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J Bacteriol, July 1998, p. 3629-3634, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
RpoS (Sigma-S) Controls Expression of rsmA, a Global
Regulator of Secondary Metabolites, Harpin, and Extracellular
Proteins in Erwinia carotovora
Asita
Mukherjee,1
Yaya
Cui,1
Weilei
Ma,1
Yang
Liu,1
Akira
Ishihama,2
Abraham
Eisenstark,3 and
Arun
K.
Chatterjee1,*
Department of Plant Pathology, University of
Missouri, Columbia, Missouri 652111;
Cancer Research Center, Columbia, Missouri
652013; and
Department of Molecular
Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan2
Received 22 January 1998/Accepted 18 May 1998
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ABSTRACT |
RpoS (sigma-S or sigma-38) controls a large array of genes that are
expressed during stationary phase and under various stress conditions
in Escherichia coli and other bacteria. We document here
that plant pathogenic and epiphytic Erwinia species, such as E. amylovora; E. carotovora subsp.
atroseptica, betavasculorum, and
carotovora; E. chrysanthemi;
E. herbicola; E. rhapontici; and
E. stewartii, possess rpoS genes and
produce the alternate sigma factor. We show that rpoS
transcription in E. carotovora subsp.
carotovora is driven from a major promoter which
resides within the nlpD gene located upstream of
rpoS as in E. coli. RpoS
E. carotovora subsp. carotovoa strain
AC5061, constructed by marker exchange, is more sensitive to hydrogen
peroxide, carbon starvation, and acidic pH than its
RpoS+ parent strain, AC5006. The basal levels of
extracellular pectate lyase, polygalacturonase, and
cellulase as well as those of transcripts of E. carotovora subsp. carotovora hrpN
(hrpNEcc), the gene for the elicitor
of the hypersensitive reaction, are higher in the RpoS
strain than in the RpoS+ parent. Likewise,
compared to AC5006, AC5061 causes more extensive maceration of celery
petioles. Our findings with the RpoS mutant and strains
carrying multiple copies rpoS+ DNA reveal that
rpoS positively controls rsmA expression. We also present evidence that supports the hypothesis that the RpoS effect
on extracellular enzyme levels, hrpNEcc
expression, and virulence manifests itself by the modulation of
rsmA expression.
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INTRODUCTION |
Bacteria have the remarkable
capacity to quickly adapt to various stresses including nutrient
limitations, temperature extremes, acidic conditions, osmolarity
extremes, and toxic chemicals. Under such conditions bacteria generally
deploy sigma factors to activate specific sets of genes. In
Escherichia coli and various other bacteria, several
alternate sigma factors such as sigma-24, sigma-28, sigma-32,
sigma-38, and sigma-54 have been identified (reference 14 and references cited therein). Of these, sigma-38
(also called KatF, RpoS, and sigma-S) is involved in the expression of
a number of genes that are activated during postexponential growth or
upon nutrient limitation or exposure to acidic pH (12, 36).
In addition, sigma-S contributes to bacterial resistance to the toxic effects of hydrogen peroxide (1, 6), to the production of certain secondary metabolites (31), and in some instances to the virulence of animal pathogens, for example in Salmonella
typhimurium (8, 9). Several studies with soft-rotting
Erwinia spp. have disclosed that virulence factors are
expressed during postexponential growth and are subject to global
regulation (2, 4, 5, 11, 13, 15, 25, 28, 34, 35). These
features prompted the notion that such virulence factors may also be
controlled by sigma-S. To test this idea we have undertaken studies
with rpoS genes of Erwinia species. Here we (i)
document the occurrence of rpoS homologs and the production
of the alternate sigma factor; (ii) show that transcription of
rpoS is mainly driven by a promoter located within the
nlpD gene upstream of rpoS; (iii) report that RpoS of Erwinia carotovora is required for the bacterium to
cope with carbon starvation, hydrogen peroxide toxicity, and acidic pH;
and (iv) document that RpoS controls the expression of rsmA, a global negative regulator gene. We also present data that demonstrate that RpoS affects extracellular enzyme production, E. carotovora subsp. carotovora hrpN
(hrpNEcc) expression, and virulence by modulating rsmA expression.
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MATERIALS AND METHODS |
Bacterial strains, plasmids and media.
Bacterial strains and
plasmids are described in Table 1. The
strains carrying antibiotic markers were maintained on Luria-Bertani (LB) agar containing appropriate antibiotics. The wild-type
Erwinia and other enterobacterial strains, described in our
previous report (5), were maintained on LB agar.
