGEMENTS

I am indebted to Dr. Neil Bowlby for performing EPR spectroscopy and
for helpful advice. I thank Gerald Babcock (Dept. of Chemistry, Michigan
State Univ.) for use of his EPR machine and James Tiedje and William Holben
(Dept. of Crop and Soil Sciences, Michigan State Univ.) for use of their
Betascope. I appreciate the gift of antibodies from Nathan Nelson, Richard
Malkin, and James Guikema. I thank John Shanklin for his assistance with
irnmunoblotting. This work was supported in part by DOE. grant DE-FG02-

90-ER 20021 (to L.M.) and M.S.U./R.E.F. (to L.M.).

 

 

 

 

97
REFERENCES

Anderson SL and McIntosh L (1991) Light-activated heterotrophic grth of
the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring
process. J. Bacteriol. 173:2761-2767.

Arnon DI, Tsujimoto HY and Tang GM-S (1981) Proton transport in
photooxidation of water: a new perspective on photosynthesis. Proc. Natl. Acad.
Sci. USA 78: 2942-2946.

 

Barry BA and Babcock GT (1987) Tyrosine radicals are involved in the
photosynthetic oxygen-evolving system. Proc. Natl. Acad. Sci. USA
84:7099-7103.

Bryant DA, Rhiel E, De Lorimier R, Zhou J, Stirewalt VL, Gasparich GE,
Dubbs JM and Snyder W (1990) Analysis of phycobilisome and photosystem I
complexes of cyanobacteria. In: Current Research in Photosynthesis, vol. II.
Baltscheffsky M, ed. Dordrecht, Netherlands: Kluwer Academic Publishers, pp.
1-9.

Chitnis PR, Reilly PA and Nelson N (19893) Insertional inactivation of the
gene encoding subunit II of photosystem I from the cyanobacterium
Synechocystis sp. PCC 6803. J. Biol. Chem. 264:18381-18385.

 

Chitnis PR, Reilly PA, Miedel MC and Nelson N (1989b) Structure and
targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I

from the cyanobacterium Synechocystis sp. PCC 6803. J. Biol. Chem.
264: 183 74-18380.

Choquet Y, Goldschmidt-Clermont M, Girard-Bascou J, Kiick U, Bennoun P
and Rochaix J-D (1988) Mutant phenotypes support a trans-splicing mechanism
for the expression of the tripartite psaA gene in the C. reinhardtii chloroplast.
CeII 52:903-913.

Chua NH, Matlin K and Bennoun P (1975) A chlorophyll-protein complex
lacking in photosystem I mutants of Chlamydomonas reinhardtii. J. Cell Biol.
67:361-377.

Cook WB and Miles D (1990) Anomalous electron transport activity in a
Photosystem I-deficient maize mutant. Photosynth. Res. 24:81-88.

Debus RJ, Barry BA, Babcock GT and McIntosh L (1988) Site-directed
mutagenesis identifies a tyrosine radical involved in the photosynthetic
oxygen-evolving system. Proc. Nat]. Acad. Sci. USA 85:427-430.

 

98

Ewert GD and Smith GD (1989) Purification and properties of soluble
hydrogenase from the cyanobacterium Anabaena cylindrica. Arch. Biochem.
Biophys. 268:327-337.

Feinberg AP and Vogelstein B (1983) A technique for radiolabelling DNA
restriction endonuclease fragments to high specific activity. Anal. Biochem.
132: 6-13.

Fish LE, Kiick U and Bogorad L (1985) Two partially homologous adjacent
light-inducible maize chloroplast genes encoding polypeptides of the P700
chlorophyll a-protein complex of photosystem I. J. Bio]. Chem. 260.1413-1421.

Fork DC and Heber UW (1968) Studies on electron-transport reactions of
photosynthesis in plastome mutants of Oenothera. Plant Physiol. 43:606-612.

Girard J, Chua NH, Bennoun P, Schmidt G and Delosme M (1980) Studies
on mutants deficient in the photosystem I reaction centers in Chlamydomonas
reinhardtii. Curr. Genet. 2:215-221.

Girard-Bascou J, Choquet Y, Schneider M, Delosme M and Dron M (1987a)
Characterization of a chloroplast mutation in the psaAZ gene of
ChIamydomonas reinhardtii Curr. Gene t. 12:489-495.

Girard-Bascou J (1987b) Mutations in four chloroplast loci of Chjamydomonas
reinhardtii affecting the photosystem I reaction centers. Curr. Genet.
12:483-488.

Golbeck JH, Parrett KG, Mehari T, Jones KL and Brand JJ (1988) Isolation
of the intact photosystem I reaction center core containing P700 and iron-sulfur
center Fx. FEBS Lett. 228: 268-272.

Golbeck J H and Bryant DA (1991) Photosystem 1. Curr. Topics Bioenerg.
16:83-177.

Goldschmidt-Clermont M, Girard-Bascou J, Choquet Y and Rochaix J-D
(1990) T rans-splicing mutants of Chlamydomonas reinhardtii. M01. Gen. Genet.
223:417-425.

Gregory RPF, Raps S and Bertsch W (1971) Are specific chlorophyll-protein
complexes required for photosynthesis?. Biochim. Biophys. Acta 234:330-334.

Henry RL, Li M and Guikema JA (1990) A comparison of the PSI polypeptide
organization of soybean and the cyanobacterium, Anacystis nidulans. In:
Current Research in Photosynthesis, Vol. II. Baltscheffsky M, ed. Dordrecht,
Netherlands: Kluwer Academic Publishers, pp. 567-570.

 

 

99

Herrmann F (1971) Genetic control of pigment-protein complexes I and Ia of
the plasmid mutant enzalba-l of Antirrhinum majus. FEBS Lett. 19:267-269.

Haj PB, Svendsen I, Scheller HV and Maller BL (1987) Identification of a
chloroplast-encoded 9-kDa polypeptide as a 2[4Fe-4S] protein carrying centers
A and B of photosystem I. J. Biol. Chem. 262:12676-12684.

Jansson C, Debus RJ, Osiewacz HD, Gurevitz M and McIntosh L (1987)
Construction of an obligate photoheterotrophic mutant of the cyanobacterium
Synechocystis 6803. PIant Physio]. 85:1021-1025.

Knecht DA and Dimond RL (1984) Visualization of antigenic proteins on
western blots. Anal. Biochem. 136:180-184.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227:680-685.

Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of
photosynthetic biomembranes. Methods Enzymol. 148:350-382.

Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein
measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.

Lundell DJ, Glazer AN, Melis A and Malkin R (1985) Characterization of a
cyanobacterial photosystem I complex. J. Biol. Chem. 26&646-654.

Miles CD, Markwell JP and Thornber JP (1979) Effect of nuclear mutation in
maize on photosynthetic activity and content of chlorophyll-protein complexes.
Plant Physio]. 64: 690-694.

Moller BL, Smillie RM and Hayer-Hansen G (1980) A photosystem I mutant
in barley (Hordeum vulgare L.). Carlsberg Res. Commun. 45:87-99.

Norris JR, Uphaus RA, Crespi HL and Katz JJ (1971) Electron spin
resonance of chlorophyll and the origin of signal I in photosynthesis. Proc. Nat].
Acad. Sci. USA 68:625-628.

Ohad 1, Kyle DJ and Hirschberg J (1985) Light-dependent degradation of the
QB-protein in isolated pea thylakoids. EMBO J. 421655-1659.

Oka A, Sugisaki H and Takanami M (1981) Nucleotide sequence of the
kanamycin resistance transposon Tn903. J. M01. Biol. 147:217-226.

Rutherford AW (1988) Photosystem II, the oxygen evolving photosystem. In:
Light-Energy Transduction in Photosynthesis: Higher Plant and Bacteria]

 

 

 

100

Models. Stevens SEJr and Bryant DA, eds. Rockville, MD: American Society of
Plant Physiologists, pp. 163-177.

Rogner M, Nixon PJ and Diner BA (1990) Purification and characterization of
photosystem I and photosystem 11 core complexes from wild-type and
phycocyanin-deficient strains of the cyanobacterium Synechocystis PCC 6803. J.
Biol. Chem. 265:6189-6196.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A
laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory Press.

Sandmann G, Scherer S and Edger P (1984) General aspects of location and
interaction of respiratory and photosynthetic electron transport in blue-green
algae. In: Compartments in Algal Cells and Their Interactions. Wiessner W,
Robinson D and Starr RC, eds. Berlin: Springer-Verlag, pp. 207-217.

Sandmann G (1986) Formation of plastocyanin and cytochrome c-553 in
different species of blue-green algae. Arch. Microbiol. 145:76—79.

Smart LB and McIntosh L (1991) Expression of photosynthesis genes in the
cyanobacterium Synechocystis sp. PCC 6803: psaA-psaB and psbA transcripts
accumulate in dark-grown cells. Plant Mol. Biol. 17:959-971.

Southern EM (1975) Detection of specific sequences among DNA fragments
separated by gel electrophoresis. J. M01. Biol. 98:503-508.

Taylor LA and Rose RE (1988) A correction in the nucleotide sequence of the
Tn903 kanamycin resistance determinant in pUC4K. Nucl. Acids Res.
16:358-358.

Towbin H, Staehelin T and Gordon J (1979) Electrophoretic transfer of

proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some
applications. Proc. Natl. Acad. Sci. USA 76:4350—4354.

Vieira J and Messing J (1982) The pUC plasmids, an M13mp7-derived system
for insertion mutagenesis and sequencing with synthetic universal primers. Gene
19:259-268.

Vieira J and Messing J (1987) Production of single-stranded plasmid DNA.
Methods Enzymol. 153:3-11.

Westhoff P, Alt J, Nelson N, Bottomley W, Bunemann H and Herrmann
RG (1983) Genes and transcripts for the P700 chlorophyll a apoprotein and
subunit 2 of the photosystem I reaction center complex from spinach thylakoid

 

 

 

 

101
membranes. Plant Mol. Biol. 2:95-107.

Williams J GK (1988) Construction of specific mutations in photosystem II
photosynthetic reaction center by genetic engineering methods in Smechocystis
6803. Methods Enzymol. 167: 766-778.

Wynn RM and Malkin R (1988) Interaction of plastocyanin with photosystem I:
a chemical cross-linking study of the polypeptide that binds plastocyanin.
Biochemistry 27:5 863-5 869.

Wynn RM, Omaha J and Malkin R (1989) Structural and functional properties
of the cyanobacterial photosystem I complex. Biochemistry 28:5554-5560.

Zhao J, Warren PV, Li N, Bryant DA and Golbeck JH (1990) Reconstitution
of electron transport in photosystem I with PsaC and PsaD proteins expressed
in Escherichia coli. FEBS Lett. 276: 175—180.

Zilber AL and Malkin R (1988) Ferredoxin cross-links to a 22 kD subunit of
photosystem 1. Plant Physiol. 88:810-814.

 

 

 

 

 

 

CHAPTER4

Genetic inactivation of the psaB gene in synechocystis sp. PCC 6803 disrupts

assembly of photosystem I

Published as Smart, LB. and McIntosh, L. Plant Molecular Biology (in press).

INTRODUCTION

The electron transfer components of PSI in plants, algae, and
cyanobacteria appear to be bound by three polypeptides, PsaA, PsaB, and PsaC
(Golbeck and Bryant, 1991). The PsaC protein has a molecular weight of
approximately 9 kDa and is much like a ferredoxin with two [4Fe-4S] centers
(Oh-oka et a1., 1988). The PsaA and PsaB proteins are homologous and form
a heterodimer to bind the electron transfer components P700, A0, A1, and Fx
(Golbeck and Bryant, 1991). We are using directed mutagenesis to investigate
the biogenesis of the PSI reaction center and to probe some of the critical
structure/function relationships in the core polypeptides. The unicellular
cyanobacterium Synechocystis sp. PCC 6803 is well-suited for the genetic
manipulation of PSI, because it is readily transformable, integrates DNA by

homologous recombination, expresses bacterial drug-resistance genes, and may

102

 

103
be grown heterotrophically (Anderson and McIntosh, 1991; Williams, 1988).

Targeted inactivation of the psaA gene in Synechocystis 6803 by selection and
segregation under light-activated heterotrophic growth (LAHG) conditions has
been described (Smart et al., 1991, see Chapter 3). In addition, targeted
inactivation of the psaB gene in the filamentous cyanobacterium Anabaena sp.
ATCC 29413 has been reported (Toelge et al., 1991).

This communication describes the targeted inactivation of the psaB gene
and full characterization of the effect of that mutation on the biogenesis of the

PSI reaction center.

RESULTS AND DISCUSSION

Fragments from plasmids pLSl3 and pLSlS (Smart and McIntosh, 1991,
see Chapter 2) were used in the construction of a plasmid (pLS1315K) in which
the Synechocystis 6803 psaB gene is interrupted by a gene cassette conferring
kanamycin resistance (KmR). The insert region of pLS1315K, as well as a
partial restriction map of the Synechocystis 6803 psaA-psaB operon, are shown
in Figure 4.1. Synechocystis 6803 was transformed with pLSI315K and
selection and segregation for KmR colonies was performed under LAHG
conditions, as previously described (Smart et a1., 1991, see Chapter 3). Since
pLSl315K is not able to replicate in Synechocystis 6803, and this bacterium has
an active homologous recombination system, KmR colonies would most likely be
the product of a gene replacement event, as depicted in Figure 4.1. This was

confirmed by Southern analysis of genomic DNA from one of the KmR colonies

 

 

104

psaA psaB

VWMM/i]

 

 

 

 

5’X K N K BKHS K EH NNK 3’
X X
pLS1315K
1 kb R
I______J Km
8 H BXS

Figure 4.1. Partial restriction map of the psaA-psaB operon from Synechocystis
6803 and of plasmid pLSl315K. Boxes represent coding regions. The
kanamycin resistance gene (KmR) was taken from the plasmid pUC4K (Vieira
and Messing, 1982; Taylor and Rose, 1988). Crosses represent possible regions
of homologous recombination. Restriction sites are: X, Xba I; K, Kpn I; N,
N00 1; B, BamH I; H, Hind III; S, Sph I; E, EcoR I.

 

105

(BDK8) that had been serially-streaked from single colonies at least five times
(Figure 4.2A). RNA was isolated from BDK8 and subjected to northern
analysis using a fragment of the psaA gene as a probe. A transcript of
approximately 3.7 kilobases (kb) in length accumulates in BDK8 to wild-type
levels and shows a similar pattern of degradation as wild type (Figure 4.2B).
This transcript would be of sufficient length to encode all of PsaA and appears
to terminate at approximately the position of the KmR-cassette insertion.
The phenotype of BDK8 indicates that there are major changes in the
composition of the thylakoids of this mutant. There is a decrease in the
chlorophyll per cell (Table 4.1), allowing the turquoise blue color of the
phycobilisomes to predominate (not shown). Likewise, there is a lower
proportion of chlorophyll to protein in the membranes of BDK8 (Table 4.1).
The BDK8 mutant is sensitive to continuous light above z3 umol'm'z-S'I.
BDK8 cells show very low rates of oxygen evolution in the absence of an
artificial electron acceptor (Table 4.1). However, in the presence of
2,6-dichlorobenzoquinone (DCBQ), an artificial acceptor of electrons from
PSII, rates of oxygen evolution are wild type, when expressed per cell. Electron
paramagnetic resonance (EPR) spectroscopy revealed a dark-stable feature of
approximately wild-type intensity with a linewidth of 2 milliTesla (mT) centered
at g=2.0043, typical of signal II from tyrosine YD“ in PSII (Figure 4.3). Signal
1, from P700”r in PSI, is absent in the illuminated sample (Figure 4.3). The PSI
proteins PsaA, PsaB, PsaC, and PsaD were undetectable by western analysis of

BDK8 membranes (Figure 4.4A,B,C). However, the PSII reaction center

 

 

 

106

 

 

 

   

A E - 3 B
e § s cg!)
Q § § - i9
5 8 5 8 E 8 E 8
kbp
kb
23.1 - 23.1 -
C It
9.4- 9.4-
6.6- 6.6- .
4.4- 4.4-
3:8: 33;
-
0.6- 0.6-

 

Figure 4.2. Autoradiographs of Southern and northern blots of wild type and
BDK8. A. Southern blot of genomic DNA from wild type (WT) and BDK8
digested with the restriction enzymes indicated and probed with a 1.4-kb Kpn I
fragment encoding the 3’-portion of psaA and the 5 ’-portion of psaB. The Xba
I digests were run on a separate gel. 5 pg of DNA was loaded in each lane. B.
Northern blot of RNA from LAHG-grown wild type cells and from BDK8,
separated using an agarose-formaldehyde gel and probed with a 1.8-kb Kpn I
fragment encoding most of psaA Size markers were ethidium bromide-stained
fragments from an RNA ladder (Gibco-BRL). DNA and RNA were isolated as
in Smart and McIntosh (1991, see Chapter 2).

