GENETIC AND PHYSICAL PROPERTIES OF
F77 IN SALMON-ELLA PULLORUM M835
Thesis for the Degree of Ph. D.
MICHIGAN STATE UNWERSITY
PAUL W. STEFFLER
1971
This is to certify that the
GENETIC AND PHYSICAL PROPERTIES OF
F77 IN SALMONELLA PULLORUM M835
thesis entitled
presented by
Paul W. Stiffler
has been accepted towards fulï¬llment
of the requirements for
PhOD.
Date Februaryl 1972
Q7639
degreein Microbiology 5
Public Health
Major professor
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ABSTRACT
GENETIC AND PHYSICAL PROPERTIES OF
F77 IN SALMONELLA PULLORUM M835
BY
Paul W. Stiffler
Recent conjugation experiments with Salmonella
pullorum indicated that the F—prime factor F—cysE+ rfa-
EXEEf (F77) isolated from Salmonella typhimurium transfers
the g, pullorum chromosome from one origin in two direc—
tions (42). This origin was different than the origin in
g, typhimurium from which F77 transfers only in the clock-
wise direction. Therefore, experiments were performed to
investigate and compare the genetic and physical properties
of F77 in g. pullorum and g. typhimurium.
In this study, however, the F-prime factor, F77, was
found to transfer the g. pullorum chromosome from two
different origins, both in the clockwise direction. The
genetic loci studied appeared to be in the same relative
position as they are in g, typhimurium. The primary origin
of transfer was from cysE as O-cysE—ilv-thr-pro. The
secondary origin of transfer was from a locus between
pyrD and trp as O-trprgysB-his. The recombination
Paul W. Stiffler
frequencies for selected markers transferred from the
secondary origin were 10 to 100 fold less than for
selected markers transferred from the primary origin.
The trpï¬gysB genes appeared to be inversed compared to
those in S, typhimurium. The reduction in transfer of
intact F77 factors, the increase in recombination fre-
quencies of selected donor recombinants, and the stability
of donor ability suggested that F77 converted from the
autonomous state to a stable association with the donor
chromosome. The donor carrying a spontaneous mutation in
the cysteine gene of F77, designated F77gy§§f, transferred
only from the secondary origin between pygg and Egg as
O-trpécysB-his, with the same relative frequency as F77.
This strain also displayed extreme stability of donor
ability.
Since the overall recombination frequencies for
selected markers were 10 to 100 fold lower than expected
for F-prime mediated chromosomal transfer, an experiment
was designed to select a donor with increased transfer
ability. It appeared that S. pullorum donors carrying
F77 were homogeneous with regard to F77, suggesting that
F77 was able to transfer the host chromosome from either
origin of transfer.
Electron micrographs of the S. pullorum recipient
showed no unusual surface structures while the S, pullorum
donors appeared to have at least 15 sex-pili per bacterium.
Paul W. Stiffler
The physical basis for the stability of F77 and
F77gy§§f in Salmonella pullorum was determined. The F77
factor was isolated in the autonomous state from the donor
M88300. F77 was no longer autonomous in a derivative of
M88300, designated M8830, which transferred the chromosome
at a higher frequency. The F77gy§§f in 8. pullorum M8831
and F77 in S, typhimurium 8A532 were isolated from the
autonomous state. In 8. pullorum M8830, F77 appeared to
exclude the PO-Z-like plasmid molecule, not phage P35,
while F77gy§§f and phage P35 in M8831 did not. Neither
F77 nor F77gy§§f nor phage P35 excluded the PO—l plasmid
molecule. The F77 and F77gy§§f factors appeared to have
sedimentation coefficients of 705 and molecular weights
of approximately 51 x 106 daltons. It was concluded that
F77 forms a very stable association with the chromosome
of 8. pullorum, while F77cysE- does not.
GENETIC AND PHYSICAL PROPERTIES OF
F77 IN SALMONELLA PULLORUM M835
BY
Paul W. Stiffler
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
1971
This thesis is dedicated to
my wife Lois
ii
ACKNOWLEDGMENTS
I wish to acknowledge Dr. Delbert E. Schoenhard for
his challenge, guidance, and encouragement in my scientific
development during the course of this study. His interest
in me as an individual is sincerely appreciated.
I would also like to acknowledge Mr. Stuart Pankratz
for his help and advice in preparing the electron micro-
graphs appearing in this thesis and Dr. William L. Olsen
for the helpful discussions in regards to the physical
isolation of the plasmid DNA.
During the course of this study, I was supported
financially in part by a departmental assistantship.
iii
INTRODUCTION .
LITERATURE REVIEW
Part
I. Conjugation.
Fertility factor
TABLE OF CONTENTS
(E)
Electron microsc0py
High frequency recombination donors
(Hfr)
Intermediate donors
II.
F-prime Donor Strains
Primary Feprime donors
Secondary F-prime strains
F—prime)
Sex factor affinity locus (sfa)
Aberrant donor strains derived from
F-prime strains
Chromosome-transfer mediated by
F—prime factors .
III.
Sex Factor (F).
IV.
Specific pair formation .
Effective pair formation.
Chromosome and conjugal
mobilization
Chromosomal or conjugal
factor transfer
Recombinant formation.
V. Extrachromosomal
MATERIALS AND METHODS .
Bacteria
BacteriOphage
Media
DNA.
iv
fertil
Stages of Bacterial Conjugation
Donor Strains Harboring More Than One
ity
fertility
Page
ubNN
\lflmm
O
10
ll
l4
14
16
18
19
22
25
28
28
28
35
RESULTS
Part
I.
II.
III.
Iv.
VI.
Page
Chemicals . . . . . . . . . . . 36
Buffers and dialysis . . . . . . . 36
Mutagenic treatment. . . . . . . . 36
Presence of F factor . . . . .
Isolation and characterization of donor
strains of 8. ullorum . . . . . . 41
Techniques of—bacteriaI mating . . . . 45
Linkage analysis—-scoring unselected
markers . . . . . . . . . . . 47
Kinetic analysis-—time of entry of '
genetic markers . . . . . . . . 47
Cross streak method. . . . . . . . 47
Poisson distribution for selecting
fertile donor strains . . . . . . 48
Lysogenization of S. pullorum M835 by
phage P35 . . . . . . . . . . 48
Electron microscopy. . . . . . . . 49
Radioactive labeling and counting . . . 50
Preparation of bacterial lysates . . . 51
Dye-buoyant density gradient centri-
fugation. . . . . . . . . . . 52
Sucrose density gradients. . . . . . 53
. . . . . . . . . . . . . . . 54
Counterselection of Donors Carrying F77. . 54
Origins of Transfer and Genetic Loci
Mapped with Donors Carrying F77 . . . . 57
Matings using the donor M8830 . . . . 57
Zygotic induction . . . . . . . . 71
Spontaneous Mutation of F77. . . . . . 80
Matings using the donor M8831 . . . . 80
Determination of a Homogeneous or
Heterogeneous Donor POpulation Carrying
F77 and Selection for Donors with Increased
Fertility. . . . . . . . . . . . 81
Poisson distribution test. . . . . . 81
Matings using the donor M8832 . . . . 85
Electron microscopy of 8. pullorum
strains . . . . . . . . . . . 86
Evidence of F77 Converting to a more
Stable Association with the Host Chromosome 97
Isolation and Characterization of Plasmid
DNA. 0 O O O O O O O O O O O O 98
DISCUSSION 0 o o o g I o o o o o o I
Part
I. Counterselection of Donors Carrying F77.
II. Origins of Transfer and Genetic Loci
Mapped with Donors Carrying F77 . . .
Matings using the donor M8830 . . .
Zygotic induction . . . . . . .
III. Spontaneous Mutation of F77. . . . .
IV. Determination of a Homogeneous or
Heterogeneous Donor POpulation Carrying
F77 and Selection for Donors with
Increased Fertility . . . . . . .
Poisson distribution test. . . . .
Matings using the donor M8832 . . .
Electron microscopy of 8. pullorum
strains . . . . . . . . . .
V. Evidence of F77 Converting to a more
Stable Association with the Host
Chromosome . . . . . . . . . .
VI. Isolation and Characterization of Plasmid
DNA. 0 O O O O O O O O O O 0
SUMMARY . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . .
vi
Page
116
116
116
116
118
121
124
124
124
125
126
127
131
135
Table
1.
10.
11.
LIST OF TABLES
Characteristics of Salmonellae recipient
strains . . . . . . . . . . .
Characteristics of Sa1mone11ae F-prime
strains . . . . . . . . . . .
Chemicals and sources . . . . . .
Growth requirements of pyrB, g, Q, E, and
§_mutants . . . . . . . . . .
Amino acid pools used for the determination
of auxotrOphic mutants . . . . . .
Partial characterization of 8. pullorum,
g, typhimurium, and E. coli' . . . .
Recombination frequencies and gradients of
marker transfer in crosses with S.
pullorum donors and recipients . . .
Analysis of inheritance of unselected donor
markers in recombinants from the
M8830 x M8374 crosses . . . . . .
Analysis of inheritance of cysteine
auxotrOphy and streptomycin sensitivity
of the donor in M8830 x M8374 crosses .
Analysis of inheritance of unselected donor
markers in recombinants from the
M8830 x M8390 crosses . . . . . .
Analysis of inheritance of unselected donor
markers in recombinants from crosses with
M8830 and the S. pullorum recipients
M8391 and M8392 . . .
vii
Page
29
30
37
4O
42
44
58
59
62
68
72
Table Page
12. Analysis of inheritance of unselected donor
markers in recombinants from the
M8830 x M8104 crosses . . . . . . . . 77
13. Analysis of inheritance of unselected donor
markers in recombinants from the
M8831 x M8104 crosses . . . . . . . . 82
14. Analysis of inheritance of unselected donor
markers in recombinants from the
M8832 x M8374 crosses . . . . . . . . 87
15. Analysis of inheritance of unselected donor
markers in recombinants from the
M8832 x M8104 cross. . . . . . . . . 9O
viii
Figure
l.
10.
11.
12.
Partial
LIST OF FIGURES
linkage map of the 8. ullorum
chromosome showing the reIative
position of the genetic markers . .
Partial linkage map of the S. typhimurium
chromosome showing the relative position
of the genetic markers . . . . . .
Time of
entry of the pyrD+ gene from
M8810 x M883 and M8901 x M883 matings
Time of
entry of various markers from
M8830 x M8374 matings . . . . . .
Time of entry of various markers from
M8830 x M8371 matings . . . . . .
Time of entry of various markers from
M8830 x M890 matings. . . . . . .
Time of entry of various markers from
M8830 x M8390 matings . . . . . .
Time of entry of various markers from
M8830 x M8392 (A) and M8830 x M8391 (B)
matings . . . . . . . . . . .
Time of entry of att P35+ from the M88301 x
M881 mating. . . . . . . . . .
Time of
M8830
Time of
M8831
Time of
M8832
entry of various markers from
x M8104 matings . . . . . .
entry of various markers from
x M8104 matings . . . . . .
entry of various markers from
x M8374 matings . . . . . .
ix
Page
32
34
56
61
65
67
7O
74
76
79
84
89
Figure
_13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Time of entry of various markers from
M8832 x M8104 matings . . . . .
Electron micrographs of 8. pullorum mating
types . . . . . . . . . . . .
Evidence for F77 converting to a more
stable association with the host
chromosome. . . . . . . . . . .
Isolation of plasmid DNA from S. ullorum
M835 derivatives and S. typhimurium .
Neutral sucrose gradient of plasmid DNA
from S. typhimurium. . . . . . . .
Neutral sucrose gradient of plasmid DNA
from 8. pullorum M835 derivatives . . .
Neutral sucrose gradient of plasmid DNA
from 8. pullorum M835 derivatives . .
Neutral sucrose gradient of plasmid DNA
from 8. pullorum and S. typhimurium . .
Neutral sucrose gradient of plasmid DNA
from S. pullorum and S. typhimurium .
The partial linkage maps of 8. pullorum
and S. typhimurium . . . . . . .
Page
92
94
100
102
105
107
110
112
114
123
INTRODUCTION
A conjugation system for Salmonella pullorum has been
elucidated by Godfrey (42). His data suggest that the 8.
pullorum chromosome exists as a single closed circular
linkage map very similar to the linkage map of Salmonella
typhimurium. The only obvious differences are an inversion
of the trpjgysB genes and a possible transposition of the
thr gene.
Godfrey (42) observed that the S. pullorum donor
M8809 carrying F77, an F-prime sex factor isolated from
‘S. typhimurium carrying the S. typhimurium chromosomal
genes gysE+ rfa- pyrE+ (K. E. Sanderson and Y. A. Saeed,
personal communication), appears to transfer the host
chromosome from one origin in two directions. This origin
is not as reported in S. typhimurium where F77 transfers
the host chromosome in only the clockwise direction (K. E.
Sanderson and Y. A. Saeed, personal communication).
Therefore, it was decided that the biological and
physical prOperties of F77 in S. pullorum and S. typhimurium
should be investigated and compared.
LITERATURE REVIEW
Part 1
Conjugation
The process of bacterial conjugation was discovered
and described by Lederberg and Tatum in 1946 (59, 60).
This process is responsible for the unidirectional transfer
of genetic material which occurs upon cellular contact
between two bacterial cells of Opposite mating types. The
cell donating its genetic material contains a fertility
factor termed F (21, 58). The recipient cell lacks F and
is termed F“. During conjugation there is a low frequency
4 to 10-5) but
of mobilization of chromosomal markers (10-
a high frequency of F transfer (.5 to l) to F- recipient
cells (5, 29, 46, 58). Other genetic elements exist which
can transfer themselves infectiously and promote chromosome-
transfer such as colicinogenic (C01) and resistance trans-
fer (R) factors (26).
Fertility factor (F). The F factor is an antonomous,
covalently closed double strand DNA molecule (35, 36, 94)
6 daltons
with an estimated molecular weight of 45 x 10
(35, 37). The F+ factor gene load is approximately 2%
of the total bacterial genome and is large enough to carry
2
approximately 100 genes (99). Among the known functions
coded for by F genes are: sexual transfer or conjugal
fertility (45, 46, 58, 71, 99) sex factor replication (66,
99), superinfectious immunity (64, 99), F—pilus formation
(13, 14, 71, 93), growth inhibition of certain phages
(71), f+ antigen (74), and receptor sites for a group of
male—specific phages (13, 14, 20, 26, 27, 67, 93, 99).
Rupp and Ihler (82), Ohki and Tomizawa (69), and
Ihler and Rupp (50) inferred that the unique labeled strand
of Hfr or F—prime DNA transferred during conjugation is
due to the asymmetric transfer of a specific strand of
sex-factor DNA with a 5' nucleotide at the origin of
transfer. Vapnek and Rupp (94) conclusively showed that
only the denser strand of the sex factor DNA with a 5'
end at the origin, is transferred to the recipient where
its complementary strand is synthesized resulting in a
covalently closed sex factor DNA molecule. These results
show that DNA synthesis associated with mating occurs in
both the donor and recipient cells (94). The rolling
circle model of DNA synthesis (39) provides a working
model for this asymmetric strand distribution. The sex-
factor strand that is not transferred to the recipient
during conjugation synthesizes its complementary strand
in the donor during mating and also forms a covalently
closed double-stranded molecule (94).
Electron microscopy. The electron microscope is a
_ valuable tool for visualizing the sex-pili (F-pili)
formed on bacterial cells harboring the sex factor (F).
Typical Salmonella typhimurium and Escherichia coli
donors have 2—3 F—pili per cell. Donor specific DNA and
RNA phage "stain" the F-pili by adsorbing to them (14, 27,
93). The DNA donor specific phage adsorb to the tip of
the F-pili (20, 67) and the RNA donor specific phage ad-
sorb to the sides of the F—pili (14, 20, 27, 67). The
recipient cells have no sex—pili and therefore do not
adsorb donor specific phage.
