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Previous Article | Next Article 
Journal of Bacteriology, March 2001, p. 1688-1693, Vol. 183, No. 5
Marine Biology Research Division, Scripps
Institution of Oceanography, La Jolla, California 92093-0202
Received 10 October 2000/Accepted 13 December 2000
We are currently investigating the role of ToxR-mediated gene
regulation in Photobacterium profundum strain SS9. SS9 is a moderately piezophilic ("pressure loving") psychrotolerant marine bacterium belonging to the family Vibrionaceae. In
Vibrio cholerae, ToxR is a transmembrane DNA binding
protein involved in mediating virulence gene expression in response to
various environmental signals. A homolog to V. cholerae
ToxR that is necessary for pressure-responsive gene expression of two
outer membrane protein-encoding genes was previously found in SS9. To
search for additional genes regulated by ToxR in SS9, we have used RNA
arbitrarily primed PCR (RAP-PCR) with wild-type and toxR
mutant strains of SS9. Seven ToxR-activated transcripts and one
ToxR-repressed transcript were identified in this analysis. The cDNAs
corresponding to these partial transcripts were cloned and sequenced,
and ToxR regulation of their genes was verified. The products of these
genes are all predicted to fall into one or both of two functional
categories, those whose products alter membrane structure and/or those
that are part of a starvation response. The transcript levels of all
eight newly identified genes were also characterized as a function of
hydrostatic pressure. Various patterns of pressure regulation were
observed, indicating that ToxR activation or repression cannot be used
to predict the influence of pressure on gene expression in SS9. These results provide further information on the nature of the ToxR regulon
in SS9 and indicate that RAP-PCR is a useful approach for the discovery
of new genes under the control of global regulatory transcription factors.
In Vibrio cholerae, the
transmembrane DNA binding protein ToxR and the associated membrane
protein ToxS are best known for their central role in the environmental
regulation of virulence gene expression (15, 37, 38, 50).
However, both ToxR and ToxS are widely distributed proteins among the
Vibrionaceae, and their homologs have been found in eleven
members of the family (32, 33, 41, 44, 55). Many of these
additional species are human or fish pathogens. Vibrio
parahaemolyticus, for example, is a major cause of gastroenteritis
associated with seafood consumption (26). Other
ToxR-containing bacteria are Vibrio fischeri, a bioluminescent bacterium associated with the light organs of certain fish and squid (16, 46), and Photobacterium
profundum strain SS9, a deep-sea bacterium originally isolated
from amphipod crustaceans (4, 14). Since many of the
species containing ToxR are nonpathogenic to mammals, and since the
ToxR-regulated virulence genes in V. cholerae (but not the
toxRS operon) are largely acquired by horizontal gene
transfer (27, 51), the ToxR regulatory system is not likely to have first evolved for mammalian colonization. However, little is known about its role outside of virulence in higher organisms.
Alkaline phosphatase gene fusion studies and toxR mutant
analyses indicate that the V. cholerae ToxR regulon includes
over 20 genes (20, 25, 29, 37, 42, 43). All but one of these genes require ToxR for expression, with the remaining gene being
repressed by ToxR. These genes can be divided into those that do or do
not depend on the transcription factor ToxT, whose gene expression is
activated by ToxR and ToxS, along with TcpP and TcpH, another pair of
membrane-localized proteins related to ToxR and ToxS, respectively
(21). The ToxT-dependent branch of the regulon includes
the expression of toxin and colonization genes associated with the
CTX Outside of V. cholerae, much less is known about the ToxR
regulon. In V. parahaemolyticus and Vibrio
vulnificus, ToxR is needed for hemolysin gene expression
(32, 34). SS9 ToxR and V. vulnificus ToxR, like
V. cholerae ToxR, regulate the inverse expression of two
OMPs (32, 55). In the case of SS9, the genes encoding these OMPs have been isolated. SS9 ompL is ToxR activated,
and its product is 50% identical and 63% similar to V. cholerae OmpU (47, 54), whereas SS9 ompH
is ToxR repressed, and its product is 30% identical and 53% similar
to V. cholerae OmpT (5, 33). Western analysis
indicates that proteins similar to OmpL are present in V. fischeri, Vibrio furnissii, Photobacterium angustum, and Photobacterium leiognathi, as well as in V. cholerae (55). So a minimum conserved unit for the
ToxR regulon among diverse members of the Vibrionaceae could
include ToxRS and one or two omp genes.