 

 

 

 

 

 

 

107

Table 4.1. Chlorophyll determinations and Oz evolution assays“.

 

 

 

 

WT-LAHG BDK8

Chlorophyll quantification
Whole cells

ug chl/(mlODm) 0.81:0.31 0.55 $0.11
Membranes

ug chl/mg protein 99:04 7.3:0.4
Oxygen evolution
1 mM DCBQ

umol 07/(mg chl:h) 832: 194 11663285

umol 07/(IOD730h) 674 641
no added acceptor

umol Oz/(mg chl:h) 5371-79 236:65

 

aWT-LAHG data taken from and assays performed as in Smart et a]. (1991, see
Chapter 3).

 

 

 

108

 

 

.. , BDK8
" MEMBRANES

Six." . ,

dH ”
, 3" — Illuminated
3.“; ooooo Dark-Ad apted

g=2.0043
I I I I I I I I I I l I
330 332 334 336 338 340

H (mT)

Figure 4.3. Room temperature EPR spectra of BDK8 membranes. Solid line is
the spectrum taken while illuminating the cavity with a high-intensity
microscope lamp. Dashed line is the spectrum taken after dark-adaptation.
Spectrometer conditions were: power, 20 mW; field modulation, 4G; time
constant, 200 ms; sweep time, 200 3; gain, 2.5 x 106; sample concentration, 0.6
mg chl/ml.

 

 

109

.9 sage as. .33 a .8 swam 5
atom 33 3303053: Ea 836a 32» scenes: .8? as mw<m-mmm $2 d as m 28%

8338 263 2:2on d emu < £28m 8m .25 some 5 332 863 Q: 83 £8on
8 sea—mm new AD M283 Damn Am Beans 03m .2 $523 mama wfifiemOoB 868:5
wMQm 9a. .6 Gm<+§3 RS 25 game? .3 8x955 25 ES 858%
m .86on 38982: anE can .03<¥E 652-; Co 3032582: .3. 0.5mm

eonfiomoe mm 88
Ha mm6§.mdm s8 was: a
mo 385:8 3st AD BEN
firs FREE 803 805 .AmV
-Em: Boa even—0mm 68>» 838m

8'3

ps.--8

5..
«NF «NF

 

 

 

 

 

110

protein PsbA was detected at near wild-type levels on immunoblots of BDK8
membranes (Figure 4.4D).

The psaA and psaB genes encode homologous proteins, suggesting that
one arose by duplication of the other (Fish et a1., 1985). In particular, residues
that may be important for the dimerization of the reaction center are conserved
between the two polypeptides. These are: two conserved cysteine residues in
each of the polypeptides that probably serve as ligands to the [4Fe-4S] center
FX (Golbeck and Bryant, 1991) and a series of conserved leucines spaced seven
residues apart in each protein, which may form a leucine zipper (Kossel et al.,
1990; Webber and Malkin, 1990). Thus, the possibility exists that homodimers
of either PsaA or PsaB may form. The insertional mutation in BDK8 blocked
expression of the psaB gene, but a stable transcript of sufficient length to
encode PsaA did accumulate to near wild-type levels. However, no PsaA
protein could be detected by immunoblotting, and no functional P700 was
detected by EPR spectroscopy. These data strongly suggest that stable PsaA
homodimers do not form.

The phenotype of BDK8, a mutant in psaB, is nearly identical to that of
ADK9, a mutant with an insertional mutation in psaA that also blocks
expression of psaB (Smart et a1., 1991, see Chapter 3). Both mutations block
stable assembly of PSI, but retain near wild-type levels of functional PSII. A
similar phenotype was observed in a mutant of Anabaena variabihs ATCC
29413 in which psaB was insertionally inactivated (Toelge et a1., 1991) and in

chemically-generated mutants of psaB in Chlamydomonas reinhardtii

 

 

 

 

111
(Girard-Bascou, 1987). The strain BDK8 is also extremely light-sensitive, like

ADK9, probably due to lethal photo-inhibition of PSII, left unprotected by the
loss of all of the PSI chlorophyll. However, BDK8 can grow in very dim light
(#8 umol-m'z's'l) in the presence of glucose. We have used the genetic
inactivation of PSI to an advantage in the study of site-directed mutations of
PSII, since the biochemical purification of PSII complex is greatly simplified in
mutants lacking PSI. The development of PSI' recipient strains for the
mutagenesis of psbA and psz will be described elsewhere (Smart et al.,
submitted; see Chapter 5).

The ability to recover mutants with psaB genetically inactivated indicates
that site-directed mutagenesis of this gene is possible. We have initiated
experiments to mutagenize residues in PsaB thought to play roles in
coordinating electron transport components and/or in reaction center

dimerization and structure.

ACKNOWLEDGEMENTS

I wish to thank Dr. Neil Bowlby for performing EPR spectroscopy and
for helpful discussions and G. Babcock (Dept. of Chemistry, MSU) for use of
his spectrometer. The PsaA/B antibodies were the gift of J. Guikema (Kansas
State Univ.). The PsaC and PsaD antibodies were the gift of J. Golbeck (Univ.

of Nebraska).

 

 

 

 

 

 

112
REFERENCES

Anderson SL and McIntosh L (1991) Light-activated heterotrophic growth of
the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring
process. J. Bacteriol. 173:2761-2767.

Fish LE, Kiick U and Bogorad L (1985) Two partially homologous adjacent
light-inducible maize chloroplast genes encoding polypeptides of the P700
chlorophyll a-protein complex of photosystem I. J. Biol. Chem. 260:1413-1421.

Girard-Bascou J (1987) Mutations in four chloroplast loci of Chlamydomonas
reinhardtii affecting the photosystem I reaction centers. Curr. Genet.
12:483-488.

Golbeck JH and Bryant DA (1991) Photosystem I. Curr. Topics Bioenerg.
16:83-177.

Kossel H, Dory I, Igloi G and Maier R (1990) A leucine-zipper motif in
photosystem 1. Plant M01. Biol. 15:497-499.

Oh-oka H, Takahashi Y, Kuriyama K, Saeki K and Matsubara H (1988) The
protein responsible for center NE in spinach photosystem I: isolation with
iron-sulfur c1uster(s) and complete sequence analysis. J. Biochem. 103:962-968.

Smart LB and McIntosh L (1991) Expression of photosynthesis genes in the
cyanobacterium Synechocystis sp. PCC 6803: psaA-psaB and psbA transcripts
accumulate in dark-grown cells. Plant Mol. Biol. 17:959-971.

Smart LB, Anderson SL and McIntosh L (1991) Targeted genetic inactivation

of the photosystem I reaction center in the cyanobacterium Synechocystis sp.
PCC 6803. EMBO J. 1&3289—3296.

Taylor LA and Rose RE (1988) A correction in the nucleotide sequence of the
Tn903 kanamycin resistance determinant in pUC4K. Nucl. Acrds Res.
16:358-358.

Toelge M, Ziegler K, Maldener I and Lockau W (1991) Directed mutagenesis
of the gene psaB of Photosystem I of the cyanobacterium Anabaena variabilis
ATCC 29413. Biochim. Biophys. Acta 1060:233-236.

Vieira J and Messing J (1982) The pUC plasmids, an M13mp7—derived system
for insertion mutagenesis and sequencing with synthetic universal primers. Gene
19:259-268.

Webber AN and Malkin R (1990) Photosystem I reaction-centre proteins

 

 

113

contain leucine zipper motifs. A proposed role in dimer formation. FEBS Lett.
264:1-4.

Williams J GK (1988) Construction of specific mutations in photosystem II

photosynthetic reaction center by genetic engineering methods in Synechocystis
6803. Methods Enzymol. 167:766-778.

 

 

 

CHAPTERS

Genetic manipulation of the cyanobacterium synechocystzs sp. PCC 6803:

development of strains lacking PSI for the analysis of mutations in PSII

Submitted for publication as Smart, L.B., Bowlby, N.R., Anderson, S.L., Sithole,

I., and McIntosh, L.

INTRODUCTION

The study of structure and function relationships in the PSII reaction
center has been aided in recent years by the resolution of the three-dimensional
structure of the bacterial reaction center (Deisenhofer and Michel, 1989; Allen
et a1., 1987). The conservation of overall structural and functional components
has enabled accurate predictions of the corresponding architecture of PSII
(Michel and Deisenhofer, 1988). However, the non-oxygen-evolving bacterial
reaction center provides few clues about the organization of the oxygen
evolving complex (OEC) of PSII and the mechanism of water oxidation. In
contrast, a wealth of biochemical, biophysical, and physiological data have been
collected describing the enzymatic and chemical properties of the four Mn

cluster that forms the active site of the DEC (Diner and Joliot, 1977;

 

115

Ghanotakis and Yocum, 1990). This Mn cluster serves to accumulate the
oxidizing equivalents needed for the oxidation of water in a linear series of S-
states driven by four single-photon events (Kok et a1., 1970; Forbush et a1.,
1971). By using molecular genetics, precise determination of the amino acids
necessary for the coordination of this unique and crucial structure may be
possible.

Study of the structure and function of the photosystems has been
accelerated by the use of the transformable unicellular cyanobacterium
Synechocystis sp. PCC 6803 (Debus et a1., 1988a; Vermaas et 21., 1988a). This
strain is naturally competent and has an active homologous recombination
system, making it very amenable to molecular genetic manipulation (Williams,
1988). Synechocystis 6803 may also be grown photoheterotrophically in the
presence of DCMU and glucose (Williams, 1988). Photoheterotrophic growth
conditions negate the selective advantage conferred by the presence of wild-
type PSII and have allowed the segregation and recovery of directed mutations
in PSII reaction center genes (Debus et 31., 1988a; Metz et a1., 1989; Nixon and
Diner, 1992; Vermaas et al, 1988a).

Synechocystis 6803 lacks a trait found in chloroplast thylakoid
membranes, which allows for easy purification of PSII: lateral heterogeneity of
PSI and PSII (Andersson and Anderson, 1980). In chloroplasts, PSII is
concentrated in the stacked membranes, while PSI is confined to the unstacked
regions (Andersson and Anderson, 1980). Published protocols for the

purification of PSII from Synechocystis 6803 rely on selective detergent

 

116

solubilization, sucrose density centrifugation, and/or a series of chromatographic
separations (Burnap et al., 1989; Noren et a1., 1991; Rogner et a1., 1990),
techniques that can reduce or destroy OEC activity, especially in mutants (N.
Bowlby, unpublished results). Current techniques for the purification of PSII
have served well for certain types of analysis of site-directed PSH mutants
(Debus et a1., 1988a; Nixon and Diner, 1992) and detection of the EPR
multiline spectrum from the 82 state of the Mn cluster has been reported
(Noren et al., 1991). However, a method yielding highly purified and active
PSII with a minimum of biochemical manipulation would be highly beneficial

for the analysis of mutants in the OEC.

To facilitate such studies, we have embarked on a genetic approach to
the elimination of PSI, a detrimental contaminant of PSII preparations, in
Synechocystis 6803. Since there is no effective inhibitor of PSI activity, cells
must be grown heterotrophically to eliminate the selective advantage conferred
by wild-type PSI. Synechocystis 6803 may be grown in complete darkness using
glucose as a carbon source, requiring only a brief (5 min) pulse of light every 24
h (Anderson and McIntosh, 1991). By selecting under these light-activated
heterotrophic growth (LAHG) conditions (Anderson and McIntosh, 1991), we
have successfully inactivated the genes encoding the reaction center proteins of
PSI, psaA and psaB (Smart et a1., 1991, see Chapter 3; Smart and McIntosh,
1992, see Chapter 4). These mutants were devoid of any detectable PSI

complex, but still accumulated functional PSII at wild-type levels.

 

Here we describe the development of recipient strains for the

 

117

mutagenesis of the PSII reaction center genes psbA and psz. These strains,
D1'/PSI' and D2'/PSI' have mutations in psaA that prevent assembly of PSI. In
order to avoid problems associated with the segregation of a mixture of wild-
type and mutant psbA or psz sequences, the regions of the chromosome
targeted for mutagenesis have been deleted. In Synechocystis 6803, psbA is
present in three copies (Jansson et a1., 1987), therefore, deletions were
introduced into all three copies by replacing those regions of the chromosome
with three different drug-resistance cassettes (I. Sithole, N.R. Bowlby, J.
Sinclair, G.T. Babcock, L. McIntosh, in preparation). Likewise, psz is present
in two copies (Williams and Chisholm, 1987), into both of which deletions were
introduced. The rationales for attempting particular mutations in the psbA or
psz genes will be discussed elsewhere (Sithole et a1., in preparation).
Characterization of the strains D1'/PSI' and D2'/PSI' also gives us insight into

the biogenesis of PSII and aspects of cyanobacterial physiology.

MATERIALS AND METHODS
Materials

Chemicals and antibiotics used were of high purity and were obtained
from Sigma Chemical Co. (St. Louis, MO) or Research Organics (Cleveland,
OH). Restriction and other enzymes were purchased from New England
Biolabs (Beverly, MA). Radioactive isotope ([a-32P]dATP) was purchased from
Amersham (Arlington Heights, IL), nitrocellulose membrane from Schleicher

and Schuell (Keene, NH), and bacto—agar from Difco (Detroit, MI).

 

 

 

118

Strains and growth conditions

All studies were performed using a glucose-tolerant strain of the
cyanobacterium Synechocystis sp. PCC 6803 (Williams, 1988). The mutant
ADK9, an insertional inactivation mutant of psaA, was previously described
(Smart et a1., 1991, see Chapter 3). The mutant 3ApsbA, with all three copies
of psbA deleted, will be described elsewhere (Sithole et al., in preparation).
Cells were grown in liquid BG-11 medium with 5 mM glucose or on solid BG-
11 with 5 mM glucose and 1.5 % purified Bacto-Agar under LAHG conditions,
as previously described (Anderson and McIntosh, 1991). Transformation of
Synechocystis 6803 was performed essentially as described (Williams, 1988).
For analysis of mutants, cells were grown in carboys containing 15 l of medium,
were harvested using a continuous flow rotor ( DuPont Sorvall, Wilmington,
DE), and were frozen at -70°C in BG-11 with 15% v/v glycerol. When
appropriate, media were supplemented with antibiotics in the following
concentrations: chloramphenicol, 10 mg/l; erythromycin, 5 mg/l; spectinomycin,

20 mg/l; kanamycin, 5 mg/l; and gentamycin, 1 mg/l.

Nucleic acids

All nucleic acid manipulations were performed using standard
procedures (Sambrook et a1., 1989), unless otherwise stated. DNA was isolated
from Synechocystis 6803 using a modification of the procedure described by
Golden et a1. (1987) for the isolation of RNA from Synechocystis 6803. The

aqueous phase obtained after vortexing the cells with glass beads, buffer,

 

 

119

phenolzchloroform, and detergents was extracted twice with chloroform, then
purified over a cesium chloride gradient with ethidium bromide by
centrifugation in a Beckman vTi50 rotor at 45,000 RPM. for 18 h. The
ethidium-bromide-stained DNA band was recovered, extracted with isopropanol
to remove ethidium bromide, and ethanol precipitated. Hybridization
conditions and preparation of radiolabeled probes were previously described
(Smart and McIntosh, 1991, see Chapter 2). The probe for pszZ was a 0.5—kb
Kpn I/Sma I fi'agment from plasmid pRD655 (Debus et 31., 1988a). The pszI
probe was a 3.5-kb Hind III fragment from plasmid pRD1219 (Debus et a1.,
1988a). A 1.8-kb Kpn I fragment from pLSlS (Smart and McIntosh, 1991, see

Chapter 2) was used to probe for psaA.

