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GENE cmNG-HUA YANG.
1970 ‘

 

   
     

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0-169

 

 

ABSTRACT

INHIBITION OF THE SYNTHESIS OF DEOXYRIBONUCLEIC
ACID IN YERSINIA PESTIS DURING PRODUCTION
OF VIRULENCE ANTIGENS

 

BY

Gene Ching-hua Yang

Virulent and potentially virulent cells of Yersinia
(Pasteurella) pestis, the causative agent of bubonic plague,
were shown by others to produce virulence or V and W antigens
(VW+) but remain static at 37 C during aeration in enriched
Ca++ -deficient medium containing 0.02 M Mg++. In this
environment, which simulates mammalian intracellular fluid_
VW+ cells possessed a functional cytOplasmic membrane as

14 32

judged by concentration of C-isoleucine release of P,

and consumption of oxygen at rates comparable to those of
dividing cells cultivated with Ca++. Furthermore, the rates
of protein and ribonucleic acid synthesis were essentially
identical in both dividing and static VW+ cells and also in
mutant VW’ organisms. However, the synthesis of deoxyribo-
nucleic acid (DNA) ceased in static cells about 4 hr after
the removal of Ca++. During this period of time, which

. . ++
corresponded to one generation in the presence of Ca ,

Gene Ching-hua Yang

the static VW+ cells completed one chromosomal replication
as judged by a two-fold increase in content of DNA and a
corresponding degree of resistance to irradiation with
ultraviolet light. These findings agree with the results

of an independent study which disclosed that the typical
static cell possesses at least twice the number of visible
nuclei that were observed by direct staining to exist within
dividing cells. The static and growing organisms contained
essentially identical levels of DNA polymerase.

Accordingly, the static organisms appeared to be unable
to initiate septation or to synthesize new DNA following
termination of an initial replication that was in progress
when Ca++ was removed. These metabolic blocks appear to
be caused by thermo-inactivation of the membrane-associated
proteins that are required for the initiation of DNA replica-

tion and the formation of septae.

INHIBITION OF THE SYNTHESIS OF DEOXYRIBONUCLEIC

ACID IN YERSINIA PESTIS DURING PRODUCTION

 

OF VIRULENCE ANTIGENS

BY

Gene Ching-hua Yang

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

1970

DEDICATION

This thesis is respectfully dedicated to my mother,
Wang Lan, who instilled in me the importance of education,
and to my wife, Alice, whose patience and understanding

made this achievement possible.

ii

ACKNOWLEDGMENTS

The author wishes to thank all with whom he has
associated during the course of his study in this country.

I wish to express my most sincere appreciation to
Dr. Robert R. Brubaker, my major advisor, not only for his
inspiration and guidance throughout this investigation but
especially for his assistance in taking samples for me on
some cold Michigan winter mornings.

I am grateful to Dr. John A. Boezi, Department of
Biochemistry, for the supply of necessary material and sug-
gestions in deoxyribonucleic acid polymerase studies. The
technical assistance of Mrs. Prudence J. Hall in the amino
acid uptake experiments is also appreciated.

Special thanks are also extended to Drs. Harold L.
Sadoff, David H. Bing, Ralph N. Costilow, Peter Hirsch,
and Richard L. Anderson for their interest in this project

and helpful criticisms.

iii

TABLE OF CONTENTS

DEDICATION . . . . . .

ACKNOWLEDGMENTS . . . .

LIST OF TABLES . . . .

LIST OF FIGURES . . . .

LIST OF APPENDICES . . .

Chapter

I.

II.

III.

INTRODUCTION . . .

LITERATURE REVIEW .

Taxonomy and Morphology
Carbohydrate Metabolism

Growth and Virulence

Virulence Determinants

Fl+ Determinant .
VW+ Determinant .
P+ Determinant .
Pu+ Determinant .
PI+ Determinant .

Control of Replication

METHODS . . . . .

Bacterial Strain .

and

Cell Division

Cultivation and Culture Media . . . . .

Measurement of Growth

Characterization of Virulence Determinants
V and W Antigen Production . . . . .
Pesticin I Production .

Pigmentation . .

Measurement of Amino Acid Uptake
Measurement of Oxygen Uptake .
Leakage of Radioactive Compounds

Protein Synthesis .

iv

Page
ii
iii
vi
vii

ix

Chapter

RNA Synthesis . . . . . . . . .
DNA Synthesis . . . . . . . .
Sensitivity to Ultraviolet Irradiation. .
Quantitative DNA Determination . . .
DNA Polymerase Assay. . . . . . .
Effect of Ca++ on Initiating Growth of
Static Cells. . . . . . . . . .

IV 0 RESULTS 0 O O O O O C O O O O O 0

Characterization of Virulence Determinants
Growth Pattern. . . . . .
Effect of Antibiotics on the Growth of VW+
Cells . . . . . . . . . . . .
Comparison of Regulatory Capacity of
Cytoplasmic Membrane . . . . . . .
Uptake of Amino Acid . . . . . . .
Uptake of Oxgen. . . . . . . . .
Direct Measurement of Membrane Leakage .
Comparison of Rates of Macromolecular . .
Comparison of DNA Polymerase Activity . .
Sensitivity to Ultraviolet Light. . . .
Quantitative DNA Determination . . . .
Mode of Inhibition of DNA Replication . .
Effect of Ca++ on the Growth of Static
Cells . . . . . . . . . . .
Thermal Induction of Growth Inhibitor in
VW+ Cells. . . . . . . . . . .

V. DISCUSSION. 0 O O O O O O O O O 0

VI 0 SUMMARY 0 O O O O O O O O O O O 0

LIST OF

APPENDIX.

REFERENCES 0 O O O O O O O O O O

Page
22
22
23
24
24
25
27

27
27

31

36
36

4O
40
45
51
54
54
58
58
66
76
78

89

Table

1.

LIST OF TABLES

Page
Characterization of virulent Determinants in
Strains of Y. pestis and Y. pseudo-
tuberculosis . . . . . . . . . . . . 28

Effect of Inhibitors of DNA Synthesis on the
Growth of VW+ Yersinia pestis . . . . . . 37

 

Comparison of Rates of Macromolecular Synthesis

in 109 VW' and VW‘ Y. pestis Cultivated in

the Presence (0.0025M) and Absence of Ca++ . . 48
Control Experiment of DNA Polymerase Assay . . 51

DNA Content of Y. pestis Cells Cultivated in the
Synthetic MedIum for 12 hr. . . . . . . . 55

vi

LIST OF FIGURES

Figure Page

1. Growth of Y. pestis Strain EV76 in Modified
Synthetic Medium of Higuchi, Kupferberg
and Smith at 37 C . . . . . . . . . . 29

2. Growth of Y, pseudotuberculosis Strain PBl in
in Modified Synthetic Medium at 37 C . . . . 32

 

3. Effect of Antibiotics on the Viability of
Growing and Static VW+ cells of Y, pestis
Strain EV76 . . . . . . . . . . . . 34

4. Incorporation of 14C-L-isoleucine into the
Internal Pool of Growing and Static VW+
Cells of Z, pestis Strain EV76 . . . . . . 38

5. The Consumption of Oxygen by Static and Growing
Cells of Y. pestis Strain EV76 . . . . . . 41

6. Release of 32P at 37 C from VW’+ Cells of Y,
pestis Strain EV76 preloaded with KH32PO4
at 6 C . . . . . . . . . . . . . 43

7. Rates of Synthesis of Macromolecules by Static
and Growing Cells of Y. pestis Strain EV76 . . 46

8. DNA Polymerase Activities of Growing and
Dividing Cells of X. pestis Strain EV76 . . . 49

9. Sensitivity to Ultraviolet Light of Growing and
Static Cells of Z, pestis Strain EV76 . . . 52

10. Per cent rate of DNA Synthesis in Static and
Growing Cells of VW+ Z. estis Strain EV76
Following Temperature sh1ft up to 37 C . . . 56

11. Effect of Ca++ on the Growth of vw+ Cells of
Y, pestis Strain EV76 at 37 C . . . . . . 59

12. Effect of Ca++ on the Growth of VW+ Cells of

Z. pestis Strain EV76 after Ca++-starvation
for 8 or 12 hr at 37 C . . . . . . . . 61

vii

Figure Page

13. Brief Thermal Treatment on the Growth of VW+
Cells of Y, pestis Strain EV76 . . . . . . 63

viii

LIST OF APPENDICES

Appendix Page

A. Modified Synthetic Medium of Higuchi,
Kupferberg, and Smith . . . . . . . . 89

ix

CHAPTER I

INTRODUCTION

Investigation of the metabolism of obligate intracel-
lular parasites has yielded considerable information regard-
ing the reasons why these organisms are restricted to growth
within the host cell. The finding that chlamydiae and
viruses evidently lack the ability to produce adenosine
triphosphate (ATP) can itself explain why these organisms
are restricted to an intracellular existance (97, 116).

