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thesis entitled

Studies on Cold-Regulated Gene Expression
in Rhizobium meliloti

 

presented by

Marcia Deane Lehmann

has been accepted towards fulfillment
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M. S . degree in Microbiology

 

 

    

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Date NOV. 4, 1994

 

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MSU Is An Affirmative Action/Equal Opportunity Institution
Warts-oi

 

STUDIES ON COLD-REGULATED GENE EXPRESSION IN
IUILZOBUIHIJKELJIKITI

BY

Marcia Deane Lehmann

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree

MASTER OF SCIENCE

Department of Microbiology and Center for Microbial Ecology

1994

ABSTRACT

STUDIES ON COLD-REGULATED GENE EXPRESSION IN RHIZOBIUM
MELILOTI

BY

Marcia Deane Lehmann

Rhizobium.meliloti strain 1021 (Rm 1021) was grown at various
temperatures; growth rate decreased with a decrease in
temperature. SDS—PAGE analysis of protein synthesis showed 8
up-regulated and 8 down-regulated proteins after a shift from
300C to 150C. Rm 1021 was mutagenized using Tn5-luxAB to
identify cold-induced genes. Temperature shift from 300C to
100C revealed two mutants, Rm 5—5-6 and Rm 3—16-6, which
exhibited an increase in light emission. Timing and degree
of induction was characterized at various temperatures. Both
Rm 5—5—6 and Rm 3—16—6 were induced by a shift from 300C to
200C or 150C, but not 250C. A shift to 100C resulted in
delayed induction. Cloning and sequencing of genomic regions
flanking the TnS—luxAB insertion in Rm 3—16—6 identified the

cold—regulated gene as a 16S rRNA gene.

ACKNOWLEDGEMENTS

It is impossible to thank everyone who has helped me
during my graduate work. This is, at best, an attempt to
thank the major contributors. I would like to thank Pyung Ok
Lim, who started the project. Frans de Bruijn, Sylvia
Rossbach, and Maria Schneider answered questions concerning
Rhizobium meliloti and protocols. Peter Wolk and members of
his lab, especially Todd Black, Yuping Cai, and Jeff Elhai,
answered my many questions about luxAB and the photonic
camera. Mike and my labmates, Sarah Gilmour, Todd Black,
Julia Bell, Nancy Artus, Dave Horvath, Brett McLarney, Kathy
Wilhelm, Ann Gustafson, Eric Stockinger, Kevin O'Connell,
Stokes Baker and Lenny Bloksberg, as well as Sue Hammar in
the Grumet lab, have answered questions about almost
everything else.

As my major professor, Mike Thomashow kept me on track
and pulled me through in the end. My committee members,
Frans de Bruijn, Dennis Fulbright, and Wendy Champness, were
helpful, tough, honest and fair. I also want to thank the
Department of Microbiology and the Center for Microbial
Ecology. And of course a big thanks to my family, my

friends, and Bill. Thanks again, everyone.

iii

TABLE OF CONTENTS

Content Page
List of tables V
List of figures vi

Chapter One
Introduction 1
Bibliography 7
Chapter Two
Studies (n1 Cold—Regulated Gene Expression le Rhizobium

meliloti 10
Bibliography 50

iv

LIST OF TABLES

Table Page
Chapter Two
1 Strains and plasmids used 16
2 Generation times of Rm 1021 in TY and GT8 media at
various temperatures 21
3 Light emission at 300C and 100C in mutant carrying
Tn5—luxAB insert 30

LIST OF FIGURES

Figure Page
Chapter Two

1 Growth after shift of Rm 1021 from 300C to lower

temperature 22
2 Arrhenius plot of Rm 1021 steady state growth rates at

decreasing temperatures 23
3 1D gel analysis of Rm 1021 35S—labeled proteins at

300C and 150C 25
4 Map of the suicide vector, pRLlO62a 26
5 Stability of V. fischeri and V; harveyi luxAB 27
6 Light emission from cold—regulated mutants at 300C and

100C 29
7 Detection of Tn—luxAB insertions in Rm 5—5—6 and Rm

3-16-6 32
8 Induction of luxAB insertion in Rm 5—5-6 and Rm 3—16—6

upon shift from 300C to lower temperatures 33
9 Fold induction after shift from 300C to 370C 35
10 Sequence comparison between Tn5-luxAB insertion site

in Rm 3-16-6 and R. meliloti l6S rRNA 36
ll Detection of 168 rRNA copies 37
12 Sequence identity between E. coli rrnB and Rm 3—16—6

rRNA operons 39
13 Steady state growth rates of Rm 1021 and Rm 3—16-6 at

decreasing temperatures 41

Vi

INTRODUCTION

Prokaryotes experience Imnur environmental stresses,
including extremes in temperature, pH, osmolarity, and
nutrient—limitation. Bacteria are often able to adapt to
harsh conditions and the mechanism can involve changes in
gene expression. The general subject of this thesis regards
microbial acclimation to low temperature.

Cold shock response in Escherichia coli.

E. coli exhibits an ability to rapidly adjust to shifts
up or down in temperature between 370C and 200C. Cultures
shifted within this range assume a new steady state growth
rate almost immediately with reported periods of adjustment
varying from zero to ten minutes (10, 15, 17). Total protein
synthesis reaches its new rate almost immediately; however,
synthesis rates of individual proteins exhibit a transient
increase or decrease then reach a steady state synthesis rate
after approximately 20 minutes (15).

Temperature downshifts outside the 370C to 200C range
require a much longer period of adjustment before resuming
growth. For example, E.coli experiencing a shift from 370C
to 100C generally exhibits a 4 hour lag before assuming the

steady state growth rate for 100C (12). An earlier study

2
(17) reported a slightly more complex situation in which the

4 hour lag was followed by a period of rapid growth which
gradually declined to the new steady state rate of growth.
After a shift from 370C to 50C, the bacteria are unable to
initiate protein synthesis and the culture no longer doubles
(2, 4, 6).

Cold shock proteins in E. coli.

Overall, the rate of protein synthesis decreases when a
culture is cold shocked, but some proteins are produced, at
least transiently, at.ea higher level. These upregulated
proteins are known as cold shock proteins (CSPs). CSPs were
first identified by comparing the quantitative levels of
proteins at different temperatures using 2—D gel
electrophoresis (9). Thirteen CSPs were identified, 12 of
which were found to be upregulated 2— to 10—fold (12). Nine

of the 12 have been identified as polynucleotide

phosphorylase (PNP), NusA, initiation factor 2a, initiation

factor 23, RecA, dihydrolipoamide acetyltransferase, pyruvate

dehydrogenase (lipoamide), H-NS, and DNA gyrase subunit A
(11, 12, 13). Together these proteins have a wide range of
activity, such as formation of acetyl CoA for the
tricarboxylic acid cycle (dihydrolipoamide acetyltransferase
and pyruvate dehydrogenase), recombination and induction of
the SOS response (RecA), DNA supercoiling (GyrA), degradation
of single stranded RNA (PNP) thermoregulation of
transcriptbma of the pilin genes (H—NS), termination of

transcription (NusA), and binding tRNAfmet to the 305

3

ribosomal subunit for initiation of translation (initiation
factors a and B) (8, 11, 12, 13).