Chl and protein analysis

Chl was extracted from whole cells or from membranes using methanol
and was quantified using the extinction coefficients of Lichtenthaler (1987).
Cells were broken using a bead beater (Biospec Products, Bartelsville, OK),
and membranes were prepared essentially as previously described (Smart et a1.,
1991, see Chapter 3). Membrane proteins were prepared for electrophoresis as
described by Wynn et a1. (1989) and were separated by 10% SDS-PAGE using
the buffers of Laemmli (1970). Proteins were transferred to nitrocellulose and
immunoblotting was performed as described by Towbin et a]. (1979) and Smart
et a]. (1991, see Chapter 3), respectively. Antibodies raised to the PsaA/B

proteins from Synechococcus have been described (Henry et a1., 1990), as have

 

 

 

 

 

 

120

antibodies raised to a portion of the D1 protein from Amaranthus hybridus
(Ohad et a1., 1985). Antibodies raised to the D2 polypeptide from spinach

were previously described (Vermaas et al., 1988b).

EPR spectroscopy

EPR spectroscopy was performed at room temperature using a Bruker
ER200D spectrometer as previously described (Smart et a1., 1991, see Chapter
3). Spectrometer conditions were: power, 20 mW; modulation amplitude, 0.4
mT; time constant, 200 ms; and sweep time, 200 s. Illumination was provided

by a high-intensity microscope lamp. Dark-adaptation was for at least 10 min.

RESULTS

Plasmid constructs and transformations

We used different strategies to create the two recipient strains for
mutagenesis of psbA and psz. In the case of the D2-recipient, we started
with a PSI~ mutant, ADK9 (Smart et a1., 1991, see Chapter 3), and sequentially
introduced deletions into both c0pies of psz. To create the D1-recipient
strain, we inactivated the psaA gene in a strain, 3ApsbA (Sithole et al., in
preparation), which had deletions in all three copies of psbA

The first step in constructing the D2-recipient was the deletion of the
pszZ gene. The strain ADK9 was transformed with the plasmid pRD655CmR

(Debus et al., 1988a), in which most of the pszZ gene had been replaced by a

 

 

 

 

 

121

gene encoding resistance to chloramphenicol. Colonies resistant to kanamycin
and chloramphenicol were selected and streak-purified from single colonies to
accomplish gene segregation. DNA was isolated from one of these colonies,
ADK9Apsz2, and was subjected to Southern analysis using the 3’-portion of
the pszZ gene as a probe (Figure 5.1A). Since these cells were found to be
segregated for the deletion of pszZ (Figure 5.1A), they were then transformed
with the plasmid pSLA1219szR (Figure 5.2A). This plasmid was a
modification of pRD1219 (Debus et al., 1988a). In pSLA1219szR most of the
psz] gene had been replaced by a 2.0-kb BamH I—fragment containing a gene
encoding resistance to gentamycin (Yin et a1., 1988), originally purified from the
plasmid pRleO7, then subcloned into the BamH I site of pUC119 (Vieira and
Messing, 1987), yielding pUC119-gen. Colonies resistant to chloramphenicol,
kanamycin, and gentamycin were selected and steak-purified from single
colonies to allow for segregation. DNA was isolated from one of these colonies
to assess whether there had been segregation of the psz] deletion by
Southern analysis (Figure 5.1B). This strain was designated D2'/PSI'.

The strain 3ApsbA was generated by targeted deletion of psbA2 in a
strain with deletions in psbA] and psbA3 (Debus et a1., 1988b), resulting in a
strain with genes encoding resistance to chloramphenicol, spectinomycin, and
erythromycin in the chromosome (Sithole et al., in preparation). In order to
inactivate PSI genetically in this strain, we constructed a plasmid, pLS1813G,
with a portion of the psaA gene replaced by a BamH I-Kpn I fragment from

the plasmid pUC119-gen encoding resistance to gentamycin (Figure 5.2B). The

 

 

 

122

1kb

2.6- .

 

Figure 5.1. Autoradiographs of Southern blots of genomic DNA from wild-type
and mutant cells. Lanes are: 1, 3, 5, wild type; 2, ADK9Apsz2; 4, D2‘/PSI’; 6,
D1'/PSI‘; 5 ug DNA per lane. Panels are: A, pszZ probe, DNA digested with
Hinc II; B, psz] probe, DNA digested with BstE II; C, psaA probe, DNA
digested with Kpn I. Autoradiographs were exposed for different lengths of
time.

 

 

 

123

 

A pst
psb D 1 V///////,l

 

 

EZZE
H TT HE 3,
.I———|—|.——l-h

 

. "" a r—-""—1 pSLA1219zRGm
H 070')

(B) camR (B)

psaA psaB

lf/Z///A7//L[/A U////¢7//////l

 

 

 

N K N K BK K NNK
5’ . . 3’
. 1

Kfi—‘(M K K pLS1813G

Figure 5.2. Partial restriction maps of plasmids used for mutagenesis and of
portions of the Synechocystis 6803 chromosome. A. Map of the pszl-pst
region and of the insert region from the plasmid pSLA1219:GmR. B. Map of
the psaA-psaB operon and of the insert region from the plasmid pL81813G.
Thick lines represent regions of the Synechocystis 6803 chromosome. Hatched
boxes represent coding regions. The cross-hatched boxes represent the
gentamycin resistance gene cassette. Crosses depict possible sites of
homologous recombination. Restriction sites in parentheses were destroyed in
the cloning process. Restriction sites are represented by: H, Hind III; T, BstE
II; E, EcoR I; B, BamH I; N, Nco I; K, Kpn I.

 

 

 

 

 

 

124

strain 3ApsbA was transformed with pL81813G and colonies resistant to
chloramphenicol, erythromycin, spectinomycin, and gentamycin were selected
and streak-purified from single colonies to accomplish segregation. DNA was
isolated from one of the resistant colonies and was subjected to Southern
analysis to assess whether there had been segregation of the psaA mutation

(Figure 5.1C). This strain was designated D1'/PSI'.

Characterization of D1'/PSI' and DZ'IPSI'

Analysis of the strains D1'/PSI' and D2'/PSI' included chl determinations,
immunoblotting, and EPR spectrosc0py. Chl determinations performed on
whole cells revealed very low levels of chl per cell in strain D2'/PSI', relative to
WT-MIXO (wild-type cells grown under mixotrophic conditions, light in the
presence of glucose) or WT-LAHG (wild-type cells grown under LAHG
conditions) (Table 5.1). The chl levels in strain D1'/PSI' were also lower than
in WT-LAHG (Table 5.1), but greater than in D2'/PSI°. The ratio of chl to
protein in isolated membranes follows the same trend (Table 5.1). Both strains,
D1‘/PSI' and D2'/PSI‘, grew at near wild-type rates under LAHG conditions, but
did not grow in continuous light above #3 umolm'Z-S'1 (data not shown).
Also, both cell types exhibited the turquoise-blue color seen previously in cells
lacking PSI (Smart et a1., 1991, see Chapter 3; Smart and McIntosh, 1992, see
Chapter 4).

To determine if any PSI or PSII reaction center proteins were

accumulating in the recipient strains, membrane proteins were subjected to

 

 

 

 

 

125

Table 5.1. Chlorophyll content of whole cells and thylakoid membranes.

 

 

 

WT- WT- D1'/PSI' D2‘/PSI'
MIXO‘ LAHG‘
Whole cells
ug chl/(mlOD730) 3.66:0.63 08110.31 0.31:0.06 0.15 -_*:N.D.
Membranes
ug chl/mg protein 43.1:7.2 99:04 5.5 $0.1 3.2-$0.3

 

'Data taken from Smart et a]. (1991, see Chapter 3).

ND, not determined.

 

126

irnmunoblotting. Antibodies raised to the PsaA/B polypeptides did not detect
the PSI reaction center proteins in membranes from either D1'/PSI' or D2'/PSI‘
(Figure 5.3A). Likewise, antibodies raised against D1 did not detect that
protein in membranes from either of the two recipient strains, but did display
apparent non-specific cross-reaction to a protein of av33 kDa (Figure 5.3B).
However, when antibodies raised to the D2 polypeptide were used, a reduced
amount of D2 protein was detected in membranes from the strain D1'/PSI'
(Figure 5.3C). Almost no D2 was detected in membranes from the strain D2'
/PSI' (Figure 5.3C). Protein >55 kDa that was recognized by antibodies raised
to D2 may represent dimers of D2 or may be protein exhibiting non-specific
cross-reaction.

EPR Spectroscopy is a sensitive and quantitative method for detecting
redox components in the two photosystems. We collected room temperature
EPR spectra from membranes of both recipient strains; conditions under which
one would expect to detect both signal I (from P700+ in PSI) and signal 11 (from
tyrosine YD”r in PSII). The spectra from both recipient strains include a
prominent feature centered at g=2.00425 with a linewidth of 1 mT and a
lineshape and low intensity (Figure 5.4) that clearly indicate it does not arise
from tyrosine YD+ (g=2.0045, AHPP==2 mT) (Barry and Babcock, 1987). This
feature is seen under illumination and in the dark and is of approximately equal
intensity in samples from the two strains (Figure 5.4). There are no light-

induced signals in the spectra from either recipient strain (Figure 5.4).

 

 

127

123123123
kDa

95.5 -

lax. -..
55.0-!

43.0 -

36.0 -

29.0- ‘

18.4 -

 

Figure 5.3. Immunoblots of membrane proteins isolated from wild type, D1'
/PSI‘, and D2‘/PSI'. Lanes are: 1, wild type; 2, D1'/PSI'; 3, D2'/PSI'; 150 ug
protein per lane. Antibodies used for the three panels recognized: A, PsaA/B;
B, the D1 polypeptide; C, the D2 polypeptide. Molecular weight standards
were prestained proteins purchased from Diversified Biotech (Newton Centre,
MA).

 

128

   
   

       
  

Dt /PS I
.fga,#}%u.&y§%é\§~hM-fifi5‘ . . p." -_‘ {W}: ‘yéigJii‘sa‘MWIIWksxgfixgfiut?
:. . z. ,9 “ '
dtol g=2.00425 """
—d D27PS r , |
35%;;
‘ ‘ .I. i» \ .
. :..’.;..'-',.5‘Mo".~ :‘ ' , . : I '
2‘», xx??? :" ‘59 " ’3 '
.J
.3 .
, — Illuminated
------ Dark-Adapted
I I I I I I l I I I I I
330 332 334 336 338 340

H (mT)

Figure 5.4. Room temperature EPR spectra of thylakoids from D1'/PSI' and
D2'/PSI'. The solid lines are the spectra obtained under constant illumination.
The dotted lines are the spectra collected after dark-adaptation. Spectra were
normalized to approximately equal chl concentrations. The magnetic field (H)
is expressed in milliTesla (mT). The vertical position of each spectrum in the
figure is unrelated to the scale of the vertical axis.

 

 

 

 

 

 

129

DISCUSSION

The purification of active PSII is essential for the analysis of site-directed
mutations targeted at the OEC. The process of purification of PSII from
Smechocystis 6803 is complicated by the lack of lateral heterogeneity of PSI
from PSII that is found in chloroplasts (Andersson and Anderson, 1980). We
have taken a genetic approach to eliminate PSI from mutants with site-directed
changes in PSII, by generating recipient strains in which PSI has been

genetically inactivated and deletions have been introduced into the targeted

 

PSII reaction center genes. By deleting the regions targeted for mutagenesis,
problems associated with segregating a mixture of wild-type and mutant
sequences are avoided. This is the first published use of the gentamycin
resistance gene (Yin et a1., 1988) for selection of mutations in a
cyanobacterium. This gene adds another option for manipulation of the
cyanobacterial chromosome. We plan to transform the strains D1'/PSI' and D2“
/PSI' with plasmid DNA encoding psbA2 or psz], respectively, including the
desired site-directed mutation. The DNA used for the transformation will
overlap the deleted regions and, since the plasmid vector cannot replicate in

the cyanobacterium, will integrate into the Synechocystis 6803 chromosome by

 

homologous recombination (Williams, 1988). An adjoining drug-resistance gene
will be used to select for integration of the transforming DNA. This type of
mutagenesis strategy allowing for rapid recovery of homozygous mutants has
been described previously (Nixon and Diner, 1992).

Our previous analysis of isolated membranes from strains ADK9 or

 

 

 

 

 

 

 

130
BDK8 yielded EPR spectra with PSII signals of wild-type intensity and

essentially free of other contaminating signals (Smart et 31., 1991, see Chapter
3; Smart and McIntosh, 1992, see Chapter 4). Thus, we are quite confident
that the inactivation of PSI does not significantly alter the stable assembly of
PSII in Smecbocystrls 6803. The strains D1'/PSI' and D2'/PSI‘ give us some
insight into the biogenesis of PSII. Genetic inactivation of the two copies of
psz, which encode the D2 polypeptide, also prevented accumulation of the
D1 polypeptide. However, targeted deletion of the three copies of psbA, which
encode D1, caused only partial reduction of the accumulation of D2. The D2
protein that did accumulate in D1'/PSI' did not assemble into functional PSII
complex, since we were unable to detect EPR signal II or oxygen—evolving
activity in the presence of DCBQ, an artificial electron acceptor from PSII
(data not shown). For the most part, our observations concerning PSII
biogenesis in PSI' strains correspond with those reported for strains containing
PSI. When both copies of psz were inactivated in a strain containing PSI, the
D1 polypeptide and other PSII proteins failed to accumulate (Vermaas et 31.,
1988b; Yu and Vermaas, 1990). In a mutant with the three copies of psbA
inactivated, the proteins CP43, Cyt b,,.,, and the 33-kDa protein accumulated to
levels only slightly lower than wild type, while the accumulation of the D2 and
CP47 proteins was greatly reduced (Jansson et 31., 1987; Nilsson et a1., 1990).
The difference in accumulation of D2 observed for strain D1'/PSI' and a D1'
strain containing PSI may be the result of different membrane isolation

techniques or due to different antisera. Our results reaffirm previous

 

 

 

 

 

 

131

observations that both D1 and D2 are crucial for stable assembly and
accumulation of PSII complex.

Assays of chl accumulation per cell serve well in estimating the
accumulation of PSI or PSII reaction center proteins (Smart et 31., 1991, see
Chapter 3; Smart and McIntosh, 1992, see Chapter 4) and may be used as a
quick and easy assay for initial characterization of mutants. This would be
expected, since cyanobacteria do not contain peripheral chl-binding antennae

proteins. The chl content in the D1‘/PSI' cells is reduced relative to ADK9 or

 

BDK8, cells lacking only PSI (Smart et 31., 1991, see Chapter 3; Smart and
McIntosh, 1992, see Chapter 4). The chl content in D2‘/PSI' is about half that
of D1'/PSI'. The higher chl levels in D1'/PSI' may reflect the accumulation of
low levels of D2. The low amount of chl in D2‘/PSI' may be bound to CP43
and CP47. We did not examine the accumulation of CP43 or CP47 in these
strains. The chl levels in both strains D1'/PSI‘ and D2'/PSI' are higher than one
would expect for cells that lack both PSI and the PSII reaction center. The
elevated chl levels may be the result of free chl that is trapped in membranes
or chl in some stage of synthesis or degradation, which may or may not be

bound to protein.

 

We have shown previously that cells with mutations which inactivate the
PSI reaction center genes exhibit normal assembly of stable PSII complex
(Smart et 31., 1991, see Chapter 3; Smart and McIntosh, 1992, see Chapter 4).
However, these strains are extremely light-sensitive, and, therefore, must be

grown in very dim light ('63 umol-m'Z-S'l) or under LAHG conditions. The

 

 

 

 

 

 

132

genetic inactivation of PSII does not appear to have relieved that light-
sensitivity, suggesting that there is some other light-induced component creating
toxic elements or that respiration cannot function to synthesize ATP in light >3
pmolm‘zsl.

The EPR spectra from D1'/PSI‘ and D2'/PSI' contained neither signal I,
from P700+, nor signal 11, from tyrosine YD+ in D2. The prominent feature,
common to spectra from both strains, was not light-induced. One possible
explanation is that this feature may have arisen from a semi-quinone in the
cytochrome bdf complex (G. T. Babcock, personal communication).

The strains D1'/PSI' and D2‘/PSI' represent the first mutants in a
photosynthetic organism with both photosystems genetically inactivated. As
well as serving in the process of site-directed mutation of PSII genes, these
strains may be particularly well-suited for other studies. In cyanobacteria, the
Cyt bdf chain is shared between photosynthesis and respiration (Scherer, 1990).
The coordinate regulation of electron flow between these pathways is poorly
understood. These strains may be appropriate for the study of respiration in
cyanobacteria, since the interaction with the photosystems has been eliminated.
The membranes from D2'/PSI' are essentially free of photosynthetic proteins, so
they may serve as excellent starting material for the purification of the

cytochrome b6/f complex.