Plasmodia and Rickettsia, on the other hand, can produce

 

 

ATP via the tricarboxylic acid (TCA) cycle (95, 117, 119);
therefore the restriction of these organisms to an intra-
cellular location is due to other factor(s). It has been
suggested that a lack of membrane regulatory capacity is
the primary cause of this restriction since the cytoplasmic
membranes of rickettsial or plasmodial cells are freely
permeable to various phosphorylated organic compounds

(6, 91). However, the recent finding of Myers, Provost
and Wissman (92) demonstrates that rickettsial cell mem-
branes are easily damaged by the standard procedure for
purification of these parasites from tissue culture cells.
This observation may indicate that leakage of metabolites

observed by previous workers is the result of unavoidable

damage associated with the process of preparation. The
search for a suitable explanation for restriction to living
mammalian cells thus continues.

Moulder (91) suggested that the dependence of the
obligate intracellular parasites on the host cell and the
highly restricted ability of these organisms to parasitize
such cells are presumably closely related phenomena. There-
fore, gaining an understanding of one of these mechanisms
should greatly facilitate the elucidation of the other. In
order to minimize the difficulties associated with the
cultivation and purification of obligate intracellular
parasites, one line of investigation has focused on the
metabolic study of facultative intracellular parasites of

the genus Yersinia (Pasteurella). These organisms exhibit

 

distinct responses in synthetic media which simulate the
intra- and extracellular environments of mammalian tissue.

When cells of wild type Z, pestis or X. pseudotuber-

 

culosis are incubated at 37 C in an enriched chemically

+

+ but no added Ca+ ,

defined medium containing 0.02 M Mg+
they produce the V and W antigens (vwi) of Burrows and
Bacon (22), but remain static. This medium simulates
mammalian intracellular fluid with respect to the ion
concentrations of magnesium and calcium (70). However,

the addition of Ca++

(0.0025 M) to such a medium promotes
cellular division but suppresses the production of V and

W antigens (16, 53). Mutants which lack the ability to

produce V and W antigens are avirulent and do not require
Ca++ for growth at 37 c (21).

Brubaker (8) suggested that bacterial-stasis with
the expression of the V and W antigens, and cell division
with apparent repression of these antigens may reflect
metabolic patterns which are essential for intra- and
extracellular growth, respectively. Therefore, identifica-
tion of the nature of the metabolic block in static VW+
cells, which occurs upon cultivation in the simulated
intracellular environment, should not only lead to a greater

understanding of virulence in Yersinia but should also be

 

valuable in elucidating the metabolic blocks of some obligate
intracellular parasites during in_vitro cultivation.
A definition of the physiological block in static

VW+ cells is presented in this dissertation.

CHAPTER II

LITERATURE REVIEW

Taxonomy and Morphology

 

The genus Pasteurella comprises the etiological agents

 

responsible for pasteurellosis (hemorrhagic septicemia),
plague, pseudotuberculosis, and tularemia. Although these
agents share superficial morphological, staining, and
biochemical properties, there are several fundamental dis-
tinctions between them. These include differences in nutri-
tional requirements (88), antigenic prOperties (118), and
lack of deoxyribonucleic acid (DNA) homology except between
organisms of plague and pseudotuberculosis (97). Due to
these differences, it has been suggested by several workers

(79, 87) that a separate genus, Yersinia, be established

 

for the organisms of plague and pseudotuberculosis. Stocker

(108) pointed out that this new genus Yersinia is taxono-

 

mically related to the Enterobacteriaceae. His conclusion

was based on the findings that Yersinia and Salmonella

 

share antigenic properties (67) and that bacteriophages of

Yersinia also lyse species of Escherichia, Shigella and

 

Salmonella (52, 77, 104, 108). Martin and Jacob (84) and

 

Lawton, Morris and Burrows (76) demonstrated the transfer

of the F-lac episome from E. coli to Y. pestis and Y.
pseudotuberculosis, respectively.
Y. pestis was isolated by Yersin in 1894. The organism

is a gram-negative ovoid bacillus, 0.5 to 0.8 micron wide and

1.5 to 2.0 microns long (118). Unlike Y. pseudotuberculosis,

 

this organism lacks flagella and is not motile (88). Y.
pestis is divided into three physiological varieties:

orientalis, antiqua and mediaevalis, based on the organism's

 

 

ability to ferment glycerol and to reduce nitrate to nitrite

(118).

Carbohydrate Metabolism

 

Due to a deficiency of g1ucose-6-phosphate dehydro-
genase (7, 89 90), Y. pestis utilizes the Enbden-Meyerhof
pathway almost exclusively to ferment hexoses (101).
Gluconate is readily metabolized by a combination of
Weimberg and Wolochow (78) reported that Y, pestis decar-
boxylates pyruvate to acetate, which is then completely

oxidized to CO via the TCA cycle (34, 35, 36). Anaerobically

2
grown cells are unable to oxidize acetate; however, upon
aeration for a brief period of time, anaerobically grown

cells become adapted to aerobiosis and then oxidize acetate

readily.

Growth and Virulence
Virulent strains of Y. pestis are usually defined as

those having average lethal doses for mice and guinea pigs

of less than 10 cells following subcutaneous or intra-
peritoneal injection (20). Due to its high degree of
pathogenicity, Y. pestis has been used by many workers
as a model for the study of virulence.

The optimum temperature for in_yi£52 cultivation of
Y. pestis is 27 C for the majority of pathogens. At room
temperature, these organisms have less complex nutritional
requirements than at 37 C (23, 30, 50, 55).

The influence of culture conditions on the loss of
virulence in Y. pestis has frequently been noted. Fukui
e£_al. (40) found that cultivation in aerated broth medium
at 37 C consistently resulted in a loss of virulence.

This process of attenuation was attributed to selective
conditions favoring growth of avirulent mutants which
normally persist in the virulent inoculum (40, 94). Several
effective treatments, at least for the initial 24 hr incuba-
tion period, have been shown to prevent this population
shift. These processes include lowering the incubation
temperature to 26 C (39), addition of spent culture fil-
trate of avirulent mutants, adjustment of the initial
culture medium to pH 7.8 (94), or the addition of compounds
such as NaHCOB, pyrimidine (3), 2.4 dinitro phenol, potas-
sium iodide, salicylate ions and biliverdine which all act
to selectively retard the growth of VW- organisms (28, 110).
The shift to avirulence may also be prevented by addition

of Ca++, Sr++, Zn++ (53), KCN, potassium oleate and sodium

deoxycholate which act by selectively stimulating the growth
of VW+ cells (111). As noted by Surgalla, Andrews and
Cavanaugh (110), all of these substances with the exception
of NaHCO3 and pyrimidine, could directly interact with cell
membrane. The CO2 requirement for the 12:31359 cultivation
of virulent, but not avirulent, strains was also confirmed
by Burrows and Gillett (23) in nutritional experiments
utilizing chemically defined solid media.

Fukui EE.§£' (38) showed that the phenotypic expression
of virulence was repressed when virulent cells were grown
at temperatures below 26 C. This repression, however, was
reversed by raising the temperature to 37 C for 6 hr.
Further investigation revealed that restoration of virulence
required vigorous aeration and a high concentration of an
adequate nitrogen and energy source (93). Other findings
presented by Fukui, Lawton and Mortlock (39) indicated that

the expression of maximal virulence required d§_novo

 

synthesis of ribonucleic acid (RNA) and protein, but not
DNA. These results appear to be consistent with the finding
of Baugh, Andrews and Surgalla (3), that pyrimidines are

required for retention of virulence during aeration at 37 C.

Virulence Determinants

 

Compared to the intensive and fruitful studies on
general bacterial genetics, the effort devoted to the genetic
study of virulence determinants of pathogens has been minimal.

The major difficulty has been in finding an adequate host

and parasite system which would permit the use of the
mutant approach to study virulence properties. The problem,
in part, has been caused by lack of laboratory facilities
suitable for this type of research. In spite of such
limitations, a number of virulence determinants have been

discovered in I. pestis in the last 20 years.

Fl+ determinant

 

Baker 32 31. (2) isolated several protein antigens
from the surface envelope of virulent Y, pestis. Strains
that are genetically capable of synthesizing fraction one
(Fl) of these capsular antigens are designated F1+. Mutants
which lack the ability to produce this antigen (Fl-) are of
reduced virulence in guinea pigs but not mice (109). The
F1 antigen is produced at 27 C and 38 C (21), but its
accumulation on the cell surface only occurs at 37 C. This
seems to indicate that the Fl+ state is determined by two
gene loci--one determining Fl antigen production, the
other its expression as the surface envelope. Spontaneous
mutation from Fl+ to F1- occurs readily, but no case of
back mutation has yet been reported. The role of the F1
antigen is unknown. However, Burrows (21), and Englesberg
93 31. (33) found that F1+ strains were more resistant to
phagocytosis than F1- strains. This statement is challenged
by the recent finding of Janssan and Surgalla (66) who
showed that both virulent and avirulent cells were phago-

cytized at equal rates in mice.

