From the Department of Food Science and Technology,
Department of Molecular Biotechnology, Institute of Biotechnology,
Chonnam National University, Kwang-Ju 500-757, the
§ Department of Environmental Science, Hankuk University of
Foreign Studies, Yongin, Kyunggi-Do 449-791, and the ¶ Department
of Microbiology, Chonnam National University Medical School,
Kwang-Ju 500-190, South Korea
Received for publication, November 22, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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Elastase activity of Vibrio
vulnificus was highly dependent on growth phase, reached a
maximum during the stationary phase, and was regulated at the level of
transcription. The stationary phase production of elastase in
crp or rpoS mutants, which were constructed by
allelic exchanges, decreased about 3- and 10-fold, respectively.
However, the promoter activity of vvpE encoding elastase
was unaffected by those mutations in the log phase when analyzed using
a vvpE-lux fusion. A primer extension analysis revealed
that the transcription of vvpE begins at two different sites, consisting of putative promoter L (PL) and promoter S (PS). The
PL activity was constitutive through the log and stationary phases,
lower than the PS activity, and unaffected by the crp or
rpoS mutations. The transcription of PS, induced only in
the stationary phase, was dependent on RpoS. The mutation in
crp reduced the activity of PS; however, the additional
inactivation of crp did not influence the PS activity in
the rpoS mutant, indicating that CRP exerted its effects
through PS requiring RpoS. These results demonstrate that
vvpE expression is differentially directed by PL and PS
depending on the growth phase and elevated by RpoS and CRP in the
stationary phase.
The pathogenic marine bacterium Vibrio vulnificus is
the causative agent of food-borne diseases, such as life-threatening septicemia and possibly gastroenteritis, in individuals with underlying predisposed conditions. The mortality from septicemia is very high
(>50%), and death may occur within 1-2 days after the first signs of
illness (1). Several potential virulence factors including an
endotoxin, a polysaccharide capsule, iron-sequestering systems, a
cytolytic hemolysin, an elastase, a phospholipase A2, and other exotoxins have been identified for V. vulnificus (for a
recent review, see Ref. 2).
Among the putative virulence factors is an elastolytic
metalloprotease. Elastase, with a broad substrate specificity
including biologically important host molecules, has been suggested to
be an important virulence factor of various human pathogenic bacteria (3, 4). The characteristics of the elastase of V. vulnificus as a potential virulence factor have been studied primarily using the
purified protein in animal models (5-7). Injection of purified elastase reproduced many of the observed aspects of disease
caused by V. vulnificus, including dermonecrosis,
destruction of tissues, edema, and ulceration. However, when the
isogenic mutant deficient in the elastase was compared with the
parental strain for virulence, it appeared that elastase is less
important in the pathogenesis of V. vulnificus than would
have been predicted from examining the effects of administering
purified proteins to animals (4, 8). One possible explanation for this
contradiction is that the expression of vvpE encoding
elastase may not be sufficient at least under the conditions used;
hence the effects of the inactivation of the vvpE on the
virulence of the pathogen were not apparent. This possibility strongly
underscores the necessity of understanding the regulation of
vvpE expression. However, no promoter(s) of the
vvpE gene have been identified, and the molecular mechanisms by which the bacterium modulates the expression of the vvpE
gene have not yet been characterized. This lack of information on the regulatory characteristics of the elastase gene makes it difficult to
understand how the expression pattern and level of elastase varies
spatially and temporally during infection with viable V. vulnificus.
Accordingly, the influence of the growth phase on the expression of the
vvpE of V. vulnificus was examined in the present study. V. vulnificus isogenic mutants, which lack either the
cyclic AMP receptor protein
(CRP,1 a gene product of
crp) or both CRP and RpoS ( Strains, Plasmids, and Culture Media--
The strains and
plasmids used in this study are listed in Table
I. Escherichia coli strains
used for plasmid DNA replication or conjugational transfer of plasmids
were grown in Luria-Bertani (LB) broth or on LB broth containing 1.5%
(w/v) agar. Unless noted otherwise, V. vulnificus strains
were grown in LB medium supplemented with 2.0% (w/v) NaCl (LBS). When
required, appropriate antibiotics were added to the media as
follows: 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 10 µg/ml
tetracycline.