The compositions of King's B (KB) medium, LB medium, minimal salts
medium, nutrient gelatin agar, polygalacturonate-yeast
extract agar,
and M9 medium have been described previously (
23,
27,
30).
To test the effects of osmolarity on
rpoS expression,
bacteria were grown in TY medium (1% [wt/vol] tryptone, 0.5%
[wt/vol]
yeast extract) either without NaCl or with 0.1 or 0.25 M
NaCl.
When required, antibiotics and drugs were supplemented as follows
(concentrations are in micrograms per milliliter): ampicillin,
100;
kanamycin, 50; nalidixic acid, 50; spectinomycin, 50; and
tetracycline
(TC), 10. Media were solidified by the addition of
1.5% agar.
The compositions of agarose media for semiquantitative assays of
enzymatic activities were described previously by Chatterjee
et al.
(
4).
Enzyme assays.
The preparation of enzyme samples for pectate
lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease
(Prt) assays and the assay procedures were described previously
(4, 27).
DNA techniques.
Standard procedures were used in the
isolation of plasmid and chromosomal DNAs, transformation, restriction
endonuclease digests, gel electrophoresis, and DNA ligation
(30). Southern hybridizations were carried out as described
by Cui et al. (5). Restriction and modifying enzymes were
obtained from Promega Biotec (Madison, Wis.). The Prime-a-Gene DNA
labeling system of Promega Biotec was used for labeling DNA.
RNA isolation and Northern blot analyses.
Bacterial cultures
were grown at 28°C in different media and harvested for total RNA
extraction at the Klett values indicated in the figure legends. The
procedures for RNA isolation and Northern blot analysis have been
described previously (21). The 875-bp HpaI-MluI fragment from pTB2 (Table 1) was used
as the rpoS probe, the 779-bp
EcoRV-SmaI fragment of pAKC924 was used as the
hrpNEcc probe (25), and the 183-bp
NdeI-SalI fragment of pAKC882 was used as the
rsmA probe (24).
S1 nuclease protection assay.
Ten picomoles of primer rpoS1
(5'-TGCCGACAGCAGCTGTATTGCTG-3'; complementary to the base
positions 
417 to 440 from the translational start codon) was end
labeled by polynucleotide kinase (Promega Biotec) and
[
-32P]ATP (New England Nuclear Life Science Products,
Boston, Mass.). The end-labeled probe was amplified by PCR using
end-labeled primer rpoS1, the opposing unlabeled primer rpoS2
(5'-TGGCAACAACCGATGCCACACGCG-3'; corresponding to the base
positions 633 to 610 from the translational start codon), and
pAKC942 as the template DNA. The conditions of PCR, hybridization, S1
nuclease digestion, and analysis of products were as described
previously (19).
Western blot analysis.
Bacterial strains were grown at
28°C in LB medium to a Klett value of ca. 180. Cells were collected
by centrifugation (4,000 × g, 10 min, 4°C),
resuspended in TESP buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride), and sonicated with a
Braunsonic 1510 sonicator (B. Braun Biotech Inc., Allentown, Pa.) at a
100-W output. The samples were centrifuged at 15,000 × g for 15 min at 4°C, and the supernatants were collected.
The protein concentration of the cell lysates was determined by using
the bicinchoninic acid (Pierce Corp., Rockford, Ill.) method with
bovine serum albumin as a standard. Double-strength sodium dodecyl
sulfate (SDS) loading buffer (100 mM Tris-HCl [pH 6.8], 200 mM
dithiothreitol, 4% SDS, 0.2% bromophenol blue, 2% glycerol) was
added, and the samples were boiled for 5 min. Proteins were
fractionated by 0.1% SDS-12% polyacrylamide gel electrophoresis and
transferred to a NitroBind nitrocellulose membrane (Micron Separations,
Inc., Westboro, Mass.). The blots were probed with antibodies raised
against E. coli sigma-38 (14) and visualized
with goat anti-rabbit antibody conjugated with alkaline phosphatase
(Promega Biotec).
Construction of RpoS strains by marker
exchange.
The plasmid pAKC941 (Table 1), wherein rpoS
was inactivated by inserting an 
-Spc cassette (29) at the
HpaI site (Fig. 1), was transferred into AC5006 and AC5070
by using helper plasmid pRK2013 (10). Transconjugants were
selected on minimal salts agar containing sucrose and supplemented with
spectinomycin. Isolates that were Spcr and Tcs
were selected for further studies. The marker exchange was confirmed by
Southern blot hybridization as well as Northern and Western blot
analyses.
