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Journal of Bacteriology, December 1998, p. 6306-6315, Vol. 180, No. 23
State Scientific Research Institute of
Genetics and Selection of Industrial Microorganisms, Moscow 113545, Russia,1 and
Institut Jacques Monod
(Centre National de la Recherche Scientifique, Université
Paris 7), 75251 Paris Cedex 05, France2
Received 21 July 1997/Accepted 1 October 1998
Two Escherichia coli genes, expressed from multicopy
plasmids, are shown to cause partial induction of prophage Temperate bacteriophages possess a
dual mode of existence, able either to lyse sensitive cells or
to form stable lysogens in which the phage genome is propagated as
prophage by the bacterium, with phage lytic functions repressed by one
or several phage repressors (28). For this reason, temperate
phages have been attractive models for studying the mechanisms
governing biological decisions: the choice between lysis and lysogeny
after infection of a sensitive host and the choice of the established
lysogen at each generation to continue propagating the prophage or to
enter the lytic cycle.
The demonstration that, for certain lysogens, the vast majority of the
population could be induced to enter the lytic cycle by UV irradiation
was a major discovery, dispelling lingering doubts about the reality of
the phenomenon of lysogeny (19, 20). Since that time, the
combined efforts of many researchers over several decades have
established the mechanism of lysogenic induction by UV irradiation and
other DNA-damaging treatments. Under these conditions, the SOS response
is induced; the RecA protein, activated by single-stranded DNA formed
near DNA lesions, promotes autoproteolytic cleavage of certain prophage
repressors as well as of the bacterial SOS repressor LexA (18, 28,
38, 39). The SOS response and SOS-inducible temperate phages have been described in a number of bacteria, including such distantly related species as Escherichia coli and Bacillus
subtilis. In those cases tested, the SOS response is also the
major pathway of spontaneous induction, which, under laboratory
conditions at least, is severely reduced in recA mutant
lysogens (28).
It has often been suggested that lysogenic induction via the SOS
response is a selective mechanism permitting the phage to use its
host's detection system for DNA damage in order to leave a severely
damaged cell which is likely to die, like a rat leaving a
sinking ship. If so, there may well be other conditions in which the phage, using other host detection mechanisms to sense imminent cell
death, would similarly choose to enter the lytic pathway. Furthermore,
many temperate phages are not SOS inducible yet still exhibit
spontaneous induction, even in recA hosts. Well-known examples in E. coli are Mu and P2; host functions involved
in their induction have not been identified.
For E. coli, a large number of stress responses have been
described, including those permitting the cell to sense changes in
temperature, osmolarity, pH, or nutrient availability and adjust its
pattern of gene expression appropriately. In theory at least, it is
possible that for each stress response there is a set of phages tuned
in to the cell's detectors in such a way that, if cell survival seems
threatened, the phage is induced.
In the present work we looked for E. coli genes, the
amplification of which leads to RecA-independent induction of prophage Bacterial strains and bacteriophages.
The bacterial
strains used in this study, all derivatives of E. coli K-12,
are listed in Table 1, together with the
bacteriophages. Standard techniques were used for growth of bacteria
and bacteriophages, phage crosses, generalized transduction with
P1vir, and lysogenization (23, 30). All
recA strains were checked for UV sensitivity and lack of
growth of
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
RecA-Independent Pathways of Lambdoid Prophage
Induction in Escherichia coli
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
in
recA mutant lysogens. One is rcsA, which
specifies a positive transcriptional regulator of the cps
genes, which are involved in capsular polysaccharide synthesis. The
other is dsrA, which specifies an 85-nucleotide RNA that
relieves repression of the rcsA gene by histone-like protein H-NS. Genetic contexts known to increase Cps expression also
cause RecA-independent induction: the rcsC137 mutation, which causes constitutive Cps expression, and the lon and
rcsA3 mutations, which stabilize RcsA. Lambdoid phages 21, 
80, and 434 are also induced by RcsA and DsrA overexpression in
recA lysogens. Excess cI repressor specifically blocks
induction, suggesting that induction involves repressor
inactivation rather than repressor bypass. RcsA-mediated induction
requires RcsB, the known effector of the cps operon,
whereas DsrA-mediated induction is RcsB independent in stationary
phase, pointing to the existence of yet another RecA-independent
pathway of prophage induction.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
. The selection used produced clones of the host genes
rcsA and dsrA, which when overexpressed cause
induction of and other lambdoid prophages in recA
lysogens. Both genes code for positive regulators of capsular
polysaccharide synthesis. Other genetic alterations leading to capsular
polysaccharide overproduction also cause RecA-independent induction. From complementation studies, we conclude that DsrA
participates in prophage induction via two different mechanisms.