Measurement of Cell Growth and Elastase Activities--
Cultures
of V. vulnificus strains grown at 30 °C under aeration
and 5-ml samples were removed at the indicated times for determination of cell density and elastase activity. Growth was monitored by measuring the A600 of the cultures, and
measurement of the A600 of the diluted cultures
was done once the A was above 0.8. Cultures incubated for
12-16 h (A600 = 1.2) were harvested, and the
enzyme activities in the stationary phase were determined according to the procedures described previously (4). Averages and S.E. values were calculated from at least three independent determinations.
General Genetic Methods--
Procedures for the isolation of
plasmid DNA and genomic DNA and for transformation were carried out as
described by Sambrook et al. (9). Restriction and
DNA-modifying enzymes were used as recommended by the manufacturer (New
England Biolabs, Beverly, MA). DNA fragments were purified from agarose
gels using the Geneclean II kit (Bio 101, Inc., Vista, CA). Primary DNA
cloning and manipulation were conducted in E. coli DH5 Construction of crp::nptI Cartridge--
Plasmid
pUC::crp, which carries a V. vulnificus
chromosomal DNA fragment of 1.7 kb and contains the entire
crp gene,2 was
inactivated in vitro by the insertion of nptI
encoding for aminoglycoside 3'-phosphotransferase and conferring
resistance to kanamycin (10). For this purpose, a
HindIII-SalI fragment of the crp open
reading frame from pUC::crp was subcloned into pCVD442 (11), forming pKC9964. The 1.2-kb DNA fragment carrying nptI was isolated from pUC4K (Amersham Pharmacia
Biotech) and then inserted into a unique SphI site
present within the open reading frame of crp in pKC9964. The
resulting construct, pKC9965, is a derivative of pCVD442 and was
maintained in E. coli SY327 Generation of crp::nptI Mutant or crp::nptI
Conjugation was conducted using methods previously described (4, 13).
The desired transconjugants that showed a green colony formation on
thiosulfate citrate bile salts agar supplemented with kanamycin,
maltose (0.5%, w/v), neutral red (30 µg/ml), and sucrose (6%, w/v)
were selected. The transconjugants that were ampicillin-sensitive due
to the absence of the pKC9965, were confirmed for the presence of
nptI in the crp gene of the mutants by PCR using
a pair of primers, CRP9901 (5'-TACCTACTGGCCATGATCGATG-3') and CRP9902
(5'-CGGAATCTGAGAGGGTTTAGT-3') (see Fig. 2B). The V. vulnificus mutants chosen for further analysis were named KC74 for
the crp mutant and KC84 for the crp rpoS double mutant.
Construction of a vvpE-luxAB Transcriptional Fusion--
The
parent plasmid, pHK0011, carries a 3.2-kb
HindIII-BamHI fragment of promoterless
luxAB from Mini-Tn5 luxAB (14) in the broad host
range vector pRK415 (15) digested with HindIII (see Fig.
4A). The 697-base pair upstream regulatory region of
the vvpE from pKC980 (4) was amplified by PCR using the two
primers VVPE0002 (5'-GAGGTACCCCAAATGATTACTGATTTTCCC-3') and VVPE0003
(5'-CTTCTAGAAGACGGACACCATTTCTGCAGC-3') and inserted into pHK0011
digested with KpnI and XbaI to yield pHS0011.
Measurement of Cellular Luminescence--
pHS0011 was
transferred into ATCC29307 and the isogenic mutants by conjugation.
Cultures were grown to log phase (A600 = 0.6) or
stationary phase (A600 = 1.2), and then 1-ml
samples taken from each culture were diluted 100-fold with
phosphate-buffered saline (pH 7.4) and placed into cuvettes. A decanal
stock solution (0.3%, v/v) was prepared by adding decanal to a 1:1
mixture of water and ethanol. After adding 10 µl of the decanal stock
solution to cuvettes with the cells, the cellular luminescences were
measured with a Lumat model 9501 luminometer (Berthold, Wildbad,
Germany) and expressed using the arbitrary relative light units of the instrument.
