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LIBRA 1:. Y 7
Mlchlgan ”1‘ {"912}:
University V

This is to certify that the
thesis entitled
CHARACTERIZATION OF THE DEOXYRIBONUCLEIC
ACID OF VEGETATIVE CELLS OF
AZOTOBACTE’R VI NELAZVDI I

presented by

Eric S. Berke

has been accepted towards fulfillment
of the requirements for

Ph. D. . Microbiology and
Jegree 1n Public Health

 

 

@745 14,564? J ‘ [Hz/A

Major professor _/-' :1

 

Date September 27, 1972

 

0-7639

 

ABSTRACT

CHARACTERIZATION OF THE DEOXYRIBONUCLEIC
ACID OF VEGETATIVE CELLS OF
AZOTOBACTER VINELANDII

 

BY

Eric S. Berke

A study of the DNA of Azotobacter vinelandii was

 

made. Vegetative cells of A. vinelandii were found to

contain 6.7 x 10'-14

grams of DNA per cell and two stain—
able nuclear bodies, while cysts contained one half as
much DNA and only one nuclear body. The minimal DNA con—
tent per Azotobacter cyst is thus an amount eight times
that of the E. ggli genome.

Melting curves of A. Vinelandii DNA when plotted
on normal probability paper show a bimodality, indicating
two components with different guanine + cytosine (G+C)
contents. Both components are in about equal concentra—
tion in the cell, one having 61% G+C and the other 69%
G+C.

DNA renaturation experiments on A. vinelandii
DNA have been shown in this study to deviate from second
order kinetics, unlike typical bacteria. Analysis of

the data resolved the renaturation curve into the sum of

 

—i—i

Eric S. Berke

two second order curves, characterized by Cot values

l/2
(the initial concentration of DNA times time at the 50%
renaturation point) of 1.3 and 8.9 (corrected for G+C
effects). This corresponds to unique nucleotide sequence
lengths which are 0.4 and 3.1 times as long as that of

9 daltons,

the E. col; genome, i.e. 1.2 X 109 and 7.9 X 10
respectively.

Deviation from second order kinetics has also
been substantiated by the discrepancy between unique
sequence sizes obtained from initial renaturation rates

and Cot values on A. vinelandii DNA. Initial renatur-

1/2
ation rates of this DNA have indicated a unique sequence
size about half the value of that obtained from the
Cotl/Z value of the total Azotobacter renaturation. This
rate has been shown to correspond to the expected initial
rate when two components of Cotl/Z values of 1.3 and 8.9
are present.

The slow component of the analyzed renaturation

curve (Cot = 8.9) was concentrated by allowing the

‘ 1/2
; renaturation to proceed to 88% completion and fractionat-
i ing the DNA on an hydroxylapatite column. The single-

‘ stranded DNA, unrenatured at the time of fractionation,

} consists mainly of the slow component (approximately

‘ 85%) since the greater portion of the fast component
=l.3) has renatured by the time of fractionation.

(Cotl/Z

 

Eric S. Berke

This concentrated slow component was then allowed to
renature to completion. Values for unique sequence size
were obtained from this renaturation curve. The curve
could also be resolved into two components of Cotl/Z
values of 1.3 and 8.9 respectively, as was the case for
the total Azotobacter renaturation.

Attempts to separate the two G+C components of
A. vinelandii DNA using CsCl density gradients, NaI
gradients and both in combination with a relaxation
centrifugation technique were only partially successful
since the peaks showed considerable overlap. This was
attributable to a shearing of the DNA which was inherent
in the purification procedures and a model is presented
to account for the poor separation.

A number of models are devised to describe the

possible organization of the DNA within the cell.

CHARACTERIZATION OF THE DEOXYRIBONUCLEIC
ACID OF VEGETATIVE CELLS OF

AZOTOBACTER VINELANDII

 

BY

Eric S. Berke

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Microbiology
and Public Health

1972

Dedicated to my wife, Nancy

ii

 

ACKNOWLEDGMENTS

I wish to thank Dr. Harold Sadoff for his excellent
guidance and for the many stimulating discussions we have
had during the course of my graduate work. I am grateful
to Dr. Roy J. Britten for the opportunity to visit his
laboratory to become acquainted with the techniques of DNA
renaturation studies. I also wish to acknowledge the
informative discussions I have had with Dr. Donald Brenner
concerning this work. Lastly, I wish to thank Brian Berke
for his technical assistance in preparation of the figures
in this thesis for publication.

Financial assistance from the National Institute of
Health and the Department of Microbiology, Michigan State

University, is gratefully acknowledged.

DEDICATION . . .

ACKNOWLEDGMENTS. .

LIST OF TABLES .

LIST OF FIGURES. .

INTRODUCTION.

LITERATURE REVIEW .

TABLE OF

The Azotobacteriaceae.

 

DNA--Historical Survey
Methods of Studying DNA

EXPERIMENTAL METHODS

0

Strains and Cultivation . . . . .
Determination of DNA Content per Cell . .

Nuclear Staining
Preparation of DNA.

DNA Melting Curves.
Preparation of DNA for Renaturation . . .
Renaturation Techniques . . .
Hydroxylapatite Fractionation of Double and

Single Stranded DNA . .
Density Gradient Centrifugation . . . .
Analysis of G+C Contents. . . . . . .

RESULTS . . .

CCONTENTS

o a a o o o 0

DNA Content per A. vinelandii Cell
Number of Stainable'NUclear Bodies per Cell

DNA per Nuclear Body

DNA Melting Curves.

DNA Renaturation Experiments . . . . .

Density Gradient Centrifugation . .

iv

Page

ii

 

 

 

Page

DISCUSSION I O O O O O O O O O O O I O 57
Melting Curves . . . . . . . . . . . 58
DNA Renaturation . . . . . . . . . . 59
Density Gradient Centrifugations . . . 61

Models for Cellular Organization of the DNA . 62
Approaches to Resolving the Validity of the

Final Model . . . . . . . . . . . 65
SUMMARY . . . . . . . . . . . . . . . 67
LIST OF REFERENCES. . . . . . . . . . . . 69
APPENDICES . . . . . . . . . . . . . . 74

A. A Model for the Estimation of G+C Content
Differences of the Two Components of
A. vinelandii . . . . . . . . . . 76

 

B. Some Aspects of the Theory of Renatura-
tion of DNA. . . . . . . . . . . 85

C. Physiological Studies of Encystment in
Azotobacter vinelandii . . . . . . . 89

 

 

 

Table

l.

 

LIST OF TABLES

DNA content per cell of A. vinelandii

 

vi

'Page

27

LIST OF FIGURES

Figure Page

 

1. DNA melting curve for E. coli, Ag, aeruginosa
and a mixture of the two DNAs. . . . . . 30

 

2. DNA melting curve for A. agilis and A.
vinelandii DNA. . . . . . . . . . . 32

 

3. 'Normal probability plot of DNA melting data
for E. coli, Ag. aeruginosa and a mixture of
the two DNAs . . . . . . . . . . . 34

 

 

4. Normal probability plot of DNA melting data

 

for A, agilis and A, vinelandii DNA. . . . 36
5. Kinetics of reassociation of E. coli DNA . . 39

6. Kinetics of reassociation of A. vinelandii

DNA . . . . . . . . . . . . . . 43

 

7. A comparison of the reassociation kinetics
of A. vinelandii with the sum of two ideal
second order reaction kinetic curves . . . 45

 

8. Kinetics of reassociation of fractionated A.
vinelandii DNA enriched for the slowly
renaturing component. . . . . . . . . 49

 

9. CsCl density gradient centrifugation of
sheared and "unsheared" A. vinelandii DNA. . 53

 

lO. CsCl density gradient centrifugation of a
mixture of E. coli and Ag. aeruginosa DNA
sheared and "unsheared" . . . . . . . 56

 

 

11. Models for the possible organization of DNA
in A. vinelandii. . . . . . . . . . 64

 

12. The theoretical DNA denaturation curve for
a mixture of two DNAs with a large differ-
ence in G+C content. . . . . . . . . 79

vii

 

Figure

13.

14.

The theoretical DNA denaturation curve for
a mixture of two DNAs with an intermediate
difference in G+C content . . . . . .

The theoretical DNA denaturation curve for
a mixture of two DNAs with a small differ-
ence in G+C content . . . . . . . .

viii

Page

81

83

 

 

 

 

INTRODUCTION

Azotobacter vinelandii is a nitrogen-fixing soil

 

organism capable of encystment (39, 46, 53). The encyst-
ment process can be induced by growth on B-hydroxybutyrate
(BHB), butanol and other related compounds (25). Growth

of A. vinelandii vegetative cells on BHB results in a

 

final cell division (41), loss of flagella, accumulation
of poly-Bchydroxybutyrate and formation of outer cyst
layers to form a mature cyst. Cysts are thick-walled,
spherical, resting cells which are highly resistant to
ultraviolet light, sonication, and dessication (47).
Although both cysts and spores are eubacterial resting
cells, cysts differ from spores in that they are not
heat resistant, contain no dipicolinic acid (46) and loss
of calcium has no effect on their viability and dessica-
. tion resistance.

The suspension of cysts in glucose and nitrogen-
free salts solution (Burk's buffer) stimulates germination
and outgrowth to form vegetative cells. Such cells are
large (3 x 1 pm), rod-shaped, gram negative, highly
motile and obligately aerobic.

The life cycle of A. vinelandii has been compared

 

to sporulation in Bacillus species and has been used as

 

a model system for studying cellular differentiation.
DNA levels during encystment have been investigated (41)

and the DNA content of the resting Azotobacter cyst has
14

 

been determined to be 3.4 x 10- grams while vegetative
cells have 2 to 4 times this amount (41).
Similar DNA contents per cell from synchronous

cultures of A. vinelandii cells have been reported by

 

Zaitseva (54). Muller and Kern (35) found comparable

levels of DNA in radiation resistant mutants of Azotobacter

 

chroococcum.

 

Presumably the cyst contains the minimal amount of
DNA needed for the cell to survive. Nuclear staining (41)
has shown the cyst to have one stainable nuclear body,
probably corresponding to one genomic unit.

This amount of DNA is about 8 times that of the

E. coli genome. It is unlikely that A. vinelandii has a

 

range of biochemical capabilities 8 fold greater than
E. coli and this fact, coupled with the high levels of
DNA per cell, has led to the present study into the nature

of the DNA of A. vinelandii.

 

A number of other reports have suggested that the

DNA of A. vinelandii might differ from other bacteria.

 

Mishra and Wyss (33, 34) have reported difficulty in

obtaining A. vinelandii mutants. Gunter and Kohn (19)

 

have shown A. agilis to exhibit multiple—hit survival

curves when subjected to x-irradiation, unlike other

, 1 _
-..”... —--L - - ._r _. if —= _-_— ._

bacteria which show single-hit curves. Pochon (39) has

described the Azotobacter "nucleus" to be vessicular in

 

nature and similar to the yeast nucleus in appearance.
‘It was the aim of this research to characterize

the DNA of A. vinelandii in terms of: G+C content, unique

 

nucleotide sequence size and renaturation kinetics.