Results obtained for the induction of cIind
mutants suggest several possible mechanisms.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
bio10 (or imm434
bio10) phage, as described elsewhere (42). The
rcsC137 and lon strains were mucoid on
Luria-Bertani (LB) plates at 37°C and 30°C, respectively. The
rcsA3 mutant formed mucoid colonies at 30°C on M9 plates
but not on LB plates.
TABLE 1.
Bacterial strains and bacteriophages
Media and chemicals.
Permanent bacterial stocks were stored
in 20% glycerol at 
70°C; working stocks were maintained on LB agar
at 4°C for up to 2 weeks. Except where otherwise stated, cells were
grown in LB medium (23). M9 (23) and GT
(24) plates were as described previously. Solid media were
supplemented with 1.5% agar (Difco). Soft agar contained
0.2% MgSO4 with 0.8% agar. Eosin-methylene blue (EMB)
galactose plates (23) were used to screen for
Gal+ colonies. Plates containing 50 µg of
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal) per ml and 50 µg of
isopropyl--D-thiogalactopyranoside (IPTG) per ml were
used to distinguish between Lac+ and Lac
colonies. Transducing phages were grown in NZCYM medium, as
described by Sambrook et al. (29). Bacterial strains
carrying the mini-Mu element pEG5005 and helper phage
Mucts62 were induced in LB medium with CaCl2 (20 mg/ml) and MgSO4 (10 mg/ml), as described by Groisman and
Casadaban (14). The following antibiotics were added, when needed, at the indicated concentration: ampicillin (Ap), 50 µg/ml (to
maintain plasmids) or 20 µg/ml (to measure infective centers); chloramphenicol (Cm), 20 µg/ml; kanamycin (Km), 10 µg/ml;
streptomycin (Sm), 10 µg/ml; and tetracycline (Tc), 10 µg/ml.
Dilutions of cells and phages were in 0.8% saline.
Plasmid construction.
The plasmids used in this work are
listed in Table 2. Isolation of plasmid
and phage DNA and routine nucleic acid manipulations were as described
previously (29). pUC19- and pGEM-7zf()-derived plasmids
were constructed by restriction-enzyme digestion of the transducing
phage and plasmid DNA followed by ligation with T4 DNA ligase. pDR406
and pDR416 were prepared in vivo after heat induction of strain C600,
which carries both the mini-Mu element pEG5005 (Kmr) and a
Mucts62 prophage, as described elsewhere (14).
The resulting mixed phage lysate was used to transduce strain MT2 to
Kmr on EMB galactose indicator plates containing Km to
screen Gal+ colonies.
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Apr
mucoid transformant of XL1-blue was isolated, and plasmid DNA was
extracted. pDR500 had the expected enzyme restriction pattern (Fig.
1A). pDR501 was constructed by insertion
of 
Tn903 from pUC4K (37), cut with
HincII, into the EcoRV site of pDR500.
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3B3 from the Kohara collection (miniset 343) and from pUC19
were digested with EcoRI and PstI, mixed, and
ligated. A Lac Apr mucoid transformant of
XL1-blue was isolated, and the plasmid was extracted.
