Download Selfish genes, plasmids, phage: Altruistic bacteria

BIMM 122 – Lecture Notes #6B
Selfish genes, plasmids, phage; altruistic and selfish bacteria
Dr. Milton Saier
Programmed Cell Death (PCD): (1) aids development, (2) facilitates genetic exchange,
(3) eliminates defective cells, (4) may allow development of new types of antibiotics, (5)
can occur in response to growth inhibition, antibiotic treatment etc.
Addiction Modules: Programmed cell death and anti-death
Several types: (1) Proteic killer systems, (2) antisense RNA killer systems (usually block
translation), (3) holin-autolysin systems, (4) restriction-modification systems, and (5)
pore-antipore systems
Proteic Addiction Modules: Characteristics
2 genes: 2nd gene: a stable toxin (~120 aas)
1st gene: an unstable antitoxin (~80 aas)
Responsible for (1) death upon plasmid or prophage withdrawal and (2) programmed cell
death upon starvation (i.e., post-segregational killing, or stress-induced suicide).
1.
2.
3.
4.
5.
6.
One operon, 2 adjacent genes  2 small proteins.
The antitoxin gene is always upstream of the toxin gene.
The antitoxin inhibits the toxin by directly binding to it.
A specific ATP-dependent protease degrades the antitoxin.
Transcription is autoregulated by the antitoxin or antitoxin-toxin complex.
Some chromosomal addiction modules are activated by phage infection by
inactivating the antitoxin, thereby altruistically sacrificing the infected cell to
protect the population as a whole.
Example #1: CcdAB of plasmid F (a 95 kbp conjugative plasmid of E. coli)
1 copy of the plasmid is present per chromosome: It therefore needs postsegregational killing.
ccd: couples cell division; controls cell death
There are 2 other operons on plasmid F that encode additional post-segregational killing
systems. They use antisense RNA. Thus one plasmid/episome has three different selfish
gene cassettes.
Characteristics
1. The ccd operon is negatively controlled by CcdAB.
CcdAB bind to the DNA, overlapping the promoter.
CcdA is degraded by the Lon protease (multimeric; ATP-dependent);
degradation is slow (1/2 life, 20-30 min).
2.
CcdA binds CcdB directly, blocking its toxic activity.
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3. CcdB binds to gyrase: [(GyrA)2 (GyrB)2]. A mutant GyrA (R462C) is resistant to the
 DNA
 ATP
killing action of CcdB.
4. Resistant mutants are in GyrA or GroE (a chaperonin).
5. The last 3 aas of CcdB bind GyrA  reversible inhibition.
6. The CcdB-Gyr complex blocks passage of RNA (& DNA?) polymerases. A
cleaved Gyr-DNA complex is stabilized.
7. Crystal structure: CcdB is a dimer. It inserts into the cleft of GyrA.
Example #2: Phd-Doc (Prevents host death)-(Death on curing)
P1 phage can lysogenize E. coli  a low copy # plasmid (not integrated). It is lost with
 = 10-5/cell/generation. The plasmid is the prophage.
Both daughters get the plasmid due to (a) a partition system and (b) the addiction module.
Phd is destroyed by the ClpPA protease.
Example #3: MazEF: An E. coli chromosomal addiction module.
It is negatively controlled by ppGpp: activated by (a) the stringent response to amino
acid starvation (RelA-mediated) and (b) the stringent response to carbon starvation
(SpoT-mediated).
1. RelA is activated in response to amino acid starvation by free tRNAs.
2. SpoT is activated in response to carbon limitation; both make ppGpp.
3. Operon structure: p relA mazE mazF t
(MazEF are homologous to PemIK of plasmid R100)
4. Other addiction modules are encoded on the E. coli chromosome; they are
activated by other conditions.
5. MazE is inactivated by ClpAP.
6. Antibiotics that inhibit transcription (Rif) or translation (Chl) also activate mazEF
for death.
7. DNA damage can activate mazEF.
8. Thymine-less death is mediated by mazEF.
9. Toxins can activate mazEF.
Example #4: MqsRA: A toxin-antitoxin (TA) system in E. coli for programmed cell
arrest, biofilm formation and persister cell production (Wang, X. and T.K. Wood. Appl.