+ .
VW determ1nant

 

The search for a second virulence determinant stemmed
from the finding that Fl- strains remain virulent in mice.
Burrows and Bacon (22) demonstrated that all virulent strains
produce V and U antigens, and may thus be termed VW+.
Mutants which are unable to produce these gene products
(VW-) are avirulent for mice and for guinea pigs. The V
and W antigens were partially purified by Lawton, Erdman
and Surgalla (74) who reported that the former was a protein
and the latter was a lipoprotein; both were easily extract-
able from the culture medium. Virulent strains always
produce these two antigens together and mutants producing
one without the other have not been isolated. Most of the
nutritional requirements and culture conditions previously
described under Growth and Virulence are found to be associ-
ated with production of V and W antigens (28, 38, 39, 94).
Janssen and his coworkers showed that the VW+ determinant
is associated with survival and multiplication of virulent
cells within fixed macrophages of the host reticuloendo-
thelial system (65, 66). The mutation rate from VW+ to VW'
is 10-4 per bacterium per generation (54).

The inhibitory effect of Ca++ on the production of V
and W antigens in_viE£9_was noted by Brubaker and Surgalla
(16). Lawton (73) had earlier demonstrated that maximal
production of V and W antigens occurred at 37 C in aerated

+

enriched medium containing 0.02 M Mg+ , but no added Ca++.

10

When VW+ cells were grown under these conditions, they
became elongated (16) but remained static (53). These
virulent static cells could initiate growth in the absence
of Ca++ if the culture medium was adjusted in such a way
that its ionic strength was increased and the osmolarity
was decreased (8). Brubaker and Surgalla (15) isolated

an avirulent mutant which retained all other virulence
characters but did not require Ca++ for growth at 37 C.
This result clearly established the fact that the Ca++

requirement is an essential virulence property.

P+ determinant

 

Virulent strains grown on solid synthetic media con-
taining hemin form dark pigmented colonies (P+). Mutants
which form light,non-pigmented colonies (P—) on the same
medium are avirulent (63). Colony-pigmentation readily
occurs at 26 c but not at 37 c. Like the F1+ and vw+
determinants, no back mutation from P- to P+ has yet been
observed. However, the full virulence of P- cells can be
restored if Fe++, Fe+++, or hemin is simultaneously injected
with cells into the mice (64). Therefore P- strain is
usually described as potentially virulent cells (16). The
role of iron is unknown, but it has been suggested that
the ability to acquire iron in_yiyg is associated with the
expression of virulence. To date, it is still unknown

whether the P+ determinant is directly concerned with

virulence or indirectly related to some other virulence

ll

determinant. Recently, Surgalla and Beesley (112) have
develOped a new technique for detection of colony pigmenta-
tion by substituting Congo red for Hemin. They suggested
that basic residues of protein or mucopolysaccharide of P+
cells may serve as the binding sites for chromogens. The
mutation rate from P+ to P- is 10-5 per bacterium per

generation (11).

Pu+ determinant

 

Purine auxotrophs (Pu-) of Y. pestis are of reduced
virulence since the availability of purines in host cells
is limited (19). This nutritional requirement for purines
as a virulent determinant has also been characterized in

Salmonella typhi (l9), Klebsiella pneumoniae (41), and

 

 

Bacillus anthracis (62). Concomitant injection of purines,

 

 

but not iron, restored full virulence of Pu- strains in
mice (21). Mutants which were incapable of synthesizing
inosine monOphosphate by the dgbngyg_pathway retained a
considerable degree of virulence in mice, whereas strains
which could convert inosine monOphosphate to guanosine

monophosphate were completely avirulent (10).

PI+ determinant

 

At least two types of bacteriocinwlike substances are
produced by wild type Y. pestis. The first, designated
pesticin I, inhibites the growth of certain strains of Y.

pseudotuberculosis (5, 51), E. coli, and pesticin-deficient

 

12

(PI-) mutants of Y. pestis (13). Brubaker and Surgalla
(13, 14) demonstrated the presence of a second type of
pesticin, pesticin II, which is also produced by Y. pseudo-
tuberculosis. The activity of pesticin I, but not pesticin

II, can be supressed by Fe+++, Mg++, inorganic phosphate,

 

hemin and an acid soluble metabolite (termed pesticin I
inhibitor) produced by the pesticin I forming (PI+) cells.
Whether these cations and hemin affect pesticin I directly
or indirectly by either stimulating the production or
activation of pesticin I inhibitor is still unclear.
Suppression of the activity of pesticin I by Fe+++ or
pesticin I inhibitor can be reversed by the addition of
either Ca++ or chelating agents (14). Brubaker and his
coworkers (4, 17) found that all PI+ strains are capable
of synthesizing coagulase and fibrinolysin, whereas PI-
strains are devoid of these two biological activities.
Since fibrinolysin and coagulase are important invasive

factors of pathogens, they speculated that the PI+ deter-

minant is another bona fide virulence determinant. This

 

suggestion was confirmed in a later report (12) by the
isolation of an avirulent mutant, strain G 32, which was
positive for all known virulence determinants except PI_.
Simultaneous injection of iron also restored the full
virulence of the PI- avirulent mutant in mice (21).

The optimum temperature for pesticin I production is

26 C (5). Brubaker (11) found that most pesticin I-sensitive

13

strains are PI P+. When the organisms mutate to PI- P-,
they concomitantly lose sensitivity to pesticin I. As a
result of this correlation, pesticin I was used as the

selective agent to estimate the mutation rate of P+ to P-.

Control of Replication and Cell Division

 

Although the biochemical nature of the control of cell
division is not well understood, the sequential events of
this process have been characterized in E. coli as follows:
1) the cell replicates DNA and forms two sets of nuclei;

2) the cell elongates and the newly formed chromosomes are
progressively segregated; 3) a "septum" is formed near the
equatorial plane; 4) two daughter cells, each containing a
complete genome,physically separate from each other, and
the new cycle is re-initiated. For organisms which have
short generation times, new rounds of replication are
initiated before the occurrence of division (24).

Helmstetter (48) divided the entire cycle into a
three step process, I+C+D, representing the periods required
for synthesis of an "initiator complex" (I), completion of
a round of replication (C), and that occurring between the
end of a round of replication and the following cell divi-
sion (D). He showed experimentally that the length of the
C+D period for E. coli growing exponentially at a defined
temperature is constant. Accordingly the generation time
of this organism in a particular medium is determined by

the period I.

14

The "initiator complex" appears to comprise at least
two pieces of heat labile membrane-associated protein,
which also function in the regulation of nuclear segregation
(32, 72, 101). Initiation of a new round of replication in
cells with a short generation time is not affected by the
completion of the previous round (49, 81), but is regulated
by a crucial value of the cellular mass/DNA ratio (29, 82) ,
or the availability of chromosome attachment sites (72).

Evidently, the process of cellular division in gram-
positive and gram-negative organisms is different. In the
former, cell wall synthesis is initiated equatorially and
peripherally at the same time. Thus the new portions of
the daughter cells develop adjacently, and the old ends of
parental cells remain intact but are gradually pushed apart
(83) . The formation of cross wall appears to be associated
with mesosome (98) . In gram-negative cells, on the other
hand, the insertion of new wall material occurs along the
entire cell surface. The peripheral wall and cytoplasmic
membrane grow inwards, resulting in a pinching-off effect.
However, under special conditions of growth and fixation,
the formation of septae have been demonstrated in cells of
E- g (107). The role of mesosomes in the cell division
0f <Fawn-negative organisms has not been characterized (98) .

The initiation of septum formation, under normal con-
ditiOrls, is obviously coordinated with DNA replication. If
DNA SYnthesis is arrested during the process of replication,

the Viability of the cells declines rapidly. On the other

15

hand, if the inhibition occurs after completion of a round
of replication, the cells divide once and then die (24, 49,
85). These observations suggest a very appealing mode of
regulation; namely, that septation does not occur in cells
which cannot complete replication of their chromosomes,
whereas cells which have completed this replication can
divide without further DNA synthesis. In this manner each
daughter cell is assured of a complete genome.

Under abnormal growth conditions, however, some
‘mutants of E, coli and B, subtilis deviate from this regu-
latory pattern (44, 57, 86). For example, when thermosensi-
tive mutants of E. 321; strains CRT 115 and PAT 84 are
incubated at 40 C, they continue to synthesize DNA and
other macromolecules but do not divide. As a result, multi-
nuclear filaments are produced (57). On the other hand,
strain PAT 421, while unable to synthesize DNA at the
impermissive temperature, can still form a septum to one
side of the chromosome. Consequently some anucleated daugh-
ter cells are produced (56). It would appear from these
observations that cellular division and DNA replication are
not coordinated.