High frequency recombination donors (Hfr). Inte—
gration of the F+ factor into the bacterial chromosome
results in an Hfr strain (46) which transfers its chromo—
some in a specifically oriented and linear way. Due to
random separation of the mating pairs the frequency of
inheritance of donor markers in recombinants is highest
if the marker is located near the beginning of the Hfr
chromosome and lowest if it is near the end of the Hfr
chromosome with the sex factor always the last marker to
be transferred (47, 54). Early markers are transferred
at a frequency of 10"1 to 10-2. Integration of F+ into
the bacterial chromosome is believed to occur by reciprocal
crossover between the circular F+ factor and the circular
bacterial chromosome resulting in a linear insertion of
the F+ factor into the bacterial chromosome (18). The
bacterial chromosome remains circular, only slightly
larger due to the presence of the integrated F+ factor.
Curtiss and Renshaw (29) described two classes of
F+ donors according to their ability to give rise to
stable Hfr derivatives. Type II F+ donors fail to give
rise to detectable frequencies of stable Hfr derivatives
because the association between F and the chromosome is
transient. They suggested three possibilities that may
be responsible for the existence of Type II F+ donor
strains. First, stable F integration may not occur either
because a bacterial enzyme(s) specifically necessary for
F integration is absent or because the F attachment site
on the membrane is altered, which in effect prevents pair-
ing and/or exchange between F and the chromosome. Second,
stable F integration may occur without causing the expres-
sion of the usual Hfr phenotype. Third, stable F inte-
gration resulting in chromosome lethality may occur
because of the absence of a functional bacterial enzyme(s)
necessary for complete integration of F into the chromo—
some or for the circularization of the F episome.
Intermediate donors (Fjprime). Abnormal detachment
of F from the Hfr state results in the removal of a seg-
ment of the bacterial chromosome with it. These sex
factors are called F-prime factors and can transfer the
chromosome at a high frequency and with the same orien-
tation as the parental Hfr strain (3, 51).
Broda 33 31. (16) and Scaife (85) proposed that F—
prime factor formation is the result of a reciprocal
crossover between two chromosomal sites on either side of
the integrated F or between a site in the integrated F and
the chromosome. This model is essentially the reverse of
Campbell's Model (18) of episome integration. A recipro-
cal crossover between two chromosomal sites results in an
F-prime factor carrying segments from both distal and
proximal regions of the ancestral Hfr chromosome (16).
A reciprocal crossover between the chromosome and inte-
grated F factor results in F prime factors carrying seg-
ments of the Hfr chromosome that are transferred proximally
(62) or distally (11, 51, 62).
Part II
F—prime Donor Strains
Primary F—prime donors. The data of genetic experi—
ments demonstrate that cells in which F—prime factors arise
are haploid and the genes deleted from the chromosome are
now on the F—prime factor (11, 78, 85, 87). These F—prime
donors are designated as primary F-prime donors (11) and
they transfer the F-prime factor to F— recipients at nearly
100% efficiency but chromosomal genes not carried on the
F-prime factor are transferred at random with frequencies
4 5
of 10‘ to 10’ per donor cell (11, 85, 87).
The results of treatment of primary F-prime donor
cells with acridine orange (AO) indicates that the F-prime
factor carries a gene(s) necessary for cell survival.
Consequently viability during AO treatment depends upon
integration of the F'prime factor into the host chromosome
(11, 16, 85, 87). Since the chromosome contains a
deletion of the prrime segment, integration must take
place at different regions on the bacterial chromosome by
undergoing "non-allelic" pairing and recombination (48).
This results in a class of aberrant donors which transfer
the chromosome from new origins and possibly in the Oppo-
site direction (11, 85, 87).
Secondary Feprime strains. When a primary F-prime
factor is transferred to a F- strain, partial diploidy
results. These F-prime strains can mobilize the chromo-
some with the polarity of the parental Hfr and are called
secondary F-prime donors (3, ll, 51). The early papers
describing F-prime factors (3, 51) actually were describ-
ing secondary F-prime factors. Approximately 10% of the
secondary F—prime donors transfer the chromosome while
the other cells continue to transfer only the F-prime
factor (3).
Sex factor affinity locus (sfa). Adelberg and
Burns (3) proposed that the infectious F+ factor has a
low affinity for the chromosome and no preferential site
of attachment. Following the rare event of Hfr formation,
an F-prime factor is formed resulting in a primary F-prime
strain (3). Following curing with AD the cell giving rise
to this primary F-prime factor can be reinfected with F+
or the primary F-prime factor and these new donors are
capable of relatively high frequency of oriented chromosome-
transfer (3). This is due to the recognition of the site
on the chromosome at which the F+ factor had originally
integrated. They (3) inferred that this "sex factor
affinity" (sfa) locus results by a reciprocal exchange
during F-prime formation. Broda (15) suggested that there
exists specific regions on the chromosome at which F+ inte-
gration occurs to form Hfrs. Now, it is known that there
are E. coli Hfrs with origins all around the chromosome
indicating a random distribution of these sites for inte—
gration (19, 92). There are Hfr strains of S, typhimurium
and Salmonella abony with points of origin in at least 17
different regions (83). The distribution appears to be
random over the 45—138 min region of the 138 min map but
there is no report of Salmonella Hfrs with an origin in
the 0 to 45 min region. There is an abundance of Hfr
strains of E. coli in this region.
There is no explanation for the numerous sites of
chromosomal homology with F+ that allows the rare occur—
rence of Hfr formation which arise by chance attachment
of the F+ factor to the host chromosome (3).
Aberrant donor strains derived from F—prime strains.
Transposition and inversion Hfr strains resulting from
primary F-prime factor re—integration into the host chromo-
some are known (11). The transposition Hfrs result from
re—integration in a different site. The direction of
transfer can either be the same as the parental Hfr or in
the opposite direction (11). Inversion Hfrs result from
the inversion of the F-prime factors which re-integrate
into their normal site and transfer the chromosome in the
Opposite direction (11).
A secondary F—prime donor can be constructed from
mating a primary F—prime derivative of an inversion Hfr
and a non-inverted isogenic F“ strain of the inversion
Hfr. Therefore, a crossover between a non-inverted seg—
ment on the F-prime factor and the homologous segment on
the chromosome gives mobilization in one direction, and a
crossover between an inverted segment on the F-prime factor
and the non—inverted homologous segment on the chromosome
gives mobilization in the other direction (11).
The sites of reciprocal exchange in transposition
Hfr formation and the polarity of exchange is definitely
nonrandom (11). These sites of pairing are the result of
mutual recognition between regions of fortuitously similar
nucleotide sequences. The probability of pairing is a
function of the extent of the similar sequences. The
relative orientation of the nucleotide sequences involved
is the determining factor for the direction of polarity.
10
Chromosome-transfer mediated by Fjprime factors.
Chromosomal transfer mediated by F-prime factors requires
a region of homology for synaptic pairing (76, 86). A
reciprocal crossover occurs within this region of pairing.
The denser single strand of sex factor DNA (94) breaks
between its origin and terminus (86, 76, 94). The origin
with the free 5' nucleotide is the lead end in chromosome-
transfer (82, 69, 94). The terminus is the most distal
segment transferred (3, 11).
After specific pair formation (76, 77), there is a
delay of 8 to 10 min for initiation of transfer of chromo-
somal markers in F-prime strains compared to their analo-
gous Hfr strains. The rate of chromosome—transfer is the
same for both F—prime and Hfr strains.
There are also secondary F-prime male strains known
as Type I or Type II (76). Type I donors give 3 to 10
times as much F—prime transfer as chromosome—transfer, but
Type II donors give higher frequencies of recombination
for chromosomal markers and proximal F—prime markers.
Type I donors change to Type II donors after storage at
5 C for several weeks followed by subculture in minimal
medium (76). Type II donor strains must have a higher
frequency of crossing over between the F-prime factor and
chromosome.
The F-prime donors of S. typhimurium carrying at
least the trp Operon are similar to Type II males (84).
11
Independent transfer of both chromosome and F-prime factor
occurs either extremely rarely or not at all (84), unlike
the high frequency of independent transfer in E. coli
(86).
Part III
Donor Strains Harboring More Than One
Sex Factor (F)
Clark (22) isolated a double male strain of E. 29;;
K12 by crossing two Hfr strains. The resulting recombinant
is a haploid monokaryotic Hfr containing two chromosomally
integrated sex factors. This strain is viable, stable, and
transfers its genetic material to recipients in the form
of two non-homologous linkage groups. Any given cell
appears to transfer one or the other linkage group, but
not both.
Echols (34) reported that an F—prime strain harbor—
ing Fgglf excludes or destroys a superinfecting Figgf
episome. Maas (64) isolated an Hfr recipient that com—
pletely excludes a superinfecting Figgf episome.
Bastarrachea and Clark (8) experimentally synthesized
a strain of E, 32;; K12 harboring three sex factors. The
donor is an F-30 merodiploid and the recipient is an F-
phenocopy of the strain harboring two integrated sex
factors (22). Chromosome-transfer is detected from both
origins due to the two integrated F factors in addition
12
to the autonomous transfer of the F—30 merogenote. F-30
is lost spontaneously.
Maas and Goldschmidt (65) isolated a recombination
deficient (EEEE) Hfr strain containing a mutation most
likely in the integrated F factor which permits the co-
replication of an integrated and a free F factor. The F
factor is F'lgg. They did not find a wild type strain
harboring two free F factors or one free and one inte—
grated. Palchoudhury and Iyer (75) found a chromosomal
mutation (DNA-ts43) that leads to termination Of DNA
synthesis at 42 C which permits the cohabitation of two
F-prime factors at the permissive (31 C) temperature. This
lack of entry exclusion and intracellular incompatibility
of one F—prime factor for another may result from an
alteration in the membrane for the membrane—replication
complex which is unstable at 42 C.
Joset 23 El- (56) isolated an Hfr strain following
ultraviolet (UV) treatment Of an Hfr strain that transfers
the chromosome in the Opposite direction and from a new
origin. The Ra-l Hfr strain also gives rise to RaF+
(F+) cells spontaneously (61). Further experiments by
Low (61) indicate that the Ra-l Hfr culture actually gives
rise to the cells transferring from the second site
(Ra-2 Hfr) by a detachment Of the F from its Ra—l site
of integration and reassociation at the Ra-2 origin.
Certain Ra—Z Hfr cells can transfer the chromosome like
13
the Ra-l Hfr. The mating properties Of the normal F+ in
cured RaF+ cells, RaF+ in a normal F- cells and an F+
revertant from the Hayes Hfr in cured RaF+ and normal F-
cells are those of the strains now harboring them. Low
(61) concluded that the E. gal; K12 RaF+ strain (including
Ra-l and Ra-2) carries a normal sex factor but possesses
chromosomal irregularities which give rise to the mating
behavior characteristic Of the Ra system in which there
is a preference Of RaF+ to integrate into one Of two
specific chromosomal loci and only rarely in other sites
around the chromosome. There apparently exists a segment
Of chromosomal DNA having sufficient homology with the F
factor to allow reciprocal crossover and integration.
Kahn (57) presented an elaborate scheme for the
evolution Of a chromosomal locus responsible for two
directional chromosomal transfer from one origin based
upon a tandem duplication Of Col V in the host chromo-
some.
Devries and Maas (33) described the isolation Of
double male strains in E. gel; by mating various F—prime
donor strains and a EEEQI Hfr recipient and selecting for
recombinants which can act as early donors Of both markers.
These recombinants may be mutants in which the incompati-
bility barrier has been lost or which have two integrated
sex factors. Further analysis indicates that all Of the
selected recombinants are indeed double Hfr strains. The
14
F-prime factor integrates into a region Of the chromosome
homologous to the chromosomal genes carried by the F-prime
factor. Insertion is in the same direction as the F-prime
mediated chromosomal transfer Of the parental strain. An
exception is the Observation Of a strain which transfers
the chromosome in the Opposite direction. This means that
the orientation of chromosomal genes on the episome is
Opposite to that Of the corresponding genes on the chromo-
some. It makes it unlikely that a reciprocal crossover
takes place between homologous genes Of chromosomal origin.
The insertion process has a high degree Of specificity
which is evident by the constancy Of the resulting double
Hfr strain, even in the absence of an intact bacterial
recombination system (33).
Part IV
Stages of Bacterial Conjugation
Specific pair formation. De Haan and Gross (31)
defined specific pair formation as a donor-recipient cell
union that is stable during gentle dilution. Curtiss
23.2l' (27) published electron micrographs Of presumed
specific pair formation. The F+, F-prime and Hfr donor
cells possess F-pili (14) which react with specific f+
antiserum and are the sites of attachment Of F donor-
specific phage. It is generally believed that the
presence Of donor pili is essential for specific pair
15
formation (13, 14, 27, 93). It can be seen in electron
micrographs that donor—specific RNA phage which attach to
the sides Of the sex—pilus outline the F-pili that appear
to be making contact with the recipient cell (27, 93).
The removal of F-pili by blending results in the temporary
loss of ability for the donor to form specific pairs but
is regained upon resynthesis Of the F-pili (13, 27).
Curtiss EE.E$- (27) suggested that donor cultures
which are grown anaerobically prior to mating have a
higher mean number of F—pili per cell, longer F-pili, a
higher probability Of forming specific pairs with F-
cells and a faster rate Of initiation Of chromosome-
transfer than cells grown aerobically. A rich medium is
superior to a completely synthetic medium (27). During
periods of starvation, amino acid auxotrophic donor cells
lose their F-pili, the ability to adsorb donor—specific
phage, the ability to form specific pairs with F- cells
and they become more recipient—like (27). Certain transfer—
defective mutations affect donor pili formation; these
mutants cannot form specific pairs or transfer genetic
material to recipients (l, 70, 71). Therefore, it appears
that the F-pili act like grappling hooks and are necessary
for specific pair formation with recipient cells (7).
Normal donors do not mate with DNA-deficient minicells
isolated from F+ or F-prime minicell producing strains
(24). The fact that a class Of donor pili—less mutants
16
have recipient abilities like the donor from which they
arise (70) implies that the mere presence Of F-pili is
not sufficient to prevent donor:donor specific pair for-
mation (26). From studying another class Of donor pili-
less mutants which have recipient ability like a normal
recipient strain, Ohtsubo (70) localized single mutations
of the F-factor which apparently affect the synthesis Of
a regulatory product that permits the synthesis Of both
donor pili and some other product necessary for preventing
donor:donor matings.
Another explanation for why donor cells form specific
pairs with recipient cells may be that the donor cell
possesses a cell surface structure that is responsible for
donor exclusion which is nonantigenic or associated with
the cell membrane (26). The only known antigenic struc—
tures that differ between donors and recipients are the
f+ (donor pili) and i+ (somatic pili) antigens in donors
(26).
Specific pair formation can occur in the absence Of
all energy metabolism on the part Of either or both
parents (30).
Effective_pair formation. Effective pair formation
is defined as the process by which specific pairs estab-
lish cellular connection through which genetic material
can be transferred (26). The nature Of the conjugation
bridge has not been established unequivocally. The
direct relationship Of donor ability and the presence Of
l7
vaili (13, 14, 27, 28, 93) indicates that the F-pili
play some vital role either as the conjugal bridge or in
the formation Of the conjugal bridge.
Brinton 22.31' (14) suggested that the F-pili are
very similar tO non-sex specific I—pili with respect to
gross physical structure. The F—pili have an axial hole
of 2.0-2.5 mu in diameter, running the length of the.
pilus, thus providing the space for the passage Of DNA
through the pilus. During conjugation there does not
appear to be significant transfer of any material other
than DNA (44). However DNA has not yet been isolated in
F-pili. Rosner 25.21' (81) found no detectable transfer
of B-galactosidase during matings between F+ and F- cells.