Environ. Microbiol. 77, 5577-5583 (2011). Toxin-antitoxin systems influence biofilm
and persister cell formation as well as the general stress response. Note: Persister cells are
a small fraction of a population that are arrested for growth and demonstrate resistance to
various antibiotics. They favor diversity in biofilms. MqsR is an interference RNase that
cleaves mRNAs at GCU sites. MqsA binds DNA sites via a Helix-Turn-Helix motif in its
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C-terminal domain while binding MqsR in its N-terminal domain. DNA binding directly
regulates mqsRA expression as well as expression of other stress related genes, one of
which is cspD encoding, a DNA replication inhibitor. MqsRA also regulates the General
Stress Regulatory (GSR) System by controlling expression of rpoS (σS), allowing cell
survival under conditions of starvation and stress. Thus TA systems help mediate stress
responses. There are 37 known TA systems in E. coli and 88 in Mycobacterium
tuberculosis. While appearing to be functionally redundant, each may allow responses to
differing but overlapping stresses via different mechanisms. Of the E. coli interference
RNases, MqsR cleaves mRNAs at GCU, MazF at ACA, YafQ at AAA and ChpB at
AC(A/G). Both non-degraded mRNAs and partially degraded mRNAs may be active.
Example #5: The bacterial peptide, TisB, is involved in persister cell formation in E.
coli. TisB and its analogs form multi-state ion-conductive pores in planar lipid bilayers
with all states displaying similar anionic selectivity. TisB analogs differing by ±1
elementary charges show corresponding changes in ion selectivity. Probing TisB pores
with poly-ethylene glycols revealed only restricted partitioning even for the smallest
polymers, suggesting that the pores are characterized by relatively small diameters.
Gurnev et al. (FEBS Letters, 586, 2529-2532, 2012) suggested that TisB forms clusters of
narrow pores that are essential for its mechanism of action in arresting growth and
promoting the formation of non-growing persister cells. TisB homologs are found in
several enterobacteria.
TisB is a toxic peptide of 29aas; overexpression causes cessation of growth and induces a
stress-response. A number of membrane protein genes are expressed, leading to cell
death. Part of the programmed response to DNA damage leads to increased accumulation
of TisB which slows or stops bacterial growth, probably allowing DNA repair before
cells continue to grow (Wagner and Unoson 2012).
TisB is part of the SOS-response regulon, controlled by LexA. The sRNA, IstR-1,
inhibits toxicity by sequestering the standby ribosome binding site for tisB; as the levels
of IstR-1 decrease, this site opens, and ribosomes are able to bind to initiate translation
further downstream. It is therefore a type 1 toxin/antitoxin (TA) system, where
expression of the proteinaceous toxin is controlled by an antisense sRNA.
Competition experiments between isogenic strains with or without the TisB/IstR-1 region
revealed that in the presence of DNA-damaging agents, deletion strains were
disadvantaged and were almost extinct by 4 days (Wagner and Unoson 2012).
Example #6: Antideath proteins
1. Phage  encodes an “Antideath” protein, RexB.
2. RexB inhibits ClpPA (specifically ClpP).
3. RexB prevents death by both Phd-Doc (Pl lysogenic phage loss) and MazEF
(starvation induced).
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4. RexB is one of the few genes expressed from the phage chromosome under
lysogenic conditions.
5. rexB is a “survival” gene for phage .
Example #7: Eukaryotes
Similar programmed cell death/anti-death systems are found in animals and their viruses
(i.e., Bcl-2 family proteins: “executioners and pardoners”).
Cowpox virus encodes in its genome an anti-death protein which blocks caspase-type
proteases that mediate apoptosis of animal cells.
Thus, the death modules of bacteria and their phage are similar to those of eukaryotes and
their viruses. The evolutionary precursor of programmed cell death in animals was
probably of prokaryotic origin.
