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Journal of Bacteriology, September 1999, p. 5193-5200, Vol. 181, No. 17
Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139
Received 30 September 1998/Accepted 16 June 1999
The competence and sporulation factor (CSF) of Bacillus
subtilis is an extracellular pentapeptide produced from the
product of phrC. CSF has at least three activities: (i) at
low concentrations, it stimulates expression of genes activated by the
transcription factor ComA; at higher concentrations, it (ii) inhibits
expression of those same genes and (iii) stimulates sporulation.
Because the activities of CSF are concentration dependent, we measured the amount of extracellular CSF produced by cells. We found that by
mid-exponential phase, CSF accumulated to concentrations (1 to 5 nM)
that stimulate ComA-dependent gene expression. Upon entry into
stationary phase, CSF reached 50 to 100 nM, concentrations that
stimulate sporulation and inhibit ComA-dependent gene expression. Transcription of phrC was found to be controlled by two
promoters: P1, which precedes rapC, the gene upstream of
phrC; and P2, which directs transcription of
phrC only. Both RapC and CSF were found to be part of
autoregulatory loops that affect transcription from P1, which we show
is activated by ComA~P. RapC negatively regulates its own expression,
presumably due to its ability to inhibit accumulation of ComA~P. CSF
positively regulates its own expression, presumably due to its ability
to inhibit RapC activity. Transcription from P2, which is controlled by
the alternate sigma factor Response to high cell density
(quorum sensing) is widespread in bacteria, regulating many diverse
processes. In Bacillus subtilis, sporulation and the
development of genetic competence (the natural ability to import
exogenous DNA) are stimulated as cells grow to high cell density. As
with other gram-positive bacteria, cell density control in B. subtilis is mediated by extracellular peptides (4, 13, 26,
31).
The development of genetic competence is stimulated by two peptide
factors, ComX pheromone, a modified 10-amino-acid peptide (15), and the competence and sporulation factor (CSF), an
unmodified five-amino-acid peptide (32) (Fig.
1). ComX pheromone and CSF stimulate
transcription of the srfA operon (15, 32). The
srfA operon encodes a small protein ComS, which is required
for competence development (3, 7), and the surfactin
biosynthetic enzymes (18). Transcription from the
srfA promoter is activated by the phosphorylated form of the
response regulator ComA (5, 19-21, 28, 38), and activity of
ComA is controlled by a kinase, ComP, and a phosphatase, RapC (Fig. 1).
ComP, a membrane-bound histidine protein kinase (39), is
required for activation of ComA. RapC, a putative aspartyl-phosphate
phosphatase (24), inhibits activation of ComA
(32). ComX pheromone stimulates gene expression, apparently by stimulating the kinase ComP (15, 33), whereas CSF
stimulates gene expression, apparently by inhibiting the phosphatase
RapC (32) (Fig. 1). ComX pheromone most likely works at the
cell surface, and CSF is actively transported into the cell by the oligopeptide permease Opp (also known as Spo0K), where it interacts with intracellular receptors (14).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
An Autoregulatory Circuit Affecting Peptide
Signaling in Bacillus subtilis
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
H, increased as cells entered
stationary phase, contributing to the increase in extracellular CSF at
this time. In addition to controlling transcription of
phrC, H appears to control expression of at
least one other gene required for production of CSF.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Model of the regulation of synthesis of and response to
RapC and CSF. CSF is synthesized as a precursor protein PhrC with a
signal sequence and putative peptidase cleavage sites. Pre-CSF is
cleaved to produce the active CSF pentapeptide (ERGMT). The
transcription factor ComA is activated (phosphorylated) by the
membrane-bound histidine protein kinase ComP and by ComX pheromone,
which probably activates ComP. ComQ is needed for production of the
active ComX pheromone. CSF is transported into the cell by the
oligopeptide permease (also known as Spo0K), where it stimulates
expression of genes activated by ComA~P, probably by inhibiting
activity of the phosphatase RapC, which is a negative regulator of
ComA~P. Results presented in this report indicate that ComA~P
activates transcription of both rapC and phrC.