Construction of rpoS-lacZ fusions and
-galactosidase assay.
A series of rpoS-lacZ
transcriptional fusions were constructed by cloning sequences from
nucleotides (nt) 736 to +24, from nt 736 to 429, and from nt
429 to +24 into promoter probe vector pMP220 (32) to
produce pAKC943, pAKC944, and pAKC945, respectively. The orientation of
the cloned fragments in these constructs was determined by restriction
mapping. -Galactosidase assays were carried out as described by
Miller (23), and the units of activity are expressed as
A420 units per min per
A600 unit.
Sensitivity to environmental stress.
The RpoS
strain and its parent were grown overnight in M9 medium at 28°C in a
shaker incubator. Stationary-phase cells were collected by
centrifugation and resuspended in the desired media (A600 of 1.0) and exposed to the indicated
stress. For H2O2 treatment cells were washed
and resuspended in 0.9% NaCl to an A600 of 1.0. H2O2 was added to a final concentration of 5 mM, and the mixture was incubated for 10 min (1, 18). To
find out the effects of starvation, cells were washed and resuspended
in M9 medium containing 0.025% glucose and incubated for 100 h.
For acid tolerance determination cells were grown overnight in LB broth
and resuspended in LB medium buffered with MES
(morpholineethanesulfonic acid; final concentration, 5 mM; pH 3.0) and
incubated for 3 min (1). After exposure to the respective
stresses in the incubation media at 28°C in a shaker incubator, the
bacterial suspensions were diluted and appropriate dilutions were
plated on LB agar.
Plant tissue maceration.
The celery petiole assay has been
previously described (28). The extent of tissue maceration
was visually estimated.
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RESULTS AND DISCUSSION |
Evidence for the occurrence of rpoS homologs.
Calcutt et al. (3) determined the nucleotide sequence
of rpoS and DNA flanking this gene for
E. carotovora subsp. carotovora strain 71 (hereafter strain 71). Their data showed that (i) the nucleotide
sequence of the rpoS structural gene is 81% identical to
that of the corresponding E. coli gene; (ii) the
Erwinia gene could encode a protein of 331 amino acid
residues that is 89 to 91% identical to RpoS of E. coli; and (iii) the putative nlpD gene is located
upstream of rpoS, as in E. coli. To
ascertain if homologs of strain 71 rpoS occur in other
Erwinia species, we conducted Southern blot hybridizations
using an internal fragment of strain 71 rpoS (the 875-bp
HpaI-MluI fragment; Fig.
1) as the probe. The data in Fig.
2 show that strain 71 rpoS DNA
hybridized with genomic fragments of all tested strains of
Erwinia species as well as E. coli,
Yersinia enterocolitica, and S. typhimurium.
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FIG. 1.
(A) Restriction map of the 4.5-kb DNA segment of
E. carotovora subsp. carotovora 71 containing rpoS, nlpD, and proS genes.
This genetic organization is deduced from the nucleotide sequence data
of Calcutt et al. (3). The locations and directions of the
genes are indicated by arrows. The site of -Spc insertion in the
HpaI site, resulting in the inactivation of rpoS,
is indicated. The 875-bp HpaI-MluI fragment was
used as the probe in Northern and Southern blot hybridizations. E,
EcoRI; H, HincII; Hp, HpaI; M,
MluI; P, PstI; S, SspI. (B) Nucleotide
sequence of the SspI-HpaI fragment containing
parts of nlpD and rpoS genes of strain 71. The
asterisk and +1 indicate transcriptional and putative translational
start sites, respectively. The putative 10 and 35 regions and some
restriction enzyme sites are shown. The numbers on the right refer to
the positions of the nucleotides.
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FIG. 2.
Southern blot hybridization of
HincII-digested chromosomal DNAs of wild-type strains of
Erwinia, Salmonella, Yersinia, and
E. coli with rpoS of E. carotovora subsp. carotovora 71. Lane 1, E. carotovora subsp. carotovora 71; lane 2, E. carotovora subsp. atroseptica Eca12; lane 3, E. carotovora subsp. betavasculorum
Ecb11129; lane 4, Erwinia chrysanthemi EC16; lanes 5 and 6, Erwinia amylovora E9 and Ea321; lane 7, Erwinia
rhapontici Er1; lane 8, Erwinia herbicola EH105; lane
9, Erwinia stewartii Es1; lane 10, S. typhimurium LT2; lane 11, Y. enterocolitica 8081v; and
lane 12, E. coli AE908.