In addition to completion of chromosome replication,
septation also requires an accumulation of a heat sensitive
membrane-associated protein which may be different from the
components of "initiator complex" (61, 105). So far, this

hypothetical protein can only be demonstrated indirectly.

l6

Septum formation is also regulated by the biosynthesis
of cell walls. Inhibition of peptidoglycan formation by
cycloserine results in the inhibition of cell division of
Erwinia (45). This inhibition, however, can be overcome

by the addition of Ca++, Mn++

, D-alanine and pantoyl lactone.
The mechanisms whereby these compounds promote growth are

not understood with certainty.

CHAPTER III

METHODS

Bacterial Strain

 

Unless otherwise stated, Y, pestis strain EV76 was
used throughout the course of this investigation. Strain
EV76 possesses all of the virulence determinants of the
species except P+. A VW- mutant was selected on the magner
sium oxalate (MGOX) agar of Higuchi and Smith (54). Stock
cultures were preserved in buffered glycerol at -20 C as

described by Brubaker (8).

Cultivation and Culture Media

Following incubation for 2 days at 26 C on slopes of
blood agar base (BAB), the organisms were centrifuged at
4 C for 10 min in a Sorvall RC2—B refrigerated centrifuge
at 27,000 x g, washed once with 0.033 M potassium phosphate
buffer, pH 7.0 (phosphate buffer), and inoculated at a
concentration of about 3 to 5 x 108 cells per ml into a
250 or 500 ml Erlenmyer flask containing 20 or 50 ml of a
modification of the chemically defined medium of Higuchi,
Kupferberg, and Smith (53). The composition of this
synthetic medium is described in Appendix A of this disser-

tation. Cultures were aerated at 37 C on a model BB wrist

l7

18

action shaker (Burrell Corp.) for specific intervals. To
permit the growth of VW+ cells, 0.0025 M CaCl2 was included
in the medium. Appropriate steps, including routine plating
on BAB and MGOX agar, were taken to assure that cultures

were not contaminated or overgrown with VW- cells.

Measurement of Growth

 

Optical absorbance was determined at 620 nm with a
Beckman DU spectrophotometer using appropriate uninoculated
medium as blank. Assays of viable cells were performed by
spreading 0.1 ml of culture, appropriately diluted in the
phosphate buffer, on BAB plates; thelatter were incubated

for 2 days at 26 C.

Characterization of Virulence Determinants

 

V and W Antigen Production

 

The immunodiffusion technique of Lawton, Fukui and
Surgalla (75) was used to estimate these antigens qualita—
tively. The overnight culture of strain EV76 aerated at
37 C in an enriched medium containing 0.02 M Mg++ but no
added Ca++ with a concentration of 109 cells per ml, was
used as a source of antigens. The monospecific antisera
against V or W antigen were kindly provided by Dr. M. J.

Surgalla.

l9

Pesticin I Production

Cells of Y, pestis strains EV76 and Y. pseudotuber-

 

culosis strain PBl/+ (a PI_ strain) were spotted on a BAB
plate and incubated at 26 C for 2 days. In order to kill
the producer cells, the plate was eXposed to chloroform
vapors for about 10 min, and then overlaid with 5 ml of

sterile 4% BAB containing 0.1 M CaCl 0.5% Ca-

2!
ethylenediaminetetraacetic acid (Ca-EDTA) seeded with

5

10 cells of Y. pseudotuberculosis strain PBl/O (a pesticin—

 

sensitive strain). The zones of inhibition were observed

after incubating the plate at 37 C for 24 hr.

Pigmentation

 

The technique of Surgalla and Beesley (112) was used.
The agar medium contained 1% heart infusion broth, 2% agar
(BBL), 0.01% Congo red and 0.2% Dugalactose. Colony
pigmentation was observed after 2 days of incubation at

26 C.

Measurement of Amino Acid Uptake

 

Cells of strain EV76 (VW+), incubated in synthetic
medium (with or without Ca++) at 37 C for 12 hr, were
washed, resuspended in phosphate buffer containing 0.025

l4C-L-isoleucine (2 uC

M potassium gluconate and 10 uM
per umole), and continuously incubated at 37 C in a model
G76 gyrotory water bath shaker (New Brunswick Scientific).

Samples of 1 ml were withdrawn at brief intervals, immediately

20

filtered through a 0.45 u pore millipore filter membrane,
and the residue was washed twice with 3 ml of prewarmed
phosphate buffer. After the filters had been dried at 80

C for at least 60 min, the radioactivity was measured in

a Mark I liquid scintillation spectrometer (Nuclear Chicago
Corp.). The counting fluid consisted of 10 ml of a mixture
of 0.4% 2,5-diphenyloxazole (PPO) and 00.5% 1,4-di-[2-(5-
phenloxazolyl)]—benzene (POPOP) in toluene. The control
experiments indicated that there was no significant quench-
ing and the counting efficiency was constant in all of the

samples.

Measurement of Oxygen Uptake

The standard manometric technique of Umbreit, Burris,
and Stauffer (113) was employed. Cell suspensions were
prepared with a procedure similar to that just described
except that the cells were suspended in distilled water.
The reaction mixture consisted of 0.1 ml of Dngluconate
(o.1M), 0.3 ml of potassium phosphate buffer (0.5 M, pH
7.0), and 2.5 ml of cell suspension. The center well con-
tained 0.1 ml of 10 N KOH. Prior to introduction of sub-
strate, organisms were shaken for 30 min in the buffer in

order to reduce the endogenous metabolism.

Leakage of Radioactive Compounds
The ability of dividing and static cells to retain

internal compounds containing 32F was determined by

21

cultivating the VW+ organisms for 12 hr at 26 C in the
synthetic medium containing KngPO4 (0.4 uC per mole).
Following this process of preloading, the organisms were
washed 3 times in non-radioactive phosphate buffer contain-
ing 0.07 M NaCl in order to remove unchangeable isotope

and then incubated in the synthetic medium, in the presence
or absence of Ca++, at 37 C. Samples of 1 ml were then
removed at 90 min intervals, filtered through 0.45 u pore
membrane filters, and, after drying, the radioactivity in

the filtrate was estimated on the aluminum planchets with

a model 447 gas flow counter (Nuclear Chicago Corp.).

Protein Synthesis

 

The synthesis of protein was estimated by measuring

l4C-L-isoleucine into cold trichloro-

the incorporation of
acetic acid precipitable fraction. In order to increase

the specific activity of radioactive isoleucine, cells that
had been cultivated in the synthetic medium for 12 hr at

37 C were resuspended in the prewarmed fresh medium contain-
ing 0.15 mM l4C-isoleucine (1.3 uC per umole) instead of

the normal concentration. Samples of 1 ml were removed at
10 min intervals from the cell culture, mixed with 1 m1 of
cold 10% trichloroacetic acid containing 50 ug per ml of
non~radioactive isoleucine and incubated at 4C for at least
30 min. The contents were filtered through 0.45 u millipore

membrane filters and washed with 5 ml of cold 5% trichloro—

acetic acid. The membranes were mounted on aluminum

22

planchets, dried, and the radioactivity was estimated in

the gas flow counter.

RNA Synthesis

 

Essentially a procedure similar to the estimation of
protein was used to determine the synthesis of RNA. In this

case, 0.44 uM l4

C-uracil (1.0 uC per umole) was added to

12 hr cultures without changing the medium. In order to
minimize the incorporation of radioactivity into DNA, non-
radioactive cytosine was added to the culture at a concentra-
tion of 20 ug per ml (71). Samples were collected and the

radioactivity was determined by the procedures used for

estimation of protein synthesis.

DNA Synthesis

 

Due to the inability of wild type Yersinia to accumulate
thymine or thymidine, the synthesis of DNA was determined
with l4C-uracil by a pulse labeling procedure similar to
those of Goss, Deitz and Cook (43). Radioactive uracil
(1 uC per umole) at a concentration of 0.05 mM was added
at various intervals to the cultures undergoing incubation
at 37 C. Samples of 1 ml, withdrawn at 10 min intervals,
were added to the tubes containing 0.1 ml of 5.5 N NaOH;
the latter were sealed with parafilm (Marathon) and incubated
for 18 hr at 37 C. The contents were neutralized and pre-

cipitated by adding 0.1 m1 6 N HCl and 1.2 ml cold 10%

trichloroacetic acid containing nonvradioactive uracil

23

(50 ug per ml) and incubated at 4 C for at least 30 min.
The samples were filtered, washed with 5% trichloroacetic
acid and the radioactivity was determined either by a gas
flow or liquid scintillation counter as described in the
preceding experimental methods. RNA but not DNA is
hydrolyzed by this procedure (102), thus the radioactive
uracil of RNA appeared in the filtrate and the radioactive

thymine of DNA remained on the membrane filter.