The ToxR regulation of omp gene expression contains
similarities and differences with the well-characterized
ompFC control by OmpR and EnvZ in Escherichia
coli (19, 30). The cytoplasmic amino-terminal DNA
binding domain of ToxR shares significant identity with the OmpR family
of regulatory proteins (38), although, unlike OmpR, ToxR
lacks a phosphoacceptor domain and is not believed to function as a
two-component system response regulator. Also like ToxR, OmpR has been
found to be a global regulator of gene expression in both E. coli and Salmonella enterica (see, for example, references 3, 31, 49, and 57).
ToxR regulation in SS9 is responsive to environmental cues different
from those for V. cholerae ToxR. Whereas the V. cholerae ToxR circuit is controlled by medium salinity and amino
acid content and also by temperature and pH in the case of the ToxT
branch (37), SS9 ToxR modulation of ompL and
ompH expression is controlled by hydrostatic pressure.
ompL transcription is optimal at atmospheric pressure, while
at 28 MPa ompL expression is low and ompH
transcription is derepressed (4, 11, 12, 54, 55). Thus,
the ToxR system in the piezophilic deep-sea bacterium SS9 appears to
have evolved mechanisms for responding to the changes in pressure that the organism encounters as it is moved up or down within the water column.
In order to further assess the nature of the ToxR regulon in SS9, we
have chosen to use an RNA arbitrarily primed PCR (RAP-PCR) approach.
This technique uses random oligonucleotide primers to create a unique
cDNA fingerprint for a given microorganism in a particular
physiological state, thus providing a powerful tool for assessing
differential gene expression in prokaryotes. It has recently been used
to examine microbial responses to stress induction, soil growth
conditions, and host infection (7, 10, 18, 56). Here we
report on using RAP-PCR to reveal differences in gene expression
between wild-type and toxR mutant strains of SS9 both at
atmospheric pressure and at 28 MPa, the SS9 pressure optimum. These two
different conditions were selected because we were interested in
uncovering additional genes influenced by ToxR in SS9 as well as
examining the nature of high-pressure gene regulation by ToxR. Eight
additional SS9 ToxR-regulated genes are described along with their
significance to understanding the nature of the SS9 ToxR regulon.
Bacterial strains and plasmids.
P. profundum SS9
strains DB110 and TW30 were used in these studies. DB110 is a
rifampin-resistant derivative of wild-type SS9 (11), and
TW30 contains a 478-bp deletion within the toxR gene of
DB110 (55). Strains were aerobically cultured in 2216 medium (28 g/liter; Difco Laboratories, Detroit, Mich.) at 15°C. Anaerobic growth of SS9 cultures was performed at 0.1 or 28 MPa (9°C)
in 2216 medium supplemented with glucose (22 mM) and 0.1 M HEPES, pH
7.5. High-pressure growth of SS9 cultures was performed as previously
described (8). Plasmid pDB1231 contains the SS9 toxRS operon from pTW15 (50) cloned into the
BamHI-XhoI site of the broad-host-range vector
pKT231 (2). The antibiotic kanamycin was used at a
concentration of 200 µg/ml during the cultivation of TW30 with pDB1231.
RNA preparations.