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To detect if
rpoS genes are expressed in these
Erwinia species, cell lysates of bacteria were subjected to
Western blot analysis.
All the tested
Erwinia strains
produced protein species of about
38 kDa that cross-reacted with
polyclonal antibodies raised against
E. coli RpoS (data
not shown). Since this cross-reacting material
was absent in the
extract of an RpoS
E. carotovora subsp.
carotovora strain, we concluded that the
38-kDa protein
represents the RpoS species. These data, taken
along with the results
of Southern blot analysis (Fig.
2), establish
the occurrence of active
rpoS alleles in these plant-pathogenic
and plant-associated
bacteria.
The 5' end of the
rpoS transcript of strain 71 was localized
by an S1 nuclease protection assay at the A residue at base
525
relative to the translational start site (Fig.
3; also see Fig.
1). The calculated sizes
of the
rpoS transcripts presumed to be
initiated from this
start site matched well to the 1,600-base
size determined by Northern
blot assays (Fig.
4A). Upstream of
the
putative
rpoS transcriptional start site, there are a
10
(TATTCT) element and a
35 (TTGATT) element,
which are highly
similar to the consensus
E. coli
sigma-70 promoter. The results
of the S1 nuclease protection assay also
indicated that this putative
promoter is located within the coding
region of
nlpD in strain
71, as is the major
rpoSp1 promoter of
E. coli (
17).
To confirm
that this promoter is actually functional in
E. carotovora subsp.
carotovora, we made the following
transcriptional
rpoS-lacZ fusions:
an
SspI-
HpaI fragment corresponding to nt
736 to
+24 in pAKC943;
the upstream region, i.e., the
SspI-
PvuII fragment from nt
736
to
429 in
pAKC944; and the downstream region, i.e., the
PvuII-
HpaI
fragment from nt
429 to +24 in
pAKC945 (Fig.
1 and Table
1).
E. carotovora subsp.
carotovora AC5006 carrying each of these
plasmids or the
promoter probe vector, pMP220, was grown in LB
broth to a Klett value
of ca. 180, and
-galactosidase activity
was assayed. AC5006 carrying
pAKC943 produced 3,694 Miller units
of
-galactosidase activity, and
AC5006 carrying pAKC944 produced
5,568 Miller units. By contrast,
AC5006 carrying vector pMP220
produced 155 Miller units of
-galactosidase activity. The high
expression levels of the
rpoS-lacZ fusions in these constructs
and the location of
the consensus sigma-70 promoter strongly suggest
that the major
rpoS promoter (rpoSpM) is present within nt
561
and
525
(Fig.
1). The transcription of
rpoS from this promoter
was
stimulated during postexponential growth and by medium osmolarity
(data
not shown), as in
E. coli (
12,
22).
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FIG. 3.
S1 nuclease mapping of the putative transcriptional
start site of rpoS. Strain 71 was grown in LB medium to a
Klett value of ca. 200 for RNA isolation. Lane 1, 100,000 cpm of
end-labeled DNA probe with 20 µg of total RNA; lane 2, 100,000 cpm of
end-labeled DNA probe without RNA. The nucleotides on the left refer to
the nucleotide sequence beyond the 5' end. The asterisk denotes the A
residue at which transcription was presumed to be initiated.
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FIG. 4.
Northern blot analysis of rpoS (A) and
hrpNEcc (B) mRNA in E. carotovora subsp. carotovora AC5006 (lane 1) and its
RpoS derivative, AC5061 (lane 2). Total RNA was extracted
from bacteria grown in minimal salts medium supplemented with sucrose
(0.5% [wt/vol]) to a Klett value of 200. Each lane contained 10 µg
of total RNA.
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AC5006 carrying pAKC945 produced 887 Miller units of
-galactosidase
activity, which is about sixfold higher than the levels
produced by
bacteria carrying promoter probe vector pMP220 but
much lower than the
levels produced by bacteria carrying either
pAKC943 or pAKC944.
These data suggest that there may be another
weak promoter
(rpoSpW) behind rpoSpM. This conclusion is supported
by the S1 nuclease
protection assay, which revealed the presence
of weakly protected bands
within nt
166 and +19 (data not shown).
It is noteworthy that in
E. coli,
rpoS transcription is mainly
driven
by a single major promoter, rpoSp1, which is homologous
to the strain
71 rpoSpM promoter. Although there is a second putative
promoter about
251 bp downstream of rpoSp1 (
17), this promoter
apparently
does not play a significant role in the expression
of
rpoS
in
E. coli. A very similar situation probably occurs in
strain 71 since by Northern blot analysis we did not detect an
rpoS transcript smaller than 1,600 bases (Fig.
