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ISSN: 1040-841X (print), 1549-7828 (electronic)

Crit Rev Microbiol, Early Online: 1–12

! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/1040841X.2013.804030

REVIEW

mazEF-mediated programmed cell death in bacteria: ‘‘What is this?’’

Bhaskar Chandra Mohan Ramisetty1, Bhargavi Natarajan1,2, and Ramachandran Sarojini Santhosh1
1
School of Chemical and Biotechnology, SASTRA University, Thanjavur, India and 2Department of Biotechnology, Indian Institute of Technology
Madras, Chennai, India
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Abstract Keywords
Toxin–antitoxin (TA) systems consist of a bicistronic operon, encoding a toxin and an antitoxin. Bacterial stress physiology, Escherichia coli,
They are widely distributed in the prokaryotic kingdom, often in multiple numbers. TAs are extra cellular death factor, persistence,
implicated in contradicting phenomena of persistence and programmed cell death (PCD) in toxin–antitoxin systems
bacteria. mazEF TA system, one of the widely distributed type II toxin–antitoxin systems, is
particularly implicated in PCD of Escherichia coli. Nutrient starvation, antibiotic stress, heat History
shock, DNA damage and other kinds of stresses are shown to elicit mazEF-mediated-PCD.
ppGpp and extracellular death factor play a central role in regulating mazEF-mediated PCD. The Received 29 January 2013
activation of mazEF system is achieved through inhibition of transcription or translation of Revised 1 May 2013
mazEF loci. Upon activation, MazF cleaves RNA in a ribosome-independent fashion and Accepted 7 May 2013
subsequent processes result in cell death. It is hypothesized that PCD aids in perseverance of Published online 24 June 2013
the population during stress; the surviving minority of the cells can scavenge the nutrients
released by the dead cells, a kind of ‘‘nutritional-altruism.’’ Issues regarding the strains,
reproducibility of experimental results and ecological plausibility necessitate speculation. We
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review the molecular mechanisms of the activation of mazEF TA system, the consequences
leading to cell death and the pros and cons of the altruism hypothesis from an ecological
perspective.

Introduction mean suicide of the organism with no conceivable benefit.

Nevertheless, there are several studies indicating PCD-like
The objectives of life forms are colonization of a habitat and
phenomena in different bacterial species grown in different
its propagation to other habitats. In general, every ‘‘organ-
conditions. The long held perception of bacterial immortality
ism’’ employs pro-survival strategies irrespective of the
is challenged by numerous reports of PCD in bacteria. Recent
prevailing conditions. In fact, the occurrence of regulated
findings suggest a ‘‘multicellular-like’’ behaviour among
metabolic activities to make more of their own kind is the
some bacteria (Dunny et al., 2008). Phenomena like quorum
fundamental difference between living and non-living beings.
sensing and biofilms that support the ‘‘multicellular-like’’
However, certain individual cells of multi-cellular organisms
behaviour are well studied (Hammer & Bassler, 2003; Henke
die by inherent mechanisms called as programmed cell death
& Bassler, 2004; Waters & Bassler, 2005; Waters et al.,
(PCD). It is well established and accepted that PCD is a
2008). Bacteria could form complex multicellular-like struc-
normal physiological phenomenon, in which the cells die due
to activation of an intrinsic cell-death system, so as to benefit
tures on prolonged incubation. 13
20
Toxin–antitoxin (TA) systems found in many bacterial
the organism as a whole. It is usually initiated by one or more
genomes are shown to mediate PCD and are therefore
mechanisms/triggers. In eukaryotes, two forms of PCD,
attractive targets for novel anti-microbial drugs since they
autophagy and apoptosis, are significant and essential for
are thought to ‘‘kill from within’’ (Amitai et al., 2004, 2009;
their normal development and survival. For example, during
Engelberg-Kulka et al., 2009). However, it should be noted
frog morphogenesis, the cells present in the tail undergo PCD
that TA systems are implicated in contradicting phenomena;
resulting in degeneration of the tail (Hensey & Gautier, 1998).
persistence (Gerdes & Maisonneuve, 2012) and PCD. Also,
Virus infected cells of animals are killed through PCD to
the presence of numerous TA systems in prokaryotes makes
restrict the spread of infection (O’Brien, 1998). In free living
this idea complicated to venture. mazEF TA system is
and unicellular organisms, PCD phenomenon would literally
particularly implicated as a mediator of PCD in Escherichia
coli and is the focus of this review. In spite of many
researchers working on the mazEF system, contradicting
observations have led to diverse implications and specula-
tions. Here, we discuss in brief, the details of type II TA
Address for correspondence: Bhaskar Chandra Mohan Ramisetty, AP II,
School of Chemical and Biotechnology, SASTRA University, Thanjavur, systems, the mechanism of mazEF-mediated PCD and debate
613402, 613402 India. E-mail: ramisettybcm@biotech.sastra.edu on the pros and cons of this phenomenon.
 2 B. C. M. Ramisetty et al.
Toxin–antitoxin systems and the mazEF system
Toxin–antitoxin systems, initially discovered in plasmids,
were thought of as extra-chromosomal genes that were
responsible for post-segregational killing, so as to maintain
plasmid integrity (Gerdes et al., 1986). However, several
chromosomal genes have also been identified as TA loci.
They are widely distributed in the prokaryotic genomes and
are often present in multiple copies (Anantharaman &
Aravind, 2003; Pandey & Gerdes, 2005). The toxin interferes
with the essential metabolic processes of the cell and is
therefore harmful. The antitoxins neutralize the toxin through
different mechanisms. TA loci are classified into three types
based on the mode of neutralization of the toxin by the
antitoxin (Gerdes et al., 2005; Yamaguchi & Inouye, 2011).
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Antitoxins of Type I TA system are antisense RNAs that bind
to toxin mRNA through complementary base pairing and
inhibit translation of Toxin, for example hok-sok (Faridani
et al., 2006). Type II TA systems are most common, with at
least eight families based on homology (Anantharaman &
Aravind, 2003; Gerdes et al., 2005; Pandey & Gerdes, 2005).
In this case, the antitoxin neutralizes the toxin through direct
protein–protein interactions, for example relBE (Gotfredsen
& Gerdes, 1998). Type III TA systems were recently
discovered and are characterized by RNA–protein interaction,
where RNA antitoxin binds to proteic toxin to bring about
neutralization, for example toxIN (Fineran et al., 2009).
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A typical Type II TA system consists of two genes in a
single operon; usually the gene encoding the toxin is located
downstream to that of the gene encoding the antitoxin which
is under the control of a common auto-regulated promoter.
The toxins are usually globular in structure and have a longer
half-life. On the other hand, the antitoxins are labile and have
a shorter half-life since they are subject to degradation by
cellular proteases. The dynamic nature of auto-regulation and
difference in half-lives of toxin and their cognate antitoxin are
vital for the expression and functioning of the TA systems.
