103
FEMS Microbiology Ecology 74 (1990)103-120
Published by Elsevier
FEMSEC 00285
When nutrient limitation places bacteria in the domains of slow
growth: metabolic, morphologic and cell cycle behavior
William Chesbro
’, Michael Arbige ’ and Robin Eifert
‘ Department of Microbiology, University of New Hampshire, Durham, NH. Genencor Inc.. San Francisco, CA
and ’Lederle Biologicals, Pearl River, N Y , V.S.A.
Key words: Chronic starvation; Stringent response; Toxin secretion; Cell cycle
1. SUMMARY
Growth systems appropriate for studying mass
transfer in different bacterial environments are
reviewed. Fed batch and recycling fermentors are
suited to modelling nutrient limitation and slow
growth. Use of these two growth systems reveals
the existence of three growth rate regions, or domains, defined by maintenance energy demands,
nucleotide regulation, metabolism, and physiologcal behavior. They are exemplified in Escherichia
coli by domain-dependent synthesis of attachment
antigens, heat-labile toxin, and inducible enzymes.
Distribution of the bacterial population among
cell cycle stages changes with growth rate domain
because lengths of the stages differ in their dependence on growth rate. This produces subpopulations whose ratios vary with growth rate and that
are likely to differ in both molecular composition
and stress resistance.
2. INTRODUCTION
In a hospitable milieu containing enough nutrients to saturate their uptake systems, bacteria
Correspondence to: W.Chesbro, Department of Microbiology,
University of New Hampshire, Durham, NH 03824,U S A .
must surmount at least two metabolic barriers to
reach their maximum growth rate: (i) obtaining
energy, essentially ATP, at faster rates to fuel the
demands of more rapid anabolism although their
catabolic pathways are substrate saturated; (ii)
increasing their rate of chromosome replication
although the rate of DNA chain extension has
become limited by the temperature of the environment rather than by availability of intermediates.
They solve (i) by shifting to spill, or overflow,
catabolic paths [l]. This increases the rate of ATP
production at the expense of a reduced yield of
ATP per mol catabolic substrate [2]. They solve
(ii) by starting new rounds of chromosome replication before previous rounds are completed [3,4],
producing chromosomes with multiple replication
forks. Simultaneous multiple chromosome replications then bypass the limitation set on single
chromosome replication by the fixed rate of chain
length extension.
In the less hospitable and more usual circumstances of nutrient limitation (i.e. starvation
in some degree of severity), with long doubling
times ensuing, bacteria must solve a different version of the energy provision problem: that of
capturing sufficient energy substrate and extracting from it the maximum yield of ATP. The
energy demand is exacerbated at long doubling
times by an increased need for maintenance energy that rises in abrupt steps to require 70% or
more of the cell’s energy flow [5,6].
0168-6496/90/$03.500 1990 Federation of European Microbiological Societies
104
The nature of the chromosome replication
problem also changes when bacteria are nutrient
limited. The continuous, proportionate increases
in all the cell's properties that define balanced,
exponential growth [7] become more punctate in
slower growing, chronically starved cells. Gaps
from which chromosome replication is absent develop in the cell growth cycle, the proportions
between the lengths of cell cycle stages change
[8,9], and the changed proportions are increasingly
wrong (in Escherichia coli) for provision of adequate cytoplasmic volume, through lateral wall
extension, to both accommodate the nucleoid and
allow mid-cell placement of the transverse (division plane) wall [lo].
T h e stress of nutrient limitation evokes elevated
levels of the regulatory nucleotides CAMP and
guanosine 5'-diphosphate 3'-diphosphate (ppGpp)
[11,12] which drive adaptations to the stress, including adaptations to the problems of energy
provision and transverse wall placement. Adapting
to the starved state results in populations with an
average chemical and molecular composition
markedly different from exponentially growing,
nutrient-sufficient populations (131, along with an
altered morphology [14]. Although some of the
differences in the chemical makeup of the two
populations undoubtedly arise as direct consequences of nutrient limitation, observations will
be presented that point toward another part of
these differences arising from nucleotide regulation which, in correcting the problem of inadequate cytoplasmic volume for both nucleoid separation and mid-cell placement of the nascent
division plane, compels changes to take place in
the proportions of subpopulations of cells in different cell cycle stages. The molecular makeup of
cells inherently varies between the stages. Consequently, changing proportions among the subpopulations in different cell cycles stages would be
expected to change the average composition of the
total population.
3. CHOOSING A GROWTH SYSTEM TO
STUDY NUTRIENT LIMITATION
To anyone used to the batch culture of heterotrophs in concentrated substrate solutions, an in-
terdivision time ( 7 ) of 2 h can be a limit and
larger values of T irrelevant [15]. TO those who
routinely employ the chemostat, over 95% of the
microbial growth rate range seems to have been
surveyed when a specific mass growth rate, p , of
0.05 h-' is reached, i.e. a T of approximately 14 h,
since the upper limit of p in bacteria is near 2.0
h-' and the lower limit is 0 h-'. But for those
who consider environments in which dissolved
organic carbon can have a half-life of 6000 y [16],
either value for T is unthinkably short.
The study of nutrient-limited, slowly growing
bacterial populations in the laboratory requires
choosing a growth system whose mass transfer
characteristics will yield ranges of T containing
values relevant to the study. This requirement,
although obvious, is frequently not met [5,6,10,
12,131. A model of mass transfer has been published [12] that permits identifying a number of
laboratory and industrial growth systems, as well
as a limited number of natural systems, whose
mass transfer characteristics determine the batterial growth curve the system will yield. Although
the model seemed simplistic when it was propused
and clearly has marked limitations, it gives a &gree of insight into the behavior of slowly growing
bacteria when combined with the growth rate domain concept [5,10,17-191 discussed in following
sections. The basis for the model is consideration
of a locus of indefinite volume that contains
bacterial cells and nutrients, followed by an assessment of whether or not this locus permits
biomass (cells) and nutrients to enter or leave.