LITERATURE REVIEW

The Azotobacteriaceae
Much of the early work on the family Azoto—

bacteriaceae was concerned with the groups' ability to

 

fix nitrogen. Although other organisms such as blue-

green algae, purple and green bacteria and some Clostridium

 

and Desulfovibrio species also fix nitrogen, few incorporate

 

 

as much as the Azotobacter species (21). Six species of

 

Azotobacter have been described in the literature: Azoto-

 

bacter vinelandii, Azotobacter beijerinkii, Azotobacter

 

chroococcum, Azotobacter insignis, Azotobacter macrocyto-

 

 

genes, and Azotobacter agilis. Although these six species

 

are all classified as Azotobacter, there is considerable

 

evidence to indicate that they should not all be grouped
together. DeLey and Park (16) have shown by analysis of
DNA base ratios and DNA hybridization among the strains
that the Azotobacteriaceae should really be divided into
3 groups. The vinelandii-beijerinkii-chroococcum group
consists of organisms having a range of 65.7 i 0.8% G+C

and hybridizable with PSeudomonas putida DNA to the extent

 

of 40-50%. The insignis—macrocytogenes group has G+C
contents ranging from 57-58.6%. These strains are 50-60%
hybridizable with Ag. putida DNA. The third group consists
of the insignis strain which has 52.9 i 0.4% G+C and is

4

about 30% hybridizable with Ag. putida DNA. DeLey has
suggested that only the first group be designated Agggg-
bacter while the other two groups should be termed Azomonas
and Azotococcus respectively.

A. vinelandii, the organism used in this study,
was first isolated in 1904 by Lipman (26). The organism
is a large rod or peanut—shaped cell with an average size
of 3 x l um. It is highly motile by peritrichous flagel-
lation (4), produces a soluble pigment which fluoresces
apple—green under ultraviolet light (16) and undergoes a
life cycle involving a dessication resistant, resting
cell known as a cyst.

Relatively little research has been carried out
on the DNA of A. vinelandii. Olson and Wyss (37) frac—
tionated the nucleic acids from dormant and germinated
cysts of Azotobacter on methylated albumin-kieselguhr
(MAK) columns and noted changes in DNA configuration
after germination. Pochon (39), in light microscope
studies, showed the nuclear body of A. vinelandii to
possess a number of chromatinic granules making up a
vessicular nucleus. During cell division the nuclear
material takes the form of two or four large rods which
divide with the cell and then fragment to reform the

vessicular nucleus.

 

 

DNA——Historical Survey

 

In the late 1800's, F. Miescher (32), while study—
ing the cell nucleus in animal cells, was the first to
isolate and chemically characterize pure DNA from salmon
sperm. The purines and pyrimidines comprising DNA were

isolated in the late 1800's by Kossel (5) and later the

 

deoxyribonucleotides and deoxyribonucleosides corresponding
to the four bases were isolated and characterized. The

tetranucleotide model for DNA structure was relatively

 

long—lived. It was suggested in 1909 (20) and disproved
in 1948 (21). Briefly this model represented nucleic
acids as high molecular weight compounds composed of a
large number of tetranucleotides in sequence similar to
the amino acids in a protein.

In 1944, Avery, MacLeod and McCarty (3) showed
DNA to be the agent responsible for Pneumococcal trans-
formation and in so doing established the chemical nature
of genes. Soon thereafter the composition of DNA was
further elucidated by the observations of Vischer gE_gA.
(48) that adenine and thymine and guanine and cytosine
showed equal molar proportions (i.e. adenine and thymine
concentrations were always equal as were guanine and
cytosine concentrations). The present model for DNA
structure, commonly called the Watson-Crick structure

(49), can be summarized as follows:

l. The amount of adenine equals the amount of
thymine and the amount of guanine equals the
amount of cytosine.

2. Adenine pairs only with thymine and guanine
pairs only with cytosine.

3. The DNA molecule consists of two right-handed
helical polynucleotide chains of opposite
polarity (i.e. internucleotide linkage in one
strand is 3' to 5' and 5' to 3' in the other).

4. Bases are on the inside of the chain and a
purine on one strand always pairs with a
pyrimidine on the other.

5. The distance between base pairs is 3.4 A and

there are ten bases per turn of the helix.

Methods of Studying DNA

 

Since the Watson-Crick model, many techniques have
been developed to study and Characterize the DNAs of
various organisms. Native DNA molecules undergo a process
called denaturation in the presence of a number of agents,
Viz increased temperature, addition of hydroxyl or hydrogen
ions and a large variety of organic reagents. Denatura-
tion involves the transition of native double-stranded
DNA to single—stranded DNA through hydrogen bond breakage
between purine-pyrimidine pairs. A considerable amount

of information about the DNA of an organism can be

 

 

 

obtained from a DNA melting curve (thermal denaturation
curve). Typically this technique involves the heating of
a solution of DNA (at neutral pH) from room temperature to
some higher temperature (usually about 100C) until no
further strand separation is observed. Melting curves
are carried out in an ultraviolet spectrophotometer where
the change in absorbance at 260nm is used to monitor the
strand separation. As strand separation proceeds, the
absorbance at 260mm increases due to the "unstacking" of
the bases in the molecule. Melting curves are sigmoidal
in shape and are characterized by the position of their
midpoints (Tm, the temperature at which half the DNA is
denatured) and the steepness of the slope at this point.
Changes in ionic strength of the solvent or addition of
various denaturing agents will shift the position of the
TM for any DNA. Marmur and Doty (29) have shown that

there is a linear relationship between the base composi—

tion (G+C%) and T Thus the G+C content of a DNA can

M‘
be determined by obtaining its TM in a solvent of known
ionic strength.

Another parameter obtainable from a DNA melting
curve is the degree of hyperchromicity exhibited by the
DNA sample. The hyperchromic shift as it is called is
represented by the percentage of absorbancy (at 260nm)

increase seen following denaturation. This parameter is

also dependent on the G+C content of the native DNA,

with DNAs of higher G+C contents having lower hyperchromi—
cities (e.g. a DNA of 63% G+C has a hyperchromicity of
29% while a DNA of 51% G+C has a hyperchromicity of 39%).

DNA melting curves may also be analyzed by the
method of Knittel (22). This technique involves plotting
thermal melting data on normal probability paper.

Gaussian distributions when plotted in this way give a
straight line. Melting data yields a straight line and
allows for accurate determination of Tm and a rapid determ—
ination of the standard deviation of the compositional
distribution of the DNA. Such plots may also be used to
reveal compositional heterogeneity if a broken line is
obtained.

Russel gE_gA. (40) have described another technique
which allows for the calculation of the base composition
of nucleotide sequences which melt in different portions
of the DNA melting curve. Briefly the method consists
of obtaining ultraviolet wavelength scans from 245 to
280nm at a number of temperatures along the melting curve.
A formula based on the molar extinction coefficients of
AT and GC pairs at various wavelengths, is used to
determine the base composition of DNA melting during
any temperature increment along the melting curve.

Among the most recent techniques employed in DNA
research is the study of the kinetics of the reassocia-

tion of DNA. This technique was first employed by

10

Britten (9) and others in the middle 1960's to show the
presence of repeated nucleotide sequences in the DNAs
of many higher organisms. DNA, when denatured to its
single-stranded state and incubated at fairly high salt
concentrations, will reassociate (return to its double-
stranded form). The process involves the random colli-
sion of single—stranded fragments until a complementary
sequence of base pairs is encountered and bonding between
the base pairs results in double—stranded DNA. DNA
renaturation (reassociation) follows second order kinetics
(see Appendix B). Marmur gE_gA. (30) originally explored
the conditions necessary for efficient reassociation and
others (9, 50, 51) have further refined the techniques.
The requirements for reassociation are:
1. An adequate concentration of cations (renatur-
ation is blocked below 0.01 M sodium ion),
2. An incubation temperature high enough to
weaken intrastrand secondary structure
(optimal renaturation temperature is about
25C below the temperature for dissociation
of DNA) ,
3. An incubation time and DNA concentration suf-
ficient to allow an adequate number of col—

lisions to take place,

 

11

4. A homogeneous population of DNA fragments
about 500 nucleotides in length (the larger
the fragment the faster the rate).

A DNA renaturation experiment would thus involve,

DNA purification, shearing to small fragment size (about
500 nucleotides), dialysis against a suitable buffer,
denaturation of DNA and the monitoring of the reassocia—
tion of the strands. The reassociation of DNA can be
assayed in a number of ways, each dependent on some
detectable difference between double (reassociated) and
single (unreassociated) stranded DNA. For example, one
of the most commonly used techniques is ultraviolet
spectrophotometry (30). This method is based on the fact
that dissociated (denatured) DNA absorbs more ultraviolet
light than reassociated DNA (at 260nm). Thus renaturation
reactions are carried out in water jacketed thermal
cuvettes in a spectrophotometer and the absorbance at
260nm is monitored. This technique has the advantage of
requiring little DNA (about 80 ug).

Another commonly used technique takes advantage
of the fact that single and double stranded DNAs can be
separated on an hydroxylapatite (calcium phosphate)
column (9). Thus renaturation is followed by removing
samples from a DNA solution which is renaturing and
fractionating the double and single stranded DNA on

hydroxylapatite. This procedure provides an estimate

12

of how much DNA has renatured. This technique has the
advantage that the DNA concentration can be increased
greatly over the spectrophotometric method so that slow
renaturations can be Speeded up. It also allows for
fractionation of DNA species of different renaturation
rates.

A third technique which may be used is the DNA—
agar method (31). Long strands of DNA are physically
immobilized on a support substance and radioactivity
labeled fragments of single stranded DNA are added.
Following various incubation times the unbound labeled
fragments are washed away and the bound radioactivity
is assayed by standard techniques.

DNA renaturation kinetics can be characterized
as follows:

1. The renaturation reaction follows second

order kinetics,

2. The maximum rate of renaturation occurs 25C

below the dissociation temperature of the
'DNA,

3. Decreasing the molecular weight of the

fragments, decreases the rate of the reaction,

4. Renaturation rate decreases as the genetic

complexity of the source increases (i.e. as

the unique sequence size increases),

 

l3

5. The rate is very dependent on ionic strength

below 0.4 M sodium ion.