Under normal conditions, the antitoxin is translated several
folds higher than the toxin to maintain a high antitoxin-to-
toxin ratio, resulting in continuous neutralization of the toxins
(Yamaguchi & Inouye, 2011; Yamaguchi et al., 2011). This is
because the Shine–Dalgarno (SD) sequence of the antitoxin is
very efficient as opposed to the inefficient SD sequence of the
toxin. Also, the TA complexes repress the transcription of
their genes, therefore ensuring auto-regulation of the gene
system (Gerdes, 2000). Thus, auto-regulated transcription and
differential translation of these TA genes compensates for the
loss of antitoxin by proteolytic cleavage. The relative
concentration of antitoxin to that of toxin is the limiting
factor in switching the operon ON or OFF (Christensen-
Dalsgaard et al., 2008; Overgaard et al., 2008, 2009). When
the amount of antitoxin is low relative to the toxin, the
promoter is switched ON ensuring the production of large
number of antitoxins to neutralize the toxin. If stress affects
translation, the reduced production and continued proteolysis
of the antitoxin will result in low antitoxin-to-toxin ratio. A
consequent increase in the concentration of free toxin could
theoretically be deleterious to the cell.
Most of the toxins are sequence-specific endoribonu-
cleases (also called mRNA interferases) that cleave mRNA.
Crit Rev Microbiol, Early Online: 1–12
Some cleave RNA that is being translated (associated with
ribosomes) and some cleave untranslated RNA, that is, their
action may either be translation/ribosome dependent or
independent (Christensen-Dalsgaard et al., 2008; Yamaguchi
& Inouye, 2009). However, the extent and consequences of
such mRNA cleavage during stress are unclear. It is generally
believed that the TA systems play a role during stress, more
appropriately in suboptimal growth conditions. Various
researchers have implicated TA systems in stress tolerance,
PCD, plasmid stabilization, persister cell formation, growth
arrest, stabilization of genomic parasites and as addictive
genomic debris and selfish alleles (de la Cueva-Mendez,
2003; Hayes, 2003; Inouye, 2006; Jensen & Gerdes, 1995;
Magnuson, 2007; Van Melderen & Saavedra De Bast 2009).
Recent advancements emphasize physiological relevance of
chromosomal Type II TA systems in two directions: as ‘‘stress
tolerance factors’’ (Gerdes et al., 2005) or as ‘‘mediators of
Programmed Cell Death’’ (Engelberg-Kulka et al., 2006). In
other words they can be classified as ‘‘beneficial to the
individual cell’’ or ‘‘detrimental to the individual cell.’’ It has
been shown that the presence of at least one of the TA systems
of Mycobacterium smegmatis is essential for its survival
(Frampton et al., 2012). Stress tolerance known to be
mediated by the relBE system and persister cell formation
mediated through the mqsR-mqsA system (Kim & Wood,
2010), both involving induction of metabolic-stasis, can be
considered advantageous. On the other hand, altruistic PCD
enabled through mazEF system is a disadvantage at the
cellular level.
mazEF TA system is well-characterized and widely found
in the bacterial kingdom including pathogenic bacteria. mazE
and mazF are chromosomal genes belonging to the rel operon
of E. coli strain MC4100, and were first described as
‘‘addiction modules’’ (Aizenman et al. 1996). It was found
that overexpression of MazF leads to decrease in CFU (colony
forming units), thus acting as a toxin. MazE is a labile protein
that is degraded by ClpAP serine protease. It neutralizes
MazF, thus acting as the antitoxin. The genetic architecture of
mazEF system is that of a typical type II TA system with
mazE located upstream of mazF, under a common promoter.
There are three promoters; P1, P2 and P3 that have been
identified upstream of mazE. The protein products MazE and
MazF form a complex that inhibits the transcription of the
genes from the P2 and P3 promoters (Marianovsky et al.,
2001). The genetic architecture and transcriptional autoregu-
lation of the mazEF system is shown in Figure 1.
mazEF-mediated PCD
The pioneering work of Engelberg-Kulka and co-workers has
demonstrated that the presence of mazEF system induces a
loss of viability in E. coli on exposure to stress (Aizenman
et al., 1996). Reduction in CFU is due to the fact that when
the cell is challenged with various stress conditions, the
inhibition of transcription or translation causes a reduction in
Antitoxin concentration rendering the toxin free. MazF, the
toxin, is a sequence-specific mRNA interferase that cleaves
mRNAs at ACA or ACU sites in E. coli (Zhang et al., 2003).
It was concluded that MazF is a ribosome independent mRNA
interferase since it could cleave ACA sequences even outside
 DOI: 10.3109/1040841X.2013.804030
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Figure 1. Genetic architecture of mazEF system: The rel operon of E. coli MC4100 consists of four genes relA, mazE, mazF and mazG. Transcription
from P2 and P3 promoters lead to production of MazEF mRNA and translation produces MazE and MazF protein products. MazE and MazF form a
complex in a 4:2 stoichiometry. MazE2-MazF2-MazE2 complex represses transcription of these genes. MazE is constitutively cleaved by ClpAP and the
MazF released therein cleaves RNA leading to cell death.
the open reading frame. Cleavage of mRNAs reduces
translation rates and prevents synthesis of essential proteins
needed for metabolism and survival. Consequently, cell death
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ensues resulting in dramatic loss of CFU upon induction of
stress. However, similar reduction in cell viability was not
observed in a mazEF null mutant. This phenomenon was
named as the ‘‘mazEF-mediated PCD.’’
Several theories have been proposed to account for the
benefits of this seemingly wasteful process to a unicellular
organism. Primarily, it is said to prevent spread of phage
infections (Engelberg-Kulka et al., 2004) and it is also
proposed that the release of nutrients by the dead majority of
the cellular population might be utilized by the surviving
minority (Aizenman et al., 1996). Other roles include
maintenance of genomic stability, aid in biofilm formation
(Amitai et al., 2009; Mutschler et al., 2011) sporulation
(Adler et al., 2001) and in the development of Myxococcus
xanthus (Nariya & Inouye, 2008). Among all of these
proposed mechanisms for functioning of TA system, the
generation of free MazF and the mechanism by which it
causes lethality form the two central aspects of mazEF-
mediated PCD. Each aspect is achieved by multiple ways as
shown in Figure 2. In the following sections, we describe the
stress conditions observed to trigger PCD, factors involved in
mediating it and the consequences of free MazF Toxin.