Furthermore, we show that RapC negatively regulates it own synthesis,
presumably by dephosphorylating ComA~P. CSF, in contrast, was shown
to positively regulate transcription of itself and rapC,
presumably by inhibiting the phosphatase activity of RapC.
H, the spo0H gene product, activates
transcription of phrC.
CSF has a remarkable constellation of activities for a five-amino-acid peptide (14, 32). At relatively low extracellular concentrations (1 to 5 nM), CSF stimulates expression of srfA by inhibiting the phosphatase RapC. However, high concentrations of CSF (>20 nM) inhibit expression of srfA, perhaps by inhibiting activity of the kinase ComP. In addition to its effects on expression of ComA~P-controlled genes, high concentrations of CSF (>20 nM) can stimulate sporulation under some conditions (14, 32). CSF appears to stimulate sporulation, at least in part, by inhibiting the activity of the phosphatase RapB (23), an aspartyl-phosphate phosphatase that dephosphorylates Spo0F~P (25), an essential component of the phosphorelay that controls sporulation (1, 4, 10).
Because CSF has these different activities at different extracellular concentrations, we were interested in characterizing the regulation and production of CSF. Production of mature CSF involves several steps, starting with transcription and translation of phrC, the gene encoding the precursor of CSF (32). The 40-amino-acid primary product of phrC has a signal sequence and putative peptidase cleavage sites, indicating that a 11- to 25-amino-acid peptide is exported via a Sec-dependent pathway (24). Steps after initial processing that result in release of the mature pentapeptide are not yet known.
We have measured the extracellular concentration of CSF produced during growth and compared this to the transcriptional control of phrC. phrC is in an operon with rapC (24). We found that transcription of the rapC phrC operon is activated by high cell density through ComA~P and that rapC and phrC regulate their own expression (Fig. 1). RapC, by negatively regulating ComA~P, is part of a homeostatic autoregulatory loop. PhrC (CSF) stimulates ComA activity and positively regulates its own expression. Furthermore, we show that by mid-exponential phase, CSF is at concentrations that stimulate competence gene expression and that as cells enter stationary phase, the extracellular concentrations of CSF reach levels approaching 100 nM, concentrations that are known to stimulate sporulation and inhibit early competence gene expression.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Strains construction.
B. subtilis strains used are
listed in Table 1; unless otherwise
indicated, all are derived from the parent strain JH642 and contain
trpC and pheA mutations. Strains were constructed by transformation with chromosomal DNA by standard protocols
(8). 
codY was transferred by linkage to
unkU::spc (30). The presence of the codY mutation was confirmed by PCR analysis of
chromosomal DNA.
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Construction of lacZ fusions. Three lacZ transcriptional fusions were constructed to analyze regulation of the rapC phrC operon. To analyze expression from the P1 promoter, a 699-bp fragment (fragment 1 [Fig. 2A]), from 446 bp upstream to 253 bp downstream of the start codon of rapC, was cloned upstream of lacZ and integrated into the chromosome at amyE. The P1 fragment was amplified by PCR using Vent polymerase (New England Biolabs) and primers rapC3 (5'-AGAAGCTTACGGTGACATTTGGCTG-3') and rapC4 (5'-GGGATCCAATTCAGACAGGCTTGGC-3') with restriction sites (underlined) added at the ends. This PCR fragment was subcloned between the HindIII and BamHI sites of pKS2 (15) making plasmid pGK47. pGK47 was linearized and transformed into wild-type B. subtilis, selecting for Neor transformants. The transformants were screened for an amylase-deficient phenotype to confirm that the plasmid had integrated into the chromosome at the amyE locus.
