Download Bacterial Senescence, Programmed Death, and Premeditated Sterility

Bacterial Senescence, Programmed Death,
and Premeditated Sterility
Single-cell analyses of oxidative damage suggest senescence underlies
the loss of culturability among stationary-phase bacteria
Thomas Nyström
hen bacteria enter stationary
phase, various regulatory networks are activated, enhancing
the capacity of those cells to
withstand environmental stresses.
Despite these resources, however, cells in stationary phase gradually lose their ability to reproduce on standard nutrient plates. This loss in
plating efficiency is described in microbial textbooks as the death phase of the bacterial growth
cycle.
Several decades ago, this loss of culturability
was assumed to be a consequence of stochastic,
starvation-induced deterioration. Indeed, several microbiologists during the 1960s and 1970s
considered their studies of the phase following
starvation of bacterial cells as equivalent to se-
W
• Stationary-phase bacterial cells become nonculturable either because they senesce and deteriorate, are programmed to be refractory to
growth until conditions become more favorable, or are programmed to die.
• Oxidative stress is a key factor limiting stationary-phase survival when there is an increased
demand for oxidation management in cells subjected to growth arrest.
• When growth ceases, E. coli cells are subject to
a sudden burst of oxidative damage, namely
carbonylation that damages proteins, that is
particularly dominant among those cells that
become nonculturable.
• Pronounced oxidative damage to proteins in
deteriorating bacterial cells suggests that there is
a great deal in common between bacterial senescence and aging in higher organisms.
nescence and analogous, in some respects, to
aging and death in higher organisms.
However, other researchers subsequently
challenged that view, arguing that starvationinduced sterility is the culmination of a genetically programmed pathway. According to one
such hypothesis, the failure of a bacterial cell
to produce a colony on a standard nutrient plate
may not mean that the cell is dead or has
irreversibly lost its capacity to reproduce. Instead, it is possible that such cells become
temporarily nonculturable or that the plate
conditions are harmful for growth-arrested
cells. According to an alternative hypothesis,
some bacterial cells are genetically programmed to die. Here we will assess these two
models in several ways, including by considering
population heterogeneities, specific mutants
with altered survival characteristics, and
protein damage that occurs in stationaryphase cells.
Some Bacteria May Be Programmed
Either To Stop Growing or To Die
Advocates of the notion that some bacterial
cells are programmed to refrain from growing call such sterile but intact cells “viable
but nonculturable” (VBNC), and they suggest that stationary phase or suboptimal
(low) temperatures are conditions that commonly trigger cells to become VBNC. Further, they say that cells are genetically programmed to form VBNC in a manner that is
analogous to spore formation of differentiating bacteria. This program results in
highly resistant survival forms that, under
Thomas Nyström is
Professor of Microbiology at the Department of Cell
and Molecular Biology, University of
Göteborg, Göteborg, Sweden.
Volume 71, Number 8, 2005 / ASM News Y 363
FIGURE 1
contrast to the VBNC hypothesis, nonculturable bacteria are regarded as moribund, if not outright dead. According
to this hypothesis, the suicide program
is functionally linked to toxin-antitoxin
(TA) operons. Bacteria harbor several
TA loci that can be activated to potentially mediate programmed cell death
upon nutritional stress and growth arrest (Fig. 1).
Several Genes Appear To Affect
Stationary-Phase Culturability
Some genes induced early after bacterial
cells stop their growth affect the rate at
which such stationary-phase cells lose
the ability to form colonies on nutrient
agar plates. Most of these genes exhibit
positive effects. Thus, when those genes
are absent, the cells lose culturability at
an accelerated rate, and they encode
proteins with specific roles in protecting
the cell against external stresses, such as
heat, oxidants, osmotic challenge, and
exposure to toxic chemicals. Sigma factor ␴S is involved in inducing many of
these genes. However, other global regulators of transcription, including the
Schematic representation of different models for the loss of cultivability of stationary
heat shock sigma factor ␴H, the extraphase bacteria. 1. Stochastic deterioration of a subpopulation of cells entering stationary
phase. 2. Programmed death by starvation-induced activation of suicide modules, such
cytoplasmatic stress sigma factor ␴E,
as toxin-antitoxin operons. 3. Loss of culturability by programmed formation of VBNC
and the oxidative stress response regu(viable but nonculturable cells). Note that model 1 and 2 both argue that the cells
lators OxyR and SoxRS, work with ␴S
becoming non-culturable upon starvation are the ones that are moribund and will
eventually die whereas model 3 predicts that the nonculturable cells are the ones that
during stasis. Further, cells lacking one
will survive long term starvation and growth arrest. See text for details.