The unique sequence length of the DNA of an
organism can be obtained from the results of a renatura—
tion experiment. The % renaturation is plotted on semi—
logarithmic paper versus Cot (the initial DNA concentra-
tion in moles of nucleotides/liter times time).* The Cot
value at which 50% reassociation has taken place is termed

the Cot and is characteristic of each organism. This

1/2
value is indicative of the minimal length of the unique
sequence of the organism's DNA or in the case of most
bacteria the genome size. A. ggii DNA is used as a
standard Since its genome size has been measured quite
accurately by Cairns (13). The shape of a renaturation
curve is important in determining whether an organism
contains repeated base sequences in its DNA. NOn—repeated
DNA, characteristic of most procaryotes, gives the typi-
cal second order S-shaped plot. Encaryotic DNAs have been
shown to deviate from second order kinetics. This is
easily visualized in most eucaryotes where the curve is
biphasic. For example, calf DNA shows a biphasic renatur-

ation curve (10). Forty percent of the calf DNA has

already reassociated at a Cot value of 2. Little

 

*

As a general rule, dissociated DNA of OD260 = 2
renatured for 1 hour in 1 SSC at the optimal re—
naturation temperature gives a Cot of l.

l4

renaturation takes place between values of 2 and 200.
The remaining 60% has a Cotl/Z of about 3000. The rapidly
reassociating fraction of calf DNA has a Cotl/Z of 0.03
as compared to 3000 for the slow fraction. Thus the
concentration of DNA sequences which reassociate rapidly
is 100,000 times the concentration of those which
renature slowly. Therefore 40% of the calf genome con-
sists of unique sequences repeated about 100,000 times.
To date all procaryotic DNAs studied have shown no
redundant DNA, while all eucaryotic DNAs have contained
repeated DNA sequences.

The function of repeated sequences in DNA is
unknown. A number of possible roles have been suggested.
Multiple copies of a gene would allow for higher rates
of synthesis. This might be necessary for the synthesis
of structural proteins found in large amounts in the cell,
or for ribosomal RNA. Multiple similar copies could pro-
duce a class of proteins which were similar such as
antibody proteins. Britten (8) has also suggested the
possibility of highly repetitive DNA sequences having a
regulatory role. Finally, Crick (10) has proposed that
repeated DNA sequences play a structural role by inter-
acting with the chromosomal proteins in eucaryotes.

Initial rates of a renaturation reaction can be

used to obtain unique sequence sizes on non-repeated DNA.

 

15

Gillis (18) and Brenner (6) have determined genome sizes
for a number of bacteria using this method.

Density gradient centrifugation is of value in
studying DNA (42), since the buoyant density of DNA in
CsCl is directly proportional to its G+C content. This
technique is used to determine the G+C contents of an
unknown DNA and to effect the separation of DNAs of vary-
ing G+C contents. A modification of the CsCl buoyant
density centrifugations is the density gradient relaxa—
tion method as described by Anet (1). This technique
takes advantage of the facts that the "steepness" of the
gradient formed is dependent on the Speed of centrifuga-
tion and that CsCl diffuses rapidly relative to the dif-
fusion of DNA. Operationally, centrifugation is first
performed at a high speed to form the gradient and
separate and concentrate the 2 DNA species. The rota-
tional velocity is then brought to the lower value to
form the desired density gradient. The final gradient
is quickly formed following change over to the slower
speed. DNA molecules in such a gradient seek their new
densities after relaxation (slow speed) and thus the time
required for a density gradient run is shortened. Anet
(2) has also described the use of sodium iodide gradients
with and without ethidium bromide to separate DNA,

mixtures. Ethidium bromide intercalates in DNA and

l6

lessens its buoyant density. Anet was thus able to
separate more efficiently mixtures of DNAs of different
G+C contents, since high G+C DNAs take up less ethidium

bromide than lower G+C DNAs.

EXPERIMENTAL METHODS

Strains and Cultivation

 

A. vinelandii (ATCC 12837) was grown from a single
isolated cyst from a laboratory stock culture. Cells were
grown on Burk's nitrogen—free medium plus 0.5% glucose at
30C on a rotary shaker with 100-200 milliliters of culture
medium per 500 milliliter flask. Larger batches were
grown in a 3 liter fermenter (New Brunswick Scientific
Co., New Brunswick, New Jersey) or in a 100 liter pilot—
plant fermenter (Stainless and Steel Products Co., St.
Paul, Minn.). Cells were harvested in a continuous—flow
centrifuge (The Sharples Corp., Philadelphia, Pa.).

Burk‘s nitrogen-free medium as described by Wilson

and Knight (52) is made up of the following ingredients:

KH21304 0.2 gm/l
KZHPO4 0.8 "
MgSO4°7H20 0.2 "
CaSO4'2H20 0.1 "
FeSO4'7H20 5.0 mg/l
Na2M004'2H20 0.25 "

Glucose was dissolved in distilled water and
sterilized by filtration.

A. coli K12 and Pseudonomas aeruginosa were grown

 

on nutrient broth in shake flasks at 30C.

17

18

Determination of DNA Content

per Cell

Three methods were employed in determining the

 

DNA content of A. vinelandii cells. These were grown in
Burk's medium to late log phase and cell numbers Were
assessed by direct counts in a Petroff—Hauser counting
chamber and by dilution plating. In the first method, the
culture was chilled, cells harvested and washed by centri-
fugation in 0.1M tris- 0.1M NaCl buffer (pH 9) and resus-
pended in the same buffer. Cold 10% sodium dodecyl sulfate
(SDS) was added to give 1% SDS and the suspension was
stirred for 20 minutes in an ice bath. An equal volume of
cold water-saturated phenol was added and the mixture
stirred for another 20 minutes, after which the aqueous
and phenol layers were separated by centrifugation. The
aqueous layer was precipitated with cold ethanol, the
precipitate resuspended in 0.1M NaCl and DNA assayed by
the diphenylamine assay of Burton (12).

A second method involved extraction of a known
number of cells with 10% trichloroacetic acid (TCA) at
95C for 15 minutes and DNA levels determined by the
Burton method.

The third method estimated DNA per cell using a
modification of the Schmidt-Thannhauser nucleic acid
fractionation (42). Cells were harvested as before and

washed twice with Burk's buffer (Burk's medium without

19

glucose). The cells were resuspended in 7% cold TCA

(30 ml) and were stirred for 20 minutes in the cold. The
mixture was centrifuged and the pellet reextracted with
30 ml 10% TCA. The pellet obtained from centrifugation
was suspended in 5 ml of water and 20 ml of 95% ethanol
and centrifuged. One extraction with 25 ml of 100%
ethanol, and three extractions with ethanolzether (321)
were carried out at room temperature with stirring. The
precipitate was then collected by centrifugation and
hydrolized for 15 hours at 37C in 1N KOH. Following
hydrolysis the solution was neutralized with HCl and the
DNA precipitated with 1 volume of 5% TCA and the precipi-
tate washed twice with 5 ml 5% TCA by centrifugation.

The final pellet was extracted twice with 5% TCA at 90C
for 15 minutes, cooled and centrifuged. Supernatants
contained the DNA and were assayed by the diphenylamine
method of Burton, an inorganic phosphorus assay and the

indole assay.

Nuclear Staining
Nuclear staining of A. vinelandii cells was per-
formed by a modification of the procedures of Piekarski
(38). Cells were air dried on clean slides and fixed
in 70% ethanol for 1 hour. Hydrolysis of cellular RNA
was then carried out by incubation in IN HCl for 30

minutes at 60C. After washing with water the cells were

20

stained with Giemsa stain for 5-10 minutes. Concentrated
Giemsa stain is prepared by mixing 0.5 grams Giemsa with
33 ml glycerol and 33 ml methanol and heating for 2 hours
at 60C. Diluted Giemsa is used for staining and consists
of one drop of concentrated Giemsa stain per 1 ml 0.1 M

phosphate buffer pH 6.8. With this technique cell cyto—

plasm appears sky blue and nuclei dark purple.

-Preparation of DNA

 

Cells were harvested in late log phase, washed
and frozen. A modification of the Marmur technique (28)
for DNA purification was used with two additional RNAse
incubations (100 ug/ml RNase for 1 hour at 37C) and a
pronase treatment using 100 ug/ml of the enzyme. The
pronase was self-digested for two hours at 37C to rid it
of DNAse prior to treating the DNA overnight at 37C.

DNA purity was monitored by observation of ultra—
violet spectra where the 260/280 nm ratio was required to
be close to 2. DNA was assayed by a number of methods
including Burton's diphenylamine assay (12), Ceriotti's
indole assay (14) and a modification of the Schmitt-
Thannhauser inorganic phosphorus assay of Schneider (44).
RNA was assayed by the orcinol method for pentose (11)
and protein was assayed by the method of Lowry (27). DNA
was considered pure when RNA and protein levels were
less than 1% of the total preparation. DNA preparations

were stored at ~20C.

21

DNA Melting Curves

 

All DNA melts were carried out in 0.1 x SSC*
buffer. DNA solutions with an absorbance of approximately
0.5 (at 260 nm) were placed in jacketed cuvettes (Arthur
Thomas Co., Philadelphia, Pa.) of 1 cm light path for
temperature control during observation. The cuvettes were
placed in an Hitachi DB spectrophotometer and the temper-
ature controlled by an insulated circulating water bath
(Sargeant Co., Detroit, Mich.). The temperature of the
solution in the cuvette was monitored using a YSI therm-
ister thermometer with the probe cemented into the
cuvette plug (Yellow Springs Industries, Yellow Springs,
Ohio). The absorbance at 260 nm was read and the temper-
ature raised quickly to about 10C below the onset of the
melting region. The temperature was then raised in
approximately lC increments, allowing 10 minutes for
equilibration at each temperature. The optical density
at 260 nm was read at each temperature and corrected for
thermal expansion (Vt/V25C)' Data were plotted on both
rectangular coordinate paper and on normal probability
paper by the method of Knittel (22). Scan melts used in
the method of Russel (40) were carried out as above and
at each temperature values for optical density at 245 to

280 nm in 5 nm increments were obtained.

 

*ssc buffer is 0.15M nac1 plus 0.015M sodium
citrate pH 7. -

22

Preparation of DNA for
Renaturation

 

Shearing of DNA was accomplished by two passages
through a French pressure cell (American Instrument Co.,
Silver Springs, Md.) at 15,000 lbs/in2. To minimize DNA
denaturation during the shearing process, the DNA was
dissolved in 5 x SSC buffer and the pressure cell was
cooled in ice before use. Following shearing DNA samples

were dialyzed to equilibrium against 0.1 x SSC buffer.

Renaturation Techniques

 

The reassociation of DNA was assayed spectro-
photometrically. sheared DNA in 0.1 x SSC buffer was
denatured by heating at 100C for 5 minutes. The DNA
was immediately diluted with an amount of 10 x SSC buffer
which yielded a final concentration of 2 x SSC buffer and
an optical density of 1.5 - 2.0 at 260 nm. The DNA was
then transferred to a water—jacketed cuvette preheated to
the desired renaturation temperature in the spectrophotom—
eter. The total time necessary for mixing and transfer
of the DNA to cuvettes was about 2 minutes. Optical
densities at 260 nm were read every 5 minutes for the
first hour (for initial renaturation rates) and every

half—hour thereafter.