RNA Purification and Northern Dot Blot Analysis of vvpE
Transcript--
Total cellular RNAs were isolated in different growth
phases using a Trizol reagent kit (Life Technologies, Inc.). For
a Northern dot blot analysis, a series of reactions was performed
according to standard procedures (9) with 20 µg of total RNA. A
1.2-kb HindIII-HindIII DNA probe representing the
internal sequences of the vvpE was labeled with
[ Primer Extension Analysis--
Primer extension experiments were
carried out with SuperScript II RNase H Growth Phase-dependent Expression of vvpE--
The
elastase activities of the ATCC29307 culture were analyzed at the
indicated time intervals (Fig.
1A). Elastase activity appeared in the exponential phase of growth and reached a maximum in
the stationary phase. The level of elastase activity increased about
10-fold in the stationary phase. This growth phase variation of
elastase possibly occurs either at the transcriptional level or
post-transcriptional level of vvpE expression.
The relative levels of the vvpE mRNA in the same amount
of total RNA isolated from ATCC29307 showed that the vvpE
mRNA levels increased as the bacterial culture entered the
stationary phase (Fig. 1B). This result suggests that the
increase in the level of elastase activity in the stationary phase was
correlated with the increase in the mRNA level of the
vvpE gene. This increase in the vvpE mRNA
level could be the result of an increase in the rate of mRNA
transcription initiation or increased mRNA stability. However, when
the total RNAs were separated by electrophoresis and hybridized with
the vvpE DNA probe, only one band corresponding to the full
size of the vvpE mRNA deduced from the DNA sequence (about 2.0 kb) appeared (data not shown). These results indicated that
the increased level of transcription initiation plays the major, if not
sole, role for the increased elastase activity on the entry of the
V. vulnificus cells into the stationary phase.
Construction and Confirmation of V. vulnificus crp Mutant or crp
rpoS Double Mutant--
The crp mutants KC74 and KC84 were
constructed by allelic exchange and confirmed by PCR (Fig.
2). The PCR analysis of the genomic DNA
from ATCC 29307 or KP101 with primers CRP9901 and CRP9902 produced a
1.2-kb fragment (Fig. 2B), whereas the genomic DNA from the
crp::nptI mutants resulted in an amplified DNA
fragment approximately 2.4 kb in length. The 2.4-kb fragment is in
agreement with the projected size of the DNA fragment containing
wild-type crp (1.2 kb) and the nptI gene (1.2 kb). The mutants exhibited a slow growth and no fermentation of many
sugars such as maltose and D-galactose, which is consistent
with the phenotypes of a typical crp mutant. To determine
the stability of the insertional mutation, KC74 or KC84 strains
were grown overnight without kanamycin selection. The inserted
nptI DNA was stably maintained, as determined by the
maintenance of kanamycin resistance and by the generation of the
appropriately sized DNA fragment by PCR (data not shown).
Effect of crp Mutation on Production of Elastase--
ATCC29307
and the crp mutant KC74 were grown to the stationary phase,
and the elastase activities of each culture were compared. Although the
general pattern of elastase production was similar in the two strains,
in the KC74 strain the level of elastase was lower (Fig.
3A). Whereas elastase activity
was present at about 30 units in the wild-type strain, the residual
level of elastase activity in KC74 corresponded to approximately
one-third of that in the wild type. The reduced production of elastase
suggested that CRP acts as a positive regulator in vvpE
expression. To characterize the role of CRP in more detail, the effects
of glucose and cAMP exogenously added to the wild type were studied.
During the stationary phase, the elastase activity in the wild-type
culture supplemented with 0.5% glucose alone was reduced to the level
of the KC74 but was restored by the addition of glucose and cAMP (1 mM) together (data not shown). These results indicate that
the expression of vvpE in V. vulnificus is under
the positive control of cAMP·CRP at least in the stationary
phase.
Effect of rpoS Mutation on Production of Elastase--
When the
rpoS mutant KP101 was compared with its parental wild type
during stationary growth, it produced much less elastase, and the level
of elastase activity was almost 10-fold less than that of the wild type
(Fig. 3A). This result indicated that the major portion of
elastase was produced by the RpoS-dependent promoter. Taking these results together led us to conclude that vvpE
is under the positive control of both CRP and RpoS during stationary growth. However, it was apparent that vvpE was not
completely repressed, even poorly expressed, in the absence of the
active gene product of rpoS. This suggested the existence of
at least one more promoter for vvpE, which is expressed in a
RpoS-independent manner in the stationary phase.