Sensitivity to Ultraviolet Irradiation

 

The procedures of ultraviolet (UV) sensitivity tests
were similar to those of Howard-Flanders, Simson and Theriot
(58). Cells that were incubated in the synthetic medium at
37 C for 12 hr were washed and re-inoculated into phosphate
buffer at a concentration of l to 5 x 107 cells per ml. UV
irradiation was performed in a box fitted with a shutter
with a standard glass petri dish containing 6 ml of cell
suspension (2mm in depth) with light from a 30 watt General
Electric germicidal lamp at a distance of 50 cm from the
surface of platform. The cell suspension was constantly
swirled at 26 C during the irradiation period. Dilutions
for plating were made in the buffer under a dark environment
and the inoculated plates were incubated at 26 C for 2 days.
Intensity of the light, as estimated by comparing data from
E. 3913 strain K12 with those of Howard—Flanders, Simson

and Theriot (58) was about 13 ergs per mm2 per sec.

24

Quantitative DNA Determination

 

A modified procedure of Copeland (25) was used to
extract DNA from the cell suspension. The nucleic acids
of the cell suspension were precipitated by adding 1
volume of cold 10% trichloroacetic acid and incubated at
4 C for 30 min. After the acid was decanted following
centrifugation, the precipitates were resuspended in 1
volume of l N KOH and incubated at 37 C for 15 to 18 hr.
The hydrolysates were neutralized and precipitated by
adding 4/10 volume of a cold acid mixture (0.28 ml 1 N
HCl, 0.62 ml 10% trichloroacetic acid and 0.1 ml H20) and
incubated in an ice bath for 1 hr. Pellet and supernatant
fluid were separated by centrifugation at 27,000 x g. DNA
content of the pellet was determined by the method of Giles
and Myers (42) using highly polymerized calf thymus
(Calbiochem) as standard. Total cell counts were determined

by use of a Petroff-Hausser counting chamber.

DNA Polymerase Assay

 

The modified procedures of Richardson (96) were
employed. The reaction mixture contained 60 ul of 0.35 M

glycine buffer (pH 9.2). 3.5 x lo‘2

M MgClz, 5 mM 2-
mercaptoethanol; 30 ul of 2-deoxythymidine-5-triphosphate
(dTTP), 2 deoxycytidine-S-triphosphate (dCTP), 2-
deoxyguanosine-5-triphosphate (dGTP), 0.1 umole each,
3H-Z-deoxyadenosine-S—triphosphate (37 uC per umole;

3H-dATP), 0.1 umole; 30 ul of calf thymus "activated"

25

DNA; and the appropriate volume of extract and H20 to make

a total volume of 300 ul. The reaction mixture was incubated
at 37 C for 30 min and the reaction was terminated by adding
5 ml of cold 10% trichloroacetic acid containing 1% sodium
pyrophosphate. After incubating in an ice bath for 10 min,
the mixture received 2 drops of carrier DNA (salmon sperm)
with agitation and was then filtered through a 0.45 u pore
Scheicher and Schuell nitrocellulose filter membrane. The
membrane was dried and the radioactivity was determined

with a liquid scintillation counter. The control deter-
minations which consisted of a series of reaction mixtures
lacking enzyme extract, dTTP, and "activated" DNA, resper
tively, were treated similarly. The calf thymus "activated"
DNA was prepared by partial enzymatic degradation of DNA
(96). The reaction mixture contained 0.2 ml calf thymus

DNA (2.3 mg per ml); 0.1 ml pancreatic deoxyribonuclease

(5 x 10"3 ug per ml; DNAse); 0.1 ml tris buffer, pH 8.0

(0.5 M); and 0.4 ml H20. The mixture was incubated at 37

C for 15 min and was then heated at 80 C for 15 min to
terminate the DNAse activity. The degraded DNA was kept
frozen until use. Protein was estimated by the Folinv
phenol method of Lowry 33 a1. (80) using bovine serum albumin

as standard.

26

Effect of Ca++ on Initiating Growth
of Static Cells

VW+ cells were preincubated at 26 C for 12 hr in
synthetic medium on a model R25 gyrotory shaker (New

Brunswick Scientific), resuspended in prewarmed fresh

medium and then incubation was continued at 37 C. At time

intervals, 0.0025 M CaCl2 was added to the individual cell

cultures and 1 ml samples were withdrawn for the estima-

tion of viable cells.

CHAPTER IV

RESULTS

Characterization of Virulence
Determinants

 

 

The results of Table 1 confirmed that Z.‘pestis strain
EV76 produced V and W antigens and pesticin I but was non-

pigmented, whereas X. pseudotuberculosis strain PBl/+

 

produced V and W antigen but not pesticin I. Since the
temperature-dependent nutritional Ca++ requirement is only
associated with VW+ character (8), the results indicated
that both organisms were suitable for use in the subsequent
study. The results also indicated that Ca++ -independent

mutants of strains EV76 and PBl/+ were VW-.

Growth Pattern

 

A lag period of about 6 hr occurred before growth of
+ . . . .

VW cells of Y. pestls commenced 1n cultures contalnlng
Ca++ (Fig. 1). Following further incubation for 6 hr, such
organisms had entered the exponential growth phase whereas
stasis was completed by this time in parallel cultures lack-
ing added Ca++. Accordingly, cells used in subsequent experi-
ments were preincubated for 12 hr in order to permit the

expression of maximum phenotypic differences related to

growth and stasis. The generation times of growing VW+

27

28

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29

Fig. 1. Growth of Y, pestis strain EV76 in the modified
synthetic medium of Higuchi, Kupferberg and Smith following
inoculation at a level of 3 x 108 cells per m1; (. ) VW+
cells without added Ca++, ((3 )‘VW+ cells plus 0.0025 M
Ca++, (‘) VW- cells without added Ca++, and (A) W—

cells plus 0.0025 M Ca++.

 

 

OPTICAL DENSITY (620 nm)

30

 

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31

and VW" cells, as estimated from Figure l, are 4 and 3 hr
respectively.

The finding of Higuchi, Kupferberg and Smith (53)
was verified that the synthetic medium was toxic to Y.
pestis at inocula of less than 108 cells per ml (not illus-
trated). This toxicity was not observed in the case of Y.

pseudotuberculosis, which could initiate growth without lag
7

 

from inocula of 10 VW+ cells per ml (Fig. 2). Unlike VW+
cells of Y. pestis, those of Y, pseudotuberculosis were able
to undergo about 3 divisions in Ca++~deficient medium before
growth ceased. This phenomenon was even more pronounced
when a high inoculum was used (not illustrated). The VW-
cells of both species did not show the Ca++-requirement for

growth. This observation is consistent with the results

shown in Table 1.

Effect of Antibiotics on the Growth
of vw+ Cells

 

Penicilin was introduced into the cultures in order
to show with certainty that stasis was a property of all
VW+ organisms within the population rather than a function
of growth with concomitant lysis. As shown in Figure 3,
static VW+ cells were not significantly influenced by as
much as 250 units of penicillin per ml of medium, whereas
organisms growing in the presence of Ca++ and penicillin
were rapidly killed. These results demonstrate that there
was no significant turnover within the population of static

VW+ cells.

32

Fig. 2. This figure represents the repetition of the experi-

ment of Figure l with Y. pseudotuberculosis strain PBl; the
7

 

inocula were 2 x 10 cell per ml-

 

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Fig. 3. Effect of antibiotics on the viability of growing
and static VW+ cells of Y. pestis strain EV76; (.) static
cells without addition, ( O ) growing cells without addition,
(I ) static cells plus 250 units of penicillin per ml of
medium, ( [1) growing cells plus 250 units of penicillin

per ml of medimn, (ll) static cells plus 25 units of
streptomycin per ml of medium, and (A) growing cells plus

25 units of streptomycin per ml of medium.

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36

Limited attempts to obtain selective killing of divid-
ing or static VW+ organisms by the use of other antibiotics
with known modes of action were only partially successful.
For example, attempts to inhibit synthesis of DNA with
mitomycin C and nalidixid acid failed to kill the cells of
Y. pestis in the synthetic medium at concentrations of 10
and 100 ug per ml respectively (Table 2). However, static
VW+ cells were extremely sensitive to as little as 25 units
of streptomycin per m1 (Fig. 3).

Comparisons of Regulatory Capacity
of Cytoplasmic Membrane

 

 

The previous reports of Brubaker (8) and Surgalla,
Andrew and Cavanaugh (110) suggested a distinction in mem-
brane function of VW+ dividing and static cells. This

speculation was tested by the following experiments.

Uptake of Amino Acid
14

 

C-L-isoleucine was used for the measurement of amino
acid uptake since this amino acid apparently is not cataliz-

able by Yersinia cells (9). Essentially no difference in

 

ability to accumulate l4C—isoleucine was observed between

static and dividing organisms (Fig. 4).

Uptake of Oxygen

 

Since gluconate is readily metabolized by Y, pestis
(see LITERATURE REVIEW), this substance was used as sub-

strate for the measurement of the rates of oxygen uptake.