Hanna Engelberg-Kulka1 and Gad Glaser2 (1999)
ADDICTION MODULES AND PROGRAMMED
CELL DEATH AND ANTIDEATH
IN BACTERIAL CULTURES
Ann Revi Microbiol
Vol. 53: 43-70
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ABSTRACT
In bacteria, programmed cell death is mediated through “addiction modules” consisting of two genes. The
product of the second gene is a stable toxin, whereas the product of the first is a labile antitoxin. Here we
extensively review what is known about those modules that are borne by one of a number of Escherichia
coli extrachromosomal elements and are responsible for the postsegregational killing effect. We focus on a
recently discovered chromosomally borne regulatable addiction module in E. coli that responds to
nutritional stress and also on an antideath gene of the E. coli bacteriophage λ. We consider the relation of
these two to programmed cell death and antideath in bacterial cultures. Finally, we discuss the similarities
between basic features of programmed cell death and antideath in both prokaryotes and eukaryotes and the
possibility that they share a common evolutionary origin.
Acronyms
Terms
For we are merely the leaf and the husk
The great death, contained in each of us,
That is the fruit around which everything revolves.
R. M. Rilke
The Book of Hours (1902)
INTRODUCTION
Programmed cell death, defined as an active process that results in cell suicide, is recognized as an essential
mechanism in multicellular organisms. Generally, programmed cell death is required for the elimination of
superfluous or potentially harmful cells (for reviews, see 49, 79). In eukaryotes, programmed cell death is
classically known as apoptosis (55), a term that originally defined the morphological changes that
characterize cell death. Today, the phrase “programmed cell death” has evolved to refer to any form of cell
death mediated by an intracellular death program, no matter what triggers it and whether or not it displays
all of the characteristic features of apoptosis (for review, see 49).
In bacteria, programmed cell death is mediated through a unique genetic system. It consists of a pair of
genes that specify for two components, a stable toxin and an unstable antitoxin that prevents the lethal
action of the toxin. Until recently, such genetic systems for bacterial programmed cell death have been
found mainly in Escherichia coli on low–copy-number plasmids and are responsible for what is called the
postsegregational killing effect; that is, they are responsible for the death of plasmid-free cells. When
bacteria lose the plasmid(s) (or other extrachromosomal elements), the cured cells are selectively killed
because the unstable antitoxin is degraded faster than the more stable toxin. Yarmolinsky and colleagues
have called such plasmid-borne pairs of genes “addiction modules,” because they cause the bacterial host to
be addicted to the continued presence of the “dispensable” genetic element (60, 121). Addiction modules
are responsible for the lethal consequences of plasmid withdrawal. Along with other very precise
mechanisms for preventing plasmid loss (replication control, plasmid partition, and resolution of
multimers) (for reviews, see 48, 80, 117), the stability of low–copy-number plasmids in the host bacterial
cells is maintained by the mechanism for killing plasmid-free bacteria provided by the addiction modules.
Two different classes of addiction modules have been identified in bacteria: (a) systems in which both
products, the stable toxins and the unstable antidotes, are proteins [named by Jensen & Gerdes proteic killer
gene systems (51)] and (b) systems in which, again, the stable toxin is a protein synthesized from a stable
mRNA but the antidotes are small unstable antisense RNA molecules (37). In cells harboring plasmids
bearing such addiction modules, the antisense RNAs prevent the translation of the stable toxin-encoding
mRNAs. However, in plasmid-free cells, the unstable antisense RNA is degraded, allowing the translation
of the toxins and the subsequent death of the plasmid-free cell (105).
Here we focus on proteic addiction modules. We do not review the addiction modules specifying for
antitoxins that are antisense RNAs (belonging to the hok/sok family) because they have recently been
extensively reviewed elsewhere (36). Among the proteic addiction modules we discuss are the best
characterized systems of extrachromosomal elements including ccdAB of plasmid F, kis/kid of plasmid R1,
pemI/K of plasmid R100, parDE of RK2/RP4, and phd-doc of prophage P1 (Table 1). This topic has been
partially reviewed by Jensen & Gerdes (51) and by Couturier and colleagues (20). Here we particularly
focus on a recently discovered regulatable addiction module located on the E. coli chromosome, on an
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antideath gene of bacteriophage λ, and on their relation to programmed cell death and antideath in bacterial
cultures. Finally, we discuss the similarities of programmed cell death and antideath in prokaryotes and
eukaryotes and the possibility that they share a common evolutionary origin
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.