CspA and CspA-like proteins.

The major CSP in E. coli, CspA (CS7.4), is induced 100—
fold in response to cold shock; it is undectable at 370C, but

is the most abundantly synthesized protein at 100C (7, 11,

12). Within 30 minutes of temperature shift, CspA is
detectable (7). CspA levels increase during the cold shock—
induced lag period (11), then decrease to a level
approximately 20% of the maximum level (7). No temperature

has been determined as the threshold for induction. Fourteen
degree temperature shifts from 420C to 280C, 370C to 230C,
and 240C to 100C all resulted in the induction of CspA (10),
but shifting from 240C to 100C resulted in the strongest
induction. Shifting from 370C to 100C resulted in even
higher levels of CspA than the shift from 240C to 100C (10).
Together these results indicate that the level of induction
is a factor of both the magnitude of the temperature shift
and the final temperature itself.

CspA appears to be a DNA-binding protein based on
sequence identity to the human protein YB-l; YB—l binds to
the CCAAT—containing "Y box" (5, 23). Binding of a Y box
factor to the cis—acting Y box in a promoter has been shown
to activate transcription (20). CspA has been shown to bind
to the CCAAT sequence and hns, cspA, recA, nusA, pup, and

gyrA contain at least one ATTGG sequence (11, 13). Thus, it

4
is possible that CspA is a transcriptional activator which is

auto-regulated (11, 13, 19).

Recently, three other genes have been identified in E.
coli that encode proteins with 79, 70, and 45% amino acid
identity to CspA (14). These proteins have been designated
CspB, CspC, and CspD, respectively. Curiously, while CspB is
induced by cold shock, CspC exhibits no response to cold and
CspD is actually inhibited by cold shock (14).

CspA-like proteins exist in other prokaryotes as well.
For example, Bacillus subtilus contains 21 protein, CspB,
which is induced by a cold shock (22). CspB protein
demonstrates 61% amino acid identity to CspA, while the DNA
sequences show 60% identity (22). CspB demonstrates 43%
identity to the human Y—box factor binding site (22, 23).
The Gram positive bacterium Streptomyces clavuligerus has a
7.0 kDa protein which is 56% identical and 80% similar to
CspA (1).

Cold shock proteins in Rhizobium spp.

Three arctic strains of Rhizobium spp. were isolated
from arctic legumes and compared to three temperate strains,
(one being R. meliloti A2), in regard to their heat and cold
shock responses (3). After a shift from 250C to -20C for
arctic strains and 300C to —20C for temperate strains, 17—24
cold shock proteins were present in arctic strains, while
temperate strains had 18—22 cold shock proteins.
Quantification was not reported. An 11.1 kDa protein was

found tx> be abundant after temperature downshifts, with

5

levels increasing with decreasing temperature, similar to
CspA. Whether this protein is a CspA—like protein is not yet
known.

Regulation of the cold shock response in E. coli.

The mechanism(s) responsible for regulating the cold
shock response has yet to be defined. There is evidence,
however, that the cold shock response may involve the action
of (p)PpGpp. (p)ppGpp levels have been shown to decrease
with a decrease in temperature (10, 16, 18). The fact that
induction of the stringent response prior to temperature
downshift resulted in the repression of CspA, NusA, RecA,
PNP, dihydrolipoamide acetyltransferase and pyruvate
dehydrogenase (10), suggests that (p)ppGpp may negatively
regulate CspA, which in turn regulates the other ppGpp—
repressed cold shock proteins. A mutant unable to synthesize
detectable levels of ppGpp (relA spoT), had a much higher
steady state level of CspA at 240C than the wild type (12).
At 300C, however, CspA levels in the mutant did not differ
from the wild type (21) . Thus, CspA is not regulated by
(p)ppGpp alone.

Ribosomes have been proposed to act as sensors of heat
and cold shock based on the upregulation of heat shock or
cold shock proteins after addition of antibiotics which
target the ribosome (21). In particular, heat shock proteins
were found to be induced by kanamycin, puromycin and
streptomycin. Ten of the fourteen CSPs, including CspA, were

induced by a second set of antibiotics which target the A

6

site of the ribosome, including chloramphenicol,
erythromycin, fusidic acid, spiramycin, and tetracycline
(21). Although (p)ppGpp and/or ribosomes may be involved in
regulation of the cold shock response, the mechanism(s)

remains to be elucidated.

BIBLIOGRAPHY

Sci.

B IBLIOGRAPHY

Ay-Gay, Y., Y. Aharonowitz, and G. Cohen. 1992.
Streptomyces contain a 7.0 kDa cold shock like protein.
Nucleic Acids Res. 20:5478.

Broeze, R.L., C.J. Solomon, and D.H. Pope. 1978. Effects
of low temperature on in vivo and in Vitro protein
synthesis in Escherichia coli and Pseudomonas
fluorescens. J. Bacteriol. 134:861—874.

Cloutier, J., D. Prevost, P. Nadeau, and H. Antoun.
1992. Heat and cold shock protein synthesis in arctic

and temperate strains of rhizobia. App. Env. Micro.
58:2846—2853.

Das, H.K., and A. Goldstein. 1968. Limited capacity for
protein synthesis at zero degrees centigrade in
Escherichia coli. J. Mol. Biol. 31:209—226.

Didier, D.K., J. Schiffenbauer, S.L. WOulfe, M. Zacheis,
and B.D. Schwartz. 1988. Characterization of the cDNA
encoding a protein binding to the major
histocompatibility complex class II Y box. Proc. Natl.
Acad. Sci. USA. 85:7322—7326.

Friedman, M., P. Lu, and A. Rich. 1969. Ribosomal
subunits produced by cold sensitive initiation of
protein synthesis. Nature. 223:909—913.

Goldstein, J., N.S. Pollitt, and M. Inouye. 1990. Major
cold shock protein of EScherichia coli. Proc. Natl. Acad.
USA. 87:283—287.

Goransson, M., B. Sonden, P. Nilsson, B. Dagberg, K.
Forsman, K. Emanuelsson, and 8.3. Uhlin. Transcriptional
silencing and thermoregulation of gene expression in
Escherichia coli. Nature. 344:682—685.

Herendeen, S.L., R. van Bogelen, and F.C. Neidhardt.
1979. Levels of major proteins of Escherichia coli
during growth at different temperatures. J. Bacteriol.
139:185—194.

10.

11.

12.

13.

14.

15.

l6.

17.

18.

19.

20.

8

Jpnes, P.G., M. Cashel, G. Glaser, and P.G. Meidhardt.
1992. Function of a relaxed—like state following
temperature downshifts in EScherichia coli. J.
Bacteriol. 174:3903—3914.

Jpnes, P.G., R. Krah, S.R. Tafuri, and A.P. WOlffe.
1992. DNA gyrase, CS7.4, and the cold shock response in
Escherichia coli. J. Bacteriol. 174:5798-5802.

Jones, P.G., R.A. Van Bogelen, and F.C. Neidhardt. 1987.
Induction of proteins in response to low temperature in
Escherichia coli. J. Bacteriol. 169:2092-2095.