Silver (89) and Silver 22.2l (90) found essentially no
RNA or protein transferred during conjugation.
Ohtsubo (70) isolated donor-defective mutants
possessing F-pili that are able to form specific pairs.
This argues for genetic functions Of the F factor that
may be necessary for effective pair formation and/or for
chromosome or F—factor transfer (26).
Curtiss (26) prOposed a model for effective pair
formation based on available data and some hunches as
follows. After specific pair formation involving an
interaction between the recipient cell surface and the
tip of a donor pilus, the pilus is withdrawn into the
donor cell, with the expenditure of energy, so as to
18
achieve wall-to-wall contact between donor and recipient
cells. Formation Of a conjugation tube can then occur
either by use of a component Of the donor cell wall or
membrane, or possibly by the hole in the donor cell sur—
face remaining after withdrawal Of the pilus.
Chromosome and conjugal fertility mobilization.
This step prepares the circular donor chromosome and/or
sex factor for linear sequential transfer. This process
may occur during specific and effective pair formation or
after effective pair formation (26, 27).
Jacob and Brenner (52) and Jacob 32 3E. (53) prOposed
that chromosomal mobilization is initiated in the donor
parent upon receiving a contact stimulus from the F-
parent. They proposed that chromosome-mobilization is
related to vegetative chromosome replication which can be
controlled by two chromosomal loci. One locus specifies
the synthesis Of an initiator probably a protein (53),
and the other a replicator that recognizes the initiator
and controls the direction of sequential chromosome—
replication. They apply this two loci replication control
model to autonomous F factors as well and suggest the
simultaneous loss Of these functions when F integrates
into the chromosome. In their model they prOpose that the
contact stimulus received from the recipient parent
triggers the synthesis Of the F-specified initiator, which
acts to cleave the circular chromosome at F allowing for
the linear sequential transfer Of the DNA (52, 53).
19
However their model (52, 53) is based on the transfer Of
double stranded DNA from the donor to the recipient.
The rolling circle model for DNA replication Of
Gilbert and Dressler (39) suggests the transfer Of a
single strand Of DNA resulting from a cleaving Of a
single strand at the site of F integration. The sex
factor can be inserted into the E. 93;; chromosome with
the origin facing either direction (69, 82). Therefore,
depending on the orientation Of the inserted sex factor,
either strand Of the E. 99;; chromosome can be attached
(to a particular strand Of F.
Chromosomal or conjugal fertility factor transfer.
Chromosomal and conjugal fertility factor transfer is the
process of transferring the genetic material from the
donor to the recipient cell. Currently it is believed
that only a single strand Of DNA is transferred during
conjugation (12, 24, 39, 44, 69, 82, 94). The rolling
circle model for chromosome replication Of Gilbert and
Dressler (39) is an ideal explanation for chromosomal
transfer during conjugation. They postulate that repli-
cation begins by nicking one strand Of the chromosome at
a specific point. This may be at the origin of the
autonomous or integrated sex factor tOO. Then the Open
strand with the exposed 5' terminus attaches to a cell
membrane site for replication or to a site at the con-
jugal bridge for transfer. As this strand (positive) is
20
peeled Off and transferred, it is replicated in the
recipient. The nontransferred strand (negative) remains
closed. The positive strand is transferred to the
recipient as a template and is replicated as short
pieces by 3' to 5‘ growth of DNA and joined by the
ligase. The negative strand receives its complementary
strand simultaneously during the peeling away of the old
complementary strand in the normal manner.
Curtiss EE.§£° (26, 28) described experiments
utilizing recombinant production and zygotic induction
of prophage from different combinations of donor and
recipient strains which can or cannot ferment the avail—
able carbohydrate source to determine that chromosome~
transfer depends upon active metabolism in the donor to
initiate chromosome-transfer and active metabolism in the
recipient to control the rate of chromosome-transfer.
The conclusions of Bonhoeffer and Vielmetter (12)
that chromosomal transfer is independent of DNA synthesis
in the Hfr parent and dependent on DNA synthesis in the
F_ parent is at odds with other published data and con-
clusions on the role of DNA synthesis during bacterial
conjugation. From the use of DNAts mutations in F+, F',
Hfr donors and minicell (6) recipients, it is known that
the amount of DNA synthesized in the donor parent is equal
to the amount of DNA transferred to the minicells
(R. Curtiss, R. L. Seigel, D. R. Stallions, and G. Van
21
Denbos, Bacteriol. Proc., P. 35, 1970). Therefore, DNA
transfer during conjugation is accompanied by DNA syn—
thesis in the Hfr parent and is not dependent on DNA
synthesis in the F- parent. They believe that this DNA
synthesis in the donor during transfer is under separate
control from vegetative chromosome replication. Stallions
and Curtiss (91), by using DNAts mutants, concluded from
a reinvestigation of the experiment of Bonhoeffer and
Vielmetter (12) that chromosome-transfer from donors to
recipients unable to replicate DNA at 42.5 C during vege-
tative growth occurs at normal frequencies when the mating
is conducted at 42.5 C. Therefore some stage in haploid
recombination formation is adversely affected in DNAts
recipients mated at the temperature restrictive for DNA
synthesis (91).
Marinus and Adelberg (66) studied different DNAts
mutations located in at least two different genes on the
chromosome with one of the 8 mutations present in each of
8 mating pairs. They (66) demonstrated that genetic
transfer occurs normally in DNAts F“ strains mated at
42.5 C. Therefore, DNA synthesis in the F“ parent is not
required for genetic transfer. They concluded that
vegetative replication of the chromosome and transfer
replication of F are separate processes with the former
requiring at least two gene products which are non-
essential for the latter (66).
Q
_ 0‘
MAC
5 e"!
'3‘ P
“M
51';
t0
re
Ci
PI
22
Curtiss 2E.3l?(28) and Cohen 35.3E. (24) showed
that DNA synthesis in the Hfr donor can force transfer of
several percent of the donor chromosome to the recipient.
Also F and short F~prime factors can be transferred.
However, effective homologous pairing between the re—
cipient chromosome and episome is necessary for transfer
of longer F-prime factors and for chromosome-transfer
mediated by F and F—prime factors (28). The F‘ parent
winds in the donor chromosome with the expenditure of
energy (28). This process ensures pairing of homologous
regions of the donor and recipient for recombination.
Recombinant formation. Recombinant formation re—
quires synaptic pairing of homologous regions of the donor
and recipient chromosomes following chromosome—transfer
to enable crossovers to take place which are necessary
for the integration of transferred markers. This is
followed by reassortment of the donor and recipient
genetic information which yields new combinations of
genetic information. Finally there is segregation of
recombinant chromosomes from nonrecombinant chromosomes.
Pittard and Walter (79) and Curtiss g: il- (28)
reported that the homologous pairing of donor and recipient
chromosomes is necessary for the initiation of recombinant
production.
23
The coinheritance of two donor markers in the same
recipient depends upon the distance in transfer time be-
tween them. Linkage of less than 50% indicates a random
coinheritance of the two markers (55). There is random
coinheritance of proximally unselected markers that are
more than 15 to 25 min of transfer time from the selected
distally transferred marker. As the distance decreases,
the frequency of coinheritance approaches 100%. Also, the
coinheritance of distally transferred unselected markers
with proximally transferred selected markers drops below
50% (or random linkage) when more than ten minutes of
transfer time separates the two markers (55).
Pittard and Walker (79) and Glansdorff (40) con-
cluded that genetic exchange almost always occurs near
the origin of F-prime or Hfr chromosome transfer and the
only significant exclusion in inheritance of donor markers
occurs with markers less than 1 min of transfer time from
the origin. Glansdorff (40) also found that two or more
very proximally located markers may give the idea of being
transposed when they are actually pseudotranspositions
based on their kinetics of transfer.
Several models have been proposed to explain this
low recovery of very early markers (26, 38, 40, 79).
Walker and Pittard (97) reported that low recovery of
very early donor markers in recombinants is not caused
by the presence of sex-factor DNA at the leading end of
donor DNA transferred during conjugation when using an
24
isogenic Hfr phenocopy as the recipient. They found that
recombination frequencies for a selected allele is as low
as when a female strain is the recipient. These results
do not rule out the possibility that a piece Of sex factor
DNA forms the lead end or origin of the DNA transferred by
conjugation.
None of the recombination defective (£397) mutants
studied has an effect on chromosomal mobilization and
transfer in Hfr donors, but in the recipient strain they
are unable to perform the functions necessary for haploid
recombination (7, 100).
The model of recombinant formation proposed by
Curtiss (26) based on the available data rules out a OOpy-
choice type of recombination event. He proposed that the
double stranded recipient chromosome undergoes regional
melting at sites of single strand breaks to separate the
complementary strands. The single stranded donor DNA then
interacts with the recipient chromosome at these sites of
regional melting. The effective homologous pairing may
occur then by insertion of a portion of the single stranded
donor DNA in place of the like strand of the recipient
chromosome. Breakage then occurs in the other strand to
produce a segment of inserted single stranded donor DNA.
Synthesis of the strand complementary to the integrated
donor strand proceeds in a 3' to 5' direction along the
template, and when it is completed covalent bonds form
25
between the ends of the polynucleotide strands. The re-
sulting structure contains regions composed of parental
donor and recipient DNA synthesized prior to mating with
the donor segment composed of one strand synthesized prior
to mating and one strand synthesized during recombination.
The majority of the recombinant chromosome is the double
stranded recipient DNA synthesized prior to mating.
Part V
Extrachromosomal DNA
Novick (68) defines "extrachromosomal element" as
any hereditary unit that is physically separate from the
chromosome of the cell and an independent replicon.
Classifying an extrachromosomal element as either an
episome or plasmid has met with considerable controversy
recently (49, 68). Hayes (49) prefers to classify extra-
chromosomal elements as transmissible and nontransmissible
plasmids. Transmissible plasmids include those extra—
chromosomal elements which can transfer themselves via
conjugation and those which can transfer genetic units
not linked to themselves. Nontransmissible plasmids
cannot bring about their own transfer but can be trans-
ferred in association with a sex factor.
Many of the different transmissible plasmids have
been isolated and characterized. These plasmids have all
been isolated as covalently closed double stranded DNA
26
molecules. To facilitate direct isolation of the plasmid
from its natural host, Bazaral and Helinski (10) adapted
the procedure of Radloff _e__t_ Si' (80) employing ethidium
bromide (EtBr) in a preparative CsCl density gradient.
Radloff £5. 11;. (80) used the dye EtBr, which intercalates
between the base pairs of a double stranded DNA molecule
causes a 12 degree unwinding of the helical structure.
Waring (98) reported that the maximum amount of ethidium
bromide that can bind to unwinding double stranded DNA
is one molecule per every four or five base pairs. The
supercoiled covalently closed plasmid molecules bind much
less EtBr at saturating concentrations. Therefore, the
unwinding double stranded DNA, both open circular and
linear forms, bind much more dye resulting in a decrease
in buoyant density. When the DNA-dye complexes are
centrifuged to equilibrium in a CsCl density gradient,
the supercoiled covalently closed plasmid molecules will
hand lower in the tube at a greater density than the open
Circular and linear forms.
Neutral sucrose gradients are used to determine the
Sedimentation coefficients of supercoiled DNA molecules
( l7) . When these supercoiled molecules are centrifuged at
pH values greater than 12, the molecules sediment at a
f7‘as.ter rate. This is due to the more compact structure
of the denatured supercoiled molecules (95) .
27
Bazaral and Helinski (10) have determined that
ColEl supercoiled DNA has a molecular weight of 4.6 x 106
and a sedimentation coefficient of 235 in neutral sucrose.
Olsen and Schoenhard (73) showed that the PO—l and PO-2
Ellasmids of Salmonella pullorum M853 have molecular weight
(Df 1-5 x 106 and 45 x 106 daltons respectively and sedi-
Huentation coefficient in neutral sucrose of 175 and 655
r e spectively .
MATERIALS AND METHODS
Bacteria. Salmonella pullorum strain M835, desig-
nated wild type, was selected from the stock collection of
Dr. D. E. Schoenhard as the prototype organism from which
auxotrophic recipient strains were derived (Table l) . The
donor strains used for this investigation are listed in
Table 2. E. pullorum strain M853 was used as an indicator
Strain for the zygotic induction experiment and testing
lYa’sogenic derivatives of M835. Escherichia coli AB312
Was used for prOpagation of M82.
The genotypic and phenotypic symbols suggested by
Demerec e_t_:_ El- (32) were used.
The partial linkage maps of E. pullorum and E.
EXEhimurium depicted in Fig. l and Fig. 2 respectively,
Show the relative position of the relevant markers and the
EDCDint of origin and direction of transfer of the donor
strains referred to in this investigation.
Bacteriophage. The temperate phage P35 described
by Olsen (72) was induced from E. pullorum M835 by zygotic
induction. M82 was the donor-specific RNA bacteriophage.
Phage were propagated and titered by a modification
0
f a procedure described by Adams (2) . Log phase bacterial
28
EOHHOEHnmwu maawcoeamm u «m “SOHOHHOQ maamcoEHmm u mzm
29
acmumecmm .m .x mnmum mmmnums ammuxm mmvaam
moams mJMMI mums» Hmmmo Humww «cams
cams mlmmw mn>Hw Hmmso Himww Namz
Hams Haupm mu>aa Human Hmew cams
Amaze mmmz mumae Hausa Hmmso Hwaw mmmz
Amaze mmms House Houwm Hmmxo Himmw hams
Hams Housm Hmmxm Himmw mmmz
mam: III Hmmxo HJmmH Hams
ahmms x ommms Hmmxo Himmw anon mewm mamms
45mm: x ommmz Hmmwo Huwrw HJmMI Himwm Hmmmz
ekmmz x ommmz Hmmmw ambit anus» Himmm ommms
mmmms alam HJwHH Himmw Himml Hmem eemmz
58mm: III waww stuns Himwm mewm Humms
58mm: drama Hu>aa slush Himml Human anuum mmmms
enuncoosom .m .a aroma Humxm Hmem mmmz
.mmu no GHOHMO mumxuma owumcom ucm>maom .Oc cï¬muum
m.mcflmuum unmwmaomu OMHHmcoEHmm mo mowumwuouomuwao .~ w~&m&
3O
Esfluseflnmmu maamcoEHmm n <m “EDHOHHOQ waamcoï¬amm u m2m
acmumecmm .s lemme +muxm sees +mmwwtm\ommmmxo mmvuums mmmam
omms x ommmz lemme +muxm ummu +mmwosm\anuum mu>Hfl Hmmxw Husma scams
Aucmeflummxm
coauoofluumflv
commeomv ommmz Abbey +muwm new“ +Mmmmum\aouwm Hmmxo asses Nmmms
Acowumuse .
msomcmucommv oommmz AlmmNOnnmv mmuNm mmmu tumultM\HQuNm HmmNO Husma Hmmmz
oommms lapse +mumm new“ +mmwoum\floumm Hmmwm Husma ommms
mmms x mmmmm Akkmv +muxm new“ +mmmoum\aauxm Hmmxo Husma oommms
nomms lanes +muusm\~u up «swan Husmfl Hemmo Humuo oamms
.mmu no cflmwuo
mumxume oaumcmm ucm>mamm
.Oc cwmuum
m.mcwmuum weï¬umum mmHHchEHmm mo moeumï¬umuomumso
.N manna
31
Figure 1. Partial linkage map of the E. ullorum chromo-
some showing the relative position of the
genetic markers. The F-prime factors are indi-
cated in the expanded portion.
32
- + + -
pyrE+ rfa cysE F77 cysE rfa pyrE+
N
pro
ilv
thr
trp
cysB trp
F71
his
Figure l
Figure 2.
33
Partial linkage map of the E. typhimurium
chromosome showing the relative position of the
genetic markers. The F-prime factors are indi—
cated in the expanded portion.
34
thr
+
£33'IE
r fa- ilv pro
4.