23

Hydroxylapatite Fractionation of Double
and Single Stranded DNA

 

Sheared DNA in 0.14 M sodium phosphate buffer,
pH 7 was renatured in a sealed vial. The sample was
placed on a thermal jacketed hydroxylapatite column
(Biorad Laboratories, Richomnd, Calif.) maintained at the
temperature of renaturation and equilibrated with 0.14 M
sodium phosphate buffer, pH 7. The sample was washed
through the column with 2 ml portions of the same buffer
to stOp the renaturation and fractionate double and single
stranded DNA. The absorbances of the eluates was moni-
tored at a wavelength of 260 nm. When the absorbance of
column eluates had returned to a basal level, the column
was washed with 0.4 M sodium phosphate buffer pH 7 in 2 ml
aliquots and monitored as above. A fractionation of double
(renatured) and single (unrenatured) stranded DNA was
obtained by this method. Single stranded DNA was eluted
in the 0.14 M phosphate buffer and double stranded DNA

was eluted from the column with 0.4 M phosphate buffer.

Density Gradient Centrifugation

 

Cesium chloride (CsCl) density gradient centrifu-
gation was carried out by the method of Ganesan and
Lederberg (17). Optical grade CsCl was used (Schwartz-
Mann Bioresearch, Orangeburg, N.Y.) and the average
density in the tubes was 1.72 grams/m1. DNA in SSC

buffer was added in amounts from about 75 to 200 ug. A

 

24

total of 4.5 ml of CsCl was pipetted into 5 ml capacity
nitrocellulose centrifuge tubes and layered with mineral
oil. Tubes were spun in an SW39 head in the Beckman

Model L refrigerated ultracentrifuge at 30,000 rpm for

80 hours at 15C. Tubes were pierced, drops collected,
diluted with distilled water, and the absorbance at 260 nm
monitored.

An alternate method involved centrifugation at
41,000 rpm for 24 hours at 15C in the SW 50L head.
Fractionation was performed as above.

Both of these methods were tried using the density
gradient relaxation method of Anet (l) in an attempt to
effect better separation of the DNA components.

Sodium iodide gradients with ethidium bromide
(2) were also used in combination with the density gradient
relaxation technique (1). In this case DNA samples were
spun in a type 50 angle head at 50,000 rpm for 20 hours
followed by a centrifugation at 35,000 rpm for 50 hours
at 15C. Average density of the gradient was 1.55 grams/ml

and a total of 5.6 mls per polyalymer tube was used.

Analysis of G+C Contents

 

G+C contents were analyzed spectrophotometrically
by the method of Hirshman (20). In this method DNA is
heated above the temperature at which it is completely

denatured and the absorbance values at 240 to 280 nm in

25

10 nm increments is determined. From these data one can

calculate the G+C content of the denatured DNA.

RESULTS

DNA Content per A. vinelandii Cell

 

DNA content per cell was determined by three
methods: a phenol DNA extraction, a hot TCA extraction
and a modification of the Schmitt-Thannhauser fractionation

with the DNA supernate assayed by three different DNA

methods. The phenol extraction gave a value of 6.7 x 10-14

grams per cell (corrected for a yield of about 70%). Hot

14

TCA extraction gave 6 x 10_ 'grams/cell. The third method

14

gave results of 6.6 x 10_ grams (Burton diphenylamine

l4

assay), 6.7 x 10- grams (inorganic phOSphate assay) and

6.3 x 10_14 grams (indole assay) per cell (Table 1).

Number of Stainable Nuclear
Bodies per Cell

 

 

The number of nuclear bodies per cell were
determined from the same cultures used for DNA assay by
the nuclear staining technique described. Cells in early
exponential phase were found to contain as many as four
nuclear bodies per cell while late exponential cells had
two nuclear bodies. Cysts always contained one nuclear
body. The amount of DNA in the cyst is presumably the
minimal amount of DNA necessary for maintenance of the

cell.

26

27

TABLE l.-—DNA content per cell of A. vinelandii.

 

 

DNA Extraction Method DNA Assay Method DNA/Cell

l. Phenol Diphenylamine 6.7 10-14*

2. Hot TCA Diphenylamine 6.0 10-14

3. Schmitt—Thannhauser Diphenylamine 6.6 10—14
Schmitt—Thannhauser Inorganic phosphorus 6.7 10”14
Schmitt—Thannhauser Indole 6.3 10—14

 

*
Corrected for estimated yield of 70%.

28

DNA per Nuclear Body

 

From the amount of DNA per cell and the number of
nuclear bodies per cell, the amount of DNA per nuclear
body was calculated to be about 3.3 x 10_14 grams. This

represents a value about 8 times that of the A. coli

genome.

DNA Melting Curves

 

DNA melting curves were obtained as described.
The results are shown in Figures 1 and 2. All curves
appear as typical S-shaped curves when plotted on
rectangular coordinates except for the mixed melt of
A. SEA; (G+C = 51%) and Ag. aeruginosa (G+C = 68%) in
Figure l. The hyperchromic shift of A. agilis (Figure 2)
is low probably due to low purity.

Figures 3 and 4 are sama data plotted on normal
probability paper by the method of Knittel. Figure 3
shows the melting curve of an approximately equal mixture
of the DNA of A. ggAA and Ag. aeruginosa. The normal
probability plots in Figures 3 and 4 are straight lines
only in the case of A. ggAA, A.agilis, and Ag. aeruginosa.
The mixed melt as expected is biphasic as is the A.
vinelandii plot (Figure 4). While the mixed melt has a
plateau region where no DNA is melting the A. vinelandii
plot shows a break at about 54% of the total absorbance

increase.

29

Figure l.-—DNA melting curve for A. coli, 5. aeruginosa
and a mixture of the two DNAs. _E_:. coli DNA (m) ,
Ps. aeruginosa DNA (I—.) and an approximate equal
mixture of the two DNAs (fl). See'text for ex-
perimental details.

 

 

30

 

 

|.5

L4!-

_ - _
3. 2. l.

EcomN ....4 woz<mmomm< w>_._.<.._mm

LO

 

80

TEMPERATURE (°c).

 

75

31

Figure 2.--DNA melting curve for A. agilis and A. vinelandi

 

DNA A. a ilis DNA (...-7 and A. vinelEndiTL DNA
(OI—u. . See text for experimental details.

 

32

 

fi

l
l
80

TEMPERATURE (° C)

 

l
_L
75

 

70

E . L _
4 3 2 I.

a: com 2 moz<mmomm|< mated".

 

 

l.5
I.O

 

33

Figure 3.--Normal_probability plot of DNA melting data for
E. coli, Ag. Aeruginosa and a mixture of the two
DNAS. A. colTDNA (H), Ps. aeruginosa DNA
(H) and a mixture of the—Ewo DNAs H).
See text for details.

 

 

34

 

 

—_d4—__

I
84 86 88

l
78 80 82

l
76
TEMPERATURE cm

I
72

 

 

0.0l

— _—-—
0 00000
I. 23456

wmdmmoé

70"

_ b
O O
8 9

moz<mm0mm< ...zw omwn.

.
9
9

99.99

 

35

Figure 4.-—Normal.probabi1ity plot of DNA melting data for
A. a 1115 and A. vinelandii DNA. A. agilis (Inn—u.)
and A. Vinelandii (~5. See text for experi-
mental details.

 

INCREASE

PERCENT ABSORBANCE

0.0|

5

20

3O
4O
50
60
7'0

80
9O

99

99.99

36

 

 

I

l

l

 

 

7O 72

74

76 78 so 82
TEMPERATURE (°C)

84

86 88

37

An estimate was made of the approximate differ—
ence in the G+C contents of the two DNA components of the
A. vinelandii melting curve by setting up a model for a
two component mixed DNA melt (see Appendix A). Two
straight lines were drawn on probability paper with slopes
approximately equal to the slopes of the two components in
the A. vinelandii melt. These represented melting curves
of homogeneous DNAs with different G+C contents. By vary—
ing the distance between the lines (increasing or decreas—
ing G+C differences) and graphically adding them, the
following was obtained: with two lines far apart (i.e.
large difference in G+C contents) there was obtained a
curve like the mixed melt with Ag. aeruginosa and A. ggAA.
As the hypothetical lines were moved closer together the
plateau diminished and finally disappeared giving a
biphasic curve resembling the A. vinelandii melting curve.
Finally by moving the curves even closer the line became
indistinguishable from a straight line. By this method
of analysis the G+C contents of the two components of the
A. vinelandii melting curve appears to differ by 7 to 11%
G+C . Since the hyperchromicity of the low G+C component
is greater than the high G+C component, the 54-46% split
seen on the melting curve is really more closely repre-
sented by two components in about equal amounts.

By using scan—melts on the DNA of A. vinelandii

and A. coli calculations were possible to determine the

38

% G+C melting between various temperature increments on
the melting curves. Calculations for A. coli extrapolated
to a % G+C of about 51%, a value consistent with the true

G+C percent of the organism. The Azotobacter calculations

 

however gave anomalous results in that only two tempera-
ture increments gave positive values which could be plotted
while three increments gave values which were negative.

A line drawn through the two positive increment points
extrapolated to about 69% G+C, as compared to 65% G+C for

A. vinelandii.

 

DNA Renaturation Experiments

 

DNA from A. coli and A. vinelandii was renatured.

 

A. ggAA_was used as a standard since the genome size of
the organism is well documented (13) and renaturation
kinetic data is also available (10). Figure 5 shows the
renaturation values of A. ggAA superimposed on an ideal
second order reaction plot with the same Cotl/Z value.

The two curves show good agreement. The Cotl/Z value for
A. ggAA AAA from this curve is about 2.9 in 2 x SSC buffer
at the Optimal renaturation temperature in this buffer
(72C). Using the correction factor for salt concentra-
tion given by Britten (7), a value of about 8 was

obtained for the Cot of A. coli in 1 x SSC buffer.

1/2
This value agrees well with those already reported (10,

50).

 

39

Figure 5.--Kinetics of reassociation of A. coli DNA. A.
coli data points (0 0 O) . and ideal second order
kinetic curve (_) . See text for experimental
details.

40

 

 

l l l l

O O O O
N <1" to a)

N0 IlVHfllVNBH .LNBOHBd

 

IOO

IO

LO

0]

0.0l

Co‘l’

41

Figure 6 shows the renaturation curve of A.

vinelandii compared to an ideal second order reaction

 

with the same Cotl/Z value. These curves do not super-

impose well. The A. vinelandii DNA appears to renature

 

faster in the early part of the curve and is well below
the ideal curve, while it seems to renature slightly
slower than ideal in the last part of the curve. The A.

vinelandii renaturation curve was analyzed graphically

 

to obtain the best fit for the experimental curve from the
sum of two ideal second order curves. 'In this analysis

it is assumed that the two ideal second order components
contributed equally to the reaction. This is substantiated
by the melting curve results which showed two components

in about equal concentrations. The two best fitting curves
thus obtained have Cot1/2 values of 1.1 and 6.8 respec—
tively in 2 x SSC. Figure 7 shows these two curves and

their sum compared to the total A. vinelandii renaturation

 

curve. The unique sequence sizes of the two components of
the renaturation curve after correction for G+C effects
(45), gives values of about 1.2 x 109 daltons (0.4 times
the size of the A. ggAA genome) and 7.9 x 109 daltons
(3.1 times the size of the A. ggAA_genome).

