Ema 1 01:35,;

ABSTRACT

CHROMOSOME TRANSFER IN SALMONELLA PULLORUM MEDIATED BY
F-PRIME FACTORS CARRYING SALMONELLA GENES

By

Otis Webester Godfrey

When F-prime factors carrying Salmonella genes are introduced into

 

Salmonella pullorum they are able to initiate transfer of the chromosome.

 

A partial linkage map of §, pullorum has been derived using this genetic

system. The linkage map of §, pullorum compared with that of Salmonella

 

typhimurium appears to have an inverted cysB trp region, and a transposed

 

 

thr locus. The FT59 (pyrB+) factor was isolated from Salmonella abony»
and the FT71 (trp+) and FT77 (cysE+ pyrE+ rfa+) factors were isolated

from §, gyphimurium. Both the FT59 and FT71 factors in §, pullorum mobi-

 

lize the chromosome in the opposite direction than when in S, typhimurium.

 

The FT77 factor in g, pullorum appears to localize in an area between the
pro and ilv loci and to transfer the chromosome in two directions. There

appears to be good fine structure homology between g, typhimurium and §,

 

pullorum since crosses between them mediated either by transduction or
conjugation yield recombination frequencies which are analogous to intra—

species crosses.

CHROMOSOME TRANSFER IN SALMONELLA PULLORUM
MEDIATED BY F-PRIME FACTORS CARRYING

SALMONELLA GENES

BY
2"!"

Otis W. Godfrey

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

1969

ACKNOWLEDGEMENTS

I wish to express my sincerest appreciation to Dr.
Delbert E. Schoenhard who has made this work possible. It
is only by his interest and motivation that this work was
completed and my scientific education continued.

During the course of this study, I was supported in
part by a departmental assistantship.

This thesis is dedicated to Marie, Steven and

George.

ii

TABLE OF

LIST OF TABLES . . . . . . .

LIST OF FIGURES . . . . . .

INTRODUCTION . . . . . . . .

LITERATURE REVIEW

Part

I.

II.

III.

IV.

VI.

Early History . .

Chromosome Mobilization

The F factor .
"F" fimbriae .

CONTENTS

Nature of the conjugal tube . . .
Model of chromosome mobilization

Transfer replication
Chromosome mobilization

donors . . .
Role of female

Gene pseudoinversion

Recombination . .

by rec—

Recombinationless mutants . . . .

Segregation . .

Episome Chromosome Interaction . . .

Campbell's model

Primary F—prime strains . . . . .
Secondary F-prime strains . . . .

Consequence of episome integration

Conjugal Systems in Other Genera . .

Phylogenetic Relation between Escherichia

 

coli and Salmonella typhimurium .

 

iii

Page
vi

viii

13

13
14

15
15
l6
l6
17

18

21

Part

VII.

Salmonella pullorum . . . . . . . . .

 

MATERIALS AND METHODS

RESULTS

Part

I.

II.

III.

IV.

Chemicals . . . . . . . . . . . . .
Bacteria . . . . . . . . . . . . .
Media . . . . . . . . . . . . . . .
Mutagenic treatment . . . . . . . .
Determination of UV sensitivity . .
Bacteri0phage sensitivity . . . . .
Technique of bacterial mating . . .
Scoring unselected markers . . . .
Cross-streak method . . . . . . . .
Transduction . . . . . . . . . . .

Feasibility of a Conjugation System in
Salmonella pullorum . . . . . . . .

 

Ultraviolet sensitivity . . . . . .
HYbrj-ds O O O O O O O O C O O O O 0
Recipient nature of S. pullorum . .

Isolation and Characterization of Donor
Strains of S. pullorum . . . . . .

Isolation of donor strains . . . .
Gene transfer . . . . . . . . . . .
UV stimulation of gene transfer . .

Stability of F factors in S. pullorum

Episome transfer . . . . . .

Enrichment of Donor Strains for Increased

Fertility O C I O O O O O O O O O I

Temperature sensitive episome . . .

Fluctuation method for the isolation
of donor strains . . . . . . . .

Replica-plating . . . . . . . . . .

Mapping Studies . . . . . . . . . . .
Prolonged matings . . . . . . . . .
Linkage analysis . . . . . . . . .

Kinetic studies . . . . . . . . . .

iv

Page

23

24
24
24
32
33
33
34
37
37
38

39

39
42
42

45
45
51
53
53
55
55
55

57
6O

60
60

67
72

Part

V. Transduction

VI.

S. pullorum .

VII.

DISCUSSION

Part

I.

II.

 

0

Linkage Map of S. pullorum .

Comparison of Linkage Maps S.

to S. typhimurium . . . . .

III.

IV.

SUMMARY . . . .

LITERATURE CITED

pullorum

Orientation of the cysB trp Region in

Criteria of Conjugation in S. pullorum

Chromosome Mobilization directed by the
FT77 Factor

Chromosome Mobilization in S. pullorum .

Page

78

87

88

94

98

103
104
110

111

ll.
12.

13.

14.

15.

16.

LIST OF TABLES

Characteristics of Salmonella pullorum
recipient strains . . . . . . . . . . . .

 

Characteristics of Salmonella strains . . .

 

Characteristics of Escherichia coli strains.

 

Donor strains of Salmonella pullorum . . . .

 

Conjugation frequencies between Salmonella
pullorum and Salmonella typhimurium . . .

 

 

Inheritance of Unselected markers by re-
combinants selected from a cross between
SBl72 and M8367 O O O O O O O O O O O O 0

Recipient ability of Salmonella pullorum . .

 

Partial characterization of S. pullorum,
S. typhimurium and S. coli . . . . . . .

 

 

Gene transfer . . . . . . . . . . . . . .

Stability of episomes in Salmonella

pullorum . . . . . . . . . . . . . . . .

Episome transfer in Salmonella pullorum . .

 

 

Attempt to enrich donor strains . . . . . .

Fertility of F-prime donors in crosses with
different recipient strains of Salmonella

pullorum . . . . . . . . . . . . . . . .

Gene transfer by a Salmonella typhimurium

strain possessing the FT71 (trpT factor).

 

 

Gradient of transfer . . . . . . . . . . . .

Linkage analysis of the M8810 x M8374 and the
M8807 x M8369 matings . . . . . . . . . .

vi

Page

25
26
27

28

43

44

46

49

52

54

56

59

61

63
64

68

Table

l7.

l8.

19.

20.

21.

22.
23.

24.

Occurrence of unselected donor markers
the cross M8806 x M8369 . . . . . .

Occurrence of unselected donor markers
the cross M8809 x M8369 . . . . . .

Occurrence of unselected donor markers
the cross M8808 x M8369 . . . . . .

Summary of time of entry experiments .

Transduction of various markers to 8.

Bullorum O O I I O O O O O O O I 0 O
Cotransduction of trp—3 and cysBl . .

Orientation of the cysB trp region . .

Recombinant analysis . . . . . . . . .

vii

Page

70

71

73

83

85
86
89

91

LIST OF FIGURES

 

Figure Page
1. Linkage map of Salmonella typhimurium . . . . . 29
2 0 UV senSitiVj-ty O O O I O O O O O O O O O O O O 40

3. Time of entry from the M8807 x M8369 mating . . 74
4. Time of entry of the episomal trp+ gene . . . . 76

5. Time of entry from the M8809 x M8369 mating . . 79

6. Time of entry of the episomal cysE+ gene . . . 81
7. Linkage map of Salmonella pullorum .. . . . . . 96

 

8. Linkage map of Salmonella pullorum and
Salmonella typhimurium . . . . . . . . . . . 99

 

 

viii

INTRODUCTION

Jacob, Brenner and Cuzin (62) proposed a model for
chromosome mobilization during conjugation which was an ex—
tension of their replicon model. They pr0posed that the
Hfr chromosome results from the integration of the F factor
into the bacterial chromosome. Once the F factor is inte-
grated into the chromosome, its replication is controlled
by the host cell chromosome.

Campbell prOposed a specific insertion model with
high predictive value (19). He suggested that the episome
becomes associated with the bacterial chromosome by a single
reciprocal crossover which integrates the episome linearly
into the chromosome. The frequency at which the episome
integrates into and detaches from the chromosome is a func-
tion of the degree of homology between the two replicons.
The Campbell model predicts that an F—prime factor is formed
if a crossover occurs between sites bracketing the inte—
grated F+, rather than at the original sites. Scaife and

Pekhov (100) demonstrated that an Escherichia coli strain

 

possessing an F-prime factor carried a deletion on its
chromosome corresponding to the chromosome genes carried by

the F-prime factor.

The chromosomal fragment carried by a F-prime factor
confers a high affinity for the homologous region of the
chromosome causing frequent integration at that site (98).
The frequency of chromosome transfer is determined by the
frequency of donor crossover between the F-prime factor and
the chromosome (99). Consequently, in a culture of inter-
mediate donors approximately 10% of the cells transfer the
chromosome with the same orientation of the parental Hfr.
Integration of a sex factor is usually studied by observing
its ability to mobilize the bacterial chromosome during
conjugation.

Bacterial conjugation occurs in several genera, but
most experimentation has involved either S. Sgli_K-12 or

Salmonella typhimurium. Though the guanine to cytosine

 

ratio of S. typhimurium and S. coli K-12 are the same, 50%,

 

 

(118) there is a considerable difference in genetic fine
structure. Both low transduction frequencies (31) and poor
$3.!SE2 nucleic acid hybridization (81) between the two
genera have been used to demonstrate the lack of fine struc-
ture homology. Divergence of the two species at the chromo—
somal level is evidenced by the inversion of the pygngygS
EEE region (60,95,96,97). Otherwise, the sequence of genes

in S. typhimurium is very similar, if not the same as that

 

found in S. coli K-12 (95,105).

Transduction between Salmonella pullorum and S. typhimuri-

 

 

Sm_was reported by Schoenhard in 1963 (117) and has been demonstrated

many times since then. The transfer of an F-lac+ episome
from S. coli to S. pullorum and back again, as well as be-
between S. pullorum was described by Robinson in 1964.(117).

Since then, F—prime factors carrying S. typhimurium genes

 

have been reported (95). It seemed clear that the time was
right to develop a conjugation system in S. pullorum by use

of F-prime factors isolated from and carrying S. Syphimuri-

 

E§_genes. If this conjugation was successful then it would
be possible to determine the gross structure of the S.
pullorum chromosome. To these ends this research was done.

S. pullorum unlike most Salmonella species is non—

 

motile and does not produce H28. It is a slow growing
species which has been classified as a group D Salmonella
(somatic antigens 9 and 12) in the Kaufman White Schema
(34). S. pullorum is the causative agent of fowl typhoid
and is commonly isolated from chickens (body temperature 41-

43C) while S. typhimurium is usually isolated from mice

 

(body temperature 36-38C). However, there is a high degree
of fine structure homology between S. pullorum and S.
typhimurium as shown by the high transduction frequencies

obtained from interspecies crosses (117).

LITERATURE REVIEW

Part I. Early History

In 1946 Joshua Lederberg, a Ph.D. student of Tatum,
discovered conjugation in bacteria. He assumed that the two
parental types of bacteria were equal partners, and the fu-
sion between them led to the formation of fully diploid zy-
gotes. The wild type of _ E; 59;; ’ strain used in
this work was labeled K-12 (71).

Employing streptomycin sensitive and resistant

strains of 1“"”E:'c01i " ’ K-12, Hayes (51) found that the

 

viability of one of the strains was essential to the fer-
tility of the cross. From this experiment, he concluded
that conjugation was a heterothallic system in which recom-
bination is mediated by a one-way transfer-of genetic materi—
al from the donor to the recipient bacterium. This one-way
transfer was further substantiated when Hayes (52) demon-
strated that pre-treatment of donor cells with ultra vio—
let light increased the yield of recombinants as much as
50-fold while pre-treatment of the recipient cells de—
creased the yield of recombinants.

In bacterial conjugation a donor is characterized by

the presence of an autonomous, transmissible genetic element

which is designated F, for fertility (20). There are three
types of unique genetic elements that confer donor ability
on the bacterium.within which they reside. Luria (76)
termed these genetic elements "conjugans," they are the F
(fertility) factors, the CF (colicinogenic) factors and the

RTF (resistance transfer) factor.

Part II. Chromosome Mobilization

The F factor. The F factor is a small piece of de—

 

oxyribonucleic acid (DNA) with a molecular weight of
4.5x107 daltons, which is comparable to about 2% of the

S. coli ' chromosome. The F factor exists as a cir-

 

cular piece of double stranded DNA (41,42). The DNA of the
F factor can code for 40-80 genes, but only a few gene func-
tions are presently known.

It would appear that the F factor contains few if
any chromosomal genes, since there is no preferential origin
and direction of F-mediated gene transfer as is the case
with F—prime containing males (2). However, since 40% of
the DNA of the F factor hybridizes with chromosomal DNA from

S, coli, ' ‘ (35), it would appear that the two repli-

 

cons are phylogenetically related. The largest fraction of
F DNA (9/10) has a GC ratio of 50% like the chromosome of

Escherichia, Shigella and Salmonella. The other fraction of

 

 

F DNA (1/10) has a GC ratio of 44% (35,93).

The autonomous male fertility factor may be elimi-
nated from the cell by treatment with acridine dyes (57).
However, the acridines do not inhibit the replication of
the F-factor when it is integrated into the host cell chro-
mosome (56). It is thought that the acridine dyes prefer-
entially inhibit multiplication of the autonomous F—factor
(107).

Only a few gene functions have been directly re-
lated to the presence of the F-factor in a bacterial cell.
The most actively studied F-gene function has been the bio-
synthesis of a specialized fimbriae designated as "F" fim-

briae (18).

"F" fimbriae. Fimbriae, first referred to as

 

"pili" (17) are a widely distributed class of bacterial sur-
face appendages. Two types of fimbriae have been reported
(110). They are structural and "F" fimbriae. "F" fimbriae

are rods 2-10x103

nm in length with a diameter of 8.5nm and
an axial hole 2.0—2.5nm in diameter. They are generally
longer and thicker than structural fimbriae and they often
have a knob of variable shape on their distal end (82).
"F" fimbriae are recognized by their selective adsorption
of a male specific bacteriophage (111).

Male specific phage were first isolated by Loeb in

1960 (72) as a phage that would form plaques on lawns of E-

coli K-12 donor strains but not on recipient strains (F').

 

However, Hourichia and Adelberg (58) were unable to demon-

strate plaque formation in strains of Proteus mirabilis har-

 

 

boring the F+-factor, but they could show an increase in
phage titer when these cells were infected with the male

specific phage.

Nature of the conjugal tube. The nature of the

 

union between conjugation partners and the extent of the
material transferred is not entirely clear. Bridges between
cells consisting of cytoplasmic extensions 100 to 300 nm in
width have been repeatedly observed by Anderson in elec-
tron microscope preparations of conjugating cells (3).
Brinton, Gemski and Carnahan (18) argued that these cyto-
plasmic extensions were artifacts since these bridges were
frequently seen joining male cells. They suggested that

the true conjugal bridges were the "F" fimbriae.

If "F" fimbriae are involved in bacterial conju—
gation and phage penetration then conjugation should inter-
fere with phage invasion and phage invasion should disrupt
bacterial conjugation. Silverman et al. (104) have per-
formed this type of experiment and their results indicated
that conjugation and phage penetration were mutually ex-
clusive.

If the large diameter bridges described previously
by Anderson are the true conjugal bridges, then one might

expect extensive transfer of protein and RNA through them

during conjugation. If "F" fimbriae of 2.0-2.5 nm in dia-
meter are the conjugal bridges, then the passage of mole-
cules having a Stokes radius greater than 2.5nm.would be
restricted. Rosner (92) did not find a significant trans-
fer of B-galactosidase during mating between F+ and F- cells. Sil-
ver (103) found that the transfer of labeled ribonucleic acid
(RNA) or protein from Hfr to F‘ cells did not exceed the
lower limit of detection of about 1%.? '. Thus the trans-
fer of material other than DNA, if it occurs, appears to be
relatively minor in bacterial conjugation. Since chromo-
somal DNA has not been found in "F" fimbriae (113) the na-
ture of the union between the two conjugation partners re-

mains unanswered.

Model for chromosome mobilization. Jacob, Brenner

 

and Cuzin in 1963 (62) proposed a model for chromosome mo-
bilization during conjugation which was a simple extension
of their replicon model and is currently the model in vogue.
They proposed that the Hfr chromosome results from the fu-
sion of the F factor and the bacterial chromosome. Once the
F factor integrates into the chromosome, its replication is
controlled by the host cell chromosome. This integrated F
factor is thought to be in juxtaposition with both the plas-
ma membrane and the "F" fimbriae. The event of conjugation
triggers the start of a new round of replication beginning

at the "F" replicator, such that the origin, which is the

lead point in transfer, is duplicated first. Genetic trans-
fer is coupled necessarily with DNA replication. Conse-
quently the transferred molecules are synthesized at the
time of mating. One replica is driven into the female cells
by the same forces which ensure DNA replication.

The Hfr donor transfers its genes in a polarized
fashion beginning at the origin (location of F factor) and
proceeding sequentially along the linkage group until the
integrated sex factor is reached and is itself transferred.
Random chromosome breakage during transfer terminates the
transfer process and results in a higher number of early
markers near the origin being transferred than late markers
(64).

Jacob and Wollman (64) followed the appearance of a
series of donor markers in recipient cells as a function of
time by separating the mating pairs at various times with
a Waring Blendor. By this procedure,called interrupted mat-
ingjthey were able to translate the distance between genes

into time units.

Transfer replication. The predominance of evidence

 

seems to suggest that DNA synthesis in the male is neces-
sary for chromosome transfer. Barbour (6) found that F'lac
transfer was severely restricted in the presence of Nali-
dixic Acid, which is a known inhibitor of DNA replication.

Gross and Caro (48) studied chromosome transfer in Hfr

10

males using quantitative autoradiography and concluded that
the DNA is replicated prior to or during transfer. They
also concluded from the intensity of beta tracks that it
was double stranded DNA that was transferred during conju-
gation. Freifelder (40) also demonstrated that the DNA
transferred during conjugation was replicated prior to or
during transfer. He mated a thymine-requiring cell in the
presence of S-bromouracil and was able to show the presence
of 5-bromouraci1 in the recombinant by virtue of its sensi-
tivity to ultraviolet light. However, Cohen et a1. (24)
demonstrated the presence of single stranded DNA in "mini

cells" when they were mated with F+ donor cells of S. coli

 

K-12.