CYSE rfa
F7 7 cysE
cysB
tr? F71
tr
his p
Figure 2
35
cells in aerated L broth were infected with phage at a
multiplicity of infection (m.o.i.) of 0.1. The infected
cells were incubated 18 hr with aeration by shaking at
37 C. One ml of chloroform was added to the culture,
followed by vortexing the culture for one—half min. The
chloroform treated cells were then reincubated at 37 C
with aeration by shaking for 30 min. The bacterial debris
was then removed by centrifugation for 10 min at 8000 x g.
The phage in the supernatant fluid were stored over chloro-
form at 4 C. The phage were titered by assay of the number
of plaque forming units (pfu) per ml by the soft agar over—
lay technique (2).
EEEEE. The E minimal medium described by Vogel and
Bonner (96) was supplemented with L-amino acids at a final
concentration of 20 ug/ml and D-glucose (Pfanstiehl) at
0.4% (w/v) for the growth of amino acid auxotrophs.
L broth and L agar (1.5% Difco agar) containing 10 g
Of tryptone (Difco), 5 g of yeast extract (Difco), and
10 g of NaCl per liter of deionized distilled water were
employed for routine cultivation.
When used, dihydrostreptomycin sulfate was added
to a final concentration of 1200 ug/ml in minimal media.
Bacto SIM medium (Difco) was used to detect sulfide
and/or indole production.
For radioactive labeling, the bacteria were grown
overnight in TCGU broth containing 0.1 M tris (hydroxy-
methyl)aminomethane(Tris)—hydrochloride pH 7.4, 0.4%
36
vitamin—free casamino acids, 25 ug/ml of deoxyadenosine,
7 ug/ml uridine, and 0.4% glucose which was autoclaved
separately and added prior to use. When labeling with
14C-thymidine, 25 ug/ml of deoxyguanosine were added
in addition to the TCGU broth.
Bacterial cultures were checked for specific anti-
gens with antisera (Difco, Salmonella O antiserum group D
factor 9 for E. pullorum and Difco, Salmonella H anti-
serum i for E. typhimurium).
Chemicals. The general chemicals used were
reagent grade. Special chemicals are listed in Table 3.
Buffers and dialysis. TM buffer (Tris-maleic) was
made in deionized, distilled water which contained: 0.05 M
tris (hydroxymethyl)aminomethane(Tris)-hydrochloride and
0.05 M maleic acid, pH 6.0. The general buffer TES was
made in deionized, distilled water which contained: 0.05 M
tris (hydroxymethyl)aminomethane(Tris)-hydrochloride,
0.005 M (ethylenedinitrilo)tetraacetic acid (EDTA), and
0.05 M NaCl, pH 8.0.
Dialysis was performed using sterile dialysis tubing
which had been boiled in 0.5 M EDTA pH 7.0 for 10 min, and
then autoclaved in 0.05 M Tris, pH 8.0.
Mutagenic treatment. The uridine mutation was in-
duced by N—methyl-N'nitro-N—nitrosoguanidine (NTG) follow-
ing the method recommended by Adelberg, Mandel and Chen
8
(4). Five ml of logarithmic phase cells (2 x 10 cells/
ml) growing in E minimal broth were collected on a
37
Table 3. Chemicals and sources.
Chemicals
Source
N-methyl—N'—nitro—N—
nitrosoguanidine (NTG)
Ethidium bromide (EtBr)
Lysozyme (crystallized
egg white)
Cesium chloride (CsCl)
Bovine albumin fraction V
(BSA)
Brij 58
Antisera
2,5(diphenyloxazole)—benzene
(PPO)
1,4,-bis 2(4-methyl—5-
phenyloxazole)-benzene (POPOP)
Aldrich Chemical Company
Milwaukee, Wisconsin
Calbiochem
Los Angeles, Calif.
Armour Pharmaceutical Co.
Kankakee, Illinois
Schwarz—Mann
Orangeburg, New York
Pentex Incorporated
Kankakee, Illinois
Emulsion Engineering Co.
Elk Grove, Illinois
Difco Laboratories
Detroit, Michigan
Packard Instrument Co.
Downers Grove, Illinois
38
millipore filter and resuspended in 10 ml of TM buffer
pH 6.0 containing 100 ug of NTG/m1. The suspension was
incubated at 37 C for 20 minutes with aeration. A 1 m1
aliquot portion was filtered to remove the excess NTG,
and then resuspended in 10 ml of E minimal broth supple—
mented to permit the growth of uridine mutants. The
suspension was incubated with aeration for five gener-
ations.
Enrichment for the desired mutant was by the
penicillin treatment described by Gorini and Kaufman (43).
Ten ml of the NTG treated suspension (5 x 108 cells/ml)
were centrifuged and the pellet resuspended in 1 m1 of
E minimal broth. A 0.1 ml aliquot portion of the re-
suspended pellet was added to 10 m1 of E minimal broth
supplemented with 10% sucrose, 0.5% glucose, and 0.01 M
M9804 and the growth requirements of the parental cell
type. The culture was grown with aeration for 3 hr
followed by the addition of 2000 units/ml of Penicillin G.
Additional incubation was done at 37 C without aeration
for 4 hr until approximately 50% of the cells had become
Spheroplasts. Then the action of penicillin was stopped
by chilling the culture in an ice bath. The culture was
centrifuged and the pellet resuspended in 10 m1 of E
minimal broth properly supplemented to permit growth of
the uridine mutants. Following the second cycle of
Penicillin enrichment, the cells were plated on L agar
Plates and the uridine mutants isolated by replica plating
39
to selective media. The uridine mutants were then repli-
cated to selective media on which they were characterized
(101) as shown in Table 4.
The histidine (EEEeS) and arginine (Eggfl) mutations
were induced in M883 by NTG according to the method de—
scribed by Glover (41). Five ml of logarithemic phase
cells (2 x 108 cells/ml) growing in L broth were centri—
fuged and the pellet resuspended in 5 ml fresh L broth.
NTG was added to a final concentration of 30 ug/ml and the
suspension allowed to incubate at 37 C for 15 min with
aeration. The cells were washed twice in E minimal broth
and resuspended in 5 ml of E minimal broth. A 0.1 ml
aliquot portion was added to 5 ml of L broth and incubated
37 C overnight with aeration to allow expression of the
mutations. The cells were pelleted, washed in E minimal
medium and resuspended in E minimal broth supplemented
with the amino acid requirements of the parental strains
and allowed to incubate at 37 C for 3 hr with shaking.
Enrichment for the induced mutations was by the penicillin
treatment of Gorini and Kaufman (43) as previously de-
scribed. Following the completion of the penicillin
enrichment treatment the cells were iced, pelleted by
centrifugation in a Sorvall RC-2 centrifuge at 5 C and
resuspended in 3 ml of E minimal broth. One—tenth ml
aliquot portions Of diluted resuspended cells were plated
on L agar plates to permit the growth of approximately
200 colonies per plate.
40
Table 4. Growth requirements of pyrB, E, E, E and E
mutants.
Carbamyl Dihydro— Uracil
aspartic orotic Orotic or
Locus acid acid acid Uridine
(CAA) (DHOA) (OA) (U)
pyrB + + ++ +++
C - + +++ +++
D — - +++ +++
E - — - +++
F
- — - +++
41
These isolated colonies were picked to fresh L agar
plates and spread in patches to serve as master plates for
replica plating to pools shown in Table 5 for the identifi—
cation Of the induced mutations.
The EEETI mutation was further studied and identi-
fied as EEEEl- The mutant strain grows on citrulline but
not on ornithine.
Presence of F factor. The method of Schleif (88)
was used to test for the presence of the F'prime sex
factor. The donor specific RNA bacteriophage M82 was
streaked down the center of an L agar plate and allowed
to dry. The bacteria being tested were streaked across
the bacteriOphage. Bacteria harboring F showed a greatly
reduced number at the intersection of the M82 streak;
F- cells showed no reduction in number.
Isolation and characterization of donor strains of
8. pullorum. The F77 factor was isolated from E. typhi-
. . - +
murium by Sanderson and carries the cysE+ rfa pyrE
genes. The origin and direction of chromosome—mobilization
by F77 in E. typhimurium are shown in Fig. 2.
The F77 factor was introduced into E. pullorum by
mating E. typhimurium SA532 with M883. Log phase cultures
of the donor (1 x 108 cells) were mixed with the recipient
(l x 108
cells) and impinged upon a millipore membrane
filter. The filter was removed to pre—warmed L agar
plates at 37 C. The membranes were then inserted into
42
Table 5. Amino acid pools used for the determination of auxotrophic
mutants.a
Pool number 1 2 3 4
5 phenylalanine leucine serine glutamate
6 tryptophane isoleucine glycine arginine
7 histidine valine cysteine proline
uridine
8 aspartic acid methionine threonine lysine
a . . .
Each pool was supplemented with leuc1ne and cysteine.
43
either 1 ml of E minimal broth with glucose and agitated
in fluted test tubes on a Vortex Jr mixer to remove the
mating pairs from the membrane and interrupt the mating,
or placed into 1 ml of E minimal broth plus glucose in a
13 x 100 mm test tube and vibrated for 15 sec in an
apparatus described by Low and Wood (63). The mating
mixture was then diluted 1:3 in E minimal broth and 0.1 m1
aliquot portions were dispensed into 3 ml L soft agar
overlays and poured over the surface of supplemented E
minimal agar plates to allow growth of only the desired
recombinant type. After incubation at 37 C for 96 hr, the
recombinants were re—streaked on the same kind of selective
medium and reincubated at 37 C for 72 hr. Isolated colo—
nies were then picked and inoculated into 3 ml of L broth
and incubated at 37 C for 12 hr with aeration and then
tested for sensitivity to M82 phage. The tubes contain-
ing M82 sensitive bacteria were then subcultured into SIM
media to detect hydrogen sulfide, or indole production.
The cultures appearing to be E. pullorum were then
streaked for isolation on L agar and incubated at 37 C
for 24 hr. Individual colonies were then tested for their
response to Group D antisera and auxotrOphic requirements.
The donor M88300 resulted from these manipulations. A
EPartial characterization of E. pullorum, E. typhimurium
and E. coli is shown in Table 6.
44
I I + I AHOO .m
+ I I + Eseuoeflsmmu .m
I + I I Esuoaaom .m
H msoum m Houomm a moonm m
m mHHwOOEHmm o maaocoEHmm waoocfl m m
mooscoum moosooum Emflcmmuo
Esummï¬ucm ou omOOQmmm
.HHOO am can Esï¬usaflnmwu xm .EOHOHHOm nm mo OOHDMNHHmuomumno aneuumm .m manna
45
A routine check of the parental auxotrOphic require-
ments, M82 sensitivity and donor fertility of M88300,
revealed an isolate with increased fertility, designated
M8830, and an isolate with a cysteine requirement,
designated M8831.
M8901 is a recombinant isolated from a mating of
M8830 x M890.
All donor strains constructed during this investi-
gation were stable with respect to the F-prime factor when
stored on L agar plates for periods up to 4 months at 4 C.
L broth cultures were less stable when stored at 4 C for
over 3 months.' Therefore, spontaneous curing of the F—
prime in E. pullorum donors was not a problem.
Techniqges of bacterial mating. A modification of
the millipore filter matings described by Godfrey (42) was
employed for routine interrupted matings. The donor and
recipient cells were grown overnight in L broth at 37 C
with aeration. Following a 1:20 dilution into fresh L
broth, the recipients were incubated at 37 C for 3 hr with
aeration and the donors incubated at 37 C for 3 hr with—
out aeration. The mating mixture contained a ratio of l
donor to 10 recipients at a final concentration of l x 108
donors. The mating mixture was impinged upon pre—wet
Inillipore HA 0.45 u, 25 mm sterile filters. The zero
time was taken when the cells were drawn onto the milli-
pore filter. The filter was placed immediately upon a
“nxoist prewarmed L agar plate and incubated for the desired
46
period at 37 C. Following incubation, the millipore
filter was removed from the agar surface to a 13 x 100 mm
sterile test tube containing 1 ml of E minimal medium
with glucose, and shaken for 15 sec with an apparatus
described by Low and Wood (63) to separate conjugal pairs.
Further dilutions were made in E minimal broth with glu—
cose. One—tenth ml portions were pipetted from the mating
mixture dilutions into tubes containing 3 ml of E minimal
soft agar, 0.75%, kept at 45 C. The tubes were shaken
and then the mixture was poured over the surface of E
minimal agar plates selective for specific recombinants.
The plates were incubated 96 hr at 37 C. Donor cells
were counterselected by omitting an amino acid required
of the donor from the E minimal agar plates.
Zygotic induction experiments were carried out
exactly like millipore filter matings described above
except that following interruption of mating pairs, the
mating mixture was diluted into sterile physiological
saline. One-tenth ml portions were transferred to melted
L soft agar overlays containing 0.2 m1 of logarithmic
phase M853 which served as the indicator strain. The L
soft agar overlays were then poured over the surface of
L agar plates. Following incubation at 37 C for 24 hr,
1Zhe phage titer for each time interval was calculated.
To allow for the putative 40 min latent period of
tile phage growth cycle, the diluted mating mixture that
47
was plated at each time interval was reincubated at 37 C
for 40 min. One-tenth m1 portions were then plated as
described above.
Linkage analysiSv—scoring unselected markers. The
selected recombinant colonies were purified by restreaking
them on the same type of selective minimal agar medium.
After incubation for 96 hr at 37 C, isolated colonies were
picked and spread as patches onto the same type of selec—
tive minimal agar medium. Following 72 hr incubation at
37 C, these patch plates served as master plates for
replica plating to various types of selective minimal media
agar plates to determine linkage of the selected markers
to unselected markers.
Kinetic analysis--time of entry of genetic markers.
The kinetic studies were done to demonstrate that the
gradients of transfer were due to F-prime mediated chromo-
somal transfer and not to random F+ type of chromosomal
transfer. The time of entry of the genetic markers was
determined by interrupting millipore filter matings at
10 min intervals.
Cross streak method. This technique is based on a
modification of the procedure of Berg and Curtiss (ll).
A.loopfull of logarithmic phase donor cells of approxi—
Jnately 2 x 108 cells/ml was streaked across the dried
Jaine (0.02 ml) of the logarithmic phase recipient tester
Stzrain already applied to the surface of selective E
miLnimal agar plates. Twelve donor cultures could be
48
tested this way. The plates were incubated for 96 hr at
37 C. The recombinants were scored and the donor strains
yielding the most recombinants were selected for use in
further mating studies.
Poisson distribution for selecting fertile donor
strains. A logarithmic phase culture of approximately
2 x 108 donor cells/m1 was diluted to 10 cells/ml and
0.1 ml aliquot portions were added to 3 ml of L broth and
incubated overnight at 37 C with aeration. When there
was no growth in at least 37% of the broth tubes, there
is an average of one cell per tube. A loopful of the
donor cultures was tested by the cross streak method of
bacterial mating. The appearance of recombinants was
taken as evidence for the selection Of a fertile donor
strain arising from one cell.
Lysogenization of S. pullorum M835 bny35. Logarith—
mic phase cultures of E. pullorum M835 were infected with
a high m.o.i. with phage P35 and incubated overnight at
37 C with aeration. A loopful of the overnight culture
was streaked for colony isolation on an L agar plate and
incubated at 37 C for 24 hr. Isolated colonies were sub-
cultured into 3 ml of L broth and incubated at 37 C with
aeration through early log phase. A loopful of each cul—
11ure was spotted on a fresh lawn of M853 indicator strain
Sensitive to P35 and incubated at 37 C for 8 hr. Lysis
01E M853 occurred if the culture tested was lysogenic for
49
P35. Lysogenization had no effect on auxotrophic require-
ments or mating type.
Electron microscopy. Photographs of various 8.
pullorum donor and recipient strains were printed from
developed Estar thick base plastic film (3% x 4 in) that
had been exposed in a Philips EM300 electron microscope.