37

 

 

 

 

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38

l4C-L-isoleucine into the internal

Fig. 4. Incorporation of
pool of growing (0) and static (. ) VW+ cells of Y. pestis
strain EV76 suspended in neutral potassium phosphate buffer

plus gluconate ions.

39

 

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40

On the basis of dried weight, the rate of oxygen uptake
determined for VW+ growing cells was slightly greater than
that obtained for VW+ static cells (Fig. 5). This difference
did not appear to be significant because the size of the
static cell is larger than the dividing cell (16). The
difference would therefore be reduced if the rate of oxygen

uptake was computed on a per cell basis.

Direct Measurement of Membrane Leakage

No differences were observed between the rates of
release of non-exchangeable 32? by static and dividing VW+
cells (Fig. 6). In separate experiments it was found that
both static and dividing cells released 260 nm absorbing
substance at equal rates (data not shown).

These observations suggested that the cytoplasmic
membranes of static organisms retained considerable regu-
latory capacity. Furthermore, it appeared that a consider-
able amount of macromolecular synthesis occurred in static
cells as judged by the acute sensitivity to streptomycin
and release of macromolecules at a rate equivalent to that
of dividing organisms. Accordingly, a study of macromolecular
synthesis was initiated.

Comparison of Rates of Macromolecular
Synthesis

 

 

Essentially no difference in rates of RNA synthesis by
static and dividing cells was observed (Fig. 73). Protein

was synthesized at slightly reduced but not significantly

41

Fig. 5. The consumption of oxygen expressed in terms of
microliters per mg of dry weight, by washed static and
growing cells of Y. pestis strain EV76; ( O ) static cells
plus gluconate, (A) static cells less gluconate, (O )
growing cells plus gluconate, and (A) growing cells less

gluconate.

OXYGEN UPTAKE

250

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MINUTES

 

43

Fig. 6. Release of 32P from VW+ cells of Y. pestis strain

32PO4- at 26 C containing 180,000 cpm

per 108 organisms; (. ) optical density of static cells,

EV76 preloaded with KH

( 0) optical density of growing~ cells, (A) radioactivity
released from static cells, and (A) radioactivity released

from growing cells.

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different rates in static cells than in dividing cells

(Fig. 7A). However, the rate of DNA synthesis in static
cells was markedly reduced as compared to that in the divid«
ing organisms (Fig. 7C). Comparative rates of macromolecular
synthesis, corrected from the data illustrated in Figure 7,
are shown in Table 3. To obtain an explanation to account
for this selective inhibition of DNA synthesis, the follow- L 1

ing series of experiments was conducted.

 

Comparison of DNA Polymerase Activity E
DNA polymerase, which is presumably necessary for

chromosome replication, was examined to see if a defect in
this enzyme activity was the major cause of inhibition of
DNA synthesis in static organisms. The results of Figure 8
indicated that both VW+ and VW- cells possessed the same
levels of DNA polymerase, whether or not Ca++ was present
in the growth medium. The validity of the assay method is
demonstrated in Table 4. ‘In the absence of enzyme of dGTP,
very low amounts of radioactivity were incorporated into
the trichloroacetic acid precipitable fraction. About an
equal amount of radioactivity was incorporated into the
complete system and that lacking "activated" primer DNA.
This finding appeared to indicate that the crude cell-free
extracts of VW+ static and growing cells already contained
degraded primer DNA and that additional "activated" DNA

was not required for DNA polymerization.

46

Fig. 7. Rates of synthesis of macromolecules by static and.

growing cells of Y. pestis strain EV76; (. ) VW+ cells
without added Ca++, (O ) VW+ cells plus 0.0025 M Ca++,

(A) W. cells without added Ca++, and (A) VW- cells plus
0.0025 M Ca++. A - incorporation of l4C-L-isoleucine into
protein; B - incorporation of l4'C-uracil into ribonucleic
acid; and C - conversion of l4C-uracil into thymidine

triphosphate and subsequent incorporation into deoxyribo—

nucleic acid.

 

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49

Fig. 8. DNA polymerase activities of growing and dividing
cells of Y. pestis strain EV76; (. ) VW+ cells without
added Ca++, (o ) vw+ cells plus 0.0025 M Ca++, (A) vw"

cells without added Ca++, and (A) VW“ cells plus 0.0025

M Ca++.

 

 

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51

TABLE 4. Control experiment of DNA polymerase assay.

14

- a
Components in C-dATP 1ncorporated

 

 

 

the assay system VW+ + Ca VW+ _ Ca
Complete system 2389 2295
Less enzyme 303 313
Less dTTP 334 379 11%
Less "Activated DNA" :.
(calf thymus) 2116 2064 II
aActivity expressed as cpm per 10 ug of protein 30"1 ‘
min. (4 x 105 cpm equivalent to l nmole of d-ATP incor-

porated).

Sensitivity to Ultraviolet Light

 

As an indirect approach to determining the number of
nuclei per cell, the UV sensitivity of the static and divid-
ing cells were compared. As expected, the results showed
that static cells are more resistant to UV radiation than
dividing cells (Fig. 9). The VW+ dividing cells, cultivated
with Ca++, were more resistant to UV radiation than were
VW- cells, but were less resistant than were VW’+ static
cells.

Since the preceeding experiment had demonstrated that
both static and dividing cells contained equal levels of
DNA polymerase (Fig. 8), the distinct responses to UV
radiation appeared to reflect a quantitative gradient of

nuclear substance in the static and dividing cells.

52

Fig. 9. Sensitivity to ultraviolet light of growing and
static cells of Y. pestis strain EV76; (. ) VW+ cells

grown without Ca++, ( O ) VW+ cells grown with 0.0025 M
Ca++, (‘) VW- cells grown without Ca++, and (A) VW-

cells grown with 0.0025 M Ca++.

 

 

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Accordingly, the DNA concentration of these cells were

compared by chemical determination.

Quantitative DNA Determination

 

The results of diphenylamine assays revealed that
static cells contained more than twice the amount of DNA
than that present in dividing cells (Table 5). This result
was consistent with microscopic observation that static
cells contained more nuclear staining bodies than did

dividing cells (P. J. Hall, personal communication). The

 

fact that dividing VW+ cells did not contain more nuclei
than did VW- dividing cells, but were more resistant to

UV radiation than the latter, may be related to the reports
of Surgalla (109) and Brubaker (8) that production of V
and W antigens may be regulated by episomal determinants
which can often promote resistance to UV (31, 59, 103).

The foregoing experiments demonstrated that after 12
hr incubation at 37 C, the static cells were not synthesiz-
ing DNA, but had already accumulated twice the amount of
DNA as had the dividing cells. In order to determine the
cause of of the termination of DNA synthesis, the rates of
DNA synthesis prior to the 12 hr incubation period were

examined.

Mode of Inhibition of DNA Replication

 

A culture of VWfi organisms which had been incubated at

26 C for 12 hr in synthetic medium was redistributed into

‘1'?

p. 1.01.; s‘hl‘b 01.1w.

55

TABLE 5.«—DNA content of Y. pestis cells cultivated in the
synthetic med1um for 12 hr.

 

 

Added Ca++ ug of DNA per
Cell type (0.0025 M) 109 cells
vw+ 0 42.32
vw+ + 18.89
vw‘ 0 13.52
vw‘ + 17.92

 

*
Determined by diphenylamine assay.

“sum—“liv—

several flasks. In order to minimize the chance of con-
tamination during a long period of incubation the kinetics
of DNA synthesis was determined from these flasks at inter-
vals during incubation at 37 C. The absolute rates of
synthesis in VW+ dividing cells were similar in all of
these experiments. The per cent rate of DNA synthesis was
calculated by taking the value of dividing cells at each
interval as 100%. The results, as illustrated in Figure

10, indicated that both growing and static cells synthesized
DNA at almost equal rates during the first hr of incubation.
Subsequently, the rate of synthesis in static cells became
progressively reduced. Termination of synthesis in static
cells occurred after about 4 hr of incubation; a period
which was incidentally equivalent to one generation time

of VW+ cells growing in the presence of Ca++ (Fig. 1).

56

Fig. 10. Per cent rate of DNA synthesis in static (.) and
growing (0 ) cells of VW+ Y. pestis strain EV76 cultivated

at 37 C.

 

57

 

 

 

 

I00. o—o— o-——"r
80 '-
, Q
g 60 I 4
< .
M 1
g 40 '
2014. . -I
0 ‘\’_J’
3 6 9

I ‘_ up“! .'

58

Effect of Ca++ on the Growth
of Static Cells

 

When VW+ cells were plated on MGOX agar, no colonies
were observed after 40 hr of incubation at 37 C. However,
colonies appeared when the plates were further incubated

at 26 C for 2 days (Brubaker, personal communication).