Chromosome mobilization by rec' donors. Implicit

 

in the model of Jacob et a1. (62) is the concept that
chromosome transfer during conjugation is mediated by an
integrated sex factor. Thus, chromosome transfer by F+
cells is due to a small fraction of the cells being Hfrs.
However, Clowes and Moody (23) did find chromosome transfer
mediated by certain conjugans (F, F-prime, coIVQ and c013)
in recombinationless (rec') donor strains. In these strains
the chromosome was transferred at a decreased level and
there was no preferential origin of transfer even with F-
prime strains. Furthermore, Curtiss and Stallions-(28)

found that only about 10% of the recombinants formed in

ll

F+x1f'matings were due to stable Hfr donors. Thus it ap—
pears that chromosome mobilization does not depend entirely
on recombination or even transient association of the two

replicons; conjugans and chromosome.

Role of female. Some 11 years ago, Fisher (38)

 

published experiments which demonstrated that only the male
cell required an available source of energy during mating;
therefore, the female was assumed to play a passive role in
conjugation. It is currently believed that this conclusion
was wrong and that the female actively participates in DNA
transfer during conjugation.

Freifelder (40) mated Hfr and F'lac male strains of

S. coli K412 5’ with purine requiring recipients. He

 

found that mating in the absence of purine markedly reduced
the yield of recombinants and concluded that DNA transfer
required some, as yet unknown, function of the female. Bon-
hoeffer et a1. (13) found that the donor genome is not
transferred to a temperature sensitive recipient at the
restrictive temperature. He concluded that the recipient
strain contained a component which was necessary for DNA
synthesis in the recipient and for transfer of DNA during
conjugation. Spelina (106) mated synchronously dividing
Hfr cells with randomly dividing F' cells and found that re-
combinant formation was not influenced by the time in the

division cycle at which the male cells were taken. When

12

synchronously dividing female cells were used with randomly
dividing Hfr cells, the yield of recombinants was greatly
influenced by the time in the division cycle of the female.
The highest yield of recombinants occurred with females
which were in the middle of the division cycle. This cor-
responded nicely with Helmstetter's data (54) which indi—
cated that DNA replication began when bacteria were in the

middle of cellular division.

Gene pseudoinversion. Pittard and Walker (89) em-

 

ploying both proximal and distal unselected markers found
that an obligatory interaction occurred between the donor
and recipient DNA molecules in the region immediately adja-
cent to the lead end of the transferred DNA. It has been
shown also that markers transferred early during conjuga-
tion are integrated at a lower frequency than more distal
markers (74,45). Thus, the true order of genes located
near the origin has to be deduced by an obligatory analysis
of the genetic constitution of several classes of recom-
binants. The explanation for the "pseudoinversion" of
genes located near the origin is thought to be due to the
presence of F-DNA on the lead region of the transferred
DNA (27). The F portion of lead segment has little homology
with the recipient chromosome thus exerting an anti-pairing
effect and reducing effective synapsis of the respective

alleles. This would necessitate that the obligatory

13

recombination event must occur at a more distal locus, thus
reducing the recombination frequency of genes located 1-2
min from the origin as compared to genes located 5 min from
the origin (74,23). After the lead region of the donor DNA
has recombined with the recipient chromosome it is assumed
that the female winds in the DNA. This would in part assure

effective synapsis of the respective alleles.

Part III. Recombination

The central problem of bacterial conjugation seems
to be the very process of recombination itself. One might
reasonably ask how two DNA molecules get close enough to
enable switching of exact and identical nucleotide sequen-
ces. The phosphate groups on the DNA are all negatively
charged under the normal physiological conditions of the
cell. If the DNA strands did pair closely enough to ex-
change bonds, strong forces of repulsion would have to be
overcome. Further, the phosphate ester bonds are rela—
tively stable bonds, and considerable energy would be re-

quired to break and reform them.

Recombinationless mutants. Since it seemed likely

 

that enzymes participate in the events leading to the for-
mation of the completed recombinant DNA structure, Clark
and Margulies (22) undertook the isolation of mutants in

which one or more of the hypothetical recombinant enzymes

14

would be defective. Two mutants were isolated that were un-
able to form recombinants with suitable Hfrs. These mutants,
designated rec", were found to be much more sensitive than
the parental strain to the effects of ultraviolet light.
Flanders (59) found a definite correlation between the rec‘
mutation and the inability of rec" strains to excise pyri-
midine dimers and repair other DNA lesions. Oppenheim and
Riley (85) also demonstrated that enzymes participate in
recombinant formation. They showed that gene integration

in bacterial conjugation involved a physical association

mediated by covalent bonds of parental DNA molecules.

Segregation. The measurement of segregation fre-

 

quency in merozygotes gives information about the fate of
the donor genome from the moment of its insertion until the
moment of pure recombinant formation. Bresler (16) was not
able to demonstrate essentially haploid clones until at
least three to nine generations after termination of mating.
He assumed that the exogenote was replicated with the endo-
genote and gradually integrated. However, Wood (115) found
widely different segregation patterns depending on the Hfr
strain employed. Thus, during conjugation there is a time
period during which a transient merozygote exists.

In 1951, Lederberg et al. (70) discovered the presence

of stable merozygotes in an F“ strain of S: coli K-12.

 

This strain multiplied mainly as a partial diploid cell,

15

segregating a few haploid recombinants at a low frequency.
This strain was thought to possess a defective recombinase.
Low (75) studied the inheritance patterns of cells from a
Hfr x F‘ rec“ mating and found that the rec- zygotes and
their progeny retained the alleles of both parents. The
behavior of these rec’ zygotes bears a remarkable similari—
ty to the stable merozygotes previously described and it is
becoming clear that usually the rec’ recombinants are not

normal.

Part IV. Episome Chromosome Interaction

Campbell's Model. In 1962, Campbell proposed a

 

specific insertion model with high predictive value (19).
Since then, the model has been extensively tested in sev-
eral systems and extensive experimental evidence has been
obtained in support of it. Campbell suggested that the
episome becomes associated with the bacterial chromosome
by a single reciprocal crossover at which time the episome
integrates linearly into the chromosome. The mechanism
that detaches an episome :jjs" exactly the reverse of that
causing insertion, and seems to be a function of loop for-
mation which allows synapsis followed by crossing over.
The frequency at which the episome integrates into or de-
taches from the chromosome is a function of the degree of

homology between the two replicons. This model accounts

16

for the rare alteration between the F+ and Hfr states of

the F factor (64).

Primary F-prime strains. The Campbell model predicts

 

that an F-prime factor is formed if a crossover occurs be—
tween sites bracketing the integrated F+, rather than at
the original sites. Such an event would yield a circular
F-prime factor and a chromosome with a deletion correspond-
ing to the chromosome fragment incorporated by the F—prime
factor. Scaife and Pekhov (100) demonstrated that an S.
ggli strain possessing an F-prime factor carried a deletion
on its chromosome corresponding to the chromosomal genes
carried by the F-prime factor. This parental strain pos-
sessing the F-prime factor is termed a primary F-prime
strain; it is an F-prime strain descended directly from an
Hfr . Primary F-prime strains would be expected to lack

a region of homology between the replicons and thus chromo-
some transfer would be randomly oriented and the recombinant
yield would be of the order found with the F+ factor.
Scaife and Pekhov (100) demonstrated this with the primary

F-prime strain that they originally isolated.

Secondary F-prime strains. F-prime factors are

 

transferred autonomously to the recipient during conjuga-
tion, and they are converted to intermediate donors uwhiCh
are designated secondary F—prime strains . The chromosomal

fragment carried by an F-prime factor confers a high

l7

affinity for the homologous region of the chromosome in the
intermediate donor, and this affinity results in frequent
integration at that site. Consequently, a culture of inter-
mediate donors contains approximately 10% of cells which
are able to transfer the chromosome with the same orienta-
tion as the parental Hfr. The other cells continue to
transfer the F-prime factor in the autonomous state (29,99).
A secondary F-prime strain when compared to the parental
Hfr will show a delay in time of transfer of a marker equal
to that needed for transfer of the F-prime factor (98,100).
This delay is defined as the lead time, whereas dead time

is defined as the time interval from contact formation un-

til gene transfer.

Consequence of episome integration. It is implicit

 

in Campbell's model that the episome is linearly integrated
into the chromosome. Pittard (87) found that when the sex
factor is integrated at a site between two cotransducible
genes, their linkage is markedly reduced. Mapping studies
also indicated that the F-prime factor is integrated by in-
sertion into the chromosome. This conclusion was drawn
from the analysis of chromosomal markers transferred by an
F-prime strain (98,100).

If an episome integrates by insertion into the chro-
mosome, then the insertion within a gene should inactivate

that gene. Beckwith et al. (9) employed this concept to

18

direct the transposition of an Hfr derivative. They em-
ployed the S. gel; strain that was sensitive to T6 phage

and possessed a temperature sensitive F'lac replicase.

They grew this strain in the presence of T6 phage at the
elevated temperature with lactose as the only energy source.
Employing this procedure, they were able to isolate Hfr de-
rivatives with the episome integrated in the region coding
for T6 phage receptor sites. The rationale of this experi-
ment was two-fold: (1) once integrated the replication of
episomal genes would be accomplished by the host chromosome
replicase system and (2) integration within a gene coding
for a phage receptor site would inactivate that gene and

the cell would then be phenotypically resistant to the phage.
In other experiments Beckwith et a1. (9), employing the tem-
perature sensitive sex factor isolated by Cuzin and Jacob
(29), were able to isolate both clockwise and counterclock-
wise transposition Hfrs. These experiments demonstrated
that a gene inversion and/or transposition was not neces-

sarily a lethal event.
Part V. Conjugal Systems in Other Genera

The predominance of work done with bacterial conju-

gation has employed fertile strains of S: coli.

 

K-12. Conjugal systems have been established in a number
of other strains and genera of bacteria by application of

the basic concepts gleaned from the K-12 system.

l9

Chromosomal markers have been transferred by mating

E. coli ‘ . K-12 Hfr donors with Sg’typhimurium j'

(7), Salmonella typhosa (8), and Shigella (76,102) recipi-

 

 

 

ents. Such matings result in an apparent lower frequency

of chromosomal transfer than with S. coli Hfr x S. coli F"

 

 

matings. Usually the former type matings result in the for—
mation of unstable partial diploids (7,44).

Genetic compatibility by sexduction does not require
that the interacting organisms display a similarity in DNA
base composition, since the transferred material does not
need to exchange with the resident chromosome (35). A num-
ber of conjugans have been transferred from S,

coli K-12 to strains of Shigella, Salmonella, Serratia and

 

 

Proteus at a high frequency (2). In Serratia and Proteus
strains infected with the S. coli F-factor, the DNA band
profile obtained by density-gradient centrifugation shows

a minor band identical to S. coli DNA. If the F-exogenote

 

is lost from these strains either spontaneously or by curing!
the satellite DNA band disappears (35). This allows a
unique way of isolation and study of S. 29;; conjugans.

Shigella x Shigella matings do not yield recom-
binants, even when Shigella cells are used which had re-
ceived the F-factor from S. 3211' This might possibly be
due to the lack of homology between the F-factor and the

Shigella chromosome (76).

20

The F—factor from S. coli was first introduced into

Salmonella abony, which subsequently served as a donor of F

 

to other Salmonella strains. S. abony strains possessing

 

the F+ factor were mated with S. abony recipients and
yielded recombinants at a frequency similar to that found
in S. coli matings. Since chromosome transfer could be

demonstrated Makela (78) attempted to isolate Hfr Salmonella

 

donors from F+ strains which had been subjected to ultra
violet light and replicating onto a recipient. All attempts
were futile using this procedure. Hfrs were isolated by

Sib selection, and in many cases a high percentage of the
apparent Hfrs transferred the F-factor at a high frequency.
From an S. SSggthfr (SW1444) an F-prime factor FT59 was
isolated. This factor when introduced into other Sglmf
onella strains transferred the chromosome in the same
direction and with the same orientation as the Hfr from
which it was isolated (79,80).

Schneider and Falkow (102) developed another method
of Hfr selection. They obtained an Hfr by terminal marker
selection in a cross between an Hfr S. 2211 and a Shigella
flexneri recipient. The resultant Shigella Hfr had the
same gene sequence as S. 9211-

Conjugation and recombination have been reported to

occur in Pseudomonas aeruginosa (73). The conjugan respon-

 

sible for these events (FP) behaves very much like the F-

factor of S. coli K12. A mating system also has been

21

discovered in Pasteurella pseudotuberculosis (68). A strain

 

of Vibrio cholera which produces a bacteriocin that kills

 

vibrios has been found to yield recombinants when mixed

with certain Vibrio cholera strains which lack the deter-

 

minant for bacteriocin production (12). In 1956 Belser and
Bunting (10) studied what they thought was a conjugation

system in Serratia marcescens. However, in 1963, DuSh-

 

man (33)proved that their results were mainly the consequence
of syntrophism.

Part VI. Phylogenetic Relation
Between Escherichia coli and

Salmonella typhimurium

 

 

Even though the guanine to cytosine ratio of.S;
typhimurium and S,_ggli are the same, 50%, and they have
approximately the same gene sequence (96,95,109), a consider-
able divergence in fine structure homology has been detected
by fine structure analysis as indicated by the inability to
transduce genes between the two genera (31). These genetic
results :»results: earlier in vitro nucleic acid hybridiza-
tion studies (81), which demonstrated poor homology between
the two genera. The corresponding genes are not truly
allelic, although enough chromosomal homology exists to al-
low genetic exchange when larger units are transferred by

conjugation (109,95).

 

In both S. typhimurium and S. coli the trp-cysB-pyrF

genes are localized within a short segment of the chromosome

22

but are, comparatively speaking, inverted (95). If
evolution proceeds by the integration of new genetic
information into simpler clustered sequences, then a
diffusion of the clusters must ensue. Since no known
genes other than gySB are located between £52 and pySF

in both S. typhimurium and S. coli then strong selec-

 

tive pressure must have existed during evolution for
maintenance of the EEEIEYEB‘EYEF cluster in these
species (105).

Ino and Demerec (60) suggest that S. 2911 and

S. typhimurium have diverged in two distinct fashions

 

that are of potential evolutionary significance. Di-
vergence at the intra-genic level is indicated by low
transduction frequencies between the two genera and
divergence at the chromosomal level is demonstrated by
the inversion of the SgpfgygB-EXSF region (97). This
knowledge of gene orientation is important because of
its bearing on models of gene transcription and regu-
lation. It is also important to realize that the en-
vironment would be eXpected to play a large role in
diffusion, inversion and/or transposition of gene

clusters.

23

Part VII. Salmonella pullorum

 

S. pullorum is commonly isolated from chickens
and is the causative agent of fowl typhoid. It is slow

growing in comparison to other Salmonella species. It

 

is classified as a group D Salmonella strain by the

 

Kauffman White Schema and has an antigenic structure of 9,
12. The somatic factor 12 is the presumed receptor for
PhageP22.

Schoenhard (117) was able to transduce genes from

S. typhimurium to S. pullorum employing the PLT-22 phage,

 

thus indicating fine structure homology between the species.
Robinson and Schoenhard (91) demonstrated that S. pullorum
could accept and transfer conjugans at a high frequency

with S. coli K-12 donor and recipient strains.

MATERIALS AND METHODS

Chemicals. N'-methy1-N'-nitro-N-nitrosoquanidine

 

(NTG) was obtained from Aldrich Chemical Company, Milwau-
kee, Wisconsin. Sodium azide was obtained from Distilla-
tion Products Industries, Rochester, New York. All other

chemicals employed were reagent grade.

Bacteria. S. pullorum, strain M835, was selected
as the prototype organism from the stock collection of
Dr. D. E. Schoenhard; it was designated wild type. S.
pullorum, strain M835, and auxotrophic mutants derived

from it are described in Table l. S. typhimurium and S.

 

coli K12 strains used in this experiment are described in
Tables 2 and 3 respectively. The donor strains of S.
pullorum which were isolated during this investigation

are listed in Table 4. The linkage map of S. typhimurium

 

is shown in Figure l. The chromosomal distribution of
relevant markers and the point of origin and direction
of transfer of the various male strains used in this in-

vestigation are located on the linkage map.

Media. The E minimal medium was that described

 

by Vogel and Bonner (112). Sterile D-glucose was added

to a final concentration of 0.5%. The A minimal medium (119)

24

25

Table 1. Characteristics of Salmonella pullorum recipient

strains.

 

 

 

 

Strain Mating Relevant Genetic Origin
No. Type Markersa or Ref.
M535wb F: cysAl cstl 1eu-l (66)
M818 F_ 1eu-l M835
M8350 F_ strAl M818
M8351 F_ strAl tyr-l M8350
M8352 F_ strAl ilv-2 M8350
M8353 F_ strAl ser-l M8350
M8354 F strAl met-1 M8350
M5355 F- strAl trp-l M5350
M8356 F: strAl leu-2 M8350
M8357 F_ strAl thy-1 M8350
M8358 F_ strAl pro-2 M8350
M8359 F_ strAl thr—2 M8350
M8360 F_ strAl ara-l M8350
M8361 F_ strAl xyl-2 M8350
M8362 F_ strAl gal-2 M8350
M8363 F_ strAl glyAl M8350
M8364 F_ strAl his-1 M8350
M8365 F_ strAl his—l pro—1 M8364
M8366 F_ strAl his-l pro-l thr-l M8365
M8367 F_ strAl his-l pro-l thr-1 ilv—1 M8366
-M8368 F_ strAl his-l pro-l thr-l ilv-l azi—l M8367
M8369 F_ strAl his-l pro-1 thr-l ilv-1 gal-1 M8367
M8370 F_ strAl his-l pro-1 thr-1 ilv—l gal-l xyl-l M8369
M8374 F_ strAl pro-1 thr-l ilv-l gal—l M8369
M836 F cysAl cstl leu—l trp-2 M835
M837 F: cysAl cstl leu-l his-2 M835
M881 F_ leu—l cysEl M818
M882 F_ 1eu-1 cysEl glyA2 M881
M890 F_ leu-l cysEl ilv-3 strA2 M881
M8100 F_ leu-l cysBl M818
M8103 F_ leu-l cysBl trp-3 M8100
M8104 F_ 1eu-l cysBl trp-3 his—3 M8103
M8105 F 1eu-l cysBl trp-3 his-3 gal-3 strA3 M8104

 

aWhere possible, we have used the same symbols as
Sanderson (95) and have followed the conventions suggested
by Demerec et al. (30) for genotypic and phenotypic symbols.

bW = wild type (streptomycin sensitive).