To analyze expression from the P2 promoter, a 743-bp fragment (fragment 2 [Fig. 2A]), from 619 bp upstream to 124 bp downstream of the start codon of phrC, was cloned upstream of lacZ and integrated into the chromosome at amyE. This fragment extends two base pairs beyond the stop codon of phrC, resulting in the introduction of an additional copy of phrC. lacZ fusions with downstream junctions internal to phrC had
-galactosidase activity
similar to the fusion with the additional copy of phrC (data
not shown). The P2 fragment was amplified by PCR using Vent polymerase
and primers rapC5 (5'-GGAATTCTACGTGGAGCAGGAAAC-3') and phrC4 (5'-GGGATCCTCTTACGTCATTCCTCTT-3'),
with restriction sites (underlined) added at the ends. This PCR
fragment was cloned between the EcoRI and BamHI
sites of pKS2, making plasmid pGK52. pGK52 was introduced into
wild-type B. subtilis as described above for pGK47.
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10 and phrC::erm
mutant JMS751 (14) was transformed with pGK50, selecting for
Cmr transformants. The resulting transformant has
lacZ fused to the entire operon, including a wild-type copy
of phrC.
Media. Cells were grown at 37°C in defined S7 minimal medium with glucose (1%), glutamate (0.1%), and required amino acids (Trp and Phe), and MOPS (morpholinepropanesulfonic acid) buffer was used at 50 rather than 100 mM (12, 37). Casamino Acids were added to 0.1% when indicated.
Primer extension analysis.
The transcription start site for
the rapC P1 promoter was determined by primer extension
analysis. Total RNA was prepared from strain JMS682 (a wild-type strain
with amy::srfA-lacZ), which was grown
in defined minimal medium until 2 h after the end of exponential
growth. The RNA was purified using an RNeasy Maxi kit (Qiagen)
according to the manufacturer's instructions; 40 µg of this RNA was
analyzed by primer extension as described previously (34),
with the following exceptions. The primer, RM2
(5'-ACTTCGGCATCCGGCACGCTGAATG-3'), which corresponds to +126
to +102 relative to the start of transcription, was radiolabeled with
polynucleotide kinase and [
-32P]ATP, and the
unincorporated nucleotides were separated from the primer on a
MicroSpin G-50 column (Pharmacia). Primer extension reactions and
sequencing reactions with the RM2 primer were run on an 8%
polyacrylamide gel, and radioactivity was detected by autoradiography.
-Galactosidase assays.
-Galactosidase specific
activity was measured essentially as described previously (12, 15,
16) and is presented as (A420 per
minute per milliliter of culture per unit of optical density at 600 nm
[OD600]) × 1,000.
Extracellular factor assays.
The activity of extracellular
factors was assayed essentially as described previously (15,
32). Briefly, cells containing a lacZ fusion were
grown to low cell density (OD600 of ~0.1), and 0.25 ml of
culture was mixed with an equal volume of a test sample, either
conditioned medium, partly fractionated signaling peptides, or
synthetic CSF. Samples were incubated at 37°C for 70 min
(15), and -galactosidase specific activity was measured. Conditioned medium (culture supernatant) was prepared by growing cells
in defined minimal medium, then removing the cells by centrifugation, and subjecting the supernatant to filtration through a
0.2-µm-pore-size filter. Synthetic CSF was resuspended in defined
minimal medium containing 50 µg of bovine serum albumin per ml prior
to mixing with cells.
Quantitation of extracellular CSF.
Cells were grown in
defined minimal medium, samples (7 ml) were taken at different times
during growth, and cells were removed as described above. To separate
CSF from ComX pheromone in the culture supernatant, the pH was adjusted
to 2 with trifluoroacetic acid (TFA), and each sample was run over a
1-ml C18 reverse-phase column (Sep-Pak Plus; Millipore)
that had been equilibrated with aqueous 0.1% TFA. After being washed
with 3 column volumes of 0.1% TFA, CSF was eluted with 3 column
volumes of buffer containing 0.1% TFA and 11% acetonitrile. To assay
the column eluate for CSF activity, samples were dried in a Speed-Vac
to remove TFA and acetonitrile and then resuspended in minimal medium
containing 50 µg of bovine serum albumin ml. The samples were then
diluted, and CSF activity was measured (as described above), using
strain BAL125 (phrC::erm srfA-lacZ).