La Teana, A., A. Brandi, M. Falconi, R. Spurio, C.L.
Pon, and C.O. Gualerzi. 1991. Identification of a cold
shock transcriptional enhancer of the EScherichia coli
gene encoding nucleoid protein H—NS. Proc. Natl. Acad.
Sci. USA. 88:10907—10911.

Lee, S.J., A. xie, W. Jiang, J.-P. Etchegaray, P.G.
Jones, and M.Inouye. 1994. Family of the major cold—shock
protein, CspA (CS7.4), of Escherichia coli, whose

members show a high sequence similarity with the
eukaryotic Y-box binding proteins. Mol. Micro. 11:833—
839.

Lemaux, P.G., S.L. Herendeen, P.L. Bloch, and F.C.
Neidhardt. 1978. Transient rates of synthesis of
individual polypeptides in E. coli following temperature
shifts. Cell. 13:427—434.

Mackow, E.R., and F.N. Chang. 1983. Correlation between
RNA synthesis and ppGpp content in Escherichia coli
during temperature shifts. Mol. Gen. Genet. 192:5-9.

Ng, H., J.L. Ingraham, and A.G. Marr. 1962. Damage and
derepression in Escherichia coli resulting from.growth
at low temperatures. J. Bacteriol. 84:331—339.

Pao, C.C., and B.T. Dyess. 1981. Stringent control of
RNA synthesis in the absence of guanosine 5'—
diphosphate-3'-diphosphate. J. Biol. Chem. 256:2252—2257.

Tanabe, H., J. Goldstein, M. Yang, and M. Inouye. 1992.
Identification of the promoter region of the Escherichia
coli major cold shock gene, CspA. J. Bacteriol.
174:3867—3873.

Tafuri, S.R., and A.P. Wolffe. 1990 Xenopus Y—box
transcription factors: molecular cloning, functional
analysis, and developmental regulation. 1990. Proc.
Natl. Acad. Sci. USA. 87:9028—9032.

21.

22.

23.

9

van Bogelen, R., and P.G. Meidhardt. 1990. Ribosomes as
sensors of heat and cold shock in Escherichia coli.
Proc. Natl. Acad. Sci. USA. 87:5589—5593.

Willimsky, G., H. Bang, G. Fischer, and M.A. Marahiel.
1992. Characterization of cspB, a Bacillus subtilus
inducible cold shock gene affecting cell viability at

low temperatures. J. Bacteriol. 174:6326-6335.

Wistow. G. 1990. Cold shock and DNA binding. Nature.
344:823—824.

CHAPTER 2

Studies on cold-regulated gene expression in
Rhizobium.meliloti

lO

ABSTRACT

Rhizobium meliloti strain 1021 (Rm 1021) was grown at various
temperatures to determine generation time. Growth rate was
found to be a linear function of temperature between 15 and
300C. Above 300C and below 150C, however, growth was
"restricted". Protein synthesis at 300C and 15°C was
evaluated by SDS—PAGE. Analysis indicated that 8 proteins
were up—regulated and another 8 down—regulated in response to
a temperature downshift of 300C to 150C. To identify genes
induced by low temperature, Rm 1021 was mutagenized using a
Tn5-luxAB reporter transposon containing a kanamycin
resistance gene for selection of mutants. The luxAB gene
cassette encodes the light—producing luciferase enzyme of
Vibrio harveyi. Mutants were shifted from 300C to 100C for
six hours and screened for increased light emission. Two
mutants, designated Rm 5—5—6 and Rm 3—16—6, exhibited 20— and
100-fold increase in light emission at low temperature and
were selected for further study. The timing and degree of
induction was characterized at various temperatures between
300C and 100C. Overall patterns of induction were the same;
both Rm 5—5-6 and Rm 3—16—6 were induced by a shift from 300C

to 200C or 150C, but not 250C. A shift to 100C resulted in

11

12

delayed induction. Cloning and sequencing of the genomic
regions flanking the Tn5—luxAB insertion in Rm 3—16-6

identified the cold—regulated gene as a 16S rRNA gene.

INTRODUCTION

When E. coli experiences a change in temperature within
a range in which growth is not restricted (370C to 200C), the
culture immediately assumes the steady-state growth rate of
the post—shift temperature (17). In contrast, cold shocks
from 370C to 100C exibit a four hour lag before assuming the
new growth rate, implying a change in cellular composition
when growing outside the "normal" range (21, 22). At 50C, E.
(fllLi is unable to initiate protein synthesis, ribosomal
subunits accumulate, and cellular growth stops (5, 9, 14).

The proteins involved in response to cold shock have
been best characterized in E. coli. The major cold shock
protein, CspA, demonstrates loo—fold induction after a
thirteen degree temperature downshift. (15). CspA shows
sequence identity to known transcriptional activators which
bind to a CCAAT-containing "Y box" within a promoter (12,
38). CspA also has CCAAT binding activity (21). Thus, CspA
may be a transcriptional activator. CspA-like proteins have
been identified in Ikufillus subtilus and Streptomyces
clavuligerus (2, 37). 2D gel analysis of the cold shock
response has shown the presence of an additional twelve
proteins that are induced some 2— to 10—fold in response to

low temperature (15, 24). Ten of these proteins have been

13

14

identified (23, 24, 26), some of which are involved in either
transcription or translation.

Little is known about the cold shock response in
Rhizobium spp. A study of three arctic and three temperate
strains of Rhizobium spp. revealed the presence of 17—24 and
18-22 cold shock proteins, respectively (6). The identity of
the genes and the mechanism for the regulation is unknown.

The overall goals of my research project were to
describe more fully R. meliloti growth at low temperature and
to isolate, identify, and characterize the expression of one
or more cold—regulated genes using R. meliloti strain 1021
(Rm 1021). The data indicate that the growth rate of Rm 1021
is a linear function of temperature between 15 and 300C, but
that below 150C, growth is "restricted." The upregulation of
8 polypeptides, and downregulation of 8 polypeptides, was
found to occur when Rm 1021 cells were shifted from 300C to
150C. 'Dmo genes that were cold-regulated were tagged by
transposon mutagenesis and the patterns of induction were
examined. Both Rm 3—16—6 and Rm 5—5—6 were induced by
temperature shifts from 300C to 20, 15, or 100C, although
induction at 100C was delayed. The identity of one was
determined to be a 16S rRNA gene. This appears to be the

first description of a cold—regulated rrn gene in bacteria.

MATERIALS AND METHODS

Bacteria and plasmids. Table I describes the bacterial
strains and plasmids used in this study.

Media and Culture Conditions. R. meliloti strains, (Rm
1021, Rm 3-16—6, Rm 5—5—6 and Rm C-30) were grown at 300C in
either TY (4) medium adapted to 0.5 g/l CaC12—2H20, or GTS

(25) medium, generally supplemented with 50 ug/ml

streptomycin. E. coli strains were grown at 370C in LB
medium (34). Strains containing plasmids were generally
supplemented with 50 ug/ml kanamycin. (Tryptone and yeast

extract were purchased from Difco, St. Louis, MO.)
Tri-Parental Matings. Transposon :mutagenesis was
accomplished via tri-parental matings, adapted from the
method of De Bruijn and Rossbach (10). Rm 1021, E. coli
containing pRL1062a and E. coli containing pRK2013 were grown
to late log phase. 100 ul of each culture was placed in the
center of a TY plate, allowed to dry, and incubated at 300C
overnight. The resulting cell mass was scraped off with a
loop and resuspended in 1 ml of TY medium and the dilutions
plated on TY plates containing 200 ug/ml kanamycin and 250
ug/ml streptomycin. Plates were incubated at 300C for 3—4

days and screened for light emission.