Restriction-enzyme and hybridization analysis confirmed the structure
suggested by the genetics (Fig. 1B). Plasmids pDR200 (rcsA+) and pDR300 (dsrA+
dsrB+) were constructed by subcloning in pUC19
isolated HincII and HincII-EcoRI
fragments, respectively, from pDR100. It is known that E. coli K-12 strains become mucoid when they carry several copies of
the rcsA gene (36). pDR300
(dsrA+ dsrB+), like pDR200
(rcsA+), increased capsular polysaccharide
synthesis, and the transformants formed slime colonies, but the pDR300
(dsrA+ dsrB+)-mediated effect was
less pronounced and depended on the strain. We used C600 to select
mucoid transformants. pDR200 (rcsA+) and pDR300
(dsrA+ dsrB+) had the expected
0.6-kb and 1.1-kb fragments, respectively. It should be noted that
pDR300, in addition to the dsrA gene, also carries the
adjacent dsrB gene. Both code for small RNAs, as described
by Sledgjeski and Gottesman (31). The restriction map for
the region containing rcsA, dsrA, and
dsrB is shown in Fig. 1B; it is similar to that published
earlier (17, 31, 34). Plasmid rcsA and
dsrA genes were disrupted in vitro by inserting the
HincII fragment from pUC4K, containing Tn903,
into pDR200 (rcsA+) cut with EcoRV
and pDR300 (dsrA+ dsrB+) cut
with Bsu36I and blunted with Klenow polymerase. Nonmucoid Apr Kmr transformants of C600 were selected,
and their plasmid DNA was extracted. The resulting plasmids, pDR201
(rcsA::Tn903) and pDR301 (dsrA::Tn903 dsrB+),
had the expected restriction-enzyme cleavage patterns (Fig. 1B).
To define the physical limits of the dsrA+ and
dsrB+ regions that confer the phenotype
observed, we cloned the isolated 1.1-kb HincII-EcoRI fragment of pDR100 in pGEM-7zf()
digested with SmaI and EcoRI. The resulting
plasmid, pDR12, was digested to completion with SphI or
NsiI as demonstrated by electrophoresis. Then pDR12 was
digested again with XbaI or HindIII, and DNA
deletion with exonuclease III was carried out as described in the
Erase-a-Base system from Promega Biotec. The endpoints of the deletions
were determined by sizing the linearized DNAs on agarose gels.
To clone the cI gene, + DNA was digested
with BglII and mixed with pUC19 digested with
BamHI. Lac Apr transformants of
XL1-blue were checked for lambda immunity. Restriction analysis
confirmed the genetic conclusion; plasmid pDR1 carries the fragment of
DNA from 35,711 to 38,103 kb (5) (Fig.
2).
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Tn903) cut with
ScaI and into the bla gene of pDR300
(dsrA+ dsrB+), and pDR301
(dsrA::Tn903
dsrB+) was partially digested with the same enzyme
because of the presence of a second ScaI site outside
of the bla gene. Restriction analysis of the selected
plasmid confirmed insertion of the cat cassette in the
bla gene.
Transfer of mutation from plasmid to chromosome.
The
insertion mutation constructed in plasmid pDR301 was transferred to the
chromosome as described by Parker and Marinus (26). Plasmid
pDR301 (dsrA::Tn903
dsrB+) with a disrupted dsrA gene was
introduced into C600 Hfr, and the resulting strain was used as a donor
in conjugation experiments with N99 as the recipient. Smr
Kmr colonies were checked for sensitivity to ampicillin. P1
transduction confirmed that the
dsrA::Tn903 allele was linked to
hisG::Tn10 (3% cotransduction) as
expected for insertions in the rcsA dsrA region.
Halo test and quantitative measurement of prophage induction. (i) Halo test. Lysogenic bacteria, grown overnight in LB medium at 30°C from single colonies on LB plates, were spotted on a lawn of the indicator strain LE392 in soft agar, and the plates were incubated overnight at 30°C. Herein, the term induction refers to appearance of a halo of lysis or of plaques within the spot.