Both collodion and formvar coated grids were used. The
Optimum stain was 0.5% phosphotungstic acid (PTA) at
pH 7.5.
An overnight L broth culture of the bacteria to be
examined was impinged upon a millipore HA 0.45 u, 25 mm
membrane filter. The filter was placed in a 13 x 100 mm
test tube containing 3 ml of sterile, distilled water.
The test tube was gently shaken to wash the cells Off of
the filter. The cell suspension was diluted to about
2 x 108 cells/ml and a drop was placed on a grid for
viewing.
To show the attachment of the RNA containing male—
specific phage M82 to the sex—pili of E. pullorum, the
phage were added at a m.o.i. of 100 to the cells after
they had been washed off the millipore membrane filter
in a 13 x 100 mm test tube containing distilled water.
The mixture was allowed to sit at room temperature for
approximately 30 min before placing a drOp of the sample
On the grid for viewing.
To photograph mating pairs, 1 x 109 donors were
miexed with 1 x 109 recipients in L broth and incubated
50
15 min at 37 C in a New Brunswick G76 gyrotory shaker at
a Speed setting of 3. The mating pairs were then impinged
upon a millipore HA 0.45 u, 25 mm membrane filter and.
incubated for 15 min at 37 C on a prewarmed moist L agar
plate. The mating pairs were washed from the filter into
1 ml of distilled water in a 13 x 100 mm test tube by
gently swirling the test tube. M82 phage were added at a
m.o.i. of 100 and the mixture incubated 20 min at 37 C.
The mixture was then stored in the cold until used (never
more than 1 hr). A drOp of the sample was placed on the
grid for viewing.
Radioactive labeling and counting. The 3H—thymidine
(15 Ci/m mole) was purchased from Calbiochem, Los Angeles,
Calif., and l4C-thymidine (57 mCi/m mole) was purchased
from Amersham/Searle, Chicago, Ill. The cells growing in
TCGU broth containing deoxyadenosine were labeled with
3H-thymidine at l uCi/ml of broth or l4C-thymidine at
0.125 uCi/mi of broth supplemented with deoxyguanosine.
To allow maximum incorporation of the 3H-thymidine, E.
pullorum cultures were incubated 12 hr at 37 C with
aeration. E. typhimuirum cultures were incubated 6 hr at
37 C with aeration. The E. pullorum M835 derivatives used
in labeling experiments incorporated approximately 70% of
‘the added 3H-thymidine. The E. typhimurium strains incor-
FXDrated 25% of the added 3H-thymidine and l4C-thymidine.
The scintillation mixture used for cell radioactive
C=<Dunting contained 1.35 gm of 2,5(diphenyloxazole)-benzene
51
(PPO) and 27 mg of l,4,—bis 2(4—methy1-5—phenyloxazole)—
benzene (POPOP) per liter of toluene. Ten m1 of this
mixture were added to vials containing the dried radio-
active samples on filter paper and counted in a Packard
Model 2002 Tri—Carb Liquid Scintillation Spectrometer.
Preparation of bacterial lysates. The lysate
preparation procedure of Clewell and Helinski (23) was
modified for isolation of plasmid DNA. Cells were grown
in 30 m1 of TCGU broth containing radioactive thymidine
at 37 C with aeration. The labeled cells were pelleted
by centrifugation in a Sorvall RC-2 at 10,000 rpm for
15 min at 5 C. The pellet was resuspended in 1 ml of
cold 25% sucrose in 0.05 M Tris, pH 8.0, transferred to
a polycarbonate test tube and plunged into an ice bath.
Two-tenths m1 of lysozyme (5 mg/ml in 0.25 M Tris, pH 8.0)
were added and the mixture iced for 5 min, followed by the
addition of 0.4 ml EDTA made 0.25 M at pH 8.0 and another
5 min on ice. The cells were then lysed by the addition
of 1.6 ml of the "lytic mixture" containing 1% Brij 58,
0.4% sodium deoxycholate, 0.0625 M EDTA and 0.05 M Tris
pH 8.0. The mixture was again iced for 15 min. Lysis
was completed by transferring the mixture to Beckman
cellulose nitrate test tubes and subjected to 5-7 cycles
of freeze—thawing. Freezing was accomplished by plunging
the cellulose nitrate tubes into an ethanol—dry ice bath
followed by thawing in a 45 C water bath. Lysis was
complete when the mixture turned from Opaque to transparent,
52
and increased in viscosity. This crude lysate was then
transferred to a small plastic centrifuge tube and centri-
fuged in a Sorvall RC2—B at 20,000 rpm (48,000 x g) for
30 min at 5 C. The pellet contained approximately 95% Of
the chromosomal DNA leaving the plasmid DNA in the
supernatant fluid. The supernatant fluid was now re-
ferred to as the cleared lysate.
Eye-buoyant density equilibrium centrifugation.
A modified procedure of Bazaral and Helinski (10) was
used to isolate plasmid DNA in a CsCl-EtBr solution.
Three ml of the 3H—thymidine or l4C-thymidine labeled
cleared lysate from a 30 ml TCGU supplemented culture were
mixed with 2.7 ml of TES, 0.5 ml of ethidium bromide
(5 mg/ml TES) and 6 gm of anhydrous CsCl (final density
of 1.54 gm/ml). The mixture was poured into a poly-
allomer tube that had been pretreated by boiling 15 min
in TBS buffer and soaked in 100 ug BSA/ml TES for 1 hour.
The mixture was then covered with a layer of sterile
light mineral oil and the tube capped and centrifuged in
a Type 50 rotor at 44,000 rpm for 30 hr at 15 C in the
Beckman model L3-50 ultracentrifuge. 1
Approximately 60 fractions (12 drops each) of
0.1 ml were collected directly into autoclaved 12 x 75 mm
polypropylene tubes by puncturing the bottom of the poly-
allomer gradient tube with a #24 gauge needle. Five ul
samples of each fraction were spotted on 3/4" squares of
ow
‘ b
I u
.1“
53
Whatman #1 filter paper, washed in TCA, ethanol and
anhydrous ether, dried and counted as described above.
The plasmid peak fractions were pooled and dialyzed
overnight at 5 C in the dark in TBS buffer to remove the
EtBr and CsCl.
Sucrose density gradients. A 0.15 ml sample of
E. pullorum or 0.3 ml sample of E. typhimurium cleared
lysate or pooled, dialyzed fractions was layered directly
onto a 5.2 ml linear 20-31% neutral sucrose gradients
made in 0.005 M EDTA, 0.5 M NaCl and 0.05 M Tris, pH 8.0.
Centrifugation was in a SWSOL rotor at 50,000 rpm for 90
min at 15 C. Approximately 32 fractions of 0.17 ml each
(8 drops) were collected from the bottom of the tube by
using a Beckman fraction recovery system. The fractions
were collected directly onto 3/4" squares of Whatman #1
filter paper, dried under a heat lamp, washed successively
in 250 ml of cold 5% TCA, 95% ethanol and anhydrous ether.
The filter paper fractions were then dried, and placed in
vials containing 10 m1 of toluene scintillation fluid and
counted as described above. For cosedimentation experi—
ments, two samples from the dialyzed pooled fractions in
the amounts described above were layered on top of each
other on the neutral sucrose gradients and centrifuged
as described above for the single samples.
RESULTS
Part I
Counterselection of Donors Carrying F77
The F77 transmissible plasmid in E. typhimurium
transfers the host chromosome in the clockwise order
O—cysE—ilv-thr-pro. Godfrey (42) observed that F77
transfers the host chromosome of E. pullorum from one
origin between Elz_and pgg in two directions (Fig. 1).
A very stable mutant of E. pullorum M881, which
harbors a pyEEl mutation, was selected and designated
M883. An isolate of M883 infected with F77 was desig—
nated M88300. A stable derivative of M88300 which trans—
ferred the host chromosome at a higher frequency was
designated M8830 and used for the mating experiments
described in this thesis.
To map the pyEEl mutation, the donor M8810 carrying
F71 and the recipient M883 were mated and PyrD+ recombi—
nants were selected. The F71 transmissible plasmid carry-
ing Egpf mobilizes from gap in a counterclockwise
direction in E. pullorum (42). The pyEE gene was mapped
in E. pullorum (Fig. 3) in the same relative position it
54
Figure 3.
55
Time of entry of the pyrD+ gene from M8810 x M883
(A) and M8901 x M883 (B) matings. Matings took
place on millipore filters and transfer was
interrupted at various times. A 0.1 ml of the
mating suspension (1 x 106 donor cells) was
plated at each time interval on media selective
for PyrD+ recombinants. The selective media was
supplemented with leucine and cysteine. Histidine
(A) and isoleucine (B) auxotrOphy were used for
counterselection.
Number of Recombinants per 0.1 ml x 10"4
10
56
+
pyrD (A)
?—_‘?‘_?——‘O
+
pyrD (B)
10
20 30
4O
Mating Time (min)
Figure 3
50 6O
57
mapped in E. typhimurium, which was approximately 10
minutes counterclockwise from the trp Operon. To deter-
mine that pyrD was not located near the origin of transfer
for F77 in E. pullorum, the M8901 x M883 mating served as
the control.
Part II
Origins of Transfer and Genetic Loci
Mapped with Donors Carrying F77
Matings using the donor M8830. The recombination
frequencies and gradients of transfer determined from
matings between the donor M8830 and various E. pullorum
recipients are listed in Table 7.
It appeared from the matings with M8830 x M8374
(Table 7) that F77 transfers the E. pullorum chromosome
in the order O-ilv-thr-pro.
From the analysis of the linkage data (Table 8) and
kinetic studies (Fig. 4) of the M8830 x M8374 matings, it
was concluded that the linear arrangement and orientation
of these selected genes in E. pullorum was O—ilv—thr—pro.
The data on the incidence of coinheritance of the donor
M8830 gyEEl auxotrophic marker and streptomycin sensi-
tivity gene are presented in Table 9. There was more
coinheritance of the donors streptomycin sensitivity gene
than the gygEl gene.
The results from the M8830 x M8369 matings (Table 7)
were interpreted as a gradient of transfer continuous
58
Table 7. Recombination frequencies and gradients of marker transfer in crosses
with E. pullorum donors and recipients.
Recombination
Cross Counter~ Length of Selected frequency Relative
selection mating (min) recombinant (per initial frequency
donor input)
'4' .—
M88300 x M5374 pyrDl 6o 11v+ 3.9 x 10_2 1.00
Thr+ 7.5 x 10__6 .22
Pro 1.5 x 10 .05
+ ..
M5830 x M5374 pyrDl 60 Ilv+ 1.2 x 10_g 1.00
Thr+ 6.7 x 1o_5 .54
Pro 1.8 x 10 .15
M5830 x M5369 pyrDl 60 11v: 1.3 x 10:; 1.00
Thr+ 7.0 x 10__5 .54
Pro+ 1.8 x 10__5 .14
His 0.7 x 10 .05
M5830 x M5371 pyrDl 60 Ilv: 8.8 x 10:: 1.00
Thr+ 7.4 x 10_6 .84
His 4.2 x 10 .05
M5830 x M590 pyrDl 6o Cys 6.5 x 10:: 1.00
Ilv 1.1 x 10 .17
+ -
M5830 x M592 pyrDl 60 CysE 6.8 x 10 4 1.00
M5830 x M5390 pyrDl 60 Cyss+ 6.0 x 10:: 1.00
11v: 1.3 x 10_5 .22
Thr 1.2 x 10 20
M5830 x M5104 pyrDl 60 Trp++ 1.4 x 10:: 1.00
CysB 1.0 x 10__S .71
His .7 x 10 .50
M5831 x M5369 pyrDl 60 11v: 4.8 x 10:? .80
Thr+ 7.0 x 10_6 .12
pro+ 6.0 x 10_6 1.00
His 1.5 x 10 25
M5831 x M5374 22:01 60 Ilv: 6.3 x 10:: .93
Thr+ 1.8 x 1o_6 .27
Pro 6.8 x 10 1.00
M5831 x M5104 pyrDl 60 Trp++ 2.0 x 10:: 1.00
CysB 1.5 x 10__6 .75
813+ 2.4 x 10 .12
M5832 x M5374 pyrDl 60 Ilv 4.7 x 10'4a 1.00
M5832 x M5104 pyrDl 60 His+ 4.2 x 10'63 1.00
M5832 x M5374 pyrDl 60 11v: 1.7 x 10:; 1.00
Thr+ 8.8 x 10_6 .52
Pro 1.5 x 10 .01
M5832 x M5104 pyrDl 60 Trp++ 6.3 x 10:: 1.00
CysB 5.1 x 10_7 .81
815* 2.7 x 10 .04
8Initial Observation
59
Table 8. Analysis of inheritance of unselected donor
markers in recombinants from the M8830 x M8374
crosses.a
Selected phenotype
Unselected b
phenotype 918+ 858+ 1189
Ilv Thr Pro+
+
Ilv - 26.5 10.8
Thr+ 18.5C - 16.5
Pro 8.1 15.2 —
aUridine auxotrOphy used for counterselection and
60 min mating period.
bThe number of recombinants analyzed.
c .
The results are g1ven as percent.
Figure 4.
60
Time of entry of various markers from M8830 x
M8374 matings. M8830 was mated with M8374 on
millipore filters and transfer was interrupted
at various times. A 0.1 ml of the mating sus-
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for Ilv ,
Thr+ and Pro+ recombinants. The selective media
were supplemented with leucine and cysteine.
Uridine auxotrOphy was used for counterselection.
Number of Recombinants per 0.1 ml
1100
1000
900
700
600
500
400
300
200
100
61
T l
I—
_ , I pro
I —-I
I
I I
0 I
l I
I I
I I 1’
I I ' I I I I I
10 20 3O 40 50 6O
Mating Time (min)
Figure 4
62
Table 9. Analysis of inheritance of cysteine auxotrOphy
and streptomycin sensitivity of the donor in
M8830 x M8374 crosses.a
Selected phenotype
Unselected
phenotype 329b 219 253+
Ilv+ Thr Pro
CysE- 1.2C 2.7 <1
Streptomycin
sensitivity 11.5 3.2 9.5
a60 min mating.
bThe number of recombinants analyzed.
c .
The results are g1ven as percent.
63
from an origin near ilv and in a clockwise direction as
O-ilv-thr-pro—his.
From the analysis of the gradient of transfer
(Table 7) and kinetic studies (Fig. 5) of the M8830 x
M8371 matings it was concluded that the orientation of
transfer was O—ilv-thr'his.
The recombination frequencies of the CysE+ recombi-
nants observed from the matings of M8830 x M890 and M8830
x M892 were identical (Table 7). It was not possible to
study the El! and £25 loci in M892 due to its extremely
slow growing nature. M890 was useful in determining a
gradient of transfer for gyEE and 112 (Table 7).
Figure 6 shows the time of entry of the gyEEf and
Eizf genes from the M8830 x M890 mating. It appeared that
the gyEEf gene was transferred approximately 10 min after
initiation of mating regardless of whether transferred as
a plasmid or chromosomal marker and the Ele gene was
transferred after 25 minutes.
The mating M8830 x M8390 was very useful in demon—
strating a gradient of transfer of cysE ilv thr (Table 7).
This recipient was similar to M892, but was derived as a
recombinant from an M8830 x M8374 mating and had the
typical E. pullorum growth rate, unlike the slower grow-
ing M892.
The analysis of the linkage data (Table 10) and
kinetic studies (Fig. 7) from the M8830 x M8390 matings
Figure 5.
64
Time of entry of various markers from M8830 x
M8371 matings. M8830 was mated with M8371 on
millipore filters and transfer was interrupted
at various times. A 0.1 m1 of the mating sus—
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for Ilv ,
Thr+ and His+ recombinants. The selective media
were supplemented with leucine and cysteine.