15

16

Table 1 Strains and plasmids used.

 

Strain or plasmid

Description Sourc

 

E. coli DHSa

R. meliloti 1021

R. meliloti 3—16-6
R. meliloti 5—5—6

R. meliloti C—30

pRL1063a

pRL1062a

pRK2013

pAGla

supE44 AlacUl69 ($801acZAM15)
hst17 recAl endAl
gyrA96thi—1 relAl

SmR, Mos”, Moc‘
derivative of SU47

Rm 1021 mutagenized with pRL1062a
Rm 1021 mutagenized with pRL1062a

Rm 1021 mutagenized with pRL1062a

Suicide vector carrying Tn5
with promoterless
luxAB from Vibrio fischeri

Suicide vector carrying Tn5
with promoterless

luxAB from Vibrio harveyi
ColEl KmR tra(RK2)

Tn5—luxAB and flanking DNA
from Rm 3-16-6 (Apa I digest)

e or reference
34

29

This study
This study
This study

40

C.P. Wolk

13

A.M. Gustafson

 

17
Screening of Mutants. Mutants obtained by triparental

mating were screened for light emission at 300C and 100C.
After exposure to n-decanal for two minutes, light emission
was quantitated for one minute with a Hamamatsu photonic
camera (model c1966—20, Photonic microscopy, Oak Brook, IL).
Light emission was measured at 300C, the aldehyde allowed to
dissipate in a sterile environment, and the plates incubated
at 100C for 6 hours before being screened again. Mutants
that emitted light at 100C, but not 300C, were picked and
rescreened using the same method.

Stability of V; fischeri and V; harveyi luxAijroteins.
2 ul of overnight cultures were pipetted onto filter squares
on TY plates. Plates were incubated at 300C overnight and
filters were transferred to fresh plates and incubated at
100C for 6 hours. Filters were then transferred to fresh
plates at room temperature for O, 15, 30, and 45 minutes.
The plates were exposed to aldehyde for 2 minutes then
screened for light emission for 1 minute using a Hamamatsu
photonic camera.

Genomic DNA Analysis. Total DNA from Rm 1021, Rm 5-5—6
and Rm 3-16—6 was isolated by a method adapted from Rossbach
(32). One and a half ml of an overnight culture was
centrifuged for 3 min. The pellet was washed with 1 volume
1M NaCl, 25mM potassium phosphate and centrifuged 3 minutes.
The pellet was resuspended in 350 ul TE, 25 ul 20% SDS, 25 ul
pronase (10 mg/ml) and 100 ul sterile distilled H20. After

incubating for 1 hour at 370C, the DNA was passed three times

18
through a sterile syringe with an 18 gauge needle. DNA was

purified by 2 phenol extractions with a ten minute
centrifugation, followed by 2 phenol/chloroform extractions
with five minute centrifugations and 1 chloroform extraction
followed by a 2 minute centrifugation. Two volumes of 100%
ethanol were added and mixed by careful shaking. The DNA was
spooled out with a pasteur pipette and dissolved overnight in
100 ul sterile distilled water.

Kpn I and Sma I (New England Biolabs, Beverly, MA) were
used separately to digest Rm 1021, Rm 5—5-6 and Rm 3—16—6.
Digests were incubated according to manufacturer's
specifications. DNA fragments were separated on a 0.7%
agarose gel. DNA was transferred to a nylon filter (nytran)
using a posiblotter (Stratagene, La Jolla, CA).
Hybridization was accomplished using 32P—radiolabeled luxAB
fragments from pRL1062 or the 5' 16S DNA flanking genomic
fragment from pAGla as a probe for quantitating to number of
Tn5-luxAB insertions or confirming the 16S copy number,
respectively.

Protein synthesis. Rm 1021 was grown at 300C to an
OD500 of ~0.3. Before shifting to 150C, 1 ml of culture was
added to a disposable 15 ml culture tube and labeled with 353
methionine (5 ul/ml) for 10 minutes at 300C. 500 ul of 10
mg/ml l—methionine was added to stop labeling. Cells were
centrifuged for 15 minutes, washed with 1 ml of 25 mM Tris/ 1
M NaCl, centrifuged for 5 minutes and resuspended in 100 ul

25 mM Tris/10 mM MgClg, 5 ul 6 mg/ml lysozyme, 5 ul 1 mg/ml

l9

DNase I. Cells were incubated at 300C for 30 minutes, 110 ul
of a cracking buffer containing 100 mM NaOH, 1% SDS and 10 mM
EDTA was added, cells were boiled for 2 minutes then
centrifuged for 30 minutes. To precipitate proteins, 1.3 ml
of acetone was added to the supernatant, placed at —200C for
2 hours then centrifuged for 30 minutes. The resulting
pellet was resuspended in SDS sample buffer. After the
remaining culture was incubated at 150C for one hour, 1 ml
was removed, labeled with 358 methionine for 30 minutes at
150C and labeled proteins were purified as above. Proteins
were separated by SDS-PAGE (27).

Induction in TY medium. Idght emission was measured
using a Berthold luminometer (Lumat LB9501, Wallac Inc.,
Gaithersburg, MD). 0.1% n—decyl aldehyde (D—7384, Sigma, St.
Louis, MO) was buffered with 2% BSA (initial fractionation by
heat shock, 98—99%, A3803, Sigma, St. Louis, MO) and 50 ul
was added to 5 ul of culture. Light emission was measured in
triplicate and the values for each set were averaged and
adjusted for background and O.D. Fold induction was defined
as light emission at low temperature divided by light
emission at 300C.

Growth rates. Culture growth rates were determined for
wild type and mutant strains Rm 3—16-6 and Rm C—30 in
triplicate. Twenty—four hour old cultures grown at 300C were
diluted into fresh medium to give a final O.D.6oo of 0.1
before incubation at lower temperatures. Growth rates were

established by measurement of increased O.D. over time.

 

RESULTS

Growth rates of Rm 1021 at low temperatures.

Steady state growth rates at various temperatures were
determined for the R. meliloti wild type, Rm 1021, in TY and
GT8 media (Table 2). As expected, growth rate decreased with
a decrease in temperature. Interestingly, when compared to
growth in TY medium, Rm 1021 in GT8 medium exhibited a slower
growth rate at 300C, but a faster growth rate at 100C.

Growth lags were not observed upon shift of Rm 1021
cultures from 300C to 100C, or any of the higher temperatures
tested in either TY (Figure 1) or GTS media (not shown).