(ii) Quantification of induction efficiency.
The efficiency
of lysogenic induction was determined by calculating the ratio of
infective centers to viable lysogens. Exponentially growing lysogens, washed to eliminate free phage, were mixed with indicator
strain B7218 in soft agar and plated onto GT plates containing 20 µg
of ampicillin/ml, as described by Moreau et al. (24). Plates
were incubated overnight at 37°C. Ampicillin prevents uninduced
bacteria from multiplying and releasing phage during overnight
incubations but does not prevent induced lysogens from forming
infective centers (25). This technique was used with Aps lysogens.
receptor).
The cultures, washed to eliminate free phage, were diluted 50- to 100-fold in the same medium and incubated at 30°C with aeration. At
various times, samples were assayed for total viable cells and the
optical density at 590 nm was measured. Several drops of chloroform
were then added, and the free phages were mixed with LE392 indicator
bacteria in soft agar and plated on LB plates. Plates were incubated
overnight at 37°C.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Screening for clones of E. coli DNA that cause
RecA-independent induction.
To screen for genes which, when
overexpressed, can induce prophage, we took advantage of the
characteristics of the MT strains constructed by Toman et al.
(35). These strains carry a defective prophage with the
N, cI, and cro genes. The
gal operon is fused to the cro gene (and the
normal gal promoter is deleted). When cI repressor is
expressed, the strains are Gal. If cI repressor is
removed, the cro-gal operon is transcribed. The Cro protein
then represses the cI gene and prevents turning off of
cro-gal, so the strain becomes locked in a Gal+
configuration. The strains thus guard a "memory" of a past
induction event. We used strain MT2, which carries a recA
mutation and normally displays a much lower frequency of
Gal+ colonies, <106, than the frequency,
103 to 104, in the
recA+ strain MT1.
precA phage
DNA. Four plasmids carried the recA gene. The other two
plasmids, pDR406 and pDR416, were reintroduced into MT2 and also into a
B6905() (recA) lysogen. Both plasmids caused induction, as judged by the appearance of Gal+ clones in
MT2 and by the formation of a halo of lysis when B6905() (recA) transformants were spotted on a sensitive lawn.
Excess RcsA or DsrA causes RecA-independent induction of .
The two plasmids, pDR406 and pDR416, in addition to inducing in
strains MT2 and B6905() (recA), conferred a mucoid
phenotype. Furthermore, in MT2 the mucoid phenotype was restricted to
the Gal+ colonies, presumably reflecting the requirement
for GalE enzyme to synthesize UDP-Gal, a precursor of colanic acid
(21). Since this made scoring of MT2 difficult, we tested
induction by monitoring the formation of a halo of lysis.
3B3, which carries the rcsA gene. Both plasmids carried the rcsA region. Furthermore, the restriction map of the
cloned inserts accorded with that reported by Kohara et al.
(17) for the region from 2,030 to 2,040 kb (Fig. 1). The
2.0-kb PstI fragment from pDR416 containing rcsA
and dsrA was subcloned into pUC19. The resulting
plasmid, pDR500, caused mucoidy and induced prophage in B6905()
(recA) cells. When the rcsA gene of
pDR500 was interrupted by Tn903 (Fig. 1A; see
Materials and Methods), the resulting plasmid, pDR501, caused mucoidy
and induced . However, this plasmid carries the dsrA
gene, overexpression of which can derepress chromosomal rcsA
expression (reference 31; see below).
To determine conclusively the role of RcsA and DsrA in the induction
observed, we recloned the rcsA and dsrA genes
from Kohara phage 3B3, making plasmids pDR200
(rcsA+) and pDR300
(dsrA+) (Fig. 1B). Both plasmids caused mucoidy
and induced prophage in B6905() (recA) cells.