 

Although Ca++ was found to promote the growth ofVW+ cells

.o‘ 1""—
v

at 37 C (53) it was not certain that this cation could

initiate division in cells which had remained static at

V .o\

 

37 C for long periods of time. I E
Further study showed that when‘VW+ cells were cultivated
in the Ca++-deficient medium and aerated at 37 C for 2 to 6
hr, the addition of Ca++ initiated growth after a significant
lag period (Fig. 11). The length of this period was in pro-
portion to the time of Ca++-starvation and the total amount
of growth of the starved cells was considerably decreased.
Growth could not be initiated by supplementation of Ca++ if
the cells were starved for Ca++ for periods longer than 8 hr
(Fig. 12). However, decreasing the temperature of incubation
to 26 C in the absence of Ca++ permitted growth to occur
following a short lag period (Fig. 11F, 12).

Thermal Induction of Growth Inhibitor
in VW' Cells

 

Witkin (120) has proposed a model for filament forma-
tion in Escherichia coli B in which UV action on the
chromosome was assumed to promote formation of a division

inhibitor. By analogy, attempts were made to demonstrate

59

Fig. 11. Effect of Ca++ on the growth of VW+ cells of Y.
pestis strain EV76 at 37 C. Cells were preincubated in
synthetic medium lacking added Ca++ for 12 hr at 26 C and
then incubation was continued at 37 C with addition of Ca++
(0.0025 M) at 0 hr (A), 2 hr (B), 4 hr (C), 6 hr (D), or

was not added (E); growth following reduction of temperature

to 26 C after 6 hr is shown in F.

 

OPTICAL osusnv (620 nm)-

1.0

0.1

1.0

60

 

 

 

 

-r—_r—fiIIIII

: ' ' ki—H
:- / !
E l
: / I
"' 3 0— 0’0
. ’0’ 1 I
C . / 0 9
O
- 4
.. / -I

 

a 12 Is ‘ 20 24
HOURS

In. lie-.1... Lug.

61

Fig. 12. This figure represents the repetition of the
experiment of Figure 11 except that VW+ cells were starved
for Ca++ for 8 hr (A) or 12 hr (B) at 37 C; (.) continuous
incubation at 37 C without further alteration, ( O) addi—

+

tion of Ca+ (0.0025 M), and (‘) reduction of temperature

to 26 C.

 

VIABI. CELLS PER MI.

III

62

 

I 7 Illll

 

 

 

I YIIIIIII

 

 

 

 

 

 

63

Fig. 13. Growth of VW+ cells of Y. pestis strain EV76
following preincubation at 26 C for 12 hr and treatment in
Afor 30 sec at 58 C (0), 55C (I), 53 C (A). and
not treated (A ); the cells were then incubated at 26 C
except for one culture ( O) which was incubated at 37 C.
In B the cells were preincubated at 37 C and were retained

at 37 C (Q) or were incubated at 26 C (O ).

VIABLE CELLS PER ML

64

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

    

 

 

I I I I I — I I I I I
I0 I:
IO _A /,,.4 : B a.
E 3 — //O:
h——- . —-I — --—I
- - 9
9 " 2
IO : : E / :
’5/9 E _. I
.I- I : 8— _
— _. I0 I l I L I
8 ‘ I I I I I
IO 1 I I I I D a
: I I F I I (.4.
: /. JIOB 8? I I I I
9 , I I I I I
'0 E 5 I0 OLF 1
E : E A
e r -
IO I I I I I l09
‘ I I I I I _.__. E E
E : : E
._._ . I. I I
e I e_ 1
IO . I I I I I I0 I I l I I
O 8 I6 24 O 8 IS 24

HOURS

- | - -..:T:'m"‘—1TCT:i-- P
l
I

65

the possible existence of an analogous growth inhibitor in
VIII+ cells which could be induced by heat. The vw+ cells
were grown at 26 C for 12 hr in synthetic medium without
Ca++ and were then heated for 30 sec at various temperatures;
incubation was subsequently continued at 26 C. The results
of this determination, shown in Figure 13, indicated that
the brief treatment with heat did not result in inhibition
of growth at 26 C. Incidentally, organisms apparently were

quite sensitive to heat since treatment at 58 C for 30 sec ;

 

was completely bactericidal. B

CHAPTER V
DISCUSSION
Compounds which can interact with cytoplasmic membranes I

are known to influence selectively the rates of growth of

both vw+ and vw" organisms (see LITERATURE REVIEW). Further- I

 

 

more, Brubaker (8) reported that a high concentration of Na+ g
and a low ratio of ionic strength to osmolarity were toxic
to VW+ organisms. This set of conditions, which may have
been responsible for the lag period shown in Figure 1, was
assumed to promote a disruption of normal membrane function.
In view of these findings, as well as the reports noted
previously concerning the loss of metabolites from obligate
intracellular parasites (see INTRODUCTION), a study designed
to detect the possible functional defects in the cytoplasmic
membranes of static X: pestis cells was initiated.

If the amount of 260 nm absorbing material or pre-
loaded 32P released by dividing VW+ cells had been less
than that released by static cells, a loss of regulatory
capacity of the cytoplasmic membranes of the latter would
have been suspected. The reverse case would have suggested
that the static cells were metabolically inactive, at least
with regard to macromolecular synthesis. However, as shown

32

in Figure 6, the rates of release of P by both types of

66

67

cells were identical suggesting that significant turnover

of organic phosphate occurred in the static and dividing
cells and that these compounds were retained by a functional
cell membrane.

In another direct test of membrane function, isoleucine
was accumulated by static cells at a rate commensurate with
that of growing organisms (Fig. 4). The finding that one I
amino acid can be accumulated by static organisms does not,

of course, prove that the remaining required amino acids or

 

other substances were similarly transported. However, the !
observation that both static and dividing cells could

synthesize protein at comparable rates (Table 3) indicated

that stasis is not caused by decreased rates of uptake of

amino acids. The rates of the energyudependent synthesis

of RNA and protein in dividing and static cells were similar

(Table 3) indicating that stasis is not caused by a limita-

tion in energy supply. The similar rates of 02 consumption

(Fig. 5) are consistent with this conclusion.

The major difference noted between the dividing and
static cells was that the latter failed to synthesize sig-
nificant amounts of DNA (Table 3). This finding in itself
would not be surprising if the rates of RNA and protein
synthesis were similarly reduced as occurs when stasis is
a consequence of or analogous to a shift-down to a minimal
medium or to one lacking some critical nutrient (82). Con-

tinued RNA and protein synthesis presumably accounts for

68

the previous observation that static organisms are larger
than dividing cells (16).

Figure 8 shows that inhibition of DNA synthesis in
static cells is not due to a lack of the polymerizing
enzyme. The possibility that inhibition of DNA synthesis
was due to deficiency in the formation of deoxynucleoside
triphosphates (dNTP) was not tested. However, thermo-
inhibition of DNA synthesis in certain E, coli mutants was
not due to this reason (37).

Selective inhibition of DNA synthesis has also been
characterized in several thermosensitive mutants of E, coli
and E. subtilis (37, 44, 57). At high temperatures, these
mutants stop DNA synthesis either immediately or progres-
sively but continue to synthesize RNA and protein. As a
result, the cells become elongated and fail to divide. These
mutants contain normal levels of DNA polymerase, synthesize
the four dNTP (37), and repair UV damage normally (26). Due
to their inability to complete a round of DNA replication,
the viability of these mutants declines rapidly.

Despite the similarities shared by these mutants and
the static cells of X. pestis, the fundamental differences
between the two are that the latter appears to have com-
pleted a round of DNA replication before the sensitivity
of DNA synthesis to high temperature becomes apparent
(Table 5),and also the cells remain viable even in the

absence of DNA replication (Fig. 11E). It is conceivable,

 

69

therefore, that the cause of stasis is a block in the
process of septation with concomitant termination of DNA
synthesis. However, thermosensitive mutants defective in
septum formation usually continue to replicate DNA for
several generations and form long multi-nuclear filaments
(57). Therefore, by analogy, it seems unlikely that the
termination of DNA synthesis in static cells is due to the
inhibition of septation alone. Rather it appears as if
there is another block at the reinitiation of DNA replica-
tion.

Initiation of a new round of replication appears to
be associated with four factors: 1) the availability of
an initiator complex which includes the initiator pro-
replicator and replicator described by Lark (72); 2) with
exception of fast growing organisms, the completion of an
old round of replication; 3) nick formation (creation of
3' hydroxyl ends of DNA by endonuclease action) and the
initial breakage of one chromosome strand so as to permit
strand separation and initiation of replication (68, 72);
4) and the crucial value of cellular mass/DNA ratio (29,
82).

The finding that static cells have completed a round
of replication obviously eliminates the second factor as
being associated with inhibition of replication. Nick
forming ability by static cells was not tested directly.