An Arrhenius plot is often used to show the relationship
between the specific growth rate constant (k) and temperature
(19). Prokaryotes typically have a "normal" range in which
the growth rate decreases linearly with a decrease in
temperature. Growth outside this range is "restricted" (17,
19). An Arrhenius plot of Rm 1021 from 370C to 100C
indicated a linear relationship between 300C and 150C with a
calculated temperature characteristic (u) of about 102,800
J/mol (~24,600 cal/mol) (Figure 2). Outside of the 30 to

150C range, growth became restricted; i.e., the k value fell

20

 

21

Table 2. Generation times of Rm 1021 in TY and GT8 media at
various temperatures.

 

 

Temperature TY medium GTS medium
370C 2.0 hr i 0.7 hr
300C 2.4 hr i 0.1 hr 3.6 hr 1 0.6 hr
250C 5.5 hr i 0.6 hr
200C 9.5 hr 1 0.7 hr 8.0 hr i 0.4 hr
150C 21.5 hr i 0.9 hr
100C 80.3 hr i 10.5 hr 32.3 hr i 2.4 hr

 

Flasks of media were inoculated with overnight cultures of Rm
1021 to an O.D.600 of ~ 0.1. In each case, three replicate
cultures were used. Generation times were calculated from
growth rate during exponential phase.

22

 

 

 

 

1 I I I I I r I I r r I I T j
. 25o+
.
- 200 .
g 150 -
‘° 1oc -
=2
0
1 1 n I n l l 1 u l l 1 l L j
—6 -4 -2 0 2 4 6 8
HOURS

Figure 1. Growth after shift of Rm 1021 from 30°C to lower
temperature. Overnight cultures of Rm 1021 grown in TY

medium at 300C were diluted into fresh TY medium to an
O.D.5oo of ~0.1 and incubated at 30°C until the cultures
reached an optical density of ~0.3. The cultures were
transferred to either 25, 20, 15, or 100C and O.D.5oo
measurements were taken at the indicated times. Plus, 300C.
Diamond, 250C. Circle, 200C. Triangle, 150C. Square, 100C.

 

23

 

 

 

 

10 ’ ' I I ' I r a
e a
z ..
< 1
E! 1 1
to q
2: i
C> I
L) 4
:1
E: .01 a
O :
m 1
w I

001 . I .4 l . 1 .
112 33 3A 35 36

1000/ ABSOLUTE TEMPERATURE

Figure 2. Arrhenius plot of Rm 1021 steady state growth
rates at decreasing temperatures. Steady state growth rates
at each temperature were measured and used to calculate the
growth rate constant, k (h’l), which is the inverse of the
generation time. Each data point is labeled with the growth
temperature, in Celsius. Dashed line indicates the expected
growth rate constant if growth was not restricted.

24

below that predicted by extrapolation from the linear region
of the Arrhenius plot.

Protein synthesis.

Changes in gene expression were found to occur in Rm
1021 upon ea shift from 300C t1) 150C (Figure 3). In
particular, the synthesis of at least 8 rmflypeptides was
upregulated in response to the temperature shift and the
synthesis of at least another 8 proteins was downregulated by
temperature downshift.

Comparison of V; fisheri luxAB and V3 harveyi luxAB as
reporter genes to "tag" cold-regulated genes.

Rm 1021 was mutagenized with Tn5 derivatives carrying
luxAB genes from either V. fischeri or V. harveyi (Figure 4)
(39). Mutants were screened at 300C and 100C and any that
appeared to be upregulated in response to low temperature
were isolated and rescreened.

Initially Rm1021 was mutagenized using luxAB from VA
fischeri. Unexpectedly, the majority of the mutants
demonstrated a dramatic increase in light emission upon shift
from 300C to 100C. Five such isolates are shown in Figure 5
(spots 1-5); light emission of cells grown at 300C (panel B)
was much less than that of cells incubated at 100C for 15
minutes (panel C). This result, however, was in apparent
conflict with the protein synthesis study indicating only
modest changes in gene expression in response to low
temperature (Figure 3). Thus, it was suspected that the

increase in light emission might be due to an increase in

 

25

Rm 1021

30°C 15°C

 

Figure 3. 1D gel analysis of Rm 1021 358-1abe1ed proteins at
30°C and 15°C. An Rm 1021 culture was grown in TY medium at
30°C. At an O.D.5oo of ~0.3, half the culture was 35S—
labeled at 30°C. The other half was transferred to 15°C for
2 hours, then 35S-labeled. 35S-labeled proteins were
extracted from the cells and run on a polyacrylamide gel.
Exposure of the 15°C lanes was increased to allow comparison
of overall protein expression. Long arrows indicate
upregulated proteins. Short arrows indicate downregulated
proteins.

26

pRL1062a

 

Smol

Figure 4. Map of the suicide vector, pRL1062a. pRL1062a
carries the luxAB genes from V. harveyi. pRL1063a (not
shown), which carries luxAB from V. fischeri, varies only in
the luxAB region. Enzyme sites of interest are indicated.

27

 

 

 

 

 

Figure 5. Stability of V. fischeri and V. harveyi luxAB.
Mutants containing luxAB from V. fischeri or V. harveyi were
compared to determine the relative stability of luxAB. (A) 1,
2, and 3 are V. fischeri mutants with putative constitutive
expression; 4 and 5 are V; fischeri mutants which were
apparently induced by a shift to 10°C; 6 and 7 are V; harveyi
mutants which demonstrated no change in light emission upon
temperature shift. Plates were incubated overnight at 30°C
(B), shifted to 10°C, then placed at room temperature for 0
min (C), 15 min (D), 30 min (E), or 45 min (F).

28
LuxAB protein stability at low temperature as opposed to

cold—induced expression of the genes. The V. harveyi LuxAB
protein is known to be considerably more stable than the V.
fischeri LuxAB protein (30). Indeed, the half life of the V.
fischeri protein in Rm 1021 at 30°C must be less than 3
minutes as the light emission decreased more than 10 fold
within 15 minutes of returning cold—treated cells to 30°C
(Figure 5d).

If jprotein. instability' accounted. for tflna results
obtained with the V. fischeri luxAB transposon, then it was
possible that the transposon carrying the luxAB gene from V.
harveyi might alleviate the problem. Indeed, when the V.
harveyi luxAB transposon was used, most of the inserts gave
constitutive light emission at 30°C and 15°C. An example of
such a mutant is shown in Figure 5 (spot 6).

Transposon tagging of cold-regulated genes.

The Tn5 derivative carrying the luxAB from V; harveyi
was used for transposon mutagenesis. Mutants were grown at
30°C, then transferred t1) 10°C for 5 tx>'7 hours. The
screening of approximately 17,000 mutants resulted in the
isolation of two mutants, designated Rm 5-5—6 and Rm 3—16—6,
carrying a transposon insert that displayed cold regulation
(Figure 6). Rm 3—16—6 and Rm 5—5—6 exhibited an approximate
100- and 20-fold induction, respectively, upon a temperature
shift from 30°C to 10°C for 6 hours (Table 3). Rm C—30 was
selected as a control due to its constitutive expression.