Inactivation of rcsA (plasmid pDR201; rcsA::Tn903) or of dsrA
(plasmid pDR301; dsrA::Tn903)
abolished both phenotypes. The dsrB gene, of unknown
function, is present on the dsrA+ and
dsrA::Tn903 plasmids but does
not induce . Furthermore, it is not required for DsrA-mediated
induction: reducing the size of the insert in plasmid pDR12 by the
Erase-a-Base system showed that the dsrA gene alone was
sufficient for RecA-independent induction (Fig.
3).
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in a recA lysogen.
We verified that the induced phage was indeed and not some hitherto
unknown prophage. First, the induced phage was unable to grow on a lysogen. Second, transformants of the B6905 (recA) nonlysogen with plasmids pDR200 (rcsA+) and
pDR300 (dsrA+) produced no lysis zone on a lawn
of the indicator strain.
Effect of RcsA or DsrA overproduction on other lambdoid
phages.
To determine whether the induction was strain or phage
specific, we analyzed the effect of RcsA and DsrA overproduction on another recA mutant lysogenic for various lambdoid phages.
Plasmids pDR200 (rcsA+) and pDR300
(dsrA+) were introduced into four lysogens of
the recA strain B7244, and the ability of the resulting
strains to produce halos on a sensitive lawn was observed. Lysogens
having phages , 21, 80, and 434 and both plasmids produced halos,
but those without any plasmid did not. We conclude that overproduction
of either RcsA or DsrA causes RecA-independent induction of all
four of these phages and that induction is not strain specific.
Quantification of RecA-independent induction by RcsA or DsrA
overproduction.
RcsA is known to be a transcriptional activator of
the cps genes, which are involved in the synthesis of
colanic acid, the capsular polysaccharide of E. coli. RcsA
is normally limiting for the transcription of these genes, and its
overexpression results in a mucoid phenotype (10, 12, 13,
34). The closely linked dsrA gene codes for a small,
85-nucleotide RNA molecule which, when overexpressed, derepresses
rcsA transcription (overcoming repression by H-NS)
(31). In addition to RcsA, the Cps regulon includes the RcsB
and RcsC regulators, which form a sensor-effector pair. In response to
an unknown environmental signal, the membrane sensor RcsC is thought to
phosphorylate RcsB, thereby increasing expression of the Cps regulon
(10, 12, 33) (Fig. 4).
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pDR210 (rcsA+
bla::cat), pDR310 (dsrA+
bla::cat), pDR211
(rcsA::Tn903
bla::cat) and pDR311
(dsrA::Tn903 bla::cat)were introduced into the
tester strains and analyzed for inducibility. All conditions leading to
high level of Cps expression also caused RecA-independent induction of
(Table 3). Our results do not
contradict those of Gottesman et al. (11), who observed no
influence of lon mutation on the frequency of prophage
induction, because the level of RcsABC-mediated induction is several
orders of magnitude lower than that of induction via the RecA pathway.
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+ prophage induction
caused by RcsA or DsrA overproduction, but lytic phage development can
also result when cI mutants arise, contributing to the
number of infective centers. To distinguish between induction and
mutation, we periodically monitored the levels of free phage in growing cultures, scoring c+ and c
separately. c+ and c phages can
be recognized because they form turbid and clear plaques, respectively.
Both rcsA+ and dsrA+ that
were cloned on plasmids, like the rcsC137, rcsA3,
or lon mutation, stimulated prophage induction (Table
3). The results confirm that the genes regulating the synthesis of
capsular polysaccharide affect prophage stability. It is interesting
that RcsA overproduction causes an increase in free phage in
exponential phase whereas DsrA overproduction produces the maximum
free-phage titer on entry to the stationary phase of growth.
The mucoid phenotype of strains carrying rcsA+
and dsrA+ plasmids could reduce phage
readsorption, thereby apparently increasing the free-phage titer.