Uridine auxotrOphy was used for counterselection.
65
NI
OH x as H.o Mom mucmcwnsooom mo nonEdz
2
lO
Mating Time (min)
Figure 5
Figure 6.
66
Time of entry of various markers from M8830 x
M890 matings. M8830 was mated with M890 on
millipore filters and transfer was interrupted
at various times. A 0.1 ml of the mating sus-
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for CysE
and Ilv+ recombinants. The selective media
were supplemented with leucine. Uridine
auxotrOphy was used for counterselection.
Number of Recombinants per 0.1 ml x 10-2
35
3O
25
20
15
10
67
+
cysE (M82—)
1 l l
+
cysE (M82+)
10 2O 30 4O
Mating Time (min)
Figure 6
50
6O
68
Table 10. Analysis of inheritance of unselected donor
markers in recombinants from the M8830 x M8390
crosses.a
Selected phenotype
Unselected
Phen°type 200b 196+ 181+
CysE Ilv Thr
+
CysE - 33.7 6.1
11v+ 20C - 13.3
Thr+ 4.5 8.7 -
aUridine auxotrOphy used for counterselection and
60 min mating period.
bThe number of recombinants analyzed.
c .
The results are g1ven as percent.
Figure 7.
69
Time of entry of various markers from M8830 x
M8390 matings. M8830 was mated with M8390 on
millipore filters and transfer was interrupted
at various times. A 0.1 ml of the mating sus-
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for CysE ,
Ilv+ and Thr+ recombinants. The selective media
-were supplemented with leucine. Uridine auxo-
trOphy was used for counterselection.
7O
N
OH x as H.o you musmcHnEooom mo Hoosdz
Mating Time (min)
Figure 7
71
led to the conclusion that the linear arrangement and
orientation of transfer of the selected genes was
O-gysE-ilv-thr.
The analysis Of the linkage data of the M8830 x
M8391 and M8392 matings (Table 11) further suggested link-
age Of gyEE to Ely. BEE and pgg. Combining the data from
parts A and B of Table 11, one is able to construct a
linkage map as O-cysE-ilv-thr—pro.
Figure 8 is a composite of time of entry studies
with M8830 and the recipients M8391 and M8392. These data
confirmed the gene order based on the linkage data pre-
sented in Table 11.
Zygotic induction. M8830 was made lysogenic for P35
as described in the Materials and Methods section and
designated M88301. This donor was mated with the non—
1ysogenic recipient M881 on millipore filters as described
in the Material and Methods section. The apparent time
of entry of the iEE.E§§f marker was at approximately 45
min after initiation of chromosomal transfer by F77
(Fig. 9).
The matings of M8830 x M8104 demonstrated a gradient
of transfer of trp>cysB>his (Table 7). From the analysis
of the linkage data (Table 12) and kinetic studies (Fig.
10) from the M8830 x M8104 matings, it was concluded that
the linear arrangement and orientation of the selected
markers was O—trpecysB-his. Therefore, I inferred that
there was also an origin of transfer for F77 between pyrD
Table 11.
72
Analysis of inheritance of unselected donor
markers in recombinants from crosses with
M8830 and the E. pullorum recipients M8391
and M8392.a
A. Mating:
M8830 x M8391
Unselected phenotype
Selected phenotype
b
284 + 179+
CysE Ilv
CysB+ — 40.7
Ilv+ 23.3C —
Pro+ 3.1 7 2
B. Mating: M8830 x M8392
Unselected phenotype
Selected phenotype
38410+ 269+
CysE Thr
CysE+ — 5.9
Thr+ 6.0C —
Pro+ 1.0 1.5
aUridine auxotrOphy used for counterselection and
60 min mating period.
bThe number of recombinants analyzed.
c .
The results are given as percent.
Figure 8.
73
Time of entry of various markers from M8830 x
M8392(A) and M8830 x M8391(B) matings. The
matings were done on millipore filters and
transfer was interrupted at various times7 A
0.1 ml of the mating suspension (3.4 x 10 donor
cells) was plated at each time interval on media
selective for CysE+, Ilv+ and Thr+ recombinants.
The selective media were supplemented with
leucine. Uridine auxotrOphy was used for
counterselection. Symbols: M8830 x M8392-C)
and [J , M8830 x M8391— . and I .
Number of Recombinants per 0.1 m1 x 10—2
35
3O
25
20
15
10
74
+
cysE (B)
cysE+(A)
Mating Time (min)
Figure 8
Figure 9.
75
Time of entry of 225.222? from the M88301 x
M881 mating. M88301 was mated with M881 on
millipore filters and transfer was interrupted
at various times. A 0.1 m1 of the mating sus-
pension (3 x 102 donor cells) was mixed with
M853 in an L soft agar overlay and poured over
the surface of an L agar plate.
Plaque Forming Units
35
3O
25
20
15
10
76
+
att P35
(+40 min)
20 4O 6O
Mating Time (min)
Figure 9
80
77
Table 12. Analysis of inheritance of unselected donor
markers in recombinants from the M8830 x M8104
crosses.a
Selected phenotype
Unselected
phen°type 153b 221 + 229+
Trp+ CysB His
Trp+ - 92 3.5
CysB+ 98c — 3.9
His+ 2 3 -
aUridine auxotrOphy used for counterselection and
60 min mating period.
bThe number of recombinants analyzed.
c .
The results are given as percent.
78
Figure 10. Time of entry of various markers from M8830 x
M8104 matings. M8830 was mated with M8104 on
millipore filters and transfer was interrupted
at various times. A 0.1 ml of the mating sus-
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for Trp ,
CysB+ and His+ recombinants. The selective
media were supplemented with leucine. Uridine
auxotrOphy was used for counterselection.
Number Of Recombinants per 0.1 ml
79
100 I I l I l
trp+
75h ..
+
cysB
50 L _
25 — _
his
0 l I I
0 10 20 30 40 50
Mating Time (min)
Figure 10
6O
80
and trp cysB which transferred in the clockwise direction
as O-trpécysB-his. The high degree of linkage of cysB and
trp (Table 12) was expected as it had been shown that
they are co-transducible in E. pullorum (42).
Part III
Spontaneous Mutation of F77
The donor M8831 was chosen for further study after
its accidental isolation. This donor strain appeared to
be identical to M88300 except that it was auxotrophic for
gygE due to an apparent spontaneous mutation of the F77
gygE gene. Hereafter, this mutant transmissible plasmid
will be designated F77gy§E..
Matings using the donor M8831. The recombination
frequencies and gradient of transfer for the various
selected markers from the crosses of the donor M8831 and
various E. pullorum recipients are listed in Table 7.
The gradient of transfer and recombination frequencies
for the M8831 x M8369 and M8831 x M8374 matings differed
considerably from those of M8830 and the same recipients
(Table 7).
There were no CysE+ recombinants from M8831 x M890
and M8831 x M892 matings.
The gene order based upon the gradient of transfer
from the M8831 x M8104 matings was trp-cysB—his (Table 7).
81
It was evident from Table 13 that the trp and cysB
genes were very closely linked but have little linkage to
his and that the order of gene entry was O—trpecysB-his
(Fig. 11).
These data substantiated the data previously ob-
served for these three genes in the M8830 x M8104 matings
(Table 12, Fig. 10). Therefore, I inferred that F77gy§Ef
transferred the host chromosome from an origin similar to
or identical to the origin of transfer for F77 between
pyrD and trp, also in the clockwise direction.
Part IV
Determination of a Homogeneous or Heterogeneous
Donor POpulation and Selection for Donors
with Increased Fertility
After observing the ability of the donor M8830 to
transfer its chromosome from possibly two origins and at
moderate frequencies, I attempted to select donor strains
that might transfer at a higher frequency and/or exclu—
sively from only one origin.
Poisson distribution test. I employed the Poisson
distribution test as described in the Materials and Methods
section. There was growth in 49 of 100 tubes of L broth
following diluting and dispensing the M8830 culture.
According to the Poisson distribution if there is no
growth in 37% of the inoculated tubes, then there is on
the average one bacterial cell per tube. I considered
82
Table 13. Analysis of inheritance of unselected donor
markers in recombinants from the M8831 x M8104
crosses.a
Unselected Selected phenotype
phenOtYPe 192b 186+ 186+
Trp+ CysB His
Trp+ - 90 1.6
CysB+ 92C - 1.6
His+ 3 3 —
aUridine auxotrOphy for counterselection and 60
min mating.
bThe number of recombinants analyzed.
c .
The results are g1ven as percent.
83
Figure 11. Time of entry of various markers from M8831 x
M8104 matings. M8831 was mated with M8104 on
millipore filters and transfer was interrupted
at various times. A 0.1 ml of the mating sus-
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for Trp ,
CysBI, and His+ recombinants. The selective
media were supplemented with leucine. Uridine
auxotrOphy was used for counterselection.
Number Of Recombinants per 0.1 ml
84
175 I I I
1501—
125»-
100%-
75s-
50—
25—
Mating Time (min)
Figure 11
85
that the growth in the tubes inoculated with M8830 to have
arisen from one bacterial cell. Each of the 49 broth cul—
tures was tested for sensitivity to the bacteriophage M82.
Forty-eight of the 49 cultures were sensitive. These 48
cultures were cross-streak mated with the recipients M8374
and M8104 and Ilv+ and His+ recombinants reSpectively were
selected.
The data collected after 48 hr of incubation at 37 C
indicated that there might be three populations of donor
cells: one pOpulation transferring from both origins, and
two other populations with each transferring from only one
of the two origins. After 96 hr of incubation at 37 C,
the donor strains which appeared to transfer from only one
of the two origins then appeared to be transferring from
both origins. Therefore, I concluded that the donor M8830
was a homogeneous population carrying F77 which could
transfer from both the origin near gygE and between EXEE
and Egp, each in the clockwise direction.
Matings using the donor M8832. The donor M8832,
isolated from M8830 during the Poisson distribution experi—
ment transferred the iiyf and Eigf genes initially at a
frequency 4X and 3X respectively (Table 7), that pre-
viously observed with the donor M8830. Table 7 shows
that during subsequent matings, the recombination fre—
quencies for the various selected markers in mating with
M8832 x M8374 dropped.
86
From the analysis of the linkage data (Table 14)
and kinetic studies (Fig. 12) of the M8832 x M8374 matings
it was concluded that the linear gene arrangement and
orientation of transfer was O—ilv-thr-pro.
From the analysis of the gradient of transfer from
the M8832 x M8104 matings (Table 7), it was concluded that
the linear arrangement of these selected markers was
O-trp-gysB-his.
The linkage data (Table 15) and kinetic studies
(Fig. 13) of the M8832 x M8104 matings confirmed the
suggested orientation of gene transfer as O-trp-gysB-his.
Electron microscopy of S. pullorum strains. The
overall recombination frequencies and linkage of genetic
markers from E. pullorum matings with chromosomal trans—
fer mediated by F77 and F77gy§Ef were lower than ex—
pected (29). To see if the E. pullorum donors and/or
recipients had any apparent cell surface structures that
might be responsible for poor mating pair formation and
therefore reduction in chromosomal transfer, preparations
of the cultures were scanned in the electron microscope.
The recipient M8374 is shown in Fig. 14A. It looked
normal with respect to gross bacterial cell surface struc—
tures.
The gross cellular appearance of the donor strain
M8830 is shown in Fig. 14B. The average number of sex-
pili per donor cell in E. pullorum is about 15. The donor
M8831 and M8832 were identical in appearance to M8830.
87
Table 14. Analysis of inheritance of unselected donor
markers in recombinants from the M8832 x M8374
crosses.a
Selected phenotype
Unselected
PhenOtYPe 212b 198 140
Ilv+ Thr+ Pro+
11v+ — 17.4 6.2
Thr+ 12.6C - 9.4
pro+ 7.2 8.6 —
aUridine auxotrOphy used for counterselection and 60
min mating period.
bThe number of recombinants analyzed.
CThe results given as percent.
Figure 12.
88
Time of entry of various markers from M8832 x
M8374 matings. M8832 was mated with M8374 on
millipore filters and transfer was interrupted
at various times. A 0.1 ml of the mating sus-
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for I1v+,
Thr+ and Pro+ recombinants. The selective media
were supplemented with leucine and cysteine.
Uridine auxotrOphy was used for counter-
selection.
89
#-
pro
20
15 ~
N
_
0
l
Ioa x HE H.o mom mDCMGHQESOom mo umnEoz
6O
50
4O
3O
20
10
Mating Time (min)
Figure 12
90
Table 15. Analysis of inheritance of unselected donor
markers in recombinants from the M8832 x M8104
cross.a
Unselected Selected phenotype
Phen°type 114g 68 + 53+
Trp CysB His
Trp+ - 89.7 1.9
CysB+ 94.7C - 1.9
His+ 15.8 5.8 -
aUridine auxotrOphy used for counterselection and 60
min mating period.
bThe number of recombinants analyzed.
CThe results given as percent.
91
Figure 13. Time of entry of various markers from M8832 x
M8104 matings. M8832 was mated with M8104 on
millipore filters and transfer was interrupted
at various times. A 0.1 ml of the mating sus-
pension (3.4 x 107 donor cells) was plated at
each time interval on media selective for Trp ,
CysB+ and His+ recombinants. The selective
media were supplemented with leucine. Uridine
auxotrOphy was used for counterselection.
Number of Recombinants per 0.1 ml
70
6O
50
4O
30
20
10
92
Mating Time (min)
Figure 13
Figure 14.
93
Electron micrographs of E. pullorum mating
types. The cells were prepared for viewing by
washing them off a millipore filter into
sterile saline. The bar in each picture repre—
sents 0.5u. A. Recipient, M8374. Magnifi—
cation is 65,000 x. B. Donor, M8830. Magni-
fication is 65,000 x. C. Mating pair, M8830 x
M8374. Magnification is 31,500 x.
94
RU
95
96
97
The E. pullorum donor and recipient in Fig. 14C
might actually represent effective pair formation. It
appeared that many of the sex-pili of the donor make con-
tact with the recipient cell.
In all cases, the male specific RNA bacteriophage
M82 were found adsorbing to the sex-pili of the donor
cells.
Part V
Evidence of F77 Converting to a More Stable
Association with the Host Chromosome
The M88300 strain was checked regularly by the
cross—streak method for its ability to transfer markers
and from each test the isolate transferring at the high-
est frequency was chosen as the donor for mating experi-
ments. The recombinants from these matings were analyzed
for their sensitivity to M82. Those recombinants receiv—
ing F77 were sensitive and indicated that F77 was in the
autonomous state. Upon stable association or integration
of F77 and the host chromosome, the recombinants would
only receive the sex factor as the terminal marker charac-
teristic of Hfr mediated chromosomal transfer. Inte—
gration of F77 should lead to a reduction in the number
of M82 sensitive recombinants when selection was for a
marker transferred very early. Concomitant with this
transition, one should observe an increase in frequency
98
of selected donor markers in recombinants and a stability
of donor ability in the donor strain studied.
It was pointed out in the Materials and Methods
section that the E. pullorum donor strains harboring F77
were quite stable with regard to donor ability measured
by M82 sensitivity.
Figure 15 shows the decreasing percentage of
selected CysE+ recombinants remaining M82 sensitive and
an increasing relative recombination frequency of Ilv+
recombinants during an eight-month period study of M88300 x
M890 matings.
Part VI
Isolation and Characterization
of Plasmid DNA
Lysates prepared from E. pullorum donor strains
M8830 and M8831, recipient strain M883 and E. typhimurium
8A532 and SAl466, a donor and recipient, respectively,
were subjected to equilibrium centrifugation in a solution
Of CsCl-EtBr to determine whether the F77 DNA could be
separated from chromosomal DNA. With lysates of both the
donor and recipient strains of E. pullorum a more dense
sedimenting fraction characteristic of closed circular
DNA (80) and a less dense fraction characteristic of Open
circular DNA and linear DNA were Observed (Fig. 16A).