The activity of the column eluates was compared to the activity of
known concentrations of synthetic CSF. CSF activity was linear in this
assay from 0.05 to 1.5 nM CSF.
phrC). This sample was fractionated and prepared as
described above and then tested for CSF activity. Greater than 95% of
the CSF activity added to the conditioned medium was present in the
column fraction (data not shown). Conditioned medium from the
phrC strain had no activity in this assay in the absence of added CSF (data not shown and reference 32).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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lacZ fusions to the rapC phrC regulatory
regions.
The rapC phrC operon is predicted to have at
least two promoters. Immediately upstream of rapC is a
promoter, P1, with the recognition sequence for the vegetative sigma
factor, A (Fig. 2A). The location of the start of
transcription for the P1 promoter was determined by primer extension
analysis, using a primer internal to rapC (Fig. 2B). Just
upstream from this promoter is a potential binding site for the
transcription factor ComA. This sequence matches the consensus binding
site for ComA (17) in 10 of 12 positions (Fig. 2B). In
addition to the A-dependent promoter, there is a
H-dependent promoter, P2, upstream of phrC
(2), internal to rapC (Fig. 2A). Expression of
phrC should be controlled by both P1 and P2.
Regulation of rapC by cell density and ComA.
Transcription from P1 increased as cells grew to high cell density in
defined minimal glucose medium, as judged by accumulation of
-galactosidase specific activity from the P1-lacZ fusion
(Fig. 3A). Indeed, expression of
P1-lacZ in cells at low density increased in response to the
addition of conditioned medium from cells at high density (data not
shown). This pattern of expression is similar to that of the promoter
for the srfA operon (15, 32, 33), which is
activated by the phosphorylated form of the transcription factor ComA
(reference 28 and references therein). This pattern of expression and the presence of a putative ComA binding site suggest
that P1 is activated by ComA~P.
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-galactosidase from P1-lacZ
was reduced to ~1% of that in wild-type cells (Fig. 3A).
comP encodes a histidine protein kinase (39) that
is required for activation (phosphorylation) of ComA and full
transcription from ComA-dependent promoters (Fig. 1) (19,
39). As expected, a null mutation in comP also reduced
expression from P1-lacZ (Fig. 3A). ComP activity is
stimulated by an extracellular signaling peptide, the ComX pheromone
(Fig. 1) (15, 33). Expression of P1-lacZ was
reduced in a mutant (comQ) unable to make the ComX pheromone
(Fig. 3A). Interestingly, expression from P1 was lower in the
comQ (pheromone) mutant than the comP (kinase)
mutant, indicating that ComP could have an inhibitory role, perhaps as
a phosphatase, in addition to its stimulatory role. Alternatively, ComQ
could have a role in stimulating ComA-dependent gene expression that is
independent of ComP. The mutations have a similar effect on expression
of srfA (33).
In addition to ComP and ComX pheromone, expression of genes activated
by ComA~P is also affected by CSF, the oligopeptide permease (Opp)
that transports CSF into the cell, and the putative phosphatase RapC,
which is a negative regulator of ComA~P (Fig. 1) (14, 15, 32,
33). We found that transcription from P1 was affected by all of
these components. A null mutation in rapC caused increased
transcription from P1 such that -galactosidase specific activity was
approximately threefold greater throughout growth than that in
wild-type cells (Fig. 3B). This result indicates a negative
autoregulatory loop; that is, RapC negatively regulates (indirectly)
its own expression, presumably by inhibiting accumulation of
ComA~P (Fig. 1) (32). In contrast, a null mutation in
phrC, which eliminates production of CSF (32),
caused decreased transcription from P1 (Fig. 3B), indicating a positive
autoregulatory loop. That is, CSF (phrC) stimulates its own
expression, most likely by inhibiting activity of the phosphatase RapC,
leading to increased accumulation of ComA~P and increased
transcription of the rapC phrC operon from P1 (Fig. 1).