Indeed, adsorption to these cells was slower (data not shown). However,
similar free-phage curves were obtained with -resistant
recA() lysogens, in which readsorption was prevented (data not shown). Furthermore, the number of infective centers, which
should not be significantly influenced by phage readsorption, showed a
similar 10- to 100-fold increase in recA lysogens bearing rcsA+ or dsrA+ plasmids
or carrying an rcsC137, rcsA3, or lon
allele (Table 3). The growth rates of the various strains used differed
little, although mucoid strains reached stationary phase at a lower
concentration (data not shown). We thus conclude that
rcsA+ or dsrA+ plasmids
increase the free-phage titer not by changing cell vigor, phage burst
size, or adsorption efficiency but rather from genuine prophage induction.
Neither pDR210 (rcsA+) nor pDR310
(dsrA+) significantly increased the
concentration of c in the culture medium, showing that the burst of c mutants is independent of RcsA and DsrA.
This once again implies that the observed increase in the concentration of c+ free phage reflects true induction of
+ prophage, since other factors would be expected to
affect c+ and c titers similarly.
Genetics of RcsA- or DsrA-mediated induction.
To clarify the
role of the rcsA, dsrA, and rcsB genes
in RecA-independent induction, we carried out complementation tests in which lysogenic strains B7252() (rcsA recA),
B7307() (dsrA recA), and B7253() (rcsB
recA) were tested for their ability to induce the prophage in the
presence of rcsA- and dsrA-bearing plasmids
(Table 4).
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c+ free phage in rcsA and
rcsB mutants carrying a dsrA+
plasmid. This observation is difficult to reconcile with the hypothesis that DsrA RNA causes prophage induction only through its
positive regulation of rcsA transcription (antagonizing
repression by H-NS). It suggests that in the late phase of cell growth,
an increased level of DsrA RNA induces prophage via an
RcsA-independent pathway.
The levels of c in all experiments were similar (data not shown).
Effect of repressor overproduction on RcsA-mediated
induction.
If the RcsABC system provides a repressor bypass for
the various phages, overproduction of repressor would not be expected to affect induction. If, on the other hand, some component of the
RcsABC system (or a gene product induced by these activators) interacts
directly with the various phage repressors, then repressor overproduction should reduce induction frequency. Using a multicopy plasmid pDR1 (cI+), we overproduced repressor in recA rcsC137() and recA
lon() lysogens. Induction was completely abolished
(data not shown). This suggests that RcsA-mediated induction involves
repressor inactivation. The same plasmid in recA lon strains
that are lysogenic for the heteroimmune phages 21, 80, and 434 had
no effect on induction. Similar specificity has been shown for SOS
induction, where excess cI, although preventing induction, does not saturate the system since 434 repressor can still
be inactivated (1).
Influence of cIind mutations on induction by RcsA and DsrA.
To clarify the mechanism of prophage
induction by RcsA and DsrA, we tested the inducibility of mutant
cIind prophages, described by Gimble and Sauer
(8). The repressors of all cIind
mutants usedGR185, EK117, DY125, and GE112have a defect
in RecA-stimulated proteolysis, but all except GE112 (mutated in the
cleavage site) can carry out self cleavage, at various efficiencies,
under alkaline condition in vitro (9). Since the
cIind mutants all carried the b2
deletion, covering the att site, we crossed them with
imm434 plac5 to obtain cIind
plac5 att+ recombinants, which we tested for inducibility
(Table 5).
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DY125 plac5 in exponential phase increases two- to tenfold when the cells carry pDR210 (rcsA+) or pDR311
(dsrA+), as judged by the frequency of infective
centers and the level of free phage. This observation shows that the
action of RcsA and DsrA on the prophage is not inhibited by the
cIind mutation. Similar results were obtained
with GR185 plac5 and EK117 plac5 (data not shown). However, the
action of RcsA was lowered in the case of prophage GE112 plac5
(Table 5). As mentioned above, the repressor of this phage is mutated
in the cleavage site and differs from the other cIind
proteins in that it does not undergo self cleavage under alkaline
condition in vitro (9). This result suggests that prophage
induction in exponential phase is by repressor inactivation, possibly
reflecting the action of an alternative RecA coprotease.