Relationship between CRP and RpoS in Regulation of vvpE--
To
examine whether the activation by CRP is through the
RpoS-dependent promoter or RpoS-independent promoter, the
level of elastase activity in the crp rpoS double mutant was
determined. The elastase activity in the crp rpoS double
mutant KC84 was present at 4 units (Fig. 3A). This level of
elastase activity was much lower than those reached by the wild type or
crp mutant KC74 but was indistinguishable from that in the
rpoS single mutant KP101 (Fig. 3A). The
expression of vvpE remained low unless the functional gene
product of rpoS was provided and was not significantly
affected by the additional inactivation of crp. This
observation indicated that activation of vvpE by CRP in the
stationary phase is mediated through the promoter whose activity
depends on RpoS.
Complementation of crp and rpoS Mutation--
For the
complementation test, plasmid pKC0004 or pHS0001 was constructed by
subcloning crp or rpoS, respectively, into
pRK415. The elastase activities of both KC74 (pKC0004) and KP101
(pHS0001) in the stationary phase were restored to levels comparable
with the wild-type level of ATCC29307 (Fig. 3B). Therefore,
the decreased elastase activity of KC74 and KP101 resulted from the
inactivation of functional crp or rpoS rather
than any polar effects on any genes downstream of crp or
rpoS.
The elastase activity in KC84 containing pHS0001 was restored to a
level similar to that in KC74, whereas the repressed level of elastase
in KC84 was not restored at all by the introduction of pKC0004 (Fig.
3B). This indicated that CRP was able to activate vvpE only when RpoS was present, supporting again our
previous hypothesis that CRP exerts its effects on vvpE
expression through the RpoS-dependent promoter.
Expression of vvpE in the Log Phase--
In the log phase, the
elastase activities of the wild type and its isogenic mutants were too
low to be precisely compared. To ensure accurate sensitivity, a
vvpE-luxAB transcriptional fusion was employed to determine
the elastase activity of the cultures in the log phase. For the
ATCC29307 strain containing pHS0011, luminescence activity was present
at about 90,000 relative light units (Fig.
4B). After cells were
introduced with pHS0011, the expression of luminescence in the
crp or rpoS mutant and the crp rpoS
double mutant cells did not differ significantly, and the levels of
luminescence in the mutants were comparable with that in the wild type.
Apparently, the promoter activity of vvpE in the log phase
cells of V. vulnificus was not dependent on RpoS and not
activated by CRP.
However, the levels of luminescence decreased in the crp or
rpoS mutant and the crp rpoS double mutant in the
stationary phase when compared with that in the wild type. The
magnitude of the decrease in luminescence in the mutants was similar to
the decrease in elastase activities, which were determined directly
(Fig. 4C). The luminescence of the wild type is much higher
in the stationary phase than in the log phase (Fig. 4, B and
C). These results indicate that the upstream region of
vvpE used for construction of the vvpE-luxAB
fusion is sufficient for the stationary phase induction and the CRP and
RpoS dependence of vvpE. These results also reconfirm the
previous observation that vvpE expression is mainly
regulated at the level of transcription.
Identification of Transcriptional Start Sites of vvpE with crp,
rpoS, or crp rpoS Background--
A single reverse transcript was
identified from the RNA isolated from the log phase cells of both the
wild type and all mutants tested (Fig.
5). This indicated that a transcriptional
start site, P1, located 61 base pairs upstream of the translational
initiation codon, was used for the transcription of vvpE in
the log phase. The putative promoter that constitutes P1 was named PL
to represent the log phase promoter. Apparently, the PL activities,
determined based on the intensity of the bands of the reverse
transcripts, were unaffected by the inactivation of crp and
rpoS. This observation is consistent with the results
obtained from the vvpE expression studies with a
vvpE-lux fusion, as presented above.