The ability of cells to respond to CSF depends on the oligopeptide
permease, Opp (14, 33). An opp null mutation
decreased expression of P1-lacZ to ~6% of that in
wild-type cells (Fig. 3B), similar to effects on expression of
srfA (32). This effect is much greater than that
of the phrC null mutation and indicates that the loss of the
oligopeptide permease has pleiotropic effects, perhaps causing other
phosphatases of the Rap family to be hyperactive and dephosphorylate
ComA~P (32).
CSF and the control of rapC transcription. CSF is known to have two effects on expression of the ComA-controlled gene srfA. Low concentrations of CSF stimulate, and high concentrations inhibit, transcription of srfA (14, 32). We found that CSF also had these two effects on transcription from P1 (Fig. 4), indicating that this regulation was not specific for srfA. When added to cells at low cell density, relatively low concentrations of CSF (2 to 5 nM) stimulated transcription from P1 ~2.5-fold in our standard assay (Fig. 4). The maximum potential induction caused by CSF may be partly masked by the inhibitory activity of CSF.
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Regulation of rapC by CodY. Under some growth conditions, the transcription factor CodY represses expression of several genes known to be activated by ComA~P. In growth medium containing complex mixtures of amino acids, expression of srfA and rapA is inhibited during exponential growth and induced upon entry into stationary phase. The inhibition during exponential growth is mediated by CodY (29).
We tested the effects of amino acids and a codY null mutation on expression from rapCP1. The presence of casamino acids in the growth medium greatly reduced expression of P1-lacZ during exponential growth, and this effect was relieved in a codY null mutant (Fig. 5). The codY mutation had no noticeable effect on expression from P1 in the growth medium without Casamino Acids (data not shown). Despite the ability of CodY to repress rapC when Casamino Acids were added to the medium, codY did not affect expression of rapC during growth in nutrient sporulation medium (data not shown), similar to previous findings on rapA expression (30). CodY has been shown to bind to the srfA promoter region in vitro (29), and although a binding sequence has not been defined, it seems plausible that CodY also binds to the rapC promoter region.
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Regulation of P2, the H-dependent promoter upstream
of phrC.
The H-dependent promoter upstream
from phrC was identified previously in a search for
promoters transcribed in vitro by RNA polymerase containing the
alternate sigma factor H, the spo0H gene
product (2). We found that in defined minimal medium,
expression of P2-lacZ was relatively constant during
exponential growth and increased as cells approached stationary phase
(Fig. 6). Expression was virtually
undetectable in a spo0H null mutant (Fig. 6A), consistent
with previous findings (2). Increased expression from P2 as
cells approach stationary phase is most likely due to an increase in
levels of H protein (9, 40). Expression from
P2 did not appear to be regulated by cell density, as a comA
null mutation had little or no effect on expression of
P2-lacZ (Fig. 6A), nor did addition of conditioned medium to
cells at low density (data not shown).
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Regulation of phrC transcription.
Transcription of
phrC is control by both the ComA-regulated promoter P1, and
the H-dependent promoter P2 (Fig. 2A). To understand how
these two promoters affect the regulation of phrC, we
constructed a lacZ fusion [(P1, P2)-lacZ] that
is controlled by both promoters. This was done by integrating a plasmid
containing a phrC-lacZ fusion into the chromosome at the
phrC locus.
-galactosidase of an otherwise wild-type strain (Fig.
7A). Expression of the (P1,P2)-lacZ fusion in a comA null mutant
increased after entry into stationary phase, presumably due to the
activation of H and increased transcription from P2.
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Production of extracellular CSF.
