Microbiology (2003), 149, 1061–1071
DOI 10.1099/mic.0.26032-0
Role for the major outer-membrane protein from
Vibrio anguillarum in bile resistance and biofilm
formation
Su-Yan Wang, Johan Lauritz, Jana Jass3 and Debra L. Milton
Correspondence
Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden
Debra Milton
[email protected]
Received 1 October 2002
Revised 15 January 2003
Accepted 17 January 2003
Vibrio anguillarum, a fish pathogen, produces a 38 kDa major outer-membrane porin, which may be
involved in environmental adaptation. The gene encoding the 38 kDa porin was cloned and deleted.
The deduced protein sequence was 75 % identical to that of the major outer-membrane protein
(OMP), OmpU, from Vibrio cholerae. LacZ expression from an ompU : : lacZ transcriptional gene
fusion was increased 1?5-fold in the presence of bile salts and was decreased 50- to 100-fold in a
toxR mutant compared to that in the wild-type, showing that ompU expression is positively regulated
by ToxR and induced by bile salts. Similar to a toxR mutant, an ompU mutant showed a slight
decrease in motility, an increased sensitivity to bile salts and a thicker biofilm with better surface area
coverage compared to that of the wild-type. When ompU was expressed under a ToxRindependent promoter in the toxR mutant, the phenotypes for bile resistance and biofilm formation,
but not motility were complemented to that of the wild-type. In rainbow trout, the ompU mutant
showed wild-type virulence via immersion into infected seawater and intraperitoneal injection. The
ompU mutant produced two colony morphologies: opaque, which did not grow at 0?2 % bile, and
translucent, which grew at 2 % bile. The translucent ompU mutant strain produced a second major
OMP that was induced by bile. All ompU mutants showed variations in the amount and length of
smooth LPS. In V. anguillarum, OmpU is not required for virulence, possibly due to a second OMP
also critical for resistance to bile; however, outside of the fish host, OmpU limits the progression of
biofilm formation.
INTRODUCTION
Vibrio anguillarum causes a terminal haemorrhagic septicaemia in marine fish and is associated with high rates of
mortality, resulting in great economic losses within
aquaculture (for reviews see Actis et al., 1999; Austin &
Austin, 1999). V. anguillarum is suggested to constitute part
of the microflora of marine fish as well as part of the normal
microflora of the aquatic environment (Muroga et al., 1986;
Oppenheimer, 1962; West et al., 1983). In each of these
environments, the bacterium likely utilizes its external
membrane to protect its intracellular contents from
damaging agents or conditions.
The outer membrane consists predominately of phospholipids, LPS and proteins, either integral membrane proteins or lipoproteins, which make up almost 50 % of the
3Present address: The Lawson Health Research Institute, 268
Grosvenor Street, London, Ontario, Canada N6A 4V2.
Abbreviation: OMP, outer-membrane protein.
The GenBank accession number for the sequence reported in this
paper is AY157299.
0002-6032 G 2003 SGM
outer-membrane mass (Koebnik et al., 2000). The outermembrane protein (OMP) profiles of 10 serotypes of
V. anguillarum, although varying in the minor proteins,
contain one major OMP in the molecular size range of
35–42 kDa (Simón et al., 1996). The major OMPs of
serotype O1 and O2 are classified as general diffusion porins
with similar functions to that of OmpF and OmpC from
Escherichia coli (Davey et al., 1998; Simón et al., 1996).
Furthermore, the major OMP from serotype O1 exhibits
weak cation selectivity and possesses a moderate surface
charge (Simón et al., 1996). The N-terminal amino acid
sequence suggests that these two major OMPs are possible
homologues of OmpU from V. cholerae (Okuda et al., 2001;
Wang et al., 2002).
In Vibrio cholerae and Vibrio fischeri, OmpU homologues
play a role in protecting the bacterium from bile salts
(Aeckersberg et al., 2001; Provenzano & Klose, 2000). In V.
cholerae, bile resistance is suggested to be an early step in the
evolution of this bacterium as an intestinal pathogen.
OmpU has previously been suggested to play a role in
adherence of V. cholerae during intestinal colonization
(Sperandino et al., 1995), but conflicting results have also
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
Printed in Great Britain
1061
S.-Y. Wang and others
been reported (Nakasone & Iwanaga, 1998). However,
neither of these studies were done using an ompU null
mutant. A recent study utilizing a null ompU mutant
suggests that OmpU is not essential for colonization or
virulence factor production (Provenzano et al., 2001).
Furthermore, bile was shown not to affect membrane
permeability via the OmpU pore. Thus, the role of OmpU in
the virulence of V. cholerae is suggested to provide a means
for nutrient uptake in the presence of bile without compromising membrane integrity (Wibbenmeyer et al., 2002).
In this study, the gene encoding the major OMP from V.
anguillarum serotype O1 was cloned and designated ompU.
A strain carrying a null mutation showed a significant
decrease in bile resistance, but no decrease in virulence.
However, a second OMP was found that was induced by low
concentrations of bile and could be involved in bile
resistance. In addition, OmpU was shown to have a new
role in limiting biofilm formation.
Interestingly, in V. fischeri, a symbiont of the light-emitting
organ of the squid, OmpU is suggested to play a role in the
initiation of colonization of the light organ. In addition, a
null mutation in the V. fischeri ompU gene also showed an
increased sensitivity to membrane-disrupting agents such as
bile. However, this observation may indicate the importance
of OmpU in maintaining membrane integrity as opposed to
survival within the intestinal environment (Aeckersberg
et al., 2001).