This produces four types of loci, representing the
possible combinations of open and closed mass
transfer. Figure 1 provides examples of these four
archetypes of mass transfer and the bacterial
growth kinetics expected in each archetype.
The nutrient closed-biomass closed system, the
ordinary seeded medium contained in a closed
vessel, gives the 7-phase growth curve generally
taken to be representative of the growth of
bacterial populations. The bacterial behavior best
observed in this system is behavior exhibited in
steady-state exponential growth and the system is
unsuited to the study of transients, or slowly g r o w
ing cells, because of the rapidity of growth rate
changes in the system. It is a system simple t o
105
GROWTH
SYSTEM
EXAMPLES
Is
PATTERN OF
POPULATION
GROWTH
nutrient closed,
biomass closed
batch bioreactors:
test tube and flasks
often the “7-phase”
sigmoid curve
nutrient open,
biomass open
chemostat; some other forms
of continuous culture;
caniage of luminous bacteria
by some fish
may be in
steady state at
constant td
nutrient closed,
biomass open
common in nature:
bacterial biofilms on
particulate substrates
attached cells at
shortest t d h
system, detached
cells at longest td
nutrient open,
biomass closed
fed batch bioreactors:
recycling fermentor.
host-containedbacteria
td increases
progressively for
indefinitely long
period
9
t d denotes the mass
doubllno time In h
Fig. I, Simple model of mass transfer into and from a bacterial
environment of indefinite volume.
e ~ ~ p vlory studying phenomena that occur in
exponential growth, and probably for this reason
has been the preferred starting point in most
laboratory studies in microbiology. However, it is
difficult to identify any natural environment with
these mass transfer characteristics, and this probably has a bearing on why most laboratory studies
are difficult to relate to bacterial behavior outside
the laboratory.
The nutrient open-biomass open system most
frequently used in the laboratory is the chemostat.
Chemostats produce a population in exponential
growth with a reasonably narrow dispersion of c
among cells of the population when c is shorter
than 14 h. At longer r, its dispersion becomes too
great for studies requiring a homogeneous population [13,20]. The chemostat is the system of choice
for studying exponential growth at r shorter than
14 h, and for studying transients to and from
starvation. Perhaps the clearest example of a natu-
rally evolved chemostat is the cheek pouch of the
fish Photoblepharon in which it maintains an apparently monoclonal population of luminescent
bacteria [21].
The nutrient closed-biomass open system occurs in nature wherever a particle of substrate is
surface colonized by bacteria capable of metabolizing it. Bacteria attached to the substrate will be
at the nutrient-sufficient, maximum growth rate
for the system while detached bacteria will be
starving at the minimum growth rate.
The nutrient open-biomass closed combination
is best suited of the four for examination of slowly
growing, chronically starved populations. Cells
held within the system are subject to a continuously dwindling rate of nutrient supply per unit
biomass, causing the specific growth rate, p , to
dwindle continuously. This situation occurs naturally wherever bacteria are a biofilm on a nondegradable surface and likely also when pathogens
or symbiotes remain contained inside a host cell
or macrostructure. In the laboratory, fed batch
and recycling fermentor systems belong to this
class, as do illuminated reactors for growing phototrophs (such units are basically fed-batch reactors by virtue of the way light energy is supplied).
The fed batch system is simpler to operate, but
because the system is ultimately limited by the size
of the reactor vessel (except for illuminated reactors for growing phototrophs), the practical longer
limit to the mass doubling time ( f d ) range it can
produce is about 40-50 h. The recycling fermentor, however, has no theoretical limit to the t ,
range it can produce.
A recycling fermentor can be constructed from
any of the instrumented fermentor units commonly available by adding an inflow line for
pumping sterile medium at the desired rate from a
reservoir and a tubing loop leading from the reactor to a tangential flow membrane filter. Sterile
medium is continuously pumped to the fermentor.
At the same time, fermentation broth is pumped
to the membrane filter. A portion of the broth is
continuously filtered through the membrane at a
rate equal to the rate at which sterile medium is
being added to the fermentor and the balance of
the broth, containing the cells, returned across the
membrane’s surface to the fermentor.
106
4. THE GROWTH RATE DOMAIN CONCEPT
4.1. Progressive nutrient limitation is marked by the
onset of global changes in growth rate and yield at
particular mass doubling times
In all the experiments to be described, the
culture was grown in a recycling fermentor at
30°C in a glucose-limiting minimal medium with
the pH kept at 6.8-7.0. The pattern of anaerobic
growth of Escherichia coli B under these conditions is shown in Fig. 2. Paracoccus denitrificans
[5], Bacillus polymyxa [lo], Bacillus licheniformis
[ 13,191, Clostridium beijerinckii [22] and Clostridium C7 (this Volume; Ross et al.) all produce
similar patterns of growth in the fermentor. The
pattern is the same aerobically and anaerobically
for the facultative species, and similar patterns are
observed with the aerobe Paracoccus denitrificans
and the two anaerobic clostridia.
The growth curve in Fig. 2 has two inflections,
separating the curve into three growth rate regions. After seeding, the culture goes to its maximum p and shortest t,. The critical nutrient in the
2
1.(r
1.8
1.4
Q
0.8
0)
E 0.4
02
0
-0.
-$O
.