or several of the primary oxidative-defense proteins become non-culturable
prematurely during starvation. In addiappropriate conditions, resuscitate to become
tion, in a Salmonella population lacking both ␴E
culturable again (Fig. 1).
and ␴S, nearly all cells lose culturability within
VBNC bacteria loom as a concern for public
the first 24 h of stationary phase, but the plating
health experts who conduct risk assessments.
efficiency of these mutants is completely preThus, if pathogenic gram-negative bacteria,
served under anaerobic stationary-phase condisuch as Vibrio cholerae, Vibrio vulnificus, Saltions. Also, the loss of culturability of wild-type
monella, and Escherichia coli, enter a VBNC
E. coli cells during the first 10 days of stasis can
state, they could readily evade detection but
be counteracted by limiting oxygen availability.
could pose serious health risks if and when they
Apart from inducing stress-defense genes and
again become infectious following, for example,
regulons, entry into stationary phase of E. coli
temperature shifts, passage through animals, or
other environmental changes that would trigger
cells is accompanied by a shift in metabolic
active growth.
activities. Specifically, the synthesis of glycolysis
Advocates of the alternative hypothesis about
enzymes, phospho-transacetylase, and acetate
genetic programming suggest that starved, nonkinase increases, whereas synthesis of many triculturable bacterial cells are the products of a
carboxylic acid (TCA) cycle enzymes decrease,
specifically orchestrated suicide program. In
reflecting a dependence on the two-component
364 Y ASM News / Volume 71, Number 8, 2005
Studying Stressed Bacteria with Art and Music in the Background
While a student, Thomas Nystrom imagined himself becoming
a pianist and painter. But an inner
voice directed him to study chemistry and biology as an option to
becoming a high school teacher in
case those artistic designs fell
short of his expectations. “I
thought it was pretty much an OK
way to waste my time, waiting for
the big breakthrough as an artist
or musician— until I took microbiology for the first time,” he says,
recalling that early excursion into
the sciences.
One look into a microscope
convinced him that the creatures
he saw were “the coolest thing
ever. I don’t know why, really,
but the cylindrical shape of an E.
coli or Bacillus cell strikes me as
one of the most beautiful forms of
life in its perfect simplicity,” he
says. “To see them swim and divide under the microscope still
gives me goose bumps.”
Deferring his artistic ambitions,
Nystrom pursued microbiology at
Goteborg University in Goteborg,
Sweden, where he now is a professor and researcher in the department of cell and molecular biology. “I have a deep interest in the
limitations of life, that is, why,
exactly, the molecular design of
organisms fails to support life under conditions deviating too much
from the optimal and when the
organisms become old,” he says.
“I started off studying marine
vibrios dying of starvation for
carbon and energy, and then went
on to the more genetically tractable bacteria, E. coli. In the course
of these studies, it dawned on me
that starving and dying bacteria
displayed many of the same signs
of deterioration and tribulations
as aging model organisms used in
gerontology.”
From that realization, his next
moves in studying oxidative stress
in bacteria took shape almost on
their own. “Many genes were argued to be pace setters in the aging
process, especially genes linked to
oxidative stress defense,” he says.
“This prompted me to start developing techniques to study oxidative damage to proteins on the
single-cell level.’’
Nystrom has applied these techniques to study bacteria, yeast,
plants, and mouse stem cells, revealing novel mechanisms underlying how cells cope with oxidative damage, including the
involvement of “gerontogenes” in
some of these processes. A major
goal of his research is to decipher
the molecular basis for the apparent tradeoff, or antagonism, between activities devoted to proliferation and survival. “How is the
environment affecting this
tradeoff, and is the design of such
systems determining the survival
potential of the bacterial cells?
“Why do I care?” he asks. “I
am honestly at a loss here. I could
give a large number of examples
of possible applications about the
importance of this knowledge in
fighting chronic infections, in understanding and possibly treating
age-related diseases, but none of
this would truly explain why I do
this. I just want to know how it
works, I guess!’’
Nystrom, 45, is the younger of
two sons of Bertil and Ingrid, a
banker and language teacher. His
older brother, like their father,
“became a master Jedi of the fi-
nancial world.” Nystrom was
born in Lund but grew up in the
small town of Linkoping. He exhibited a fledgling interest in biology as a youngster on summer
visits with his grandparents in
southern Sweden where the long
days were filled with “exploring,
collection of butterflies and beetles, and making reproductions of
them in oil or watercolor.”