The primer extension analysis performed with RNA prepared from
stationary cells revealed two different transcriptional start sites;
one was identical to Pl, and the other, P2, was 3 base pairs apart from
P1. The promoter S (PS), consisting of P2 and representing the
stationary phase promoter, was not expressed in the log phase and was
induced only when cells entered the stationary phase. In contrast to
P1, the band corresponding to P2 was not detected with the RNA from the
rpoS mutant or crp rpoS double mutant and was
reduced in its intensity with the RNA from the crp mutant.
This observation indicates that the induction of PS is entirely
dependent on RpoS, and CRP activates PS only in the presence of
functional RpoS. However, the activity of PL, which was much lower than
that of PS, appeared to be constitutive throughout the log and
stationary growth phases.
Consequently, the transcription of the vvpE of V. vulnificus was initiated by two different types of promoters, PL
and PS, in a growth phase-dependent manner. The basal level
of expression of vvpE was directed by PL, independent of
RpoS and CRP, and remained low throughout the log and stationary growth
phases. In addition to this basal level, more vvpE
expression was induced by PS in the stationary phase, which was under
the control of both CRP and RpoS. This differential utilization of two
promoters may permit precise levels of elastase in response to
modifications of the environment and the growth stage.
There have been several different lines of evidence leading to the
hypothesis that elastase is an important, if not essential, component
of virulence for V. vulnificus. Previously, the function of
elastase during an infectious process, rather than the artificial system of injecting purified proteins, has been examined by
constructing isogenic elastase mutants of V. vulnificus and applying the molecular version of Koch's
postulates (4, 8). When the isogenic vvpE mutants were
compared with the parental strain for virulence in animal and cell
culture models, the mutants did not show any significant differences in
any aspect of the disease process. The major problem to be addressed is
the discrepancy between these infection experiments and the studies
that primarily relied on injection of proteins into animals. However,
it is important to note that the expression of vvpE encoding
elastase of V. vulnificus remains at a low level under
certain conditions, such as in the log phase of growth. Although other
explanations are possible, the lack of significant difference in
virulence between the vvpE mutant and wild-type parent could
be related to this low level of elastase expression in the conditions
used. This possibility, which remains to be proved, strongly
underscores that the expression pattern and level of putative virulence
attributes during an infectious process must be examined to identify
their roles in pathogenesis.
In the present study, it was found that the expression of
vvpE encoding elastase of V. vulnificus is
regulated as a function of the growth phase and reaches a maximum in
the stationary phase. It has been previously reported that
Enterobacteriaceae undergoes a global modification of their gene
expression pattern at the onset of the stationary phase (16, 17). As a
result, the bacteria acquire tolerance to a number of chemical and
physical stresses, such as extreme temperatures, oxidative
agents, hyperosmotic tension, and nutritional starvation. The
key regulator for the expression of the genes responsible for this
increased tolerance is RpoS ( This report has shown that the expression of the V. vulnificus elastase gene is dependent on CRP. Besides regulating
the synthesis of many catabolic enzymes, CRP regulation has also been
observed in the synthesis of the toxin proteins of several pathogenic
bacteria (24-26). It was found that CRP affects the
RpoS-dependent expression of vvpE at PS. There
are several possible ways for CRP to affect PS. One is by binding
directly to PS to stimulate open complex formation by the RpoS-RNA
polymerase holoenzyme. Because purified V. vulnificus CRP is
unavailable, in vivo footprinting was performed to identify
the CRP binding region in the vvpE upstream region (27).
Dimethylsufate methylation-protection footprinting revealed no
difference in the protection pattern in the upstream region of
vvpE between KC74 and its parental wild type (data not
shown). In agreement with this, no convincing consensus sequences for CRP binding are identified in the upstream region of vvpE
(Fig. 6.). These observations suggest the
possibility that CRP activates PS indirectly by increasing the cellular
level of either RpoS or an unidentified third component(s) in the
stationary phase. The unidentified component(s) may then modulate the
level of RpoS or bind directly upstream of the vvpE to
activate the PS.
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s, a gene product
of rpoS), were constructed, and the promoter activities of
the vvpE in these mutants were then analyzed. The vvpE promoters were also mapped through a primer extension
analysis of the vvpE transcripts in different growth phases.