0
L
10 20 30 40 50 60 70 80 90 100
HOURS
Fig. 2. Characteristic bacterial growth pattern given by
Eschertchio colt B relA spoT growing anaerobically in a recycling fermentor in a glucose-limiting minimal medium held at
pH 6.8-7.0 and 30 O C. A nutrient upshift was made 70 h after
seeding the reactor by increasing the nutrient inflow rate
4-fold.The shaded zone contains the second growth rate region
which separates the first and third regions by growth rate
changes under bacterial control at constant nutrient inflow
rate.
reactor is rapidly consumed and the growth rate
becomes dependent on the transfer rate of fresh
nutrient into the reactor; the rate drops abruptly
to a t , of about 20 h, producing the first inflection, and the apparent yield coefficient, Ys (g
biomass/mol carbon-energy substrate consumed)
falls by about 50%. The decrease in Ys is canonically interpretable as a diversion of about 50% of
the cell's available energy from growth processes
to maintenance processes [6]. The volumetric
growth rate, dX/dt (g biomass increment /h/ l),
in this second region (shaded in Fig. 2) proceeds
at a constant, linear rate determined by the constant rate of nutrient flow to the bioreactor. Because the specific growth rate, p ( g biomass increment/g biomass per h), is derived by dividing
dX/dt by X (g biomass/l, the instantaneous value
of biomass in the fermentor) and X continuously
increases, p is required to fall continuously, while
its reciprocal, t , , consequently increases linearly.
That is, after the first inflection in the growth
curve td lengthens in direct parallel with biomass
increase. At a t , of 50-55 h, the second inflection
occurs in populations which are wild-type in the
stringent response genes. The stringent response is
enabled at this point and dX/dt drops abruptly as
a result of the inhibition of ribosome synthesis.
This results in the calculated t , lengthening
abruptly to about 100 h. At the same time, Ys
decreases by a further 50%, interpretable as a
further diversion of energy to maintenance
processes. After the transition to the stringent
response state, linear growth in this third region
resumes at a lower volumetric rate and t , continues to lengthen indefinitely unless the nutrient
flow rate to the bioreactor is changed.
Figure 2 illustrates the effect of a 4-fold upshift
in the nutrient supply rate administered in the 3rd
region. The culture rapidly returned to the t d and
Ys values of the 1st region and then recapitulated
the passage into the second region. The volumetric
growth rate, dX/dt, in this recapitulated second
region was four-times that of the earlier 2nd region, but the calculation of td and the Ys showed
them to be the same. That is, when the culture
returned to the t, range of the second region, it
returned to the Same regulational and mass transfer behavior.
107
corresponding amount of biomass being synthesized, which increases sharply in the two domains
of slower growth. In domain 3 the increase in
maintenance energy demand is clearly associated
with the onset of stringent regulation [ll] and
editing of translation [6]. Escherichia coli subjected to a 4-fold upshift in the rate of nutrient
supply to the fermentor in domain 3 (Fig. 2)
returns in growth rate to the domain 1 range and
the domains are quickly retraversed. In such an
upshift the cellular level of ppGpp returns to that
of the preceding domains [111.
In E. coli, the sudden drop in dX/dt when it
enters domain 2 defines a t, boundary at which
energy is diverted abruptly from biosynthesis to
maintenance. In B. licheniformis re1 - growing
aerobically and in a phenotypically wild type P.
4.2. Maintenance energy and nucleotide regulation
in the three growth rate domains
In each of the three growth rate regions just
described, the population showed markedly different levels of regulatory nucleotide, mass transfer
behavior, catabolic paths, morphology, and enzyme inducibility, making each region a distinct
domain bounded by particular growth rates. Table
1 summarizes the characterizing features of the
domains found so far. The most characteristic
feature of each domain is the cellular level of the
regulatory nucleotide guanosine 5’-diphosphate
3’-diphosphate, ppGpp.
Another distinguishing characteristic is the apparent fraction of the energy substrate required to
meet the maintenance energy demand, i.e. the
amount of energy substrate consumed without a
Table 1
Bacterial metabolism, morphology and mass transfer relationships in three growth rate domains corresponding to different rates of
nutrient provision
Characteristic
Growth rate domain
1
2
3
Nominal id’ range
Nutritional status
Regulatory
nucleotides
(14 h
Sufficient
cAMP and ppGpp at basal
levels
Mass transfer fluxes
YEz reaches a maximum;
MED’ reaches a minimum;
spill catabolism may
occur; exclusive use of
favored catabolic
substrate
Exploitation
20-60 h
Chronic starvation
cAMP at maximum
level; ppGpp level
rising
YE 50%of maximum;
MED 50% of energy
flux; no spill
catabolism;
multiple use of
catabolic substrates
Adaptation
>1Wh
Severe chronic starvation
cAMP elevated; ppGpp
at level enabling the
stringent response
YE 25% of maximum;
MED 75% of energy
flux; no spill catabolism;
multiple use of
catabolic substrates
Disbursed
Exhausted
Minimal
Minimal
Species/strain dependent
Species/strain dependent
D period becomes a
larger fraction of T
D approaches a
constant fraction of
as td increases
Relationship to the
environment
Energy
Accumulation
reserves
Decreases with increasing
Attachment
( E . coli)
ld
Species/strain dependent
Enzyme/toxin
secretion (E. coli,
B. lichenijormis,
Closiridium strain 0)
Cell cycle stages
D period becomes a
smaller fraction of T
as f d increases
-
-
as f d increases
~
is the mass doubling time; T is interdivision time.
YE is the biomass yield in g dry weight per mol energy-limiting substrate.
MED is the maintenance energy demand as a fraction of the cellular energy flux.
I td
-
-
Maintenance
T
108
denitrificans [13,23], dX/dt falls at a more constant rate without a sudden change between domains 1 and 2 to indicate an abrupt increase in
maintenance energy demand. However, the maintenance energy demand increases continuously to
reach levels similar to those reached in E. coli in
domain 2. The reason for the increased maintenance energy demand of domain 2 remains unknown.
