California State University, Northridge
EPISOMAL TRANSFER IN E. coli
1\
A study of F' lac+ transfer under conditions
of continuous cultivation
A
thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Larry
~resi
January, 1973
,---------- - - - - - - - - ·- - - - - --- -----------------------------1
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The th...EJsis of Larry Baresi is approved:
Committee Chairman
California State University, Northridge
January, 1973
ii
ACKNOWLEDGMENTS
i
To Dr. Charles R. Spotts I would like to extend my deepest
!
I appreciation
for his assistance, guidance, and criticisms in the
I development of this thesis. Further thanks is extended to
I Dr. Donald Bianchi and Dr. Charles Weston for serving on my committee
Iand for their assistance.
I am indebted to the California State University, Northridge
J
I Foundation for
I
financial assistance; to Dr. Pat Zamenoff for the
j strains used and to Phil Goodman for his assistance in photographing
I the
I
apparatus.
I
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Lastly, I would like to express my gratitude to my sister
I Mrs.
L. Touhy for typing this thesis and to my parents Mr. and Mrs.
1
i
L Baresi for supporting my education.
I.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS
• • • • e • • • a •
1t
a a • a • • a a a • • a • a • a • a • • e a • a a a
iii
LIST OF TABLES AND FIGURES .••.•••••••••••••••••••••• v
ABSTRACT
..............................................
INTRODUCTION
vi
1
...........................
METHODS AND f.1ATERIALS
ORGANISr.1S
7
7
10
tviEDIA •••••••••••••••••••••••••• • ••• • • • • • • • • • • • •
CONTINUOUS CULTIVATION
11
Tl1EORY ••••••••••••••••••••••••••• • · • • • • • • • 11
12
APPARATUS
.....................
••••••••••••••••••••••••••••••
'II
••
.............................
HETHODS
GLUCOSE ASSAY
CELL WEIGHT
POPULATION At~ALYSIS
YIELD CONSTANT
A.~ALYTICAL
•••
li
•••••••••••••••••••••••••
U ~fax·····~········
.....................
RESULTS
STEADY STATES OF LB2 AND LB4 •.••••••••••.••••••.
SPONTANEOUS LOSS OFF' lac+ ..•••••••...••••••••
TRANSFER OF F' lac+ AT VARIOUS
DILUTION RATES
TRANSFER OF F' lac+ AT VARIOUS
INITIAL F '/F- RATIOS
•••••••
'll
•••••
tl
............................... .
13
13
18
18
19
19
21
21
21
22
27
•.....•••••.•••••••••••••••• 28
46
POPULATION DISADVANTAGE
•
RA1''E OF F'
..... .................. .
........................................ 49
DISCUSSION
BIBLIOGRAPHY
TRA.t~SFER
•
•
II
9
A
a a • a • • a a a a a a a a e ••
iv
S
e a·. a a a • • a a a a e a a • e
58
r·--------------·---------------- ------- --- ---------- - - -- ·---- ---------- ----------------- ----- ----- -
---------------~------~-------------~-----------,
I
I
J
LIST OF TABLES AND FIGURES
TABLE
1
BACTERIAL STRAINS USED
2
SUMMARY OF EXPERIMENTS ••••••.•••.•••••••.•••••• 40
9
FIGURES
1
PI CfURE OF CHEMOSTAT • • • . • • • . . • • • • • • . • • • • • • • • • • • 15
2
DIAG~1
3
STEADY STATES FOR LB2 AND LB4
4
6
SPONTANEOUS LOSS OF F'
26
+
F' lac TRANSFER AT D = 0.53 hr. -1 . ............. 30
F' lac+ TRfu~SFER AT D = 0. 268 hr. -1 . ............ 32
7
F' lac+ TRfu~SFER AT D = 0. 09 hr. -1 . ............. 34
8
F' lac+ TRANSFER AT LB4/LB2
9
F' lac+ TRANSFER AT LB4/LB2
.............
= 0.006 ............
10
INITIAL F' lac+ TRANSFER AT
VARIABLE DILUTION RATES
....... , ...........
5
11
12
OF CHEMOSTAT .••••••.•••••••••••.•••.••. 17
..................
.........................
= 0.09
24
36
38
43
INITIAL F' lac+ TRANSFER AT
VARIABLE LB4iLB2 RATIOS
45
+
F' lac TRANSFER AT D = 0. 237 hr. -1 . ............ 48
...................
v
r-------------·-·-· --- . . · ·------·- . -
-··
-~--· ~ ~----·
-· -~- ·-~-------···---- -----~~--~------~~
.
·~--
...... ~- .... ~
ABSTRACT
Il
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!
TRANSFER IN E. coli
EPISO~~L
I
I
A study of F' lac+ transfer under conditions
of continuous cultivation
by
Larry Baresi
I
Master of Science in Biology
1
January, 1973
I
Episomal transfer was observed between donor and recipient
I
! strains
I
I
of Escherichia coli while under conditions of continuous
-
-
cultivation in the chemostat.
I donor
The effects of the initial recipient/
ratio and the generation time of the recipient cells on rate
and extent of transfer were determined.
It
was found that cellular
replication does not affect the rate of conversion whereas the
recipient/donor ratio drastically alters the rate of recipient conversion.
Finally it was found that the number of episomes per donor
cell was around 1.3.
This study indicates that it is possible to
study genetic transfer quantitatively under conditions of continuous
cultivation.
vi
'
-----'------,
i
I
The transfer of genetic material between bacteria was first
The
process which they described, called conjugation, involved the transfer of genetic material directly from one bacterial cell to another
while in cellular contact.
At first, conjugation was observed only
between strains of the species E. coli; however in recent years this
process has been detected in an ever increasing number of bacterial
species.
It has now been shown not only to occur between various
strains of E. coli but also between strains of Salmonella, Pasturella,
Rhizobi~~,
and Vibrio.
Conjugation is now known to occur between
E. coli and strains of Shigella, Salmonella, Serratia, Proteus, and
even between E. coli and Pasturella, members of two different
families.
(Reviews of conjugation between various bacteria can be
found in Curtiss, 1969; Hayes, 1968; Adelberg and Pittard, 1965;
Jacob and Wollman, 1961).
Recognition of the mechanisms involved in conjugation stems
from the work of Lcderberg, Cavalli, and Lederberg (1952) and Hayes
(1952 a,b).
The presence of an autonomous genetic element, the F-
factor or fertility factor, is necessary for conjugation.
Cells that
'
contain the F-factor are called
F+ , male, or donors; while those that
lack it are called F-, female, or recipients.
Except in the specific case ofF', to be described shortly,
only a very small percentage of chromosomal DNA is transferred during
the conjugation of F+ and F- cells.
.'
,.... . .
~~-~--"-~~--.-~--~-----
, __,._ _ _ _ _ _ _ ,.
·-
~--~-~-- --·~---
, _ _ ._ ..
0
~--
--·-·----
--~---·-···
-- ••••
---~--
1
The F-factor itself, however,
.......
~
-------
'
-~------- ---~------·-·~----"~-----·-=····--·--·-- ---~--~--~------~~--------------
2
,---------·--·--------~----·-----·····----·~·-····------·-·~---------------.--
1 is
transferred from F+ to F- with high efficiency resulting in the
+
conversion of the recipient F cells into F cells.
(1952) were the first to describe this conversion.
Lederberg et al
They mated sus-
+
pected F and F cells and then reisolated the F by selective
streaking.
By this method they found that under their conditions 10%
j of recipient cells were converted to F+ within one hour.
In the
l subsequent years it was found that the frequency of transfer of the
+
F..:factor from F to F cells varied depending on the strain and
environmental conditions used (Curtiss, 1969; Curtiss and Charamella,
1966).
For example, Curtiss (1969) found that conversion ofF
to
+
F was enhanced if the cells were grown in a supplemented instead of
a synthetic medium and if donors were grown anaerobically instead of
aerobically prior to mating.
Cairns (1963 a,b) has found the F-factor to be a closed circular
molecule of DNA.
According to at least one report F-factors may
exist in multiple copies up to four per cell which segregate according
to a regular non-random distribution during cell division (Scaife,
1967).