CM581, 82, 90, 100-105 isolated by B. Klooster.

6
2

I .mcflmnum Ncoao .m mum QUHQB
«momm cam mmmm mo coauomoxm 0:» suns ssaussasmwu .m 0H6 cmnanummo maamupm Haas

 

 

 

msamm mamHmMas sound um measmz
cmfiuumm QMmmo um mmaamz
cmEuumm mummmo um wmaamz
cmsnnmm soanoum um mmaems
casuumm Hmoaommo um amasmz
casuumm omammo um owasms
scams aloud slugs Human moaumuu um madam:
ammmm moanduu um ooaemz
um omems
Amps 6650065 +5 amsmm
comuoocom A+omn mumm Mmmov thM\mmmmm>o mmVIumE .m mmmdm
oomnmocmm A mus mnmm mm>m+awxv mth\ommmm>o mmwlumfi .m mmmfim
ism .6mv cemumscmm A+musa +mmsm +mumv msBM\msH->Hfl swamnsm mammmo Nmamuu .5 macaw
Asa .6mc somumocmm x+aupv Haem\msan>afl oaamusm mammao «mamas .m amosm
Ammo casunmm A+manv omBh\Homnmum ammuuwm assumes .m ommmm
Amps cmsuumm x+mummcmmBM\ .m mmmm
16V :msuumm +0ma mum\moancuo .m ammmm
samumocmm maaumm Hum mmmam
newswocmm mHMHOm Hum mmmam
newsmccmm maaumm sum ammam
amsnumm NRNHMmMB songs Hum «samm
unsymmm omuamm mmomfln mum mamsm
oocmnwmmm oUHSOm mumxumz some .02
oaumsmw usm>mamm mcflumz :Hmnum

 

 

 

m.msflmuum waamcoEHmm m0 moaumflumuomumnu .m manna

27

Table 3. Characteristics of Escherichia coli strains.

 

 

 

 

Mating Relevant Genetic
Strain No. Type Markers
AB257 Hfr met
AB3ll Hfr thr leu
AB312 Hfr thr leu +
AB785 F' met/F-lac
3349 F; his ile/F-his
W6 F_ met
AB113 F_ his leu thr
MSE311a F thr leu

 

aM8E311 isolated as an M82 insensitive mutant of
AB311.

28

.hommz Eonm oo>flumom

 

 

In- 61+mupv Hasm\~-muu 6-65s Husma Human Hammo .m oammz
mam: x mmmam A+mmu +mumm +mmxov s596\ausma NSSHS Hmmso .m momma
mmms x mmm<m A+mwu +mumd +mm>0 +mev mssm\ausma maaam Hmmso .m momma
6mm: x sawsm x+mu0c H556\mudnu Husma Human Hamso .m sommz

mmmz x mmmm A+mnsmv mmBM\Husma Hemmo Amman .6 mommz
5mm: x ommmm 1+mflnv omem\man: Husma Hemmo Hammo .m mommz
5mm: x mvmm +mflsumxmumas H-565 Human Hammo .m sommz
mmmz x 6mmmm +omH mumxausma Hamso Hammo .m mommz
mmmz x mmsma 06H sxansma Hemmo Hammo .m momma
mmms x emmmm + Husma Hemmo Hammo +m Hommz
mmmz x 63 Husma Hemso Hamso +m comm:
mswumz mane mnmxuoz mmwe .oz cflmuum
Eoum Um>flumo owuocww “co>mamm mcflumz

 

 

 

.EsHoHHnm mHHmGOEHom mo mcflmnum Hocoo .v magma

29

Figure 1. Linkage map of the S. typhimurium chromosome
showing the points 3f origin andvdirection
of transfer of the various male strains used.
The Hfr strains are indicated on the circle
and the F—prime factors in the expended
portion.

 

FT59
PyrB
P‘u A 'h'
" em

1222 136$A534 _ ,0

 

SA 536
strA x
E
3 3 1..., gal K
arg E 102
91
A
‘ SA 535 I cys

65 frp

his

Illa-“'-

F180

 

Linkage Map ‘01

Salmonel lo typhimurium
mm‘m’efl Wm

31

lacking citrate was used for the detection of carbohy-
drate utilization. Filter sterilized carbohydrates

were added to a final concentration of 0.2%. Where
necessary, amino acids were added to a final concen-
tration of 25 ug/ml. L broth and L agar containing 10g
of tryptone (Difco), Sg of yeast extract, and 109 of

NaCl per liter of distilled water were employed for
routine cultivation. Nutrient broth consisting of 8g
nutrient broth (Difco) and 5g NaCl per liter of distilled
water was employed in dilution and plating of mating mix-
tures. Difco agar was used at 1.5% final concentration
unless otherwise stated. MacConkey Agar Base (Difco) and
Levine Eosin Methylene Blue Agar without lactose (BBL)
were routinely employed for the detection of carbohydrate
fermentation. The pH was adjusted to 7.1 and after auto-
claving, a filter sterilized carbohydrate solution was
added to a final concentration of 1%. The glucose con-
centration of the various carbohydrate sources was deter-
mined by the Glucostat (Worthington). Bacto SIM Medium
(Difco) was employed for the detection of sulfide and
indole production. The medium described by Ball (5) was
employed for the detection of motility. Dihydrostrepto-
mycin Sulfate was added to a final concentration of 1200

pg/ml in minimal media and 250 ug/ml in complete media.

32

Mutagenic treatment. Mutants isolated during

 

the course of this work were induced by NTG following
the method recommended by Adelberg, Mandel and Chen (1).
Five ml of logarithmic phase cells (2 x 108 cells/ml)
growing in E minimal broth were washed by filtration

and then resuspended in 10 ml of TM buffer pH 6.0 con-
taining 100 ug of NTG/ml. The suspension was incubated
for 20 min at 37C with shaking and then 1 ml was fil-
tered to remove the excess NTG. These treated cells were
resuspended in 10 m1 of E minimal broth properly supple-
mented to permit growth of the desired mutant type and
incubated with aeration for at least five generations.

Also penicillin treatment as described by

Gorini and Kaufman (47) was employed to enrich for the
desired mutants. Ten m1 of the NTG treated suspension
(5 x 108 cells/ml) were centrifuged and the pellet re-
suspended in 1 m1 of E minimal broth. A 0.1 ml aliquot
of the resuspended pellet was placed in 10 ml of E mini-
mal broth supplemented with 10% sucrose, 0.5% glucose,

0.01 M MgSO and other growth requirements of the parental

4
cell type. The culture was grown with aeration for two
generations or 3 hr and Penicillin G then added to a final
concentration of 2,000 units/ml. The suspension was in-
cubated at 37C without shaking. After 4 hr incubation,
when about 50% of the cells had become spheroplasts, the

action of penicillin was stOpped by chilling and the

33

culture centrifuged. The pellet was resuspended in 10 m1
of E minimal broth properly supplemented to permit growth
of the mutant type. After two to three cyles of penicil-
lin enrichment the cells were plated on L agar and the

mutants isolated by the replica plating technique.

Determination of UV sensitivity. Logarithmic
8

 

phase cells (1 x 10 cells/m1) growing in L broth were
centrifuged and then resuspended in an equal volume of
A minimal broth. A 3 m1 sample of cells was placed in
an open glass 60 cm2 petri dish at a distance of 48 cm
from a horizontal, 30-watt General Electric G30T8 germ-
icidal lamp emitting primarily at a wavelength of 253.7
nm. The suspension was shaken during eXposure to UV
light and then immediately diluted and plated on L agar.

The plates were incubated at 37C for 24 hr. Extreme care

was taken to avoid photoreactivation.

Bacteriophage sensitivity. Sensitivity or in-

 

sensitivity to the bacteriophage M82, a donor-specific
RNA bacteriOphage isolated by A. J. Clark, was used to
indicate the presence or absence, respectively, of the
F factor in the tested culture. The following procedure
was employed to detect sensitive donor strains. It is
based on the observation that S pullorum donor strains
infected with M82 will allow an increase in titer of the

phage to a maximum of 2 x 108 phage/ml even though M82

34
does not form plaques on donor strains of S. pullorum.
A lOOpful of an overnight culture was inoculated into a
tube containing 2 m1 of L broth supplemented with 200 ug
of CaClz/ml and previously inoculated with 103 M82
phage/ml. This suspension was incubated overnight at
37C without aeration. After incubation two drops of
chloroform were added to each tube and then each tube
was swirled. After incubation at 37C for 15 min a loop-
ful from each tube was placed onto a fresh lawn of S.
3211 AB312 growing on an L soft agar overlay on L agar
plate. Approximately 10 tubes could be tested per plate.
The plates were scored after 3hr incubation at 37C.
Sensitive strains carrying the M82 phage gave a clear
zone of lysis 1 cm in diameter; whereas, strains without
M82 phage produced no clearing. Extreme care was taken

to avoid transferring any excess chloroform to the lawn

of E. coli AB312.

Technique of bacterial mating. The techniques

 

employed were essentially those previously described
by Anton EE_El- (4). Overnight aerated L broth cultures
of the donor and the recipient were diluted 1:20 in L
broth followed by incubation for 3 hr at 37C without
aeration. Five ml of the donor (1 x 108 cells/ml) were

8 cells/ml) and

mixed with 5 ml of the recipient (l x 10
the mixed suspension filtered on a pre—wet membrane fil—

ter, Millipore HA 0.45M, 25mm. The filter was immediately

35

placed on a prewarmed L soft agarjplate, 0.75% agar and
incubated for 3 hr at 37C. After incubation the milli-
pore filter was removed from the agar and placed into
the ' tube containing 2 ml of nutrient broth, and
agitated with a Vortex Jr. Mixer for 60 sec to resuspend
the cells. Further dilutions were made in nutrient
broth. One tenth ml aliquots were pipetted from the
various dilutions of the mating mixture into tubes con—
taining 3 m1 of E minimal soft agar, 0.75%, kept at 45C.
The tubes were mixed and then poured onto E minimal agar
plates which were supplemented when necessary. The
plates were then incubated for 96 hr at 37C. Growth of
the donor strains and plate—mating were prevented by
omitting from the minimal medium a supplement required
by the auxotrophic donor strain, preferably a requirement
determined by the distal region of the donor genome, and
by adding 1200 ug/ml of dihydrostreptomycin to eliminate
the streptomycin sensitive donor strain.

Interrupted matings were performed as above ex-
cept that several 3ml portions of donor and recipient
were used and the millipore filters were removed from the
agar at appropriate time intervals. After the cells were
resuspended in nutrient broth the cell suspension was
transferred to a fluted screw cap test tube to further

agitate for 2 min to disrupt the mating pairs. Time zero

36

was taken as the time at which the cells were drawn onto
the Millipore Filter.
Centrifuge matings were conducted in a manner analogous

to the millipore matings. Ten m1 of the donor (1 x 108

cells/m1) was mixed with 10 m1 of the recipient (1 x 108
cells/ml) and the mixed suspension poured into a pre-
warmed centrifuge tube. The suspension was then centri-
fuged for 4 min at 10,800 x g and then incubated at 37C
for 30-60 min. After the prescribed time interval, the
mating suspension was agitated with a Vortex Jr. Mixer
for 2 min to resuspend the cells.

Interrupted centrifuge matings were performed as
above except that four to eight tubes containing 10 ml

8

of the donor (1 x 10 cells/ml) were poured into the pre-

warmed centrifuge tubes and then 10 m1 of the recipient

cells (1 x lo8

cells/ml) were added to each tube. The
mating suspensions were swirled and immediately centri-
fuged. The cells were centrifuged for 4 min at 10,800 x g
at 37C. Time zero was taken as l min after the start of
centrifugation. The centrifuge tubes were removed and
incubated as described previously. At appropriate time
intervals the mating cells were resuspended and 2 ml of
the suspension was transferred to a fluted screw cap test

tube to further agitate for 2 min to disrupt the mating

pairs.

37

Scoring unselected markers. Recombinant clones

 

were purified by streaking on minimal medium of the same
composition as that used for initial selection and incu-
bating for 4 days at 37C. Single colonies were then
spread into patches on minimal medium of the same compo-
sition as that used for initial selection and incubated
24-48 hr. These patches of colonies were tested for
their inheritance of unselected markers by replicating
them onto plates of medium appropriately supplemented.

In instances where streptomycin was not used for counter-
selection it was necessary to rule out cross feeding by
the donor strain and this was accomplished by replicating
the above master plates onto a minimal medium supplemented

with the growth requirements of the donor strain.

Cross-streak method. The procedure is essentially

 

that described by Berg and Curtiss (11). Young logarith-
mic phase donor cells were diluted to a concentration of
2 x 102 cells/ml in L broth. One ml aliquot portions were
then pipetted into 200 Wasserman Tubes and incubated at
37C for 8 hr without shaking. After 8 hr incubation the
cell concentration in most tubes was approximately 1 x 108
cells/ml. Approximately 0.2 ml of a log phase broth cul-
ture of the apprOpriate recipient tester stock was applied
in two parallel vertical lines to the surface of a properly

supplemented E minimal agar plate. After the streaksin~

had dried, loopfuls of the donor cultures were streaked

38

horizontally across the recipient. Using this procedure
20 cultures could be tested per plate. After 72 hr in-
cubation at 37C the number of recombinant colonies in
each streak was scored. In many instances the donors
were pretreated with NTG or UV light immediately prior

to being pipetted into the Wasserman tubes.

Transduction. Transduction studies were made with

 

P35 phage. This phage was isolated from S. pullorum by
zygotic induction. Each donor lysate was prepared after
the phage was purified by three separate plaque isolations
on the donor strain. An isolated plaque was fished into
a tube of L broth containing log phase cells of the donor

(1 x 106

cells/ml) and incubated overnight at 37C with
aeration. The phage bacterial mixture was thencentrifuged
at 10,800 x g for 10 min and the supernatent stored over
chloroform at 5C. Transduction was effected by mixing an

9 cells/ml recipient bacteria

overnight culture of l x 10
with phage at a multiplicity of infection of one. The
mixture was incubated at 37C for 15 min after which 0.1 ml
aliquot portions were pipetted into 3 m1 of E minimal

soft agar maintained at 45C and then plated on E minimal
agar, supplemented where necessary. The recipient was
tested for reversion, and the phage for contamination.

The frequency of transduction was defined as the number

of transductant colonies per absorbed or input P35 phage.

RESULTS

Part I. Feasibility of a conjugation
system in Salmonella pullorum

 

In order for a conjugation system to be develOped
in a species of bacteria it is necessary that the cells
possess the necessary enzymes to catalyze recombination
and the ability to accept, maintain and transfer fertility
factors. In addition, the discovery of a conjugation sys—
tem in another genus or species is facilitated if the

population is a homogenous recipient type.

Ultraviolet sensitivity. Since there exists a

 

definite correlation between the ability of a strain to
mediate genetic recombination and the ultraviolet light
sensitivity of the strain, the UV sensitivity of S. E217
l2£EE.MS35 was compared with that of a good recipient

strain, S. typhimurium MSTlOO. Log phase cells of each

 

strain were resuspended in minimal A broth and irradiated
with shaking at a distance of 48 cm from the lamp. From
the survival curves shown in Figure 2 it appears that S.

pullorum compared to S. typhimurium is not unduly sensi-

 

tive to UV light. This indicates that S. pullorum possesses
at least some of the enzymes required for recombination.

39

40

Figure 2. Comparative survival of S. pullorum M835
and S. typhimurium MSTlOO exposed to various
UV doses.

 

 

90'.)
o

.. sunvwmsfl FRACTION _.
l ('11 1
I 90/
/ 4
/’ ’ l
:/

 

 

,7
0. msnoo ‘
M535
.0 ‘ 10 ‘ .20 '30
uv DOSE ( sac. AT48 CM )

42

Hybrids. A number of different S. typhimurium Hfrs

 

were mated with S. pullorum recipients. These matings were
done to determine the ability of S. pullorum recipient

strains to form recombinants with S. Syphimurium Hfrs and

 

to produce recombinants that could be genetically analyzed.
The data obtained from these matings are listed in Table 5.

In the case of SA535 and 88172 good recombination frequen-

 

cies were found with both S. Syphimurium and S. pullorum
recipients. The hybrids produced in these matings were
stable, prototrophic and infertile. The latter conclusion
was based on the insensitivity to M82 phage and the absence
of F-prime factors. The hybrids obtained from the SBl72 x
-MS367 mating were analyzed for their inheritance of un-
selected donor markers and the data are presented in Table
6. There does not appear to be a high co-inheritance of
any donor markers like that previously described for some
recombination—less strains of S. 32;; (75). When the S.

typhimurium Hfrs SA534, SA536 and 219 were mated with S.

 

pullorum recipients very low recombination frequencies
were obtained. A possible explanation for this might be
gross gene transpositions in the lead region of the trans-

ferred DNA.

Recipient nature of S. Pullorum. To determine the

 

recipient nature of S. pullorum approximately 100 cells of

the recipient strains, S. coli, S. typhimurium and S. pul-

 

lorum were spread on single L agar plates. The plates were

43

Table 5. Ikxxmbinafion frequencies between S. pullorum and
S. Syphimurium cultures.