The td at which the domain 2-3 transition
occurs is independent of nutrient provision rate
and is determined solely by the status Gf the major
genes determining cellular levels of ppGpp. In
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10 20 30 40 50 60 70
HOURS
ao
90 100
HWRS
Fig. 3. Growth curves given by Escbericbio coli H10407 ?el+
and 711P307SPOTgrowing anaerobicallyin a recycling fermentor in a glucose-limiting minimal medium held at pH 6.8-7.0
and 3OOC. The shaded zones contain the second growth rate
region which separates the first and third regions by growth
rate changes under bacteria) control at constant nutrient inflow
rate.
strains wild type for these loci, the transition
occurs at a t d of 50-55 h. In relA mutants, with
ribosome-dependent ppGpp synthetase I activity
reduced [24], or a relX mutant, with ribosome-independent synthesis of ppGpp reduced [25,26],
ppGpp accumulates more slowly during chronic
starvation and the transition occurs at a t d of
65-70 h. In spoT mutants, with ppGpp pyrophosphatase activity reduced [24], ppGpp accumulates
more rapidly than in the wild type and domain 2
terminates at a t d of 20-25 h [ll].
Figure 3 illustrates the effect of these gene
differences in two enterotoxigenic E. coli, strains
H10407 rel+ and 711P307 SPOT.Domain 2 extends to a td of 55 h in the former, but only to 25
h in the latter. In terms of actual time elapsed,
H10407 rel+ requires approx. 48 h to traverse
domain 2 (i.e. accumulate enough ppGpp to cause
a sudden reduction in rRNA and ribonucleoprotein synthesis, producing the transition to domain
3) while strain 711P307 SPOTrequires only approx.
12 h.
4.3. Enzyme induction and simultaneous use of mu/tiple carbon-energy sources, including endogenom
sources, by E. coli in the two domains of Slower
growth
When E. coli B was grown in a minimal medium
containing 0.007 M glucose, 0.001 M lactose, and
0.0015 M L-tryptophan and allowed to traverse
the three domains (Fig. 4), P-galactosidase and
tryptophanase activity appeared in the cells as
they entered domain 2. Specific activities of the
enzymes rose to maximum levels as the cells passed
to domain 3 and thereafter remained roughly at
this level. The imposition of a 4-fold nutrient
upshift in domain 3 had little effect on tryptophanase activity but P-galactosidase activity fell
sharply after the upshift.
Simultaneous u t i h t i o n of multiple substrates
should be survival-positive in chronic starvation.
It appeared that at the level of starvation corresponding to the td of the domain 1-2 transition,
catabolite repression by glucose was sufficiently
reduced to allow CAMP-dependent inductions by
alternative substrates and the population became
capable of metabolizing both tryptophan and Pgalactosides along with the major carbon-energy
109
shake flasks [27]. But it is not possible to correlate
that in vivo finding with ours since, as noted in
the first section, the batch culture method (nutrient closed-biomass closed) is suited only to the
study of exponentially growing cells, and definitions of any other phase of growth in that system
remain idiosyncratic because there is no general
agreement on what characterizes ‘early stationary
phase cells’.
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rig. 4. Lnanges in p-giuacrosioase m u irypwprimas actiwry
in Escherichra coli H10407 growing anaerobically in a recycling fermentor in a glucose-limitingminimal medium containing 0.007 M glucose, 0.0015 L-tryptophan and 0.001 M lactose
held at pH 6.8-7.0 and 3OoC. A nutrient upshift was made 62
h after seeding the reactor by increasing the nutrient inflow
rate Cfold. The shaded zone contains the second growth rate
region which separates the first and third regions by growth
rate changes under bacterial control at constant nutrient inflow
rate.
source, glucose. Repetition of the experiment with
three other strains of E. coli growing on glucose
confirmed that both enzymes were simultaneously
inducible in domains 2 and 3. Thus, it seems likely
that the capability for simultaneous use of multiple substrates are present when this bacterium is
in domains 2 and 3.
Furthermore, Escherichia coli H10407 rel+ accumulated glycogen in domain 1 when growing on
glucose. Glycogen granules were visible in electron
micrographs and measurable as cell-bound glucose. The glycogen was metabolized during the 1st
10 h elapsed time that the culture was in domain 2
(data not shown). Thus, the utilization of this
carbon-energy reserve by strain H10407 commenced at a nutrient level and t, appropriate for
aiding energy-dependent adaptation to progressive
starvation. The in vitro synthesis of E. coli glycogen synthesizing enzymes has been reported to be
stimulated by CAMPand ppGpp, and the accumulation of glycogen and transcription of the biosynthetic genes in vivo found to be at their highest
rates in ‘early stationary phase cells’ grown in
4.4. Domain-related synthesis of attachment antigen
and secretion of heat-labile (LT) enterotoxin by E.
coli
Escherichia coli H10407 was grown anaerobically at 3OoC and a constant pH of 7.5 in a
glucose minimal medium. Secreted, heat-labile
toxin (LT) was sampled from the filtrate stream of
the recycling fermentor, reflecting the instantaneous, extracellular concentration of the toxin in
the bioreactor. It was quantitated both on a volumetric basis using an ELISA method [28] and as
specific activity by its action on CHO cells in
tissue culture [29].