+
The F-factor determines a specific male antigen termed F
(¢rskov and
~rskov,
1966) and leads to production of male pili which
apparently act as conjugation bridges (Brinton, Gemski, and Carahan,
1964).
It has been shown that the F-factor can alternate between a
stable autonomous cytoplasmic state and a stable integrated state
(integrated into the genome).
Factors that are capable of such
alternation are called "episomes" (Jacob and Wollman, 1958).
A cell
containing the F-factor in the integrated state is called HFr and is
characterized by the ability to donate its chromosome at a high
3
r---------~------------~------········"·-··------·-·-·-· ----·------------·-----~--~
I frequency.
The HFr is further characterized by its inability to
donate the integrated F-factor at the
transfers chromosomal genes.
1 restricted
I and
sa~e
frequency with which it
By definition, the designation p+ is
to cells that possess the F-factor in the autonomous state
is characterized by the F-factors ability to replicate indepen-
I- dently of the chromosome.
While in the autonomous state the F-factor
is transferred at high frequencies while little chromosomal material
is donated.
Some genetic elements are known which exist permanently
in the autonomous state.
1952).
i
These are called "plasmids" (Lederberg,
By definition, an episome which loses the ability to integrate
becomes a plasmid (Jacob, Schaeffer, and Wollman, 1960).
In 1959 both Adelberg and Burns, and Jacob and Adelberg described
a fertility factor that not only was transferred at a high frequency
but carried a chromosomal marker.
(F') factors.
These elements are called F-prime
It has been shown that these fertility factors arise
by a reciprocal cross over between a chromosomal site and a site
within the integrated F (and behave like the p+ factor).
Most of the F-factor transfer studies have been done in batch
culture.
These studies usually consisted of mixing p+ and p- cells
obtained from overnight batch cultures and then analyzing, several
hours later, for converted cells by the converted recipients cells
ability to donate the F-factor.
fixed volume of medium.
Batch culture refers to growth in a
In such cases the-nutrient concentration
varies continuously as the organisms grow.
As nutrients are being
depleted changes in the physiological.state of the cells occur which
i obsr~ures
the significance of any quantitative results that a.re
I
4
conditions
I conditions are necessarily short.
In their normal habitat, the intestine, E. coli cells are
continually being supplied with the necessary nutrients for growth.
I
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Because of this continuous supply the cells are able to grow
I uninterrupted
for long periods of time.
To see if genetic exchange
takes place under in vivo conditions several workers have carried out
genetic transfer studies using germ free animals (Guinee, 1965;
Kasuga, 1964; Jarolmen and Kemp, 1969; Reed, Sieckmann, and Georgi,
1969; Jones and Curtiss, 1970).
One advantage found in using germ
I
free animals was the ability to extend or lengthen the duration of the'
experiment, thus viewing genetic transfer over a longer period of time
than could have been previously done.
As one example of this type of
study Jones and Curtiss (1970) fed germ free mice food pellets that
were contaminated with F- E. coli cells.
They later reintroduced
+
into the mice F or F' E. coli and determined by bacteriological
examination of fecal samples, the genetic exchange that had taken
place in the animal's intestine.
They found that almost the entire
F population had become donors after only 6 hours and that this was
excretion of waste materials.
However, even by using animals, the
exact physiological conditions at the time that the genetic exchange
5
--1
takes place could not be defined.
The possibility arises that the
i
I
physiological conditions of the animal's intestine varies or is cyclic!
I
and is not constant. These cycles could for instance be related to
the
cons~~ption
of food by the animal or the presence of bacterial
pockets residing in the intestinal lining of the animal's gut.
To define more precisely the effect of the cells physiological
i
state on transfer and to study long term results of F+/F- mixing it
I
is necessary to maintain the cells under strictly constant and defined'
physiological conditions for long periods of time.
This is possible
by means of a technique known as continuous cultivation.
The concept
of continuous cultivation was developed by Monad (1950) and by Novick
and Szilard in 1950.
Both groups realized that a population of
organisms could be maintained under conditions of continued growth
for long periods of time if new nutrients were continually added
while waste material and cells were continually removed.
They
suggested that in order to keep the population at a constant level
one had only to limit the availability of a growth factor or an energy,
!
source.
By selecting the proper concentration of limiting substrate
old cells could be removed as fast as new ones were being formed, thus
reaching a stable popttlation which could be maintained indefinitely.
One device that accomplishes this is the Chemostat.
The Chemostat
permits the delivery to a growth vessel fresh medium in which one of
the nutrients is present at growth limiting concentrations.
The
vessel is equipped with an overflow device so that for each drop of
medium that enters, a drop of culture and waste material flows out.
The cells that are in the culture vessel grow at the expense of the _
I
6
-·
··-~·-------·---------.---------·---·-----~··----~----···------·---------~
added nutrients.
When properly adjusted, the number of new cells
being formed in the growth chamber is exactly balanced by the nwnber
l
of old cells leaving the vessel and the population size is therefore
constant.
The growth rate of the culture can be controlled by
adjusting the rate at which new medium enters the culture vessel.
Thus the chemostat keeps a culture of growing cells for indefinite
period of time,_ at constant population sizes, and under constant
physiological conditions.
I
J
I
The experiments described in this study were designed to
demostrate the dynamics of F-factor transfer while under controlled
I conditions
I
offered by use of continuous cultivation.
I and initial infection
I to change in the rate
i
Generation times
ratios were altered and analyzed with respect
and extent of F-factor transfer.
IL____ -- ....... -----···--- ----- .. - ·-- ......................................-....... .
i
1,.
I
----- ·----
-------~--~-------·------·-------·····-··----···----------------·--------------,
j
METHODS A.\lD :MATERIALS
ORGANIS~5:
All bacteria utilized in this study were derived from
Escherichia coli K-12.
Sources and genotypes .of each strain are
shmm in Table 1.
Spontaneous mutants to Strr or T1r were obtained by plating 10 9
cells on the appropriate selective medium and maintained in stock
cultures lacking streptomycin sulfate or T1 virus.
The presence of
the F' lac + in strain LBl was demostrated by its ability to ferment
lactose and by the ability to transfer this property to AB 593.
T1 virus (used for diagnosis of the T1 sensitivity of strains)
was obtained from Dr. Charles Spotts (Dept. of Biology, CSUN).
Virus
was prepared by infecting 50 ml of log phase cells of strain AB 593
in LB medium with 1 ml suspension of T1 virus.
The lysate was cleared ;
of cells and debris by centrifugation for 30 minutes at 1,000 x G
. (using a Sorvall RC-2).
The supernatant from this centrifugation
Iwas decanted and recentrifuge for 10 minutes at
I supernatant
chloroform.
12JOOO x G.
The
from the second centrifugation was decanted into a sterile ·
The T1 virus suspension was assayed by the plaque count
method as describt:d by Adams (1959).
After adjusting the concentra-
tion to 9 x to 10 PFU/ml, the stock solution was stored in a
refrigerator at 4°C.
I
l~-·-" i~
¥
· - - -··-••·
~-·-~·~ -.-v-~,"-•·~-~~·- • -·~~-~~-·- ··~ ,.,,, ~···-·~---•·•~ • •·--·~--- ~- ~-··
••«-- ,_
•---~····---··
7
______ ._ _ _ _ _ _ _ -
"~-·•-• ·------·-·••---··-·-•-••~---• ~~-i
••-
8
Table 1:
Source and genotype of bacterial strains
used.
r----1
II
DESIGNATION
I
I
AB 593
Dr. Patricia Zamenoff (Dept. of
Biological Chemistry, UCLA)
(Zamenoff, 1966)
B1 - lac - str s T s
1
Dr. Patricia Zamenoff (Depto of
Biological Chemistry, UCLA)
(Zamenoff, 1966)
B - lac - str s T1 s (FT lac + )
1
Isogenic with AB 593 but
containing F' lac+)
LB 1
Spontanous strr mutant of AB 593
B1 - lac - str r T1 s
(F-)
LB 2
Spontanous T1 r mutant of LB 1
B1 - lac - str r T1 r
(F-)
Altered LB 1 selected from LB 1
x DZ 58 mating
Bl
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II
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GENOTYPE
SOURCE
STRAIN
DZ
58
!
i
i
LB
4
lac - str r T1 s
I
(F - )
+
(F' lac )
I
lI
I
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l
!i_________________________
---·---·----------·-
,
________________
...I
10
10
~~-D-IA-:
--·-
-~-------~--------------·-
------· ______________. _____________________ . ___
--------~
The standard chemostat medium (CM) consisted of (per liter):
,
I
(NH4) 2so4 , 1 grn; MgS04 • 7H2o, 0.05 gm; Thiamine-HCL, 0.001 gm;
glucose, 0.01 gm; trace element solution, 1 ml.