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Plasmids and bacterial strains used in this study
,
and restriction mapping was used to confirm that transformants
contained the appropriate plasmids. PCR amplification of DNA was
performed using a GeneAmp PCR system 2400 (PerkinElmer Life
Sciences) and standard protocols.
pir
(12).
rpoS Double Mutant by Allelic Exchange--
To generate the
crp::nptI mutants in V. vulnificus by
homologous recombination (see Fig. 2A), E. coli
SM10
pir, tra (11, 12) was transformed with
pKC9965 and used as a conjugal donor to V. vulnificus
ATCC29307. For the construction of the crp rpoS double
mutant, V. vulnificus KP101, an isogenic rpoS
mutant of ATCC29307, was used as a recipient. The KP101, in which two
thirds of the rpoS open reading frame were deleted, was
constructed by replacing rpoS on the chromosome with
rpoS.3
-32P]dCTP using the Prime-a-gene labeling system
(Promega, Madison, WI) and used for hybridization as described
previously (4). The blots were visualized and quantified using an
image analyzer (BAS1500 model; Fuji Photo Film Co. Ltd., Tokyo,
Japan) and the TINA (version 2.0) program.
reverse
transcriptase (Life Technologies, Inc.) according to Sambrook et
al. (9). The 24-base primer used was VVPE9905
(5'-GACGTTGATTGAGTTTCATTATCG-3') located within the open reading frame
of vvpE. The primer was end-labeled with
[
-32P]ATP using T4 polynucleotide kinase (Life
Technologies, Inc.). The cDNA products were purified and resolved
on a sequencing gel alongside sequencing ladders generated with the
same primers used for the primer extension. The nucleotide sequence of
the plasmid DNA of pKC980 (4) was determined using the dideoxy chain
termination method with TopTM DNA polymerase (Bioneer,
Seoul, Korea) following the manufacturer's protocols. The gels
were then dried and exposed by the same procedure as used for Northern
dot blots.
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Fig. 1.
Growth kinetics and growth
phase-dependent expression of vvpE.
Samples removed in different growth phases, as indicated, from a
culture of strain ATCC29307 were analyzed for elastolytic protease
activity (A) and for Northern dot blot analysis
(B). Relative amounts of the vvpE transcript of
each dot were presented using the amount of the vvpE
transcript at 16 h as 100%. , cell density;
, elastase
activity.
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Fig. 2.
Diagram of allelic exchange and confirmation
of the crp::nptI mutants.
A, homologous recombination between chromosomal
crp gene from strain ATCC29307 or KP101 and pKC9965.
Dashed lines, chromosomal DNA; solid line,
plasmid DNA; open boxes, the target crp gene;
shaded boxes, the nptI gene; open
arrows, locations of the oligonucleotide primers used for
confirmation of the nptI insert; X, crossover.
B, PCR analysis of ATCC29307 and isogenic mutants generated
by allelic exchange. Molecular size markers (1-kb ladder, Promega) and
PCR products (in kb) are indicated.
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Fig. 3.
Dependence of elastase production of V. vulnificus in the stationary phase on CRP and RpoS.
A, elastase activities were determined from ATCC29307 and
each isogenic mutant as indicated. B, complementation of the
mutants with functional crp (pKC0004) or rpoS
(pHS0001) as indicated. For both panels, samples removed at
A600 of 1.2 were analyzed for elastase activity
on each bar. Error bars represent the S.E. WT, wild
type.
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Fig. 4.
Promoter activity of vvpE in
the log phase assessed using a vvpE-luxAB
transcriptional fusion. A, construction of
vvpE-lux fusion plasmid pHS0011. Filled blocks,
the lux DNA; open blocks, the vvpE
DNA; solid lines, the vector DNA used. Hybridizing locations
of the oligonucleotide primers used for the PCR are depicted by
open arrows. Transcription start sites determined by the
primer extension analysis are presented as P1 and P2. D,
DraI; E, EcoRI; K,
KpnI; X, XbaI. B and
C, cellular luminescences were determined from ATCC29307 and
the isogenic mutants as indicated. Cultures in log (B) and
stationary (C) phases of growth were used to measure
luminescences. Error bars represent the S.E. obtained from at least
three independent determinations. WT, wild type;
RLU, relative light units.
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Fig. 5.