Mid-exponential-phase cultures were
harvested and RNA was extracted using RNAzol B (Tel-Test; Friendswood,
Tex.), a guanidinium thiocyanate-phenol-based reagent, according to the
manufacturer's instructions. RNA was quantitated using a UV
spectrophotometer, and its quality was assessed on an agarose gel prior
to experiments. When necessary, RNA was treated with DNase I (Ambion;
Austin, Tex.) according to the manufacturer's recommendations.
RAP-PCR.
RAP-PCR was performed essentially as described by
Fleming et al. (18). Arbitrary 10-mer primer kits with G+C
contents of 50% (GEN1-50) and 60% (GEN1-60) were obtained from
Genosys Biotechnologies (The Woodlands, Tex.). First-strand cDNA
synthesis was performed using 200 ng of heat-denatured (10 min, 65°C)
RNA in 20-µl reaction mixtures containing the following: 200 µM
concentrations of each deoxynucleoside triphosphate, 5 mM
dithiothreitol, 50 U of Moloney murine leukemia virus-reverse
transcriptase (RT) (Ambion), 1× RT reaction buffer, and 0.4 µM
arbitrary primer. The first-strand reaction was performed in an MJ
Research minicycler (PTC-150) as follows: touchdown from 50 to 30°C
in 45-s increments followed by 1 h at 37°C. Second-strand
synthesis was performed in 30-µl reactions containing the following;
3 µl of first-strand reaction, 0.3 U of Taq polymerase
(Gibco BRL, Rockville, Md.), 20 µM concentrations of each
deoxynucleoside triphosphate, 6% dimethyl sulfoxide, 2 µM
concentrations of each primer (primer 1 is from first-strand synthesis;
primer 2 is a different randomly chosen primer), 2.5 µCi of
[ ompL RAP-PCR.
Primers ompLf (5'
CATCACTGAAGATTTCTAC) and ompLr (5' CTACAGTGTTACGTGA)
were synthesized (Integrated DNA Technologies, Coralville, Iowa)
and were designed to amplify a 513-bp internal fragment of the
ompL gene. ompLr was used in first-strand cDNA synthesis, and both primers were used in second-strand synthesis as described above.
Isolation and cloning of RAP-PCR fragments.
Following
identification of regulated fragments, bands of interest were isolated
in the following manner. The developed film was placed on the dried
gel, with phosphorescent marks used for alignment. Bands were excised
from the dried gel using a scalpel and were placed into a
microcentrifuge tube containing 50 µl of Tris-EDTA for overnight
elution at room temperature. A portion of the eluted fragment was used
in a subsequent PCR containing the original primers used in RAP-PCR
amplification. Following secondary PCR, fragments were cloned using the
TOPO-TA cloning kit (Invitrogen, Carlsbad, Calif.) according to the
manufacturer's instructions. Clones verified for the correctly sized
insert were then sequenced to determine identity.
DNA sequencing.
DNA sequences were determined by thermal
cycle dideoxy sequencing with an ABI373A automated sequencer using
fluorescently labeled terminators (Perkin-Elmer, Branchburg, N.J.) and
T7 primers. Global similarity searches were performed with the BLAST
network service (1).
Confirmation of regulation of RAP-PCR fragments by ToxR.
To
verify differential expression of the fragments identified in RAP-PCR,
the approach described by Benson et al. was used (7). This
is a quantitative RAP-PCR method that relies first on the creation of
random cDNA molecules from an RNA population, followed by gene-specific
PCR. Briefly, first-strand cDNA was created using RNA from SS9 strains
following the same RT conditions stated above, with the exception that
2 µM concentrations of random hexamers were used in place of a single
arbitrary primer. RAP-PCR was then performed, under the same conditions
as those already described, on the first-strand cDNA population by
using custom-made 18-mer primer pairs specific for each sequence being
tested. Synthesis of a PCR product of the correct size and expression
pattern was considered verified for differential expression of the
original RAP-PCR result. All quantitative RAP-PCR experiments were
performed in duplicate to ensure reproducibility.
Nucleotide sequence accession numbers.