After autoclaving
l
Ithis medium it was routinely supplemented with (per liter):
I Streptomycin sulfate,
6.6 ml.
0.05 gm; 0.5M Na2HP04 , 13.4 ml; O.SM KH2 Po4 ,
The phosphates were autoclaved separately and added along
with the Streptomycin sulfate to the CM in the chemostat reservoir
through a 0.22 u millipore filter.
The trace element solution consisted of (per liter):
Znso4 ·
7H2o, 0.44 mg; coso4 • 7H 2o, 0.024 mg; cuc1 2 • 2H 2o, 0.0135 mg;
Ii
MnS04 • H2 0, 0.0165 mg; Na 2 B5 o7 • lOH 2 0, 0.88 mg; ·peso4 • 7H 2 0,
I
24.9 mg.
!i
Basic minimal medium (BMM), used for identification of lac+
consisted of (per liter):
cells~
Minimal Broth Davis w/o glucose (Difco),
10.6 mg; lactose, 5 gm; NaCl, 0.58 gm; t-1gS0 4 • 7H 20, 0.12 grn;
Thiamine l!Cl, 0.001 gm; Agar (Bacto), 15 grn; trace element solution,
1 ml.
The lactose was autoclaved separately and added aseptically to
the sterile medium.
Levine EMB (LEMB) was prepared by modifying slightly the
standard Difco Levine EMB in order to facilitate virus absorption.
The following salts were added (per liter):
7H 2o, 0.12 gm.
NaCl, 0.58 gm; MgS0 4 _·
This medium served as the basic differental medium
for distinguishing lac- from lac+ cells.
I
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11
Difco Nutrient Agar (NA) was made according to instructions and
will be referred to as Nutrient Agar (NA).
LB medium consisted of (per liter):
Bacto-Tryptone, 10 gm;
Yeast Extract (Difco), 1 gm; glucose, 1 gm; NaCl, 8 gm; MgS04 • 7H2o,
0.2 gm; CaC1 2 , 0.22 gm.
Selective media used for isolation of strains were modifications
of the previously described media in which either Streptomycin sulfate
(50 ug/ml) or T virus (9 x 109 PFU) was added.
1
CONTINUOUS CULTIVATION:
1.
Theory
The kinetics of growth under conditions of continuous cultivation
are described by certain general formulas that were developed by
Monod (1950) and by Novick and Szilard (1950).
These formulas
describe quantitatively the various parameters involved in continuous
cultivation in terms of three growth constants:
the maximum growth
rate constant, Urn, (the maximum rate of growth at saturation levels
of substrate); t:he yield constant, Y, (the weight of organisms formed
per gram of substrate used); and the saturation constant, Ks, (the
concentration of substrate allowing !J the maximal growth rate to
occur).
I
These three growth constants can be determined under either
batch or continuous conditions and are characteristic of the strains
and culture conditions used.
I
The parameters defined by these formulas are the residual sub-
I strate
concentration in the growth chamber (5) and the concentration
of bacteria (x) under steady state conditions.
!
The residual substrate.
concentration is described by the following equation:
L ______ ._.,...... ·- ....................... - ....................................
s=
K5 (D/Um-D)
12
--~----~6~~--~~~-·~~~-
-·~---~-~~-~--"---·~"·-.~--A--~-~--"~-~-~---~--~~---~-----·------·---~~---··••••-~•o~
!
where D is the dilution rate (the fraction of the culture volume being
displaced per hour).
X= y
equation
The bacterial concentration is described by the
(Sr- s) where sr represents the substrate concentration
in the reservoir.
At defined values of Sr and D and within certain
limits the concentration of cells in the growth chamber will reach an
I equilibrium value,.
values of
x can be
By altering Sr or D, a wide range of equilibriwn
9btained at the discretion of the researcher.
Besides the formulas describing the residual substrate concentration and the bacterial concentration under steady state conditions
Monod (1950) was also able to describe a relationship that exists
I between
the dilution rate (d), generation time (td), and the specific
growth rate (u) • . Under steady state conditions Monod (1950) was able
to show that the doubling time of a culture (td) is equal to 1/D
(td
= 1/D).
Using the experimental growth equation (u
= ln
2/td)
Monod (1950) ltas then able to show that the specific growth rate
equals the dilution rate (u = ln 2/td =D).
H.
The Apparatus:
·During this study continuous cultivation was carried out in a
completely mixed gravity flow counter-balanced chemostat with a
culture voltmw of 430 ml in a 1, 000 ml vessel.
__ .pictured -in
Figur~-];~and
diagramed in
Figm:·~
This apparatus is
2; the major components
of the apparatus are identified by letters A through H.
The culture
was mixed using a magnetic stirrer maintained at a constant rate
throughout the experiments by a variable transformer.
Aeration was
accomplished by passing filter sterilized hwnidified air through a
fritted gla5s tube (A) at about 350-.300 ml/min. and kept at a constant
13
-·----~
flow rate by connecting, just prior to entry, to an outside pressure
head (B).
The temperature of the culture vessel was kept ~~.S7°C by
the circulation of water around the jacketed culture vessel.
CM was
pumped to an overflow reservoir (D) from a 18 liter glass carboy (C).
The overflow reservoir was then reconnected to the pyrex glass carboy
so the excess media could be recycled.
From the overflow reservoir,
media passed through a 3 meter coil of capillary tubing (E) (0.5 mm ID)
supplying the necessary capillary resistance to stabilize, by counter- 1
balancing, the gravity feed.
The coil was then connected to the
medium inlet through a dripper (F) which was constructed in such a
manner that no physical connection existed between the medium leaving
the dripper and that entering the vessel.
This break in the delivery
system is necessary to prevent contamination of the reservoir.
The
culture vessel (G) had openings for air, media, thermometer,
inoculating port, and overflow.
Excess material from the culture
vessel was expelled through the overflow by escaping air.
Mediu~
supplements were added to the reservoir by way of a Hydrosol Stainless
Millipore filter with a 0.22 u filter (H).
The flow rate was deter-
mined by measuring the delivery from the overflow.
When necessary
the delivery rate was changed by altering the height of the overflow
reservoir with respect to the culture vessel.
ANALYTICAL
~IETHODS:
Glucose assay
Glucose was determined by the procedure given by HcComb and
Yushok (1958), using Glucostat reagent obtained from Worthington
14
FIGURE 1;
Picture of actual apparatus used in this
study. The appratus was kindly made
available by C.R. Weston.
16
FIGURE 2:
Diagram of apparatus used with major components
lettered A through II. A- fritted glass tube;
B- pressure head: C- reservoir; D- overflow
reservoir; E- coil; F- mediwn dripper; G- culture vessel; H- millipore filter. Explanation
in text.
\
,rl
l
I
HUMIDIFIED
AIR
l
I
i
H
AIR
FILTER
B
c
WATER
JACKET
Ii
!_ ______ _
I
---------------
!
"__,___ _j
....,
"""
18
filtered glucostat reagent were incubated at 45°C for 30 minutes in
graduated test tubes.
After incubation the mixtures were cooled in
ice and diluted with cold SO% sulfuric acid to yield a final volume
The reactants were then mixed, allowed to stand at room
I
I temperature for 10 minutes, and the absorbancy of each tube measured
of 10 ml.
i
I
~--
at 540 mu on a Beckman DU Spectrophotometer.
i
Concentration of glucose
! .