As shown in Table 5, on entry to stationary phase pDR311
(dsrA+) causes a similar, severalfold increase
in the concentration of free phage with plac5 and
cIind. The effects of the plasmid were the
same on all cIind phages. DsrA RNA thus acts
on the prophage independently of cIind mutations
in the repressor gene.
The amounts of c mutants differed little among the
strains tested (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Prophage stability is coupled to cell physiology. Under
conditions in which cell viability is jeopardized, the prophage may manage to survive by entering the lytic pathway. The paradigm for
lysogenic inductionand in fact the only induction mechanism known at
present to existis the SOS response and its positive regulator, the
RecA protein. In response to DNA damage, RecA stimulates the
autoproteolytic cleavage of certain prophage repressors
(28).
Other stress responses of the bacterial cell, alongside the SOS
response, may also induce certain prophages, allowing them to abandon a
host in physiological difficulty. Under laboratory conditions, these
putative systems of lysogenic induction are likely to be inefficient
unless the right stress is applied. Here we present evidence that this
is indeed the case for E. coli. In particular, we have
demonstrated RecA-independent induction of and other lambdoid
prophages by overproduction of RcsA, a transcriptional regulator of the
cps operons involved in capsular polysaccharide synthesis
(10, 12, 13, 34), and by overproduction of DsrA, an
85-nucleotide RNA able to antagonize H-NS repression (31).
In both cases, the numbers of free phage and of infective centers in
overproducing strains were increased 10- to 100-fold compared to those
in control strains.
The synthesis of capsular polysaccharide, or colanic acid, is regulated by the two component system RcsC-RcsB, in which RcsC, a membrane protein, is similar to histidine kinase sensor proteins and RcsB, which is absolutely required for cps gene expression, is similar to DNA-binding effectors (33). RcsA, and probably RcsF as well (7), are additional transcriptional activators of the Cps regulon. The RcsA level is regulated by H-NS repression of rcsA, which is antagonized by DsrA RNA (31), and by degradation by protease Lon (34, 36).
We show here that the RcsABC-dependent system induces , 21, 80,
and 434, and its action may well extend to other lambdoid phages and
possibly also to nonlambdoid, SOS-inducible phages. It is interesting
that spontaneous induction of lambdoid phages is principally via the
SOS response, as evinced by its sharp reduction in recA
lysogens (cf. Table 3). The second mechanism seems to be via RcsABC,
since induction is essentially abolished in recA rcsB()
lysogens (cf. Table 4).
At present we have no information as to the nature of the environmental
signal transduced by the RcsABC system that stimulates capsular
polysaccharide synthesis. Nevertheless, all strains with genetic
backgrounds that resulted in increased cps gene expression also displayed RecA-independent prophage induction. These include strains overproducing RcsA or DsrA from multicopy plasmids,
lon and rcsA3 mutants in which RcsA is stabilized
(and therefore present at high levels), and the cps
constitutive mutant rcsC137, thought to phosphorylate RcsB
spontaneously. It is a curious fact that, although lon
mutants have higher cps expression than the rcsA3 mutant (3), the latter strain has a higher level of induction (Table 3). This suggests that prophage induction does not
result from transcriptional activation of a cps operon.
Prophage induction by the RcsABC system, which takes place in
exponential phase, depends on the concentration of repressor in the
cells. One can speculate that this induction results either from a
lowering of cI repressor synthesis from the promoter
PRM or from decreased repressor activity via a direct
interaction between cI and some cell protein(s) whose
synthesis induces the RcsABC system. The second hypothesis seems more
likely since the mutation in phage GE112, which blocks self cleavage
of the repressor, lowers prophage induction via RcsA. The RcsABC system
may regulate the expression of an alternative RecA coprotease. The
known homology between RcsA and the LuxR family of
transcriptional activators (34) suggests the activation of
the gene of an alternative RecA coprotease. In addition, the complete
dependence of RcsA action on the presence of an intact rcsB
gene and the effect of the rcsC137 mutation also argue in
favor of this proposition.