The analysis of physiological
responses to CSF involved addition of different concentrations of
chemically synthesized peptide to cells (14, 32). To
determine if these concentrations are physiologically relevant, we
measured the CSF activity present in culture supernatants during
exponential growth and upon entry into stationary phase. Culture
supernatant was fractionated to separate CSF from ComX pheromone. The
activity of CSF found in partly fractionated culture supernatant was an
accurate reflection of the concentration of CSF. Reconstruction
experiments were done to estimate recovery of CSF from culture
supernatant. A known amount of CSF was added to culture supernatant
from a phrC mutant (unable to make CSF). At least 95% of
the expected CSF activity was recovered following fractionation (see
Materials and Methods). Activity was compared to a standard curve
determined with known concentrations of synthetic CSF (Materials and Methods).
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H affect the accumulation of extracellular CSF,
but the effect of H on accumulation is greater than its
effect on the transcription of phrC. The accumulation of
extracellular CSF was severely reduced in a comA mutant
strain to ~7% of the level of a wild-type strain (Fig. 8). This is
similar to the effect of a comA mutation on the expression
of phrC [the (P1, P2)-lacZ fusion] (Fig. 7A).
spo0H mutants also had severely reduced levels of
extracellular CSF (Fig. 8). A spo0H mutant strain had less
than 2% of the normal amount of CSF. However, this effect of a
spo0H mutation on extracellular CSF production is more
severe than the effect on transcription of phrC (Fig. 7A),
indicating that spo0H probably is required for expression of
genes, in addition to phrC, that are involved in CSF
production. We cannot rule out that ComA also has an affect on the
expression of genes required for CSF production other than phrC, but this effect would have to be small compared to
ComA's effect on transcription of phrC.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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CSF of B. subtilis was initially identified as an extracellular peptide factor that stimulates transcription from the srfA promoter when added to cells at low cell density (32, 33). CSF is a pentapeptide produced from the C-terminal 5 amino acids of the 40-amino-acid product of phrC (32). CSF has at least three activities: (i) at low concentrations, it stimulates expression of genes activated by ComA~P; at higher concentrations, it (ii) inhibits expression of those same genes and (iii) under some conditions stimulates sporulation (14, 32). Genetic evidence indicates that CSF stimulates expression of genes activated by ComA~P by inhibiting the activity of the phosphatase RapC (32). CSF stimulates sporulation (14, 32), in part, by inhibiting activity of the phosphatase RapB, a negative regulator of the phosphorelay required for the initiation of sporulation (23). It is not known how CSF inhibits ComA-controlled gene expression, but we suspect that it might inhibit activity of the histidine protein kinase ComP. Measuring production of CSF during cell growth, we found that CSF accumulates in culture medium, reaching concentrations approaching 100 nM. These concentrations are in the range of those previously shown to cause the three different regulatory responses (14, 32).
RapC and CSF are part of an autoregulatory loop. We found that both rapC and phrC affect their own expression by affecting transcription from the upstream operon promoter P1 that is activated by ComA~P. RapC and PhrC (CSF) appear to accomplish this by affecting the level of ComA~P (Fig. 1). A rapC null mutation causes increased expression from P1 (and other ComA~P-activated promoters [32]), indicating that RapC negatively regulates its own transcription, creating a homeostatic loop. At low cell densities, CSF is secreted and diluted into the culture medium, causing the extracellular CSF concentration to be low. When CSF levels are too low to inhibit RapC activity, RapC should inhibit accumulation of ComA~P, thus decreasing transcription from the ComA~P-controlled rapC promoter. As the concentration of RapC falls, transcription from the rapC promoter would increase. This homeostatic loop should cause the level of RapC to remain relatively constant while cells are at low densities and extracellular CSF levels are below those that inhibit RapC activity.