With the lysates of the donor strain of E. typhimurium,
SA532, similar fractions were observed (Fig. 16B) but with
Figure 15.
99
Evidence for F77 converting to a more stable
association with the host chromosome. The
selected recombinants from the M88300 x M890
matings conducted over an eight-month period
were analyzed at various times for the transfer
of F77 and the donor marker ily+ gene.
100
OH x Ndm uo mmflocmswmum coï¬umsaQEoomm
AOVOI + .
0 no no no .5
O 0 q. l
5 l
A O _ _ a q
T
1
f O
T o
C CIe\HI. C RI _
.5 O .5 O .5 AU 5
.5 8 .I 2 1. .1
A.Ivmmz
ou o>fluwmcom
mucmcï¬neoomm +mn>O unwound
Feb Mar Apr May Jun Jul Aug
Jan
Mating Experiments (Months)
Figure 15
Figure 16.
101
Isolation of plasmid DNA from S. pullorum M835
derivatives and E. typhimuriumT' Three m1 of
cleared lysate of the various strains labeled
with 3H-thymidine or l4C-thymidine were centri-
fuged in CsCl-EtBr dye buoyant density gradients
in a Type 50 rotor at 44,000 rpm for 30 hr at
15 C. Fractions were collected and 5 ul samples
spotted on filter paper squares, washed in TCA,
ethanol and ether and counted for radioactivity.
Fractions 20-27 containing plasmid DNA were
pooled and dialyzed in the dark against TES
buffer at 5 C. (A) S. pullorum M883, M88300,
M8830 and M5831,+T (E) g. typhimurium 511532,
—lk4}q (C) S. typhimurium SAl466,—{1— .
Symbols: 3H-thymidIne +, l4C-thymidine 4}- .
Counts/Min
102
ï¬vl I T fJ\J r I l 9000
A
’- "I
600 _ .— 6000
400 T’ —
_ J 3000
200 —- _
l, I I l -1, I I
0 JVI I I IKV I I â€I 0
B
250 —' ‘ 250
200 -- -* 200
150 r- -7 150
100 _ a 100
50 P ~ 50
l
0'28 i I ‘ikvi i i 0
C
200 —' '4 200
150 _. — 150
100 — ‘H 100
50 - T 50
1
0 u\§ I I 1 “\II I 0
18 20 25 30 38 40 45
Fractions
Figure 16
Counts/Min
103
the recipient strain Of E. typhimurium, SAl466, the more
dense fraction was not present (Fig. 16C).
The fractions corresponding to higher density Of
each strain were separately pooled, dialyzed and layered
on 20-31% neutral sucrose gradients and subjected to
centrifugation to ascertain whether there were molecules
of different sedimentation coefficients in these fractions.
As expected, the donor strain of E. typhimurium carried a
closed circular DNA molecule (Fig. 17). Two peaks of
radioactivity appeared in the denser material from the
E. pullorum recipient M883 (Fig. 18A), and the donor
M8831 (Fig. 18C). The results with M883 were expected
since two plasmids, PO—l and PO-2, which have s values
of 17 and 65 respectively, are found in another strain
of E. pullorum, M853 (73).
The fact that a PO-2-like plasmid was present in
M8831 (Fig. 18C) but absent in M8830 (Fig. 18B) sur—
prised me. Since infection of M853 with phage P35 re—
sulted in the loss of the PO—2 plasmid (W. L. Olsen and
.D. E. Schoenhard, Bacteriol. Proc., p. 46, 1971), M883
and M8831 were infected with phage P35 to find out whether
‘the PO-2-1ike plasmid would be lost. A peak of radio-
éactivity corresponding to the PO-2—like plasmid was ob-
Eserved in a sedimentation profile made from pycnographi—
<=ally separated DNA of M8831; similar results were ob-
tlained with the recipient M883 (data not shown).
1Iunerefore, neither the F77cysE- factor nor phage P35
Figure 17.
104
Neutral sucrose gradient of plasmid DNA from
_s_. typhimurium 511532. A 0.3 m1 sample of 14c
labeled pooled plasmid DNA from the CsCl-EtBr
gradient was layered on a 20-31% neutral sucrose
gradient. The gradient was centrifuged in an
SW 50L rotor at 50,000 rpm for 90 min at 15 C.
Fractions were collected directly onto filter
paper squares and washed in TCA, ethanol and
ether and counted for radioactivity.
105
)—-.
r
_
75
O
5
ACHZ\mucuouvU
Z
25
AU
Figure 17
Figure 18.
106
Neutral sucrose gradient of plasmid DNA from
E. pgllorum M835 derivatives. A 0.15 ml sample
of JH labeled pooled plasmid DNA from the CsCl-
EtBr gradient was layered on a 20-31% neutral
sucrose gradient. The gradient was centrifuged
in an 8W 50L rotor at 50,000 rpm for 90 min at
15 C. Fractions were collected directly onto
filter paper squares and washed in TCA, ethanol
and ether and counted for radioactivity.
(A) M883, (B) M8830, (C) M8831.
107
A
OH x cflz\mucsouvmm
N
4 r0 4. 6 8 4 6
2 1 2 l 2 l
_ . e . _ _ .
r L]
T. 4|
I. LI
I 1'
ll LII.
C
r .7 p h t s
3 2 3 2 l 3 2
x cHz\mucsoov m
m
30
25
20
15
10
Fractions
Figure 18
108
excluded the PO—2 plasmid, but when F77 became associated
with the host chromosome in M8830, the PO-2-1ike plasmid
was lost.
In the sedimentation profiles of M883 and M8831 it
was observed that the area underneath the peak of the
rapidly sedimenting plasmid was larger with the material
from M8831 (Fig. 18C) than M883 (Fig. 18A). Resolution
of the broader peak of M8831 was tried by fractionating
the material into smaller samples. A definite shoulder
in the faster sedimenting material was observed (Fig. 19A).
A new M88300 donor strain was isolated as before. The
sedimentation profiles of pycnographically separated DNA
from M88300 (Fig. 198 and 20A) were very similar to the
ones found with M8831 (Fig. 18C and 19A).
A series of reconstruction experiments were done to
show that the PO-2-like plasmid of M8831 was a composite of
IF77gy§Ef and the PO-2 plasmid. Cosedimentation of F77 DNA
isolated from E. typhimurium SA532 with plasmid DNA from
£4883 resulted in the F77 DNA sedimenting with a greater
‘Ielocity than the PO—2 plasmid DNA (Fig. 21A). An s
\falue of 70 was calculated for F77 (17) corresponding to
61 molecular weight of 51 x 106 daltons (9). Cosedi-
ITientation of F77 DNA isolated from E. typhimurium SA532
‘Nflith plasmid DNA isolated from M8830 (Fig. 21B) confirmed
1the absence of PO-2 plasmid or F77 DNA. Finally cosedi-
Inentation of F77 DNA isolated from E. typhimurium SA532
Figure 19.
109
Neutral sucrose gradient of plasmid DNA from
E. ullorum M835 derivatives. A 0.15 ml sample
of §H IaBeIed pooled plasmid DNA from the CsCl-
EtBr gradient was layered on a 20—31% neutral
sucrose gradient. The gradient was centrifuged
in an SW 50L rotor at 50,000 rpm for 90 min at
15 C. Fractions were collected directly onto
filter paper squares and washed in TCA, ethanol
and ether and counted for radioactivity. (A)
M8831, (B) M88300.
-2)
3H(Counts/Min x 10
A
—II-
4—
—L
_L.
J-
—P
I—-
b ——I
by
I I I l I 1 I I I I I I
O 4 E3 12 16 20 22 38 4O 44 48 52 54
Fractions
Figure 19
12
w
l'-‘
NO
-2)
3H(Counts/Min x 10
Figure 20.
111
Neutral sucrose gradient of plasmid DNA from
E. pullorum and E. typhimurium. The samples of
labeled pooled plasmid DNA from the CsCl-EtBr
gradient were layered on a 20—31% neutral suc-
rose gradient. The gradient was centrifuged in
an SW 50L rotor at 50,000 rpm for 90 min at
15 C. Fractions were collected directly onto
filter paper squares and washed in TCA, ethanol
and ether and counted for radioactivity. (A)
E. allorum MS8300,-l- (B) §' pullorum M88300,
_—iF—v + E. typhimurium SA532,——o—-, (C) S.
pullorum M8831,-—l——, + E. typhimurium SA532,
——n-—. Symbols: 3H—thymidine—l—, 14C-
thymidine-4J——.
-2
Counts/Min x 10
_
—I)-—
_‘F"
+
Z
—I-—
«L—
Fractions
Figure 20
24
16
24
16
24
16
-2
Counts/Min x 10
Figure 21.
113
Neutral sucrose gradient of plasmid DNA from
S. pullorum and S. typhimurium. The samples
3f labeled pooled plasmid DNA from the CsCl—
EtBr gradient was layered on a 20-31% nuetral
sucrose gradient. The gradient was centri—
fuged in an SW 50L rotor at 50,000 rpm for 90
min at 15 C. Fractions were collected directly
onto filter paper squares and washed in TCA,
ethanol and ether and counted for radioactivity.
(A) E. ppllorum M883, —iF-, + S. typhimurium
SA532,.43_., (B) E. pullorum M8830,-4|— , +
S. typhimurium SA532,-{}— . Symbols:
3H-thymidine —lF—, 14C-thymidine-4D—-.
114
15
10
N OH x cflz\mucsou
5 0 4
2
6 8
l
I.—
_
ï¬
e
0
m OH x caz\mucsoo
_ _
L _
.1
Figure 21
115
with PO-2-like plasmid DNA from M8831 and M88300 resulted
in the F77 DNA sedimenting with the leading edge of the
broad PO-2—like plasmid DNA peak with either M88300 (Fig.
208) or M8831 (Fig. 20C).
A comparison of total counts of radioactivity in
plasmid DNA determined from placing an aliquot portion of
the cleared lysate of M883 and M88300 or M8831 separately
onto a neutral sucrose gradients to the total radio-
activity incorporated into cellular DNA, indicated that
there existed approximately 20% and 24%, respectively, of
the total DNA as extrachromosomal plasmid DNA in E.
pullorum.
There was about 8X as much plasmid PO-l DNA as PO—2
DNA. Therefore, there were about 170 COpies of plasmid
PO-l for each copy of the PO—2 plasmid per cell. It
appeared that there were 1-2 copies of the F77 and F77gyEEf
factor per M88300 and M8831 respectively.
DISCUSSION
Part I
Counterselection of Donors Carrying F77
The recipient M883 was chosen as the host for carry—
ing F77 because it has a very stable pyrDl mutation which
maps in the region between pro and trp in E. pullorum, as
in E. typhimurium. However, the map position of pyrDl
limits its use to short mating periods to avoid coinheri-
tance of this marker. The control mating, M8901 x M883,
indicates that pyrDl is not located within the first 60
minutes of an origin of transfer for F77.
Part II
Origins of Transfer and Genetic Loci Mapped
with Donors Carrying F77
Matings using the donor M8830. From the analysis
of the gradient of transfer, linkage and kinetic data of
the M8830 x M8374 matings (Table 7, Table 8, and Fig. 4),
I infer that F77 transfers the E. pullorum host chromosome
from the same origin and with the same orientation:
O-ilv-thr-pro, as it transfers the E. typhimurium chromo—
some (Fig. 2). This suggests that the origin of F77
116
117
transfer in E. pullorum is not as seen in Fig. 1 (42) but
in the same relative position as the origin in E. EypEE—
murium (Fig. 2).
Recall that the genotype of M8830 is $3351 gyEEl
pyrDl/F-cygE+ rfa- pyrE+, and when streptomycin sensitive
or CysEl recombinants are selected, a small number are
streptomycin sensitive and few are cysteine auxotrophs.
Thus, the use of streptomycin to counterselect the donor
is futile.
The gradient of transfer of the M8830 x M8369 matings
(Table 7), O-ilv-thr-pro-his, is like that described for
the M8830 x M8374 matings (Table 7, Table 8, and Fig. 4.).
Since pyggl is used for counterselection, the His+ re-
combinants must result from either counterclockwise trans-
fer from the origin of F77 transfer in E. pullorum, or
from a second origin of transfer.
The kinetic data from the M8830 x M8371 matings
(Fig. 5) indicate that F77 may be able to transfer the
chromosome from one origin in two directions or from two
different origins in E. pullorum. Therefore, the Ilv+
and Thr+ recombinants can result from clockwise chromo-
. . . . + .
somal transfer as in E. typhimurium, and the His recombi—
nants from either counterclockwise chromosomal transfer
from that same origin or from another origin.
The results from the M8830 x M890 (Fig. 6) matings
indicate that EXEEI is the most proximal marker trans-
ferred by F77. It has the same time of entry, 10 min,
118
whether it is transferred as the plasmid or as a chromo-
somal marker. This is about the same as reported by
Godfrey (42).
I conclude from the gradient of transfer, linkage
and kinetic data analysis of the M8830 x M8390 matings
(Table 7, Table 10, and Fig. 7) that the cysE, ilv and thr
genes of E. pullorum are in the same relative position as
they are in E. gyphimurium. It appears that F77 transfers
the E. pullorum chromosome from the same origin and with
the same orientation as in E. typhimurium.
From the analysis of the linkage and kinetic data
from the M8830 x M8391 and M8830 x M8392 matings (Table
11 and Fig. 8) it is possible to construct a linkage map
for E. pullorum as O-gysE—ilv-thr—pro, with these genes
in the same relative position as in E. typhimurium
(Fig. 2).
Eygotic induction. Figure 9 shows that the rela«
tive time of entry of the BEE EEEf locus is 45 min after
initiation of chromosomal transfer by F77. Since P35
antiserum was not used to adsorb free P35 released from
the P35 lysogenic donor M88301, the data in Fig. 9 can
be interpreted in either one of two ways: (1) the increase
in P35 titer reflects the actual transfer of the prOphage
P35 which has its attachment site near the pgg locus which
is transferred after 45 min of mating and is analogous to
att P22+ which maps between proC and proA in E. typhimurium,
119
or (2) the increase in the P35 titer is simply the result
of a one step growth curve resulting from free P35 phage
infection of M881, which happens to have a latent period
of about 45 min.
The analysis of the gradient of transfer, linkage
and kinetic data of the M8830 x M8104 matings (Table 7,
Table 12, and Fig. 10) leads to the conclusion that F77
can also transfer from an origin between EXEE and 35p in
the clockwise direction resulting in a gradient of trans-
fer of O—trp-cysB—his, which confirms the conclusion of
Godfrey (42) who inferred that trp cysB are inverted in
E. pullorum compared to E. typhimurium. Therefore, the
previously Observed E. pullorum His+ recombinants may have
resulted only from transfer from this origin between EXEE.
and 35p instead of from a random origin or by counter-
clockwise transfer from an origin of two directional
transfer near gyEE.
Therefore, it appears that F77 has the ability to
transfer the E. pullorum chromosome from at least two
different origins on Opposite sides of the host chromo—
some and both in the clockwise direction. The primary
origin corresponds to the origin of transfer mediated by
F77 at 116 min on the E. typhimurium chromosome map with
the secondary origin corresponding to a region near 45
min on the map. This suggests a situation analogous to
that reported by Clark (22) for an E. coli donor with
two integrated F factors transferring from two independent
120
origins, both in the clockwise direction, but only from
one origin at a time. At this time neither an appropri-
ate donor with counterselection by a very distal marker
nor a suitable multiple auxotrOphic recipient is available
to determine if in fact M8830 has two integrated sex
factors. Also, it may be that F77 integrates or associ-
ates initially at one of the two origins, but gives rise
either to a subpopulation which transfers from the second
origin or capable of transferring from both origins. An
Hfr population of E. ggli_which gives rise to a subpopu—
lation of Hfr donors transferring from an independent,
separate origin, with the opposite orientation of trans-
fer has been described (61). On the E. typhimurium
chromosome there are two origins of transfer in Opposite
directions near the 47 min region (83). One origin is for
~an Hfr transferring in the counterclockwise direction, the
other origin is for an F-prime and an Hfr transferring in
the clockwise direction. Since the chromosomes of E.
typhimurium and E. pullorum appear to be similar, it is
possible that E. pgllorum strains may have similar chromo-
somal irregularities or nucleotide sequence which have
sufficient homology with the cysE+ rfa- pyrE+ portion of
F77 or a sfa locus with sufficient homology with the F
portion of F77 to allow reciprocal crossovers and inte-
gration for transfer from the second origin between the
pyrD and trp loci.