Nystrom did postdoctoral research at the University of Michigan in Ann Arbor during the early
1990s, a stint he thoroughly enjoyed. “Ann Arbor was great,” he
says, praising it as “a good place
to live,” and for its “good local
beer (from Kalamazoo), a great
lab, and a great mentor in Fred
Neidhardt. I believe that he and
his approach to science, more
than anything else, made me decide to become a researcher.”
For all Nystrom’s immersion in
microbiology research, his youthful musical leanings did not disappear. He and his wife Nan, who
also is a scientist, and their two
daughters, Lina, 11, and Stella, 4,
jam together in a family rock and
roll band. He and Lina play electric guitar, while Nan plays bass.
“We need a drummer and have
hopes that Stella will take this on
eventually— but now she is
mostly dancing around and singing along,’’ he says. Although inspired by Led Zeppelin and Deep
Purple, the family band mainly
plays simpler songs while “eagerly awaiting our big breakthrough.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
Volume 71, Number 8, 2005 / ASM News Y 365
FIGURE 2
Schematic representation of the bacterial defense against starvation-induced oxidative
deterioration of proteins. Green ovals denote proteins that are activated or accumulated
during starvation and whose activity is important to slow down starvation-induced loss of
cultivability. The red oval (Acn) is aconitase, the absence of which has been shown to
retard loss of cultivability in stationary phase. GSH and ppGpp is glutathione and
guanosine tetraphosphate, respectively. The defence acts at different levels. The ArcA
dependent down regulation of reducing equivalent production and respiratory activity is
suggested to reduce generation of reactive oxygen species (ROS) during starvation
whereas the superoxide dismutases (SodA and SodB), and catalases (KatE and KatG)
further reduce ROS levels catalytically. Another line of defence is the elevation of
proteins involved in the repair and reduction of oxidized proteins. These proteins include
MsrA (peptide methionine sulfoxide reductase), GorA (glutathione reductase) in concert
with GSH (glutathione), TrxB (thioredoxin), GrxB (glutaredoxin), and the DnaK/DnaJ (heat
shock chaperones). The RelA/SpoT/ppGpp system of the stringent response is suggested to reduce protein oxidation by affecting translation fidelity (aberrant proteins are
more susceptible to oxidative attack). Soluble species of oxidized proteins appear
ordained for proteolysis. However, highly carbonylated proteins form high molecular
aggregates that are proteolysis-resistant. Such aggregates appear to inhibit protease
functions and have been suggested to contribute to the aging process in higher
eukaryotes. Abbreviations: PN, native protein; PA, aberrant protein; POX, oxidized protein.
response regulator ArcA. Repression of aerobic
metabolism during stationary phase correlates
with upregulation of arcA, according to geneexpression profiling experiments by Tyrell Conway of Ohio State University in Columbus and
his collaborators. They further suggest that
ArcA, via its sensor component ArcB, is acti-
366 Y ASM News / Volume 71, Number 8, 2005
vated in stationary phase by a mechanism encompassing redox control of the
quinone pool.
Regardless of the mechanism of ArcA
activation in stationary phase cells, this
regulator is important to culturability
because cells missing arcA lose viability
at an accelerated rate after a few days in
stationary phase. The reduced production of respiratory substrate and components of the aerobic respiratory apparatus during starvation may be a
mechanism that protects such cells
against the potentially damaging effects
of reactive oxygen species. The fact that
overproducing superoxide dismutase,
SodA, suppresses the accelerated loss of
culturability in starving arcA deletion
mutants supports this notion.
Very few gene disruptions significantly retard the loss of culturability of
stationary-phase cells. However, disrupting acnA, which encodes aconitase,
enhances the survival of stationaryphase Staphylococcus aureus cells
about 100-fold, perhaps by reducing
respiration and oxidative load.
These and other data from studies of
various mutants suggest that oxidative
stress is a key factor limiting stationaryphase survival and that there is an increased demand for oxidation management in cells subjected to growth arrest
(Fig. 2). Proponents of the programming
theories may interpret these findings differently. Thus, they could argue either
that these oxidative cues set off an adaptive VBNC response or that they enhance
or trigger bacterial suicide systems.