Approximately 4,500 mutants were shifted from 30°C to 15°C

 

29

 

30°C
10°C
Rm 5—5—6
30°C
10°C

 

Rm 3-16-6

Figure 6. Light emission from cold-regulated mutants at 30°C
and 10°C. Overnight cultures of Rm 5—5—6 (A) and Rm 3—16—6
(B) were spotted in replicate onto TY plates and incubated
overnight. The following day plates were split in half with
one half placed at 10°C and the other half returned to 30°C.
After 6 hours the halves were placed together, exposed to n—
decanal and screened for light emission.

30

Table 3. Light emission at 30°C and 10°C in mutant carrying
Tn5-luxAB insert.

 

 

 

 

Mutant Average Light Emitted Fold
(relative counts) Increase
300c 100c at 10°C
Rm 3-16-6 245 i 74 (4) 29620 i 9525 (4) ~ 120
Rm 5-5—6 265 i 75 (4) 5340 i 145 (4) ~ 20
Rm C—30 1640 i 265 (2) 1880 i 190 (2) ~ 1

 

Overnight cultures were spotted in replicate onto TY plates
and incubated overnight at 30°C. Plates were split, one half

was placed at 10°C, the other half placed at 30°C, then
placed together after 6 hours and exposed to n-decanal.
Relative light emission was measured from each culture spot.
Reported relative light emission from Rm 5—5—6 and Rm 3—16-6
is the average from four replicate spots; relative light
emission reported for Rm C-30 is the average of two replicate
spots.

 

31
for 24 hours but screening did not yield any additional cold—

regulated mutants.

number of Tn5—luxAB inserts.

Southern analysis was used to determine the number of
Tn5—luxAB insertions into the chromosome in Rm 5—5-6 and Rm
3—16—6. DNA from the mutants and wild type, Rm 1021, was
digested with either Kpn I, which has no sites within the
transposon, or Sma I, which cuts twice within the transposon
(see Figure 4). After separation of the DNA fragments by
agarose gel electrophoresis and transfer to nitrocellulose, a
32P—labeled Xba I fragment containing luxAB from pRL1062a
(see Figure 4) was used to probe for the Tn5—luxAB
insertion(s). As expected for a single insert, the probe
hybridized to only one band in each of the digests for each
mutant, (Figure 7). No hybridization was detected with the
wild type strain, Rm 1021.

Low temperature induction in TY medium.

Rm 3—16—6, Rm 5—5—6, and Rm C—30 were grown in TY liquid
culture to further examine the level of low temperature
induction. Light emission upon shifts from 30°C to various
lower temperatures was measured (Figure 8). Little to no
induction was evident after a shift to 25°C. Both Rm 3—16—6
and Rm 5—5-5 were induced by a shift to 20°C, 15°C or 10°C,
but the level of induction at 20°C and 15°C was much greater

over the time course of the experiment than at 10°C.

32

Rm 1021 Rm 5-5-6 Rm 3-16-6

Kpn I Sma I Kpn I Sma I Kpn I Sma I

 

Figure 7. Detection of Tn-luxAB insertions in Rm 5-5-6 and
Rm 3-16-6. DNA from Rm 1021, Rm 5—5—6, and Rm 3—16—6 was
digested with Kpn I or Sma I and separated on an agarose gel.
DNA was transferred to nitrocellulose and probed with 32P—
labeled luxAB from pRL1062a.

FOLD INDUCTION

FOLD INDUCTION

33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50 ..... . ..... ....r
A 25 c z
40- - 9
- - [-1
30- - B
- c:
20- - E
10- - 5
remu- a
0 III II
0 5 10 15 1 15
HOURS AT 25 C HOURS AT 20 C
50 ..... ,..r
g D I 10 C
H 40 -
H .
8 30- -
o . .
E 20- -
S 10.' '
2 1.5.44 1
. ....1.... _ O
5 10 15 0 5 10 15
HOURS AT 15 c HOURS AT 10 C

Figure 8. Induction of luxAB insertion in Rm 5—5—6 and Rm 3-
16-6 upon shift from 30°C to lower temperatures. TY medium
was inoculated from overnight cultures grown at 30°C to an
O.D.600 of ~0.1, incubated at 30°C until an O.D.5oo of ~0.3
was reached, then shifted to 25°C, 20°C, 15°C, or 10°C.
Relative light emission was measured and adjusted for optical
density. Results are the average from one experiment run in
triplicate. (A) 25°C. (B) 20°C. (C) 15°C. (D) 10°C.
Squares, Rm C—30. Triangles, Rm 3—16—6. Circles, Rm 5—5—6.

34
Expression in response to heat shock.

To determine if Rm 3—16—6 was also responsive to a
temperature upshift, light emission after heat shock was
examined. Rm 3—16—6 and Rm C—30 were grown at 30°C in TY

medium to an optical density (O.D.) of ~0.3 then shifted to

37°C. When light emission was measured at 37°C, then
adjusted for O.D., a decrease in light emission was seen
(Figure 9).

Identification of cold-regulated gene.

The gene into which the Tn5—luxAB inserted in Rm 3—16-6
was identified by cloning and sequencing. Total DNA from Rm
3—16—6 was digested with restriction enzymes that have no
sites in the transposon. The fragments were recircularized
using T4 ligase and the molecules were transformed into E.
coli. An Apa I digest yielded a 9 Kb clone, designated
pAGla, which was used for sequencing the R. meliloti genomic
DNA flanking the transposon. A BLAST search of Genbank (1)
indicated that the cloned gene had 99% sequence identity to
R. meliloti 16S rRNA (accession number D12783) (Figure 10).

Southern blots used to quantitate the number of Tn5—
luxAB insertions (see Figure 7) were used to determine the
number of 16S rRNA genes present in Rm 1021. The luxAB probe
was removed from the blot and the blot was re—probed with
32P—labeled fragment of genomic DNA flanking the transposon
in pAGla. Three bands were revealed in the Sma I digest of
Rm 1021 (Figure 11) which is consistent with the conclusions

of Honeycutt, et al. that R. meliloti has three rrn operons

35

 

lA-' j

 

 

 

 

L) ' .
53 12 f j
9‘ .
‘< .
z I
O _
i-i
E—t 1
U n
D u
a 1
z u
u cl
a q
A q
o -
I“ I
00 i l L’ I ‘
0 1 2 3
HOURSAT37C

Figure 9. Fold induction after shift from 30°C to 37°C. Rm
3-16-6 and Rm C—30 overnight cultures grown at 30°C were used

to inoculate TY medium to an O.D.6oo of ~0.1. After
incubation at 30°C until an O.D.6oo of ~0.3 was reached,
cultures were shifted to 37°C. Light emission was measured

at 0, 0.5, 1, and 2 hours and adjusted for optical density.
Adjusted light emission at 37°C was divided by adjusted light

emission at 30°C to give relative light emission. Results
shown are the average of three replicates in one experiment.
Squares, Rm C-30. Circles, Rm.3—16-6.