! was deteTmined by comparison with a standard curve.
i
The standard
curve showed linear response between 2.5 and 20 ug glucose/ml.
Cell Weight
Dry weight of bacterial cells was determined from cells grown
under batch culture conditions.
LB2 was grown in 50 ml of CM con-
taining 2% glucose in a 250 ml DeLong culture flask.
A sample of log;
phase cells was plated on NA for determination of the cell count.
Four replicated ten ml samples were taken at the same time for weight
determination.
The cells were harvested by centrifugation at 12,000
x G for 20 minutes and were then washed twice, first with 10 ml of
0. 01
ter.
r-.1
phosphate buffer at a pH 7.1 and then with 5 ml of sterile waFinally the cells were suspended in 1 ml of sterile water,
placed in prevwighed aluminum trays, and dried in an oven at 90°C
a constant weight.
Average weight determined by this method was
8.7 x lo- 13 grams/cell.
Population Analysis
Population samples were taken from the overflow after first
wiping it clean with sterile 95% ethanol.
and plated on,the various diagnostic media.
The samples were diluted,
The total population. of
19
f """"""-----------·····-······ ------- ... ,.....
I
I the culture was determined
!I of
by colony counts on NA; the toJ:al number
lactose fermenting cells was determined from colony counts on BMM.
i
I Converted cells, for example F- lac- cells having received F' lac+
I
l from
LB4 (F'lac+), were determined by colony counts on BMM in the
i
I presence
I
1
of T1 virus which was added to B~W plates at the time of
spreading.
I colonies
i
i YIELD
LB2 cells were determined by counting non-pigmented
on LEMB medimn in the presence of T1 •
CONSTANT (Y)
The yield constant of both LB4 and LB2 were determined under
Iconditions of continuous cultivation.
1
The chemostat was operated at a
very low dilution rate (for example, 0.09 hr.- 1) until a steady state
I' was achieved. The population density was measured by plate counts on
I
I NA and converted to dry weight assuming the same cell weight as under
I
i
1
batch conditions.
The concentration of glucose in the reservoir was
determined and the yield was then derived from the equation Y = x/Sr.
This formula is derived from the steady state equations; x
and
s = K5
{D/Um- D).
becomes small
s becomes
= Y (Sr-
Using these equations, one finds that as D
negligible as compared to Sr.
s can then be neglected leaving the equation Y
In such cases
= x/Sr.
U Max was determined under batch culture conditions.
LB2 and
.LB4 were gro'Wn in 250 ml DeLong Culture Flasks containing 50 ml of
CM with 0.1% glucose.
Cultures were grown overnight at 37°C in a
New Brunswick Water Bath Shaker set at 200 RPM.
They were then re-
inoculated into fresh CM supplement with 5% glucose, to give a
final cell density of 8 x 106 cells/~1.
i
t,,_~, ·~•< ~-~
<-- ••••• •• •
» , ••••••-•-•••
•
-----•·--
-
«
-••"
•
-
• • - ••
~
••
·-·~··-••"•
·•-u• -••
"""""" ••••-
.>
-~·
Cell densities were
s)
20
C.nnined
turb:dimetric~:y -:=-~eck:~n
wi-th
Do Uo Spectophotometer
lI 420 mu every 20 minutes for 3 hours during the early log phase of
I growth.
I
The U max of LB2 and LB4 cultures were found to be 1.22 hr.-
I
I and
.JI
1. 35 hr.-
1
1
1
II
respectively.
l
I
L_..__ ~-----·~-~~ ---~--~-- --··------·-----~-~-~ -----··--·---·------- --~--~~----------~--~-~-.,----------~-- __
Fo _ _ _ _ _ _ _ _ «
----~
. . - - - - - - - - - - - · - - - - - · - - · - · · - - -. . - - . .
---~J
,---------------------------------'
RESULTS
· STEADY STATES OF LB2 AND LB4:
Both LB2 and LB4 can be maintained under similar steady state
conditions.
Within 24 hours of the time of inoculation steady states
i
I
were reached and these levels could be maintained for extended periods 1
I of time.
Figure 3 shows steady states for both LB2 and LB4 under
I similar conditions.
The strains were grown separately in the
chemostat for up to 140 hours at dilution rates of 0.52 hr.-l for
LB2 and 0.49 hr.
-1
.
for LB4.
+
SPONTANEOUS LOSS OF F'LAC :
It has been previously sho\1n that cells lose F-factors
spontaneously during growth.
In order to check whether this strain
(LB4) spontaneously loses its F-factor a controlled experiment was
set up in which LB4 cells were grown alone under conditions to be
used in the subsequent experiments.
The self-curing of LB4 cells
while under continuous cultivation is reproduced in
4.
Fi~ure
LB4
cells possess an F-factor that carries a lactose gene but which would
otherwise be lac .
If the LB4 cell loses the F-factor it becomes lac
and can be differentiated from the lac+ cell by differential counts
on
LE~m.
This experiment shows that after 144 hours or 101 genera·-1
tions of steady state growth at a dilution rate of 0.48 hr.
4.1% of the LB4 cells had lost the F-factor.
'
L---------------------------- - - - - - - - - - --- -------- ---------------------- - - - - - - - --------------21
only
I
I'
1
22
"1
I
TRANSFER OF F'LAC+ AT VARIOUS DILUTION RATES:
The effect of growth rate on transfer of F'lac+ was examined by
.
i
Imixing approximately equal numbers of F'lac+ and F- cells at different!'
II dilution rates. The results of three such experiments representing Ii
I
the extremes of D capable for this organism are shown in Figures S, 6
1
and 7.
l
In each experiment the recipient LB2 strain was introduced
aseptically into a sterilized chemostat and allowed to reach a steady
state.
After a minimum of 24 hours the LB4 cells, taken from batch
culture, were introduced such that the final ratio of LB4 to LB2 cells
1
was approximately 1:1.
I is
Since the lac + locus is on the F' of LB4 and
therefore associated with the fertility factor F, it was assumed
Ithat the transfer of the lac• locus was a reflection of the efficiency
I of
F transfer.
!
At a dilution rate of 0.53 hr.-l (equivalent to a doubling ~ime
of 1.3 hours) (Figure 5) the conversion of F- to F' is very rapid
essentially reaching a equilibrium value after only 7 hours.
In this
particular experiment there is a marked loss of lactose cells after
addition to the chemostat.
This loss consisted of LB4 cells but did
not interfere with transfer as evidenced by the fact that nearly the
total lactose population at equilibrium consisted of converted cells
(LB2(F'lac+)).
At equilibrium the lactose population represent only
17% of the total cells in the culture.
Figure 6 represents the results obtained in a similar experiment
in which the dilution is maintained at 0.268 hr. -1 .
The conversion
of the F- LB2 cells is not as rapid as reported in the previous ex1 periment but still reaches a plateau some SO hours after initial start.
1~----~------~~-~-- ~~~------.--~-----·--···--··--~----------- ······ -----~-------·· -- --. --· -- ---·· -- ~- ------------------- -------~----<------- ---~----- .. -----~-------~--------~-----~-'
23
FIGURE 3:
Steady state growth of LB2 and LB4. Both
curves are plotted on same graph for comparison. Each culture was inoculated with
2 ml of cells grown for 12 hours in 50 ml
of CM supplemented with 0.1% glucose.
Each run started with 36 liters of sterile
CM. Final chemostat volume for ~F, 424
ml ; Sr = 0 . 01 gm/ 1 ; D = 0 • 52 hr. ;
G = 1. 32 hours. Final chemostat volume for
LB4, 420 ml; Sr = 0.009 gm/1; D = 0.49 hr.-1;
G = 1.40 hours.
24
•
•
..
•
I\
•
I
o:l
0
C\1
.-I
.-I
I
•
\•
IZl
C\1
j:Q
H
-
IZl
_.:jj:Q
H
0
0
ri
i• i•
I I
• •
•
I
•
•
+Q
I
I
0
co
-1
~ I
:::1
I
I
,.Gi
o
'-'I
\•
\
~~
0
H
...0
8
!
\
\\
0
I
C'J
•
\•
!