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PROTEIC ADDICTION MODULES: Definition and General Properties
The best characterized proteic addiction modules have striking organizational and functional parallels.
These include (Figure 1, Table 1): (a) a proteic addiction module harbors two adjacent genes; (b) the
product of one is a long-lived and toxic protein, whereas the product of the second is a short-lived protein
that antagonizes the toxic effect of the first; (c) the antitoxic protein is encoded by the upstream gene in the
module; (d) the toxic and antitoxic proteins are coexpressed; (e) the antitoxic protein is synthesized in
excess; (f) the toxic and antitoxic proteins are small (toxic proteins are in the range of 100–130 amino
acids, and antitoxic proteins are in the range of 70–85 amino acids); (g) the toxic and antitoxic proteins
interact; (h) the antitoxic protein is degraded by a specific bacterial protease; and (i) the addiction module is
autoregulated at the level of transcription either by a complex formed between the toxin and antitoxin or by
the antitoxin alone. Located on various extrachromosomal elements or on the E. coli chromosome, the
proteic addiction modules are quite similar in genetic structure and function; however, they rarely share
sequence homology. In addition, these addiction modules also differ in the natures of their toxic and
antitoxic proteins, in the bacterial protease that degrades the antitoxic protein, and in the cellular targets of
the toxic proteins.
Figure1 Schematic illustration of the general characteristics of proteic “addiction modules” and the fate of
their products. Addiction modules consist of two adjacent genes that are coexpressed. These two genes
specify for a stable toxic protein () and for a labile antitoxic protein ( ). In each genetic module, the
upstream gene specifies for the antitoxin and the downstream gene specifies for the toxin. (A) Under
conditions of continuous expression of the addiction module. Both products are synthesized. The antitoxins
form complexes with the toxins, thereby neutralizing them to prevent cell killing. In all known cases, the
addiction module is negatively autoregulated by the toxin-antitoxin complex at the level of transcription.
(B) Under conditions in which expression of the addiction module is prevented. For a plasmid-borne
module, this can occur by the loss of the plasmid itself and hence of the module; for a chromosomal
addiction system, this can occur by the action of the regulatory element affecting its expression. The toxin
and antitoxin molecules, synthesized before their de novo synthesis was prevented, have a different fate:
the antitoxins are degraded by specific proteases, leaving the toxins free to cause cell death.
_____________________
The ccdAB Addiction Module of Plasmid F
The first system in which a genetic element was found to be responsible for killing plasmid-free segregants
was the ccd locus of plasmid F (50, 81). The 95-kb conjugated plasmid F has a very low copy number and
is found in the cell at about one copy per chromosome. Originally, ccd stood for couples cell division, that
is, a system coupling cell division to plasmid proliferation, thereby acting as a plasmid rescue system (73,
75, 81). Today, ccd stands for control cell death (50). Of all the proteic addiction modules, the ccd locus
has been the one most studied, and it can be considered as a paradigm. The ccd locus consists of two genes,
ccdA and ccdB (also known as H and G or letA and letB), which encode the 72-amino-acid-long 8.7-kDa
protein CcdA and the 101-amino-acid-long 11.7-kDa protein CcdB (14, 75). These two proteins are
involved in the toxic-antitoxic mechanism that enables plasmid F to be maintained stably in the cell; CcdB
is toxic to the cell, and CcdA is CcdB's unstable antidote (54, 73, 75). The 41 carboxy-terminal residues of
CcdA are sufficient for its antitoxic activity (11). The F plasmid contains two additional operons, srnB
(stabile RNA degradation) (3) and flm (F leading maintenance) (62), which function independently as
postsegregational killing systems. These two killing systems are addiction modules in which the antidotes
are antisense RNAs (36). When present on an intact F plasmid, ccdAB plays a relatively minor role in
postsegregational killing; however, when present on a mini-F plasmid or when cloned with a heterologous
replicon, the presence of ccdAB results in the killing of >90% of the plasmid-free segregants (50, 73, 75).