Stationary Phase-Induced
Oxidative Damage
Key questions are whether entry into
stationary phase is associated with increased oxidative damage to cellular
constituents and, if so, whether this damage
affects the culturable or nonculturable cell fractions equally. These questions were recently addressed by analyzing a specific marker of oxidative damage to proteins in single culturable and
nonculturable cells that had been separated on
density gradients.
Protein carbonylation is one of the most commonly used biomarkers of severe oxidative damage to proteins. When proteins become decorated with carbonyls, it is a sign of probable
dysfunction. Human diseases associated with
proteins that are modified in this way include
Parkinson’s disease, Alzheimer’s disease, cancer,
cataractogenesis, diabetes, and sepsis. Compared to other oxidative modifications, carbonyls are relatively difficult to induce but are all
but irreversible within living cells.
This same kind of modification increases
among proteins within stationary-phase E. coli
cells, affecting specific proteins. By this criterion, several different cell processes appear to be
targets for stasis-induced damage, including
peptide chain elongation, protein folding and
reconstruction, large-scale DNA organization,
central carbon catabolism, and general stress
protection.
During the early stages of stasis, E. coli cells
are subject to a sudden burst of carbonylation
that affects a large number of proteins, especially aberrant isoforms. Moreover, there is a
link between ribosomal proofreading and protein carbonylation. Thus, carbonylation is drastically attenuated in mutants harboring intrinsically hyperaccurate ribosomes. Thus, elevated
carbonylation of proteins in cells entering stationary phase may be due, in part, to aberrant
proteins being more susceptible to oxidative attack. The pool of such oxidation-sensitive, aberrant proteins expands during starvation due to
a temporarily reduced fidelity of the translational apparatus.
When we used density gradient centrifugation
to separate culturable and nonculturable E. coli
cells, we detected far more protein carbonyls in
nonculturable than in culturable cells. In addition, several proteins are oxidized exclusively in
the nonculturable cell fractions, including the
histone-like DNA-binding protein H-NS, glutamate synthase (GltD), and ␤-ketoacyl [acyl carrier protein] synthetase (FabB). Other qualitative differences include significant oxidative
damage in mature ribosomes and periplasmic
proteins in the nonculturable cell fraction.
We think that this extensive, irreversible oxidative damage—not adaptive entry into a VBNC
state—is the basis for stationary-phase E. coli
cells losing their reproductive ability. Moreover,
this finding is not restricted to E. coli. Thus, for
example, stationary-phase V. vulnificus cells,
which some researchers contend become
VBNC, form a subpopulation whose members
fail to reproduce due to starvation-induced hydrogen peroxide sensitivity. However, data describing damage heterogeneity in such cells cannot distinguish between stochastic deterioration
and programmed injurious pathways.
How is this asymmetric damage generated
within a bacterial population? We now know
that the expression of many genes is altered
during progression through the bacterial division cycle. Perhaps a sudden arrest of growth
when specific gene products—such as superoxide dismutases A and B—are present at low
levels generates a damaged subpopulation of
cells. Alternatively, differences in chromosome
numbers and, consequently, gene dosage among
subpopulations of stationary phase cells could
lead to different levels in stress defense proteins
or to differential sensitivity to DNA damage.
Although we have no direct evidence to support these possibilities, we point out that the
abundance of SodA and the universal stress protein UspA is much lower in nonculturable than
in culturable E. coli cells. Moreover, the pattern
of protein carbonylation is similar in these cells
and cells lacking cytoplasmic Sod activity. Reducing cellular Sod activity mimics the elevated
expression of specific stress regulons in nonculturable cells. Thus, increased carbonylation and
subsequent loss of reproductive ability of some
cells entering stationary phase could be linked to
the relative abundance of Sod in individual
starving cells.
The Theory behind Programmed
Cell Death
Many plasmids and phage genomes harbor toxin-antitoxin (TA) loci that contribute to the
apparent stability of these episomes by selectively killing episome-free, or cured, segregants
and their progeny. The TA loci typically consist
of a downstream toxin gene and an upstream
antitoxin gene organized in an operon. The
toxin protein is stable, whereas the antitoxin is
unstable, thus explaining the postsegregational
killing of cured progeny. In addition, bacteria
harbor some TA loci on their chromosomes,
perhaps thereby providing systems to mediate
programmed cell death.
Based on experiments involving the mazEF
operon as a model TA locus, it has been pro-
Volume 71, Number 8, 2005 / ASM News Y 367
posed that nutrient starvation and growth arrest
trigger programmed cell death. This programmed
killing of part of the population may enable the
remaining cells to survive by using nutrients that
leak from their dead siblings (Fig. 1).