36

V
H

GGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTA 56
l|illl||||||||Il||||||||||||lll||l||ll||||||||||||||l|||
B: 842 GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTA 897

A: 57 CCAGCCCTTGACATCCCGATCGCGGATACGAGAGATCGTATCCTTCAGTTCGGCTG 112
ll|llllllillllllll|IIIIl||||||||||||l||lllllllllllllllll
B: 898 CCAGCCCTTGACATCCCGATCGCGGATACGAGAGATCGTATCCTTCAGTTCGGCTG 9S3
*
A: 113 GATCGGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT 168
illlllllll|||l||||||||||l|lll|i||||||||l||||||||||||l|||
B: 954 GATCGGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT 1009

A: 169 AAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGCACT 224
|l||l||l|||l||||lllllllllllllll|li|||||||l||||||l|||l|||
B: 1010 AAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGCACT 1065

A: 225 CTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTC 280
l|||||||||||||l||||Ill|||||||||||||||||llIllllllllllllll
B: 1066 CTA “CCFCACTCCPCGTGATAAGPP ‘‘‘‘‘‘‘ Am"TGGGGA‘I‘GACGTCAAGTCC’I‘C 1121

 

A: 281 ATGGCCCTTACGGGCTGGGCTACACACGTGCTACA TGGTGGTGACAGTGGGCAGC 335
Ill||||||i|||||l|l|||||||||||||||l| llllllllllllllllllll
B: 1122 ATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGC 1177

A: 336 GAGACCGCGAGGTCGACTGAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTG 391
Illillllllilllll |||||||l|||||l|l||||l|||||||i|||||l||
B: 1178 GAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTG 1233

A: 392 CAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCAGATCAGCATGCTGCG 447
llllllllll |l|||||||ll|llllllllllllll|l|||||||||||l|||
B: 1234 CAACTCGAGTNCATGAAGTTGGAATCGCTAGTAATCGCAGATCAGCATGCTGCG 1287

Figure 10. Sequence comparison between Tn5-luxAB insertion
site in Rm 3-16—6 and R. meliloti 16$ rRNA. Regions flanking
the Tn-luxAB insertion in Rm 3—16—6 were sequenced. A BLAST
search (1) revealed 99% identity to R. meliloti 16S rRNA.
Line A indicates sequence from pAGla. Line B indicates
sequence from R. meliloti 16S rRNA (accession number D12783).
Asterick indicates site of Tn—luxAB insertion.

37

Rm 1021 Rm 3-16-6

Kpn I Sma I Kpn I Sma I

 

Figure 11. Detection of 16S rRNA copies. DNA from Rm 1021
and Rm 3—16—6 was digested with either Kpn I or Sma I and
fragments were separated by agarose gel electrophoresis. DNA
fragments were transferred to nitrocellulose and probed with
32P-labeled genomic DNA from pAGla. Astericks indicate bands
which hybridized to the luxAB probe (see Figure 7). One of
the three 16S rRNA fragments present in Rm 1021 was shifted
in Rm 3—16—6, due to the Tn5—luxAB insertion.

38
(18). One of the three 16S rRNA fragments present in Rm 1021

was shifted in Rm 3—16—6, due to the Tn5—luxAB insertion.

rRNA operon structure in Rm 1021.

There have been no reports on the structure of rRNA
operons in R. meliloti. It was therefore of interest to
determine whether the 16S gene in Rm 1021 was followed by
tRNA, 23S and SS genes as is found in the rRNA operons of E.
coli and other prokaryotes such as Bacillus subtilus and
Halobacterium thermophilus (for review, see ref. 35). Cla I,
which has a restriction site within luxAB (Figure 4), was
used tr) clone a fragment containing 9 Kb (of sequence
downstream of Tn5—luxAB. This clone was then subcloned and
sections of it were sequenced. Comparison of DNA sequence
(11) indicated that the Rm 1021 16S rRNA gene was upstream of
a 23S rRNA gene, separated by approximately 1.2 Kb of spacer
region containing an alanine tRNA gene (Figure 12) (41).
Sequence obtained from regions further downstream did not
conclusively demonstrate whether there was or was not a SS
rRNA gene.

Comparison of growth rates.

Rm 3—16—6 growth rates were compared to Rm 1021 to
determine whether the presumed change in the number of
functional rRNA operons affected the growth rate at either
high or low temperature. Since Rm 3—16—6 contained a Tn5-
luxAB insertion into a cold—regulated gene, it was possible
that growth at lower temperatures might have been

compromised. No significant difference in growth rate was

39

E. coli rrnB

 

 

 

glu
P1P2 16S _ tRNA 233 SS T1T2
JJitfrr' ' h thi
ala
l6S tRNA 23S 23S
Rm 3—16—6 t1

 

 

 

Figure 12. Sequence identity between E. coli rrnB and Rm 3-
16-6 rRNA operons. Rm 3-16—6 fragments were cloned,
subcloned and sequenced 5' and 3' of the Tn—luxAB insertion
site. The resulting sequences were compared to E. coli rrnB
by GenBank searches and manual searches. Rm 3—16—6 boxed
regions indicate identity to E. coli rrnB.

4O
seen between Rm 1021 and Rm 3—16—6 at 30°C, 25°C, 20°C, or

10°C when grown in TY medium (Figure 13). However,
repetition of the experiment demonstrated a reproducible,

albeit slight, difference in growth rates at 15°C.

41

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 . . ,. ., ..
A 1 B
E .3.
g: '1 '= :51 s
o : d I
.01 1 l l l 1 I .01 nnnnn n
0 4 6 8 10 5 10 15
hours at 30 C hours at 25 C
1' l ' I 1 ' ' I I I
D
c
O
\C
a q-1 g "a
O
l n l l I
.01 L '
0 10 20 30 20 4O 60 80
hours at 20 C hours at 15 C
1 - I I I I I
E

O.D. 600

 

l . I . l I l . l

01 . .
10 20 30 40 50 60 70
hours at 10 C

 

 

Figure 13. Steady state growth rates of Rm 1021 and Rm 3-16-
6 at decreasing temperatures. Rm 1021 and Rm 3—16—6 cultures
grown at 30°C were used to inoculate TY medium to an O.D.6oo

of ~0.1. Growth rate was determined during exponential
growth. Results shown are the average from triplicate
cultures in one experiment. (A), 30°C. (B), 25°C. (C),
20°C. (D), 15°C. (E), 10°C. Squares, Rm 1021. Circles, Rm

3—16—6 .

DISCUSSION

Temperate strains of R. meliloti, such as Ihn 1021,
exhibit optimal growth between 28°C and 30°C. In the
northern regions of the U.S., however, soil bacteria are
exposed to temperature ranges from 10 to —5°C nearly half the
year. How bacteria survive and adapt to low temperatures is
largely unknown. To address this question, several aspects
of response to low temperature were examined in Rm 1021 and
two mutants containing Tn5—luxAB inserts in cold—regulated
genes.

Growth in Rm 1021 between 15°C and 30°C is a linear
function (Figure 2). At temperatures lower than 15°C or
higher than 30°C, growth is restricted. In E. coli, an
Arrhenius plot shows a linear relationship between 37°C and
21°C (17). The molecular basis for restricted growth at low
temperature is not well understood. It is known that E. coli
can not initiate protein synthesis after a shift from 37°C to
5°C (5, 9, 14). However, it is unknown whether problems in
translation are responsible for restricted growth at higher
temperatures.

As has been shown with other organisms, R. meliloti
alters gene expression in response to low temperature.