1lli/VItl::tLLOV8: JO H3:8:W1ll.\I
L------~--------~------------------------------------·-
.
II
----·--·----..----·-·····
25
FIGURE 4:
Spontaneous loss of P'. Culture started
with 2 ml of LB4 cells grown for 12 hours
in 50 ml of CH supplemented with 0.1%
glucose. Run started with 36 liters of
sterile CH. Final chemostat volume_i20
ml; Sr = 0.0088 gm/1; D = 0.485 hr. ;
G = 1.42 hours.
~-·--···------·~--
I
~-----
l07J
I
i
l
l
Il
0 .....o - o - -
---~
o-o
o-o-o
~---0
-o-o
y
·-·
•
l
i
iI
I
!I
'
I
I
I
I
I
'
I
rg
..........
<
H
ffiE-l
0
10 6
<
C:Q
It-;
0
l
p::
f:il
~
b
~
.
_.........-•---·
.------
./
;·
o--o--o LB 4 (F 1 lac+)
/.
105-1
I
•
20
40
·-
•--•--• LB 4 (F- lac-)
I
60
80
TIME (hours)
~---------
-~--
./
100
120
140
I
I
I
J
N
a-
27
r:-1:~ :pulatio~ d:-:ot-=crease -as :t di~at dilut-io~rate
I
---l
· 0.53 hr.- 1 ; there was however a decrease in the initial LB2 F- population.
The converted LB2 population reaches a population size of
I 4.9 x 106 cells/ml which represents at its maximum 35%
I population.
I
Figure 7 shows the results of a 3rd experiment in
I
.
dilution rate is lowered to 0.009 hr.
~
•
i
f
l
of the total
which the
At this dilution rate an
I equilibrium was not reached and the observed rate of conversion was
far below the values obtained for either of the previously described
I
I experiments.
Also unlike either of the ~revious two experiments
neither the total lac+ nor the total F- LB2 population was found to
have decreased.
The number of converted cells after 140 hours is
5.0 x 105 cells/ml (Table 2) which represents only 3% of the total
population.
It should be noted that at
.
.
d~lut~ons
o f 0.53 hr. -l an d
0.268 hr.- 1 , 73 and 75 generations respectively had passed before
equilibrium values were reported while at dilution rate 0.09 hr.-l
only 19 generations had occurred by the time the experiment was
terminated.
TRANSFER OF F' LAC+ AT VARIOUS INITIAL F'/F- RATIOS:
Figures 6, 8 and 9 show the population dynamics obtained in a
series of experiments in which D was. maintained constant but initial
ratio of LB4/LB2 cells was varied.
Ill
A comparison of Figures 6, 8 and
9 show that there is a marked difference in the equilibrium value
when the initial LB4/LB2 ratio is altered.
II
i
In the experiment represented in Figure 8 the LB4/LB2 ratio was
i
"------··-----~---~---·--··-·--·--------~------~----·-~-----~-----··--------.-------------~-~--------·-->-·--···-~----·~·------·------------~--J
28
--
,----------~--------------------------------------------
I about
1 to 10.
~
The results from this experiment can be compared with ;
the earlier experiment in Figure 6 in which the ratio was 1 to 1.
I both experiments there is a rapid increase
1
I cells
being converted to F' cells.
In
1_,·'
in the number of LB2 p-
It is found that in the latter
case where the F'/F- ratio is 1 to 10 the final population of con-
i
I
approaches the LB2 converted population.
It is therefore evident
that the total population is almost exclusively LB2 F-.
I
Figure 9 represents an experiment similar to the one above excepti
that the LB4/LB2 ratio is increased to greater than 1 to 100.
Under
these conditions there is a slow but steady increase in the number of
converted LB2 cells which never really reaches a plateau during the
course of the experiment.
a value of 0.7% (Table 2).
The maximum converted population reached
The total lac+ population is seen to
decrease finally approaching the converted LB2 F' population.
The data for ten experiments in which D and ratio of F'/F- were
varied are summarized in Table 2 along with a presentation of a
number of calculated population ratios.
RATE OF F'TRANSFER:
The rate at which converted LB2 cells were increased when either
the dilution rate or the LB4/LB2 ratio was altered was studied in
detail during the initial few hours after mixing and is presented in
figures 10 and 11.
I,
29
FIGURE 5:
Transfer ofF' lac at dilution rate 0.53 hr.- 1
(G = 1.30 hours) and LB4/LB2 ratio 1.1. LB2
was started with 2 ml of cells grown in batch
culture for 12 hours. This culture was infected 24 hours later with 7 ml of LB4 cells
grown in batch for 12 hours. Batch cultures
OF LB2 and LB4 were grown in 50 ml of CM
supplemented with 0.1% glucose. Final chemostat volume 422 ml; Sr = 0.011 gm/1.
0
''o
>II
\
~f\
~o-~
~
<
H
10
I
6
II
·-·
....
E-1
0
<t!
co
G-!
=·
.---·
-~~-•~--- ____ .----·~ ----
-·
~
~-
.•
'-.
p::
ri1
·~e ===
o-----
------------
o
•
o
.
•
•
II.
0
I
ffi
~
,_,
p
o-o-o TOTAL POPULATION
·-·-·
:z;
TOTAL F- POPULATION
• - • - • TOTAL lac+ POPULATION
+
• - • - • CONVERTED CELLS LB .2 (F 1 lac )
104.
l
II
!I
1_-·-·----·-------··
15
30
45
60
TIME (hours)
75
90
105
I
____________j
~
0
31
FIGURE 6:
Transfer of F' lac+ at dilution rate
0.268 hr.-1 (G = 2.57 hours) and LB4/LB2
ratio 0.9. LB2 was started with 2 rnl
of cells grown in batch culture for 12
hours. This culture was infected 24
hours latter using 2 rnl of LB4 cells
grown in batch for 12 hours. Batch cultures of LB2 and LB4 were grown in 50 rnl
of CH supplemented with 0.1% glucose.
Final chemostat voiurne 438 rnl;
Sr = 0 . 01 gm/ 1.
r
I
lI 1oLI
Ii :g
i
~"'
I ;:S
'
I
\~
'\
<\'~
0
·-~ ~·~
-
.._
-1
•
/.
•
•
:x·
./
.
•
•
~l·o--6 ·./•
ffi!:l
/·
I
I
•
:--=- . I
•
.--·
-·
.---.----· .
·~ .
• ·
"'
•
•
'
•
o- o-o TOTAL POPULATION
~
•--•--•TOTAL F- POPULATION
•--•--•TOTAL lac
+
POPULATION
• - • - • CONVERTED CELLS LB 2 (F' lac
10.5
25
50
75
100
125
+
)
150
175
20j
TIME (hours)
~
N
33
FIGURE 7:
Transfer of F' lac+ at dilution rate
0.09 hr.-1 (G = 7.66 hours) and LB4/LB2
ratio 0.8. LB2 was started with 2 ml of
cells grown in batch culture. This culture was infected 24 hours latter using
5 ml of LB4 cells grown in batch fo! 12
hours and were first centrifuged and-resuspended carefully in 1 ml of CH without glucose. Batch cultures of LB4 and
LB2 were grown in 50 ml of supplemented
with O.lgii glucose. Final chemostat
·
volume 438 ml; Sr = 0.01 gm/1.
II
I
h
~/\ 'o
l
------.. ·-·-·---.----0.
·····><·=========
. .
I
o
o
\
l
•
I ~
!"
I i=i
&1
8
0
10
o--o
6
<t:
p::J
~·
rH
0
~
~
~
~
.A
·-·------·
o-o-o TOTAL POPULATION
.A
/
• - • - • TOTAL F
• - • - . • TOTAL lac
/
POPULATION
+
POPULATION
Jli.
I
I
II
r··
10
4 J>
__,----
CONVERTED CELLS
.~:t
6
20
ii
40
LB 2 (F
1
1
60
80
1
100
1
120
1
lac + )
I
1
140
TIME (hours)
i
i
l
A- A -.A
··-----------·
(...!
.;:...