 

 

 

 

Frequency
a (Recombinants per
Mating Marker Selected Donor Input)
SA534 x MST119 pro: 7 x 10:?
trp 5 x 10_
SA534 x M5367 thr: 4 x 10_;
pro <1 x 10
SA535 x MST120 cysA: 8 x 10::
SA535 x M8363 glyA 2 x lO_7
SA535 x M8367 ilv+ 5 x 10_7
:25: i : igjg
his <1 x 10
SA536 x MST119 pro: 6 x 10::
SA536 x M8367 ilv+ 2 x 10_7
:36: i : i3:§
his <1 x 10
55172 x M8367 ilv: 3 x 10:i
:25: 3 i 133%
his 1 x 10
219 x MST120 cysA: 1 x 10:;
219 x M8363 glyA 2 x 10
M8807 x MST119 hisib <1 x 10:3
t2? 2% i: 18-8
p +C -8
M835 x MST119 thr <1 x 10

 

aProcedures for these crosses are described in
Materials and Methods.

bOn each plate plaques were observed at a frequency
of 4 x 10‘5/donor cell. One was selected, purified and des-
ignated P35 by W. Olsen of this laboratory.

cNo phage located on these plates.

dThe recombinants from each mating were found to
be both infertile and M82 insensitive.

44

Table 6. Inheritance of unselected markers by recombinants
selected from a cross between SBl72 and M8367.

 

 

Selected Phenotype
Unselected

 

 

a
Phenotype 20 20 20 20
Ilv+ Thr+ Pro+ His+
11v: -- 2/20b 2/20 0/20
Thr+ 0/20 -- 10/20 2/20
pro+ 0/20 8/20 -- 4/20
His 0/20 0/20 0/20 --

 

aNumber of recombinants analyzed.

bNumber possessing unselected marker per number
possessing the selected marker.

45

incubated 24 hr at 37C after which the develOped colonies
were replica plated onto plates of E minimal agar which

had previously been spread with 109 cells of an appropriate
donor strain. The replica plates were incubated at 37C for
48 hr and then observed. The results are presented in
Table 7. Since 74-100% of the S. pullorum recipient cells
formed recombinants with the Hfrs it may be said that S.
pullorum is a uniform recipient population in its initial

ability to mate with both S.typhimurium and S. coli Hfrs.

 

Thus it is evident that the recipient ability of S. pul-
lorum, like S. typhosa, is not confined to special cells,

as has been shown with S. typhimurium (84).

 

Since S. pullorum is able to repair UV damage, is
able to form stable and normal recombinants with S.

Syphimurium Hfrs, is a uniform recipient pOpulation, is

 

transduced with P22 phage, and accepts and transfers
episomes at high frequencies, it is a likely species for

the establishment of a conjugation system.

Part II. Isolation and characterization
of donor strains of S. pullorum

Isolation of donor strains. The male specific

 

bacteriophage M82 was used to detect the presence of the
F factor in S. pullorum. M82 does not form plaques on F+
and F-prime strains of S. pullorum. However, it is ab-

sorbed at a low frequency by these strains and appears

46

.mmmpocmm unocHQEooou may you o>auooamm gamma on ooumam moaamou swap oum3 monoumm

Ummoam>mo one

coma mumz mmflsoHoo Hmoofl>flpcfl mo monouom

.Hamm¢ Eoum om>flnm© Dogfium> 0>Hpflmgmmcfl mm: on ma Hammmzo

.mnamm Eoum pm>flumo possum> 0>flpflmcomcfl mm: :0 ma mnasmz

.Nmz on 0>Huflmcmm muoz mpcmcflneooou cosmeusm saw mo 0:02

.Uhm um .Hn am How oogmnsocfl mm3 nowsa .mumam Homo A no no
.Hn am How Cum #0 omuonsocfl can soon
A owno onmoupm mama ucmcwnfiooou comm mo mousuaso sponn A usmflano>o

.mmmsd mm: on
mufi>wpflmcmm How oopmmu mama ocflume comm Eouu mpamgHQEoomH oa ummoa “do

 

 

 

 

1| av com +man maamd Hammmz
:1 av oom +mfln wwmmz oaammmz
manmum ooH omv +ma£ maamm Hammd
cannons: vb omv +ma£ vmmmz. Hammd
:1 av oom +H£u maaamz thBmz
II Hv cow +>HH mmmmz omnaamz
magnum ooa oom +H£u mHHBmz NhHmm
magnum ooa coca +>HH mmmmz thmm
mpcocHQEoomm mucmcHQEoomm po>uomoo pmuomamm ucmflmfloom Hocoo
mo mcHEuom mmflcoaoo meumz
mafiaanmum mmacoHoo no 005552
p mo pcmo mom
.mmmmuocom unscaneoomn m>fluommmmu map Hom.Eduoaaom .m mo muflaflnm Dcofimflomm .h magma

47

to propagate despite the failure to form plaques. These
results are quite similar to those described with Pro—

teus mirabilis carrying an F-factor originating in a

 

K-12 strain.

F—prime factors which carry Salmonella genetic

 

material are labeled "FT" followed by a number; the "T"
indicates that the particular chromosomal gene on the F
factor is a Salmonella gene (95). In the case of FT80
(his+), the histidine gene may be of an S. 99;; origin.
(personal communication with P. Hartman). From one "‘~
S. ESSEX Hfr strain (SW1444) an F-prime factor
called FT59 was obtained (78). The factor carries EYES!
is readily transmissible and converts bacteria acquiring
it into donors of the same type as SW1444 (O-thr-leu--pro

------ purA). The FT71 factor was isolated by Sanderson

(94,95) from S. typhimurium SU422 (Hfr B2). This factor

 

carries the entire tryptophan Operon and converts bac—
teria acquiring it into donors of the same type as Hfr

BZ (O-his-str----leu--—-cysB). The origin and direction
of chromosome mobilization of the F-prime factors used in
this research are illustrated in Figure l.

The F+ factor was introduced into S. pullorum by
mating M835 with S. ggSS_W6 and S. SSgSy_SH634. Log
phase culturesof the donor (1 x loacells) were millipore-
mated with the recipient (l x 108cells) for 30 min at 37C.

The membranes were inserted into nutrient broth dispensed

48

in fluted test tubes and agitated with a Vortex Jr. mixer
to remove the cells from the filter and to separate the
mating pairs. The W6XM835 mating mixture was plated on

dry EMB-lac plates and the SH634XM835 mating mixture

plated on dry L agar plates. The plates were incubated

at 37C for 24 hr. S. pullorum colonies were differentiated

from S. coli W6 colonies on EMB-lac agar by their color

 

since they do not ferment lactose. S. pullorum colonies
are much smaller than SH634 colonies on L agar. S. BEEF
S953m_colonies were then picked and inoculated into tubes
of L broth, incubated overnight as a still culture at 37C
and tested for sensitivity to M82 phage. Tubes containing
M82 sensitive cells were then subcultured to SIM media and
observed for hydrogen sulfide production and motility.
These cultures were also tested for the presence of in-
dole. The cultures appearing to be S. pullorum were then
streaked on L agar and incubated 24 hr at 37C. After in-
cubation individual clones were tested for their response
to Group D antisera and their requirement for cysteine

and 1eucine. M8800 and M8801 resulted from these efforts.
In Table 8 can be seen a partial characterization of S.

pullorum, S. typhimurium, S. abony and S. coli.

 

Isolation of S. pullorum strains carrying the
F-lac+ factor was carried out by mating M835 with 8B394
and AB785. In each instance log phase cultures of the

donor and recipient were mated on millipore filters for

49

.COMDMflDcmHoMMflU How Umuflaflpo no: n :20

.coapmgflpdammm moflam SQ pocHEHmumU soapommn mummaugmn

.mmgommmu m>fiummmc u I .mmcommmu
0>Hufimom u + .moosuoz poo MHMHHmez cfl oooflnommp mum huflafluofi mo coaumcflsuouoo
poo cofluosooum maoocfl .GOHuosooum mmm mo coflpomumo map How mousomooumo

 

 

 

I :2 :2 + wmmdm
I 52 02 + vmoam
I :2 02 + ommmm
I 32 02 + vammm
I DZ 32 + wmmmm
I 32 032 + mmmm
I + + I mvmm
I + + I mmnmm
I + + I m3
+ I I I mmmz
an msouw ESHmMApgm o muflaflpoz cofluoooonm mcofiuoopoum gamuum
maamcoaamm on mmcommmm maoocH mmm

 

 

 

.. .. I .38 .m E...
ESHHSEflnmmu .m .Ncoom .m .ESHOHHsm .m mo cofluoufinmuomumso Hafiuuom .m manna

 

50

30 min and then the cells were resuspended in nutrient
broth. The suspension was then plated on A minimal agar
supplemented with cysteine, 1eucine and lactose. After
incubation at 37C for 96 hr the recombinants were re—
streaked on the same selective medium and reincubated.

The purified recombinants were then tested for H 8 pro-

2
duction, indole production, motility, slide agglutination
with group D antisera, M82 sensitivity, F-lac+ transfer
and auxotrOphic requirements for cysteine and leucine.
The outcome of this work was M8802 and M8803.

The F‘Eiif factor was introduced into S. pullorum

by mating M837 with 3349 SB890. Log phase cultures

I
of I donor and recipient were mated on millipore fil-
ters for 30 min and then plated on E minimal agar supple-
mented with cysteine and leucine. The recombinant cells
were purified and analyzed in a manner analogous to that
previously described for strains M8802 and M8803. M8804
and M8805 were isolated and characterized by this
manipulation.

A mating between SH59 and MS35 was employed to
introduce the FT59 (pySSf) factor into S. pullorum. The
mating procedure, isolation and purification procedures
are essentially the same as those previously described

for isolation of strain M8801. From this mating, strain

M8806 was isolated.

51

Donor strain M8807 carrying the FT71 (EEEI) factor
was isolated after a 60 min millipore mating between SU694
and M836. M8807 was purified in a manner analogous to
M8805. One hundred purified recombinants were tested fOr
their sensitivity to M82 phage and their ability to trans-
fer the FT7l factor. Approximately 90% were sensitive to
M82 phage but only one of the recombinants was able to
transfer the FT71 factor as indicated by the cross-streaking
method.

The FT76 and FT77 factors were introduced into S.
pullorum by mating SA523 and SA532 respectively with M882
on millipore filters. After 30 min incubation at 37C the
mating mixture was plated on E minimal agar supplemented
with glycine and 1eucine. The resultant donor strains,
M8808 and M8809, were analyzed and purified by techniques

previously described.

Gene transfer. In order to determine the ability

 

of donor strains to transfer genes to appr0priate recipient
strains, overnight aerated L broth cultures of the donor
and recipient strains were diluted 1:20, incubated 4 hr at
37C without aeration and l x 109 donors were mixed with

l x 109 recipients and mated for 3 hr on a millipore filter.
The results of these matings are shown in Table 9. It is
obvious that detectable chromosome mobilization occurred

only with F-prime strains carrying Salmonella genes and

52

.m0cmaflnmmxm H6uo>mm mo 6005660 650 ucmmmummu 6066

 

 

 

 

666:9 .moonumz 666 ma6m06062 60 663600666 606 6666000 666:» How monommooum
6-60 x 6.6 +066
-o0 x 6.0 +606
w-o0 x ~.m +>00 66mm: x 6666: 6-60 x 00 +600 66662 x 6666:
6-60 5 0V +6660 66666 x «6662
6-60 x 6.6 +606 66662 x 6666: 6-60 6 0V +065 6666: x 6666:
6-60 5 0V +6606 M666: 5 «6665
6-60 x 00 +066 6666: x 60662 .
6-60 x 0v +606 66662 x 6666: 6-60 5 00 +066
6-60 x 6.0 +060 66662 x 6666: 6-60 x 00 +606
6-60 5 0V +060
6-60 x 0 +6606 66662 x 6666: 6-60 6 0V +>00
6-60 x 0v +600 66662 x 6666: 6-60 5 0V +606
0-60 x 6.0 +606 66662 x 66666 0-o0 x 6.6 +660 6666: x mommz
6-60 5 0V +6660 6666: x 6666: 6-60 5 0V +600 66mm: x 0666:
6-60 5 0V +065 6666: x 6666: 6-60 5 0V +6660 66mm: x 0666:
6-60 5 00 +6606 66662 x 6666: 6-60 5 06 +065 6666: x 0666:
6-60 5 0V +600 66666 x 6666: 6-60 x 00 +6606 mmmmz x 0666:
6-60 5 00 +066 6-60 x 0v +066
6-60 6 0V +060 6-60 x 00 +606
6-60 x 00 +>00 6-60 x 00 +060
6-60 5 0V +606 6-60 x 00 +>00
0-60 x 6.0 +606 66666 x 6666: 6-60 5 00 +606 66662 x 0666:
6-60 5 0V +600 mmmms x mommz 6-00 x 0v +600 mmmmz x 6666:
6-60 5 0V +6660 6666: x momma 6-60 5 0V +6660 6666: x 6666:
6-60 5 0V +065 6666: x 6066: 6-60 5 0V +065 6666: x 6666:
6-60 5 0V +6606 6666: x 6666: 6-60 5 0V +6606 mmmmz x 6666:
mnoa x 0v +066 mIOH 6 av +066
6-60 x 0v +606 6-60 0 0V +606
6-60 5 0V +060 6-60 x 00 +060
6-60 5 0V +>00 6-60 5 0V +>00
6-60 5 0V +606 66662 x mommz 6-60 5 0V +606 66mm: x 66666
«HHOU HOCOQ muCMCHQEOOOm mmOHU AHHOU HOCOQ m-thCHQEOUOm mmOHU
H6HuHGH ummv Umuomamm H6HMHQH Hmmv omuomamm
606656606 mogmsomnm
.06666600 6660 .6 60669

53

at a frequency of only one recombinant per 106 - 107 donor

cell input. However, the F factors themselves were trans-
ferred at frequencies from 0.14 to 0.40 per donor cell

input.

UV stimulation of gene transfer. Log phase S.

 

pullorum donor strains M8800, M8801, M8802 and M8803 were
resuspended in A minimal broth to a concentration of 1 x

108

cells per ml and exposed to UV light at a dosage which
produced 50-75% reduction in colony forming ability.

These irradiated cells were resuspended in L broth, incu-
bated 1-3 hr at 37C and then millipore—mated with M8369
and plated on media selective for Pro+, Ilv+, His+, Thr+

and Gal+ recombinants. No recombinants were detected.

Stability of F factors in S. pullorum. One pos-
sible explanation for the low fertility of the S. pullorum
donor strains might be the spontaneous curing of the F
factor in these strains. To test this possibility, over-
night cultures of each donor were streaked on L agar and
incubated 24 hr at 37C. Individual colonies were selected
and tested either for their sensitivity to M82 phage or
the ability to transfer the F-prime factor to an apprOpriate
recipient. The results are presented in Table 10. It
appears that the episomes are very stable in S. pullorum
and thus spontaneous curing of the F-prime factors does

not account for infertility of the donor strains.

54

 

 

 

Table 10. Stability of episomes in S. pullorum.
F-prime Transfer
Number of Per Cent (Per Cent of
Colonies Sensitive to Initial Donor
Strain Episome Observed M82 Phage Cells)
M8800 F: 50 100 NTa
M5800b 5+ 160 3 NT
M8801 F + 20 100 NT
M8802 F—lac+ 20 NT 100
M8802C F-lac 85 NT 22
M8803 Ftslac 60 NT 90
M5804 F-his+ + 20 NT 100
M8805 FT80his 20 NT 100
M8806 FT59pyrB 20 100 NT
M5807d FT7ltrpi 40 NT 100
M8807 FT7ltrp+ 20 NT 100
M5807e FT7ltrp + 50 NT 36
M8808 FT77cysE 50 NT 38

 

aNot tested.

bTested after 9 months storage at room temperature.

CTested after 6 months storage at room temperature.
dTested after 1 month storage at 5C.

eTested after 6 months storage at room temperature.

55

Spisome transfer. Another possible explanation for

 

the low level of chromosome transfer may be that the donor
strains cannot transfer DNA efficiently. The data from
Table 9 demonstrates that F—prime factors are transferred
at frequencies ranging from lO-25%, but the mating periods
employed were 3 hr which is extremely long. The matings
therefore were repeated with 5 x 107 donor cells mixed
with 5 x 108 recipient cells and mated on millipore filters
for 30 min at 37C. The results reported in Table 11 show
that S. pullorum can transfer and receive F-prime factors
at good frequencies.

Part III. Enrichment of Donor Strains
for Increased Fertility

Temperature sensitive episome. One method to in-

 

crease the fertility of a donor strain would be to culture
them under conditions favoring integration of the fertility
factor into host cell chromosome. S. pullorum strain M8803
carries the Ftsiégf factor. The autonomous replication of
the mutated sex factor is normal at 37C but inhibited at
42C. This was demonstrated by the fact that 99.99% of the
overnight aerated L broth cultures of M8803 incubated at
42C were cured of the Ftslggf factor; whereas, only 10-20%
of the overnight cultures grown at 37C were cured of the
Ftslggf factor. I made the hypothesis that M8803 should

be enriched for these cells possessing the integrated sex

56

 

 

 

Table 11. Episome transfer in S. pullorum.

Frequency

(Per Cent of

Cross Episome Transferred Initial Donor)

M5802 x M5350 F-lac+ 35
M8803 x M8364 F-his + 29
M8805 x M8364 FT80(his+) 3a
M8807 x M8355 FT7l(trp + + 2
M8809 x M881 FT77(cysE+ pyrE rfa ) lla
M8806 x M8350 FT59(pyrB ) 39
AB785 x M835 F-lac+ 22

 

aM82 sensitivity used to detect presence of F-
factor in recipient.

57

factor after several subcultures in L broth each followed
by a subculture to A minimal lactose broth. Thus, 103-104
cells M8803 were inoculated into L broth and grown over—
night at 42C with aeration. A 0.1 mi aliquot of the over-
night culture containing 1 x 107 cells/ml was then inoculated
into 10 m1 of A minimal broth supplemented with 0.2% lactose
and Casamino Acids respectively. This culture was then
incubated at 42C with aeration until it reached a titer of

109

cells per ml at which time it was recycled through the
subculture sequence. Seventy to ninety per cent of the
cells plated at the end of each subculture cycle produced
lactose positive colonies. The subculture procedure was re-
peated several times. This culture of strain M8803 enriched
for fertility was then millipore-mated with M8369 and selec-
tion made for Thr+, Pro+, Ilv+, Gal+ and His+ recombinants.
In no instance were recombinants detected, but the indi-
vidual M8803 colonies still remained temperature sensitive.
No significant curing of F-Sggf factors, either F—Sggf or
FtsSggf was obtained during the course of this research,

when either Acridine Orange or Acriflavin were used to cure

the cells of the F-prime factors.