Figure 5 shows the pattern of LT secretion
across the three domains. Toxin appeared as the
culture entered domain 2 whether its measurement
was made volumetrically or specifically. The volumetric rate of secretion was constant in domain 2,
but the specific rate of secretion increased
throughout the domain (i.e. as the growth rate fell,
the rate of LT secretion slowed less than did the
general rate of cell syntheses). Both rates increased
as the culture entered domain 3. When the culture
was given a 4-fold nutrient upshift the secretion
rate of LT measured as specific activity fell (i.e.
when the upshift returned the culture to domain 1
growth rates, the rate of synthesis of general cell
constituents was upshifted more than was the rate
of LT secretion). Very similar features of LT
secretion were exhibited by E. coli 711P307 SPOT,
except that they occurred sooner in elapsed time
because domain 2 is foreshortened by the spoT
mutation.
The observation that secretion of LT is domain
2-3 dependent, at least in these two strains, explains why the toxin is generally understood to be
produced poorly in laboratory culture under
anaerobic conditions, in synthetic media,, or in
110
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C
10 20 30 40 50 60 70 80
HOURS
Fig. 5. Changes in amounts of heat-labile toxin secreted by
Escherichra coli H10407 growing anaerobically m a recycling
fermentor in a glucose-limiting minimal medium held at pH
6.8-7.0 and 3OOC. A nutrient upshift was made 63 h after
seeding the reactor by increasing the nutrient inflow rate
4-fold. The shaded zone contains the second growth rate region
which separates the first and third regions by growth rate
changes under bacterial control at constant nutrient inflow
rate. Toxin was measured by an ELISA method (0)
or by CHO
cell activity (0). The former measured volumetric secretion of
the toxin and the latter specific activity secreted.
chemostat culture. The laboratory cultures used
have been batch cultures which emphasize domain
1 behavior, or chemostat cultures wlliw are
steady-state cultures only at domain 1 f d and
infrequently at f d longer than 14 h. Consequently,
the first requirement for toxin production, that the
culture be at the appropriate growth rate in domain 2 or 3, was poorly met in batch culture and
not at all in chemostat culture.
Van Verseveld et al. [17] studied synthesis of
attachment antigens K99 and F41 by E. coli in the
three domains and found that synthesis of the
antigens occurred only in domain 1. When the
domain 1 synthesis of attachment antigen and the
domain 2 synthesis of LT are considered together,
a life cycle may be suggested for the pathogen by
considering the host enteric tract an example of
the substrate closed-biomass open case, the external environment a substrate open-biomass open
case, and applying the domain concept. The pathogen passes into the substrate-rich habitat of the
host enteric tract from a domain 3 state in the
environment and its growth rate rises to domain 1
rates. Attachment antigens are elaborated and the
bacterium proliferates until it exhausts the nutritional carrying capacity of the host habitat. Its f d
then falls to domain 2 values, synthesis of attachment antigen ceases, LT toxin is secreted and the
resultant diarrhea returns the pathogen to the
external environment to seek new hosts.
If its alternative life styles were unknown, enterotoxigenic E. coli might be diagnosed as an
r-strategist [30] and copiotroph [31,32]in the host’s
enteric tract, and a K-strategist [30] and oligotroph
[31,32] in the environment external to the host.
The growth rate domain concept plus the mass
transfer model, however, provide particulars of the
pathogen’s survival strategy, aid in selecting in
vitro techniques for studying the disease, direct
attention to the roles of regulatory nucleotides in
adapting the pathogen to its alternate environments, and show, in a slight extension of Goldman’s [33] description of some microbial species
as, “efficient nomads who spend a significant
fraction of their time as wanderers”, that “nomadic
looter” is an accurate description of the enterotoxigenic E. coli.
5 . STARVATION, CELL DIVISION AND THE
CELL CYCLE IN THE THREE DOMAINS
5.I . Experimentally distinguishing chronic starvation from starvation
Chronic starvation has been defined as nutrient
limitation and starvation as complete lack of
nutrients [34]. In the experiments performed in the
recycling fermentor, the bacterial population was
being subjected to progessively more severe
chronic starvation. To experimentally test the definitional distinction between chronic starvation and
starvation, a domain 3 population of E. coli NF161
was subjected to starvation by stopping the nutrient flow to the fermentor. Viable counts were
made before and after the nutrient shutoff. The
outcome is shown in Fig. 6. The viable count had
decreased 30% by 30 min after the shutoff. The
count then remained stable for the next 12 h. At
the end of this time, the count commenced falling
111
L
4---30%
tell death
A
3oq
:
99% c@ll death
---*
-I-. .
. . . . . . J
0 20 4 0 6 0 80 10 01 20 1 4 0 1 6 0 1 8 0
HOURS
Fig. 6. Changes in viable counts and turbidity occurring in
Escherichra coli NF161 spoT growing anaerobically in a recycling fermentor in a glucose-limiting minimal medium held at
pH 6.8-7.0and 30 C.When the culture was in domain 3. 49 h
after seeding the fermentor, nutrient inflow to the fermentor
was stopped.
with first-order kinetics and 50 h later 99% of the
population was dead.
Starvation was clearly distinguishable from
chronic starvation, not surprising in that a domain
3 population is employing 75% or more of its
energy flow for maintenance, but it was also apparent that the domain 3 population was heterogeneous in its response to starvation.
5.2. Changes in cell morphology in progressive
chronic starvation
Although it was not apparent at that point in
the study in what way morphological changes
might relate to the heterogeneous response to
starvation, other experiments had shown that cell
morphology changed as populations traversed domain 2. Escherichiu coli B cells which were
bacillary in domain 1 became coccoid after passing into domain 2 [14].The change in geometry
from cylinders to spheres allowed the domain 2
cells to retain much of the volume they had possessed in domain 1. Escherichia coli K strains,
however, became coccobacillary upon passage to
domain 2 then, as they traversed domain 2 into 3,
regained mean length in a way dependent on their
re1 genotype. Table 2 contains measurements of
mean lengths and widths of a K12 rel+ strain and
an isogenic spoT mutant. The spoT strains regained mean length much more rapidly than the
rel+ strain as both traversed domains 2 and 3. In
the SPOTstrain, ppGpp accumulates more rapidly
in the cell in domain 2 and as a result it enters the
fully stringent state, domain 3, much sooner in
elapsed time and at a shorter 1,. It seemed likely
therefore that ppGpp was acting in some way to
restore mean length to the population.
