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CLASSROOM
S Mahadevan
Survival in Stationary Phase
Department of Molecular
Reproduction, Development and
Genetics
Indian Institute of Science
Bangalore 560 012, India.
Email: [email protected]
Keywords
E. coli, GASP.
The challenges faced by microorganisms in their natural environments
are much more than what they may experience under controlled
growth conditions in the laboratory. While they are provided with
abundant nutrients and stable conditions such as temperature and
aeration in the laboratory, most microorganisms are subjected to
prolonged starvation and other types of stress in their natural habitats.
In an attempt to understand microbial physiology under conditions
seen in nature, there has been a resurgence of interest in what is
generally known as ‘stationary phase’ where laboratory cultures of
bacteria apparently cease to grow. What happens if a culture of an
organism such as Escherichia coli is left shaking in the flask without
the addition of fresh medium for several days? For most students of
microbiology, the answer is simple – death. They are taught that the
culture will enter what is known as the death phase where the curve
plunges sharply and hits zero at some point (Figure 1). Can one
verify this experimentally? This is a simple enough experiment to do
in any reasonably equipped college microbiology laboratory. All
one has to do is to keep measuring the viable count for several days
after the onset of stationary phase. If one attempts to do this simple
experiment, one will be in for many surprises.
Cell number
109
108
Stationary phase
107
Death phase
106
105
104
Exponential phase
103
Lag phase
Figure 1. A typical ‘text
book’ bacterial growth
curve.
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Contrary to expectations, it has been shown that laboratory cultures
of E. coli grown in standard Luria-Bertani medium stabilize and
show a constant cell count that is about 1% of the original titre [1].
If one started with 108 cells per ml, this will be still a million cells!
How do they survive prolonged starvation and osmotic and pH
stress? This happens in three phases.
At the onset of starvation, several physiological changes take place
in the cell. The overall size of the cell reduces, the cytoplasm is more
condensed and the peptidoglycan cell wall has a higher degree of
cross-linking. These result in higher tolerance to osmotic, pH and
temperature stress. Most of these changes are primarily due to the
induction of a large number of genes triggered by the product of the
rpoS gene that codes for the stationary phase – specific sigma factor
S. This form of microbial “differentiation” is reminiscent of
endospore formation in Gram-positive organisms such as bacilli at
the onset of starvation. But what happens to the population upon
prolonged starvation in stationary phase? How do cell numbers
stabilize? The answer to this question is more difficult, at the same
time more interesting.
Cell numbers stabilize after the majority of cells die during the death
phase. This happens after 2-3 days of starvation [2]. The survivors
of prolonged stationary phase (beyond three days) undergo a
phenomenon that has been aptly named GASP, which is an acronym
for Growth Advantage in Stationary Phase. The GASP phenomenon
is a typical case of Darwinian evolution – “survival of the fittest”.
Rather than being dormant, the population of cells in stationary
phase is highly dynamic. Mutations occur periodically and those
that confer an advantage to the cells by enabling them to grow on the
available resources from the dying cells get selected. Those cells
that carry such mutations will now be able to grow and multiply at
the cost of the dying cells – a case of microbial cannibalism. Early
stationary phase cultures may have fewer such mutations and as the
cultures age, there is progressive accumulation of more such
mutations. During prolonged stationary phase, many successive
population turnovers occur (Figure 2). Several subpopulations of
GASP mutants may coexist in aged cultures and as a result, they will
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CLASSROOM
Cell number
109
108
107
106
105
GASP2
GASP3
GASP4
GASP1
104
103
3
4
5
6
7
8
9
10
Time (days)
Figure 2. The GASP phenomenon in prolonged
stationary phase characterized by successive population takeovers.
be genetically heterogeneous compared to the starting cultures.
Interestingly, the first GASP mutation seen is in the rpoS locus that
results in a S that shows reduced activity. How such a mutation
helps the cell survive is still not clear, but it has been shown that it
leads to enhanced metabolic capabilities. The fact that the
phenomenon has a genetic basis and is not simply a case of microbial
adaptation has been demonstrated by the fact that GASP mutations
can be propagated even after switching to exponential growth. In
addition, when the mutant locus is introduced into the parent strain
that has not undergone starvation, the stain can now exhibit the
GASP phenotype right away without having to undergo selection.
A relatively simple experiment can show that starved populations of
bacteria are dynamic. One needs two isogenic strains of bacteria
that carry two different selectable markers such as resistance to
tetracycline or kanamycin (Strains A and B). These markers must be
neutral which can be verified by a simple competition experiment.
Grow the two cultures A and B separately to stationary phase in LB
medium and mix equal proportions of cells without addition of fresh
medium. Allow the cultures to grow for several days and monitor
the viable count of each strain by plating on tetracycline and
kanamycin-containing plates. Once the markers are shown to be
neutral, one can mix culture A and B that have been grown for
different days (for example 1 day old culture A with 10 day old
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culture B) and do similar competition experiments. This can be
done reciprocally with different ratios of A and B in different
experiments. If the 10-day old culture can out-compete the one day
old culture, it is an indication that the former has more GASP
mutants than the latter. This can be repeated with cultures that have
been in stationary phase for different number of days.
As the cultures age, one can also look at colonies of the survivors to
record if there are any morphological variations. If the laboratory
has a PCR machine, one can also attempt to find differences at the
genomic level by using methods such as RAPD (this involves
amplification of different segments of the genomic DNA using
random pairs of PCR primers). The genetic characterization of loci
that confer a GASP phenotype will be the real challenge. So far,
only very few such loci conferring GASP phenotype have been
characterized at the functional level [2]. Thus studying aged cultures
in the laboratory can give several hints about microbial survival in
the real world.
Suggested Reading
[1] M M Zambrano and R Kolter, GASPing for life in stationary phase, Cell, Vol.86,
pp.181–184, 1996.
[2] S Finkel, Long-term survival
during stationary phase: evolution and the GASP phenotype, Nature Micobiol. Rev.,
Vol.4, pp.113–120, 2006.
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