In the absence of CcdA, the production of the CcdB protein causes cell filamentation, induction of the SOS
pathway, and, ultimately, cell death (50, 54, 75). Similar responses were observed when the synchronous
loss of a ccdAB-bearing plasmid was induced (77, 100). The ccd-induced SOS response, but not cell
killing, requires the presence of the host enzymes RecA and RecBC (6). CcdA probably prevents the lethal
action of CcdB by binding to it, thus forming a tight complex (104).
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Studies on the ccd operon revealed the finding that it is negatively autoregulated at the level of transcription
by a complex of the antitoxic and the toxic proteins. This has become one of the characteristics of the
proteic addiction module (Table 1). Both CcdA and CcdB proteins are required for repression and binding
to the operator(s) in the ccd promoter (24, 103, 104). CcdA and CcdB bind at several sites spaced over 113
bp overlapping the ccd promoter (24, 93, 103, 104).
In bacteria that have lost the F plasmid, it has been proposed that cell death is brought about by the
differential loss of activity of the CcdA and CcdB proteins. Because the active half-life of CcdA is shorter
than that of CcdB, in newborn plasmid-free bacteria the persistence of the toxic CcdB protein would lead to
cell death (50). Later it was confirmed that cell killing in bacteria which have lost the F plasmid is indeed
based on the relative instability of the CcdA protein, which has been shown both in vivo and in vitro to be
degraded by the E. coli Lon protease and has a shorter lifetime than does CcdB (115, 116). Lon is a
multimer of identical subunits that represents a major class of ATP-dependent proteases in which the
ATPase domain and proteolytic domain are encoded within a single polypeptide (40). CcdB, the toxic
partner, prevents degradation of CcdA by Lon. Since CcdB also inhibits the ability of CcdA to enhance the
ATPase activity of Lon, it may be that Lon recognizes protein-bonding domains that become exposed when
their partner is absent (116). Lon-dependent degradation of CcdA is relatively slow, like the degradation of
the antitoxic proteins of other addiction modules by their respective proteases (115).
The best characterized cellular target of the toxic component of an addiction module is that of the ccd
system of plasmid F; this target consists of the A subunits of the E. coli DNA gyrase (GyrA). This was first
shown by genetic analysis of E. coli mutants resistant to the killing effect of CcdB (12, 74). Bernard &
Couturier (12) found that in seven independent isolates the mutation mapped in the gyrA gene. Sequencing
one of these GyrA mutants revealed an amino acid substitution of Arg462 to Cys. The fact that all of the
independently isolated CcdB-resistant mutants all map to gyrA is strong evidence that gyrA is the target of
the CcdB protein. In that same study, in a merodiploid strain, the CcdB-sensitive phenotype was found to
be dominant over the resistant phenotype. This dominance of sensitivity over resistance has also been
observed with quinolone drugs (45, 70) and indicated that rather than being a simple inhibitor of gyrase
function (12), CcdB may poison wild-type gyrase. In a separate study, Miki and colleagues (74) isolated
nine CcdB-resistant mutants and showed that three of them map in gyrA and the other six map to the groE
genes. This suggested that the GroES chaperone may be involved in the interaction between CcdB and
DNA gyrase or in CcdB folding. More recently, other E. coli mutants have been isolated that can survive
low concentrations of CcdB (78). The relation of the mutated genes to DNA gyrase, CcdB toxicity, or both
is not yet clear.