4A).
Effects of RpoS on the production of extracellular enzymes and
expression of hrpNEcc.
To analyze the regulatory
role of RpoS in E. carotovora subsp.
carotovora, we constructed an RpoS-deficient
mutant, AC5061, by a marker exchange procedure. The exchange of the
wild-type rpoS gene by inactivated
rpoS:: fragment was confirmed by Northern blot
hybridizations (Fig. 4A) as well as Western blot assay and Southern
blot hybridization (data not shown). The RpoS mutant
strain was also tested for various characteristics. The inactivation of
rpoS resulted in enhanced sensitivity to carbon starvation, acidic pH, and hydrogen peroxide (Table
2). These characteristics are typical of
RpoS bacteria (22).
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TABLE 2.
Survival of E. carotovora subsp.
carotovora AC5006 and its RpoS mutant (AC5061)
upon exposure to environmental stresses
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In the course of characterization of RpoS
strain AC5006,
we noted that the levels of extracellular Pel, Peh, and Cel were
higher
in RpoS
bacteria than in the RpoS
+ parent
strain. The results of quantitative assays (Table
3)
show that Pel-specific activity was
about twofold higher in AC5061
than in the RpoS
+
parent, AC5006. Similarly, the levels of
hrpNEcc transcripts
were higher in
RpoS
bacteria than in the RpoS
+ parent strain
(Fig.
4B). The RpoS
strain was more virulent (Fig.
5) than the RpoS
+ parent as
would be expected from the production of higher levels
of extracellular
enzymes, specially the pectinases. These observations
were unexpected
since RpoS is generally known to activate gene
expression in the
stationary-growth phase (
12). This pleiotropic
effect of
RpoS deficiency was somewhat reminiscent of the phenotype
of the
RsmA
strains of
E. carotovora subsp.
carotovora (
4,
5). We therefore
argued that RpoS
positively regulates
rsmA expression and that
the reduced
pool of RsmA in RpoS
bacteria accounts for the elevated
levels of extracellular enzymes,
the higher level of expression of
hrpNEcc, and the greater plant
tissue
maceration. The data presented below substantiate this
hypothesis.
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FIG. 5.
Plant tissue maceration induced by E. carotovora subsp. carotovora AC5006 and its
RpoS mutant, AC5061. About 2 × 108
cells were injected into the celery petiole at each inoculation site.
The inoculated celery petiole was incubated in a moist chamber at
25°C for 24 h. (A) water injection; (B) AC5006 injection; (C)
AC5061 injection.
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RpoS causes accumulation of rsmA transcripts.
The
data shown in Fig. 6 demonstrate that the
levels of rsmA transcripts were much lower in
RpoS strain AC5061 than in RpoS+ strain
AC5006. To assess the effect of rpoS gene dosage, the levels of rpoS and rsmA transcripts in the
RpoS strain, the parent strain carrying a chromosomal
copy of rpoS+, and the RpoS strain
carrying low-copy-number RpoS+ plasmid pAKC940 were
determined. Bacteria were grown in KB medium containing TC. Total
RNA was extracted from cells and subjected to Northern blot analysis.
The results were as follows. (i) The level of rpoS mRNA was
higher in the strain carrying multiple copies of
rpoS+ than in the strain carrying a single copy
of rpoS+ (data not shown). As expected, no
rpoS mRNA was detected in RpoS mutant AC5061.
(ii) The highest level of rsmA transcripts was observed in
bacteria producing the most rpoS mRNA (i.e., the strain carrying rpoS+ plasmid pAKC940) (Table
4).
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FIG. 6.
Northern blot analysis of rsmA mRNA in
E. carotovora subsp. carotovora AC5006 (lane
1) and its RpoS derivative, AC5061 (lane 2). Each lane
contained 20 µg of total RNA isolated from bacteria grown in LB
medium to a Klett value of ca. 200.
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To determine if there was a correlation between the dosage of
rpoS+ DNA and the levels of extracellular
enzymes, those bacterial
constructs were grown in minimal salts medium
containing sucrose
and TC, and culture supernatants were assayed for
enzymatic activities.
The level of Pel activity in AC5061 carrying
multiple copies of
rpoS+ DNA was 6% of the
activity found in AC5061 carrying the vector
(Table
3). A similar
effect of
rpoS+ copies occurred with Peh, Cel,
and Prt activities (data not shown).