Stress conditions that induce PCD
Several stress conditions have been reported to elicit PCD in
E. coli MC4100 strain. These include nutritional downshift,
exposure to antibiotics (inhibitors of transcription and trans-
lation), high temperature, salt stress and so on, which E. coli
is likely to face in its natural habitats (Sat et al., 2001). All the
cases directly or indirectly cause the activation of mazEF
system that leads to RNA cleavage by free MazF. The
activation of mazEF system is caused by either inhibition of
3
transcription of mazEF loci or inhibition of MazEF mRNA
translation. Amino acid starvation is one of the well-studied
stresses that are reported to elicit mazEF-mediated PCD in
E. coli (Aizenman et al., 1996). This is partly due to induction
of stringent response upon amino acid deprivation (Vinella
et al., 1996). Amino acid starvation leads to accumulation of
guanosine 3,5 bispyrophosphate (ppGpp), which reduces
global transcription (Torok & Kari, 1980). Increased amounts
of ppGpp reduces transcription of mazEF genes and therefore
MazE and MazF protein products are not produced
(Aizenman et al., 1996). Carbon source starvation also
causes ppGpp levels to rise since it activates SpoT (Murray
& Bremer, 1996). Thymine starvation induced by various
methods was also observed to reduce transcription of mazEF
genes and thereby cause PCD. This phenomenon of death
caused due to thymine starvation is called thymineless death
(TLD) and it was concluded that ppGpp might be involved in
the process, or that it could be an outcome of DNA damage
caused by absence of thymine (Sat et al., 2003).
Antibiotics that inhibit transcription or translation exert
harmful effects through induction of mazEF-mediated PCD
(Engelberg-Kulka et al., 2004; Hazan et al., 2004). The
reduced levels of transcription and translation decrease the
amount of antitoxin MazE. This combined with the fact that
MazE is degraded by cellular proteases will lead to increase
in concentrations of free MazF. It was also found that
rifampicin has more pronounced effect on mazEF system than
do chloramphenicol or streptomycin (Sat et al., 2001). The
effect of these antibiotics is observed much more quickly and
is more prominent in a minimal medium when compared to
rich LB medium. It was later found that these antibiotics
cause PCD by forming reactive oxygen species (ROS) and
that the process requires the presence of extracellular death
factor (EDF) (Amitai et al., 2009; Engelberg-Kulka et al.,
2009). High temperature (50 C), DNA damage (through
addition of nalidixic acid, mitomycin C and exposure to
 4 B. C. M. Ramisetty et al. Crit Rev Microbiol, Early Online: 1–12
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Figure 2. Regulatory pathway of mazEF-mediated PCD: Central to the process is generation of free MazF. The stress conditions initiate a cascade of
events that lead to free MazF. Transcription of mazEF genes and translation of mRNA are inhibited by antibiotics and also by ppGpp whose levels rise
when subject to amino acid starvation, thymine starvation and carbon source starvation. Increased cell density and carbon starvation lead to increased
amounts of EDF generated by ClpXP. These effects combined with degradation of MazE by ClpAP causes MazF free of MazE. The downstream events
start with RNA cleavage and selective translation of death and survival proteins. Death proteins bring about death through ROS dependent and ROS
independent pathways, aided by oxidative damage and DNA damage respectively (solid arrows – activation; dotted arrows – inhibition; STM – stress
translational machinery; EDF – extracellular death factor; ROS – reactive oxygen species).

UV radiation) and oxidative stress (caused by hydrogen et al., 2002). RelA is a protein encoded by the rel operon and
peroxide) were found to elicit mazEF-mediated PCD. A it synthesizes ppGpp from ATP and GTP or GDP (Haseltine
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generalization can be made that the wild type Escherichia coli
strain MC4100 is highly sensitive to metabolic stress
compared to its mazEF mutant.
It was observed that this kind of PCD was profound in
cultures in the logarithmic phase but not in stationary phase
(Engelberg-Kulka et al., 2004). However, deletion of rpoS, the
gene encoding stationary phase sigma factor s, permitted
MazF mediated death even during the stationary phase
(Amitai et al., 2009). Also, a difference was observed with
regard to the ability of TA systems to induce PCD in liquid
and solid media. mazEF system is able to elicit PCD in both
liquid and solid medium, unlike other TA systems namely
relBE, yefMyoeB and chpBIK (Amitai et al., 2009), which
contribute to PCD only when grown in liquid cultures. It was
hence concluded that mazEF system plays a role in biofilm
formation.
Factors involved in PCD
The phenomenon of PCD in E. coli mediated by mazEF TA
system has been long studied and several research groups
have unravelled various aspects of this phenomenon. It has
been largely accepted that PCD is a complex network of
events that has many key components. Some of the main
factors, namely ppGpp, MazG, EDF and ROS are discussed
below.
ppGpp
Guanosine 3,5 bispyrophosphate (ppGpp) plays an important
role in the stringent response that is triggered by amino acid
starvation. Amino acid starvation leads to the presence of
uncharged tRNA at the ribosomal A site, which in turn
activates RelA, also known as ppGpp synthetase I (Wendrich
& Block, 1973; Metzger et al., 1989). SpoT, also known as
ppGpp synthetase II, causes ppGpp levels to increase upon
exposure to stresses other than amino acid starvation, mainly
carbon and iron source deprivation (Murray & Bremer, 1996).
ppGpp inhibits protein synthesis (Svitil et al., 1993) and
redirects RNA polymerase towards biosynthetic operons to
enable de novo synthesis of amino acids with simultaneous
reduction in transcription of stable RNAs (rRNA and tRNA)
(Schreiber et al., 1991). It was found that ppGpp reduces the
half-life of promoter open-complexes. Since stable RNA
promoters are short lived, they are affected. However,
promoters of biosynthetic operons are long lived and there-
fore, are unaffected (Barker et al. 2001). ppGpp also alters the
competency of various sigma factors that form RNAP holo-
enzyme and leads to transcription of other stress related genes
(Jishage et al., 2002; Laurie et al., 2003). It was also predicted
that ppGpp induced transcriptional pausing occurs preferen-
tially during mRNA transcription and not during synthesis of
stable RNAs (Bremer & Ehrenberg, 1995). Base pairing with
cytosines of the non-template strand of the promoter
sequences is another proposed mechanism for ppGpp
mediated regulation of transcription, since stable RNA
promoters are characterized by GC rich sequences
(Artsimovitch et al., 2004). These studies answer the question
of how ppGpp is able to selectively enable transcription of
some genes while inhibiting others.
It was shown that mazEF promoter is a target for
repression by ppGpp. Artificial overexpression of ppGpp
inhibited the transcription from mazEF promoter and also
induced mazEF-mediated PCD (Aizenman et al., 1996). As
stated earlier, the depletion of antitoxin concentration in the
cell in conjunction with decreased rate of transcription causes
an increase in the free form of the toxin MazF, which is now
 DOI: 10.3109/1040841X.2013.804030
active to exert its harmful effects and kill the cell. It was
reported that ppGpp along with other stress conditions such as
high temperature, DNA damage by nalidixic acid, oxidative
stress, and so on only affected the induction of mazEF system.