The fraction of cells showing constrictions was
also measured, since this is a direct way to estimate the fraction of the population that is in the
latter part of the D period (the cell cycle stage
between completion of chromosome replication
and cell division) [35].The fraction of the population in this cell cycle stage changed relatively little
with domain (Table 2). This was unexpected, since
the fraction of the population in the latter part of
the D period was anticipated to become negligible
in domain 2, and vanishingly small to undetect-
Table 2
Length, width and visibly constricted fraction of Escherichia coli populations growing across a wide growth rate range
E.
Hours
coli
strain
postseeding
NF161
(SPOT)
3.5
19
54
NF859,
wild type
5
54
99.5
Nominal
mass-doubling
time ( i d )
(h)
1.5
24
130
1.3
55
210
* Biomass increasing exponentially.
Domain
Mean
1*
2
3
1
2
3
cell
length
(pWS.D.N
2.36 (0.66)
1.99 (0.88)
2.32(0.66)
2.42 (0.79)
1.78 (0.49)
1.90 (0.56)
Mean
cell
width
(pM(S.D.))
0.90 (0.09)
0.88 (0.10)
0.79 (0.09)
0.79 (0.11)
0.92 (0.14)
0.95 (0.11)
Percent of
cells showing
constrictions
Number
of cells
measured
9
8
10
6
85
129
159
62
5
90
7
121
112
able in domain 3, for reasons to be presented
shortly.
5.3. Interaction between cell stages during progressive, chronic starvation reduces mean ceN volume to
a minimum and causes a problem in completing cell
division in domains 2 and 3 requiring growth ratedependent regulation of the interaction
In a presumably genetically homogeneous
population with all cells having a uniform physiological history, heterogeneous response to stress
could still arise from subpopulations containing
cells in different stages of the cell cycle if stress
resistance varied between the stages. For this reason, and because of the unexpected constancy of
the constricted cell fraction in the three domains,
the effect of chronic starvation and progressively
slower growth rates on stages in the cell cycle was
considered.
Cell cycle stages as they are understood to
occur in E. coli provided a paradigm for relating
the domain-dependent (and likely ppGpp-dependent) changes in morphology to the cycle stages.
Figure 7 is a schematic presentation of the cell
cycle modified from that of Nanninga et al. [35].
Periods designated by the letters on the left are
identified primarily by the status of chromosome
replication. The B period is the interval after cell
division and before chromosome replication initiates, while the C period is the interval during
which the chromosome replicates. Periods on the
right in Fig. 7 refer primarily to intervals involving
formation of the division plane. The D period is
the interval from completion of chromosome replication to cell division and contains withm it a
final substage, T, in which the cell becomes visibly
constricted in advance of completing division.
A period of indeterminate length, the R period,
preceding the D period has been added. In the R
period synthesis of the precursors of the division
plane, or its assembly, or both are presumed to be
regulated in a way connected to the cell's growth
rate. The reason for supposing such regulation
must exist comes from the observation of Helmstetter and his collaborators [3,4,8] that, in their
experiments, as 7 increased from 20 min to 2 h the
C period lengthened progressively and a B period
appeared at the start of the cycle but the D period
8
8
ci
Fig. 7. Cell cycle scheme modified from [35] by addition of an
R penod in which it is supposed that the synthesis, assembly,
or both of the transverse wall precursors is under regulation by
ppGpp. Periods associated with the replication of the thomosome are represented by letters and dashed lines on the left,
and periods associated with synthesis of the transverse wall
similarly represented on the right. The extension of the lateral
wall in a bilinear manner is indicated by the series of cartoons
in the center, commencing with the new-born cell at the
bottom and progressing to cell division at the top.
remained at about the same length, 22-24 min.
Kubitschek and Newman [9] extended these observations to a 7 of 12 h. They found that the C
and B periods lengthened with lengthening 7 , with
each approaching 40% of 7 as an asymptote, but
the D period did not lengthen and remained 22-24
min long. This latter finding explains the apparent
mean shortening in length commonly observed in
populations as T extends from less than 1 h to
longer times and implies that a particular problem
develops in cell division as the growth rate falls.
Surprisingly, the finding seems to have gone unremarked in the literature on cell division from its
publication until recently [lo].
Figure 8 schematically attempts to present the
basis for the mean length of cells shortening as the
growth rate falls. In Figs. 7 and 8, bilinear growth
of the lateral wall [35] has been assumed with the
more rapid rate occurring in the D period, but it is
not critical to the model whether the lateral wall
113
extends in this way, in an exponential way, or in a
linear fashion. At a 7 of 80 min, a cell is in the D
period for about 25% of r . Thus 25% of r is
available in the D period for lateral wall extension
before the cell divides. Whatever the kinetics by
which lateral wall extension proceeds, a substantial part of the length and therefore the volume of
newborn cells is attained during the D period in
rapidly growing cultures. However, as r reaches
10 h, when the C period is completed and the
22-24 min D period begins, only about 4% of r is
available for lateral wall extension before the D
period completes and division occurs. (Since the D
period becomes an increasingly small fraction of r
as the growth rate falls, it had been expected that
in domain 3 a corresponding, increasingly small
fraction of the population, approaching indetectability, would be in the T substage of the D period,
contrary to the roughly constant and relatively
large fraction actually observed (Table 2)). With
cells dividing at a shorter mean length, newborns
are therefore shorter, and consequently the mean
length of the population decreases as its growth
rate falls.