Previous reports have shown the presence 18—22 cold shock

42

43

proteins 111 Rhizobiunl meliloti A2 and other temperate
rhizobia (6). Similarly, our SDS—PAGE analysis indicated 8
proteins are upregulated by a shift to a lower temperature
(Figure 3). Additionally, certain proteins were
downregulated. The upregulation of only eight proteins on
first consideration seems like a small number but only the
most abundant proteins are visible by SDS—PAGE analysis. The
total number' of upregulated proteins in R. meliloti,
therefore, may be much higher.

Presumably the changes in gene expression that occur in
R. meliloti in response to low temperature are involved in
cold acclimation. The identity and functions of cold—
regulated genes in Rhizobia, and their relationship to cold—
regulated genes in E. coli are unknown. Towards an
understanding of the roles of R. meliloti cold—regulated
genes in low temperature growth and survival, we used the Tn—
luxAB transposon from V; harveyi to tag cold—regulated genes.
In screening > 17,000 colonies, two mutants with insertions
into cold—regulated genes were obtained.

Although Rm 5—5—6 was not induced as strongly as Rm 3—
16—6, the overall induction patterns are similar. Both Rm 3—
16—6 and Rm 5—5—6 were induced by a shift from 30°C to 20,
15, or 10°C, but not 25°C. Interestingly, induction after a
shift to 10°C was weak and delayed compared to induction
after a shift to 20 or 15°C. The delay may be related to

restricted growth at 10°C (Figure 2).

44
The cold—regulated gene tagged in Rm 3-16—6 was

identified as a 16S rRNA gene (Figure 10). This result is
somewhat surprising. Indeed, upregulation of an rRNA
promoter at low temperature seems counter—intuitive since the
promoter of rrn genes are subject to growth rate control. In
nutritional growth rate control, rRNA promoter activity
decreases with a decrease in growth rate (16). In our
studies, however, fusion of the promoter region to luxAB
showed increased promoter activity with a decrease in growth
(data not shown). Although promoter activity was not
directly measured, a previous study demonstrated a lack of
correlation between stable rRNA and temperature—controlled
growth rate (33).

Currently, two models for growth rate control of rrn
gene promoters exist. One is the ppGpp—dependent RNA
polymerase partitioning model in which 670—RNA polymerase has
two forms. rrn promoter strength depends on the ratio of
form I, which maintains full activity, to form II, which has
greatly decreased activity due to ppGpp (3). The feedback
regulation model states that an excess in ribosomes increases
translation which is then responsible for feedback control
(7, 16, 20, 36). Control is believed to be at both the level
of rrn transcript initiation and the elongation rate of RNA
polymerase (7, 8). The partitioning model requires ppGpp to
regulate synthesis. The feedback regulation model does not
necessarily involve ppGpp, but proponents of the feedback

regulation model agree that ppGpp may be the signal molecule

45

produced by excess translation (31). After temperature
downshift, ppGpp levels decrease and RNA synthesis increases,
suggesting ppGpp may be responsible, to some degree, for
control of rRNA synthesis after cold shock (28).

Overall, comparison of growth rates revealed little
difference between Rm 1021 and Rm 3—16-6 despite the
interruption of an rRNA operon in Rm 3—16-6 (Figure 13).
This is not surprising since previous studies in E. coli have
shown that the addition or deletion of rRNA operons does not
change the number of ribosomes present (7, 8, 20). Rm 3—16—
6, with 67% functional operons, is comparable to an E. coli
strain which has 5 out of 7, or 71%, functional operons.
This E. coli strain demonstrated little to no change in
growth rates after deletion of two of its rRNA operons (8).

Interestingly, we did see a slight difference between Rm
1021 and Rm 3—16—6 growth rates at 15°C. The difference does
not appear to be due to the effects of the reporter gene,
since we see induction at 20°C without a difference in growth
rate. At this time, the significant of this observation is
unknown.

There is a discrepancy in our reported light emission
from Rm 3—16—6 and Rm 5—5—6 between solid and liquid media
after 6 hours at 10°C (Figure 6 vs. Figure 8). Initially
mutants were screened for changes in light emission after a
temperature downshift while growing on solid medium. When
light emission was measured from mutants growing in liquid

medium, the measured fold induction was dramatically reduced

46
for both Rm 3—16—6 and Rm 5—5—6. This reduction may be due

to physiological differences between bacteria growing in
liquid or on solid medium. In addition, the assay conditions
vary between cells grown in liquid and on solid medium. For
example, aldehyde is delivered as a vapor to colonies on
plates but liquid culture samples are vortexed with a
solution of aldehyde dissolved in 2% BSA. Although the BSA
acts as a stabilizer, the aldehyde is not easily suspended in
water, thus the amount of aldehyde immediately available to
the cell may be limited. Attempts to simulate solid medium
conditions, either by using agar plugs or liquid culture
spotted onto filter paper, did not result in a higher fold
induction (not shown). It is possible that either of these
methods may have interfered with light detection.

Given the fact that 1D gel analysis indicated eight
cold-regulated proteins, isolation of only two mutants seems
low. There are a number of possible explanations. Some
cold—regulated genes may be essential for survival at low
temperature. Thus, an insertion into these genes would be
lethal. Additionally, changes in light emission were
visually detected, therefore, I may have overlooked mutants
that weren't strongly induced. In E. coli, for example, most
cold shock proteins are only upregulated 2— to 10—fold (15,
24). It is likely that I would have missed a 2— to 5—fold
induction. Perhaps the most likely reason for obtaining only
two mutants was due to the time and temperature used in the

screen. Induction of Rm 3—16—6 and Rm 5—5—6 at 10°C is much

47

slower and less pronounced than at 15°C. Indeed, K.
O'Connell (unpublished data), has screened for mutants using
a shift from 30°C to 15°C for 4—6 hours and has obtained 8
cold—regulated mutants in screening a total of 4,000 mutants.
Whether the Tn5—luxAB in the mutants inserted into new cold—

regulated genes remains to be determined.

FUTURE DIRECTIONS

The data presented suggest many directions for future
work. Induction after a temperature downshift was examined
using 30°C as the preshift temperature. It is unknown,
therefore, if induction after a shift to 20°C was a function
of the absolute post—shift temperature (20°C), the change in
temperature (10°C), or both. Use of lower preshift
temperatures, such as 25°C and 20°C, would be a first step in
addressing this question.

Rm 5—5—6 and Rm 3—16—6 displayed similar patterns of
induction. It is unknown, however, if the similarity is due
to control by the same regulatory mechanisms. Secondary
transposon mutagenesis could be used to tag the regulatory
elements, followed by cloning and sequencing of the tagged
gene to reveal the identity of the regulatory gene.

Studies to date have been limited to TY or GTS media.
We have not characterized response to low temperature in the
soil. Also, we have not evaluated the effect of insertion
upon survivability in the soil. Long term soil studies need
to be done.

At this time it is not known if the response to low
temperature is cold-specific or a general stress response.

Response to other environmental stresses has not been

48

49
characterized in either Rm 3—16-6 or Rm 5—5—6. Measuring

light emission before and after other stresses, such as
osmotic shock or carbon limitation, will evaluate the

specificity of the cold-regulated genes.

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