But since reduction in CFU was observed both with and
without ppGpp upon MazF overproduction, ppGpp does not
influence MazF toxicity (Engelberg-Kulka et al., 2004). It
was also suggested that ppGpp might be involved in
mediating thymineless death (TLD) (Sat et al., 2003).
MazG
mazG is the fourth gene located on the rel operon,
downstream of mazEF and has been proven to regulate
PCD in E. coli. mazG is co-transcribed with mazEF (Gross
et al., 2006); (Zhang & Inouye, 2002). Ectopic overexpression
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of MazG reduced CFU in E. coli and reduced ppGpp levels up
to two-fold. However, when mazEFG is overexpressed, the
growth rates were not affected and ppGpp accumulation were
similar to that of the controls (Gross et al., 2006). The
enzymatic action of MazG was inhibited by MazE-MazF
complex upto 70%. MazE alone could not reduce MazG
toxicity. It was suggested that there might exist a link between
‘‘point of no return’’ (Amitai et al., 2004) and MazG. During
stringent response, increased levels of ppGpp inhibit tran-
scription from mazEF promoter. Also, existing MazE is
degraded and MazF is active to kill the cells. The model
proposed in this study suggests that a parallel impact of MazE
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degradation is the activation of MazG which then decreases
the levels of ppGpp. Therefore, transcription resumes and the
cells are rescued from the toxic effect of MazF.
EDF
EDF is a linear pentapeptide and was shown to be essential for
eliciting PCD through the mazEF system. This peptide has a
sequence Asn-Asn-Trp-Asn-Asn (NNWNN). Studies indicate
that PCD is a population phenomenon that occurs in the
presence of EDF acting as a quorum sensing molecule
(Kolodkin-Gal et al., 2007). EDF is produced in dense culture
and it acts as a signal molecule, since it is capable of causing
cell death in dilute cultures. The concentration of EDF
supposedly is an indication of cell density.
The genes zwf that encode for glucose-6-phosphate
dehydrogenase which catalyses the rate limiting step of the
pentose phosphate pathway, and ygeO that encode for an
unknown protein have been reported to play a role in EDF
production (Kolodkin-Gal et al., 2007). Deletion of zwf, in the
absence of ectopic induction of mazEF, prevented the
occurrence of PCD, as shown by high CFU and low EDF
activity. Therefore, it was proposed that EDF is a degradation
product of Zwf that has the sequence NNWDN from the 199th
to 203rd residues, similar to EDF. Since supernatants from
both DclpP and DclpX strain showed minimal EDF activity, it
has been suggested that ClpXP, a cellular protease, cleaves
Zwf and a subsequent amidation step produces EDF. Deletion
of ygeO also reduced EDF activity. On the other hand,
overexpression of Zwf in strain lacking ygeO increased EDF
activity and decreased the number of CFUs, indicating that
Zwf is the precursor of EDF and YgeO is a secondary
requirement for its production (Kolodkin-Gal & Engelberg-
5
Kulka, 2008). The zwf gene is found to be induced during
oxidative and heat stresses through interaction of SoxS with
Soxbox present in the promoter region of zwf (Griffith &
Wolf, 2004; Saavedra & Sesma, 2005). Tellurite mediated
oxidative stress was shown to increase zwf expression, which
in turn augments NADPH synthesis (Sandoval et al., 2011).
The NADPH produced due to activity of Zwf is a known
antioxidant since it was shown (in Saccharomyces cerevisiae)
to minimize ROS and it is required for activity of glutathione
reductase (Izawa et al., 1998). Indeed, levels of glutathione
have been found to progressively increase during exponential
to stationary phase transition in E. coli (Fahey et al., 1978). It
should also be noted that since pentose phosphate pathway is
a source of ribose for nucleotide biosynthesis, cleavage of
Zwf could pose additional stress. However, the causes and
consequences of Zwf degradation are not yet clear.
MazF overproduction and other stress conditions induced
by antibiotics (chloramphenicol, rifampicin and nalidixic
acid), mitomycin C and trimethoprim increased production of
EDF (Kolodkin-Gal & Engelberg-Kulka, 2008). Furthermore,
the same study also proved that production of EDF resulted in
increased PCD mediated by MazF. These observations
indicate a positive feedback that exists between mazEF
activation and EDF production. It was also reported that
production of and response to EDF in the strain MG1655 was
defective. Significant production of EDF by MG1655
required overproduction of MazF, whilst MazF overproduc-
tion in the other strains (MC4100, W3110 and K-38) induced
very high levels of EDF (Kolodkin-Gal & Engelberg-Kulka,
2008). Later, it was suggested that EDF could act in
conjunction with mazEF module to invoke bactericidal
conditions through antibiotics that were previously proven
to have only bacteriostatic effects (Amitai et al., 2009). This
effect was ROS dependent when the antibiotics affected
translation/transcription (for example, rifampicin) and ROS
independent when the antibiotics caused DNA damage (for
example, nalidixic acid).
EDF was also shown to increase the cleavage activity of
MazF and ChpBK. In a study, it was demonstrated that EDF
was able to overcome MazF inhibition by MazE. Replacement
of the terminal aspargine and the central tryptophan residues
decreased the activity of EDF, indicating that these residues
are crucial for action of EDF. However, increase in length of
the synthetic peptide by addition of extra Asn residues on
either side did not affect EDF’s influence on MazF but
decreased cleavage efficiency of ChpBK (Belitsky et al.,
2011). Based on these observations, it was suggested that
EDF directly interacts with MazF through its Trp3 placed in a
hydrophobic pocket where MazE binds through Trp73. The
structure of EDF is similar to that formed by residues 71–75
of MazE. The interactions between EDF and MazF are similar
to that of MazE and MazF. Due to these multiple functions,
EDF can be considered as a major contributor to mazEF-
mediated PCD.
Others
Although MazF inhibits translation by cleavage of RNAs, it
was found that some small proteins are still synthesised upon
activation of mazEF module in E. coli. These proteins were
 6 B. C. M. Ramisetty et al.
grouped into two classes – ‘‘death proteins,’’ which are
required for death of majority of the population and ‘‘survival
proteins,’’ which are required for survival of a subset (Amitai
et al., 2009). Some of the death proteins are ClpP (causes
cleavage of MazE and Zwf to generate EDF), SlyD (enables
cell lysis), YjiD (protects ROS sensitive death proteins) and
ElaC (involves in RNA processing). Some of the survival
proteins are YajQ, RsuA and DeoC which are primarily
involved in ROS detoxification. Recently, the mechanism
behind this selective translation was elucidated. It was shown
that induction of the mazEF system leads to cleavage of 16 s
rRNA as it harbours an ACA site. The ribosome that is
formed, therefore, no longer contains the anti-SD sequence
(Vesper et al., 2011). Simultaneous cleavage of 50 UTR
regions of mRNA that bear the SD sequence also occurs.