When the transition to domain 2 at a 1, of
12-14 h occurs and the t, abruptly increases to 20
h, the problem of the growth rate-independent
length of the D period becomes even more acute
and should raise a particular difficulty for dividing cells. The basis for the model shown in Figs. 7
and 8 is the ‘I + C + D cell sequence devised by
Helmstetter and his colleagues [8] in explanation
Comparison of cell cycle stages
at 2 interdiuision times (TI
80 min
T
7
~ ~ 6 min
0 0
I
I
iC
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
@
I
t
I
I
I
&*C
I
I
~
1
I
I
I
I
I
I
I
I
I
I
I
.:.>
..............
>
.:.,
,,,-,,-----J
?
I
I
I
I
I
I
I
I
I
I
I
1
Fig. 8. Illustration of the problem incurred in cell division as the longer limit to growth rate domain 1 is approached if D period
processes remain growth rate independent while other cell cycle processes are growth rate dependent. Notation is the same as in Fig.
7, except that the interdivision time is denoted BS T.
114
of their observations on cells with r of 2 h or less.
At growth rates that rapid, the problem of inadequate time for further lateral wall extension during the D period does not occur as it does for the
cell at a r of 10 h illustrated in Fig. 8. However,
for cells at any 7 the first part of the D period
must include whatever time is necessary for adequate separation of the completed chromosomes.
A t the longer t , of domain 1 and the beginning of
domain 2, the growth rate-dependent extension of
the lateral wall could become too slow to generate
the cytoplasmic volume necessary to accommodate both the separated nucleoids and the growth
rate-independent deposition of the transverse wall
before the cell enters the final, T substage of the D
period.
The problem this disproportion in the length of
cell cycle stages makes for dividing cells is revealed in electron micrographs of E. cofi H10407
entering, and in, domain 2 (Fig. 9A-C). In domain 1 cells there was adequate volume to accommodate the separated chromosomes while deposition of the division plane was completed (Fig. 9A).
As the growth rate approached domain 2 values
the cells shortened, becoming distinctly barrel
shaped (Fig. 9B). In the shortened cells, deposition of the transverse wall started, producing a
visible constriction in the cell periphery, before
Fig. 9. Electron micrographs of Escherichia coli H10407:(A) thm section of T-period. domain 1 cell stained by the Kellenberger
technique, nucleoids separated; (B) negatively stained domain 2 cells; (C) thin section of T-period, domain 2 cell stained by the
Kellenberger technique, nucleoids unseparated: (D) thin section of T-period. domain 3 cell stained by the Kelienberger technique,
nucleoids unseparated. cell regaining length at division. Bar corresponds to 0.5 pm. See text for discussion.
115
the replicated chromosomes were separated into
daughter nucleoids (Fig. 9C). Completion of the
division plane was mechanically prevented however because the unseparated chromosomes occupied the necessary space, and the D period was
arrested in the substage in this cell.
The concept of a minimum size requirement for
the successful division of the bacterial cell has
been considered both genetically and geometrically [36,37]. The basis of the minimum size requirement is brought to view by again considering
the disparity between the growth rate-independent
length of the D period and the growth rate-dependent length of the other periods. Cells must possess
the minimum volume necessary to accommodate
the separated nucleoids at their poles if the division plane is to be completed near their centers.
As they approach slower, domain 2 growth rates,
division is completed by the growth rate-independent, D period processes as soon as the growth
rate-dependent, lateral wall extension has increased the cell volume to this minimum.
However, as the strain H10407 population progressed into domain 3, cells blocked in the T
substage were less apparent and constricted cells
assumed an appearance more like that of domain
1 cells (Fig. 9D). Like Escherichia coli K strains
(Table 2), strain H10407 also regained length as it
proceeded through the domains. This led to postulating an R period (Fig. 7) during which regulation is likely to be acting in domain 2 and 3 cells
to restore a balance among the lengths of cell
cycle stages that would permit cells to reach sizes
larger than the minimum before dividing. The
synthesis of cell wall precursors is known to be
affected by ppGpp [38]. Results with the K strains
in which length regain was dependent on the
status of their re1 genes, along with the presence
of elevated ppGpp levels in domain 2 and 3 cells
[ll],argues strongly for this nucleotide as the R
period effector.
this was a possible source of heterogeneous response to starvation stress. An approach to estimate the proportion of a domain 3 population in
each stage was developed by taking advantage of
the observation that D period cells of many E. coli
strains exhibit chloramphenicol-resistant division
[39] apd that lysogenic h phage becomes lytic in
cells when the replicating fork of the chromosome
is blocked.
Table 3 shows the results of measuring chloramphenicol-resistant division in a spoT strain in
the three domains. In domain 3, 20-358 of the
population was estimated to be in the D period.
To measure C period cells by provoking vegetative phage in lysogenized cells, the ability of nalidixic acid to block the replicating chromosome
fork in a reversible fashion, and thus evoke the
SOS response, was first tested using strain ATCC
33311 in which lac2 is joined to the left promoter
of X [40].The strain was grown to domain 3, and
the reactor pulsed first with L-tryptophan to induce tryptophanase as a marker of cell integrity.
The reactor was next pulsed with sufficient nalidixic acid to give an initial concentration of 15
pg/ml. P-Galactosidase was measured throughout
the experiment, tryptophanase measured before
and after the tryptophan pulse, and indole was
measured in the filtrate to determine when the
inducing tryptophan pulse was exhausted.