Fluctuation method for the isolation of donor strains.

 

A modified Luria-Delbrfick fluctuation method (77) was em-
ployed to enrich for strains transferring chromosomal genes.

In each instance an overnight L broth culture of the donor

58

was diluted to a concentration of 200 cells per m1 and 1
m1 aliquots were pipetted into 100-200 Wasserman Tubes.
These tubes were incubated at 37C until there were 1 x 108
cells per m1. A lOOpful was taken from each tube and
streaked across a suitable recipient on a properly supple-
mented E minimal agar plate. The plates were incubated 72
hr at 37C and the tubes were refrigerated at 5C for the
same period. The tube showing the most recombinants was
selected and 103-104cells from this tube were inoculated
each into 100-200 tubes. Each tubes was in turn incu-
bated at 37C until the average cell concentration in the
tubes was approximately 1 x 108 cells per ml; the cells
were then mated with an appropriate recipient as described
previously. Upon completion of the enrichment cycles the
enriched strains were millipore-mated for 3 hr with the
appropriate recipient strain. An overnight culture of a
non-enriched donor strain was also mated with the same
recipient as a control. From the data in Table 12 it is
evident that there is little if any enrichment of fertility
in the donor strains. A chi square test was done on the
data of the M8807 x M8354 mating described in Table 12 to
determine if the results significantly differed from that
expected due to randomness. A x2 value of 365 was obtained
with the probability of this x2 being less than 0.005,

therefore indicating non-random fluctuation. This result

is not simply explained by the instability of the autonomous

59

Ammm

0050050 00500055 00 605050000 5000650580005

"6x6

.moo.o v m 5003 006550 050

U

.692 5003 0050050 00 0508060000050

.UOu/Hmmflo @H03 mUGMGflQEOOOH OZ

 

5

0050050 00500050 050 00 905055000 5000650580005 050 66 0050000 0508500050 060096

 

 

 

 

«.0 N oom +008 6mmmz Ao9zvhommz

0.0 m com +008 6mmmz 60mm:

0.0 m 000 +050 mmmmz 0092 comm:

m.0 m oo0 +050 mmmmz mommz

o m 660 +060 06mm: 6666:

o m 000 +050 ammmz 60mm:

o m oo0 +6606 66665 66665

e 6 oo0 +600 66062 66665

o m com +050 mmmmz Aw9zv0oomm2

o 0 com +050 mmmmz comm:

o 0 oom +606 66665 oommz

5o N oom +005 66mm: comm:

6050850005m 60060 0508500050 00060 0508500055 U000000m 050050005 00505

06009 00 005852 \60559 00 005852 005062

.0506006 00500 500050 00 0580005 .N0 00569

60

and integrated nature of the F-prime factor but rather in-
dicates a more complex association of the chromosome and

episome.

Replica-plating. Several attempts were made to

 

select g. pullorum Hfrs by exposure of the cells to ultra-
violet light and then replica plating the developed irra—
diated colonies onto a lawn seeded with appropriate
recipients. In no instance were either Hfrs isolated or

F-prime strains with increased fertility detected.

Part IV. Mapping Studies

Prolonged matings. In order to produce a linkage

 

map, 5 x 108 donor cells of g. pullorum and 5 x 108 recip-
ient cells were mixed and mated on millipore filters for

3 hr at 37C. The cells were then resuspended in nutrient
broth and aliquots of the apprOpriate dilution plated on
properly supplemented minimal media. The data are pre—
sented in Table 13. With the exception of MS807, the g.
pullorum donor strains transfer chromosomal genes at a

very low frequency. From the gradient of transfer observed
with the M8807 x MS369 mating a preliminary linkage map

was constructed as follows:

gal—l pro-l ilv-l thr—l his-l

 

3 4

1.0 .42 .11 4.1 x 10' 3.1 x 10‘

61

 

 

 

 

Table 13. Fertility of F-prime donors in crosses with different
recipient strains of Salmonella pullorum.
Recombination
Frequency*
Counter- Selected (Per Initial Relative
Cross Selection Recombinants Donor Cell) Frequency
MSSOSxM8369 cysA cst strA thr: 6 x 10:: --
pro+ <2 x lO_8 --
ilv+ <2 x lO_8 --
gal <2 x lO_ --
MSBOSXMSB63 cysA cst strA glyiA l x lO_; —-
MSSOSxMSBSS cysA cst strA trp <2 x 10 --
M8806XMS369 cysA cst thr: 1.5x10:$ 1
his+ l.8xlO_8 0.12
pro+ <2 x lO_8 --
ilv+ <2 x lO_8 --
gal <2 x lO_ -—
M8806xM8363 cysA cst strA gly+A 3 x 10 7 --
M8807xMS369 cysA cst strA gal: 3.6x10:§ l
pro+ 1.5x10_4 0.42_‘2
ilv+ 2.1x10_5 5.8x10_3
thr 7.8x10 .2x10
his: 5.8x10:2 .6x10"4
M8807xMS363 cysA cst strA gly+A 1.8x10_6 -—
M8807xMS354 cysA cst strA met 5.5x10 --
M8809xM8369 glyA strA ilv: 3.2x10:g 1
pro+ l.9xlO_7 0.59
gal+ 2.4x10_7 0.08
his+ 2.0x10_7 0.06
thr 1.4xlO 0.04
M5808xMS369 glyA strA pro: 5.2x10:; 1
ilv+ 2.8x10_8 .54
gal+ 2 x lO_7 .04
his+ 1.5x10_7 .29
thr l.9xlO .37

 

The selective media were supplemented with leucine and
all the growth factors of the particular recipient strain ex-
cept that one for which selection for independence was being

made.

The known markers carried on the F-prime factor were

added as additional supplements to the media with the exception

of the M8806 x MS369 mating.

*The recombination frequency is based on a mean of at

least five plates.

times with no significant deviation.

Each mating was repeated at least three
The donor and recipient
cultures were also checked for reversion.

62

When this map is compared with the linkage map of

S. typhimurium Figure 1, it is evident that the threonine

 

locus appears to be transposed. It is also apparent that
in S. pullorum M8807 the chromsome is being mobilized in an

Opposite direction in comparison to S. typhimurium strains

 

possessing the same FT71 factor. SU694 was mated with
MST119 and the data are presented in Table 14. These data

reconfirm the fact that in S. typhimurium the direction of

 

transfer by FT71 is O-hiSf-Ehgéd-pgg ----- gygB. The gradient
of transfer for this mating and for the other matings is
shown in Table 15.

Since co-inheritance of donor auxotrophic markers
might alter the gradient of transfer, the following eXperi-
ments were performed:

1. M8807 was mated with M8368 with sodium azide
solely used for counterselection.

2. M8810 was mated with M8374 with just the ab-
sence of histidine used for counterselection.
M8807 was mated with M8369 with just the ab-
sence of cysteine used for counterselection.

In all instances the gradient of transfer was found to be

O-pro—ilv-thr-his.

 

The FT77 and FT76 factors in S. typhimurium mobilize

 

the chromosome in the same order, 0-i1v----thr--pro--,as
Hfr KI. In the M8809 x MS369 mating the order appears to
be similar except that the threonine locus rseemg: to be

transposed. This supports the gene order previously des-

cribed for S. pullorum. The recombination frequencies

63

Table 14. Chromosome mobilization of S. typhimurium strain
possessing the FT71 (trp+) factor.

 

 

 

 

Recombination

Frequency
Counter Selected (Per Initial Relative
Cross Selection Recombinants Donor Cell) Frequency

. . + -2
SU694 x MST119 ilv his+ 2.0 x lO_6 l _4
thr+ 4.0 x lO_5 2 x lO_5

pro 1.6 x 10 8 x 10

 

aThe selective media were supplemented with tryptOphan
and all the growth factors of the particular recipient strain
except that one for which selection for independence was being
made.

64

m0.o 0.0

 

00.0 00.0 0.0

00.0 0m.o m0.o 0.0

00.0 m~.o 0m.o 00.0 0.0
0-000 0-000 0-000 0-000 0-000

0-000 0->00 0-000 . 000mm: x 000020
0:005 0:005 05000 85000055 .0 x 0+6m0 +50>0 +0>xvmh95\0l560 m<m0m 05000 85000055 .0 .v

 

 

on.o 0.0
mm. mm. 0.0
Nuo0xv.h 00.0 m0.o 0.0
00x0 o0xw 000m m.o 0.0
~- ~- NI
0-000 0-000 0-000 0-000 0-000
0-000 0-000 0-000 I 000mm: 0 000000
01005 01005 05000 8500005m .m x 0+6m0 +m0m5 +M0>0vhh95\0u560 m¢m0m 050h0 8500005m .m .m
¢.o 0.0
x x . .
m|o0 m wuo0 o m o 0
0-000. 0-000 0-000
01005 Am 902 x mmemP

 

 

0-000 0-000 00000000500 .w x 0+000v0000\m00->00 0000000 000000 Nm0000 00000000 0 .0 .m

 

 

0.0 0.0 m-o0xv.0 0.0
m-o0xm.~ m-o0xn.m 0.0
070000.0 m-o0xm.m 00.0 0.0
0 o0xo.m m-o0xm.0 0-0000.m m-o0x0.0 00.0 «0.0 0.0
00000 0-000 0-000 0-000 0->00 0-000 0-000
0-000 0->00 00000: 0 000029

0-000 0-000 0-000 00000 00000000 .m x 0+00000000\m-000 0-000 00000 04000 00000000 .0 .0

 

 

.HmmeMHH MO HGGHfiMHU .MH GHQMB

65

 

0.0 0.0
0-000 000.05
00000: 0 000020

0-000 0->00 I 00000: 0 000000
0-000 0-005 0-000 00000 00000005 .0 x 0+00000000\m-000 0-000 00000 00000 00000005 .0 .0

 

0.0 00.0 0.0
00000 0-000 0-000
00000: 0 000020
0-000 00000: 0 000000

0-000 0-000 0-005 0-000 00000 00000005 .m x 0+000500000\0-000 00000 00000 00000005 .0 .0

 

 

00000000000 00 00000

66

6 to 10-7 for F—prime

7

obtained in this mating are only 10—

to 10—8

mediated chromosome transfer and still lower, 10-
for the M8808 x MS369 mating.

From the M5805 x M8369 mating the only recombinants
obtained were for the threonine marker. If the FT80 factor
carrying the his marker does mobilize predominantly from
the histidine locus this would be good evidence for the
close proximity of the threonine and histidine loci. Re-
combinants were also formed for the gly§_marker in the
M8805 x M5363 mating. It is difficult to correlate these
results with the previously described gene sequence because
of the low recombination frequencies involved, the fact
that these genes (glyAl and Ehrfl) are not in the same re-
cipient and the fact that the origin of chromosome transfer
mediated by the FT80 factor is not known.

In the M5806 x M8369 mating recombinants were ob-
tained for only the thr-l and Eififl markers. This supports
the original assumption that threonine is transposed in g.
pullorum. In the M8806 x M8363 mating recombinants at a
relative frequency of 0.2 were obtained for the glyAl
marker.

When single auxotrOphic strains carrying 25972,
ilvfz, Ehrfl, or Eififl markers are mated with the donor
strains of g. pullorum, the same recombination frequencies

were found as with the multiple auxotroph carrying all the

mutated genes.

67

Linkage analysis. To further study the gene order

 

and to learn more about F-prime mediated chromosome trans-
fer in g. pullorum, recombinants were analyzed for their
linkage to unselected markers. In each instance the cells
were mated for 3 hr on millipore filters prior to being
plated on selective media. The recombinants were recloned
on the same selective medium and then replica-plated to
determine the relevant genotype of each recombinant.

The data given in Table 16 confirm the gene order
previously described from the prolonged
matings of M8807 x MS369. To exclude the possibility of
donor auxotrophic markers reducing the linkage between
loci, a terminal marker, hisf4, was used solely for counter-
selection. The results of this mating are given in part A
of Table 16. The linkage appears to be low, especially the
thr-l to gal—l linkage of 21%, but even this percentage of

linkage agrees with the results reported in §. typhimurium

 

Hfr x g. typhimurium Fimatings where linkage of proximal

 

unselected genes, other than those located close to the
selected locus, is 29-40%. In E. coli, however, markers
nearer the origin than the selected marker are usually 50%
linked to the selected marker (95). In part B of Table 16
it is shown that his-l is essentially unlinked to the other
markers, but this probably is the result of coinheritance
of cysteine auxotr0phy or streptomycin sensitivity from the

donor.

68

Table 16. Occurrence of unselected donor markers in re-
combinants from crosses between donor strains

 

of E;f§.;;;13 pullorum possessing FT7ltrp+ and
multiple; auxotrophic recipient strains of g.
pullorum.

 

 

A. Mating: M8810 x M8374
Counterselection: Histidine auxotr0phy

 

Selected Phenotype

 

 

Unselected
Phenotype 182: 207+ 198+ 146+
Gal Pro Ilv Thr
Gal: -- 54b 29 21
Pro+ 25 -- 34 26
11v+ 3 8 -- 33
Thr <1 1.4 3 --

 

B. Mating: MS807 x MS369
Counterselection: Cysteine auxotr0phy and streptomycin
sensitivity

 

Selected Phenotype

 

 

Unselected
Phenotype 76+ 185+
Thr His

Gal: 19 5.0
Pro+ 28 5.5
Ilv+ 33 4.9
Thr+ -— 10
His 6.6 --

 

aThe number of recombinants analyzed

bThe results are given as per cent.

69

The data from the MS806 x MS369 mating presented
in Table 17 further indicate a linkage only of Ehrfl to
hisel. The linkage once again appears to be very low and
also is probably the result of coinheritance of cysteine
auxotr0phy or streptomycin sensitivity from the donor.

This mating was repeated several times with the same results.

The linkage data from the M8809 x M8369 mating are
presented in Table 18. A recombination frequency of 1 x 10-7
for the threonine marker prevented a more thorough examina-
tion of its linkage to other markers. It is evident that
the 11201 and BEETI markers are essentially unlinked. Out
of 717 recombinants ‘ analyzed only eight or 1.1% had ac-
quired the gysfi auxotrophic marker of the donor and all
eight were isolated with the selected marker Ilv+. The
possibility that the gygfl— genotype was being masked by the
coinheritance of the FT77 factor was eliminated by the fact
that only two cysteine positive recombinants out of 78 or
2.6% possessed the FT77 factor; as indicated by the cross-
streaking method.

The low linkage of the His+ recombinants to the un-
selected markers could be the result of coinheritance of
the glyA2 marker and/or streptomycin sensitivity from the
donor or the possibility that there are two or more linkage
groups in S. pullorum. Since only 55 His+ recombinants

were observed it is impossible to differentiate between the

various possibilities.

Table 17.

70

Occurrence of unselected donor markers in recom-
binants “ from the cross MS806 x M8369.a

 

 

Selected Phenotype

 

 

Unselected b
Phenotype 132+ 52+

Thr His
Thr+ -- 12

. + C
His+ 5 __
Ilv+ <1 <1
Pro+ <1 <1
Gal <1 <1

 

aCysteine auxotr0phy and streptomycin sensitivity
were used for counterselection.

b

Number of recombinants analyzed.

c . .
Numbers given in per cent.

71

Table 18. Occurrence of unselected donor markers in recom-
binants from the cross M8809 x M8369.a

 

 

Selected Phenotype

 

 

Unselected b
Phenotype 326 240 83 55 13
Ilv+ Pro+ Gal+ His+ Thr+
d
Ilv: -- 2.9C <1 <1 0/13
Pro+ 1.5 —- 58 1.8 0/13
Gal+ <1 22 -- 1.8 0/13
His+ <1 1.3 1.2 -— 0/13
Thr_ 3.7 <1 <1 5 --
Cys 2.5 <1 <1 <1 0/13

 

aGlycine auxotr0phy and streptomycin used for
counterselection.

bThe number of recombinants analyzed.

CResults given in per cent with the exception of
threonine selection.

dRatio of threonine recombinants prototr0phic for
the unselected marker per total number analyzed.

72

The data from the M5808 x MS369 mating is presented
in Table 19. The low recombination frequencies resulted in
a relatively small number of markers being analyzed. These
data also confirm the fact that 113:1 and pro-1 are essen-

tially unlinked.

Kinetic studies. Kinetic studies were done to dem-

 

onstrate that the results of the gradient of transfer were
in fact due to increased distances between markers and the
origin and not due to some artifact of derime directed
chromosome mobilization. The entry times for chromosomal
markers and the F-factors themselves were studied by inter-
rupted mating. Chromosomal markers were analyzed from
millipore filter matings and episomal markers were analyzed
from centrifuge matings.

Figure 3 shows the time of entry of galfl, prgfl,
$1371, Ehrel and hisfl markers from M5807 x MS369 mating.
The selective medium was supplemented with 1eucine and
trypt0phan. Cysteine auxotr0phy and streptomycin sensiti-
vity were used for counterselection. This mating has been
repeated four times and the same time intervals, :1 min,
were obtained in each instance. The data in Figure 3
confirm that the gene sequence is EElTlr E5271! £1131,
Ehrfl, and hisfl.

Figure 4 shows the time of entry of the episomal

trp+ gene in a M5807 x M5355 mating and the time of entry of the

73

Table 19. Occurrence of unselected donor markers in recom-
binants from the cross M5808 x MS369.a

 

Selected Phenotype

 

Unselected

 

Phenotype 20b 20 4 20 20
Ilv+ Pro+ Gal+ His+ Thr+
+ C

11v+ —- 0/20 0/4 2/20 0/20
pro+ 0/20 -- 3/4 2/20 0/20
Gal+ 0/20 9/20 -- 2/20 0/20
His+ 0/20 1/20 1/4 -- 1/20
Thr 0/20 0/20 0/4 1/20 --

 

aGlycine auxotrophy.and streptomycin sensitivity
were used for counterselection.

bThe number of recombinants analyzed.