The sequences of the
partial gene fragments identified in this work are deposited in GenBank
under accession no. AF307974 to AF307981.
RAP-PCR analysis of ToxRS regulated sequences.
Differential
gene expression in ToxR+ and ToxR
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.5.1688-1693.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
RNA Arbitrarily Primed PCR Survey of Genes
Regulated by ToxR in the Deep-Sea Bacterium Photobacterium
profundum Strain SS9
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
genome (51) and the vibrio pathogenicity island,
which has also been proposed to represent a filamentous phage genome
(27). The ToxT-independent branch of the ToxR regulon consists of two outer membrane protein (OMP)-encoding genes,
ToxR-activated ompU and ToxR-repressed ompT
(13, 33, 37, 47).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-32P]dCTP, 1× PCR buffer, and 1.5 mM
MgCl2. Cycles for RAP-PCR were 94°C for 30 s, 40°C
for 2 min, and 72°C for 1 min for 40 cycles, with a 10-min extension
at 72°C on the final cycle. All products were stored at 
20°C for
up to 1 day prior to electrophoresis. RAP-PCR samples were heated at
92°C for 2 min after the addition of loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF). Samples
were then loaded onto a 5% denaturing acrylamide gel (20 by 42 cm)
containing 7 M urea and were electrophoresed at 1,700 V for ~2 h
until the xylene cyanol band reached the bottom of the gel. The gels
were subsequently dried, marked asymmetrically with a phosphorescent
pen for orientation, and exposed to film (Eastman Kodak, Rochester,
N.Y.) for 1 to 3 days at 80°C.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
SS9 strains
was examined in cells grown aerobically in a nutrient-rich medium at
15°C and atmospheric pressure. These conditions were employed because
the previously identified ToxR-regulated ompH and
ompL genes are well expressed in either ToxR+
(ompL) or ToxR (ompH) strains under
these growth conditions (54, 55).
) SS9 strains grown at
0.1 MPa by using RAP-PCR with primers specific to an internal portion
of the ompL sequence. As expected, a 513-bp fragment of
ompL was expressed in DB110 but not in TW30 (Fig. 1). A second control testing for
reproducibility of the technique was also performed. The first RAP-PCR
experiment was performed in duplicate using the same primer sets with
RNA that was isolated, from strains of DB110 and TW30 grown aerobically
at atmospheric pressure (0.1 MPa), on separate occasions. This
experiment yielded nearly identical results (data not shown);
therefore, to increase the numbers of primer sets examined, all
subsequent RAP-PCR experiments were performed only once.
View larger version (85K):
[in a new window]
FIG. 1.
RAP-PCR performed using ompL-specific
primers. Lane 1, DB110; lane 2, TW30. Molecular size standards are
given at the left in base pairs. The arrow indicates the 518-bp
ompL fragment.
strains can be seen in Fig.
2. Differentially expressed RAP-PCR fragments were purified from each gel and were reamplified using the
appropriate primers to both verify that the correct fragment size was
isolated and to facilitate cloning.
|
Verification of differential gene expression by ToxR in SS9.
RAP-PCR fragments were cloned and sequence analysis was performed for
further transcription analysis, as well as gene characterization. The
ToxR regulation of each of the eight identified genes was then
confirmed using gene-specific PCR primers and a new set of ToxR+ and ToxR strains. cDNA was prepared
from the strain TW30 (with a deletion of toxR) containing
either the vector pKT231 (2) or pKT231 harboring the
entire toxRS operon (pDB1231). As a control the regulation
of ompL was examined in these two strains, and as expected, ompL amplification was detected in TW30(pDB1231) but not in
TW30(pKT231) (Fig. 3A). When all of the
RAP-PCR clones were examined using this method, the pattern of
toxR-regulated gene expression matched that seen in the
original RAP-PCR analyses (Fig. 3B). Therefore, these experiments
confirmed that all eight genes are indeed regulated, either directly or
indirectly, by ToxR.
|
Characterization of the ToxR-regulated genes.