 

 

 

133
ACKNOWLEDGEMENTS

Dr. Neil Bowlby performed the EPR spectroscopy presented in this
chapter. I wish to thank G. T. Babcock (Dept. of Chemistry, Michigan State
Univ.) for the use of his spectroscopy facilities and for helpful discussions. The
PsaA/B antibodies were the kind gift of J. Guikema (Kansas State Univ.,
Manhattan, KS). The D2 antibodies were the kind gift of W. Vermaas
(Arizona State Univ., Tempe, AZ). The plasmid pRZ1107, containing the
gentamycin-resistance gene, was the kind gift of C. P. Wolk (M.S.U-D.O.E.

Plant Research Laboratory, Michigan State Univ.)

 

 

 

 

134
REFERENCES

Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of
the reaction center from Rhodobacter sphaeroides R—26: the cofactors. Proc.
Nat]. Acad. Sci. USA 84:5 730-5734.

Anderson SL and McIntosh L (1991) Light-activated heterotrophic growth of
the cyanobacterium Smechocystis sp. strain PCC 6803: a blue-light-requiring
process. I. Bacteria]. 173:2761-2767.

Andersson B and Anderson JM (1980) Lateral heterogeneity in the distribution
of chlorophyll-protein complexes of the thylakoid membranes of spinach
chloroplasts. Biochim. Biophys. Acta 593:427-440.

Barry BA and Babcock GT (1987) Tyrosine radicals are involved in the

photosynthetic oxygen-evolving system. Proc. Natl. Acad. Sci. USA
84:7099-7103.

Burnap R, Koike H, Sotir0poulou G, Sherman LA and Inoue Y (1989)
Oxygen evolving membranes and particles from the transformable
cyanobacterium Synechocystis sp. PCC6803. Photosynth. Res. 22:123-130.

Debus RJ, Barry BA, Babcock GT and McIntosh L (1988a) Site-directed
mutagenesis identifies a tyrosine radical involved in the photosynthetic
oxygen-evolving system. Proc. Natl. Acad. Sci. USA 85:427-430.

Debus RJ, Barry BA, Sithole I, Babcock GT and McIntosh L (1988b)
Directed mutagenesis indicates that the donor to P680 in photosystem II is
tyrosine-161 of the D1 polypeptide. Biochemistry 27:9071-9074.

Deisenhofer J and Michel H (1989) The photosynthetic reaction center from
the purple bacterium Rhodopseudomonas vin'dis (Nobel lecture). Angew.
Chem. Int Ed. 28:829-847.

Diner BA and Joliot P (1977) Oxygen evolution and manganese. In:
Photosynthesis IzPhotosynthetic electron transport and photophosphorylation, In
Encyclopedia of Plant Physiology. Trebst A and Avron M, eds. New York:
Springer-Verlag, pp. 187-205.

Forbush B, Kok B and McGloin M (1971) Cooperation of charges in
photosynthetic 02 evolution - II. Damping of flash yield oscillation, deactivation.
Photochem. Photobiol. 14:307-321.

Ghanotakis DF and Yocum CF (1990) Photosystem II and the oxygen-evolving
complex. Annu. Rev. PIant Physio]. Plant M01. Biol. 41:255-276.

 

 

 

135

Golden SS, Brusslan J and Haselkorn R (1987) Genetic engineering of the
cyanobacterial chromosome. Methods Enzymol. 153:215-231.

Henry RL, Li M and Guikema JA (1990) A comparison of the PS1 polypeptide
organization of soybean and the cyanobacterium, Anacystis nidulans. In:
Current Research in Photosynthesis, Vol. II. Baltscheffsky M, ed. Dordrecht,
Netherlands: Kluwer Academic Publishers, pp. 567-570.

J ansson C, Debus RJ, Osiewacz HD, Gurevitz M and McIntosh L (1987)
Construction of an obligate photoheterotrophic mutant of the cyanobacterium
Synechocystis 6803. Plant Physio]. 85:1021-1025.

Kok B, Forbush B and McGloin M (1970) Cooperation of charges in
photosynthetic 02 evolution - 1. A linear four step mechanism. Photochem.
Photobiol. 11:45 7-475.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227:680-685.

Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of
photosynthetic biomembranes. Methods Enzymol. 148:350—382.

Metz JG, Nixon PJ, Rogner M, Brudvig GW and Diner BA (1989) Directed
alteration of the D1 polypeptide of photosystem II: evidence that tyrosine-161
is the redox component, Z, connecting the oxygen-evolving complex to the
primary electron donor, P680. Biochemistry 28:6960-6969.

Michel H and Deisenhofer J (1988) Relevance of the photosynthetic reaction

center from purple bacteria to the structure of photosystem II. Biochemistry
27:1-7.

Nilsson F, Andersson B and J ansson C (1990) Photosystem II characteristics of

a constructed Synechocystis 6803 mutant lacking synthesis of the D1
polypeptide. Plant Mo]. Bio]. 14:1051-1054.

Nixon PJ and Diner BA (1992) Aspartate 170 of the photosystem II reaction
center polypeptide D1 is involved in the assembly of the oxygen-evolving
manganese cluster. Biochemistry 31:942-948.

Noren GH, Boerner RJ and Barry BA (1991) EPR characterization of an
oxygen-evolving photosystem II preparation from the transformable
cyanobacterium Synechocystis 6803. Biochemistry 30:3943-3950.

Ohad 1, Kyle DJ and Hirschberg J (1985) Light-dependent degradation of the
QB-protein in isolated pea thylakoids. BAIBO J. 4:1655-1659.

 

 

 

 

136

Rogner M, Nixon PJ and Diner BA (1990) Purification and characterization of
photosystem I and photosystem 11 core complexes from wild-type and
phycocyanin-deficient strains of the cyanobacterium Synechocystis PCC 6803. J.
Biol. Chem. 265:6189—6196.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory Press.

Scherer S (1990) Do photosynthetic and respiratory electron transport chains
share redox proteins? TIBS 15:458-462.

Smart LB and McIntosh L (1991) Expression of photosynthesis genes in the
cyanobacterium Synechocystis sp. PCC 6803: psaA-psaB and psbA transcripts
accumulate in dark-grown cells. Plant Mo]. Biol. 17:959-971.

Smart LB, Anderson SL and McIntosh L (1991) Targeted genetic inactivation
of the photosystem I reaction center in the cyanobacterium Synechocystis sp.
PCC 6803. EMBO J. 10:3289—3296.

Smart LB and McIntosh L (1992) Genetic inactivation of the psaB gene in the
cyanobacterium Synechocystis sp. PCC 6803 disrupts assembly of photosystem 1.
Plant Mo]. Bio]. (in press)

Towbin H, Staehelin T and Gordon J (1979) ElectrOphoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some
applications. Proc. Nat]. Acad. Sci. USA 76:4350~4354.

Vermaas WFJ, Rutherford AW and Hansson O (1988a) Site-directed
mutagenesis in photosystem II of the cyanobacterium Synechocystis sp. PCC
6803: donor D is a tyrosine residue in the D2 protein. Proc. Nat]. Acad. Sci.
USA 85:8477-8481.

Vermaas WFJ, Ikeuchi M and Inoue Y (1988b) Protein composition of the
photosystem 11 core complex in genetically engineered mutants of the
cyanobacterium Synechocystis sp. PCC 6803. Photosynth. Res. 17:97-113.

Vieira J and Messing J (1987) Production of single-stranded plasmid DNA.
Methods Enzymo]. 153:3—11.

Williams J GK (1988) Construction of specific mutations in photosystem II
photosynthetic reaction center by genetic engineering methods in Synechocystis
6803. Methods Enzymol. 167:766-778.

 

 

 

 

137

Williams J GK and Chisholm DA (1987) Nucleotide sequences of both psz
genes from the cyanobacterium Synechocystis 6803. In: Progress in
Photosynthesis Research, Vol. IV. Biggens J, ed. Dordrecht: Martinus Nijhoff
Publishers, pp. 809-812.

Wynn RM, Omaha J and Malkin R (1989) Structural and functional properties
of the cyanobacterial photosystem 1 complex. Biochemistry 285554-5560.

Yin J CP, Krebs MP and Reznikoff WS (1988) Effect of dam methylation on
TnS transposition. J. Mol. Bio]. 199:35-45.

Yu J and Vermaas WFJ (1990) Transcript levels and synthesis of photosystem

11 components in cyanobacterial mutants with inactivated photosystem II genes.
Plant Cell. 2:315-322.

 

 

 

 

 

 

 

CHAPTER6

Mutational analysis of the structure and biogenesis of the photosystem I
reaction center in the cyanobacterium synechocysals sp. PCC 6803

Submitted for publication as Smart, L.B., Warren, P.V., Golbeck, J.H., and

McIntosh, L.

INTRODUCTION

The photosystem 1 (PSI) reaction center represents a grand complex of
protein subunits, both membrane-bound and extrinsic, which assemble to bind
cofactors that act to capture light energy and convert it into chemical energy
(for recent reviews, see Golbeck (1992); Golbeck and Bryant (1991)). The
coordination of the synthesis and assembly of the PSI subunits, which in plants
and algae are encoded by both nuclear and plastome genes, may be regulated
at many different levels. Our goal is to gain insight into the interaction of
protein subunits that allows the formation of the protein scaffold, which binds
electron transfer components, and the roles of those elements in the chemical
reactions performed by PSI.

A heterodimer of homologous 82 - 83-kDa polypeptides, PsaA and PsaB,

138

 

139
forms the reaction center of PS1. The PsaA and/or PsaB polypeptides bind the

chlorophyll (chl) a dimer P700, and the electron acceptors A0 (a chlor0phyll a),
A, (probably a phylloquinone (Golbeck and Bryant, 1991)), and Fx (a [4Fe-4S]
center); as well as a=100 antennae chl molecules (Golbeck, 1992). A third
polypeptide, PsaC, binds the terminal electron acceptors F A and FE, both of
which are [4Fe-4S] centers (Oh-oka et a1., 1988). There are at least eight
additional polypeptides associated with PSI, some that may serve to facilitate
binding of the soluble electron carriers plastocyanin and ferredoxin (Wynn and
Malkin, 1988; Zilber and Malkin, 1988), and others with unknown functions. In
plants and algae, the genes psaA, psaB, psaC, psaI, and psaJ are located in the
plastome, while the genes encoding the other known PSI proteins are nuclear-
encoded (Golbeck and Bryant, 1991).

Although attempts to determine the three-dimensional structure of PS1
are progressing (Alrnog et a1., 1991; Witt et a]., 1988), the current resolution of
the crystal structure does not allow one to define precisely the protein
structures important for the assembly and function of the reaction center. In
order to identify residues that may serve as important structural and/or
functional components in the complex, we must rely on analysis of evolutionary
conservation and on the current body of biochemical and biophysical data. The
most convincing evidence for the roles of particular residues in the PSI reaction
center concerns possible cysteine ligands to the [4Fe-4S] center Fx (Golbeck
and Cornelius, 1986) and leucines lying in a conserved leucine zipper motif in

the PsaA and PsaB proteins (Webber and Malkin, 1990; Kossel et a1., 1990).

 

 

 

140

Cysteine residues, with few exceptions, serve as the ligands to [4Fe-4S] centers
(Palmer, 1973). There are only three conserved cysteine residues in PsaA and
only two in PsaB. Two of the cysteines in PsaA are homologous to the PsaB
cysteines, lying in a twelve-residue sequence that is conserved in all the deduced
amino acid sequences of psaA and psaB genes sequenced thus far (Golbeck
and Bryant, 1991). It has been proposed that Fx bridges the PsaA and PsaB
polypeptides and is ligated by two cysteine residues from each of the reaction
center proteins (Golbeck and Cornelius, 1986) (Figure 6.1). Immediately
adjacent to the conserved cysteine motif in both PsaA and PsaB are series of
conserved leucines, spaced seven amino acids apart and predicted to lie in a
helices (Webber and Malkin, 1990; Kossel et a1., 1990) (Figure 6.1). The
cysteine residues proposed to coordinate Fx lie in a position analogous to the
DNA-binding region of bZIP proteins (Pu and Struhl, 1991; Webber and
Malkin, 1990).

We are using site-directed mutagenesis to investigate the roles of the
aforementioned cysteine and leucine residues in the biogenesis and function of
the PSI reaction center (Figure 6.1). This genetic manipulation is most readily
accomplished using the unicellular cyanobacterium Synechocystis sp. PCC 6803,
since it is easily transformed, has an active homologous recombination system,
expresses bacterial drug resistance genes, and may be grown heterotrophically
(Williams, 1988; Anderson and McIntosh, 1991). PSI in the cyanobacteria is
very similar to that from higher plants, both in subunit composition and in gene

organization (Ho and Krogmann, 1982; Golbeck and Bryant, 1991); thus,

 

 

 

 

 

141

Stroma
O O 565 S
533 Cg2) FX/©O E o

0 Fe
§ 21%
3/ . \s

 

 

 

 

 

 

 

 

 

568 8 %> 550
O
3 ,0
PsaA 00 -- o 54, PsaB
HelixVIII561 O (D """ 000 HelileIl
oQ 0O
000 ,,,,, C000 536>M >M
554 O Q : O Q g g
a F
00 00 E S
O 0 ® 0 529 '5 E
o ----- ' o 2 H
547 © ‘ O (53 an
00 00 E §
00 C30 3
CO ,,,,, (DO 522KV >V
540 0 ® L" 00
OO OO
O , O
OO ,,,,, ®O 515
533 ® ' O
O O

 

Figure 6.1. Cartoon of Fx and portions of PsaA and PsaB. Shown is a
schematic diagram of the eighth a helices of the PsaA and PsaB proteins from
Synechocystis 6803 and the regions containing the proposed ligands to the [4Fe-
4S] center FX. Site-directed residue changes described in the text are shown
next to arrowheads. FX is depicted as a cube. Regions predicted to form a
helices are bounded by rectangles. Dashed lines represent the possible leucine
zipper interaction. Solid lines represent the cysteinyl-iron bond coordinating
the [4Fe-4S] center.

 

 

 

 

142

cyanobacteria are excellent for the study of photosynthesis. By selecting under
light-activated heterotrophic growth (LAHG) conditions (Anderson and
McIntosh, 1991), we have isolated targeted mutants in which either the psaA or
the psaB gene (which encode the PSI reaction center proteins in Synechocystis
6803) have been inactivated (Smart et a1., 1991, see Chapter 3; Smart and
McIntosh, 1992, see Chapter 4). The ability to recover mutants lacking the PSI
reaction center indicates that the psaA and psaB genes are viable candidates
for site-directed mutagenesis. Inactivation mutations have also been introduced
individually into the psaD, psaB, and psaF genes, encoding peripheral PSI
polypeptides in Synechocystis 6803 (Chitnis et al, 1989a; Chitnis et a1., 1989b;
Chitnis et a]., 1991), but those mutants exhibited essentially wild-type
characteristics. Systems for the genetic inactivation of PSI genes have also been
developed using another cyanobacterium, Anabaena variabilis sp. ATCC 29413
(Toelge et a1., 1991; Mannan et a1., 1991), and the green alga ChIamydomonas
reinhardtii (Bingham et a1., 1991; Takahashi et a1., 1991).

We will describe the first site-directed amino acid changes in a PSI
reaction center protein incorporated in vivo. Site-directed mutations targeted
to a proposed ligand of Fx and to a leucine zipper motif were incorporated into
the psaB gene, and mutants were selected under LAHG conditions. Analysis of

these mutants provides new insight into the biogenesis and structure of the PSI

reaction center.

 

 

 

 

 

 

 

 

 

143
MATERIALS AND METHODS

Materials

Chemicals and antibiotics were of high purity and were purchased from
Sigma Chemical Co. (St. Louis,MO) or Research Organics (Cleveland, OH).
Restriction and other enzymes were purchased from New England Biolabs
(Beverly, MA), bacto-agar from Difco (Detroit,MI), and nitrocellulose from
Schleicher and Schuell (Keene,NH). Radioisotopes ([a-32P]dATP and [0:35-

S]dATP) were purchased from Amersham (Arlington Heights,IL).