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This leads to formation of stress ribosomes that are capable of
translating leaderless mRNAs, and is named the stress
translational machinery (STM) (Moll & Engelberg-Kulka,
2012).
Role of ROS
It is well known that ROS are toxic to a living cell and cause
death in both prokaryotes and eukaryotes. It was reported that
mazEF-mediated PCD occurs in both ROS dependent and
ROS independent ways. In addition, it was also discovered
that antibiotics that affected transcription or translation
caused ROS dependent cell death mediated by mazEF
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system, whereas those that caused DNA damage induced
cell death in a ROS independent way (Amitai et al., 2009). On
testing protein carbonylation, it was observed that DNA
damaging antibiotics caused higher levels of carbonylation
than those that inhibited transcription and translation. MazF
overproduction had a similar effect. In addition, completely
anaerobic conditions prevented death mediated by antibiotics
that inhibit transcription and translation but not the death
caused by DNA damaging antibiotics. Addition of EDF
enhanced both ROS-dependent mazEF-mediated cell death
for rifampicin and ROS-independent mazEF-mediated cell
death for DNA damaging antibiotic nalidixic acid (Kolodkin-
Gal & Engelberg-Kulka, 2008). A contradictory observation
is that EDF has antioxidant and free radical scavenging
properties. This group of researchers suggest that the EDF
produced will initially help in eliciting PCD; however when
present at very high concentrations it scavenges free radicals
and acts as an antioxidant to prevent damage to ‘‘survival
proteins,’’ thereby helping a small sub-population to survive
and escape PCD (Gao et al., 2010). However, the claim that
the ability of mazEF-system to invoke bactericidal effects
(upon treatment with antibiotics previously known to have
bacteriostatic effects) through increased ROS production has
been challenged recently (Keren et al., 2013). This group of
researchers proved that ROS was not mandatory and probably
not responsible for killing by antibiotics; since there was no
change in survival when E. coli was treated with antibiotics in
aerobic and anaerobic conditions.
The consequences: dead or dormant?
The essential feature of PCD is that free MazF would
cause RNA cleavage leading to dramatic reduction in CFU
Crit Rev Microbiol, Early Online: 1–12
of E. coli. There have been observations that conclude that
reduction in CFU through the overexpression of MazF can be
reversed by overexpression of MazE (Pedersen et al., 2002).
This indicates that the cells were in a state of suspended
metabolism. But it was later shown that this effect cannot be
seen upon long term exposure to MazF, and that the
bacteriostatic effect becomes bactericidal, the ‘‘point of no
return’’ (Amitai et al., 2004; Engelberg-Kulka et al., 2006).
However, a study showed that TAs neither confer any benefit
(in the conditions tested) nor mediate PCD in E. coli
(Tsilibaris et al., 2007). Overexpression of mazFSeq
(Staphylococcus equorum) in E. coli induced bacteriostatis
but not death since CFUs remained constant up to 8 hours
after induction of mazFSeq (Schuster et al., 2012). A similar
growth arrest effect was observed upon over expression of
mazF from Streptococcus mutans in E. coli (Syed et al.,
2011). Despite the contradictions in the literature, we can
presume that four factors probably determine the fate of the
cells: (i) the number of free toxins, (ii) the duration of
exposure to the toxin, (iii) the extent and type of RNA cleaved
and (iv) the metabolic state of the cell.
One study shows that the translation of the non-susceptible
mRNA (ACA-less mRNA) continues upon overexpression of
mazF (Baik et al., 2009), while other mRNAs are not
translated. This would mean that the translation machinery is
not completely inactivated. The synthesis of survival
proteins and leaderless mRNA is very intriguing. In principle,
the survival proteins should, directly or indirectly, lead to
resumption of MazE synthesis and/or alleviate the effects
of MazF. If a cell is destined to die, there is no conceiv-
able benefit of synthesising survival proteins and
leaderless mRNA. It is not yet clear if the stress translational
machinery (STM) is found in the dead cells or survivors or
both.
Two cell death pathways
Recently, it was found that apart from triggering mazEF-
mediated PCD, DNA damage can cause cell death through a
different mechanism that involves membrane depolarization
and DNA fragmentation. This phenomenon in E. coli was
named as apoptotic-like-death (ALD), since it resembles
eukaryotic apoptosis. It has been suggested that ALD is part
of the SOS response as it is mediated through RecA and LexA
(Erental et al., 2012). The study, conducted in an MC4100
background, proved that mazEF-mediated PCD prevents ALD
by reducing the amount of RecA mRNA. Therefore mazEF
system prevents membrane depolarisation and DNA frag-
mentation. It was shown that EDF and one of the mazEF-
downstream genes yjiD are involved in prevention of ALD
(Erental et al., 2012).
Another parallel study showed the phenomenon called
ALD herein to occur in MG1655 with various biochemical
markers. However, cell death was caused by ampicillin and
gentamicin, apart from norfloxacin and MMC (Dwyer et al.,
2012). This is in contrast to the first study which concluded
that ALD occurs only in response to DNA damage. Therefore,
extensive study on ALD is needed to explain the differences
in these observations, and to establish the physiological need
for having two cell death mechanisms.
 DOI: 10.3109/1040841X.2013.804030
The discrepancies
Review of literature to date on mazEF-mediated-PCD raises
several unanswered questions from ecological and physio-
logical perspectives. In this section we bring to light the
various differences with regard to the mazEF loci in two
widely used laboratory strains MC4100 and MG1655. It is
important to keep in mind that MC4100 exhibits PCD
phenotype whereas MG1655 does not (Kolodkin-Gal &
Engelberg-Kulka, 2008). We discuss the differences in these
strains with respect to the integrity of relA-mazEF loci, the
promoter region of mazEF and the protease that cleaves the
Antitoxin MazE.
Genetic variations
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Monash University on 07/01/13
Few genetic differences within the relA-mazEF loci of
MC4100 strain (procured from Engelberg-Kulka’s lab)
compared to MG1655 were reported (Tsilibaris et al.,
2007). The presence of Kan resistance gene in mazG
(downstream of mazEF) in a mazEF null mutant indicates
that there may be inadvertent genetic manipulations in these
loci. In direct relation to the mazEF loci, MC4100 has been
reported to harbour relA1 mutation and is deficient in
accumulating ppGpp upon nutrient starvation (Reeh et al.,
1976). Engelberg-Kulka’s lab constructed MC4100 relAþ and
MC4100 relAþDmazEF strains (Engelberg-Kulka et al.,
1998). However, the same strains were later shown to be
For personal use only.