A null mutation in phrC causes decreased expression from the upstream operon promoter P1 (and other promoters activated by ComA~P [32]), indicating that CSF positively regulates its own synthesis. As cells grow and become more dense, the extracellular concentration of CSF increases. This is partly because the number of cells making CSF increases and partly because ComX pheromone is accumulating in the culture medium, activating ComA~P and, in turn, activating transcription of phrC. The increased concentrations of CSF should inhibit the phosphatase activity of RapC, increasing the level of ComA~P and thus transcription of the rapC phrC operon. It may be that once a level of CSF capable of partially inhibiting RapC is achieved, more CSF is synthesized to further inactivate RapC and commit cells to full activation of ComA~P-controlled genes. When added exogenously to cells at low cell density, concentrations of CSF between 2 and 5 nM cause maximal stimulation of ComA~P-controlled gene expression (14, 32). In growing cultures, this concentration range is reached during mid-exponential growth, several generations before the onset of stationary phase. These levels of CSF, in combination with ComX pheromone, contribute to the activation of ComA~P-controlled gene expression during exponential growth. One of the operons activated by ComA~P, srfA, then contributes to activation of the competence transcription factor ComK and competence development (6, 35, 36). As cells enter stationary phase, the concentration of CSF reaches a level that inhibits expression of ComA~P-controlled genes and stimulates sporulation. This inhibition is likely a reflection of an activity of the CSF pentapeptide and not due to an impurity. The inhibitory activity is found in the CSF pentapeptide synthesized chemically and in CSF purified from culture supernatants (32). Furthermore, single alanine substitutions at each position of CSF results in some peptides that retain and others that lose inhibitory activity (14). In addition, expression of one of the alanine mutant peptides in vivo from a synthetic gene encoding a six-amino-acid peptide that is predicted to have the N-terminal formylmethionine removed, results in production of an intracellular peptide that inhibits expression of ComA-controlled genes (14). The significance of this inhibitory activity of CSF for ComA~P-controlled genes is unclear. phrC mutants appear to have decreased expression of ComA~P-controlled genes at all phases of growth, based on decreased expression of promoter fusions to lacZ (Fig. 3B) (14, 32). Isolation of a mutant defective in inhibiting ComA~P-controlled genes in response to CSF will help dissect the role of this activity of CSF in the regulation of ComA~P-controlled genes.Transcriptional and posttranscriptional control of CSF production
by H.
Transcription of phrC is
controlled, in part, by RNA polymerase containing the alternate sigma
factor H (Fig. 2A). H is present at low
levels during exponential growth, and its concentration increases upon
entry into stationary phase (9, 40). The increase in active
H during the transition to stationary phase is most
likely what causes increased transcription from the phrC
promoter P2. The extracellular concentrations of CSF shortly after
entry into stationary phase (when H is most active) were
as high as 50 to 100 nM, concentrations at which CSF stimulates sporulation.
H's role in transcription of
phrC, our results indicate that it has at least one other
role in production of CSF. The levels of CSF produced in a
spo0H mutant were reduced much more severely than can be
explained by the effect of H on transcription of
phrC. This indicates that H affects
transcription of at least one other gene required for production
(translation, processing, and secretion) of the CSF peptide. PhrC has a
putative signal sequence and peptidase cleavage sites, indicating that
a secreted pre-CSF peptide of between 11 and 25 amino acids is secreted
by the Sec-dependent pathway (24). Pre-CSF is then processed
to the mature five-amino-acid form. It seems likely that there is a
specific peptidase that recognizes and cleaves pre-CSF. Such a
peptidase would be a good candidate for a H-controlled
gene product required for CSF production. The small amount of CSF made
in a spo0H mutant could be due to processing of pre-CSF by a
nonspecific or alternative peptidase or to processing by lower amounts
of the normal peptidase. The additional gene(s) involved in production
of mature CSF is probably also controlled by spo0A and
abrB, as these genes also have much greater effects on
accumulation of extracellular CSF than they do on transcription of
phrC. It will be interesting to determine if genes involved in production of the CSF pentapeptide are also involved in production of other peptide signaling factors.
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ACKNOWLEDGMENTS |
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We thank members of the laboratory for useful discussions, and especially K. P. Lemon, T. Palmer, and K. Bacon, for comments on the manuscript.
B.A.L. was supported, in part, by fellowship DRG1384 from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation, and this work was supported in part by Public Health Service grant GM50895 from the NIH.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 253-1515. Fax: (617) 253-2643. E-mail: adg{at}mit.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, September 1999, p. 5193-5200, Vol. 181, No. 17
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