CRatio of recombinants prototr0phic for the un—
selected marker per total number analyzed.

Figure 3.

74

Time of entry of various markers from M8807
x MS369 mating. MS807 was mated with M8369
on millipore filters and transfer was inter—
rupted at various times. A 0.1 ml of the
mating suspension (3 x 107 donor cells) was
plated at each time interval on media selec-
tive for Gal+, Pro+, Ilv+, Thr+, and His
recombinants. Each count is the mean of
five plates. The selective media were sup-
plemented with leucine and tryptOphan.
Cysteine auxotr0phy and streptomycin sen—
sitivity were used for counterselection.

200 '*

NUMBER OF RECOMBINANTS PER 0.1ML

 

 

ilv+

 

 

 

 

 

60 80
MATING TIME

(mm)

 

0'5?“ .

 

 

 

 

Figure 4.

76

Time of entry of the episomal tr + gene.
M5807 was mated with M8355 and transfer

was interrupted at various times. Selec-
tion was made for Trp+. M8807 was also
mated with MS369, transfer interrupted at
various times and selection made for pro+.
In both instances the cells were centri-
fuge mated. Cysteine auxotr0phy and strep-
tomycin sensitivity were used for counter-
selection.

n

 

'0,

 

 

c: s
0
6 4

AS _.0 “Mn. nhz<Z—QEOUWM

0
0

  

00:

2
"no Mum—>242,

q.‘ _." '1 -' "i" -\'T‘¥ “R’ %

(MI N.)

30

20 .
MA'HNG “ME

78

chromosomal 259+ - gene in a MS807 x MS369 mating. Trpf
was transferred at 9 min and pro* ‘ at 23 min. The trans-
fer time of the p£g+”" gene correlates well with the re-
sults above from a millipore mating. For g. typhimurium

it has been reported that the episomal gene trp+ is trans-

 

ferred at 7 min and the chromosomal gene his+ at 15 min
(94).
M8809 was mated with MS369 and the times of entry

of the following markers ilv-l, pro-l, gal-l, thr-l and

 

 

 

his-l determined. The times of entry are shown in Figure

5. This confirms the gene order described previously. The

 

time interval between prgll and ilv-l is only 27 min, while
in the M8807 x M5369 mating (Figure 3) the time interval
was 54 min. This anomaly will be taken up in the discus-
sion. The time of entry of the episomal gene EXEEf is

shown to be 7 min in Figure 6.
Part V. Transduction

Transduction experiments were undertaken to deter-
mine the fine structure homologies of selected genes in g.

pullorum and g. typhimurium. The genes chosen were those

 

carried by the F-prime factors.

The phage employed in the transduction experiments
was P35. This phage was isolated from the M8807 x MST119
mating previously described in Table 5. Since no phage

appeared in the M835 x MST119 mating also shown in Table 5,

Figure 5.

79

Time of entry of various markers with a M8809
x M8369 mating. Allconditions were the same
as described under Figure 3 except the selec-
tive media were supplemented with cysteine
and leucine. Glycine auxotr0phy and strep-
tomycin were used for counterselection.

I ‘\I;‘

- l...‘ ’l‘ ‘1- | I- 't‘liwl’ - ”I7.

---E‘J'z

NUMBER OF RECOMBINANTS PER OJML

120 '

 

 

lilv+
/ga|+

MATING TIME (MIN)

 

4o 60 so 100

Figure 6.

81

Time of entry of the episomal gy§E+ gene.
M5809 was mated with M890 and transfer
interrupted at various times. Selection
was made for cysteine prototrophy and
counterselection was made with glycine
auxotr0phy and streptomycin sensitivity.

If U '15.. II‘ ,b I I.‘ Elnls‘J .5... v- ,. 3 .z u “H E ‘1'

 

_ 0.....IIIIII..|._01I..I O. o...

m

D

‘
_
.

w m w .
.:2 so am.— mhz<z_mEOUm~_ “.0 mumiaz

_. H. .l
a mg)

i

 

15 20 25
(MIN)

10
MATING TIME

83

 

 

 

cash mmso ommz mu>HH mmmmo Husma Nanum ssuoaasm .m_m

. + momma A+mmu +muwm +mmwovnaam\ausmfi mamam Hmmmo asuoaasm .m

.a

momma anamm Hu>afl Human Huonm Human Hauum anneaazm .m m

GHEvm+Hmw cflamv+oum CHEHN+>HH mommz A+mmn +MMNm +flMvah>Bm\HlsmH mdem Hmmho Eduoaaom .m
.o

caemm+oum momma Huamm Hu>aa Hung» H-0um Human Hanum ssHoHHsm mm x

saga mus mmmmz can Huang Hauum esnoflas .m m

. + sommz A+muuvanem\mumuu Husma Human Hammo asnoHHsm .m

.m

mommz anamm Hu>afl Hung“ aloud Human Hauum asuoflasm .m.m

N3....23. CHEEh“: cflemm+oum Cg3+:me nowmz A+muucaham\mumnu Husma Humso Amman asuoaasm .m
.a

.mucmfiflummxm muucm mo wEHu mo humafidm .om magma

84

it has been assumed that the phage resulted from zygotic
induction. Exposure of this phage, P35, to anti~P22

l dilution) for 30 min at 37C reduced

rabbit serum (10-
the plaque forming titer of the phage lysate by approxi-
mately 99.9%. No reduction in phage titer was detected

with normal rabbit serum. Thus P35 is antigenically

similar to P22 phage of S. typhimurium.

 

For the transduction experiments 0.5 ml of an

10 cells/ml was

10

overnight aerated culture containing 1 x 10
mixed with an equal volume of the phage lysate (1.1 x 10
phage/ml). This suspension was incubated for 15 min at
37C and 0.1 ml aliquots were plated on minimal media. At
least five plates were used for the plating of each phage-
bacterial suspension after the proper incubation period
had elapsed.
The data are presented in Table 21. Tt appears

that the two species are homologous as judged by the re-
combination frequencies. The recombination frequencies

using the phage grown on S. typhimurium are comparable to

 

those obtained using the phage lysate from S. pullorum.
Tryptophan and cysteine B markers appear to be co-

transducible using either S. pullorum or S. typhimurium

phage lysates (Table 21, parts C and D). Sanderson found

39% linkage of cysB to trpA in S. typhimurium with P22

 

phage (95). No attempt was made to quantitate the relative

homologies of various S. pullorum and S. typhimurium genes

85

.m puma .GOflmuw>coo UHGmOOmma How Houucoow

.0 Eonm UmumHsoamo map ma mocmsvmum coauospmcmuep

.mmumam m mo mmmum>m cm Eonm cmxmu ma mumam Hmm mmacoHoo mo Hones: mmmum>m mnao

.CGMEHHME .> no uMHm m Eduwm “Hanan HmEHoc 0cm xumumcmmwm .<
mo umwm msoumcmm m mm3 mummHHGMINNm anQmm .GHE om Mow ohm um pmumndocH cam Anew»

 

 

 

 

 

IDHHU aloav Esumm panama mmmlflucm mo madao> Hmsvm cm an3 pmummuumum mummha wmmnmn
.Unm om awe om MOM wmmmaosconflumxomo mo HE\m1m nuflz pmummuumnm mm3 mpMmma wmmnm msam
III moa x m.m\o.o II II Hana mmmmmz
III moa x m.m\a.o + In +uoE «mmmz
mica x o.H woa x m.m\m.hm II + +poE wmmmz
bloa x H.H moa x m.m\m.am II II +¢mE vmmmz
suoa x m.m moa x m.m\oam n- u- +muu mmmz
muoa x ~.H moa x m.m\m.m In a: +<>su smmmz
muoa x ~.~ moa x m.m\m.ma I. u- +mmmo Hmmz
Ammmmzv EduoHHsm .m :0 pmummmum ovumma Honoo .m
nu- moa x m.m\o.o + I- +ums ammmz
nu- moa x m.m\m.o + u- +ng mmmmz
muca x m.s moa x m.m\a.v I- + +uma wmmmz
suoa x m.a moa x m.m\aoa .. + +unp mmmmz
sued x m.H woe x m.m\~m u- nu +muu mmmmz
unoa x 0.x moa x m.m\mmm u- .. +muu mmmz
mica x m.H moa x m.m\m.oa .. .. +umm mmmmz
mnoa x m.s moa x m.m\m.v I- u: #65 «mmmz
muoa x m.m moa x m.m\o.m .. .. +«sgu hmmmz
nuoa x o.m moa x m.m\qma .. .. +<>Hm momma
nuoa x m.H moa x m.m\moa I: I- +unu mmmmz
mnoa x m.H mos x m.m\o.a :u .. +mm>o Hmmz
Aomamzv Neg EDHHDEHSQNH .m :0 Umummmnm mummma Hocoa .d
pwocmsvmum owmmcm UmQH0m34 EDHmmflucm mommzo pogooamm pcwflmflomm
coHuOSUmcmuB Nmm meumz
mumHm umm Q
mucmuospmcmua

Hmnadz cmwz

 

 

.Esuoaasm .m on muwxuma mooHHm> wo coauospmcmua .HN magma

86

.cofiuomm moosumz can mamwumamz Ge pmnflnomwp mounpmooumm

 

 

 

 

 

 

.. u- n- .. sued x m.H moa x m.@n.mmfl+mm»o mu»
mm .. ma ow plea x m.m moa x m.m\mva +mmmo
om ma I- mm buoa x ¢.N moa x m.m\ema +muu moamz
ammmz Educaasm .m co pmnmmoum muMmMH Honoa .m
n- I- u- .. muoa x ~.o moa x m.m\vm +mm>o mun
mm I- as am snoa x H.H mos x m.m\mm +mm>o
he ma I- mm nuoa x H.H moa x m.m\mm +muu moamz
Acmemzv NBA ESHHSEHSQMM .m co pmummmum muMmmH Honoo .4
cOHuOSUmcmuu mmwo an» commamcd zoomswmum mmmnm UmQHOmQ< wouomamm ucoflmflomm
loo usmoumm + + muqmuospmcmua coauosomcmna meumz
mo Hmnadz mumam Hmm
mucmuospmcmue
menmz owuomammco umnEdz 2mm:

 

 

m.ammxm paw mummy mo cowuospmcmnuou .mm magma

'87

because the extent of lysogenation of each strain is not
known.

To test for stability of the transductants approxi-
mately 5 of them derived from each phage bacterial suspen-
sion were inoculated into L broth and grown overnight.

The overnight cultures were then streaked onto L agar and
the plates incubated 24 hr at 37C. The genotypes of the
transductants were determined by replica-plating and in
every instance they retained the original phenotype. Ful-
fillment of this criterion indicated stability which was
inferred to be due to integration of the transduced gene.

The phage lysates employed were tested for sterility
and the recipients tested for reversion. P22—antisera ef—
fectively reduced the recombination frequency; whereas,
normal rabbit serum had no effect (data not listed). This
reduction in recombination frequency correlates closely
with the previously mentioned phage neutralization experi-
ments. It should also be noted that deoxyribonuclease had

no effect on the transduction frequency.

Part VI. Orientation of the cysB trp
Region in S. pullorum

Sanderson (95) found that in S.typhimurium the gene

 

order was pro--gal--pyrF-cysB-trp----his; whereas, Taylor
and Trotter (109) found that in S. coli the order was

pro--gal--trpecysB-pyrF---his. This indicated an inversion

 

 

88

of the pyEF-gySB-tgp region between the two genera. Thus

a plausible explanation for the Opposite direction of trans-
fer of chromosomal genes encountered with S. pullorum
strains carrying the FT71 (tgpf) factor in comparison to

S. typhimurium strains carrying the same F-prime factor

 

would be an orientation of the EEBIEXEB'EXEF region in S.
pullorum similar to that found in S. 991$.

To determine the orientation of the EXEB EEB region
in S. pullorum with respect to pggf-gale--hi§ region sev-
eral matings were done and these results are seen in Table
22. It is evident that MS807 (FT71 (Erpf)) does not trans-
fer the gy§B+ gene at a detectable level to M8105. This is
a good indication that the EXEB EEE region is inverted in

S. pullorum in comparison to S. typhimurium. The other

 

donors were apparently not capable of transferring cysB+ or
trpf genes into M8105 and thus linkage studies could not be

done.

Part VII. Criteria of Conjugation
in S. pullorum
Since 72-96 hr incubation periods were required
for appearance of recombinants and there was a high con-
centration of cells on each plate, several precautions were
.. h.
undertaken to eliminate the p0351bility of syntrogism:
1. Either streptomycin or sodium azide in
addition to a distal auxotrophic marker

was used for counterselection in many
instances.

89

Table 23. Orientation of the cysB trp region.a

 

 

Counterselection Selected Recombination

 

Mating Markers Markers Frequency
M8807 x M5105 cysA cst cysS+ <2 x 10:3
leu strA gal 4 x 10
M8809 x M8105 glyA strA gal++ 2 x 10:;
cysg <2 x lO_8
trp+ <2 x lO_8
his <2 x 10
M8806 x M8105 cysA cst cysB+ <2 x 10-8

 

aThe selective media were supplemented with all the
growth factors of the particular recipient strain except
that for which selection for independence was being made.

bThe recombination frequency is based onthemean
number of recombinants per five plates divided by the
donor input.

90

2. In all matings a female cell with the same
genotype as the donor was millipore-mated with
each recipient. In no instance did the number
of colonies appearing exceed the normal rever-
sion rate of the recipient.

3. Cross-streaking did not show the heavy back-
ground growth indicative of syntrophism.

4. A large number of recombinants inherited un-
selected donor markers.

5. In all instances the purified recombinants
were replica-plated on a medium selective for
the growth of the donor genotypes.

6. A millipore membrane filter (HA 0.45u) was
interposed between donor and recipient bac-
teria on selective media. In the area where
the donor and recipient were separated by the
membrane filter no recombinants were observed.

An exhaustive analysis (prototrophy, stability,
infertility and M82 insensitivity) of recombinants arising
from each mating was made. The data are presented in Table
24.

More than 1800 recombinants were selected from the
previously described S. pullorum matings and in each in-
stance they were found to be prototrOphic. Because the
cells tested from the colonies arising on a minimal medium
are prototrOphic, it is concluded that genetic exchange
occurred and not some artifact created by the presence of
nutrient broth in the medium and/or syntrophism.

Stability of a recombinant marker would indicate

that the donor gene is physically integrated into the

chromosome of the recipient. To test the stability of

91,

Table 24. Recombinant analysis.

 

 

Cross

Prototrophy

Stability

 

Recombinant Number
Selected

Per Cent
Tested Prototrophic

 

Number Per Cent
Tested Stable

 

A. Prototr0phy and Stability

+

 

 

M5806 x M5367 thr+ 132 100 15 100
his 27 100 18 100
M5807 x MS369 thr: 146 100 31 100
his+ 185 100 26 100
pro+ 207 100 28 100
ilv+ 198 100 32 100
gal 182 100 15 100
M5809 x MS369 thr: 13 100 10 100
his+ 55 100 10 100
pro+ 240 100 10 100
ilv+ 326 100 10 100
gal 83 100 10 100
Recombinant a
Cross Selected M52 Sensitivity
B. Fertility
M5806 x M5367 thr: 4/12
his 5/11
M5807 x MS369 thr: 0/10
his+ 0/10
pro+ 0/10
ilv+ 0/10
ga1 0/10
M5804 x MS369 thr: 0/10
his+ 0/10
pro+ 0/10
ilv+ 0/10
gal 0/10

 

The techniques have been previously described in

Methods and Materials.

aNumber of recombinants sensitive to M52 phage per

number tested.

92

the recombinants, approximately 215 purified recombinants
were grown overnight in L broth and then streaked on L

agar and incubated 24 hr at 37C. Five colonies were
selected from each of the 215 recombinants originally iso-
lated and streaked onto properly supplemented minimal
media. In no instance was the original recombinant
genotype lost. Thus, it is concluded that the recombinants
are stable.

Infertility and M52 insensitivity of the recombi—
nants shows that the transferred gene is located on the
chromosome and not on an F-prime factor. Thirty-nine per
cent of the recombinants derived from the M5806 x M5367
mating were sensitive to M52 phage, but none had the ability
to transfer either threonine or histidine to a suitable
recipient. In the case of the M5809 x MS369 mating, 3% of
the recombinants carried the FT77 factor but none of the
50 analyzed were able to transfer the recombinant gene to
a suitable recipient. In the M5807 x MS369 mating only
one out of the 50 recombinants analyzed was sensitive to
M52 and none had the ability to transfer the recombinant
gene to a suitable recipient as determined by cross-streaking.
Since the cells were mated for 3 hr at a ratio of 1:1 and
at high cell concentrations it is reasonable to exPect as
in the case of K-12 crosses involving F+ and F-prime males,
that the sex factor would be transferred independently of

chromosomal genes to F- cells during mating.

93

Thus is may be stated that the recombinants derived
from the S. pullorum F-prime x S. pullorum F- matings are
prototrophic, stable, infertile and do not possess a high

coinheritance of the donor markers.

DISCUSS ION
Part I. Linkage Map of S. pullorum

The donor strain M5807 of S. pullorum which possesses
the FT71 (Egpf) factor gave the greatest amount of chromo—
somal gene transfer. When this strain was mated with M5369
the following gradient was obtained:

g3$-l>p£gel>i£y-l>E§£fl>§i§fl (Table 15).
Interrupted matings of M5807XMS369 demonstrated (Figure 3)
that the relative recombination frequencies of the various
markers were proportional to their time of entry. The
linkage studies from the MSBlOXMS374 mating (Table 16) also
confirmed the above gene sequence. From the gradient of
transfer, interrupted matings and linkage studies it is
readily apparent that M5807 behaves as a stable, homogen-
eous population in transferring a given set of markers at
high frequency and in a particular sequence.

This gene sequence was further substantiated by the
gradient of transfer and linkage data obtained from the M5809
X M5369 and M5806 X M5369 matings. The gene sequence as
determined by kinetic analysis, linkage/and gradient of
transfer was essentially the same irrespective of the donor

strain used; thus, a general linkage map in S. pullorum is

94

95

unequivocal. The linkage map of S. pullorum is shown in
Figure 7.