SMG negative and was found to comprise several mutations
in the relA and mazG loci (Tsilibaris et al., 2007). It should be
noted that relA and mazEF loci are very close, approximately
70 base pairs. Hence, it is highly improbable to construct a
relAþDmazEF strain using lysate of relAþmazEFþ by P1
mediated homologous recombination (Engelberg-Kulka et al.,
1998). Similarly, it is also improbable to make relAþDmazEF
using lysate of relA1DmazEF (Kolodkin-Gal & Engelberg-
Kulka, 2008). A frameshift mutation in the relA gene is found
in MC4100DmazEF strain, which has not been accounted
for. RelA and MazG are regulators of the alarmone
ppGpp which has diverse functions in bacterial stress
physiology. Since mazEF loci are between relA and mazG
loci, detailed evaluation of the integrity of these loci should
be confirmed.
Number of promoters
Another important difference between MC4100 and
MG1655 is the number of promoters reported for
mazEF loci. In MC4100, three promoters P1, P2 and
P3 (Aizenman et al., 1996) were reported while in
MG1655 only one promoter was reported (Christensen
et al., 2003). It was shown that P2 and P3 are repressed
by MazEF complex. FIS protein (factor for inversion
stimulation) was found to increase transcription of mazEF
genes by binding to a 40 region (Marianovsky et al.,
2001). Assuming that P2 and P3 promoters are genuine
and functional, the number of promoters will have a
profound effect on the total transcription from mazEF
loci. It is conceivable that the concentration of mRNA is
directly proportional to the total TA proteins given that
general translation has not been affected. It can be stated
7
that an increased amount of MazEF mRNA will result in
increased MazEF complexes. Concomitantly, the cell with
larger amounts of MazF will result in lethal conse-
quences upon induction of stress, compared to the one
with lesser MazF. To confirm this, the foremost approach
would be to ascertain the levels of MazEF mRNA and
MazEF proteins in MG1655 and MC4100. Theoretically,
the two strains are supposed to have similar amounts of
MazEF mRNA and protein. We predict, based on the
mentioned discrepancies, that MC4100 of Engelberg-
Kulka’s lab may have higher levels of MazEF mRNA
in the growth conditions used.
Protease activity
Another significant difference is the protease reported to
degrade MazE. In MG1655, it was shown indirectly by
transcriptional activation of mazEF loci, that Lon protease
degrades MazE (Christensen et al., 2003). Whereas, in
MC4100, it is reported that ClpAP degrades MazE
(Aizenman et al., 1996). In Staphylococcus aureus it was
shown that MazE is cleaved by ClpPC (Donegan et al., 2010).
At this point, studies indicate that MazE could be cleaved by
Lon, ClpAP or both depending on the condition. The number
and activity of proteases at any given time would affect the
turnover rate of the MazE which influences the promoter
activity and the neutralization of MazF. It can thus be said
that proteases have an indirect effect on the total amount of
free MazF in the cell, which in turn might induce lethal
effects upon encountering stress.
Why not in MG1655?
mazEF TA system is shown to be present and functional in
both MC4100 and MG1655 strains of E. coli. The genetic
architecture, regulation, endoribonuclease activity as well
as target sequence specificity of MazF have been reported
to be similar (Christensen et al., 2003; Munoz-Gomez
et al., 2004; Zhang et al., 2003). Yet, reproducing the PCD
phenomenon has not been a predominant success. Van
Melderen and co-workers strived to reproduce PCD in
MG1655 and MC4100 (procured from Engelberg-Kulka’s
group) strains of E. coli. However, they could neither
observe PCD nor was any benefit found in any of the
strains under the stresses examined (Tsilibaris et al., 2007).
It was shown that the E. coli strains MC4100, K38 and
W3110 strains exhibited PCD phenotype while MG1655
did not (Kolodkin-Gal & Engelberg-Kulka, 2008). This
prompted the authors to suggest that the latter strain is
deficient in one or more components essential for PCD
(for example EDF). It was explained that MG1655 may
have been selected against PCD.
It was found that serine hydroxymate induced amino acid
starvation caused a Lon-dependent activation of mazEF
transcription and also time-dependent RNA cleavage by
MazF in MG1655 strain (Christensen et al., 2003). This
indicates that the mazEF system is active in MG1655. Upon
checking EDF activity in different E. coli strains, it was found
that MG1655 showed least EDF activity. Since EDF augments
MazF activity by preventing formation of MazEF complex,
low CFU is expected upon addition of synthetic EDF.
 8 B. C. M. Ramisetty et al.
However, sensitivity of MG1655 to synthetic EDF was
significantly lesser as was represented as little decrease in
CFU after treatment (Kolodkin-Gal & Engelberg-Kulka,
2008). Hence, it is valid to conclude that MG1655 is deficient
in the production as well as is relatively resistant to exogenous
EDF.
Here, we attempt to address a possibility of mazEF
dysregulation in MC4100, based on some of the discrepancies
stated in the previous sections. Hypothetically, a cell would
contain high number of total toxins if there is an increase in
either total TA mRNA or turnover rate of antitoxin. Mutations
within the mazEF loci (promoter/operator region and ORFs)
and/or mutations in MazE cleaving protease would interfere
with the rate of transcription of mazEF loci and the turnover
rate of their products. This in turn would result in excess toxin
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Monash University on 07/01/13
proteins during steady state, which upon induction of stress
would lead to lethality.
Based on the aforementioned issues on the integrity of
relA-mazE-mazF-mazG operon, the activity and number of
proteases and the number of promoters, one qualified
speculation arises – is it PCD or hypersensitivity?
Hypersensitivity is apparently associated with relBE TA
system in a phenomenon called ‘‘delayed relaxed response.’’
In relB mutants, a type of hypersensitivity was reported upon
treatment with antibiotics (Christensen & Gerdes, 2004;
Diderichsen & Desmarez, 1980) and carbon starvation
(Mosteller, 1978). In relB101 mutants, a mutation in the
For personal use only.
relB leads to decreased half-life of RelB. It is conceivable that
a higher turnover rate of RelB causes inefficient repression by
the RelBE complex. This resulted in excessive transcription
from relBE loci leading to increased total RelE. Upon
induction of stress, the depletion of RelB would render
RelE free and would result in decreased CFU. We can ascribe
the so-called mazEF-mediated PCD to hypersensitivity
caused due to dysregulation of mazEF loci. This dysregula-
tion could be a result of increased number of promoters,
inadvertent genetic manipulations at the loci and/or hyper-
activity of protease ClpAP. Moreover, MG1655 and MC4100
strains also have other large scale differences including InDels
and SNPs (Ferenci et al., 2009; Peters et al., 2003). The
deletions fall into several groups like central intermediary
metabolism, carbon metabolism, amino acid biosynthesis etc.
Further it was shown that the basal ppGpp is higher in the
MC4100 strain relative to MG1655 (Spira et al., 2008). Based
on these observations it is also possible that the global
translation is affected resulting in alteration of mazEF TA
system regulation. Indeed, only complete genome sequencing
of the strains used might reveal genetic discrepancies
accounting for the PCD phenotype.