Strains and growth conditions

Experiments were performed using a glucose-tolerant strain of
Synechocystis sp. PCC 6803, which was acclimated for growth on solid medium
in the dark (Williams, 1988; Anderson and McIntosh, 1991). Except for tests
for autotrophy, cells were grown at 30°C under light-activated heterotrophic
growth (LAHG) conditions, as previously described (Anderson and McIntosh,
1991), except that a 10-min pulse of light from fluorescent bulbs (rather than 5-
min) was used to ensure full illumination of all cultures in the growth chamber.
Cultures were grown in BG-11 medium (Allen, 1968) supplemented with 5 mM
glucose and 5 mM N-tris-(hydroxymethyl)—methyl-Z-aminoethanesulfonic acid
(TES). 1.5% w/v purified bacto-agar (Anderson and McIntosh, 1991) and 3
g/liter sodium thiosulfate were added to solid medium. Antibiotics were added

in the following concentrations: kanamycin (Km), 5 mg/liter; spectinomycin

 

 

 

 

 

 

 

 

144

(Sp), 20 mg/liter. Transformations were performed essentially as described
(Williams, 1988), except in the case of strain AB-RCPT, which was carefully
maintained in dim light throughout the procedure, since it is very light-sensitive.
Tests for autotrophic growth were performed using both liquid and solid
medium without supplemental glucose in a chamber providing continuous light
(~20 umolm'zsl). To ensure that cells would grow in continuous light, 116.
that they were not light-sensitive, cultures were also grown in the light in
medium with glucose. For those strains that exhibited light-sensitivity, screens
were used to reduce the light intensity to approximately 3 umol'rn'zs1 . Large
cultures were grown in 15 liters of medium in carboys, bubbled with air; were
harvested using a continuous flow rotor (DuPont Sorvall, Wilmington, DE); and

were frozen at -70°C in BG-11 with 15 % v/v glycerol.

DNA manipulations

Nucleic acids were manipulated using standard methodology (Sambrook
et a1., 1989), unless otherwise stated. E. coli strains XLl-Blue (Stratagene, La
J olla, CA) and TG1 (Amersham, Arlington Heights, IL) were used. Site-
directed mutagenesis was performed using an oligonucleotide-directed in vitro
mutagenesis kit as directed by the manufacturer (Amersham, Arlington Heights,
IL). DNA sequencing was performed using Sequenase version 2 polymerase as
directed by the manufacturer (United States Biochemical, Cleveland, OH). The
polymerase chain reaction (PCR) was performed using a Perkin-Elmer Cetus

thermal cycler and AmpliTaq polymerase as directed by the manufacturer

 

 

 

145
(Perkin-Elmer, N orwalk, CT). Asymmetric PCR amplification was performed

according to McCabe (1990). Amplification products were extracted once with
chloroform to remove mineral oil. Unincorporated nucleotides were removed
by precipitating and centrifuging the amplification products with an equal
volume (100 pl) of 4 M ammonium acetate and 2 volumes of ethanol. The
method for large-scale preparation of cyanobacterial DNA has been described
(Smart, Bowlby, Anderson, Sithole, and McIntosh, submitted; see Chapter 5).
Small-scale preparations of DNA were performed on cells either scraped from
solid medium or harvested from 50—ml liquid cultures as described (Ohad and

Hirschberg, 1992).

Membrane isolation and analysis

Thylakoid membranes were isolated using a modification of a previously
described procedure (Smart et a1., 1991, see Chapter 3). Material was
maintained on ice or at 4°C throughout the procedure. Previously frozen cells
from a 15-1iter culture were centrifuged in a Sorvall GSA rotor (8000 R.P.M., 8
min), and the pellet resuspended in approximately 100 ml of break buffer (50
mM Tris, pH 8.3; 0.8 M sucrose) to which were added: RNase A (20 ug/ml),
DNase I (20 ug/ml), phenylmethylsulfonylfluoride [PMSF] (10 ug/ml), and N-
tosyl-L—phenylalanine-chloromethyl ketone [TPCK] (10 ug/ml). The cells were
broken in a Bead Beater (Biospec Products, Bartelsville, OK) at 0°C with
approximately 200 ml of glass beads using 10 cycles of 30 sec on, followed by 8

min off. The cell extract solution was decanted, and the beads were repeatedly

 

 

 

 

146

washed with break buffer, which was decanted again, until the total volume of
the extract and buffer was 800 ml. Cell debris and remaining glass beads were
pelleted by centrifugation at 6000 R.P.M. for 8 min in a Sorvall GSA rotor.
The supernatant fluid was decanted into clean tubes, then 1 M calcium chloride
and 50% w/v PEG-8000 were added to a final concentration of 25 mM and 5%,
respectively. The resulting solution was centrifuged for 25 min at 12,000
R.P.M. in a Sorvall GSA rotor. The supernatant fluid, containing most of the
phycobilisomes, was discarded, and the membrane-containing pellets were
resuspended in glycerol freeze buffer (50 mM Tris, pH 8.3; 20 mM calcium
chloride; 25% v/v glycerol). This solution was centrifuged at 18,000 R.P.M. for
30 min in a Sorvall SS34 rotor, and the supernatant fluid was discarded. The
final pellets were resuspended in glycerol freeze buffer, pooled, and frozen at
-70°C.

SDS-PAGE and immunoblotting were performed as previously described
(Smart et a1., 1991, see Chapter 3). To resolve the PsaA/B proteins, 10% SDS-
PAGE gels were used; 17% gels were used to resolve PsaC and PsaD. Protein
assays were performed using the method of Lowry eta]. (1951). Equal
amounts of protein (150 pg) was loaded in each lane. Antibodies raised in
rabbits to the PsaA/B proteins from Synechococcus were previously described
(Henry et a1., 1990). Antibodies were raised in rabbits to PsaC or PsaD protein
purified from strains of E. co]i expressing, respectively, the psaC gene from
Synechococcus sp. PCC 7002 or the psaD gene from Nostoc sp. PCC 8009 (Li

et a1., 1991). PS I activity was measured in a Rank Brothers oxygen electrode

 

 

 

147

(Cambridge, England) using a modification of methods described by Izawa
(1980). Light (from projector lamps) was passed through a copper sulfate
solution and filtered with red cellophane, providing saturating illumination.
Membranes equivalent to 2.5 pg chlorophyll (chl) were assayed in 1 ml of
buffer (50 mM Hepes, pH 7.5; 10 mM NaCl; 5 mM MgC12) at 25°C containing
1 mM KCN, 20 ,uM N1-(dichlorophenyl)-N3,-dimethylurea (DCMU), 10 ug/ml
superoxide dismutase (Boehringer Mannheim, Indianapolis, IN), 0.5 mM
sodium ascorbate, 0.1 mM 2,6-dichlorophenolindophenol (DCIP), and 1.0 mM
methyl viologen. Chl was extracted with methanol and quantified using
published extinction coefficients (Lichtenthaler, 1987). A PSI complex was
isolated from thylakoid membranes using 1% v/v Triton X-100 and differential
centrifugation, followed by sucrose density centrifugation in 0.1% Triton X-100,

as described (Golbeck and Cornelius, 1986).

ESR spectroscopy

Room temperature ESR spectroscopy was performed on a Bruker
ER200D spectrometer essentially as previously described (Barry and Babcock,
1987). Membranes were pelleted and resuspended in 50 mM Tris, pH 8.3; 10
mM N aCl; 5 mM MgC12; 7.5% w/v PEG-3400 (Aldrich, Milwaukee, WI).
Illumination was provided by a high-intensity microscope lamp. Spectra were
mathematically manipulated using an IBM PS/2 computer and SpectraCalc
software (Galactic Industries Corp.). Low temperature ESR spectroscopy was

performed using a Bruker ECS-106 X-band spectrometer equipped with an

 

 

148

 

Oxford liquid helium cryostat and temperature controller, essentially as
previously described (Zhao et a1., 1992). Illumination was provided by a 150-W

xenon lamp.

RESULTS

Recipient strain

To allow for rapid segregation of mutations in the psaB gene, a recipient
strain of Synechocystis 6803 was engineered with the region of the chromosome
targeted for mutagenesis deleted. A plasmid (pLS3531K) was constructed that
incorporates a fragment encoding kanamycin resistance (Km?) from the
plasmid pUC4K (Vieira and Messing, 1982; Taylor and Rose, 1988), flanked by
a fragment encoding the 3’-half of psaA and the 5’-half of psaB from the
plasmid pLS35 and a fragment containing the psaB 3’-flanking region from the
plasmid pLS31 (Fig 2A). Wild-type Synechocystis 6803 was transformed with
pLS3531K, and KmR colonies were selected under LAHG conditions. After
five rounds of streaking from single colonies, DNA was isolated from one of the
KmR colonies and was subjected to Southern analysis using a fragment of the
psaB gene as a probe. The pattern of hybridization confirmed the segregation
of the deletion mutation (data not shown). This strain (AB-RCPT) exhibited
the turquoise-blue color characteristic of cells lacking PSI due to genetic
inactivation of either psaA or psaB (Smart et a1., 1991, see Chapter 3; Smart

and McIntosh, 1992, see Chapter 4).

 

 

 

 

r__* 1
149

Site-directed mutagenesis

Plasmids for site-directed mutagenesis of the psaB gene were
constructed in vitro and manipulated in E. coli. Oligonucleotides were designed
to effect the desired change in the coding sequence, while also destroying or
adding a restriction site. The resulting change in digestion pattern serves as an
effective and simple means of screening for the desired mutation. Site-directed

mutations were made using the Amersham oligonucleotide-directed in vitro

mutagenesis system and single-stranded DNA generated from the plasmid

 

pLS15, which contains a 1.2 kilobase pair (kbp) Hind III fragment encoding the
3’-ha1f of psaB (Smart and McIntosh, 1991, see Chapter 2). Incorporation of

the desired mutations was verified by restriction mapping and by DNA
sequencing. A 940-bp At] II/EcoR I fragment from each mutated pLSlS

plasmid was subcloned into the At] II/EcoR 1 sites of the plasmid pLS35. The

S2 fragment, which encodes resistance to spectinomycin and streptomycin
(SmR/SpR), from the plasmid pHP45S2 (Prentki and Krisch, 1984) was cloned
into an At] 11 site in pLS31, forming the plasmid pLS31€2. This Afl 11 site lies
100 bp downstream from the termination codon of psaB in the Synechocystis
6803 chromosome. An EcoR I fragment from pLS31€2 was cloned into the
EcoR I site of pLS35 and the mutated pLS35 plasmids. This final construct,
plasmid pLS3531§2 (Figure 6.2B), contains DNA that spans the deletion in the
strain AB-RCPT and includes a selectable marker, the Q cassette, downstream
from the psaB gene. The mutated pLS3531S2 plasmids were double-checked by

restriction mapping and by DNA sequencing to verify the presence of the

 

 

150

 

 

 

 

A. WT 6803 PSaA PsaB
K///YVOOCKK4A DOOCfl///VQOCH
5.x *5 5 was.“ 5,.
X X
pL33531K
Kmn

SXB H BKE

 

 

B A B-RCPT psaA psaB
' Ia///f//x[7j m/y///7/n
K K K H 3 Km“ EHA K
5’ l r__ _1 l n I 3!

 

 

X 1: X

1 kb pL33531n

 

 

 

Srr'i/SpR

 

r
MH 8 HM)

Figure 6.2. Partial restriction maps of psaA-psaB and of plasmids for
mutagenesis. A. Map of the insert region from the plasmid pLS3531K and of
the wild-type Synechocystis 6803 psaA-psaB operon. B. Map of the insert
region from the plasmid pLS3531S2 and of the psaA-psaB region of the
chromosome in strain AB-RCPT. Hatched boxes represent the coding regions
of psaA and psaB. Crossed-hatched boxes represent the KmR cassette from the
plasmid pUC4K. The stippled box represents the S2 cassette from the plasmid
pHP45S2. The solid regions of the cross-hatched rectangle in A represent
portions of the pUC119 polylinker. Crosses indicate possible regions of
homologous recombination. The asterisk indicates the location of a site-
directed mutation. Restriction sites are: X, Xba I; K, Kpn I; H, Hind III; S,
Sph I; B, BamH I; E, EcoR I; M, Sma 1. Sites in parentheses were destroyed
in cloning.

 

151

desired mutations and the proper sequence. Plasmid pLS3531S2 and its
mutated variants were then used to transform the strain AB-RCPT, and SpR
colonies were selected under LAHG conditions.

The presence of the desired mutations in SpR colonies was verified by
three different methods, each having particular advantages. To confirm the
incorporated change in restriction pattern quickly and from small batches of
cells, small-scale preparations of DNA were used as templates for conventional
PCR amplification of the mutated region of the psaB gene. The resulting PCR
products were digested with the informative restriction enzymes, resolved by gel
electrophoresis, and visualized by ethidium bromide staining (data not shown).
To confirm the overall integrity of the entire psaB region of the chromosome
and to detect any wild-type copies of the psaB gene, genomic DNA was
digested with the informative restriction enzyme and other enzymes recognizing
sites in this region and subjected to Southern analysis, using a fragment of the
psaB gene as a probe (data not shown). Finally, to verify the proper
integration of psaB sequence back into the region deleted in the strain AB-
RCPT, this region of DNA was amplified from CsCl-gradient-purified genomic
DNA by asymmetric PCR (McCabe, 1990), and the primarily single-stranded
PCR products were sequenced directly using internal oligonucleotide primers
(data not shown). This technique would also have the capability to detect a
mixture of wild-type and mutant sequence in genomic DNA In addition to
initial verification of the mutants, DNA was isolated from samples taken from

the large cultures to be used for phenotypic characterization to confirm that

 

 

 

 

 

 

 

 

152

reversion had not occurred. In all of the cultures tested, no mixture of wild-
type and mutant sequence or reversion of mutants to wild type was ever

detected.

Characterization

Mutations were made in the psaB gene as depicted in Figure 6.1,
changing the Cys at position 565 to Ser (C5658), His (C565H), and Asp
(C565D); changing the Leu at position 522 to Val (L522V) and Pro (L522P);
changing the Leu at position 536 to Met (1536M); and introducing mutations at
both positions 522 and 536 (L522V/L536M). To serve as a control, the strain
AB-RCPT was transformed with the wild-type plasmid pLS3531S2 (Figure 6.2B),
yielding the strain WTQ. After genetic verification of each mutant strain, cells
were tested for their ability to grow autotrophically in the light (z20
umol-m'Z—S'l) without glucose. The three Cys mutants and L522P, which all
exhibited light-sensitivity, failed to grow autotr0phically (Table 6.1). The three
conservative Leu mutants, L522V, L536M, and I522V/L536M, all grew
autotrophically at near wild-type rates (Table 6.1). PS1 electron transport
activity was measured in an oxygen electrode using isolated membranes. The
rate of 02 uptake was measured in the presence of: DCIPHZ, an electron donor
to PSI; methyl viologen, an electron acceptor from PSI; and inhibitors of PSII
activity (DCMU) and cytochrome oxidase activity (KCN) (Izawa, 1980). The
rates of 02 uptake by isolated membranes from L522V and L522V/L536M were

slightly lower than from the WTQ control, while the rate of 02 uptake by

 

153

 

.msoufiwmoa 0.5 as»: “a Bob 9833. 0.5 H82 8 mo ommuo>< $-0on wEon ma?

35:3 28056 oz: Sam: am Bob 93% 58 ENE 8 mo owmee>< A105. KEQOVEQ may."

.28 you Eu can 88 BEE: NO 835.5 A3. 18:. HASDOVNO 683.,

.mcofimumaoa 0.3 “an“: um 88m @3me 9% an“: 8 we owSo>< Awagbno mEVNO 38$.

 

 

manage 8.933 3 3.15.8 oz mama
8.38.2 8533 NE SN“ 82 3% EemBSNNB
338mg 83.de $3 5%..“wa 8» 283
$38.: 8533 $2 871.33 8% >~NB
«33% Sing 32 oeaaom oz Q38
N335 Sonia E Shaw oz 580
Sagas adage 59. £385 02 mmemo
RNHE: adage $8 $338 new a;
«cabana 58 Ha mug—Sm:
a 8530 ":8 ea no 35% No No we “33m £393. £86

 

333 man no «8358an .8 23.