Initial renaturation rates were calculated for

A. coli and A. vinelandii DNA as described by Gillis and

 

 

DeLey (18). The genome size of A. vinelandii was deter—
9

 

mined to be 1.5 x 10 daltons using A. coli as a

42

Figure 6.--Kinetics of reassociation of A. vinelandii DNA.
A. vinelandii (0 0 0) and ideal second order
kinetic curve (_). See text for experimental
details.

 

 

 

 

 

 

I l l 1

IO

to

O]

 

 

O O O O O
N V (D (I)

NOIlVHfllVNBH .LNSOUBd

t

44

Figure 7.--A comparison of the reassociation kinetics of
A. vinelandii with the sum of two ideal second
order reaction kinetic curves. Ideal second Order
kinetics curve of C t% value = 1.1 (_)‘, ideal
second order kinetig curve of C t. value = 6.8
(m) , the graphic sum of thg two ideal second
order curves (nu-n.) and data points for the re-
naturation of A. vinelandii (0 Q Q) . See text
for experimental details.

 

45

s .0
oo. o. o._ _.o 5.0

 

OO

0 o o
<r co co _

NOILVHOLVNSH 1N3383d

O
N

 

 

 

 

46

standard. The genome size from the Cot1/2 value of the
renaturation curve of A. vinelandii (Figure 6) is 2.5 x 109
daltons. This discrepancy is attributable to the fact that
the A. vinelandii renaturation curve is really the sum of
two second order curves and thus the initial part of a
renaturation would reflect the rapid renaturation of the
Cotl/2 = 1.1 component. Thus calculations of the unique
sequence length based on the value obtained from the initial
rate would be low. If a calculation is made of the theo-
retical genome size for the two component system from the
weighted average of the initial renaturation rates of the

9 daltons,

two components one obtains a value of 1.4 x 10
which agrees well with the experimental value.
An additional renaturation experiment for the
purpose of determining the respective Cot1/2 values of the
two components and their G+C contents was carried out.
A; vinelandii DNA in 0.14 M sodium phosphate buffer, pH
7.0 was allowed to renature almost completely at 75C and
the double and single stranded DNA was fractionated on
hydroxylapatite columns. DNA assays on the two fractions
indicated that the DNA had been 88% renatured. From the
A. vinelandii renaturation curve and its components
(Figure 7) it was then calculated that the single stranded
DNA fraction was composed of a fast component (Cotl/Z =
1.1) to the extent of 15% of the total and a slow com—

ponent (Cot = 6.8) of 85%. The single stranded fraction

1/2

47

was then allowed to renature as before in 2 x SSC buffer
at 80C. Figure 8 shows this renaturation curve and the
theoretical renaturation curve for a 15%:85% mixture of
DNAs with Cotl/Z values of 1.1 and 6.8 respectively. The
theoretical curve coincides well with the experimental
values up to about 20% renaturation when the experimental
renaturation begins to level off. Thus the values of 1.1
and 6.8 for the Cotl/Z values of the two component system
appear to be consistent with the renaturation of the single
stranded DNA fraction.

G+C content analysis was done on the single
stranded fractions from the hydroxylapatite separation
in order to calculate the G+C contents of the two com-

ponents of the A. vinelandii renaturation. Fraction 1

 

(single stranded, non-renatured) was estimated to contain
62% G+C by the method of Hirshman. Solving the simul-
taneous equations shown below yielded the values of 60.7

and 69.3 for the respective G+C contents of the slow

 

(Cotl/2 = 6.8) and fast component (Cotl/Z = 1.1), a dif-
ference of about 8.6% G+C.

Let S = G+C% of the slow component

F = " " " fast "
GCl = " " fraction 1 from hydroxylapatite = 62%

Average G+C content of A. vinelandii = 65%

GCl = 0.15F + 0.858

S + F

= 65%

 

2

48

Figure 8.——Kinetics of reassociation of fractionated A.
vinelandii DNA enriched for the slowly renaturing
component. Theoretical renaturation curve. (—)
and renaturation data for enriched DNA (0 o 0) .
See text for experimental details.

 

49

 

 

 

IO

l.O
C° t

 

 

0 0 0 0
2 4. 6 3

ZO_._.<mD._.<zmm kzmomwn.

IOO

O.l

50

A previous renaturation of A. vinelandii and A.
ggAA DNA done in Dr. Britten's Laboratory (Carnegie Insti—
tute of Washington, D.C.) prior to the experiments just
described was analyzed by a computer program at the
Carnegie Institute. The renaturation proceeded to about
50% and the computer analyzed curve consisted of two com-
ponents with Cotl/Z values of 2.6 and 17.3. The condi-
tions under which this experiment was carried out were
somewhat different from those conducted at Michigan State
University. The DNA was sheared under 50,000 lbs/inz,
the solvent used was 0.14 M sodium phosphate buffer + 8 M
urea pH 7 (MUP) as opposed to 2 x SSC buffer, and the
temperature of incubation was 62C. The computer printout
from this experiment indicated two components: the first
consisted of 45% of the DNA and had a Cotl/2 of 2.6,while
the other component (55%) had a Cotl/2 of 17.3. This
analysis coupled with the melting curves described in
Figure 4 showing a break in the curve at about 54%,
indicated that the higher G+C component is the component
of C t = 2.6. A renaturation done with A. ggAA DNA

0 1/2
indicated a Cotl/Z of about 6. Thus these values are
comparable to those determined in 2 x SSC buffer since
the 2 components are 0.4 and 2.9 times the size of the

A. coli genome.

51

Density Gradient Centrifugation

 

The physical separation of the two components of
A. vinelandii DNA was attempted using CsCl density gradient
centrifugation. It was anticipated that these two com—
ponents could be reasonably purified to allow renatura-
tions to be done separately on each component. Figure 9
shows the results of this trial. Highly sheared A.
vinelandii gives one peak while "unsheared" DNA is just
barely resolvable into two peaks (these peaks are prob-
ably real since they have been observed in repeating the
experiment). The difference in density between the two
peaks is 0.0084 grams/cm3 which corresponds to an 8.4%
G+C content difference.

The two components of A. vinelandii could not be
separated more efficiently in other attempts and this is
believed attributable to the shearing implicit in puri—
fication of DNA. Shearing has the effect of increasing
the overlap of the two DNA peaks due to increased diffu—
sion rates of sheared fragments in the gradient and the
increased compositional heterogeneity of the fragments.
The final result is one intermediate peak. The areas
under the two initial peaks remains the same but the
width of the peaks increases with shearing. The validity
of such a model was tested by attempting to separate
mixtures of sheared and unsheared A. ggAA (51% G+C) and

Ag. aeruginosa (68% G+C) DNA on CsCl gradients. The

 

 

52

Figure 9.-—CsCl density gradient centrifugation of sheared
and "unsheared" A. vinelandii DNA. Unsheared DNA

(on—o) and sheEred 'DNA (III—I). 'See text for ex-
perimental details.

 

Q
m
l

9
.b
T

F3
N
I

O

 

ABSORBANCE AT 260 mn

C)
I

0.8 —

0.6 —

0.4 —

0.2 -'

 

O |

l l

 

 

0 IO
bottom

20

30 4O 50 60
TUBE NUMBER

70

80

top

54

results are shown in Figure 10 and the results appear to
be in agreement with the theoretical considerations.
Other methods, including relaxation centrifuga-
tion, sodium iodide gradients and a combination of these
two methods did not result in any better separation of

the two DNAs.

 

 

55

Figure 10.--CsC1.density gradient centrifugation of a:mix-
ture of A. coli and Ag. aeruginosa DNA sheared and
"unsheared". Unsheared mixture of the two DNAs
(I——I) , sheared mixture of the two DNAs (H)
and Ps. aeruginosa unsheared marker DNA (Qt-:0) .
See {Ext for experimental details.

 

 

ABSORBANCE AT 260 nm

56

 

 

 

 

 

 

 

 

 

O l l l l
IO 20 7 80 9O

0 3O 4O 50 60
bottom TUBE NUMBER top

 

DISCUSSION

The purpose of the research described in this
thesis was to characterize the DNA of A. vinelandii. A.
vinelandii contains a large amount of DNA per cell (about

14

6.6 x 10_ grams). Nuclear staining has indicated two

nuclear bodies per cell in late log phase and thus each

14 grams of DNA.

nuclear body contains about 3.3 x 10-
Stained cells were observed in the light microscope and
thus the resolution was limited. The nuclear bodies of
A. vinelandii appear vessicular when stained but the
resolution is not sufficient to distinguish the number of
vessicles present per nucleoid. Pochon (39) first des—
cribed this vessicular nucleoid and suggested that it
consisted of four vessicles. The unit of DNA comprising
the genome of A. vinelandii is thus uncertain. However
one would expect the cyst to contain one genomic unit,
which consits of one nuclear body.

The significance of such a large amount of DNA
per nuclear body in A. vinelandii is unknown. It is
unlikely that the organism has seven times the range of
biochemical capability of A. ggAA. The DNA/protein ratios

in both bacteria are comparable. These facts suggested

57

 

58

the possibility of repeated sequences in DNA or the pres-

ence of multiple chromosomes.

Melting Curves

The DNA melting curves of A. vinelandii are atypi-
cal of bacteria in that a linear relationship between
temperature and the % absorbance increase, is not obtained,
when the data are plotted on probability paper. Melting
curves of A. vinelandii DNA are bimodal, indicating two
populations with differing G+C contents. The difference
in G+C content between the two populations is estimated
at about 8% by analysis of melting curves, CsCl density
gradient centrifugation and spectrophotometric analysis
of a mixture of the two components. A. ggAA AAA, Ag.
aeruginosa, and A. agilis were used as control DNAs and
all were found to give a straight line plot on normal
probability paper. Mixed melting curves of A. ggAA and
Ag. aeruginosa DNA show a bimodality in probability plot
melting curves. The plateau region is due to the large
difference in G+C content between the two DNAs (about
18% G+C difference). The A. ggAA DNA melts almost com—
pletely before the Pseudomonas DNA starts to melt. The
plateau region thus represents the temperature span
between the end of the A. ggAA melt and the beginning of
the Ag. aeruginosa melt. DNAs with more nearly equal

G+C contents show no plateau, but only a break in the

59

curve as seen in the A. vinelandii melting curve. The

 

break in the A. vinelandii curve comes at about 54% com—

 

pletion of DNA denaturation. However, after correcting
for the differential hyperchromicities of the two G+C
components, about equal amounts of the two components
were found to be present.

The results of the scanning melts on A. vinelandii

 

DNA were not very informative. They served only to indi-

cate that A. vinelandii DNA was atypical in its denatura—

 

tion kinetics.