In this context, it is worth discussing the latest study
conducted with regard to mazEF-mediated PCD in the strain
MG1655. It was found that a ser/thr protein kinase YihE
prevented formation of ROS by inhibiting the mazEF system
(Dorsey-Oresto et al., 2013). The study observed a reduction
in CFU upon encountering stress in a DyihE strain. However,
CFU was similar to wild-type strain in a DyihEDmazEF
double mutant, when subject to the same stress. Therefore, if
this model whereby YihE inhibits MazF is true, there exists a
possibility that this line of regulation of mazEF system is
absent/minimal in the MC4100 strain.
Crit Rev Microbiol, Early Online: 1–12
Unanswered questions
What is the benefit of dying?
Ecological fitness is determined by two parameters, namely
competitive fitness and perseverance. The organism should be
able to survive through the stressful conditions of various
degrees and upon it should either outcompete or coexist with
other inhabitants. How can a phenomenon like PCD enhance
bacterial ecological fitness? One proposal is that the dead
cells would be scavenged by the small subset of survivors and
thus enhances the perseverance, referred to as ‘‘nutritional-
altruism.’’ However, for this hypothesized benefit to manifest
in reality, it would require that the dead cells lyse. But the fate
of the dead cells is inconclusive – are they lysed or remain as
ghosts? It is not yet known if cell lysis occurs in mazEF-
mediated PCD. This phenomenon was also observed when the
cells were infected with phages (Engelberg-Kulka et al.,
2005). It was reasoned that by committing suicide and
remaining as a ghost, spread of phage infection is inhibited.
This would contradict the hypothesis that dead cells could
provide nutrients under starved conditions, since formation of
ghosts indicates that PCD does not ensue in cell lysis. At this
point it is not clear as to how toxin-mediated RNA cleavage
would result in cell lysis. One of the death proteins SlyD,
synthesized as a consequence of MazF-mediated RNA
cleavage, is involved in cell lysis (Amitai et al., 2009). Cell
lysis was shown to occur due to another toxin PezT
(a pneumococcal zeta toxin), which was found to elicit
autolysis in E. coli due to its direct effects on cell wall
synthesis (Mutschler et al., 2011). However, conclusive
studies on the fate of the cell wall upon activation of
mazEF system are yet to be conducted.
The manifestation of mazEF-mediated PCD requires: (i)
exponentially growing cells, (ii) specific cell density and (iii)
specific degree of stress. The dramatic reduction in CFU upon
induction of stress is generally seen during exponentially
growing cells and not in stationary phase cells (Amitai et al.,
2009). Unlike in laboratory conditions, the cells are stressed
in their natural habitats. Hence PCD might not be as
predominant in natural habitats relative to other optimal
conditions. If PCD did occur frequently, the bacterial
population would be minimized. This drastic reduction in
population might also reduce the probability of propagation
compared to a strain with no mazEF system. Hence, PCD-like
phenomenon would not confer any competitive benefit. This
phenomenon is proposed to confer perseverance to the
population by scavenging of dead cell debris by the surviving
cells. Theoretically, this is possible if the stress is for a short
duration followed by favourable conditions for growth, which
is improbable in natural habitats. Moreover, in nature a given
species of bacteria do not usually grow in isolation. All the
microorganisms are in fierce competition for the limited
resources such as nutrients (Hibbing et al., 2010). The
nutrients released by altruism, as proposed in this case, can be
expected to benefit the competitors as well. Although it is said
that bacteria employ incompatibility strategies by which they
grow in patches (Budrene, 1985), since the surviving subset is
in dormancy; the competitors could be benefited more. This
argument is not to claim that PCD in bacteria is implausible
but to debate on nutritional-altruism.
 DOI: 10.3109/1040841X.2013.804030
There are certain other benefits apart from nutritional-
altruism that can be attributed to PCD. In some cases it
was postulated that PCD serves to eliminate the DNA
damaged cells to maintain a genetically healthy population
(Mortier-Barriere et al. 1998). The DNA released by the
dead cells upon lysis may help in the formation of the
biofilm matrix (Rice et al. 2007). It was also suggested
that mazEF-mediated PCD may help create channels that
can be used for exchange of nutrients and waste products
in biofilms (Erental et al., 2012). Since PCD is dependent
on cell density, it is also plausible that PCD occurs when
bacteria form multicellular structures and may not occur
when free living. The dead cells may provide physical
shielding of cells, in the core of multicellular-like struc-
tures, from stress.
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Function of TA systems: PCD or persistence?
Most bacteria with different life styles were shown to
harbour multiple TA systems on their genomes
(Anantharaman & Aravind, 2003; Pandey & Gerdes, 2005;
Ramage et al., 2009). Most of them are similar in regulation
and mode of action to that of mazEF system but only few
TA-mediated PCD are reported. One example of PCD is
phage abortive infection mediated through toxIN system
(Fineran et al., 2009). The istR-tisB, a Type III TA system of
E. coli, in which the toxin TisB is set free during SOS
For personal use only.
response, was found to cause killing through its effects in
the inner membrane of the cell. Live/death staining showed
the formation of ghosts. Although a reduction in translation
and transcription was observed, it was not a direct outcome
of the toxins’ action. No apparent benefit could be
ascertained to the presence of these genes during competi-
tion experiments (Unoson & Wagner, 2008). The pezTA
mediated inhibition of cell wall formation was also reported
(Mutschler et al., 2011). Many recent studies implicate TA
systems in persistor cell formation (Maisonneuve et al.,
2011; Tashiro et al., 2012). Persistence, as opposed to PCD,
is the ability of a cell to survive through severe stress, like
antibiotic stress. It was suggested that TA systems reduce
the translation machinery thus inducing metabolic stasis
rendering the cell more tolerant to antibiotics. Screening for
genes affecting persistence lead to the identification of four
mutants, which survived penicillin treatment of which two
were mapped to the hipA locus (Moyed & Bertrand, 1983).
One of them was cloned (Black et al., 1991) and it was later
shown that moderate expression of HipA, the toxin,
increased the levels of persistor cells (Falla & Chopra,
1998). These mutants showed increased persistence upon
exposure to high temperature and thymine starvation
(Scherrer & Moyed, 1988). A recent study shows that
hipBA TA system enhances the formation of biofilm (Zhao
et al., 2013). As previously mentioned, many TA systems
have been shown to increase persistence. Whether
TA systems are mediators of persistence or PCD is a
question that cannot be answered with the current set of
contradicting observations. Toxin-mediated RNA cleavage,
the type and extent of RNA cleavage and also the host cell
metabolic state may determine the consequences of TA
activation.