The evidence for the location and orientation of
the gygEl E£273 region in S. pullorum is by no means un-
equivocal. The location of this region is based on the as—
umption that the chromosome transfer mediated by the FT71
(352+) factor originates at this region and on the fact
that no gy§B1+ or Egpé3+ recombinants were obtained from
the M5806 x M5105 and M5809 x M5105 matings (Table 22).
The orientation of the gySBl Egpf3 region is based on the
fact that S. pullorum strains possessing the FT71 (£321)
factor transfer the chromosome in an opposite direction to

that of S. typhimurium strains possessing the same F-prime

 

factor. This inversion is supported by the finding that no
EXEBI+ recombinants were obtained from the M5807 x M5105
mating (Table 22). The critical test to determine both lo—
cation and orientation of the gySSl £5273 region would be

to study the linkage of the gygS} and 352:3 markers to EiSfB.
This has been attempted (Table 22) but no gy§S+ or Egpf re-
combinants were obtained. The FT21 factor carries no known
chromosomal loci, but mobilizes the chromosome with EEE

near the proximal end in S, typhimurium (116,96). It is

 

possible that S. pullorum strains possessing this F factor
might transfer the entire trp-gysB ------ his region and allow
linkage studies of those three markers. The FT21 factor has

not been introduced into S. pullorum.

Figure 7.

96

Linkage Map of S. ullorum. The F-prime fac-
tors and their direction of chromosome mObil-
ization are indicated in the expanded por-
tion. The extent of chromosome mobilization
is indicated by the length of the solid line.
No linkage analysis was done with the gene
markers in parentheses.

 

 

 

 

 

 

 

 

Linkage Map ,
of Salmonella pullorum

98

Part II. Comparison of 1' g
S. pullorum to S. typhimurium
Linkage Maps

 

When the linkage map of S. pullorum is compared

with that of S. typhimurium (Figure 8) it is readily seen

 

that the EXEB EEE region appears to be inversed between
the two species and that the threonine locus is transposed.
The transposed region probably includes the EXEB region
since M5806 which possesses the FT59 (py£B+) factor seems
to mobilize the chromosome in the transposed region. In-
version of the pygB EEE region when compared to S. Eypgif
murium or S. 39;; is indicated also as M5806 x M5369 mating
producesThr+ and His+ recombinants but no Ilv+, Pro+ or
Gal+ recombinants.

Since M5809 which possesses the FT77 (gy§S+ py£E+
‘££§f) factor mobilizes the chromosome from a region located
between ilzfl and 259:1 there may be either an extension of
the previously mentioned transposed inverted area to include
Sly gygE or another transposed area; the gySE SS! region.
The xylose marker is carried by the FT76 factor and is posi-
tioned in close proximity to the gySE locus; thus it is pos-
sible that this marker could also be transposed. When M5807
was mated with M5370 and the location of the l—l marker
determined by gradient of transfer and interrupted matings,
it was found that the gylfl marker was located between

ilv-l and pro-1. This location of xyl-l may be due either

to another transposition or to an inversion associated with

99

Figure 8. The linkage Map of S. pullorum and S. typhimurium.
The Linkage Map of the S. typhimurium is the
outer circle and the Lifikage Map of S. BEE?

lorum is the inner circle.

 

 

of

Salmonella
pullorum

 

Linkage Map .al

I Salmonella typhimurium

101

the gylfgygE-Sly, 2153, ESE segment. No linkage studies
of §y_l-l to other markers were done because of the slow
growth rates of S. pullorum on A minimal agar with xylose
as the only energy source.

Further evidence in support of the previously men-
tioned gene transpositions and/or inversion was indicated
from the 5A534XMS367 and SA536XM5367 matings (Table 5) from
which extremely low recombination frequencies for early mar-
kers were obtained. If the lead region of the donor DNA (S.

typhimurium Hfr) had little homology with the recipient DNA

 

(S. pullorum) due to a gene transposition or inversion in
the recipient, then the recipient is less likely to pull
the chromosome. Likewise, recombination of the hybrid mero-
zygote may be impaired by transposition and unquestionably
it will be impaired by an inversion.

When the gross arrangement of the genes on the S.

pullorum and S. typhimurium are compared there seems to be

 

a gene inversion and/or a gene transposition. However,
there appears to be a high level of genetic fine structure
homology between the two species as crosses between them
mediated by transduction (Table 21) yield recombination
frequencies which are analogous to intraspecies crosses.
Also recombinants formed from the crosses are exceedingly
stable. Homology was also demonstrated when 53172

was millipore—mated with M5367 for 60 min at a donor re-

cipient ratio of 1:20 yielding a recombination frequency

102

for Ilv+ which was very high (0.7 per donor cell) and the
prototrophic recombinants were exceedingly stable. A simi-
lar result was obtained in the SA535 x M5363 mating where
Gly+ recombinants were selected and also found to be very
stable (Table 5).

When S. pullorum (M5364) was mated with S. 22;;
AB311 the recombination frequency for an early marker gig-l
was very low (10'6) and the His+ recombinants were very un-
stable (Table 7). Similar results were obtained when S.
EQSS_ABZS7 was mated with S. pullorum M5369 and selection
was made for Pro+, Thr+, and Lac+ recombinants (data not
presented). These results may be due to homology differences.

When the chromosome of S. typhimurium is compared

 

with S. Egli only one case of a chromosomal rearrangement
has been reported (95), the inversion of the pygF EXEB EEE
region, but there are still numerous cases of uncertainty
about gene location in both organisms. The locus of the
operator in relation to the sequence of structural genes of
the EEE operon seems to be the same in S. Egii and S. E12217
murium (95).

The heterogeneity of gene sequence between S. pg;-

lorum and S. typhimurium may be the result of a diverse evo-

 

lution of the two species. S. pullorum is commonly isolated

from chickens (body temperature 4l-43C) and S. typhimurium

 

from mice (body temperature 36-38C).

103

Part III. Chromosome Mobilization Directed
by the FT77 (cysE+pyrE+rfa+) Factor

 

The FT77 factor appears to mobilize the chromosome
in two directions. The basis for this conclusion follows.
First, the time interval between ilyfl and EEQ‘l is 27 and
54 min with the M5809XM5369 and M5807XM5369 matings respec-
tively. If the time of entry of the episomal gy§E+ gene
(Figure 6) is used as an approximation of both the "lead"
and "dead" time then the approximate location of the ini-
tiation sites for chromosome mobilization mediated by the
FT77 factor can be determined. With the above information

the following model can be devised:

 

ilv-l pro-1
L 54 min (M5807XM5369) J
I 1
ilv-l (M5809XM5369) pro-l

41 min ,

 

 

 

%:: l4 min.l”4"

Second, the ilXIliEEQIl ratio of the gradient of
transfer with M5809XM5369 and M5807XM5369 is 0.6 (Egg/ii!)
and 0.14 (ill/BEE) respectively. Third, linkage of Slyfl
to E£251 is not significant in recombinants from the
M5809XM5369 mating but is significant in recombinants from
the M5807XM5369 matings. Fourth, linkage of Ilv+ recom-
binants to the EEETl marker from the M5809XM5369 and
M5810XM5369 matings are 3.7% and 3.0% respectively. These

observations indicate counterclockwise transfer of the

104

chromosome by the FT77 factor. Finally, when the Pro+ recom-
binants obtained from the M5809XM5369 mating are analyzed for
linkage to the unselected flél'l marker it is found that 22%
of the Pro+ recombinants are Gal+ (Table 18). This corres-
ponds very nicely to the linkage of the Gal+ recombinants to
the unselected pggfl marker (MSBlOXMS374) which was found to
be 25% (Table 16). These observations indicate a clockwise
transfer of the chromosome by the FT77 factor.

Out of 717 recombinants analyzed from the M5809X
MS369 mating only 8 (1.1%) had acquired the gySE auxotro-
phic marker of the donor and all 8 were isolated in the ini-
tial selection for Ilv+ recombinants. This linkage is pro-
bably the result of low donor crossover between the FT77

factor and the cysEl marker on the chromosome.

 

This multidirectional transfer is not a unique
situation, as Clark (21) has isolated an Hfr strain that
had two stably attached sex factors at different sites on
the chromosome. The Hfr was able to transfer its chromo-

some by one or the other sex factor but not both.

Part IV. Chromosome Mobilization
in S. pullorum
Integration of a sex factor is usually studied by
observing its ability to mobilize the bacterial chromosome
to another cell during conjugation (98). Studies concern-

ing the conditions or mechanisms of F+ or F prime

105

integration into the chromosome have yielded few clear cut
results.
Chromosome transfer mediated by cells possessing

the F+ factor in S. coli, S. abony or S. typhimurium (98)
5

 

is generally about 10- recombinants per donor cell. Simi-
lar matings in S. pullorum yielded no recombinants. Even
pretreatment of the S. pullorum F+ strains with ultraviolet
light did not produce detectable levels of chromosome trans—

fer. Only donor strains of S. pullorum possessing F-prime

factors carrying Salmonella genes were able transfer chromo—

 

somal genes at detectable levels.

The inability of S. pullorum strains possessing the
F+ or F-prime factors (containing S. 99;; genes) to trans-
fer a detectable level of chromosomal genes to S. pullorum
F‘ strains is difficult to rationalize for the following
reasons. S. pullorum can repair UV damage and forms stable

normal recombinants with S. typhimurium Hfrs at very high

 

frequencies. One possible explanation would be that the

F+ factor and the F-prime factors carrying S. 99;; genes

are preferentially modified such that they cannot physically
integrate into the chromosome of S. pullorum. Modification
of the F-Sggf factor by S. pullorum has been demonstrated

in this laboratory (43). It is not difficult to envision

preferential modification of an F—prime factor possessing

106

S. coli genes and not one carrying Salmonella genes because

 

S. pullorum appears to possess a high degree of fine struc—

ture homology to S. typhimurium and very little to S. coli.

 

 

Perhaps certain regions of S. typhimurium and S.
pullorum are less homologous than others and consequently

modification of S. typhimurium DNA also otcurs when it is

 

introduced into S. pullorum. An example of this phenomenon
may be seen in the comparative frequency of transduction of
the gySE and EEE markers. Transduction of the EygE marker
between S. pullorum is 12-fold higher than between S. 2317

lorum and S. typhimurium; whereas, a similar observation

 

with the trp marker yields equivalent transductants. This
apparent difference in homology of the cysE and trp regions

of S. typhimurium and S. pullorum may be related to the dif-

 

ference in chromosome mobilization capacity of M5809 and
M5807.

If it is assumed that the FT59 factor and the FT77
factor are modified in such a manner that they cannot inte-
grate into the chromosome of S. pullorum, then the chromo-
some transfer mediated by these factors in M5806 and M5809
respectively becomes very difficult to envision. The syn-
apsis model may be invoked as a possible explanation for the
chromosome transfer directed by these factors. This model assumes
that the F-prime factor attaches laterally to a site on the
bacterial chromosome. The degree of association of the episome

and chromosome is directly related to the relative homology

107
existing between the episome and chromosome. At the time
of mating a specific endonuclease nicks the F-prime factor
at a specific site thus initiating transfer. If the episome
and chromosome are tightly synapsed then non-random break-
age of the chromosome by the endonuclease would lead to re-
gional unwinding with the creation of single stranded re-
gions. If single stranded DNA is transferred in conjuga-
tion (24), then annealing of the DNA strand of the episome
with a single strand of chromosomal DNA would lead to trans-
fer of chromosomal DNA. This model seems to account for
the inability of F+ and F-prime factors carrying S. 99;;
genes to transfer chromosomal genes to S: pullorum and for
the low levels of chromosomal transfer mediated by FT59 and
FT77. By itself this model is inadequate to account for
the two-directional transfer. The two directional transfer
by the FT77 factor could be explained by the above plus the
fact that transfer in the opposite direction could be the
result of a theoretical "transfer replicase" switching
strands from the episomal DNA to the chromosomal DNA while
maintaining the same 5' 3' direction.

This two directional transfer by the FT77 factor
could also be the result of an inversion of the gySE, pygE
or 3:3 genes in S. pullorum. A crossover between a non-in-
verted segment on the chromosome and the homologous segment
on the FT77 factor gives mobilization in one direction, and

that crossover between an inverted segment on the chromosome

108

and the noninverted homologous segment on the FT77 factor
gives mobilization in the other direction. This phenomenon
has been observed with an Figgl factor carrying an inverted
segment (11).

According to Scaife and Gross (99) the frequency of
chromosome transfer is determined by the frequency of donor
crossover between the F-prime factor and the chromosome.

In S. typhimurium the FT77 factor mobilizes the chromosome

 

more frequently than it is transferred intact (97), indi-
cating a high frequency of donor crossover between the F-

prime factor and the chromosome. However, in S. pullorum

 

the FT77 factor transfers chromosomal genes at frequencies
of 10"6 to 10"7 per donor cell while the intact episome is
transferred at a frequency of 0.11 per donor cell.

The insertion model is another one used to explain
chromosome mobilization (19). This model is currently in
vogue due to its high predictive value and the overwhelming
data supporting it. Even though highly substantiated, the
insertion model does not account for the residual undirected
chromosome transfer by an F-légf/rec’ cell (23),Hfr selec-
tion from an F+/ rec” cell (98) and the data of Curtiss
(28) showing that gene transfer by a population of F+ cells
is markedly higher than the concentration of stable Hfrs.
in a donor population.

It could also be argued that the lack of homology

between the F factor and the chromosome is the cause of the

109
low to nondetectable levels of chromosomal transfer in S.
pullorum. It has been demonstrated that the F+ factor,
even though many integration sites are known, shows a dis—
tinct preference for certain locations on the bacterial
chromosome. Many investigators equate this preference with
the requirement for gross homology between the episome and
chromosome subsequent to integration. It must be remem-
bered that it is not known how long the F+ factor has been
in S. 991; and thus the designation of a fertility factor
being F+ or F-prime is purely academic. That is to say
that the preference of the F+ factor is possibly the result
of unknown chromosomal genes on the F+ factor and thus the
requirement of homology for the integration of the episome

into the chromosome is probably not stringent.

SUMMARY

The purpose of this investigation was to study
chromosome mobilization in a slow growing species of Sal-

monella, Salmonella pullorum. Only donor strains of S.

 

pullorum possessing F-prime factors carrying genes from

either Salmonella typhimurium or Salmonella abony were able

 

 

 

to transfer a detectable level of chromosome material. The
recombinants were prototrOphic, F', stable and did not pos-
sess a high coinheritance of donor characteristics. All
matings were conducted on millipore-membranes and strepto-
mycin was used for counterselection. The following obser-
vations were determined by gradient of transfer, interrupted
matings and unselected marker analysis. The threonine locus
appears to be located in close proximity to glycine A and
histidine. S. pullorum F-Egp strains transfer chromosomal

genes in a direction opposite to that of a S. typhimurium

 

F-Egp strain. This is possibly due to an inversion of the
EZEF‘EXEB'EEE region. S. pullorum F-gySE appears either to
transfer the chromosome in more than one direction or to
mobilize it at more than one site as indicated by the near
absence of linkage of isoleucine to either proline or galac-

tose genes.

110

 

 

LITERATURE CITED

Adelberg, E. A., M. Mandle, and G. Chen. 1965. Op-
timal Conditions for mutagenesis by N-methyl-N-
nitro-N-nitrosoguanidine in Escherichia coli K-
12. Biochem. Biophys. Res. Commun. SS:788-795.

 

Adelberg, E. A., and J. Pittard. 1965. Chromosome
transfer in bacterial conjugation. Bacteriol.
Rev. 33:161-172.

Anderson, T. F., E. L. Wollman, and F. Jacob. 1957.
Sur les processus de conjugaison et de recombin-
aison chez S. coli III. Aspecta morphologiques
en microscopie electronique. Ann. Inst. Pasteur
23:450-455.

Anton, D. N. 1968. Histidine Regulatory Mutants in
Salmonella typhimurium. J. Mol. Biol. 33:533-
546. _—

 

Ball, R. J., and W. Sellers. 1966. Improved Motility
Medium. Appl. Microbiol. Sg:670-673.

Barbour, S. D. 1967. Effect of Nalidixic Acid on
conjugational transfer and expression of episomal
lac genes in Escherichia coli K—12. J. Mol.
Biol.‘SS:373-376.

 

Baron, L. S., W. F. Carey, and W. M. Spilman. 1959.

Genetic Recombination between S. coli and S.
typhimurium. Proc. Natl. Acad. Sc1. U.S. 45:

 

Baron, L. 5., W. F. Carey, and W. M. Spilman. 1960.
Diploid heterozygous hybrids from matings between
S. coli and S. typhosa. J. Exptl. Med. 112:361-
372.

 

Beckwith, J. R., E. R. Signer, and W. Epstein. 1966.
Transposition of the lac region of Escherichia
‘coli. Cold Spring Harbor Symp. Quant. Biol. 31:
393-401. __

 

111

10.

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

112

Belser, W. L., and M. I. Bunting. 1956. Studies on
a mechanism providing for genetic transfer in
Serratia marcescens. J. Bacteriol. 13:582-592.

 

Berg, C. M., and R. Curtiss III. 1967. Transposition
derivatives of an Hfr strain of Escherichia coli
K-12. Genetics SS:503-525.

 

Bhaskaran, K. 1960. Recombination of characters be-
tween mutant stocks of Vibrio cholerae, strain
162. J. Gen. Microbiol. SS:47-54.

 

Bonhoeffer, F., R. Hosselbarth, and K. Lehmann. 1967.
Dependence of the conjugational DNA transfer on
DNA synthesis. J. Mol. Biol. 32:539-541.

Bouck, N., and E. A. Adelberg. 1963. The relation-
ship between DNA synthesis and conjugation in
Escherichia coli. Biochem. BiOphys. Res. Commun.
ll;24-28.

 

Boyer, J. 1966. Conjugation in Escherichia coli.
J. Bacteriol. Sl:1767-l772.

 

Bresler, S. E., V. A. Lanzov, and A. A. Blinkova.
1967. Mechanism of genetic recombination during
bacterial conjugation of Escherichia coli K-12.l.
Heterogeneity of the progeny of conjugated cells.
Genetics SS:105-116.