35
FIGURE 8:
Transfer of F' lac+ at initial LB4/LB2
ratio of 0.09; D = 0.233 hr.-1 (G = 2.96
hours). LB2 was started with 2 ml of
cells grown in batch culture for 12 hours.
This culture was infected 24 hours latter
using 0.6 ml of LB4 cells grown in batch
culture for 12 hours. Batch cultures contained 50 ml of 01 supplemented with 0.1%
glucose. Final chemostat volume 430 ml;
Sr = 0.01 gm 1.
1r------ ~-·
l
ll
o...._
107
o
·-·
~~.-~e- ~o--
o
.
o
.
.
o
o
o
·-
0
~•
o
I
I
rg
'--10
,~
6
.lffi
l~
E-i
Ill!
I G-1
1
;105
.•r----<~
. . . . . . . -·/ ...-----~~~::::::::-..__
-----=:::!~·-·
~~
,~
I
I
Ii
-·
!
o- o-o TOTAL POPULATION
I
i
• - • - • TOTAL F- POPULATION
• - • - • TOTAL lac
+
POPULATION
+
• - • - • CONVERTED CELLS LB 2 (F 1 lac )
JJ.
I
-----·
25
50
75
100
125
150
175
200
TIME (hours)
------·-------------·
·----·-----·--------·-----·
(1.1
0\
37
FIGURE 9:
Transfer of F' lac+ at initial LB4/LB2
ratio of 0.006; D = 0.287 hr.-1 (G = 2.4
hours). LB2 was started with 2 ml of
cells grown in batch for 12 hours. This
culture was infected 24 hours latter using
1 ml of LB4 cells grown in batch for 12
hours. Both cultures were grown in CH
supplemented with 0.1% glucose. Final
chemostat volwne 424 ml; Sr = 0.01 gm/1.
r--!
oOOo
o.-o
o
0
o
0
0
~~
---o
107
I
10
6
rg
'-..
<t!
H
&1
8
0
~
105
ti-{
•\ . . . . . . . .
•••
0
·-·
----===='~·-
..---:::::::::::.~·~
.
""~
.--------·-----
•&i
~
~
o--o-o TOTAL POPULATION
104
• - • - • TOTAL lac
+
POPULATION
,._,._,.CONVERTED CELLS LB 2 (F 1 lac + )
""
""\~
I
l
I
Il ___
25
50
75
100
TIME (hours)
--·
--~·--------·-·-··--··-
125
150
175
!
_____j
.. ···
(.r.l
00
39
TABLE 2:
Swnmary of a number of experiments with
respect to dilution rate, initial LB2~LB4
ratio, various final population sizes, and
some final ratios obtained from previously
metioned data.
a
b
= vol~e
=x
10
changes/hour
cells/ml
~----
il
DILUTION
, RATEa
INITIAL
F 1 CELLS
ADDEDb
INITIAL
LB4/LB2
FINAL POPULATIONSb
RATIOS
TOTAL
F- (LB2)
CONVERTED
converted
1
+
(LB2 F 1 lac ) initial F
0.09
16.1
0.8:1
19.2
8.3
0.50
0.03
0.03
0.099
8.6
0.5:1
16.7
9.4
1.36
0.16
0.08
0 .llt-2
10.8
0.5:1
34.0
11.5
0.91
0.22
0.03
0.268
16.0
0.9:1
14.2
2.0
4.91
0.31
0.35
0.233
1.7
0.09:1
18.9
14.8
0.46
0.~7
0.02
0.288
0.1
0.006:1
16.7
14.9
0.12
1.15
0.007
0.291
7.0
0.5:1
'14.1
7-4
2.40
0.34
0.301
11.1
0.7:1
15.8
6.8
4.9
0.44
0.31
0.499
4.0
0.3:1
15.7
14.1
1.33
0.33
0.08
0.530
20.4
1.1:1
12.7
12.0
2.10
0.10
0.17
converted
total
l
TABLE 2
~
0
41
I
------------------~--­
!---~-Figure 10 shows the initial rate of conversion for three ex-
! periments
l ratios.
I
I
differing in their dilution rates but having similar LB4/LB2l
!i
The results show that as the dilution rate is lowered from
I 0.53 hr. -1 to 0.291 hr. -1
I of
l
1
and then to 0.09 hr. -1 a lag in the number
cells being converted appears.
For dilution rate 0.291 hr.-l this
lag lasts only 30 minutes whereas at dilution rate 0.09 hr.- 1 the lag
lasts some 55 hours.
Figure 10 also shows that the initial infection rate is
'
dramatically altered when the dilution rate is dropped from 0.291 hr.-f
to 0.09 hr. -1 •
In order to derive rates, slopes of straight lines
that best fit the data where obtained from linear regression analysis
(the data is presented on linear graph paper).
This does not mean to
imply that conversion is linear but that in the initial stages it
cannot be distinguished from second order kinetics.
it is
sho\~
Using these rates
that the difference in the initial rates between dilutions
0.53 hr.- 1 and 0.291 hr.-lis small as compared to the difference
I that
lies between 0.291 hr. -1 and 0.09 hr. -1 •
For reasons to be
I explained in the discussion, it is felt that this experiment
I
I (0.09 hr.- 1)
is not comparable with results of the other experiments.
Figure 11 represents changes in the initial LB4/LB2 ratio while
the dilution rate remains the same.
When the initial ratio of LB4/LB2
is 1:0.5 we find an immediate rate of increase.
When the infection
ratio is lowered to 1:0.09 the rate of increase is again immediate
but at a much slower rate.
If the infection ratio is again lowered,
this time to 1:0.006, we find a very slow increase that starts only
1
after some i hours.
1-.~------------~•···-•~----~----~·-~~-~------·-·•-·u--.-·--»•---·•
'
••••• ••-•• •·----, ·--·-•-•• -----·-•-m••-~~·••·•-"-••---··---~- ---~------··---~-~
42
FIGURE 10:
Transfer ofF' at constant F'/F , variable
Dilut~on :ates.
Initial ~onversion ra!r
for d1lutrons of 0.53 hr. 1 ; 0.291 hr. ;
0.09 hr.- . Initial LB4/LB2 ratios about 1.
Represented is the n1rnber of LB2 cells converted (LB2 F' lac+) plotted on liDear
paper. Values for each indicated besides
each curve.
43
o- o -o D
•- • -• D
56
0.53 hr. -1·
-1
0.291 hr ..
0.09 hr.
l
I
I
-1
0
Ool5 X 104
cells/ml/hr
49
cells/ml/hr
•
7
cell s/ml/hr
~/ ~0
.
~/ :o ;/
TIHE
:o
1,.
(hours)
L. . ~"~--..--~---......,.-·--~-~~·---~--·---~--·------- . ----·------~·~·----~~---~~~~·
44
FIGURE 11:
Transfer at constant D, Variable F'/Fratio. Initial conversion rates for
LB4/LB2 ratios of 1:0.5; 1:0.09; 1:0.006.
Dilution rates of 0.291 hr.-1; 0.233 hr.-1;
and 0.288 hr.-1 respectively. Represents
the number of LB2 converted (LB2 F' lac+)
plotted on linear paper.
45
I
l
I
II
72
I!)
64
o-o-o
1:0 • .5
A-A-A
1:0.09
·-·-·
56-
1:0.006
-=:10
r-l
1><:
48
r-l
a
..........
rrJ.
H
~
20.2 x 104 ce11s/ml/hr •
40
0
~
8
~
z
0
0
32
:>
0
(f....f
0
24
~
~
~
16
0
0.8 x 104 ce11s/m1/hr.
8
•
4
8
0.02 x 104 ce11s/ml/hr .
12
16
20
I
I
24
TIME (hours)
t,_ _ _ _ _ _ _
-~-------------------~----------------------------~-------:_ !
______
46
,----------------------~- ..
----"-----------------,
I POPULATION DISADVANTAGE:
II It was often found that cells added to a chemostat
' steady
I
I
in which a
state culture had already been established were preferentially
:
1
1
I
I eliminated to some extent. This phenomenon I have termed population
disadvantage.
Figure 12 shows the results of an experiment where
the mode of infection in the chemostat had been altered.