Prophage induction in the presence of excess DsrA RNA in exponential
phase depends on the presence of intact rcsA and
rcsB genes. This indicates that the action of DsrA RNA on
the prophage in exponentially growing cells is via rcsA
transcription. However, on entry to stationary phase, as we have seen,
induction by DsrA RNA no longer depends on the RcsABC system. This
leads us to postulate the existence of a second induction pathway of
prophage by DsrA RNA, independent of both RecA and the RcsABC
system. Furthermore, this pathway is not affected by the presence of a
cIind mutation in the prophage repressor gene,
even if the normal cleavage site is mutated. DsrA action on the
prophage in this phase of growth is probably at the transcriptional
level, possibly creating a repressor bypass.
Retallack and Friedman (27) have reported that 10Sa RNA, the
ssrA gene product, binds to cI repressor,
competing with binding to the operator OR2 and reducing
repression. It is not known whether an excess of this small RNA, like
DsrA, can cause RecA-independent prophage induction. However, the
recently established role of 10Sa RNA in tagging proteins produced from
truncated mRNA molecules (16) suggests that gene regulation
is not its primary function.
DsrA RNA was originally discovered because of its ability, when overproduced, to derepress rcsA expression (31). It was shown to relieve turning off by H-NS of several operons, including rcsA and rpoS, the structural gene of sigma S (31, 32). Derepression of rcsA is clearly involved in DsrA-mediated prophage induction in exponential-phase cells, since chromosomal rcsA and rcsB mutations lead to a significant drop in the induction level (Table 4). In stationary phase, however, excess DsrA causes efficient prophage induction even in the absence of RcsA and RcsB (Table 4). Stationary-phase cells normally have high levels of both H-NS (6) and RpoS (15). DsrA increases the amount of RpoS (32) and antagonizes H-NS action (31), which itself normally decreases the amount of RpoS (2, 41). Since both H-NS and RpoS regulate a number of operons, DsrA overexpression, with a larger-than-normal increase in RpoS and less-efficient action of H-NS, could radically alter the pattern of gene expression in stationary phase. How this configuration results in prophage induction remains to be shown.
Induction of prophage via RecA-assisted repressor cleavage was an
important step forward in elucidating the molecular regulation of the
SOS response. We have presented here two new, RecA-independent systems
of prophage induction. One is observed in stationary phase in the
presence of excess DsrA RNA and probably involves sigma S and the
histone-like protein H-NS; its characterization will add to our
knowledge of gene expression in stationary phase. The other induces
prophages via RcsABC regulatory network; knowledge of this circuit
should help reveal the environmental signal inducing capsular
polysaccharide synthesis. It is likely that additional mechanisms of
lysogenic induction exist. Our search, for example, would not have
identified systems inoperative on or systems that require RecA in a
non-SOS role. A complete catalogue of lysogenic induction mechanisms
would reveal what aspects of host physiology viruses are sensitive to
and would provide new tools for elucidating the molecular signals
indicating cellular disorders.
| |
ACKNOWLEDGMENTS |
|---|
We thank S. Gottesman, R. Sauer, and Y. Kohara for generously providing bacterial strains, plasmids, and phages.
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FOOTNOTES |
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* Corresponding author. Mailing address: State Scientific Research Institute of Genetics and Selection of Industrial Microorganisms, I Dorozhny Proezd 1, Moscow 113545, Russia. Phone: (7 095) 315 12 10. Fax: (7 095) 315 05 01. E-mail: vkpm{at}vnigen.msk.su.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Bacteriology, December 1998, p. 6306-6315, Vol. 180, No. 23
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