 

154

membranes from L536M was slightly lower still, when expressed on the basis of
chlorophyll (Table 6.1). The rates of 02 uptake by isolated membranes from
C565H, C565D, and I..522P were all approximately equal and were very low
relative to WTQ (Table 6.1). The rates from these three strains were
comparable to rates measured using isolated membranes from strain BDK8, in
which the psaB gene has been inactivated (Smart and McIntosh, 1992, see
Chapter 4). However, the rates from strain C565S were over two-fold higher
than those from the other two Cys mutants, but were still significantly lower
than WTQ, when normalized to chlorophyll (Table 6.1). When expressed on a
per cell basis, the rates of 02 uptake by isolated membranes from L522V,
L536M, and L522V/L536M were comparable to WTQ, and the rates from
C565H, C565D, and L522P were all equally very low (Table 6.1). When
expressed on a per cell basis, the rate from C565S was approximately one-third
that of WT9 and was three-fold higher than those of the other Cys mutants
(Table 6.1).

The reduced rates of 02 uptake measured using membranes from
C5658, C565H, C565D, and L522P could have been the result of the lack of
stable assembly of PS1 or the assembly of PS1 with reduced activity. One way
to determine the accumulation of the chlorophyll-binding reaction center
proteins in cyanobacteria is to quantify the accumulation of chlorophyll, both in
whole cells and relative to the amount of protein in isolated membranes. The
strains C565H, C565D, and L522P all had reduced amounts of chlorophyll, both

in the whole cell measurements and in isolated membranes (Table 6.1).

155
Amounts of chlorophyll were slightly reduced in the strains C5658 and L522V,

when measured in whole cells, while the strain L522V/LS36M had near wild-
type levels of chlor0phyll per cell (Table 6.1). Chlorophyll determinations of
cells and membranes from strain L536M were consistently slightly higher than
wild-type (Table 6.1). The relative amounts of chlorophyll in the membranes
generally followed the same trends seen in the whole cell measurements (Table
6.1). The mutants C5658, L522V, L536M, and L522V/L536M were green in
color, while C565H, C565D, and L522P exhibited a turquoise-blue color (not
shown). Variability in the chlorophyll determinations may have been
introduced by differing accumulation of P811 or by the presence of free
chlorophyll.

In order to assay the accumulation of P81 proteins more precisely,
immunoblotting of membrane proteins was performed using antibodies
recognizing the proteins PsaA/B, PsaC, or PsaD (Figure 6.3). The cross-
reaction of all three antibodies to protein from strains L522V, L536M, and
L522V/L536M appeared to be approximately equal to that of wild type. All
three antibodies recognized reduced levels of protein in membranes from
C5658 relative to wild type. However, greatly reduced amounts of the PsaA/B
proteins were detected in membranes from C565H and C565D, and PsaA/B
were undetectable in membranes from L522P. The PsaC and PsaD proteins
were not detected by antibodies in membranes from C565H, C565D, or L522P.
However, the PsaD antibody did recognize a protein of approximately 11 kDa

with equal intensity in all the samples. Very faint cross-reaction to a protein

 

156

WT

w'r.

C565S
C565H
C5650
L522V
L536M
L522V/L536M
L522P

l

66 kDa _ PsaA/ B

PsaC
9 kDa - fin ‘ «- --—

 

Figure 6.3. Immunoblots of membrane proteins probed with antibodies

recognizing PsaA/B, PsaC, or PsaD. Equal amounts of protein (150 ug) were

loaded in each lane. Mr was estimated by comparison to migration of

prestained molecular weight standards (Diversified Biotech, Newton Centre,

MA) and is expressed in kiloDaltons (kDa).

 

157

with approximately the same Mr has been seen with different antiserum
recognizing PsaD (Smart et a1., 1991, see Chapter 3), suggesting the presence of
another protein with an epitope in common with PsaD.

Room temperature ESR spectroscopy was utilized to detect signal I,
from P700+ (Figure 6.4). Signal I has a typical linewidth of 0.8 - 1.0 milliTesla
(mT) centered at g=2.0025 (Norris et a], 1971) and can overlap signal II from
tyrosine YD+ in PSII (Aprz2.0 mT, g=2.0046) (Barry and Babcock, 1987).
Therefore, a spectrum. of membranes from the strain BDK8, an inactivation
mutant of psaB (Smart and McIntosh, 1992, see Chapter 4), was scaled and
subtracted from the normalized spectra of each strain, in order to remove
signal II from the spectra. The spectra from L522V, L536M, and
L522V/L536M are essentially the same as that of WT9, with a feature typical of
signal I and of approximately equal intensity. The spectrum of membranes
from strain L522P lacks any prominent features, once signal 11 has been
subtracted. The spectra of membranes from the three cysteine mutants include
a feature of similar intensity, with a lineshape, linewidth, and approximate g
value expected for signal I.

ESR spectroscopy was also performed at cryogenic temperatures to
detect signals generated by reduction of the [4Fe-4S] centers FA and F3 bound
by the PsaC protein. FA is characterized by g values of 2.05, 1.94, and 1.86,
while FB has gvalues of 2.07, 1.92, and 1.89 (Malkin and Bearden, 1978).
When both centers are reduced, their high-field and low-field signals merge,

producing a spectrum with gvalues of 2.05, 1.94, 1.92, and 1.89 (Malkin and

 

 

 

158

 
 
 
  

WT-O
522v
L536M
fl'
dH L522V/L536M

 

C5 H
C5650
L522P
I r I I j I I r I 1
331 332 333 334 335 336 337 333 339 340

H (mT)

Figure 6.4. Room temperature ESR spectra taken during illumination of
thylakoid membranes. Spectra were normalized to approximately equal protein
and equal spectrometer gain, then a scaled spectrum of BDK8 membranes
(Smart and McIntosh, 1992, see Chapter 4) was subtracted. The magnetic field
(H) is expressed in milliTesla (mT) Spectrometer conditions were: power, 20
mW; field modulation, 0.4 mT; time constant, 200 ms; sweep time, 200 s.

 

 

159

Bearden, 1978). The low temperature spectra of membranes from strains
L522V, L536M, and l..522V/L536M were essentially wild-type, while F A/FB
signals were not present in spectra of membranes from the strains C565H,
C565D, and L522P (see Appendix). Signals from photoreduced FA/FB were
present in spectra of membranes from C5658 (see Appendix) and in spectra of
P81 complex isolated from C5658 membranes (Figure 6.5). In both cases, the
FA and FE resonances were present at reduced intensity relative to those in the
spectrum of P81 complexes isolated from wild-type membranes. The g values
of the features in the C5658 spectrum are essentially the same as wild-type, as

is the ratio of the intensities of resonances from FA and F3 (see Appendix).

DISCUSSION

Site-directed mutagenesis of PSI

We have described the first in vivo site-directed amino acid changes in 3
PSI protein. This mutagenesis was rapidly accomplished using the unicellular
cyanobacterium Synechocystis 6803, which has an active homologous
recombination system (Williams, 1988) and may be grown heterotrophically
(Anderson and McIntosh, 1991). By first generating a deletion recipient strain,
site-directed mutations could be integrated into the chromosome efficiently,
avoiding the problem of segregation of a mixture of wild-type and mutant
sequences. These mutants appear to be quite stable, as we have not yet

detected reversion of any mutations. Synechocystis 6803 also has the advantage

 

 

 

160

C5658 mutant

light - dark (15K)

 

1.943

 

1.919

 

 

1.849
B 1.880

 

light — dark (298K)

8 = 2.047 1.941

 

1.924

 

 

1.884

Figure 6.5. Low temperature ESR spectra of PSI complex isolated from the
C5658 mutant. Spectra were taken at 15 K under illumination of (A) complex
frozen in the dark and (B) complex frozen under illumination. Numbers above
and below the spectra indicate the g—values of the principal resonances. The
spectrum in A was multiplied by a factor of 2 relative to that in B.

 

 

 

 

lg

 

161

of well characterized procedures for the purification of both PSI and P811
(Régner et a1., 1990; Noren et a1., 1991).

Changes of the cysteine residue at position 565 of PsaB have profound
effects on the stable assembly of P81. The mutants C5658, C565H, and C565D
all have greatly reduced accumulation of the PsaA, PsaB, PsaC, and PsaD
polypeptides, as determined by chlorophyll assays and immunoblotting. Also,
PSI activity is greatly reduced or absent in the three cysteine mutants as
assayed by 02 uptake assays and ESR spectroscopy. Assaying for the ability of
these strains to grow autotrophically is complicated by their sensitivity to
continuous light above 2'3 umol‘m'zs'l. These strains will grow in dim light
(<3 ,umolm‘zs‘l) with glucose, but the low light intensity in combination with
reduced amounts of assembled PSI may not allow autotrophic growth, even if
photosynthetic electron transport were functional in the mutants. The mutant
C5 658 does accumulate significantly more PSI than the other two cysteine
mutants, reflected in higher chlorophyll accumulation, higher 02 uptake rates
and stronger signals on immunoblots. Electron transfer to the terminal electron
acceptors in PSI, F A and F3, was detected in complexes from C5658, but the
amount of F A and F3 photoreduced is less than in wild-type complex on a
chlorophyll basis. The reduced intensity of signal I may be explained by rapid
rereduction of Pm,” due to impaired forward electron flow.

Synechocystis 6803 appears to have an active mechanism for digesting or
degrading PSI subunits that cannot form an active PSI complex. This is evident

in the L522P mutant in which none of the assayed PSI polypeptides

 

 

 

 

 

162

accumulated. This phenomenon may be sensitive to improper protein folding
or perhaps to redox status. In contrast, expression of mutant psaC in E. coli
has allowed purification of mutant PsaC protein, which may then be
reconstituted with the PSI core protein in vitro (Zhao et a1., 1990; Zhao et 3].,
1992). Using this system, substitution of an aspartate for a cysteine ligand to
either F A or F3 causes an inactive [3Fe-4S] center to form, rather than a [4Fe-
4S] center (Zhao et a1., 1992). However, the in vitro reconstitution system
requires great excess of added PsaC, PsaD, iron, and sulfur and may lack
important components and interactions integral to the natural system, and thus
may not truly reflect the in vivo situation.

The three residue substitutions at position 565 represent the non-
cysteinyl residues found to coordinate iron-sulfur centers in naturally occurring
or mutant iron-sulfur proteins. Histidine residues, in addition to cysteines,
serve as ligands to the [2Fe-28] centers in the Reiske iron-sulfur protein from
Thermus thermophilus (Cline et a], 1985), spinach (Britt et a1., 1991), and
Saccharomyces cerevrsiae (Graham and Trumpower, 1991). An aspartate, as
well as cysteines, serves as a ligand to one of the iron-sulfur centers in
ferredoxins from Desulfovibrio africanus (George et a1., 1989) and Pyrococcus
furiosus (Conover et a1., 1990). This center exists in the [3Fe-4S] state until
activated, when it reacts with Fe(II) ion to form a [4Fe-4S] center (George et
a1., 1989; Conover et 3]., 1990). Site-directed substitutions of serines for
cysteines that ligate center 1 in E. colifumarate reductase altered the

spectroscopic and electrochemical properties of the protein, but still effectively

 

 

 

 

163

coordinated the [2Fe-28] centers (Werth et a1., 1990). However, substitutions
of serine for cysteine in center 2 of the E. coli fumarate reductase or in the E.
coli dimethyl sulfoxide reductase prevented formation of [4Fe-4S] centers.
Interestingly, in the latter protein, a [3Fe-4S] center does form (Manodori et
a1., 1992; Rothery and Weiner, 1991). In addition, the [4Fe-4S] center in
activated pig heart aconitase is coordinated by three cysteine residues and
either water or hydroxyl from the solvent (Robbins and Stout, 1989). It is
important to note that all of these examples represent iron-sulfur centers with
intra-subunit ligands. The Fx center is a rare example of inter-subunit
coordination of an iron-sulfur center.

The conservative mutations introduced into the leucine zipper motif of
PsaB had no detectable effect on PSI accumulation or activity. All the assayed
characteristics of the mutants L522V, L536M, and I..522V/L536M were
essentially wild-type, including chlorophyll accumulation, 02 uptake activity,
ESR signal I intensity, protein accumulation as determined by immunoblotting,

autotrophic growth rate, and photoreduction of FA/FB as assayed by ESR

 

spectroscopy. We did not specifically assay PSI stability under conditions that
might disrupt the leucine zipper, such as elevated temperature, although PSI
complex from the L522V, L536M and L522V/LS36M mutants behaved like
wild-type complex through detergent solubilization and P81 purification (not
shown).

The residue substitutions incorporated into the leucine zipper motif have

been effective in perturbing leucine zippers in other proteins. In the case of

 

 

 

 

164

C/EBP, single and double valine for leucine substitutions disrupted
dimerization, as measured by cross-linking (Landschulz et a1., 1989). Likewise,
single and double valine substitutions in the protein Jun reduced dimerization
with Fos, as measured by co-immunoprecipitation with antibodies to either Fos
or Jun (Gentz et 3]., 1989). However, heptad repeats of leucines are also
found in ion channels and do not appear to act primarily in subunit
dimerization (McCormack et a1., 1989; McCormack et 3]., 1991). Single valine
for leucine substitutions in the DrosoPhiIa Shaker K+ channel protein altered
properties of the voltage-dependent channel opening, implying a role of the
repeated leucines in pore formation or channel gating (McCormack et a1.,
1991). The leucine zipper proteins that have been mutated so far have been,
for the most part, soluble proteins. The leucine zipper motif in the PSI
reaction center is a rare example of a membrane intrinsic leucine zipper. One
may not be able to extrapolate from the results obtained by previous study of
soluble leucine zippers when analyzing the effects of mutations in a membrane-
bound leucine zipper. The Specific interactions between leucines may be less
important in a membrane environment, or fewer leucines may be required to
accomplish dimerization. Also, it is unclear whether a similar coiled-coil
structure would form in a membrane-bound leucine zipper.

In contrast to the introduction of conservative substitutions, the
introduction of a proline at position 522 of PsaB had a deleterious effect on the
biogenesis of PSI. This mutant has virtually undetectable levels of PsaA, PsaB,

PsaC, and PsaD; no ESR signal 1; low accumulation of chlorophyll; low rates of

 

 

 

 

 

165
02 uptake; and no detectable reduced FA/FB ESR signals. In all aspects

measured, this strain has the phenotype of BDK8, an inactivation mutant of
psaB (Smart and McIntosh, 1992, see Chapter 4). The leucine to proline
residue substitution probably disrupts the secondary structure of the eighth a-
helix of PsaB, preventing stable assembly of the PSI reaction center and
accumulation of P81 proteins. However, the codon introduced in this
substitution is used only 6% of the time compared with the other codons for
proline in a survey of 29 Synechocystis 6803 genes (not shown). Low
abundance of the tRNA for this codon may also contribute to the lack of

accumulation of PsaB in strain L522P.

Conclusions

Our data indicate that the cysteine at position 565 of PsaB serves as a
ligand to the [4Fe-4S] center Fx and supports the proposal that Fx bridges the
PSI reaction center proteins (Golbeck and Cornelius, 1986; Golbeck, 1992).
Not only does Fx act as an electron transfer component, but this [4Fe-4S]
center also plays a major structural role in reaction center dimerization and PSI
complex stability. Our data do not definitively address the role of the leucine
zipper, but suggest one of the following conclusions: 1) the mutations
introduced (other than L522P) were too conservative to disrupt the leucine
zipper interaction, 2) the leucine zipper was disrupted, but plays a minor role in
PSI biogenesis, perhaps acting functionally in an as yet unmeasured capacity, or

3) the leucine zipper does not form in the PSI reaction center of Synechocystis

 

166

6803. Further mutagenesis, already in progress, will be needed to address
definitively the leucine zipper hypothesis.

The successful completion of this series of site-directed mutations in the
psaB gene invites further genetic manipulation of the PSI reaction center,
hopefully leading to a full understanding of the structure—function relationships
important for the assembly and activity of this intricate and vital protein

complex.

ACKNOWLEDGEMENTS

Patrick Warren and Dr. John Golbeck performed the low-temperature
ESR spectroscopy shown in this chapter and in the appendix. I am grateful to
Dr. Neil Bowlby for performing room temperature ESR spectroscopy and for
helpful advice and discussions. I thank Dr. Gerald Babcock (Department of
Chemistry, Michigan State University) for the use of his ESR facilities. I am
indebted to Dr. James Guikema (Kansas State University, Manhattan, KS) for
the gift of the PsaA/B antibodies and to Dr. Donald Bryant (Pennsylvania State
University, University Park, PA) for providing PsaC and PsaD protein. This
work was supported by the US. Department of Energy (#DE-FG02-90-ER
20021 to L.M.), by Michigan State University Research Excellence Funds (to

L.M.) and by the National Science Foundation (#MCB-9205 756 to J.H.G.).