In this
experiment the sterile chemostat was initially infected with LB4 and
then reinfected with LB2 after 48 hours.
This figure shows an initial
5
fast increase in the converted LB2 F' population reaching 9.7 x 10
cells/ml, and a corresponding decrease in the F- population finally
reaching 7 x 105 cells/ml.
The total population in the chemostat
was predominantly LB4 F' with the converted population reaching 6.7%.
Comparing these results with those shown in Figures 5 and 9, it
is evident that the initial population present in the chemostat has
an advantage over the population inoculated last.
In each of these
experiments there is a dramatic decrease in the infecting population.
I
L----------------..--..--~- ______ ,. ______________ -------------------------- ---------------- ---------------------------------------------------------- ______ .._____ ---"
47
FIGURE 12:
Transfer of F' lac+ at dilution rate
0. 237 hr. -1 and LB4/LB2 ratio 1. 7. LB4
was started with 2 ml of cells grown in
batch culture for 12 hours. This culture
was infected 48 hours later with 10 ml
of LB2 cells grown in batch for 12.hours.
Batch cultures of LB2 and LB4 were grown in
50 ml of Cl'vl supplemented with 0.1% glucose.
Final chemostat volume 430 ml; Sr = 0.01 gm/1.
r--
l
1
0"'--o
I
I 107
I
lrg
!......_
I<
1;-·~~0
-----·
·-·--·--·------------
IH
lffi
I~
106
I<
lt:.f1
4--4
4,..,.
~~
I~~
I
i
·---------·~ z=::::::::::::: •·---------------------•
----------...
I~-
i
.0====---i
~
4
105
o-o-o TOTAL POPULATION
I
I
I
• - • - • TOTAL F- POPULATION
• - • - • TOTAL lac
4
4-A-4
i
i
I
!
I
I.
10
20
30
40
+
POPULATION
+
CONVERTED CELLS LB 2 (F' lac )
50
60
so.
TIME (hours)
__j
!
L----------··-
70
I
~·--------~-----~----~---
~
00
DISCUSSION
Genetic transfer has been shown to occur either in batch cultures or in germ free animals.
Evidence presented in this study
showed that not only can continuous cultivation be used to study
episomal transfer but it also offers benefits not found in the
I
I
I
previous methods.
I.
I
I
I
observed could be described by the following general equation; i
II
This process is based on the donor cell's
I
to transfer extra episomal factors. However the process of
In analyzing the results it was assumed that the population
j changes
ability
self curing obscures the simple results which might be expected from
such an equation.
Self curing, a spontaneous loss of the F factors,
I
I
:
I
is known to occur and results in the loss of the donor cells' episomes/
l
I
thereby reducing them to the F- state; this process could be simply
represented by the equations Fi ---)- Fi or F; ~
Fi.
Self curing
usually occurs when the donor cell divides in such a fashion as to
leave one daughter cell with all the episomes and the other without
any.
However, controlled experiments carried out under both batch
conditions (Pat Zamenhof, personal communication) and chemostatic
conditions (Figure 4), with these strains, indicated that the donor
cells used in this study had a low level of self curing.
In view of
these results self curing was ignored.whenever discussion of the
equilibrium was undertaken.
The extent to which the F- population is converted to F+ might
be influenced by a number of factors.
49
Among these are:
(1) number
!
50
[-----·----------------------
--l
lof episomes/F+ cell; (2) differential rate of episomal replication
'
jcompared to cellular replication; (3) physiological state of the
!donor cells (affecting their ability to transfer an episome);
i (4) physiological state of the recipient cells (their ability to
receive and integrate an episome).
By means of controlled growth
conditions acheived in the chemostat any of the above factors can be
!manipulated while keeping others constant.
For instance if growth
!conditions, nutrients levels etc., are maintained at constant values
!then we can assume that the physiological state of the cells will not
Ichange.
Under these conditions it is possible to observe what effect
!the number of episomes/F+ cell has on episomal transfer by manipula1 ing the relative numbers of F- and F+ cells in the initial population.
IResults
from such experiments should then yield the number of episomes :
harbored in the initial donor population.
In a similar manner by the
I
manipulation of the growth conditions one can alter the recipient
cells' physiological state; for example, by altering the dilution
rate which changes growth rate.
Results from this type of experiment
I
I would
indicate the importance of the recipients cells state during
l! episomal
I
transfer.
In both of the above cases, the physiological
state of the donor is kept constant by using cells grown for the
same length of time in the same media and under the same conditions.
I In order for the cells to
l
grow in the chemostat they must alter their
j physiological state thereby allowing them to adapt to the new grm\'th
I conditions.
I episomal
As a result of this adaption the differential rate of
replicating to cellular replication can be altered (Schaife,
I
11967).
L ...___ ,___ ·-·····..--~-------····---··-- ···-········ -·--···-····· -·· ......•. - ····-····--·········--···-·· --·--------·-- --------------------'
51
~--~ve::~ough
I
continu:us
~=:vat
ion-:ff:rs a means by
~hich
the
lll
physiological state of both the recipient and the donor can be
I manipulated
used.
there are certain inherent complications in the method
Introduction of a second population into an already stable
chemostat leads to a condition I have termed "population disadvantage".
This term has been coined to describe the loss of cells from
j a chemostat after their entry into an already stable culture.
1
Pop-
ulation disadvantage was felt to be caused primarily by the necessity
I of the new cells to adapt
I of a
to a new physiological state as a result
decrease in substrate concentration.
The infecting population,
in going from batch cul t,_~re where the cells are usually at u max to
continuous cultivation where u is generally relatively low must adjust
physiologically to a state it will assume in the chemostat.
This is
accomplished by stepping down their metabolic activities in going
from batch to continuous conditions.
It is known from batch studies
that such changes often lead to long lag periods in growth
(Kjeldgaad, 1961; Kjeldgaad, Maal¢e, and Schaechter, 1958).
Popula-
tion disadvantage could presumably be eliminated if the added cells
were derived from continuous cultures.
This would be accomplished
by maintaining two chemostats at similar levels and infecting one
with cells from the other.
This was not done in this study due to
the lack of materials and time available for this work.
In describing the population changes that occur in the chemostat
it was assumed that all converted F cells arise by infection of F
+
cells with the initial F cells added.
+
it is further assumed that the F
After this initial infection
cells are depleted of any extra
I
52
population as a whole to increase the number of extra episomes being
J
.
made must be greater than the number loss; 4%/100 generations.
Figure 5 shows results of an experiment in which (dilution rate is
I 0.53 hr. -l) there was always a small increase in the number of
Irecipients being converted, apparently never reaching equilibrium.
I Under these conditions is suggested that episomal replication is
it
. taking place at a slightly faster rate than the rate of episomal loss. ·
If a plateau in the number of converted F- cells is ever reached
then the number of newly synthesized episomes would be just balanced
by episomal loss.
Figure 6 (dilution rate 0. 268 h1·. -l) represents an
example of a run where the number of converted cells had reached a
plateau.
This would then support the assumption that a reaction be-
tween donors and recipients was indeed the only significant activity
taking place in the chemostat.
Furthermore these experiments suggest
that the physiological state of the donor has a direct ·affect upon
the differential rate of episomal replication.
k~alysis
therefore
indicates that episomal replication is influenced by the
physiological state of the donor.
The final equilibrium value of conversion can be expressed in
two ways; (1) a final percentage value of the total population
53
~,---------~--------·--~----·-------~--.
1
(converted/total population x 100) or as a ratio of recipients con-
l
verted/number of donors initially added, each giving different kinds
of information.
If the ratio of recipients converted/donor added
was used as the measurement for the physiological state, this ratio
was fairly constant for the various runs and was not influenced
drastically by either the dilution rate of the donor/recipient ratio.
With exception of three experiments, to be discussed separately,
ratios ranged from 0.1 to 0.44 and averaged 0.3 extra episomes/F+
cell (Table 2).
The value obtained at dilution rate 0.09 hr.- 1 was
excluded for two reasons.
First it ran for only some 19 generations
as opposed to 30 generations for the other experiments, within which
time a true equilibrium might not have been reached.