That the GyrA subunit of the E. coli DNA gyrase is the target of the CcdB protein has also been revealed
by biochemical studies (12, 13, 66, 67). The E. coli DNA gyrase is a tetramer formed by the association of
two GyrA and two GyrB subunits (A2B2). This tetramer catalyzes negative supercoiling at the expense of
ATP hydrolysis (35). The GyrB subunits are responsible for ATP binding and hydrolysis. The GyrA
subunits form the catalytic core of the enzyme that enables the DNA breaking-rejoining reaction, that is,
the introduction of a transient double-strand break in the DNA, the passage of another piece of DNA
through the break, and the annealing of the double strands (10, 88). The intermediates of this breakingrejoining reaction are called “cleaved complexes.” Maki and colleagues (67) studied the supercoiling
activity of DNA gyrase and have shown that in cells overproducing CcdB both the free form of the GyrA
subunit and the tetrameric form A2B2 of DNA gyrase are inactivated. This inactivation seems to be caused
by CcdB protein binding to GyrA. Furthermore, protein CcdA is able to fully reactivate the inactivated
gyrase or the GyrA subunit. Since protein CcdB and quinolone antibiotics seemed to poison DNA gyrase
similarly, Bernard & Couturier (12) measured DNA gyrase cleavage of plasmid DNA in CcdBoverproducing cells. They found that in such cells, plasmid DNA was only partially cleaved. As in the case
of quinolone drugs, the cleavage by DNA gyrase in CcdB-overproducing cells was observed only when the
cells were treated with the strong protein denaturant SDS. Furthermore, overproduction of the CcdB protein
in a gyrA462 strain that tolerates the CcdB killing effect did not lead to CcdB-induced DNA cleavage.
Moreover, in in vitro studies, they observed that purified CcdB, like quinolone antibiotics, induces DNA
cleavage by DNA gyrase and furthermore that CcdA reverses this effect (13). While all these findings
support the notion that GyrA protein is the target of CcdB, they do not clarify the mechanism(s) of CcdB
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action on gyrase. In particular, one question remains: under biological physiological conditions, what is the
primary cause of cell killing by CcdB?
As described above, until recently, CcdB had been reported to act on DNA gyrase in two distinct modes.
According to one, CcdB inactivates DNA gyrase by forming a CcdB-DNA gyrase complex, leading to the
relaxation of supercoiled DNA (66, 67). According to the other, like quinolone drugs, CcdB poisons gyrase
by freezing an intermediate step in the breaking-rejoining reaction, which results in double-stranded DNA
cleavage in the presence of SDS (12, 13). Recently, in more detailed studies of CcdB-induced DNA
cleavage, purified CcdB was shown not to affect supercoiling (95). In fact, most CcdB-induced cleavage
occurred after many cycles of ATP-driven breakage and reunion when the DNA had become highly
supercoiled (95). Furthermore, CcdB was found to stabilize a cleaved complex of DNA gyrase and DNA
but not in the same manner as do the quinolone drugs (21, 95). For example, it was shown (21) that,
although quinolone drugs can induce cleavage in relatively short DNA molecules, DNA cleavage by CcdB
requires a DNA molecule at least 160 bp long. They also found that when linear DNA is the substrate,
CcdB cleavage of DNA requires ATP hydrolysis. This requirement for ATP hydrolysis suggested the
involvement of a strand passage event, so they proposed that CcdB and quinolones affect different
intermediate steps of the cleaved complexes; while quinolone drugs can trap a cleaved complex without the
involvement of strand passage, CcdB traps a post–strand-passage intermediate. On the other hand, like the
gyrase-quinolone-DNA complex (118), the CcdB-gyrase-DNA complex can also inhibit the passage of
RNA polymerases (21). This was shown by using an in vitro transcription assay in which the CcdB-gyraseDNA complex was found to block the transcription of the T7 polymerase. An important feature of this
process is the finding that it also requires ATP. The fact that CcdA, the antidote of CcdB, prevented CcdBinduced blocking of RNA polymerase further suggests that these in vitro results are a correct reflection of
at least part of CcdB action in vivo.
Based on the results of earlier genetic and biochemical studies, it appears that a crucial role in the CcdBGyrA interaction is played by GyrA Arg462 and by the last three amino acids of the CcdB C-terminus. The
crystal structure of a large fragment of GyrA revealed that Arg462 points into the central hole of the GyrA
dimer (76), suggesting that CcdB binds into this hole. Recently, the crystal structure of CcdB has been
determined, confirming that CcdB also exists in dimer form (63). However, based on the crystal structure
of GyrA (76), the diameter of the central hole of the GyrA dimer is a little too small; to accommodate the
CcdB dimer, the GyrA dimer must open up to some degree. To solve this problem, Couturier and
colleagues (20) proposed two possible mechanisms: either (a) CcdB interacts with GyrA before the GyrA
dimer is formed or (b) CcdB interacts with GyrA when gyrase is cycling on the DNA. When the crystal
structure of the CcdB-GyrA complex is elucidated, we shall have a better understanding of the mode of
action of CcdB.