DNA Renaturations

 

DNA renaturation experiments on A. vinelandii DNA

 

showed a deviation from the ideal second order kinetics
normally observed for A. ggAA_and all other procaryotes
thus far studied. By taking advantage of the fact that
ideal second order Cot curves have a lepe of 100 and
they are fully defined by their Cotl/2 values, it was pos-
sible to construct, by trial and error, the sum of two
second order curves to obtain the best fit to the experi-
mental curve. The two curves obtained in this manner had
Cotl/Z values of 1.1 and 6.8 respectively. These values
were corrected for G+C effects by the method of Seidler
(44) in which each % G+C above 51% increases the Cotl/2
value by 0.018%. The resulting Cotl/Z values were 1.3

and 8.9. Thus it would appear that A. vinelandii has

 

60

equal amounts of DNA of two unique sequences which are
0.4 and 3.1 times the size of the A. coli genome.

Initial rates for the renaturation of A. vinelandii DNA

 

confirmed these unique sequence sizes as did renaturation
of DNA which had been fractionated on hydroxylapatite
columns. .

When the separation of the two DNA components on
CsCl density gradients failed, the DNA fractionation
experiment was devised. This experiment took advantage
of the differential rates of renaturation of the two DNA
components. Thus at the time when the renaturing A.

vinelandii DNA was fractionated on hydroxylapatite, 88%

 

of the total DNA had reassociated. However this 88% of
the total was comprised of 96% of the fast component

(Cotl/Z
(Cotl/Z value = 6.8). The unrenatured DNA therefore

value = 1.1) and 81% of the slow component

consisted of 15% fast renaturing component and 85% slow
renaturing component. This procedure, in effect, yielded
a purification of the slow renaturing component from a
50-50 mixture to an 85-15 mixture. A renaturation of

this concentrated slow component was again resolvable into
the weighted average of two second order curves of

Cotl/Z value = 1.1 and 6.8. The deviation from the
hypothetical sum after about 15% renaturation (Figure 8)

was due to the nature of the renaturation experiment and

hydroxylapatite fractionation. Bacterial DNA

61

renaturations generally proceed to 90-95% reassociation.
However, the reaction had already proceeded to 88% renatur-
ation by the time the fractionation of renatured and
denatured DNA was performed. The additional 42% renatura-
tion (of the 12% left to renature) brought the total to

93% of the DNA renatured.

Another important factor which must be considered
in assessing the extent of the renaturation reaction is
the distinction between renaturation reactions monitored
by hydroxylapatite fractionation and those observed by
spectrophotometric means. The hydroxylapatite method
measures the fraction of DNA pieces that have reassociated
regions and thus bind to hydroxylapatite. The optical
method however measures the fraction of the DNA actually
base paired. Thus it is possible that in the fractiona-
tion experiment lengths of single stranded DNA which
eluted from hydroxylapatite at low ionic strength had

their complements in that DNA which bound to the column.

Density Gradient Centrifugations

 

Density gradient centrifugations gave mostly
qualitative information. Two approximately equal peaks
were observed varying in density which corresponded to
8.5% G+C in A. vinelandii. The model of two DNA peaks
becoming one when sheared was substantiated by the A.

coli — Ag. aeruginosa density gradient experiment.

62

Models for Cellular Organization
of the DNA

 

Three models (Figure 11) may be constructed to
describe the cellular organization of the DNA in A.
vinelandii. They are the following: the multichromosomal—-
redundancy model, the monochromosomal--redundancy model and
the multichromosomal-—non—redundancy model.

In the multichromosomal-~redundancy model each

7

nuclear body has a DNA content of about 3.1 x 10 nucleo—

6 nucleotide

tide pairs. Unique sequence sizes of 2 x 10
pairs and 1.4 x 107 nucleotide pairs correspond to the
Cot1/2 values of the two components. Thus if we consider
each nuclear body as two separate chromosomes we would

have two equal size chromosomes with eight fold redundancy
in the small sequence chromosome and no redundancy in the
large sequence chromosome.

The monochromosomal--redundancy model would consist
of one large chromosome (the nuclear body) having eight
copies of the small unique sequence and one copy of the
large.

The final model, multichromosomal--non—redundancy,
would involve one large chromosome of the higher unique
sequence size and eight small chromosomes.

Which, if any, of these models represents the
nuclear array in A. vinelandii is not now known. Nuclear
staining would tend to exclude the final model as no

small staining bodies were seen. However, if an artifact

 

63

Figure ll.--Models for the possible organization of DNA
in A. vinelandii. Drawn approximately to scale.
See text for experimental details.

 

64

O C}

'MULTICHROMOSOMAL-REDUNDANCY

MON OCHROM OSOMAL- REDUNDANCY

0000
0000

MULTICHROMOSOMAL ~NON ~REDUNDANCY

65

of the staining procedure caused clumping of chromosomes,
they would be difficult to resolve and they might even
appear as large vessicular structures.

The shearing of DNA in purification produces frag—
ments of about 5 x 104 nucleotide pairs in length and
mechanical shearing produces pieces of about 5 x 102
nucleotide pairs. Since both of these nucleotide lengths
are well below either unique sequence size, little informa-
tion would be obtained on the nuclear array of A. vinelandii
by sedimentation studies.

Approaches to Resolving the Validity
of the Final Model

 

 

The establishment of the validity of the final
model could be approached by autoradiography as employed
by Cairns to Visualize the A. ggAA chromosome (13). This
method would necessitate labeling of Azotobacter gigg-
landii DNA with tritiated thymidine to obtain high specific
activity DNA, followed by gentle lysis and radioautography.
Unfortunately this method has many difficulties. A.
vinelandii do not take up tritiated thymidine well and
high specific activities may not be obtainable. In addi-
tion, chromosome lengths in the models are greater than
A. ggAA and thus the probability of shearing is greatly
increased.

Another method sometimes used to visualize DNA

with the electron microscope is the Kleinschmidt

66

Method (23). Here DNA is allowed to absorb to a basic
protein film, such as cytochrome c. The complex is then
transferred to electron microscope specimen grids which
are shadowed with platinum. Obtaining molecular sizes

of DNA using this method is suitable for only rather small
DNAs such as viral DNAs. Since the A. vinelandii chromo-
somes are so much larger than viral and even bacterial
DNAs, it is unlikely that this method could be applied
successfully here. Even if the DNA could be prepared
without shearing its large length would be difficult to

contain in the confines of one hole in a Specimen grid.

SUMMARY

The DNA content per cell of A. vinelandii was

determined to be 6.7 x 10_14 grams. Cells have two stain—

able nuclear bodies and thus 3.3 x 10_14 grams per nuclear
body. DNA melting curves of A. vinelandii when plotted on
normal probability paper were biphasic indicating two

DNAs of different G+C contents. The two DNA components
have been shown to be about 61% and 69% G+C and are in
about equal amounts in the cell. DNA renaturation curves
for A. vinelandii deviate from second order kinetics and
were analyzed to be the sum of two ideal second order
reaction curves characterized by Cotl/Z values of 1.1 and
6.8. These values correspond to unique sequence sizes of

2.0 x 106 7

and 1.4 x 10 nucleotide pairs respectively.
Attempts to separate the two components by CsCl
density gradient centrifugation were not completely
successful and a model is provided to explain this result.
Three models are presented for the possible organization

of DNA within the cell.

67

LIST OF REFERENCES

68

10.

 

—7—1 .ngxhflyflJ, _ _ __._g7

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APPENDICES

74

 

 

 

APPENDIX A

A MODEL FOR THE ESTIMATION OF G+C CONTENT
DIFFERENCES OF THE TWO COMPONENTS

OF A. VINELANDII DNA

75

A MODEL FOR THE ESTIMATION OF G+C CONTENTS
DIFFERENCES OF THE TWO COEFFICIENTS

OF A. VINELANDII DNA

The following discussion is aimed at clarifying
the general method used to assign a range of values to
the G+C content differences between the two components of
the DNA of A. vinelandii. This method is based on a model
for a two component mixed DNA melting curve. The simplify-
ing assumption is made that the compositional distribution
of nucleotide pairs is the same in both components and
thus when normal probability plots are made the Slopes of
the lines representing the two components are the same.
By varying the distance between the lines (i.e. the differ-
ence in G+C content) and graphically adding them, three
types of summation curves are obtained. Figure 12 shows
the sum of two components with large G+C content differ—
ences. Figures 13 and 14 are sums of curves with decreas—
ing differences in G+C content, respectively. Figure 13
is similar to the experimental curve of A. vinelandii.
Thus by changing the difference in G+C content between
the two curves, the range of G+C content differences
which gives a biphasic curve as opposed to a linear or

plateaued curve. In this way a range of G+C differences

76

 

 

rr_——'T "

77

can be attributed to the components of the experimental

curve .

 

78

Figure 12.—-The theoretical DNA denaturation curve for a
mixture of two DNAs with a large difference in G+C
content.

 

 

 

79

 

 

 

 

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moz<mmomm< szomwm 9

mm<wmo§

TEMPERATURE(°C)

80

Figure 13.--The theoretical DNA denaturation curve for a
mixture of two DNAs with an intermediate difference
in G+C content.

 

81

 

 

_
-
u-I
-

 

 

0.0!

l
l0-

-
0
6

70'

_ _ _
0 0 9
8 9 9

30—
4o~
50-

_
0
2

wm<wmoz_ woz<mmowm< kzwommm

84 86 88

78 so 82
TEMPERATURE(°C)

76

74

82

Figure 14.--The theoretical DNA denaturation curve for a
mixture of two DNAs with a small difference in
G+C content.

83

 

 

 

 

 

_ —_____ _ A 8

q _ 8

I I6
8

I4

I 8
I l2
8

I -m
IS

I 7
I 16
7

I -m
I 12
7

_ _ ...—... _ _ m

m I 0 0000000 0 9 9
0. I 2345678 9 9 9
9

moz<mmomm< hzwommd 9

mm<wmoz_

TEMPERATURE(°C)

APPENDIX B

SOME ASPECTS OF THE THEORY OF

RENATURATION OF DNA

84

SOME ASPECTS OF THE THEORY OF

RENATURATION 0F DNA

Plotting DNA Reassociation
Data——the Cot curve:

DNA renaturation has been observed to be a second
order phenomena. The reassociation of a pair of compli—
mentary sequences is concentration dependent relying on
the diffusion of DNA fragments and the subsequent colli—
sion and pairing of the fragments.

The general equation for a second order reaction

is as follows:

dc 2
a? '— “KC
c = the concentration of DNA in unpaired strands
t = time in seconds after initiation of the
reaction
K' = the reaction rate constant for any particular
circumstances
CO = the total concentration of DNA in all single

stranded regions

Rearranging we have:

Integrating this equation from t = 0 to any future time,

t, and from c, the concentration of unpaired strands

85

 

 

86

to Co’ the total concentration of DNA, we then have:

Following integration and evaluation between these limits

we have:

This is an ideal second order reaction equation and when
plotted gives the typical S-shaped renaturation curve (the
"Cot" curve). Such a plot has the advantage of allowing
the comparison of reactions carried out at different con-
centrations and by plotting Cot on a logarithmic scale
measurements over a long time period may be presented.
The central 2/3 of an ideal second order reaction plotted
in this manner approximates a straight line with a slope
of 100.
Dependence of Renaturation
Rate on Complexity (unique
sequence size):

In DNAs without repeated sequences the complexity
unique sequence size and genome size are all equal. The
rate of renaturation of DNAs sheared to the same piece

size and renatured under comparable conditions is

87

inversely proportional to the unique sequence size.
This may be illustrated by the following example:

DNAl has a unique sequence size 1/2 that of DNAZ.
Both DNAs are sheared to the same size fragments. DNAs
are allowed to renature under the same conditions.