9
Perspectives
The mazEF-mediated PCD in bacteria has been enigmatic to
many researchers, ‘‘ma-ze’’ (Hebrew: what is this?). mazEF
TA system is the only Type II endoribonuclease TA system
that is implicated in PCD. Uncertainty arises as the
manifestation of PCD is dependent on strains of E. coli
used for study and ‘‘hands.’’ It is probably dependent on
meticulous experimental conditions. Here, we emphasize on
the need for further molecular and morphological validation
of mazEF-mediated PCD and propose reasons that could
account for the strain specificity of this phenomenon.
In eukaryotes, PCD is characterized by genome fragmen-
tation, loss of organelle integrity, membrane depolarization,
and so on. This phenomenon in prokaryotes does not have
proper nomenclature or methods for assessment. Although,
two studies reporting ALD describe death in bacteria with
biochemical and morphological validation (Belitsky et al.,
2011; Dwyer et al., 2012; Erental et al., 2012), it seems to us
that programming to die is not a good survival rationale that
could be adopted by free living unicellular bacteria. Death in
unicellular organisms is characterized by inhibition of
metabolism and/or loss of structural integrity. In bacteria,
many essential biological processes can be affected, which
might result in loss of CFU. On a simplistic note, the five
essential processes/structures for life to sustain are the
replication, transcriptional and translation machineries, cell
wall/membrane and the chromosome. The cell wall/mem-
brane provides protection from environment and helps
maintain optimal physiological conditions for all the bio-
chemical reactions to occur. The chromosome contains the
genetic information essential for the replenishment of all the
structural and regulatory components of the cell. The
metabolic machineries of replication, transcription and trans-
lation are the key to survival and propagation of the cell. The
loss of structural integrity of the chromosome or the cell wall/
membrane would result in cell death. The machineries for
replication, transcription and translation are replenishable
from the genetic information provided by the chromosome.
Indeed, replenishment of the metabolic machineries requires
that a minimal amount of the transcriptional and translational
machineries exist in the cell. However, the inhibition of these
machineries would result in an anabolic stasis, which may
proceed to exhaustion by catabolism and finally may result in
loss of structural integrity. The fate of a non-dividing
metabolically active cell is unclear. It is likely that the
metabolically active non-dividing cell may accumulate
harmful by-products, like ROS, resulting in harmful effects
on one or more critical components of the cell. Hence, we
presume that the prolonged stasis may also result in cell death.
Thus, prokaryotic programmed cell death can be character-
ized by irreparable damage to any of the five essential
components, caused by an intrinsic factor.
If MazF-mediated RNA cleavage has absolutely affected
either transcription or translation or indirectly caused cell
lysis or chromosome fragmentation it could be considered as
PCD. The MazF toxin inhibits translation (Christensen et al.,
2003) but does not affect transcription and replication. The
inhibition of translation is not absolute as protein synthesis
occurs, although at a low level (Amitai et al., 2009;
 10 B. C. M. Ramisetty et al.
Suzuki et al., 2005). There is no evidence supporting
degradation of chromosome or cell wall as a direct conse-
quence of MazF-mediated RNA cleavage. In recent studies of
PCD in bacteria, DNA fragmentation and/or condensation and
membrane depolarization were qualitatively analyzed (Dwyer
et al., 2012). Similar techniques should be employed in
characterization of mazEF-mediated PCD.
Although many molecular players involved in mazEF-
mediated PCD have been discovered, the evolutionarily
conserved minimal machinery required for PCD is uncertain.
To our understanding, genes mazEF, clpX, clpP, clpA, zwf,
and ygeO seem to be essential for mazEF-mediated PCD. relA
is probably dispensable as mazEF system can be activated by
inhibition of translation independent of ppGpp. Some of these
molecular players said to mediate PCD seem to have
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Monash University on 07/01/13
contradicting roles. For example, proteases ClpXP and
ClpAP are important ATP dependent cellular proteases that
degrade abnormally folded and/or tagged proteins (Gottesman
et al. 1998), and also involved in heat shock response and
other stresses (Baker and Sauer 2012, Porankiewicz et al.
1999). In our view, since ClpAP and ClpXP are responsible
for degradation of MazE and production of EDF respectively,
ClpP (the proteolytic subunit) is central to the occurrence of
mazEF mediated PCD in MC4100 strain. Recent studies on
ClpP have revealed several instances of ClpP hyperactivity.
ClpP is initially produced as a proenzyme containing 207
amino acids. It is then cleaved at the N-terminus to yield a
For personal use only.
193 amino acid mature form (Maurizi et al., 1990). An
important observation is the enhanced activity of ClpP when
treated with the antibiotic ADEP (acyldepsipeptide) (Kirstein
et al., 2009). Further analysis indicated a gating mechanism
that prevents non-specific cleavage by ClpP. This gating
mechanism, by which the activity of ClpP is controlled, is
mediated by a stretch of N-terminal residues. A mutant form
of ClpP in which 20 residues from N-terminus have been
deleted, increases its activity (Bewley et al., 2009). This
mutant ClpP is able to cleave large unfolded peptides whereas
the wild-type can act only on small peptides. It is known that
association of ClpP with the ATPases ClpX or ClpA imparts
different substrate specificities (Lee et al., 2010; Reid et al.,
2001). Put together, these observations conclude that differ-
ences in the primary structure of ClpP N-terminus alter the
specificity of the enzyme and hence it becomes hyperactive in
the absence of N-terminus. It is imperative to determine the
causes of excessive degradation of Zwf resulting in produc-
tion of EDF. BLAST analysis of ClpA, ClpX and ClpP
proteases shows primary structural differences to varying
degrees within different strains of E. coli. The amount and
specificities of these proteases may cause hyperactivation of
mazEF system as well as in EDF production. PCD hypothesis
fails to explain why ppGpp, a mediator of stringent response
required to tolerate amino acid starvation, also elicits a
PCD-like cell death (Gerdes et al., 2005). It is not yet clear if
the primary function of EDF is quorum sensing and whether it
is specific to the mazEF system. The contradictory roles of
EDF in accumulation of ROS (Amitai et al., 2009) and as
an antioxidant should be further analysed (Gao et al., 2010).
Improvising a screening technique for mazEF-induced
PCD would make it possible to isolate mutants deficient
in PCD.
Crit Rev Microbiol, Early Online: 1–12
Irrespective of the cellular organization of prokaryotes and
the mechanism of cell death, the advantages conferred by
PCD should be established. The altruism hypothesis should
hold well in the context of ecological advantage in a natural
habitat. A long-term competitive study with simulated natural
conditions, between mazEF mutant and wild type cells should
be performed. We can anticipate that these new
insights would account for the benefits of harbouring TA
systems to individual bacterial cells as well as populations.
Prior to pursuing TAs as antimicrobial drug targets
(Williams & Hergenrother, 2012), mazEF-mediated PCD
may need re-examination to rule out other possible
explanations.
Declaration of interest
The authors report no conflicts of interest.
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