We attribute this effect to
overexpression of
rsmA by
rpoS+
copies, followed by RsmA-promoted decay of the cognate transcripts
of
the extracellular enzyme genes.
We performed the following experiments to establish that the RpoS
effect was mediated via RsmA. We constructed an RpoS
RsmA
double mutant, and determined that the levels of Pel
were similar
in the mutant, AC5072, and its RpoS
+
counterpart, AC5070 (Table
3). We then transferred RpoS
+
plasmid pAKC940 or cloning vector pRK415 into the RpoS
RsmA
double mutant, AC5072. The RpoS
+
RsmA
strain, AC5070, carrying pRK415 was used as a
control. These
constructs were grown in minimal salts medium containing
sucrose
and TC, and culture supernatants were assayed for enzymatic
activities.
The levels of Pel activity were very similar in these
constructs
(Table
3). Similar results were obtained with Peh, Cel, and
Prt
activities (data not shown). These observations demonstrate that
in
the absence of a functional
rsmA allele RpoS does not have
a
significant effect on extracellular enzyme production. This
clearly
contrasts with the suppression of enzyme levels by RpoS
in the
RsmA
+ strain (see above). A straightforward
interpretation of these
observations is that the RpoS effect manifests
itself primarily
by regulating
rsmA expression.
To determine if multiple copies of the strain 71
rpoS+ gene have a generalized effect on
rsmA expression in different bacteria,
we introduced pAKC940
into
E. carotovora subsp.
carotovora
SCRI193,
E. carotovora subsp.
atroseptica
Eca12, and
E. carotovora subsp.
betavasculorum Ecb11129. Strain 71 carrying pAKC940 served
as
the positive control. The plasmid-carrying strains were grown
in LB
containing TC, and total RNA was extracted from cells grown
to a Klett
value of 100. The data in Table
4 show that
rsmA transcripts
were consistently higher in strains carrying the strain 71
rpoS allele than in cells carrying cloning vector pRK415.
Since high levels of
rsmA transcripts are only detected in
the presence of RpoS, we do not consider it merely coincidental
that
the
rsmA promoter consists of a
10 region comprising
CTAAACT
and no consensus
35 region (
5). This
10 sequence is typical
of sigma-S-dependent promoters (see, for
example, reference
7).
These observations and the
finding that the level of
rsmA expression
is higher in the
postexponential than in the exponential growth
stage (
20)
provide strong support for the hypothesis that
rsmA expression is positively affected by RpoS in
E. carotovora subsp.
carotovora. In fact, we have found
that the levels of
rsmA (
csrA)
transcripts in
S. typhimurium are reduced by RpoS deficiency (data
not
shown), raising the possibility that expression of
rsmA may
be under the control of this alternate sigma factor in other
enterobacteria
as well. We have initiated studies in collaboration with
Tony
Romeo to determine if
E. coli csrA, the
rsmA homolog, is also
controlled by this alternate sigma
factor. We should note that
expression of
rsmA occurs to
some extent in RpoS-deficient
E. carotovora subsp.
carotovora strains. Thus, the gene is controlled
by sigma-70
as well as sigma-S. In fact, this prediction is consistent
with
the observation that many sigma-S-controlled genes in
E. coli are also activated by sigma-70 (
33).
In summary, we have shown that some of the structural characteristics
of
E. carotovora rpoS and its functions are generally
similar to those in
E. coli. These similarities
notwithstanding,
our data reveal several novel features as well. For
example, the
levels of extracellular enzymes and
hrpNEcc transcripts and the
degree of plant
virulence are higher in RpoS-deficient bacteria
than in the
RpoS
+ parent. We have established that this effect
manifests itself
through the reduction in
rsmA expression.
The rationale for a
dual control of
rsmA expression by
sigma-70 and sigma-S can perhaps
be appreciated by invoking an
important housekeeping role of RsmA
as well as its function as a
regulator of secondary metabolites.
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ACKNOWLEDGMENTS |
This work was supported by the Food for the 21st Century Program
of the University of Missouri and by the National Science Foundation
(grant DMB-94-19403).
We thank Mick Calcutt for RpoS plasmids.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Plant Pathology, College of Agriculture, Food and Natural Resources, University of Missouri-Columbia, 108 Waters Hall, Columbia, Mo. 65211. Phone: (573) 882-2643. Fax: (573) 882-0588. E-mail:
achatterjee{at}psu.missouri.edu.
Journal series 12,671 of the Missouri Agricultural Experiment
Station.
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0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