It is not yet clear which of CcdB's biological effects is the primary cause of its cytotoxic effect under
physiological conditions in vivo. As discussed above, three distinct phenomena have been described as
related to the action of CcdB on DNA gyrase: the relaxation of negatively supercoiled DNA, DNA
cleavage, and interference with the passage of RNA polymerases along the DNA. Based on their
observation that plasmid DNA in CcdB-overproducing cells is extensively relaxed, Maki and colleagues
(66, 67) suggested that in vivo CcdB modulates the supercoiling activity of DNA gyrase. Because CcdB
was overexpressed in these experiments, it represented 20% of the total cell protein. Because, under normal
in vivo conditions, such high concentrations of CcdB are unlikely, it is possible that normal physiological
levels of CcdB have little effect on the cellular levels of supercoiling. In addition, it is well known that
bacterial cells are also able to control cellular supercoiling levels by altering the expression of gyrase and
topoisomerase I (71, 109). Thus, even if CcdB inhibits in vivo supercoiling by binding GyrA, bacteria may
be able to compensate for small changes in the level of supercoiling by increasing the expression of the
gyrA and gyrB genes.
Recall that DNA cleavage by CcdB was observed only when the strong protein-denaturing agent SDS was
added to the medium. Thus it is also questionable whether DNA cleavage, the second phenomenon reported
to be related to the mode of action of CcdB and GyrA, is responsible for CcdB-mediated cell killing in vivo
(12, 13). However, it does seem that the additional ability of the CcdB-gyrase complex on DNA to form a
barrier for the passage of RNA polymerase and possibly of DNA polymerase (21) may have implications
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for the bactericidal action of CcdB protein. It has been shown in vivo that CcdB can indeed inhibit DNA
replication (50) and can also induce cell filamentation (75) and the formation of anucleate cells (45). Thus,
CcdB may kill bacteria that have lost the F plasmid by trapping the DNA gyrase that is bound to the DNA,
thus blocking the passage of polymerases.
In summary, several processes have been identified for the involvement of each of the toxic and antitoxic
proteins of the ccd system. CcdB is involved in three processes: (a) it poisons the DNA gyrase complex, (b)
it interacts with CcdA, and (c) it represses its own synthesis and that of CcdA by forming a CcdA-CcdB
repressor complex that binds to the ccd promoter-operator. On the other hand, through their interaction,
CcdA inactivates the toxic CcdB. In addition, CcdA is a substrate for the E. coli protease Lon. The domains
involved in each process and the structure-function analysis of CcdA and CcdB have yet to be clarified.
Based on mutational analysis, it appears that the last three amino acids of CcdB play a key role in the
poisoning process but are not involved in its autoregulation (5). It has also been shown that a truncated
CcdA protein retaining only its 41 C-terminal residues loses its autoregulatory activity but retains its
antitoxic activity (11, 93). Thus, it seems that the autoregulatory activities of CcdB and CcdA reside in
their N-terminal regions, while the toxic and antitoxic activities, respectively, reside in their C-terminal
regions (Table 2).
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Figure2 A model for the E. coli rel maszEF-mediated cell death (A) and the anti-death effect of λRexB (B).
(A) Under conditions of nutritional starvation, the level of ppGpp increases. During amino acid starvation,
this is achieved by the interaction of the tRNA with the product of relA (18). ppGpp inhibits the
coexpression of mazE and mazF. MazF is a long-lived toxic protein, whereas MazE is an antitoxic labile
protein that is degraded by the ClpPA protease. Therefore, when the cellular level of ppGpp is increased,
the concentration of MazE is decreased more rapidly than that of MazF, and thereby MazF can exert its
toxic effect and cause cell death (2). (B) λRexB antagonizes the ClpP family of proteases. As a result, it
inhibits the degradation of the antitoxic protein MazE and thereby prevents cell death (30).
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