Since renaturation depends on the random collision
of two complimentary fragments, DNAl would renature faster
since the mate to any given fragment is diluted less by
non-mates than in the case of DNAZ. This relationship
has been shown experimentally by Britten (10) and the

data are presented below:

Nuckofide paws

II Io to2 103 I I04 I0“ 10‘ I107 Io' to” to”

 

   
   

 

   

  
  

 

 

 

I I I I I I
Calf
B , (non-repetitive-
}; TA fraction)
0
o 5. call _
3 .
m ..
3
C i I ‘ i i i I i 0'5
o _
z
8 — satellite .|-.
h
u— _
.
.
‘.f\ LIIIUJLL l I “III.“ III III I IIIIIUI IIIIIIIII I 1111111! I IIIIII I IlIIIII I IIIILIII . FIIIIII I 1111“." III '10
to" 10's 10" Io" Io‘2 0.1 I I0 100 mm mm

Cot (mole X sec/liter)

Reassociation of double stranded nucleic acids from various sources. The
genome size (25) is indicated by the arrows near the upper nomographic scale. Over
a factor of 10°, this value is proportional to the Cat required for half reaction. The
DNA was sheared (18) and the other nucleic acids are reported to have approximately
the same fragment size (about 400 nucleotides, single- stranded). Correction has been
made (19) to give the rate that would be observed at 0.18M sodium-' -Ion concentration.
No correction for temperature has been applied as it was approximately optimum in
all cases. Optical rotation was the measure of the reassociation of the calf thymus
nonrepeated fraction (far right). The MS-2 RNA points were calculated from a series
of measurements (28) of the increase in ribonuclease resistance. The curve (far left)
for polyuridylic acid + polyadenylic acid was estimated from the data of Ross and
Sturtevant (29). The remainder of the curves were measured by hypochromicity at

0 nm; a Zeiss spectrophotometer with a continuous recording attachment was

 

 

APPENDIX C

PHYSIOLOGICAL STUDIES OF ENCYSTMENT

 

IN AZOTOBACTER VINELANDII

 

BY
H. L. Sadoff
E. Berke

B. LOperfido

Reprinted from Journal of Bacteriology, 1971, 105:185-189.

88

 

 

 

 

 

 

 

 

 

.IOURNAI or BA(‘Tl~RI()I.t)(-\, .Iun. I97I.p. I85 I89

Copyright l97l American Society for Microbiology

Vol I05, No. I
I’rmtt’d in I S ,-I.

Physiological Studies of Encystment in

Azotobacter vinelandii1

H. L. SADOFF. E. BERKE.2 AND B. LOPERFIDO

Department of Microbiology and Public Health. Michigan State University. East Lansing, Michigan

Received for publication 3 August 1970

Azotobacter vinelandii. in late exponential growth phase. encysts when the glu-
cose in the medium is replaced with B—hydroxybutyrate. A final cell division then
occurs without apparent deoxyribonucleic acid (DNA) synthesis. resulting in a re-
duction from two to one nucleoids per cell and a final DNA content of 3.2 x l0 "‘4
g per cell. This is also the DNA content per cyst. A fl-hydroxybutyrate dehydroge-
nase is derepressed by the addition of the inducer and is identical to the enzyme in
acetate-grown cells in its pH optimum, Michaelis constant for substrate. tempera-
ture-activity response. and mobility during electrophoresis in acrylamide gel.

 

Cysts of Azotobacter vinelandii are resting
cells which are analogous to endospores of bacilli
in that they are considerably more resistant than
vegetative cells to deleterious physical and chem-
ical agents (I7). Cultures of this organism may
be induced to encystment by growth on agar
plates of Burk’s nitrogen-free medium supple—
mented with 0.2% n-butyl alcohol as the carbon
source (I8. 23). Alternatively. cultures may be
grown to late exponential phase in liquid medium
with glucose as the carbon source; the cells are
then induced to encystment by the replacement of
the glucose with either d-hydroxybutyrate (BH B)
or crotonate (9). This two-step process can be
carried out in relatively large culture volumes
and has been applicable to the study of the time
course of this unique cellular differentiation
process at the physiological (9) or fine structural
level (6). We have been concerned with the bio-
chemical events which occur upon the induction
of encystment and have examined the immediate
effects of the addition of BHB on cell growth.
cell division. and enzyme induction.

MATERIALS AND METHODS

Strain and cultivation. A. vinelandii ATCC l2837.
Which was used throughout these experiments. was
cultivated in Burk‘s nitrogen-free medium (22) at 30 C.
In the two-step method for cyst production (9). cultures
were grown to late exponential phase (2 X It)“ cells/ml)
with I9? glucose as the carbon source. The cells were
centrifuged from the medium aseptically, suspended in
sterile Burk‘s medium with 0.2‘} BHB us the carbon

IJournal urticlc 5|77 Irom the
Michigan State University
" Public Health Scriicc predoctoml IfLIIIICC. grunt (iM-tll‘)! I.

Agricultural [\perinicnl Station.

source. and incubated with ucrzilion at 30 C. Aqueous
solutions of both glucose and BIIB (sodium salt) were
sterilized separately by LlUIt)CILlVIng.

Preparation of cell extracts. Vegetative llnd encyst-
ing cells of A. vinelandii were suspended in 0.05 M Iris
(hydroxymethyl)uminomethune (Tris) buffer (pH 7.5)
containing It) “ M cthylenediuminetetruucetic ucid
(EDT/\). and were then disrupted in a sonic oscil-
lator (Measuring & Scientific liquipmcnt. Ltd) The
cheluting agent (EDTA) was necessary to achieve rapid
and reproducible cell rupture.

Analytical procedures. The nitrogen content of vege—
tutiie and cncysting cells was determined by the micro-
Kjelduhl analysis (19). Deoxyribonucleic acid (DNA)
was determined by the method of Burton (2). Cells
were extracted with Itl‘é trichloroacetic acid at 95 C
for I5 min. Lllld the dcoxynuclcotidcs were assayed by
using highly polymerized calf thymus DNA as a stand-
ard. Proteins were estimated spectrophotometricully
(20). Compounds containing amino sugars were assayed
by the Rondle and Morgan (l5) procedure with gluco-
samine us the standard. The I)-Ii-hydroxybutyric ucid
dchydrogenusc of encysting cells was assayed by the
procedure of .Iurtshuk ct III. (7). The rate of oxidation
of BHB was equal to the rate of reduction of nicotinti-
mide adenine dinucleotidc (NAD). measured LII 340
nm. One unit of enzyme oxidized l umolc of substrate
per min at 37 C. and specific activity was expressed in
units of protein per mg.

Photomicroscopy. lime-lapse phase-contrast photo-
microscopy of encysting A. vinelandii was carried out
with a Zeiss Photomicroscope by using slide cultures
of the organism on Burk‘s nitrogen-free tlgtll’ medium
containing 0.2% BH B. Nuclear staining of ,‘I. vinelandii
at various stages in its life cycle was performed by the
procedures of Piekarski (I-l). Direct cell counts. which
were made with a Petroff-Huuser counting chamber.
corresponded well with viable counts obtained by stund-
urd plating techniques.

Gel electrophoresis. The relative mobilities of BHB

l85

186 SADOFF. BERKE. AND LOPERFIDO

dehydrogenases in acrylamide gel electrophoresis at
pH 8.3 and 4 C were tested by the method of Davis
(4). Vertical gels of 5% Cyanogum 41 (IO by 20 by 0.3
cm) were prepared in a vertical gel electrophoresis cell
(Ii-C Apparatus Corp., Philadelphia. Pa.). Tris-glycine
buffer (pH 8.3) was employed. and a constant current
of 6 ma was maintained. Bromophenol blue. the anionic

marker. migrated to within 1 cm of the anionic end of

the gel in 1.75 hr. The BHB dehydrogenases were
stained specifically by coupling nitroblue tetrazolium
reduction to the oxidation of the substrate (16).

R ESU LTS

Growth and encystment. The two-step encyst-
ment procedure (9) was scaled-up to a 3-1iter
volume in the following manner. Cultures of A.
vinelandii were grown in lOO-ml volumes of
Burk’s medium (1% glucose) per 500-m1 Erlen-
meyer flask. When a cell concentration of2 x 103
per ml was achieved (approximately 18 hr at 30
C with shaking). 200 ml of culture was intro-
duced into 2.800 ml of the same medium at 30 C
in a 3—1iter New Brunswick fermentor. The cul~
ture was stirred at 400 rev/min and aerated at
3 liters/min. Samples of this culture were taken
at intervals for analyses of turbidity. cell count.
total nitrogen. DNA. and BHB dehydrogenase.
When the cell concentration reached 2 X 108 per
ml. the cells were harvested by centrifugation
and suspended in fresh Burk‘s nitrogen-free me—
dium containing 0.2% BH B. Stirring. aeration.
and sampling were continued through the initial
stages of encystment.

Figure 1 presents growth data for A. vinelandii
under the conditions described. After the induc-
tion of encystment by BH B. the culture appeared
to undergo one cell doubling. This was apparent
from both the turbidity and total nitrogen data
and was substantiated by time—lapse phase-con—
trast micrography (Fig. 2). A cluster of six vege-
tative cells on a slide culture was observed over
a period of 8 hr. These cells had been grown in
liquid Burk‘s nitrogen-free medium containing
glucose. They were washed once in buffer and
spread over the BHB-containing medium. The
initiation of cell division was readily apparent at
2.5 and 3.5 hr. At 8 hr, five of the cells had di-
vided. and their progeny appeared to be in the
early stages of encystment. At that time. the
sixth cell was in its final division.

Concomitant with the last cell division. an ar-
rest of nitrogen fixation occurred. The total nitro-
gen of the culture remained constant with ap-
proximately 95% in the cellular material. DNA
synthesis stopped at the time of the last cell divi—
sion. The DNA content per cell dropped from
15 x 10'” g per cell in exponentially growing
cultures to 3.4 X 10'H g per encysting cell (Fig.