Probably more
importantly however was the fact that the donors present in this
experiment were subject to extreme population disadvantage as shown
by a decrease in the LB4 population by a factor of two within the
first 24 hours.
Thus after 24 hours the number of episomes actually
available for transfer was only
~
the number added.
The value
obtained for dilution rate 0.53 hr.- 1 was also excluded from consideration due to the major reduction in the initial donor population
(a ten fold decrease within 24 hours) which was attributed to
population disadvantage at this extreme dilution rate.
Dilution rate
0.288 hr.-1 was also excluded from the average because it fell outside the values obtained for similar dilution rates.
this deviation are not known.
The reasons for
I conclude that the results presented
here indicate that the replication rate of the recipient plays a
relatively minor role in the amount of conversion since approximately
54
,---·---··--------!
--·-----.-----·---·~------------·--·----·l
I
the same number of episomes are always passed on to the recipient
1 cells.
I
Further evidence to support the idea that the dilution rate has
I
little effect on rate of conversion is found when one observes what
affect dilution rate has on the initial conversion rate.
It was
found in various experiments that the initial rate of conversion was
independent of the dilution rate and varied only with respect to the
I
II
number of donors added (Figure 10 and 11).
The only serious exception!
1
occurred when the dilution rate was very low (0.09 hr.- ). At this
I
level the exponential increase was only one-fifth that shown for
dilution rates 0.53 hr.-
phase.
1
or 0.291 hr.
-1
, indicating a long lag
The reason for this difference is probably due to the
. j tremendous metabolic adjustments placed on the entering donor cells.
I Similar results have been found by others.
Fisher (1957) reported
that starvation of a carbon source prior to mating reduced the number
of recombants forr.ted.
This reported starvation affected the donor
cells more than the recipient cells.
From this it was reported that
the energy supply of the donor is more critical to conversion than
the energy supply of the recipient.
Later Freifelder (1967) showed
that the recipient is very much involved in conversion and is
possibly an active partner in the transfer of DNA.
Because the
metabolism of the donors is involved in episomal transfer it is felt
that the stepdown observed at dilution rate 0.09 hr.- 1 was due to the
tremendous decrease in the donor's metabolic activities.
shows that some fifty-five hours had to elapse
befo~e
Figure 10
the donors
were able to participate effectively in transferring their episornes.
-··~--~----·~··-··•-·
,_;
"•
--~·-·-•
-·-·-
•m-•-N••·--•····•-~·~·•·•••••-·
, ,.,, ,, • • _,._, • - • · - - - - ,._, • • · · - - - - •
•»~··-··~· -··----- ---------~-·-----------~.J
ss
r····--------·----------- ------·----·-· .... -····- -- . -···---- -- .... - .-----·-- ........ --·· ········--------
··---------------------"'~- ---~
l
I
l
I During the time that the cells are in the process of stepping down,
I
lit is known that they are metabolically incapable of transferring
!i
i
j their episomes (Fisher, 1957).
I
I
Although the F+/F- ratio did not affect the equilibrium value,
(recipients converted/number of donors
i
I alter
1
the rate of episomal transfer.
I
I stated,
added)~
it did drastically
This was to be expected if, as
the t~ansfer was simple collision phenomenon.
The p+ /F-
1-
I ratio was altered by simply maintaining the chemostat at a constant
I
!
i dilution rate while in three successive experiments the initial
i
I recipient
I111:1,
to donor ratio was changed.
and 166:1.
The three ratios were 1:1,
In each case an increase in the ratio F'/F- was
l
Ii followed
by a decrease in the initial rate of conversion (Table 2 and
Figure 11).
We conclude from these experiments that the number of
contacts between donors and recipients determines the initial rate
of conversion.
Cavalli et al (1953) was the first to describe this
phenomenon and suggested that the conversion of recipients by donors
was a second order reaction.
A second order reaction was indicated
here by changes observed in the initial conversion rates.
For
I::~:::r::o:h:a:·:::::-::d::::.:::i:F:::r:n::;~••:A:::o:::t:::.:ate
II
are represented by first order graphing they would fit the initial
/phases of a second order kinetics).
It is of some interest to see how the results obtained in this
I study
compared with those of other· studies.
The most drastic
difference between this study and those presented by other workers
: (Lederberg
e~
al., 1952; Jacob and Adelberg, 1959; Echols, 1963;
i
!
56
~-------------------------------·----·----------
Icurtiss
et al., 1968; Smith, 1966; Jones and Curtiss, 1970) is the
1
fact that in this study the total population of recipients was never
completely converted to the donor state.
Jones and Curtiss (1970),
I
!using germ free animals, indicated that only six hours was necessary
I!before
the recipient population was completely· converted to donors.
jin general it was reported that conversion of a recipient population
I
could take from one to twelve hours depending on the conditions of
mating.
1
Results similar to those presented here were,however, reported
lby Reed, Siechmann, and Georgi (1969).
In their study they used the
!resistance transfer factor instead of the F-factor and found that
Ionly about 0,5% of the recipients were converted after some 150
!minutes.
One possible reason for the difference between the various
I studies might be linked to the conditions under which transfer was
Imediated
and the recipient-donor ratio.
Curtiss (1969),for example,
showed that complex media and anaerobic conditions were far superior
. to the use of minimal media and aerobic conditions which were the
I cond.- . un der wh" h t h"1s study was per f orme d . I n t he ma30r1ty
. .
of
I experiments the ratio of donors-recipients was always from 10 to 100
1
~t~ons
~c
I
which would greatly enhance the number of recipients converted.
I
,
i
.The
average value of 1. 3 episomes/ cell determined using
cont~nuous
cultivation is in close agreement with the results
!
j reported by Jacob and Wollman (1958).
They reported that each donor
I cell had either one or two episomes/ cell under their condition of
grO\'lth.
Jacob, Schaeffer, and Wollman (1960) later found that the
I
I
57
,----
-~-~.-------------~~-----~-~--~---··~----~---~
. -···-------
1
I number of
II depending
I The
episomes/donor can vary anywhere from one to four
on the strain and most importantly the growth conditions.
number of episomes present was found to be correlated to the
I
number of chromosomes present in the donor cell.
For example, when
donor cells are replicating at a very fast rate the number of chromosomes per cell can be as high as four.
Under these same conditions
I
I Schaife
(1967) suggested that four episomes could also be present.
I
I
At the start of this discussion it was stated that genetic
transfer had been shown to occur in both batch cultures and in germ
free animals.
With this study it can now be stated that genetic
transfer also occurs under conditions of continuous cultivation.
It was also shown that through the use of continuous cultivation:
(1) the recipient cells replication rate does not affect the rate of
conversion;
(2) the rate of conversion is drastically affected by
the donor-recipient cells ratio,
(3) the number of episomes/donor
cell was around 1.3 (range 1.16-1.44).
In the discussion of these
results it was noted that in several instances the results presented
here are in agreement with results presented by other workers.
l
t~-----··-·················- ..... ·---· ...... ····-·--·- ··-··--·-··--··--··-- ............... ·---............................_ ....--···---··-·------ --·--···-·-·.. ----··---~
l
I
BIBLIOGRAPHY
I
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Adelberg, E.A., and J. Pittard. (1965). Chromosome transfer in
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Brinton, C.C., P. Gemski, and J. Carnahan. (1964). A new type of
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l~~~-,-~~-~~-,··-··--·· ~--·---··· ..
58
59
-·-----·-1
,----------~-------~--·------------
1
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i
I
l-·-~--····-····--·----------~-·-·-·-··-··-····---------------------------·----·---------···---------·--·-···---------·----·-;
60
~~--·"·~•U•--~--..~·- .-~-·~-
..
-~·
•••·•·---~·
•-
·~
''
~--
·----.- ••· ·•
'"''' ......................... -···-- ""'"----·· ........ -···- ·---------------· -·----··----·1
I Lederberg, J. and E.L. Tatum. (1946).
I
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j
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f
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1
I
(1950).
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Theorie et
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~rskov, I.
1
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Lederberg. (1952).
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!i
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·
and F. 0rskov. (1966). Episome-carr1ed surface antigen
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I