Identification of transcription start sites
of vvpE. The transcription start sites were
determined by the primer extension of the RNA derived from ATCC29307
and the isogenic mutants as indicated. Total RNAs were prepared in the
log phase (L; A600 = 0.6) and
stationary phase (S; A600 = 1.2) of
each culture. Lanes G, A, T, and
C represent the nucleotide sequencing ladders of pKC980.
Asterisks indicate the sites of the transcription start for
P1 (G) and P2 (T), respectively. WT, wild type.
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S or
38), a
stationary phase-specific sigma factor (18, 19). Recently, a direct
role for RpoS in regulating the expression of numerous virulence
factors has been established for a number of pathogenic Enterobacteriaceae (20-22). From a standpoint of bacterial
pathogenesis, the finding that numerous virulence factors are regulated
by RpoS would be not surprising. When bacteria invade the human body, the scarcity of specific nutrients and increased stresses imposed by
the host immune defense system would be encountered. As such, the
bacteria must survive these stresses to multiply and finally result in
local damage and systemic disease. This survival often involves
coordinate expression of sets of assorted genes (23), and many of these
genes are probably regulated by RpoS.
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Fig. 6.
Sequence of vvpE upstream
region. Transcription start sites of the log phase (P1) and
stationary phase (P2) are indicated by bent arrows. The
positions of the putative 10 and
35 regions are
underlined with continuous and broken
lines for the promoters PL and PS, respectively. The ATG
translation initiation codon and putative ribosome binding site (AGGA)
are indicated in boldface. ORF, open reading
frame.
In E. coli, the cellular levels of RpoS increase at the onset of the stationary phase, but the regulation mechanisms that modulate the level of the RpoS protein appear to be complicated and are not clearly understood (for a recent review, see Ref. 28). The cellular content of RpoS is regulated at the level of rpoS transcription, translation, and RpoS proteolysis in E. coli. Although some contradictions have also been observed, it has been proposed that the transcriptional control of rpoS involves the CRP·cAMP complex as a negative regulator in E. coli. However, it has not yet been established whether CRP is involved in the modulation of the cellular level of RpoS in V. vulnificus. Experiments to further examine the effects of CRP on the level of the expression of rpoS and on the level of RpoS in V. vulnificus are now underway.
Because the expression of vvpE from PL is constitutive
through the log and stationary phases, PL would be transcribed most likely by the RNA polymerase holoenzyme with the homolog of RpoD (70) that is the major housekeeping sigma factor in
E. coli. However, the exact type of sigma factor associated
with the RNA polymerase for the transcription of PL has not yet been
determined. Several hundred base pairs upstream of vvpE were
sequenced and analyzed by comparing them with putative promoter
sequences suggested previously on the basis of homology to a consensus
from E. coli (Fig. 6). A Shine-Dalgarno site (AGGA) and
potential promoter sequences consisting of
10 and
35
segments separated by 17 nucleotides have been assigned. The assigned
sequences for
35 (TTCTGA or TGAACC) scored a 50% homology to the
35 consensus sequences (TTGACA) of the promoters recognized by the
RNA polymerase with RpoD. However, no sequence of the
10 regions
assigned with respect to either P1 or P2 revealed a reasonable homology
to
10 consensus sequences. It has been reported that the sequences
for promoters transcribed by RNA polymerase with RpoS are notoriously
weakly conserved. It is also noteworthy that the expression level of
vvpE from PL is very low, and the weak homology of the
promoter sequences to the consensus sequences may explain this low
level of expression of vvpE from PL. However, additional
work is needed to clarify whether these regions really act as RNA
polymerase recognition sites.
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FOOTNOTES |
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* This work was supported by a grant to S. H. C. from the Korea Research Foundation (2000-041-G00101), Republic of Korea.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Food
Science and Technology, Inst. of Biotechnology, Chonnam National University, Kwang-Ju 500-757, S. Korea. Tel.: 82-62-530-2146; Fax:
82-62-530-2149; E-mail: shchoi@chonnam.chonnam.ac.kr.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M010567200
2 J. H. Rhee, manuscript in preparation.
3 K.-H. Lee, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: CRP, cyclic AMP receptor protein; PCR, polymerase chain reaction; kb, kilobase(s); PL, promoter L; PS, promoter S.
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