 

 

167
REFERENCES

Allen MM (1968) Simple conditions for growth of unicellular blue-green algae
on plates. J. Phycol. 4:1-3.

Almog O, Shoham G, Michaeli D and Nechushtai R (1991) Monomeric and
trimeric forms of photosystem I reaction center of Mastigocladus laminosus:
Crystallization and preliminary characterization. Proc. Nat]. Acad. Sci. USA
88:5312-5316.

Anderson SL and McIntosh L (1991) Light-activated heterotrophic growth of
the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring
process. J. Bacteria]. 173:2761-2767.

Barry BA and Babcock GT (1987) Tyrosine radicals are involved in the
photosynthetic oxygen-evolving system. Proc. Nat]. Acad. Sci. USA
84:7099-7103.

Bingham SE, Xu R and Webber AN (1991) Transformation of chloroplasts
with the psaB gene encoding a polypeptide of the photosystem I reaction
center. FEBS Lett. 292:137-140.

Britt RD, Sauer K, Klein MP, Knaff DB, Kriauciunas A, Yu C-A, Yu L
and Malkin R (1991) Electron spin echo envelope modulation spectroscopy
supports the suggested coordination of two histidine ligands to the Rieske Fe-S
centers of the cytochrome b6f complex of spinach and the cytochrome bc]
complexes of RhodospiriHum rubrum, Rhodobacter sphaeroides R-26, and
bovine heart mitochondria. Biochemistry 30:1892-1901.

Chitnis PR, Reilly PA, Miedel MC and Nelson N (1989a) Structure and
targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I
from the cyanobacterium Synechocystis sp. PCC 6803. J. Biol. Chem.

264: 18374-18380.

Chitnis PR, Reilly PA and Nelson N ( 1989b) Insertional inactivation of the
gene encoding subunit II of photosystem I from the cyanobacterium
Synechocystis sp. PCC 6803. J. Biol. Chem. 264:18381-18385.

Chitnis PR, Purvis D and Nelson N (1991) Molecular cloning and targeted
mutagenesis of the gene psaF encoding subunit III of photosystem I from the
cyanobacterium Synechocystis sp. PCC 6803. 1 Bio]. Chem. 266:20146—20151.

Cline JF, Hoffman BM, Mims WB, LaHaie E, Ballou DP and Fee JA (1985)
Evidence for N coordination to Fe in the [2Fe-28] clusters of Thermus Rieske
protein and phthalate dioxygenase from Pseudomonas. J Biol. Chem.

 

 

168
260:325 1-325 4.

Conover RC, Kowal AT, Fu W, Park J-B, Aono S, Adams MWW and
Johnson MK (1990) Spectroscopic characterization of the novel iron-sulfur
cluster in Pyrococcus furiosus ferredoxin. J. Biol. Chem. 265:8533-8541.

Gentz R, Rauscher FJ III, Abate C and Curran T (1989) Parallel association
of Fos and Jun leucine zippers juxtaposes DNA binding domains. Science
243: 1695 -1699.

George SJ, Armstrong FA, Hatchikian EC and Thomson AJ (1989)
Electrochemical and spectroscopic characterization of the conversion of the 7Fe
into the 8F e form of ferredoxin III from Desulfovibn'o aflicanus. Identification
of a [4Fe- 4S] cluster with one non-cysteine ligand. Biochem. J. 264:275-284.

Golbeck JH and Bryant DA (1991) Photosystem 1. Curr. Topics Bioenerg.
16:83-177.

Golbeck JH and Cornelius JM (1986) Photosystem I charge separation in the
absence of centers A and B. 1. Optical characterization of center ‘A2’ and
evidence for its association with a 64-kDa peptide. Biochim. Biophys. Acta
849:16-24.

Golbeck JH (1992) Structure and function of photosystem 1. Annu. Rev. Plant
Physio]. Plant Mo]. Biol. 43:293-324.

Graham LA and Trumpower BL (1991) Mutational analysis of the
mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevrlsrae. 111.
Import, protease processing, and assembly into the cytochrome bc1 complex of
iron-sulfur protein lacking the iron-sulfur cluster. J. Biol. Chem.

266: 22485-22492.

Henry RL, Li M and Guikema JA (1990) A comparison of the P81 polypeptide
organization of soybean and the cyanobacterium, Anacystis nidulans. In:
Current Research in Photosynthesis, Vol. II. Baltscheffsky M, ed. Dordrecht,

Netherlands: Kluwer Academic Publishers, pp. 567—570.

Ho K and Krogmann DW (1982) Photosynthesis. In: The Biology of
Cyanobacteria. Carr NG and Whitton BA, eds. Los Angeles: Umvers1ty of

California Press, pp. 191-214.

Izawa S (1980) Acceptors and donors for chloroplast electron transport.
Methods Enzymol. 69413-434.

Késsel H, Dory I, Igloi G and Maier R (1990) A leucine-zipper motif in

 

169
photosystem 1. Plant Mo]. Biol. 15:497-499.

Landschulz WH, Johnson PF and McKnight SL (1989) The DNA binding
domain of the rat liver nuclear protein C/EBP is bipartite. Science
243:1681-1688.

Li N, Zhao J, Warren PV, Warden JT, Bryant DA and Golbeck JH (1991)
PsaD is required for the stable binding of PsaC to the photosystem I core
protein of Synechococcus sp. PCC 6301. Biochemistry 30:7863-7872.

Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of
photosynthetic biomembranes. Methods Enzymol. 148:350-382.

Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein
measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.

Malkin R and Bearden AJ (1978) Membrane-bound iron-sulfur centers in
photosynthetic systems. Biochim. Biophys. Acta 505:147-181.

Mannan RM, Whitmarsh J, Nyman P and Pakrasi HB (1991) Directed
mutagenesis of an iron-sulfur protein of the photosystem I complex in the
filamentous cyanobacterium Anabaena variabilis AT CC 29413. Proc. Nat].
Acad. Sci. USA 88:10168-10172.

Manodori A, Cecchini G, Schroeder I, Gunsalus RP, Werth MT and
Johnson MK (1992) [3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli
fumarate reductase by site-directed mutagenesis. Biochemistry 31:2703-2712.

McCabe PC (1990) Production of single-stranded DNA by asymmetric PCR. In:
PCR Protocols:A Guide to Methods and Applications. Innis MA, Gelfand DH,
Sninsky JJ and White TJ, eds. New York: Academic Press, Inc., pp. 76-83.

McCormack K, Campanelli JT, Ramaswami M, Mathew MK, Tanouye MA,
Iverson LE and Rudy B (1989) Leucine-zipper motif update. Nature
340:103-103.

McCormack K, T anouye MA, Iverson LE, Lin J-W, Ramaswami M,
McCormack T, Campanelli JT, Mathew MK and Rudy B (1991) A role for

hydrophobic residues in the voltage-dependent gating of Shaker K+ channels.
Proc. Nat]. Acad. Sci. USA 88:2931-2935.

Noren GH, Boerner RJ and Barry BA (1991) EPR characterization of an
oxygen-evolving photosystem II preparation from the transformable
cyanobacterium Synechocystis 6803. Biochemistry 30.3943-395 0.

 

 

 

170

Norris JR, Uphaus RA, Crespi HL and Katz JJ (1971) Electron spin
resonance of chlorophyll and the origin of signal I in photosynthesis. Proc. Nat].
Acad. Sci. USA 68:625 -628.

Oh-oka H, Takahashi Y, Kuriyama K, Saeki K and Matsubara H (1988) The
protein responsible for center NE in spinach photosystem I: isolation with
iron-sulfur c1uster(s) and complete sequence analysis. J. Biochem. 103:962-968.

Ohad N and Hirschberg J (1992) Mutations in the D1 subunit of photosystem
II distinguish between quinone and herbicide binding sites. Plant Cell.
4: 273-282.

Pahner G (1973) Current insights into the active center of spinach ferredoxin
and other iron-sulfur proteins. In: Iron-Sulfur Proteins, vol. II. Lovenberg W,
ed. New York: Academic Press, pp. 285-325.

Prentki P and Krisch HM (1984) In vitro insertional mutagenesis with a
selectable DNA fragment. Gene 29:303-313.

Pu WT and Struhl K (1991) The leucine zipper symmetrically positions the
adjacent basic regions for specific DNA binding. Proc. Nat]. Acad. Sci. USA
88:6901-6905.

Robbins AH and Stout CD (1989) Structure of activated aconitase: formation
of the [4Fe- 48] cluster in the crystal. Proc. Nat]. Acad. Sci. USA 86:3639-3643.

Rothery RA and Weiner JH (1991) Alteration of the iron-sulfur cluster
composition of Escherichia coli dimethyl sulfoxide reductase by site-directed
mutagenesis. Biochemistry 30:8296-8305.

Rogner M, Nixon PJ and Diner BA (1990) Purification and characterization of
photosystem I and photosystem 11 core complexes from wild-type and

phycocyanin-deficient strains of the cyanobacterium Synechocystis PCC 6803. J.
Biol. Chem. 265:6189-6196.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory Press.

Smart LB, Anderson SL and McIntosh L (1991) Targeted genetic inactivation

of the photosystem I reaction center in the cyanobacterium Synechocystis sp.
PCC 6803. EMBO J. 103289-3296.

Smart LB and McIntosh L (1991) Expression of photosynthesis genes in the
cyanobacterium Synechocystis sp. PCC 6803: psaA-psaB and psbA transcripts

 

171
accumulate in dark-grown cells. Plant Mo]. Biol. 17:959-971.

Smart LB and McIntosh L (1992) Genetic inactivation of the psaB gene in the
cyanobacterium Synechocystis sp. PCC 6803 disrupts assembly of photosystem 1.
Plant Mo]. Bio]. (in press)

Takahashi Y, Goldschmidt-Clermont M, Soen S-Y, Franzen L-G and Rochaix
J -D (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii:

insertional inactivation of the psaC gene encoding the iron sulfur protein
destabilizes photosystem I. Ell/[BO J. 1&2033-2040.

Taylor LA and Rose RE (1988) A correction in the nucleotide sequence of the

Tn903 kanamycin resistance determinant in pUC4K. Nucl. Acids Res.
16:358-358.

Toelge M, Ziegler K, Maldener I and Lockau W (1991) Directed mutagenesis
of the gene psaB of Photosystem I of the cyanobacterium Anabaena variabilis
ATCC 29413. Biochim. Biophys. Acta 1060: 233-236.

Vieira J and Messing J (1982) The pUC plasmids, an M13mp7-derived system
for insertion mutagenesis and sequencing with synthetic universal primers. Gene
19:25 9-268.

Webber AN and Malkin R (1990) Photosystem I reaction-centre proteins

contain leucine zipper motifs. A proposed role in dimer formation. FEBS Lett.
264:1-4.

Werth MT, Cecchini G, Manodori A, Ackrell BAC, Schroder I, Gunsalus
RP and Johnson MK (1990) Site-directed mutagenesis of conserved cysteine
residues in Escherichia coli fumarate reductase: modification of the

spectroscopic and electrochemical properties of the [2Fe-2S] cluster. Proc. Nat].
Acad. Sci. USA 87: 8965-8969.

Williams J GK (1988) Construction of specific mutations in photosystem II
photosynthetic reaction center by genetic engineering methods in Synechocystis
6803. Methods Enzymo]. 167:766-778.

Witt I, Witt HT, DiFiore D, Rdgner M, Hinrichs W, Saenger W, Granzin
J, Betzel Ch and Dauter Z (1988) X—ray characterization of single crystals of
the reaction center I of water splitting photosynthesis. Ber. Bunsenges. Phys.
Chem. 92: 1503-1506.

Wynn RM and Malkin R (1988) Interaction of plastocyanin with photosystem I:
a chemical cross-linking study of the polypeptide that binds plastocyanin.
Biochemistry 27:5863-5 869.

 

 

172

Zhao J, Warren PV, Li N, Bryant DA and Golbeck JH (1990) Reconstitution
of electron transport in photosystem I with PsaC and PsaD proteins expressed
in Escherichia coli. FEBS Lett. 276: 175-180.

Zhao J, Li N, Warren PV, Golbeck JH and Bryant DA (1992) Site-directed
conversion of a cysteine to aspartate leads to the assembly of a [3Fe-4S] cluster
in PsaC of photosystem I. The photoreduction of FA is independent of F3.
Biochemistry 31:5093-5 099.

Zilber AL and Malkin R (1988) Ferredoxin cross-links to a 22 kD subunit of
photosystem 1. Plant Physio]. 88:810-814.

 

 

 

DISCUSSION AND CONCLUDING REMARKS

This project has accomplished many of the goals established at its
inception: the isolation and characterization of the PSI reaction center genes
from Synechocystis 6803, the inactivation of those genes, and site-directed
mutagenesis of a PSI reaction center gene to study structure-function
relationships. Yet, as with nearly all scientific endeavors, many unresolved
questions have arisen in the process of addressing the initial problems. It is
also important to consider the questions that warrant future experimentation.
The following is a brief discussion of some of those questions left unresolved
and of possible experiments that may address some of the interesting problems
that remain. Many of the proposals in this section are based on speculation,

rather than definitive evidence.

Coordinate regulation of thylakoid membrane complexes
Growth of Synechocystis 6803 under LAHG conditions causes significant
changes in the balance of proteins in the thylakoid membrane. There is a

three- to four-fold decrease in the accumulation of P81, while the accumulation

173

 

 

174

of PSII remains approximately the same as in mixotrophically grown cells.
Patterns of mRNA accumulation suggest some level of post-transcriptional
regulation of P81 and P811 biogenesis, since psaA-psaB mRNA accumulation
remains approximately the same, while psbA mRNA accumulation drops
significantly. The accumulation of P81 seems to be the variable component,
rather than the levels of PSII, when Synechocystis 6803 is acclimating to
changes in its environment. Variation in PSI accumulation has also been
detected when Synechocystis 6803 is grown in different light quality, as well
(Aizawa et 31., 1992). The changes in PSI accumulation are probably caused by
differential protein stability or complex stability under conditions which favor
more or less PSI. Thus, under certain redox conditions, PSI may be more or
less stable, and its accumulation is adjusted to allow for the most efficient use
of available light energy for photosynthetic electron flow. An additional
consideration in cyanobacteria is the coordinate regulation of the accumulation
of the cytochrome b6/f complex, which is shared between photosynthesis and
respiration. The relative accumulation of these proteins under

photoautotrOphic versus LAHG conditions has not been studied extensively.

Light sensitivity of PSI mutants

Mutants of Synechocystis 6803 that lack PSI, including ADK9, BDK8,
and L522P, are sensitive to continuous light >3 umol-m'zs‘l. This suggests
that PSI and its associated pigments are important for phot0protection.

Initially the light-sensitivity was thought to be caused by photoinhibitory damage

 

 

 

175

resulting from full reduction of PSII and the quinone pool. However, mutants
that lack both PSI and P811 are also sensitive to continuous light >3
umol'mzs‘l, eliminating any role of P811 and P630 in causing photoinhibitory
damage. Thus, two possibilities come to mind: 1) there is some light-induced
component creating a toxic element in the cells, or 2) continuous illumination
>3 umol°m2°s1 prevents respiratory electron flow and ATP synthesis. A
possible toxic component may be a pigment, such as chlorophyll or carotenoid,
which is no longer associated with a photosynthetic complex and can create
toxic radicals or molecular oxygen when illuminated. The second possibility
cannot be distinguished from the first at this time, because the only mutants
that lack PSI activity also lack all of the chlorophyll associated with PSI. If one
had a mutant, which accumulated wild-type levels of P81 that was inactive in
cyclic photosynthesis and ATP production, one could test the light-sensitivity of
the respiratory pathway in vivo directly in cells with a normal complement of

chlorophyll.