Table
1 presents a summary of related genes
found within the National Center for Biotechnology Information nucleic
acid sequence databases available through the BLAST network service
(1) which showed greatest similarity to the ToxR-regulated
partial gene sequences. The P values (28)
obtained from BLASTX ranged from 6 × 1045 to 0.41.
|
Examination of ToxR regulation of the identified genes at high
pressure.
Because ToxR modulates high-pressure expression of two
OMP genes, ompH and ompL (55), it
was of interest to determine whether any of the genes identified here
are also pressure regulated. RNA was prepared from ToxR+
and ToxR strains (DB110 and TW30, respectively) cultured
anaerobically at 0.1 and 28 MPa and subjected to gene-specific PCR.
Once again, the expression of ompL was tested as a control,
and as expected, based on previous work (55), it was found
that only DB110 (ToxR+) cells grown at 0.1 MPa expressed
ompL (Fig. 4A). However, when gene expression of the RAP-PCR clones was examined as a function of
pressure and strain type, a variety of gene expression patterns emerged
(Fig. 4B and Table 1). For example, the expression of the
ToxR-repressed gene (clone 1-4) was absent in the ToxR+
strain but was present in the ToxR strain regardless of
pressure. And curiously, except for clone 2-1, all of the
ToxR-activated genes were expressed in the ToxR strain,
and in most cases this expression was highest at elevated pressure. So
the pattern of both ToxR regulation and pressure regulation was
distinct for most of the genes identified in this study from the
previously characterized ToxR-regulated ompH and ompL genes (54, 55). These results suggest that
the regulation of many of the identified genes is complex. It is worth
noting that our original RAP-PCR analyses were performed using cells cultured aerobically, while the pressure study was performed using cells cultured under anaerobic conditions, owing to oxygen toxicity at
high pressure, regardless of pressure regimen. Under these culture
conditions, the strains were incubated in sealed plastic bulbs which
contain little oxygen but contain glucose to allow for enhanced
fermentative growth. It may be that under these different physiological
conditions the reliance of many of the identified genes on ToxR is
lost.
|
Concluding remarks. This study used RAP-PCR to identify genes under the control of the global transcriptional regulator ToxR. We have identified eight additional ToxR-regulated genes in SS9, bringing the total of known ToxR-regulated genes in this microorganism to 10. To our knowledge, this is the first application of this technique for use in identifying genes under the control of a particular transcriptional regulator. Under circumstances when genome sequence information is not available and it is not possible to explore global regulation of gene expression using DNA microarrays (9) or macroarrays (48), RAP-PCR should be considered.
Many of the identified genes have similarity to genes present in the recently completed V. cholerae genome (22), although none of these genes are known to be ToxR regulated in V. cholerae. Because there is no well-conserved consensus ToxR binding sequence (13, 33) and because many of the genes identified could be indirectly controlled by ToxR, it is not known at this time whether the related genes in V. cholerae are also subject to ToxR control. Further comparisons of ToxR-regulated genes among SS9 and V. cholerae are needed to better understand the nature and evolution of this important regulon. However, it is already clear that the SS9 ToxRS system should be viewed in the context of membrane structure and nutrient acquisition and that alteration of gene expression by ToxR and hydrostatic pressure need not always be coordinated in the manner previously reported for ompH and ompL (55).| |
ACKNOWLEDGMENT |
|---|
We are grateful to the National Science Foundation Metabolic Biochemistry program (NSF 9974528) for support of this research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Center for Marine Biotechnology and Biomedicine, Marine Biology Research Division, Scripps Institution of Oceanography, 8750 Biological Grade, University of California, San Diego, La Jolla, CA 92093-0202. Phone: (858) 534-5233. Fax: (858) 534-7313. E-mail: dbartlett{at}ucsd.edu.
| |
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Abstract
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Materials and Methods
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References
|
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