J. BACTERIOL.

 

   
 

 

 

 

20 '
1.0 “ '
0.8 ’ ENCYSTING ‘
c,o.<5 — -
8
o 0.4 » -
o
0.2 ' _
0.1 - - 100
' CONTROL ‘60
' - 60
— « 40 E
ENCYSTING 2
Z
” n 20
<10
< B
-20 -IO 0 10 20 30
TIME- HOURS
FIG. 1. Growth curve ofA. vinelandii in Burk's ni-

trogen-free medium containing 1% glucose. in which
turbidity (optical density at 600 nm) or micrograms of
nitrogen per milliliter are plotted as functions of time.
Cells in late exponential growth were induced to encyst'
ment with 0.2% BHB with subsequent inhibition ofni-
trogen fixation and cell growth.

3). The latter value is precisely the DNA con-
tent per cyst of A. vinelandii. Nuclear staining
showed that cells in early exponential growth
had as many as four nucleoids. whereas late ex-
ponential-phase cells had two. Cysts contained
only one nucleoid.

An alternative procedure for initial extraction
(3) and then DNA analysis was performed to
validate the observations of DNA content per
cell. A 100-ml culture of A. vinelandii was grown
in Burk‘s medium (1% glucose) to a concentra-
tion of 1.8 x 108 cells/ml. The culture was
chilled to 4 C; the cells were harvested. washed
in 0.1 M Tris-0.1 M NaCl buffer (pH 9.0). and
resuspended in 18 ml of the same buffer. Two
milliliters of cold 10% sodium dodecyl sulfate
was added, and the suspension was stirred for
20 min in an ice bath. An equal volume (20 ml)
of water-saturated phenol was added. and the
mixture was slowly stirred for an additional 20
min. The aqueous and phenol layers were sepa»
rated by centrifugation. and the DNA in the
aqueous layer was precipitated by the addition of
three volumes of ethanol. The DNA precipitates
were collected and suspended in 0.1 M NaCl. The
total DNA recovered from 1.8 x 10‘“ cells was
840 ug. amounting to 4.7 x 10‘“ g/cell. The
amino sugar content of the DNA preparation
was used as a measure of its contamination with
cell envelope constituents (21). The DNA prepa-
ration contained an equivalent of 25 lg of glu-

 

VOL. 105. 1971

ENCYSTMENT IN A. VINELANDII 187

 

FIG. 2. Time-lapse. bright phase-contrast photomicrographs of encysting A. vinelandii. Cells were grown to late
exponential growth, and slide cultures were prepared on Burk‘s agar containing 0.2% BHB. The marker indicates 5
nm.

cosamine whereas the supernatant solution con-
tained 1860 pg of glucosamine.

The addition of BHB to late exponentially
growing cultures of A. vinelandii resulted in the
induction of a soluble B-hydroxybutyric acid
dehydrogenase which was NAD dependent. This
enzyme was not found in young cells; growing on
glucose however. within 1 hr of the addition of
BHB. a specific activity of 0.03 units/mg of pro-
tein was noted in cell extracts (Table 1). Be-
cause of the relationship of BHB metabolism to
encystment. we considered the possibility that
the dehydrogenase was a unique “encystment
enzyme” analogous to several which occur in
cells with the onset of sporulation in Bacillus
species (1, 5). A soluble BHB dehydrogenase
has been studied in cells of A. vinelandii grown
on acetate as the sole source of carbon and en-
ergy (7). We grew 3-liter cultures of the organism
in Burk’s nitrogen-free medium containing 1%
sodium acetate. harvested the cells, and prepared
extracts. The specific activity of the BHB dehy-
drogenase was 0.025 units/ mg. This enzyme was
compared to that occurring during encystment by
observing enzyme activities over the temperature
range 26 to 48 C. pH responses. Michaelis con-
stants for substrate, and relative electrophoretic
mobilities in acrylamide gels. The results of the

 

   
     

 

 

 

20 . - - . 9.2
BHB .
- 9.
j '6 CELL COUNT 0
m - O .l
8 i2 - -8.6 E
< (n
2 h e —l
O _l
a. a - 4 8.2 ‘3
z
. ' DNA/CELL 8
- 4 — 0 - 7.8 -‘
» -l
o a . . . . . 7.4
-IO 0 IO 20 so
TIME-HOURS

FIG. 3. Cell concentration and DNA content per cell
of encysting A. vinelandii as a function of time.

comparisons are presented in Fig. 4 and Table 2
and indicate that the two enzymes are very simi-
lar.

DISCUSSION

The replacement of glucose with BHB in the
two-step induction of encystment is similar in

188 SADOFF. BERKE. AND LOPERFIDO J. BACTERIOL.

TABLE 1. SpeCIfic activity of B—hydroxybutyrate
dehydrogenase upon induction ofencystment 0f
Azotobacter vinelandii

 

 

Time after addition Specific activity
of BHB (hr) (units/mg)

O O

1 0.030
l.5 0.027
2 0.023
3 0.023
4 0.021

 

many respects to a metabolic shift-down (8). At
the time of its replacement by BHB, about 50%
of the original glucose still remained in the me-
dium (9). These cells must have shifted from car-
bohydrate metabolism to lipid metabolism with the
attendant need for gluconeogenesis. Marcromolec-
ular synthesis and cell division which was in pro-
gress at the time of the induction of encystment
appeared to be completed without the initiation of
new rounds. Control cultures which were main-
tained in the glucose-containing medium continued
to divide and achieved final population levels of
1.1 :l: 10° cells/ml. Approximately l% of these
cells formed cysts.

The total nitrogen in the culture increased 1.25-
fold after induction of encystment, and the total
cell count increased 1.4-fold, a value consistent
with the 1.8-fold increase in optical density. A re-
duction in the DNA content per cell was achieved
in this process, the final level equaling the DNA
content of mature cysts. A derepression of BHB
dehydrogenase synthesis occurred, and relatively
high levels of the enzyme were present in cells
within 1 hr of induction of encystment. The spec-
ific activity of this enzyme during encystment was
equal to that in cells grown on acetate. A com-
plete cessation of nitrogen fixation took place ap-
proximately 3 hr after the addition of BH B.

The shift-down per se did not induce encyst-
ment. This metabolic state occurs after glucose
exhaustion from the medium; however, under
these conditions, relatively few cysts are produced
(9). The specificity of the inducers of encystment,
(n-butanol, crotonate, or BHB) suggest that
unique metabolic sequences may be involved in
cyst formation. In some unknown manner. the
metabolites of n-butanol, which stop normal
growth, control the formation ofcyst components.
Even though key intermediates may exist, they
must be transitory because ultimately 90% of all
exogenously added BHB is oxidized to C02 (9).

The BHB dehydrogenase which was elaborated
during encystment was identical to the enzyme
which occurred in A. vinlandii during vegetative
growth on acetate on the basis of the following

parameters. The two soluble dehydrogenases were
indistinguishable in their pH optima, Michaelis
constants, electrophoretic mobilities in acrylamide
gels, and in their activation energies. We conclude
that BHB dehydrogenase is not an encystment-
specific enzyme in the sense that protease (l) or
glucose dehydrogenase (5) is related to the
sporulation of Bacillus cereus. Rather, the en-
zyme is induced when the cells shift to the me-
tabolism of lipid materials.

The DNA content per cell of A. vinelandii var-
ied with the stages of its life cycle. Cells in mid-
exponential growth contained 15 x 10'“ g of
DNA/cell and were multinucleate. During en-
cystment a minimal value of 3.4 x 10‘" g of
DNA/cell was observed for mononucleate cells.
This latter value amounts to about 2.5% of the
dry weight of the encysting cells, assuming that
they contain 8% nitrogen. The reduction in nucle-
oids per cell after exponential growth has been
observed in other bacteria and in metabolic shift-
down experiments in which cell growth rate is al-
tered (8, IO). Cysts contain approximately 10 times

 

     

 

 

 

T i l r I I I
a, O.|0 : :
t. : ENCYSTING CELL :
E - _
=3 0.05~ -
DJ - ..

2

>N- ” ACETATE GROWN "
“Z, 0.02 - -

0.0l I I I l t 1 l
300 310 320 330 340

io” l/T

FIG. 4. Arrhenius plot of the effect of temperature
over the range 25 to 48 C 0n the activities of 0.045
units of BHB dehydrogenase from acetate grown cells
and 0.055 units from encysting A. vinelandii. The activ-
ities are expressed as equivalent units (37 C).

TABIi-i 2. Properties of B-hydroxybutyrate (BHB)
dehydrogenase oj'Azotobacter vinelandii

 

 

Source p2u0£E,i- K,“ (mM)” Rmr
Encysting cells ...... 9.0 4.0 0.38
Acetate—grown cells . . 9.0 5.5 0.38

 

“The enzyme (0.05 units) was assayed over a pH
range of 7.5 to l 1.0 in 0.05 M Tris-glycine buffer.

” Michaelis constants for either enzyme were deter-
mined with 0.02 to 0.05 units of enzyme over a BHB
dehydrogenase concentration range of0.001 to 0.03 M.

"Mobility relative to bromophenol blue in electro-
phoresis on 5% acrylamide gel at pH 8.3.

VOL. 105, 1971

as much DNA per nucleoid as does Escherichia
coli(10). Similar DNA contents for synchronous
cultures of A. vinelandii have been reported by
Zaitseva et al. (24). Muller and Kern ([3) ob—
served that the DNA contents of various radia-
tion resistant mutants of A. chroococcum ranged
from 10‘" to 19 X 10'“1 g/cell. Despite these
corroborative findings, we considered the pos-
sibility that the high values of DNA content per
cell were artifacts of the assay procedure. If the
hot trichloroacetic acid used to hydrolyze the cell
DNA also hydrolyzed a part of the cell envelope,
the values for the DNA assay might have been
inordinately high. Therefore, we extracted DNA
from 100 ml ofa culture in late exponential growth
phase and, after a standard DNA assay, calcu-
lated the DNA per cell. To detect contamination
of the DNA preparation by the cells” lipopolysac-
charide, we monitored hexosamine content of our
precipitated DNA and the aqueous phase from
which it was precipitated. Only 1.3% of the hexos-
amine-containing material originally present in
the aqueous phase was precipitated with the DNA.
Assuming a 70% yield of DNA on extraction, we
calculated that late exponential cells of A. vine-
landii contain 6.7 x 10‘H g of DNA/cell. Since
these cells contained an average of two nucleoids
per cell, the individual nucleoids must have con-
tained 3.4 x 10‘” g of DNA. This value is identi—
cal to that which we have obtained for encysting
cells and cysts oI'A. vinelandii by our DNA assay.
The relatively large mass of the A. vinelandii
nucleoid suggests that there may be considerable
redundancy in its genome and may account for
the difficulty in obtaining mutants of this orga-
nism(l I. 12).

A(‘KNO\\"I.EI)(;M EN'I‘

This investigation was supported by Public ”Ctlllh Sersice research
grant Al-01863 from the National Institute of .=\||ergy .ind InlectioUs
Diseases. National institutes of Health. Ii. B. was :i predoctoral trainee.
supported by Public Health Service grant (EM-01911 from the National
Institute o|'(ieneral Medical Sciences.

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T

7:

II.

v

«J

C

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VINELA NDII I89

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. Mishra. A. K. and 0. Wyss. 1969. An adenine requiring mutant of

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