The results are shown in Fig. 10. Biomass increase halted after the antibiotic pulse and the
level of P-galactosidase rose above a rather high
background level. The enzyme increase ceased 7 h
after the pulse. By 15 h after the pulse, washout of
Hours
postseeding
Nominal mass
doubling time
(r,,) (h)
Domain
Percent increase
in particle count
5.4. Population fractions in dvferent cell cycle stages
in domain 3 and their significance for stress resistance and mean cell composition
Changes in mean cell length suggested that the
distribution of the population among the cell cycle
stages was different in the three domains and that
5.5
12
15
28.5
41
46
1.3
15
19
80
108
124
I*
2
2
3
3
3
10
11
10
30
20
35
Table 3
Increase in particle counts of slowly growing Escherichra coli
NF161 after chloramphenicol treatment
* Biomass increasing exponentially.
116
450 1
E
1
1
0
10
20
30
40
50
60
70
80
90
100 110
HOURS
Fig. 10. Escherichia coli ATCC 33311, in which the left promoter of X is fused to lucz, pulsed in domain 3 with 0.0015 M
L-tryptophan at (a) and at (b) with sufficient nalidixic acid to produce an initial concentration of 15 pg/ml while growing
anaerobically in a recycling fermentor in a glucose-limiting minimal medium held at pH 6.8-7.0and 30 O C. See text for discussion.
117
the antibiotic through the cell recycling membrane
had lowered its level enough for growth to resume.
With the resumption of growth, P-galactosidase
activity resumed its increase in parallel with the
biomass increase. The level of tryptophanase remained constant after reaching a maximum at the
exhaustion of the inducing pulse (signalled by the
downward inflection in indole level in the filtrate
stream), indicating that lysis of the culture was
unlikely to have occurred as a consequence of
exposure to the antibiotic. The experiment demonstrated that nalidixic acid reversibly inhibited the
domain 3 population and instigated the r e c A - l e d -dependent SOS system.
Escherichia coli NF161 spoT was lysogenized
with h and the nalidixic acid experiment reprised
with this strain. Fig. 11 shows the outcome. Five h
after the antibiotic pulse was administered, phage
appeared in the filtrate and over the next 4 h the
viable count in the reactor fell by 41%. The
washout kinetics shown by phage indicated that
release of new phage had ceased by 4 h after the
pulse. At 15 h post addition of nalidixate the
culture resumed growth. To test the functionality
of the SOS system and the presence of latent
phage in this surviving population, "-methylN '-nitro-N-nitrosoguanidinewas added to the fermentor at the level of 10pg/ml. This mutagen
inflicts double strand damage throughout the
chromosome as well as at the replication fork, so
that the SOS system is activated in all cells whether
or not their chromosome has a replicating fork. As
Fig. 11 shows, the mutagen provoked general release of phage and lysis of the remaining cells.
The experiment is taken to mean that in domain 3 approximately 40% of the population was
replicating a chromosome, i.e. in the C period. The
results of this experiment taken together with the
chloramphenicol experiment, in which an average
of approximately 30%of the population was in the
D period, places 40% of the domain 3 population
in the C period, 30% in the D, and the remainder,
3056,therefore into the B period.
The experiments with nalidixate revealed heterogeneous susceptibility to this antibiotic and
probably also to others that act at the replication
fork in slowly growing, chronically starved
bacteria. This differential susceptibility would not
be apparent in cells at their minimum 7 and
maximum growth rate in a hospitable milieu containing enough nutrients to saturate their uptake
systems because these cells will multiply, initiating
chromosome replication, as pointed out in the
Introduction. Cells near their maximum growth
rates pass quickly enough from one period of the
cycle 'to the next so that an antibiotic pulse can
affect the entire population since all cells will
reach the stage of maximum susceptibility during
the course of the pulse.
The nalidixate experiments showed that as
much as 60% of the population can be immune to
at least this kind of provocation of the SOS system, and since the SOS system is the major route
for fixation of mutations in bacterial populations,
the slowly growing population is less prone to
mutagenesis from this source. The differential response of D period cells to chloramphenicol serves
as another example, in this case well known, of the
influence of cell cycle stage on response to antibiotic stress. As the experiment reported in Table
3 showed, the population fraction resistant to
chloramphenicol inhibition of cell division also
changed with domain, i.e. with the degree of
chronic starvation.
Returning to the observation on the heterogeneous response of domain 3 cells to starvation, it
is likely that one of the cell cycle stages is more
susceptible to energy loss than the others. However, the proportion of starvation-susceptible cells,
30% is too close to the population proportions of
all the three stages in domain 3 to warrant even a
preliminary assignment of starvation susceptibility
to one or another stage.
Table 1 concludes with a summary of domaindependent changes in distribution of the population among the cell cycle stages. The kinds and
proportions of protein species present alter as a
cell passes through the cycle. At the least, amounts
of precursors of the division plane will differ in B,
C, and D cells. This should be reflected in changes
in the mean protein composition of populations in
the different domains because the proportions of
B, C, D, and T period cells in the population
differ within and between domains. A consequence of this analysis is that neither qualitative
nor quantitative differences between protein
118
species found in starved populations and those
found in populations growing in a hospitable,
nutrient-replete milieu can be simply identified as
revealing stress responses to starvation because in
some degree these differences will necessarily also
be due to the different proportions of B, C, and D
cells in the starved and replete populations.
..)
ACKNOWLEDGEMENTS
Nanne Nanninga and Conrad Woldringh of the
University of Amsterdam provided a substantial
amount of their time and thoughts for us, to our
considerable benefit. The work of R. Eifert, M.
Arbige, and W. Chesbro was supported in part by
U.S. Army Research Office grant DAAL 03-86-K0018. The contributions of Robert Mooney were
as indispensable to these studies as they are to all
studies in this Department.
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