121
Figure 22 shows a comparison Of the revised linkage
map of E. pullorum with E. typhimurium.
Part III
Spontaneous Mutation of F77
The donor M8831 carrying the F77gyEEf transmissible
plasmid appears to transfer the host chromosome from only
one origin between the EXEE and E£p_genes in the clock-
wise direction (Table 7, Table 13, and Fig. 11) at a
frequency slightly greater than M8830 when mated with
M8104. Unlike F77, F77gyEE- transfers ilifv EEEf and
pggf in a random, F+ type of chromosomal transfer.
The absence of CysE+ recombinants from M8831 x M890
and M8831 x M892 matings indicates that the F77gyEEf
mutation is either identical to the chromosomal gyEEl
mutation or very near it or an overlapping deletion. The
fate of the £52- py£E+ portion is not known. The homology'
between F77gygE’ and the chromosome may be due to a
chromosomal E52 locus and the F portion of F77EX§§5 or
to limited chromosomal nucleotide sequences and the gyEE
(£525 pyEEf) material of F77EX§§5 in the region between
p152 and E£p_in E. pullorum (Fig. 22). In E. typhimurium,
there are two origins of transfer in the corresponding
region between pyrD and trp (83).
122
Figure 22. The partial linkage maps of E. pullorum and
‘E. typhimurium. The origins of transfer for F71,
F77 and F77cysE’ are shown as arrows.
123
thr
ilv pro, att P22
pyrE
rfa thr
cysE
pro, att P35
F77
Salmonella pullorum
pyrD
F77 _ pyrD
F77cysE
trp
F71
cysB
his F71
trp
his
Salmonella typhimurium
Figure 22
124
Part IV
Determination of a Homogeneous or Heterogeneous
Donor Population Carrying F77 and Selection
for Donors with Increased Fertility
Poisson distribution test. A derivative of M8830,
designated M8832, was isolated from the Poisson distri—
bution test and is a homogeneous population with regard to
the cells carrying only one type of transmissible plasmid,
namely F77, which can transfer the E. pullorum chromosome
from two different origins, both in the clockwise direction.
There seems to be a preference for transfer from the origin
near gyEE. This suggests that there is either a gradient
of control favoring mobilization at the gyEE locus or that
F77 might exist autonomously in a few cells as a suprpu-
1ation of the donor M8830 or M8832 as described for a
strain of Hfr E. 99;; (61). This suprpulation might be
responsible for transfer from the origin between EXEE.
and Egp. The former conclusion is favored since no F77
closed circular DNA was isolated from M8830 (Fig. 18B).
Matings using the donor M8832. The gradient of
transfer, linkage and kinetic data (Table 7, Table 14, and
Fig. 12) from the M8832 x M8374 matings are similar to
those observed with the M8830 x M8374 matings (Table 7,
Table 8, and Fig. 4), and further substantiates the
origin and orientation of F77 chromosomal transfer as
O-ilv-thr-pro.
125
The gradient of transfer, linkage and kinetic data
(Table 7, Table 15, and Fig. 13) from the M8832 x M8104
matings are also similar to those presented from the
M8830 x M8104 matings (Table 7, Table 12, and Fig. 10)
and M8831 x M8104 matings (Table 7, Table 13, and Fig.
11), and further substantiates existence of a second
origin for F77 transfer, also in the clockwise direction.
Electron microscopy of S. pullorum strains. Even'
though it has been possible to determine the origin and
orientation of F77 and F77gy§Ef mediated chromosomal
transfer in E. pullorum by gradient of transfer, linkage
analysis and kinetic studies, the overall recombination
frequencies for the selected markers are 10 to 100 fold
less than normally Observed with E. typhimurium donor
strains. Therefore, the recipient strain M8374 and donor
strains M8830, M8831, and M8832 were scanned in the
electron microsc0pe to look for gross surface structures
that may be implicated in inhibiting pair formation. The
recipient M8374 (Fig. 14A) appears to be free Of unusual
surface structures. The donor strains (Fig. 14B) however,
appear to have an average of 15 or more sex—pili per cell.
This is in great excess of the normal 2 to 3 sex-pili per
E. 99;; donor cell (27). The many copies of the sex-pili
per cell can result from either derepression of sex-pili
synthesis (45) or relaxed replication control (9) of the
F77 or F77cysE- factor.
126
Figure 14C shows what may be an actual mating pair.
Depressed sex-pili synthesis may result in the formation
of many nonfunctional sex-pili which can interfere with
effective pair formation and conjugation. Similarly,
multiple COpies of F77 or F77gyEE- may be responsible for
an increased number of sex-pili. Another possibility is
that multiple COpies of the F—prime factor may result in
competition for the homologous region on the chromosome
and interfere with chromosomal mobilization and transfer.
From the sucrose density gradient work it was calculated
that there are 1 to 2 COpies of F77 or F77gy§§7 in M58300
and M8831, respectively. This favors the conclusion that
there is derepressed sex-pili synthesis in the donor
strains.
Part V
Evidence of F77 Converting to a More Stable
Association with the Host Chromosome
As mentioned in the Materials and Methods section,
the E. pullorum donors carrying F77 were very stable with
regard to donor ability as measured by M82 sensitivity.
The data in Fig. 15 indicate that with time and
constant selection for a better donor, there is less
transfer of the intact F77, as measured by M82 sensi—
tivity of the CysE+ recombinants in the matings of
M88300 x M890. Concomitantly, there is an increase in
127
the recombination frequency of a selected donor chromo—
somal marker, ile° Since gygEf is a plasmid gene, the
recombinants must arise from either a reciprocal cross-
over between F77 and the donor chromosome in the F-gyEE
region or a nicking of the chromosomally associated F77
between F and gyEE. This decrease in intact F77 transfer
and increase in a chromosomal transfer is analogous to
the conversion of M88300 from the Type I F-prime donor to
M8830, a Type II F-prime donors (76, 84).
The E. pullorum M8830 donor strain also has charac—
teristics of both Type I F+ and Type II F+ E. ggli_donors
described by Curtiss and Renshaw (29). Mobilization at
the EXEE locus is like a Type I F+ donor since the F77
factor is very stable and mobilizes with a specific
orientation, but with an intermediate frequency. Mobili—
zation at the pyrD-trp locus is like a Type II F+ donor
which transfers at a low frequency which may be due to
integration of F77 into the host chromosome but cannot
eXpress itself as a typical Hfr.
Part VI
Isolation and Characterization
of Plasmid DNA
The M883 isolate of E. pullorum strain M835
apparently contains two plasmids: PO—l and PO-2 (Fig. 18A)
which are similar in size, 175 and 655 respectively, to
those found in strain M853 (73). However, the PO-2 plasmid
128
in M835 is not excluded by P35 phage as it is in M853
(W. L. Olsen and D. E. Schoenhard, Bacteriol. Proc., p.
46, 1971).
The fact that closed circular DNA molecules were
isolated from M88300 (Fig. 20A) and that the sedimentation
profiles with these molecules had a shoulder on the lead—
ing edge of the more rapidly sedimenting material indi-
cates to me that F77 is still in the autonomous state
(Fig. 19A). Confirmation of this conclusion is provided
by cosedimentation of differentially labeled DNA from
'E. typhimurium SA532 and M88300 (Fig. 20B). The broad
peak of activity extending from fractions 4 to 9 with
material from M88300 probably is due to the fact that it
is a composite of F77 and plasmid PO—2.
The M8830 mutant of M88300 which transfers from two
origins: at gyEE between py£E_and Egp, and with an inter-
mediate frequency, was suspected of being an Hfr donor in
which F77 no longer existed autonomously. This conclusion
was supported since no closed circular DNA of F77 size
was observed (Fig. 18B). The Hfr type is probably the
result of a mutation(s) in some regulatory mechanism.
The correlated loss of the PO-2 plasmid was un-
expected since the autonomous state Of neither F77 nor
plasmid PO-2 was eliminated in M8831 (Fig. 19A) or
M88300 (Fig. 19B). Since neither F77 nor plasmid PO—2
remain autonomous in M8830 this is not a case of plasmid
incompatibility as described by Novick (68) and reported
129
between ColB2 and R(f) (45). An explanation of this
observation is that F77 and PO-2 have a common control
for maintenance as independent replicons. If the mainte-
nance mechanism is mutated, then either the plasmid must
come under the control of some other replicon, e.g., the
chromosome, or abort. Because the F77 factor possesses
homology with the chromosome at the gyEE locus and
probably at the EEE and EXEE loci, it can integrate while
the PO-2 lacking known homology is lost.
The M8831 mutant of M88300 which is F77gy§Ef 5E3?
pygE? was similar to its progenitor except that mobili—
zation Of the chromosome occurred only between pyEE and
Egp. Apparently the mutation in the F77 factor was
severe enough to prevent proper pairing and integration
at the gyEE locus. Probably integration at the gyEE_locus
is a function of this gene rather than genes of the F.
Since the mutation in the gyEE locus is very stable, it
is possible that the £53 and EXEE loci are also affected.
If so, then integration between pyEE and E£p_must be due
to F homology. This may account for the low frequency
of transfer from this site. Tests of this conclusion rest
upon isolating Hfr donors from M8831, and other mutants
of M88300 with which complementation and recombination
studies can be done.
Approximately 88% of the plasmid DNA of E. pullorum
M835 derivatives exists as plasmid PO—l DNA corresponding
to 170 copies per cell. The PO—2-like plasmid DNA is
130
present in only 1 to 2 COpies per cell. The PO—l and PO—2-
like plasmids are probably cryptic plasmids as they have no
known host phenotypic expression, similar to the small
molecular weight plasmid (1.4 x 106 daltons) found by
Cozzarelli EE.§£° (25) in E. gel; strain 15. Olsen and
Schoenhard (73) observed approximately 150 COpies of the
PO-l plasmid and 1 to 2 COpies of the PO—2 plasmid per
cell of E. pullorum M853.
I conclude that the F—prime factor F77 exists in E.
pullorum as either an autonomous replicon like a Type I
F-prime donor or stably associated with the chromosome like
a Type II F-prime donor. In either case, it transfers the
host chromosome from two different origins; at gy§E_and be-
tween EXEE and 55p. both in the clockwise direction. When
the F77 factor becomes stably associated with the host
chromosome, the PO-2 plasmid is lost. The F77gy§Ef factor
transfers only from the origin between EXEE and Egp.
It exists as an autonomous replicon and the plasmid PO—2
is not lost. This suggests that there is a common control
mechanism for the autonomous replication of the F-prime
and the PO-2 plasmid. A mutation Of this control mechan—
ism may force the F77 factor to either abort or be rescued
by associating with the host chromosome due to its chromo-
somal homology. Since there are only 1 to 2 COpies of F77
or F77gy§Ef in the autonomous state per donor cell, the
numerous sex—pili per donor cell must result from de—
repressed sex-pili synthesis.
SUMMARY
The transmissible plasmid F77 carrying the cysE+
+ . .
rfa pyrE genes transfers the E. typhimurium chromosome
from the chromosomal gyEE locus at 116 min on the chromo—
some map and in the clockwise direction (Sanderson and
Saeed, personal communication).
The conclusion based on the gradients Of transfer,
linkage analyses and kinetic studies from the matings
with E. pullorum donors carrying F77 and E. pullorum
recipients is that F77 transfers the E. pullorum chromo-
some from two origins, both in the clockwise direction.
F77 transfers primarily from the origin at the cysE locus
which maps in the same relative position, 116 min, as in
E. typhimurium and secondarily from the origin between the
pyrD and trp loci which is equivalent to the 45 min posi—
tion in E. typhimurium. The E. pullorum genetic markers
studied appear to be in the same relative position as in
E. typhimurium except for the inversion of the trp cysB
loci. Presumably transfer from the origin at cysE is
due to the homology between the cysE rfa pyrE loci on
both F77 and the host chromosome. Transfer from the
origin between pyrD and trp may be due to either a sfa
131
132
locus for the F portion of F77 or a nucleotide sequence
with sufficient homology for the cysE+ rfa- pyrB+ portion
of F77. The observation with M8830 of increased donor-
capability, reduction in F77 transfer per se and an in-
crease in transfer of donor markers, leads to the con—
clusion that F77 is stably associated with the host
chromosome. The conclusion based on the results of the
Poisson distribution test is that F77 is a homogeneous
transmissible plasmid in the host cell.
The spontaneous mutant of F77 designated F77gyEEf,
transfers the E. pullorum chromosome only from the origin
between EXEQ and Egp in the clockwise direction at the
same frequency as F77. Transfer from this origin must be
due to either a chromosomal EEE locus for the F portion of
F77gy§Ef or a nucleotide sequence with sufficient nucleo-
tide sequence homology for the chromosomal portion of
F77gyEE-. The low frequency of transfer suggests that
F77EX§§5 does not form a stable association with the
chromosome.
Electron micrographs show that E. pullorum donors
harboring F77 and F77EXEE- have at least 15 sex—pili per
cell. The recipient cell has no unusual gross cell sur-
face structures. Many sex-pili appear to be involved in
mating pair formation.
Supercoiled DNA molecules can be isolated from
E. pullorum M835 derivates and an E. typhimurium donor
strain by CsCl-EtBr dye buoyant density gradient
133
centrifugation and characterized by zonal centrifugation
in a linear neutral sucrose gradient. Using these methods,
it was found that the E. ppllorum recipient M883 has two
distinct plasmid molecules designated PO-l and PO—2.
Plasmid PO-l has a molecular weight of 2.1 x 106 daltons,
sedimentation coefficient of 175 and present in about 170
copies per host chromosome. The PO-2 plasmid is nOt ex—
cluded by phage P35, and has a molecular weight of 45 x 106
daltons, sedimentation coefficient of 655 and present in
about 1-2 copies per host chromosome. The donor M8830
carrying F77 has only the PO-l plasmid molecules present.
The PO-2 plasmid appears to be excluded by F77. When the
recipient M883 is newly infected with F77 (M88300), the
PO-2-like plasmid peak appears broader and is a combi—
nation Of two plasmid species, a 655 and 705 molecule.
The donor M8831 carrying F77_cys_h;' has the PO-l plasmid
species and the broad PO—Z-like plasmid species which
also is a combination of two plasmid species, a 655 and
70s molecule.
The plasmid molecule isolated from the E. typhimurium
donor SA532 carrying F77 appears to be homogeneous and co—
sediments with the 70s plasmid molecule of the broad PO—2*
like peak Of M88300 and M8831. Further studies indicate
6 daltons and
that F77 has a molecular weight of 51 x 10
sedimentation coefficient of 705. This suggests that
F77 is autonomous in E. pullorum, but when it becomes
134
associated with the host chromosome, the PO-2 plasmid
molecule is lost. This suggests that the PO—2 plasmid
and F77 factor may have a common replication control
mechanism. A spontaneous mutation may have inhibited
replication, but F77 can associate with the chromosome
due to homology and be maintained. The lack of PO-2
plasmid exclusion by F77gyEEf suggests that the spon-
taneous mutation resulting in the mutation of the F77gy§Ef
gene has not disrupted PO-2 and F77 plasmid replication
control.
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