Comparison of PS1 to other photosynthetic reaction centers

The photosynthetic reaction centers from the purple bacteria
Rhodopseudomonas viridis and Rhodobacter sphaeroides have been
crystallized, and their three-dimensional structures have been determined (Allen
et 31., 1987; Deisenhofer et a1., 1985). This is important for the study of
oxygenic photosynthesis, because there is significant structural and functional

homology between the bacterial reaction center L and M subunits and the P811

 

 

 

 

176

subunits D1 and D2, suggesting an evolutionary relationship (Michel and
Deisenhofer, 1988). Comparison of P811 to the bacterial reaction center crystal
structure has guided mutational analysis of PSII structure and function (Debus
et a1., 1988; Vermaas et a1., 1988). Recent evidence suggests there are
photosynthetic reaction centers that are evolutionarily related to P81 in the
green sulfur bacterium Chlorobium and in the anaerobe Heliobacillus mobihs
(Trost et a1., 1992; Feiler et a1., 1992). The reaction centers in these organisms
appear to be homodimers that contain iron-sulfur centers that act in electron
transport (Nitschke et a1., 1990a; Nitschke et 3]., 1990b). The genes encoding
the reaction center proteins in Chlorobium limicola f. sp. thiosulfa tophilum have
been isolated. and characterized (Biittner et a1., 1992). There are three genes in
an operon, two of which encode reaction center proteins of 730 and 232 amino
acids. The larger protein shares some homology with PsaA and PsaB,
especially in the region containing the cysteines that bind the iron-sulfur center
Fx. The smaller protein has limited homology with PsaC, but contains similar
cysteine motifs that would bind the iron-sulfur centers FA and F B. The gene
encoding the reaction center from Heliobacillus mobilis has also been isolated
and partially characterized (Wilson et a1., 1991). This protein, with a mass of
approximately 47 kDa, is much smaller than PsaA or PsaB, but does have a
similar pair of cysteines that bind Fx. The entire twelve-residue motif
containing the cysteine ligands to Fx is not conserved in the reaction center
proteins from the two organisms. Of note, the aspartates adjacent to the

cysteines are altered, as is an arginine between the two cysteines. Interestingly,

 

177

these proteins do not contain heptad repeats of leucines, proposed to form a
leucine zipper in PSI. Although it is interesting to make broad comparisons
between these reaction center proteins and P81, only the Chlorobium protein
will be useful on a limited scale in judging which amino acids may be important
for PSI structure and function. The Heliobacillus protein appears to be too
divergent. Also, the state of purification and crystallization of P81 is far ahead
of that of either Chlorobium or Heliobacillus.

Comparisons have been made between the bacterial reaction center,
P811, and PSI using computer algorithms (Otsuka et a1., 1992; Margulies, 1991).
The authors conclude that there is significant similarity between regions of the
P811 and P81 reaction center proteins. However, the regions of similarity
discovered in the two studies do not correspond. One of the papers proposes
that there are 13 membrane-spanning helices in PsaA and PsaB, rather than
the widely accepted number of 11. Preliminary interpretation of the crystal
structure of Smechococcus PSI suggests that there may only be eight
membrane-spanning helices (J. Golbeck, personal communication). This lack of
consistency and the comparison to PSII, for which no three-dimensional
structure exists, lead to the conclusion that these computer comparisons do not
provide a strong basis for predicting which amino acids are important for PSI
structure and function. Although, they are interesting when considering the
evolutionary relationships between different types of photosynthetic reaction

CCIIICI'S.

 

 

 

 

 

 

 

178
Potential future experiments

The most interesting site-directed mutant generated in this project is
C5658. It still accumulates PSI that is capable of low temperature electron
transfer to FA and E, but reduced Fx has eluded detection to this point. Also,
preliminary time-resolved optical spectroscopy suggests that F A and F3 are not
photo-reduced at room temperature (see Appendix). Since the reaction center
does assemble, although at reduced levels, there probably is an iron-sulfur
center forming. However, that cluster may be a [3Fe-4S] center, which would
not transfer electrons at room temperature and may be EPR silent. The
reduction of FA and F3 seen at low temperature may represent the low
temperature bypass of A, and Fx that has been detected before (Setif et a],
1987). Therefore, further experiments, including EPR spectroscopy and tirne-
resolved optical spectroscopy are needed to define the nature of Fx in this
mutant and to investigate the role of Fx in room temperature and low
temperature electron transport. In addition, studies involving electron-nuclear
double resonance (ENDOR) spectroscopy, Mossbauer spectroscopy, or electron
spin echo enve10pe modulation (ESEEM) spectroscopy could further define the
nature of the iron-sulfur cluster and its ligands.

Mutations that would alter the environment and the redox potential of
Fx, without affecting PSI stability, would be ideal for the study of electron flow
through the reaction center. Amino acid changes that might accomplish this
include changing the aspartate residues adjacent to the cysteine ligands of FX,

perhaps to asparagines, serines, or cysteines. Another residue that may be

 

 

 

179

involved in determining the environment around Fx is the arginine between the
two cysteines, which might be changed to lysine or leucine. Replacement of a
cysteine ligand of the [4Fe-4S] center in ferredoxin I from Azotobacter
vinelandii for alanine by site-directed mutagenesis resulted in protein
rearrangement, allowing a different cysteine residue to act as a ligand to the
[4Fe-4S] cluster (Martin et a1., 1990). A similar approach could be attempted
with the ligands to Fx, replacing a cysteine with alanine or glycine, while
simultaneously changing a nearby amino acid to cysteine. This might force a
minor protein rearrangement in order for Fx to bind, which could alter the
environment and character of Fx. Amino acids in this region may also be
involved in the association with PsaC, so the substitutions described above may
alter electron donation to FA and FE as well.

Further mutagenesis is required to address definitively the role of the
repeats of leucines in PSI. Amino acid substitutions that are less likely to
participate in the leucine zipper interaction include: glycine, alanine, or
phenylalanine. Also more double leucine substitutions, including isoleucine or
alanine residues may be effective. A mutation, which is currently being
attempted, is the deletion of the leucine residue at position 522 of PsaB, which
would disrupt the spacing of the leucines in the array. Also, the addition of a
codon in the region predicted to form the a helix may perturb the leucine
zipper without significantly altering the secondary structure of that region. A
recipient strain of Synechocystis 6803 and plasmids for the site-directed

mutagenesis of the psaA gene have been engineered and may be used for the

 

 

 

 

 

 

 

 

180

introduction of amino acid substitutions into PsaA (unpublished data).

The investigation of structure-function relationships in the PSI reaction
center could be expanded beyond the study of Fx and the leucine zipper, but
with less likelihood of immediate success. Residues that act as ligands to P700,
A0, and A, have been proposed (Margulies, 1991; Otsuka et a1., 1992) and may
be discovered by site-directed mutagenesis. It is in the study of these electron
transfer components that comparison of PSI to Chlorobium or PSII may
provide insight for successful mutagenesis.

The understanding of oxygenic photosynthesis is vital for our continued
prosperity in a changing environment. This study has advanced the knowledge
of photosynthesis in a cyanobacterium, but our ultimate goal must be the full
understanding of photosynthesis in plants, which are vital for our survival. The
recently deveIOped system for transformation of tobacco chloroplasts Opens the
door for analysis of structure-function relationships in the plant PSI reaction
center using site-directed mutagenesis. Although the cyanobacteria serve well
for rapid mutagenesis and analysis, study of these organisms must be considered
preliminary to the study of plants. Ultimately, we may be able to alter the
photosynthetic reaction centers in a positive way, possibly increasing efficiency,
stability, or high-light tolerance. Perhaps the plant photosynthetic apparatus
could be engineered for the production of Hz to be used as fuel. Hopefully,

this study has provided some useful information that may be used some day to

accomplish these goals.

 

 

 

181

References

Aizawa K, Shimizu T, Hiyama T, Satoh K, Nakamura Y and Fujita Y (1992)
Changes in composition of membrane proteins accompanying the regulation of

PSI/PSII stoichiometry observed with Synechocystis PCC 6803. Photosynth. Res.
32:131-138.

Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of
the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc.
Nat]. Acad. Sci. USA 84:5730-5734.

Biittner M, Xie D-L, Nelson H, Pinther W, Hauska G and Nelson N (1992)
Photosynthetic reaction center genes in green sulfur bacteria and in
photosystem 1 are related. Proc. Nat]. Acad. Sci. USA 89:8135-8139.

Debus RJ, Barry BA, Babcock GT and McIntosh L (1988) Site-directed
mutagenesis identifies a tyrosine radical involved in the photosynthetic
oxygen-evolving system. Proc. Na t1. Acad. Sci. USA 85:427-430.

Deisenhofer J, Epp 0, Miki K, Huber R and Michel H (1985) Structure of

the protein subunits in the photosynthetic reaction centre of
RhodOpseudomonas viridis at 3 A resolution. Nature 318:618-624.

Feiler U, Nitschke W and Michel H (1992) Characterization of an improved
reaction center preparation from the photosynthetic green sulfur bacterium

Chlorobium containing the FeS centers F A and PB and a bound cytochrome
subunit. Biochemistry 31:2608-2614.

Margulies MM (1991) Sequence similarity between Photosystems I and II.
Identification of a Photosystem I reaction center transmembrane helix that is
similar to transmembrane helix IV of the D2 subunit of Photosystem II and the

M subunit of the non-sulfur purple and flexible green bacteria. Photosynth. Res.
29:133-147.

Martin AE, Burgess BK, Stout CD, Cash VL, Dean DR, Jensen GM and
Stephens PJ (1990) Site-directed mutagenesis of Azotobacter vinelandii

ferredoxin I: [Fe-S] cluster-driven protein rearrangement. Proc. Nat]. Acad. Sci.
USA 87:598-602.

Michel H and Deisenhofer J (1988) Relevance of the photosynthetic reaction

center from purple bacteria to the structure of photosystem II. Biochemistry
27:1-7.

Nitschke W, Setif P, Liebl U, Feiler U and Rutherford AW (1990a) Reaction
center photochemistry of Heliobacterium chlorum. Biochemistry

 

182
29: 1 1079- 11088.

N itschke W, Feiler U and Rutherford AW (1990b) Photosynthetic reaction
center of green sulfur bacteria studied by EPR. Biochemistry 293834-3842.

Otsuka J, Miyachi H and Horimoto K (1992) Structure model of core proteins
in photosystem I inferred from the comparison with those in photosystem II
and bacteria; an application of principal component analysis to detect the
similar regions between distantly related families of proteins. Biochim. Bi0phys.
Acta 1118:194-210.

Setif P, Ikegami I and Biggins J (1987) Light-induced charge separation in
Photosystem I at low temperature is not influenced by vitamin K-1. Biochim.
Biophys. Acta 894:146-15 6.

Trost JT, Brune CC and Blankenship RE (1992) Protein sequences and redox
titrations indicate that the electron acceptors in reaction centers from
heliobacteria are similar to Photosystem I. Photosynth. Res. 32:11-22.

Vermaas WFJ, Rutherford AW and Hansson O (1988) Site-directed
mutagenesis in photosystem II of the cyanobacterium Synechocystis sp. PCC
6803: donor D is a tyrosine residue in the D2 protein. Proc. Nat]. Acad. Sci.
USA 85:8477-8481.

Wilson MM, Liebl U, Blankenship RE and Vermaas WFJ (1991) Cloning of
the reaction center gene from Heliobacillus mobilis. Proceedings of the
meeting: Molecular Biology of Photosynthesis, Tempe, AZ, p. 47 (Abstract).

 

 

APPENDIX

ESR and optical spectroscopy of wild type and site-directed mutants

In order to observe reduction of the [4Fe-4S] centers FA and FE in either
isolated membranes or PSI complex, ESR spectroscopy was performed at 15 K
using a Bruker ECS-106 spectrometer equipped with an Oxford cryostat and
temperature controller (see Chapter 6). The low temperature ESR spectrum
of wild-type PSI complex that was frozen in the dark includes resonances
originating from reduced FA (g=2.046, 1.944, and 1.852) or reduced FB
(g: 2.067, 1.927, and 1.881) (Figure A1, top panel). When the wild-type PSI
complex was frozen under illumination, the high-field and low-field resonances
from FA and F3 merge, yielding a spectrum with features at g=2.048, 1.940,
1.921, and 1.885 (Figure A.1, bottom panel). The low temperature ESR
spectra of isolated membranes from the mutants L522V, L536M, and
L522V/L536M frozen under illumination have essentially wild-type character
(Figure A.2, panels A, C, E, and G). The spectrum of membranes from C5658
has resonances with reduced intensity, relative to those of wild type, while

reduced FA or PE signals were not present in the spectra of membranes from

C565H, C565D, or L522P (Figure A.2, panels B, D, F, and H). The low

183

 

184

temperature spectra of membranes isolated from C5658 include weak F A and
F3 signals, when illuminated at 15 K (Figure A3, panel A). When C5658
membranes are frozen under illumination, the high- and low-field resonances
merge, but the intensity of the mid-field feature from F3 appears to be very
weak (Figure A3, panel B). The spectrum of membranes from C5658 that
were chemically reduced using dithionite includes FA and FE features that are
more intense than those observed upon photoreduction, suggesting that
electron transfer to FA and PB may occur at reduced efficiency (Figure A3,
panel C). Finally, 85% of the transient, flash-induced absorption change at 820
nm, reflective of reduction of P700, of PSI complex purified from membranes of
C5658 decays with a halftime of 4 us, while the remaining 15% decayed with a
longer halftime, on the order of ms (Figure A4, panel A). This indicates
impaired forward electron transfer through Fx, resulting in a rapid back-
reaction (perhaps from A1) and rereduction of P700. With wild-type PSI
complex, the flash-induced absorption change decays with a halftime on the
order of tens of ms, reflecting the back-reaction of FA/FB' with P700 (Figure A4,

panel B).

Acknowledgements
ESR and optical spectroscopy was performed by Patrick Warren in the

laboratory of Dr. John Golbeck, Department of Biochemistry, University of

Nebraska.

 

185

 

 

 

 

 

 

 

 

 

Wlldtype 2.046
light - dark (15K)
g=2.067 1.944
1.881
1.927
1.852
g=2.048
light - dark (298K)
1.940
1.921
1.885
I l I l T
3000 3200 3400 3600 3800

Figure A.1. Low temperature ESR spectra of wild-type PSI complex. The top
spectrum was collected during illumination after the sample was frozen in the
dark. The bottom spectrum was collected under illumination after the sample
was frozen under illumination. The gvalues are indicated above and below
features in the spectra. The magnetic field (horizontal axis) is indicated in
Gauss.

186

Survey of Site-Directed Mutations

 

 

 

 

Wildlypc
1.94
1.92
1.88

C

L522V
E

L536M
G

L522V/LS36M

 

 

 

 

 

 

C5658
WW
C565H
[ w .7.
C565D
L522P

 

Figure A.2. Low temperature ESR spectra of membranes isolated from wild-
type and mutant cells. The spectra were collected under illumination after the
sample was frozen under illumination.

 

 

187

C5658 Thylakoid Membranes

Light - Dark (l5K)

L94

 

 

 

 

 

 

 

 

L92
1.89 1.86
Light - Dark (298K)
L94
L89
1.92
Chemically Reduced
1.94
i

1.92 1.89

 

 

Figure A.3. Low temperature ESR spectra of membranes isolated from C5658.

The spectrum in A was collected during illumination after the sample was

frozen in the dark. The spectrum in B was collected under illumination after

the sample was frozen under illumination. The spectrum in C was collected I
after chemical reduction with dithionite in the presence of 0.1 M glycine, pH

10. The gvalues are indicated above and below the spectra.

188

Optical Studies of C5658 Complex

A B r,

C5658 PSI Complex Wildtype PSI Complex

AI/IS x 10'3

 

 

50 us

 

Figure A4. Optical spectroscopy of P81 complex isolated from C5658 and wild
type. The transient, flash-induced absorption change was measured at 820 nm
using a double beam spectrometer. The measuring beam was supplied by a
Schwartz Electro-Optics titanium-sapphire laser pumped by a 5 W CW argon-
ion laser (Spectra-Physics) and was split, one beam passing through the sample
to a detector, the other passing directly to a matching detector. The signals
were amplified and subtracted using a DC-coupled differential comparator
(Tectronix model 11A33, 150 MHz) and were digitized with a Tectronix DSA
601 oscilloscope. Samples were excited using a 2.3 MW nitrogen laser (PTI)
operating at 1 Hz and contained 0.1 mM DCIP and 5mM ascorbate in 0.1 M

Tris, pH 8.3.

 

 

 

 

 

 
 

 

 

 

 

 

 
 

 

 

 
 
 
 

 

 

LIBRQRIES

I

899

III

 

II

 

II

I

II

MICHIGQN STQTE UNIV.