However, these purported benefits of cell
death for the survivors have not been convincingly proved. Moreover, data from Kenn Gerdes’
group at the University of Southern Denmark in
Odense suggesting that the TA loci lock cells in a
growth-arrested mode rather than killing them
challenge the TA-based concept of programmed
cell death.
The biochemical functions of the toxins
points to them having beneficial, rather then
detrimental, roles such as restricting macromolecular synthesis in stationary-phase cells. This
mode of macromolecular shutdown can be fully
reversed by producing the cognate antitoxins.
Accordingly, it is unlikely that the TA loci are
responsible for increased damage or death in
stationary-phase cells. However, these findings
do not rule out the possibility that triggering
other bacterial systems can kill cells under specific damaging or stressful conditions. For instance, suicide systems may have evolved as a
defense mechanism to inhibit the spread of
phage in clonal bacterial populations, according
to Hanna Engelberg-Kulka and her colleagues at
the Hebrew University-Hadassah Medical
School in Jerusalem, Israel.
Conclusion
Proponents of stochastic deterioration argue
that aerobic metabolism might be the Achilles’
heel of starving E. coli cells and that oxidative
damage leads to the loss of culturability. If starvation and oxidative damage proceed for an
extended period, the nonculturable cells become
moribund and then irreversibly lose essential
life-supporting activities. This argument appears to be consistent with the finding that stationary-phase sterility in E. coli is rapidly fol-
lowed by a collapse in membrane functions. The
reduced culturability of starving rpoS, rpoE,
oxyR, sodAB, katEG, and arcA mutant cells
simply means that these mutants die at an accelerated rate. Associating nonculturability with
carbonylation further favors the deterioration
over the VBNC hypothesis.
However, bacterial cells may well become
reversibly nonculturable, meaning specific conditions could rescue such cells. For example,
apparently sterile, starving gram-positive Micrococcus luteus cells can be resuscitated in the
presence of Rpf, a protein encoded and produced by the organism itself, according to Douglas Kell of the University of Wales in Aberystwyth. However, these, and other examples, do
not support the VBNC hypothesis, which holds
that nonculturability is an inducible, genetically
programmed capacity of cells to ensure survival
under adverse environmental conditions.
Data showing that supposed toxins lock cells
in a growth-arrested mode without killing them
challenges the notion that bacteria undergo programmed cell death during stationary phase.
The biochemical functions of TA loci point to an
advantageous, rather than a seemingly purely
detrimental, role for these toxins in restricting
macromolecular synthesis during starvation.
Thus, John Postgate’s early notion about bacterial senescence appears to be the most viable
among contemporary models to explain how
some bacteria become nonculturable. Indeed,
based on our damage analysis along with efforts
to identify which bacterial cell proteins and
other functions deteriorate during stationary
phase, it appears that the pathways of bacterial
senescence and mandatory aging in higher organisms have a great deal in common. However,
whether stasis-induced deterioration in bacteria
is a purely stochastic phenomenon, and whether
genetically programmed pathways contribute to
damage heterogeneity in starving populations of
bacteria, are unsolved questions.
SUGGESTED READING
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Desnues, B., G. Gregori, S. Dukan, H. Aguilaniu, and T. Nyström. 2003. Differential oxidative damage and expression of
stress regulons in culturable and nonculturable cells of Escherichia coli. EMBO Rep. 4:400 – 405.
Dukan, S., A. Farewell, M. Ballestreros, F. Taddei, M. Radman, and T. Nyström. 2000. Proteins are oxidatively carbonylated
in response to reduced transcriptional or translational fidelity. Proc. Natl. Acad. Sci. USA 97:5746 –5749.
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Dukan, S., and T. Nyström. 1998 Stasis results in increased and differential oxidation of cytoplasmic proteins leading to
developmental induction of the heat shock regulon. Genes Dev. 12:3431–3441.
Engelberg-Kulka, H., B. Sat, M. Reches, S. Amitai, and R. Hazan. 2004. Bacterial programmed cell death systems as targets
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of RNA polymerase. Microbiol. Mol. Biol. Rev. 66:373–395.
Kell, D. B., and M. Young. 2000. Bacterial dormancy and culturability – the role of autocrine growth factors. Curr. Opin.
Microbiol. 3:238 –243.
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codon-specific cleavage of mRNAs in the ribosomal A site. Cell 112:131–140.
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