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stress response sigma factor sigma-B (56). We found that sigma-B
activity was increased at the T[-1] time point in the sigH mutant
background, consistent with previous findings that expression of
sigma-B-dependent genes is increased in sigH mutants (45, 56).
RNA from ytxG was higher in the sigH mutant due to the fact that the
promoter controlled by sigma-B was more active in the absence of
sigma-H (56). We detected this regulation in the two types of
microarray experiments: ytxG was induced by overexpression of sigma-H
but showed a higher level of expression in a sigH mutant strain (Table
(Table1).1). This example shows that one cannot necessarily assume
that a gene that is regulated by sigma-H will behave as expected in the
sigH+ versus sigH mutant experiment and demonstrates the importance of
performing both types of microarray experiments. yvyD is also known to
be controlled by both sigma-H and sigma-B (11) and had a pattern of
expression similar to that of ytxG (Table (Table11).
Identification of genes that are strong candidates to be regulated
directly by sigma-H.
In addition to the previously described genes activated by sigma-H, we
found 26 operons containing 54 genes that showed dependence on sigma-H
for expression in the microarray experiments and had a potential
sigma-H promoter within 200 bp of the start codon (except yojLM, for
which the promoter was 292 bp upstream) (Table (Table22 and Fig.
Fig.2).2). In 23 of the operons there was a promoter predicted by
an HMM. Promoters for the other three operons were uncovered by using
the pattern search algorithm on the SubtiList website
(http://genolist.pasteur.fr/SubtiList/). In our analysis of genes
that were differentially affected in the microarrays, we inferred
direct regulation by sigma-H if a good sigma-H binding site was located
upstream of the gene. It seems likely that most of the other genes are
indirectly regulated by sigma-H. Many of the known or putative
functions of the 54 genes that may be directly controlled by sigma-H
highlight the critical role of sigma-H in adaptation to nutrient
deprivation. Several genes under direct sigma-H control appear to be
involved in adaptation to nutrient deficiency. Many of the proteins in
this class are known or predicted to be secreted and could be used to
modify the extracellular environment (Table (Table2).2). Proteins
such as Vpr (extracellular serine protease) (50) could be used to
scavenge for food in the extracellular environment by degrading
proteins. In addition to Vpr there are other secreted proteases that
are indirectly regulated by sigma-H, AprE and NprE, which also could be
used to digest extracellular material. In addition there is a putative
secreted nuclease, YhcR, that could be used to degrade nucleic acid
that could also be used for food. (A recent study of E. coli has shown
that DNA can be used as a sole source of carbon for the cell
[16].) Another group of genes that could provide alternative
nutrients to the cell are transporters. For example, gltP encodes a
glutamate transporter and is predicted to be directly regulated by
sigma-H. The yhaQ gene product has similarity to ABC transporter ATP
binding proteins, likely has a role in transport, and appears to be
directly regulated by sigma-H. Lastly, genes such as ccdA and the qcr
operon encode proteins that are involved in the synthesis of cytochrome
c and the cytochrome bc complex, respectively. These proteins are
involved in the electron transport chain and may be up regulated by
sigma-H in response to nutrient-limiting conditions in an attempt to
generate energy. We also find the expression of resABC, which is also
required for cytochrome c synthesis, to be indirectly regulated by
sigma-H. Expression of resABC was previously shown to be induced upon
entry into stationary phase by a putative sigma-A promoter (53).
TABLE 2.
TABLE 2.
Newly identified sigma-H-regulated geneslegend
Expression of the ccdA operon was previously known to coincide with the
time that sigma-H is fully active (33). The ccdA operon transcript
has been mapped by primer extension, and the authors indicated that
sigma-A was likely to drive transcription of the ccdA operon. We have
identified a potential sigma-H promoter that overlaps the putative
sigma-A promoter. Thus, sigma-H could be responsible for additional
regulation of ccdA. ccdA mutant strains are deficient in sporulation at
a very late stage (48), similar to what is observed with spoVS, a
gene controlled by sigma-H.
The best-known role of sigma-H is to activate sporulation. Many of the
known sigma-H-controlled genes are involved in the signal transduction
pathway involved in the initiation of sporulation (kinA, spo0F, and
spo0A) and in the early stages of sporulation (spoIIAA, spoIIAB, and
sigF). We find that an additional histidine kinase implicated in
controlling the initiation of sporulation, kinE, is controlled by
sigma-H (27). We also searched the MICADO
(http://locus.jouy.inra.fr/cgi-bin/genmic/madbase/progs/madbase.op
erl) and JAFAN (http://bacillus.genome.ad.jp) databases to
determine if any of the newly identified direct sigma-H targets display
a sporulation defect when mutated. Two genes, ymaH and yoeA, are
reported to be defective in endospore formation when mutated.
Recently, it was shown that natural isolates of B. subtilis can form
multicellular aerial structures that may be important for the dispersal
of spores and that sigH is required for the formation of these
structures (5). Two genes required for the formation of these
aerial structures, yveQ and yveR, are thought to be part of a large
operon responsible for exopolysaccharide production. We find that genes
that are members of the yve operon that is involved in fruiting body
formation, yveKLMNOPQRST-yvfABCDEF, are dependent on sigma-H.
Three additional groups of newly identified sigma-H-controlled genes
are worth mentioning: transcription factors, cell wall binding
proteins/autolysins, and proteins involved in detoxification. Sigma-H
was already known to regulate three transcription factors, sigma-A,
Spo0A, and sigma-F. Two additional putative transcription factors show
sigma-H-dependent gene expression, ykoM (MarR family transcriptional
regulator) and yttP (TetR/AcrR family). How these putative
transcriptional regulators contribute to gene expression will be an
interesting avenue of future investigation.
The cell wall binding proteins affected by sigma-H are of interest
because sigH mutants are unable to form the asymmetric septum during
sporulation. With the exception of ftsA and ftsZ, we do not find that
the expression of any known cell division genes is affected. We do find
three genes that show dependence on sigma-H, yojL (similar to major
autolysins lytE and lytF), yrvJ (similar to N-acetylmuramoyl-l-alanine
amidase), and yuxL (similar to acylaminoacyl-peptidase), which are
likely to be involved in the modification of the cell wall and possibly
in the formation of the asymmetric septum. Interestingly two of the
previously identified sigma-H-controlled genes, dacC and spoVG, may
also be involved in cell wall modification (36, 38).
Alternatively, these gene products may be involved in generating
nutrients for the cell by digesting cell wall material. Conversely, the
major autolysins lytC, lytD, and lytF (yhdD) appear to be indirectly
controlled by sigma-H and are up regulated in the sigH mutant
presumably via activation of sigma-D in the sigH mutant cells (see
below).
We found four genes that are predicted to be involved in adapting to
changing environmental conditions. They are yoeA (similar to multidrug
efflux), yojM (similar to superoxide dismutase), ywfF (similar to
efflux protein), and bsaA (putative glutathione peroxidase). These
proteins likely provide protective properties to the cell and could