However, the finding that cell-free extract of static cells

70

already contained primer DNA (Table 4) indicates that
endonuclease activity was present within static cells. Due
to lack of information concerning the normal values of
cellular mass and DNA in the growing cells, it is difficult
to evaluate whether an abnormality of the mass/DNA ratio
could lead to inhibition of reinitiation of replication.
However, one might expect to observe reinitiation of DNA
synthesis after termination of replication because static

cells apparently synthesized RNA and protein (mass) at

w-s‘mm'fi.‘ul_ flu !
. l

normal rates (Table 3). The discovery that static cells
did not reinitiate DNA synthesis after 4 hr incubation
(Fig. 10), suggests that the fourth possibility is not
the major cause of replication inhibition. This leaves
an alteration of the first factor as being the most likely
explanation for stasis.

As reported by Hirota, Ryter and Jacob (57), Helmstetter
(48), and Smith and Pardee (105), the cell membrane-associated
protein required for cellular division and the initiator
complex of cell replication are both heat labile. Therefore,
raising the temperature from 26 to 37 C could possibly
result in the inactivation of both systems. Similarly, the
same phenotypic lesion may be responsible for the inhibi-
tion of septation and initiation of replication since it is
still unclear whether the replicator and the site of septum
formation are the same cell membrane components (98).

Inouye and Guthrie (60) reported that a membrane—associated

71

protein of E. 391i apparently coordinated the cell division
and replication through the membrane. Furthermore, it has
been shown in gram-positive organisms that mesosomes serve
as the sites of chromosome attachment and septum formation
(98, 99). These findings may suggest that the same membrane
component is responsible for initiation of replication and
septation. On the other hand, Smith and Pardee (105) demon—
strated that heat labile proteins required for cell division

and DNA replication were different since heat inactivation

‘1'}?3” shun-naming ..__
. ~. 1.; .

did not inhibit these two processes to the same degree.

The foregoing discussion leads to the prediction that
cell division would be observed if static cells were cultiv
vated under unrestricted conditions (such as addition of
Ca++ or temperature shift back to 26 C) prior to the synthesis
of new DNA. The best method to demonstrate this would, of
course, be to show that one cell division could occur when
the synthesis of DNA was blocked by thymine starvation or
antibiotics such as mitomycin C or nalidixic acid. However,
thymine auxotrophs which retained all other genetic charac-
ters of the VW+ cell could not be obtained and attempts to
use nalidixic acid and mitomycin C were not successful
(Table 2).

An alternative explanation exists. Although attempts
to induce a thermo«dependent inhibitor of growth were not
successful (Fig. 13), the possibility that such an inhibitor

was produced after a longer period of incubation at 37 C was

72

not excluded. The existence of a growth inhibitor may
account for the observation that the period of delay between
addition of Ca++ to static cells and commencement of division
increases in proportion to the length of the period of Ca++
starvation (Figs. 11 and 12). When the concentration of
this hypothetical inhibitor exceeds the threshold level,
Ca++ might no longer be able to initiate growth. In this LA

case, it is necessary to assume that the phenotypic expres-

sion of this inhibitor is temperature dependence and that

'1.r

 

its production is inhibited by Ca++, since growth was E
observed if Ca++ was included in the initial culture medium I
or if the temperature was reduced to 26 C. Assuming that
the presumptive inhibitor is freely permeable, it seems
likely that spent culture medium of static organisms would
also inhibit the growth of VW- cells at 37 C. This phe-
nomenon may have been observed in the experiments reported
by Brubaker and Surgalla (15) where static VW+ cells
inhibited the growth of streptomycin-resistant VW- organ«
isms in the presence of antibiotic.
At present it is difficult to define with certainty
the role of Ca++ in the process of cell division. Grula
and Grula (45) reported that Ca++ and Mn++ could overcome
the inhibitory effect of cycloserine. Burgoyne, Wagar and
Atkinson (18) found that Ca++ was required for the action
of endonuclease in rat liver nuclei. As discussed previ-

ously, the priming of DNA synthesis in static cells seems

73

to be normal,thus the possibility that Ca++ is required for
endonuclease activity seems unlikely. Considering that
this cation is mostly known for its function in the stabi-
lization of protein structure (46, 115) and that the effect

+ ++
and Sr

of Ca++ in Yersinia cells is replaceable by Zn+
(53), it seems possible that these divalent cations are
required to stabilize the subunits of heat sensitive initi-
ators of cell division and DNA replication (57); they could
also be required for the binding of membrane-associated

replicator and septum forming site(s). These possibilities

cannot be distinguished on the basis of current knowledge.

It is known that V and W antigens are readily secreted

by static cells cultivated in the Ca++—deficient medium
(16), and that addition of Ca++ results in suppression of
the release of V and w antigens (14) with concomitant
growth. These findings, as well as the fact that free W
antigen presumably originates from the cell wall or mem-
brane (8) suggests that these antigens are membrane con-
stituents which are essential for cell replication and
division. In this case, Ca++ would be required to main-
tain the structural integrity of the cell membrane at high
temperature.

If these considerations are valid, a qualitative or
quantitative difference between static and dividing cells
would be detectable by using the doublevlabeling technique

of Inouye and Guthrie (60) or by means of fractionation of

 

74

membrane proteins. Moreover, monospecific antibody against
V or W antigen, should, in the presence of membrane com-
ponents of VW- cells, fix complement providing that these
proteins are not inhibitory to complement activity. Further
investigation of this kind should clarify the roles of V
and W antigens.
Ogg gt El° (94) reported that spent culture filtrates L
of VW’ mutants promoted the growth of VW+ organisms. Simi— 3
larly, Delaporte (27) found that UV irradiated populations 1
of filamentous g, 921;, which evidently lack the ability to I
form septum (114), could be induced to divide in the presence
of diffusable substances from normal growing cells. Sub-
sequent study by Adler gt'gl. (1) indicated that these
division-promoting substances consisted of a heat stable
and a heat labile cellular constituent. To some extent,
these findings support the notion that static cells lack
functional sites of DNA replication or cell division.
Assuming that this concept is correct, a very obvious
question one may ask is, then, why do VW+ cells undergo
bacteriostasis when introduced into the simulated mammalian
intracellular environment? This question is difficult to
answer at present, but, as stated by Janssen and Surgalla
(66), only VW+ cells seem to be capable of multiplying
within fixed macrophages. It therefore seems likely that
the ability of these cells to release V and W antigens
and to undergo bacteriostasis is essential to intracellular

growth.

75

The findings reported in this dissertation may relate
to the similar situation that occurs in obligate intracel-
1ular parasites where, by definition, the expression of
stasis in Z£E£2.is constitutive. The failure of some of
these organisms to grow ip_yitgg is undoubtedly caused by
an inability to generate endogenous high energy compounds
or to maintain physiological concentration gradients (91).
However, these limitations do not always exist as illus-
trated by rickettsial parasites (see INTRODUCTION). The
phenomenon of stasis in these organisms may thus be analogous

to that in Yersinia.

 

CHAPTER VI
SUMMARY
1. Potentially virulent VW+ cells of X. pestis EV76

cultivated in simulated mammalian intracellular conditions ITH

were static but possessed functional cytoplasmic membranes

 

as judged by concentration of l4C-isoleucine, release of fi'
3. I

P, and consumption of oxygen at rates comparable to those
of dividing cells cultivated with Ca++.
2. The rates of protein and RNA synthesis were
essentially identical in dividing and static cells. However,
the synthesis of DNA ceased in static cells about 4 hr after

the removal of Ca++. During this period of time, which
corresponded to one generation in the presence of Ca++, the
static cells completed one chromosome replication as judged
by a two-fold increase in content of DNA and a corresponding
degree of resistance to irradiation with UV light.

3. The termination of DNA synthesis in static cells
was not due to a deficiency in DNA polymerase, but rather
due to an inability to initiate a new round of chromosome
replication.

4. Apparently the bacteriostasis of VW+ cells is

caused by thermo-inactivation of membrane-associated proteins

76

77

required for the initiation of DNA replication and the

formation of septae.

, 'nr—f

 

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Appendix A. Modified synthetic medium of Higuchi, Kupferberg

and Smith (53).

 

 

 

 

 

Constituent g per L Constituent g per L
Amino Acids Vitamins

L-Glutamate 12.00 Thiamine HCl 0.001
L—Phenylalanine 0.40 Ca—panthothenate 0.001 *r
L-Methionine 0.24 Biotine 0.0005

L-Valine 0.80 Other Additions ,.
L-Leucine 0.20 D-xylose* 10 0 I
L-lysine HCl 0.20 Phenol red 0.01 ‘
L-Proline 0.80
L-Threonine 0.16 Salt Components Molarity
Glycin 2.00

DL—Alanine 0.40 KZHPO4 0.025
L-Tyrosine 0.20 Citric Acid 0.01
L-Argine HCl 0.20 K-Gluconate 0.01

L-Isoleucine 0.50 NH4-acetate 0.01

L-Cysteine Hc1* 0.175 MnCl2 0.000001
L-Tryptophane* 0.04 FeCl2 0.0001

MgCl2 0.02

 

*
Separately sterilized.

solution).

(Vitamins combined as a Single

89