 

Brinton, C. C. 1959. Non-flagellar appendages of
bacteria. Nature 183:782-786.

Brinton, C. C., P. Gemski, and J. Carnahan. 1964.
A new type of bacterial pilus genetically con-
trolled by the fertility factor of Escherichia
coli K-12 and its role in chromosome transfer.
Proc. Natl. Acad. Sci. U.S. SS;776-783.

 

Campbell, A. M. 1962. Episomes. Advan. Genet. SS:
101-145.

Cavalli, L. L., J. Lederberg, and E. M. Lederberg.
1953. An infective factor controlling sex com-
patibility in Bacterium coli. J. Gen. Microbiol.
S:89-103.

Clark, A. J. 1963. Genetic analysis of a "double
male" strain of Esdherichia coli K 12. Genetics
SS:105-120.

 

 

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

113

Clark, A. J., and A. D. Margulies. 1965. Isolation
and characterization of recombination-deficient
mutants of Escherichia coli K 12. Proc. Natl.
Acad. Sci. U.S. SS:451-459.

 

Clowes, R. C., and E. F. Moody. 1966. Chromosomal
transfer from "recombination deficient" strains
of Escherichia coli K-12. Genetics SS:7l7-726.

 

Cohen, A., W. D. Ficher, R. Curtiss, and H. I. Adler.
DNA isolated from Escherichia coli Minicells
mated with F+ cells. Proc. Natl. Acad. Sci. U.S.
Slz6l-68.

 

Curtiss, R. 1964. A stable partial diploid strain
of Escherichia coli. Genetics SS:679-694.

 

Curtiss, R. 1968. Ultraviolet induced genetic recom—
bination in a partially diploid strain of Escher-
ichia coli. Genetics SS:9-54.

 

Curtiss, R., and L. J. Charamella. 1966. Role of
the F' parent during bacterial conjugation in
Escherichia coli. Genetics S£:329-330.

 

Curtiss, R., and D. R. Stallions. 1968. Probability
of F Integration and frequency of stable Hfr Donors
in F+ Populations of Escherichia coli. Bacteri—
ol. Proceedings. 55.

 

Cuzin, F., and F. Jacob. 1963. Integration reversible
de l'épisome sexuel F'chez Escherichia coli K-12.
Compt. Rend. 257:795-797.

 

Demerec, M., E. A. Adelberg, A. J. Clark, and P. E.
Hartman. 1966. A proposal for a uniform nomen-
clature in bacterial genetics. Genetics S£:61-76.

Demerec, M., and N. Ohta. 1964. Genetic analyses of
Salmonella typhimurium x Escherichia coli Hybrids.
Proc. Natl. Acad. Sci. U.S. SS:317-323.

 

Drabble, W. T., and B. D. Stoker. 1968. R Factors
in Salmonella typhimurium: Pattern on Transduc-
tion by Phage P22 and Ultraviolet protection Ef-
fect. J. Gen. Microbiol. SS.lO9-123.

 

Dushman, M. B. 1963. Origin of Prototrophic Variants
in mixtures of Auxotrophic Mutants of Serratia
marcescens. J. Bacteriol. SS:ll73-1181.

 

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

114

Edwards and Ewing. 1955. Identification of Entero-
bacteriaceae. Pg. 52-60. Burgess Publ. Co.,
Minneapolis, Minn.

Falkow, 5., and R. V. Citarella. 1965. Molecular
homology of F-merogenote DNA. J. Mol. Biol. SS:
138-151.

Falkow, S., H. Schneider, L. S. Baron, and S. B.
Formal. 1963. Virulence of Escherichia-Shigella

genetic hybrids for the guinea pig. J. Bacteri-
ol. SS:1251—1258.

 

Fink, G. R., and J. R. Roth. 1968. Histidine Regu-
latory Mutants in Salmonella typhimurium. J. Mol.
Biol. SS:547-557.

 

Fisher, K. W. 1957. The nature of the endergonic
processes in conjugation in Escherichia coli K-12.
J. Gen. Microbiol. SS:136-l45.

 

Freifelder, D. 1966. Replication of DNA during
F'lac Transfer. Biochem. Biophys. Res. Commun.
SS:576-581.

Freifelder, D. 1967. Role for the Female in Bac-
terial Conjugation in Escherichia coli. J. Bac-
teriol. 225396-402.

 

Freifelder, D. 1968. I. Studies on Escherichia
coli Sex Factors. J. Mol. Biol. SS:31-38.

 

Freifelder, D. 1968. II. Studies on Escherichia
coli Sex Factors. J. Mol. Biol. SS:95-102.

 

Friedman, B. A. 1966. Host controlled modification
of an intermediate sex factor in bacterial con-
jugation. M.S. Thesis, Michigan State University,
East Lansing.

Gemski, P., J. A. Wohlhieter, and L. S. Baron. 1967.
Chromosome transfer between Escherichia coli Hfr
strains and Proteus mirabilis. Proc. Natl. Acad.
Sci. U.S. SS:1461-1466.

 

 

Glansdorff, N. 1967. Pseudoinversions in the chromo-
some of Escherichia coli K-12. Genetics SS:49-61.

 

Gillespie, D., M. Demerec, and H. Itikawa. 1967.
Appearance of Double Mutants in Aged Cultures of

S. t phimurium cysteine-requiring donors. Genetics
9:453-442. -

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

115

Gorini, L., and H. Kaufman. 1961. Selecting Bac-
terial Mutants by the penicillin method. Science
131:604—605.

Gross, J. D., and L. G. Caro. 1966. DNA transfer in
bacterial conjugation. J. Mol. Biol. SS:269-284.

Haan, P. G. De, and J. Gross. 1962. Transfer delay
and chromosome withdrawal during conjugation in
Escherichia coli. Genetic Res., Camb. S:251-272.

 

Haan, P. G. De, and A. Stouthaner. 1963. F-prime
transfer and multiplication of sexduced cells.
Genet. Res., Camb. 5:30-41.

Hayes, W. 1952. Recombination in Bact. coli K-12;
unidirectional transfer of Genetic Material. Na-
ture, Lond., 169:118.

Hayes, W. 1952. Genetic Recombination in Bact. coli
K-12: Analysis of the stimulating effect of
Ultra-violet Light. Nature, Lond., 169:1017.

Hayes, W. 1964. The genetics of bacteria and their
viruses. John Wiley and Sons Inc., New York.

Helmstetter, C. 1967. Rate of DNA synthesis during
the division cycle of S. coli B/r. J. Mol. Biol.
SS;417—427.

Herman, R. K. 1968. Effect of Gene Induction on fre-
quency of Intragenic Recombination of Chromosome
and F-Merogenote in Escherichia coli K-12. Genetics
58:55-67.

 

Hirota, J. 1960. The effect of acridine dyes on
mating type factor in Escherichia coli. Proc.
Natl. Acad. Sci., U.S. £S:57-64.

 

Hirota, J., and Iijima, T. 1957. Acriflavine as an
effective agent for eliminating F-factor in
Escherichia coli K-12. Nature, Lond., 180:655-656.

 

Horiuchi, K., and E. Adelberg. 1965. Growth of male-
specific bacteriophage in Proteus mirabilis har-
boring F-genotes derived from Escherichia coli.

J. Bacteriol. SS:123l-1236.

 

 

Howard-Flanders, P. H., and L. Theriot. 1966. Mutants
of Escherichia coli K-12 defective in DNA Repair

and in Genetic Recombination. Genetics 53:1137-
1150 . ‘-

 

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

116

Ino, I., and M. Demerec. 1968. Enteric Hybrids II.
Salmonella typhimurium-Escherichia coli Hybrids
for the trp-cysB-pyrF Region. Genetics. SS:167—
176.

 

Ippen, K. A., and R. C. Valentine. 1967. The sex
hair of Escherichia coli as sensory fiber, conju-
gation tube, or mating arm? Biochem. Biophys.
Res. Commun. 31:674-675.

 

Jacob, F., S. Brenner, and F. Cuzin. 1963. On the
regulation of DNA replication in bacteria. Cold
Spring Harbor Symp. Quant. Biol. SS:329-348.

Jacob, F., and E. L. Wollman. 1956. Recombinaison
génétique et mutants de fertilité chez Escheri-
chia coli. Compt. Rend. 242:303-306.

 

Jacob, F., and E. L. Wollman. 1961. Sexuality and
the Genetics of Bacteria. Academic Press, Inc.,
New York.

Johnson, E. M., S. Falkow, and L. S. Baron. 1964.
Recipient ability of Salmonella typhosa in genetic
crosses with Escherichia coli. J. Bacteriol. S1:
54-60.

 

 

Kline, B. C. 1968. Accumulation of sulfite by a
sulfate-using revertant of Salmonella pullorum
and biochemical characterization of its cysteine-
requiring parent. Ph.D. Thesis, Michigan State
University, East Lansing.

 

Krishnapillai, and L. S. Baron. 1964. Alterations
in the mouse virulence of S. typhimurium by gene-
tic recombination. J. Bacteriol. S1:598-605.

 

Lawton, W. D., B. C. Morris, and T. W. Burrows. 1968.
Gene transfer in a strain of Pasteurella pgeudo-
tuberculosis. J. Gen. Microbiol. 52:25-34.

 

 

Lederberg, J. 1949. Aberrant heterozygotes in Esch-
erichia coli. Proc. Natl. Acad. Sci. U.S. SS:
178-184.

 

Lederberg, J. 1951. Streptomycin resistance: a
genetically recessive mutation. J. Bacteriol. SS:
549-5500

Lederberg, J., and E. L. Tatum. 1946. Novel geno-
types in mixed cultures of biochemical mutants of

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

117

bacteria. Cold Spring Harbor Symp. Quant. Biol.
SS:113-114.

Loeb, T. 1960. Isolation of bacteriophage specific
for the F+ and Hfr mating types of Escherichia
coli K-12. Science 131:932-933.

 

Loutit, J. 5., L. E. Pearce, and M. G. Marinus. 1968.
Investigation of the mating system of Pseudomonas
aeruginosa strain I. Genet. Res., Camb. SS:29-36.

 

 

Low, B. 1965. Low recombination frequency for
markers very near the origin in conjugation in
Escherichia coli. Genet. Res., Camb. Sz469—473.

 

Low, B. 1968. Formation of Merodiploids in matings
with a class of Rec' recipient strains of Esch-
erichia coli K-12. Proc. Natl, Acad. Sci. U.S.

ngl60-167.

Luria, S. E., and J. W. Burrows, 1957. Hybridization
between Escherichia coli and Shigella. J. Bac-
teriol. 2§;461-476.

Luria, S. E., and M. Delbrfick. 1943. Mutations of
bacteria from virus sensitivity to virus resis-
tance. Genetics SS:491-511.

Makela, P. H. 1963. Hfr males in S. abony. Genetics
gg: 423-429 .

Makela, P. H. 1965. Inheritance of the O antigens
of Salmonella Groups B and D. J. Gen. Microbiol.
SS:57-65.

 

Makela, P. H. 1966. Genetic determination of the O
antigens of Salmonella groups B (4,5,12) and C
(6,7).

McCarthy, J. M., and E. T. Bolton. 1963. An ap-
proach to the measurement of Genetic Relatedness

among organisms. Proc. Natl. Acad. Sci. U.S.
SS:156-163.

Meynell, E., G. G. Meynell, and N. Datta. 1968.
Phylogenetic relationships of Drug-Resistance
Factors and other transmissible Bacterial Plas-
mids. Bacteriol. Rev. SS:55—83.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

118

Miyake, T. 1962. Exchange of Genetic Material be-
tween S. typhimurium and S. coli K-12. Genetics
S1:1043-1052.

 

Okada, M",T. Watanabe, and T. Miyake. 1968. On the
Nature of the Recipient ability of Salmonella
typhimurium for foreign Deoxyribonucleic Acids.
J. Gen. Microbiol. SS:241-252.

 

 

Oppenheim, A., and M. Riley. 1966. Molecular re-
combination following conjugation in Escherichia
doli. J. Mol. Biol. SS:331-357.

 

¢rskoy, I., and F. ¢rskov. 1960. An Antigen termed
F occurring in F+ S. coli. Act. Pathol. Micro-
biol. Scand. SS:37-4l.

Pittard, J. 1965. Effect of integrated sex factor
on transduction of chromosomal genes in Escher—
ichia coli. J. Bacteriol. SS:680-685.

 

Pittard, J., J. Loutit,and E. A. Adelberg. 1963.
Gene Transfer by F‘ strains of Escherichia coli
K-12. 1. Delay in initiation of chromosome
transfer. J. Bacteriol. SS:1394-1401.

 

Pittard, J., and E. M. Walker. 1964. Conjugation in
Escherichia coli: Recombination events in ter-
minal régfan of transferred DeoxyribOnucleic Acid.
J. Bacteriol. SS;1656-l663.

 

Ptashne, M. J. 1965. Replication and host modifica-
tion of DNA transferred during bacterial mating.
J. Mol. Biol. SS:829-838.

Rdbinson, M. J., and D. E. Schoenhard. 1964. Trans-
fer of F'lac to Salmonella pullorum. Bacteriol.
Proceedings. 147.

Rosner, J. L., E. A. Adelberg, and M. B. Yarmolinsky.
1967. An Upper Limit on B-Galactosidase transfer
in Bacterial Conjugation. J. Bacteriol. 21:1623-
1628.

Round, R., R. Nakaya, and A. Nakamura. 1966. Molecu-
lar nature of the drug—resistance factors of the
enterobacteriacae. J. Mol. Biol. SZ:376-393.

Sanderson, K. E. 1965. F-prime factors of Salmonella
typhimurium. Heredity. SS:166.

 

 

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

119

Sanderson, K. E. 1967. Revised Linkage Map of Sal-
monella typhimurium. Bacteriol. Rev. 31:354-372.

 

Sanderson, K. E., and M. Demerec. 1965. The linkage
map of Salmonella typhimurium. Genetics. 51:897-
913.

 

Sanderson, K. E., and C. A. Hall. 1969. F—Prime
Factors of Salmonella typhimurium and an Inver-
sion between S. typhimurium and Escherichia coli.
Submitted to Genetics.

 

 

 

Scaife, J. 1967. Episomes. Ann. Rev. Microbiol.
SS:602-638.

Scaife, J., and J. D. Gross. 1963. The mechanism
of chromosome mobilization by an F -prime factor
in Escherichia coli K-l2. Genet. Res., Camb. 4:
328-331. "

 

Scaife, J., and A. P. Pekhov. 1964. Deletion of
chromosomal markers in association with F-prime
factor formation in Escherichia coli K-12.
Genet. Res., Camb. S:495-498.

 

Schneider, H., S. Falkow, and L. S. Baron. 1963.
Genetic homology between Shigella flexneri 2a
and S. coli. Genetics. SS:907-928.

 

Schneider, H., and S. Falkow. 1964. Characteriza-
tion of an Hfr strain of Shigella flexneri. J.
Bacteriol. SS:682-689.

 

Silver, 5. D. 1963. The transfer of material during
mating in Escherichia coli. Transfer of DNA and
the upper limit on the transfer of RNA and pro-
tein. J. Mol. Biol. S:349-360.

 

Silverman, P., S. Rosenthal, and R. Valentine. 1967.
Mutant male strains with an altered nucleic acid
pump. Biochem. Biophys. Res. Commun. 31:668-673.

Somerville, R. L. 1966. Tryptophan operon of Esch-
erichia coli: Regulatory behavior in Salmonella
typhimurium cytoplasm. Science V61. 154, pp.
1585—1587. 1966.

 

 

 

Spelina, V., and J. Starke. 1968. Kinetics of Re-
combinant formation in synchronized cultures of
Escherichia coli. J. Bacteriol. SS:2442-2443.

 

107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

120

Stouthamer, A. H., P. G. DeHaan, and E. J. Bulten.
1963. Kinetics of F+ curing by acridine orange
in relation to the number of F-particles in Esch-
erichia coli. Genet. Res., Camb. 1:305-317.

 

Taylor, A. L., and M. S. Thoman. 1964. The genetic
map of Escherichia coli K-12. Genetics. 50:659-
677. ——

 

Taylor, A. L., and C. D. Trotter. 1967. Revised
linkage map of Escherichia coli. Bacteriol. Rev.
SS:332-353.

 

Thornly, M. J., and R. Horne. 1962. Electron micro-
scopic observations on the structure of fimbriae,
with particular reference to Klebsiella Strains,
by the use of the negative staining technique. J.
Gen. Microbiol. SS:51—56.

Valentine, R. C., and M. Strand. 1965. Complexes of
F-Pili and RNA bacteriophage. Science. 148:511-
513.

Vogel, H. J., and D. M. Bonner. 1956. Acetylorni-
thinase of Escherichia coli. Partial purifica-
tion and some prOperties. J. Biol. Chem. 218:
97-1060

 

Wendt, L. W., K. A. Ippen, and R. Valentine. 1966.
General Properties of F-Pili. Biochem. Biophys.
Res. Commun. SS:375-380.

Woolman, E. L., and F. Jacob. 1955. Sur 1e méchanisme
du transfert de matérial génétique au cours de la
recombinarson chez Escherichia coli K-12. C. R.
Acad. Sci. Paris 339:2449.

 

Wood, T. H. 1967. Genetic Recombination in Esch-
erichia coli: Clone Heterogeneity and the kinetics
of segregatiOn. Science 157:319-321.

 

Zinder, N. D. 1960. Sexuality and Mating in Sal-
monella. Science 131:924-926.

Schoenhard, D. E. 1963. Transduction of Salmonella
ullorum by FLT-22 phage. Bacteriol. Proceed-
ings. pg. 30.

 

Belozersky, A. N., and A. S. Spirin, 1960;J31The
Nucleic Acids, by E. Chargaff and J. N. DaVidson,
Ed. (Academic Press, New York, Vol. 3, p. 147).

121

119. Hartman, P. E., S. R. Suskind, T. Wright and A. E.
Koziuski. 1962. "Principles of genetics, labora-
tory manual." John Hopkins University, Baltimore.

"lllllllllllllllllllls