METHODS
Strains, plasmids, phage and growth conditions. Bacterial
strains and plasmids are described in Table 1. E. coli SY327 (lpir)
was used for transformation after subcloning fragments into the
pDM4 or pNQ705-1 suicide vector. All plasmids to be conjugated
into V. anguillarum were transformed into E. coli S17-1 (lpir),
which was used as the donor strain. Plasmid transfers from E. coli to
V. anguillarum were done as described previously (Milton et al.,
1996). E. coli XL-1 Blue was used for bacteriophage l infections and
for most transformations.
Table 1. Bacterial strains and plasmids
Strain or plasmid
E. coli
SY327
S17-1
XL-1 Blue
V. anguillarum
NB10
SY10
SY12
Plasmids
pBluescript
pBSOmpU-AC
pBSOmpU-8
pDM4
pDMOmpU2
pDM8
pDM8-OmpU1
pNQ705-1
pDM35-OmpU1
pSup202P
pSup-OmpU
pMMB208
pMMB-OmpU1
pToxR
1062
Genotype and/or relevant markers
Reference or source
D(lac pro) argE(am) rif malA recA56 lpir
thi pro hsdR hsdM+ recA RP4-2-Tc : : Mu-Km : : Tn7 lpir
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 D(lac-pro) [F9proAB lacI q
lacZDM15 Tn10(Tetr)]
Miller & Mekalanos (1988)
Simon et al. (1983)
Stratagene
Wild-type, serotype O1, clinical isolate from the Gulf of Bothnia
NB10 derivative containing a toxR deletion
NB10 derivative containing an ompU deletion of bp 966–1778
Norqvist et al. (1989)
Wang et al. (2002)
This study
Apr; ColE1 origin
Apr; pBluescript containing a 560 bp PCR fragment from ompU (bp 1008–1567)
Apr; pBluescript containing a cloned fragment with the ompU gene
Cmr; suicide vector with an R6K origin (pir-requiring) and the sacBR
genes from Bacillus subtilis
Cmr; pDM4 derivative containing ompU bp 725–965 fused to bp 1779–2011
Cmr Tcr; (ColE1 origin, RP4 mob+), pSup202 derivative (Simon et al., 1983)
containing a promoterless lacZ gene used for transcriptional gene fusion studies
Cmr Tcr; pDM8 derivative containing an ompU : : lacZ transcriptional
gene fusion (bp 400–919)
Cmr; suicide vector that contains an R6K origin (pir-requiring)
Cmr; pNQ705-1 derivative containing an
ompU : : lacZ transcriptional gene fusion (bp 400–919)
Cmr Tcr; pSup202 derivative (Simon et al., 1983) (ColE1 origin, RP4 mob+)
with a polylinker cloned into PstI within the Apr gene
Cmr Tcr; pSup202P derivative containing
the ompU gene and its promoter (bp 442–2011)
Cmr; IncQ lacI q Ptac polylinker from M13mp19
Cmr; pMMB208 derivative containing the
ompU gene lacking its promoter (bp 921–2011)
Cmr Tcr; pSup202P derivative containing the toxR gene and its promoter
Stratagene
This study
This study
Milton et al. (1996)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
This study
Croxatto et al. (2002)
This study
McGee et al. (1996)
This study
Milton et al. (1992)
This study
Morales et al. (1991)
This study
Wang et al. (2002)
Microbiology 149
OmpU affects bile resistance and biofilm production
E. coli was routinely grown in Luria broth, which contains Bacto
Tryptone (10 g l21), Bacto yeast extract (5 g l21) and sodium chloride
(10 g l21). For V. anguillarum, Trypticase soy medium (TSB) from
BBL was used for routine growth and the cultures were grown with
aeration at 24 ˚C. Biofilm growth medium was minimal M63 salts
(Silhavy et al., 1984) supplemented with 1 % (w/v) NaCl, 1?5 % (w/v)
Casamino acids, 1 % (w/v) glucose, 1 mM MgSO4 and 10 mg thiamine
ml21. Antibiotic concentrations for V. anguillarum were tetracycline at
5 mg ml21 and chloramphenicol at 10 mg ml21.
PCR conditions, DNA manipulation and sequencing. PCR was
performed as described previously (Wang et al., 2002). Unless otherwise stated, all conditions for the various DNA techniques were as
described by Sambrook et al. (1989). Reaction conditions for the
DNA-modifying enzymes and DNA restriction enzymes were as suggested by the manufacturers. Double-strand DNA sequencing was
performed using a Seq464 Automated Sequencer and a Thermo
Sequenase Cy 5.5 Terminator Cycle Sequencing Kit (Amersham
Pharmacia Biotech).
Cloning of the ompU gene. Previously, the amino-terminal
sequence of the major OMP from V. anguillarum serotype O1 was
sequenced and shown to be 75 % identical to that of OmpU from V.
cholerae (Wang et al., 2002). Using the amino-terminal sequence of
V. anguillarum OmpU and an internal amino acid sequence from V.
cholerae OmpU, two degenerate inosine-containing oligonucleotides
were designed and used to PCR amplify a 560 bp DNA fragment
from the V. anguillarum ompU gene. Primer OmpU-A [59GGTGAGCTCTAIAAICAIGA(TC)GG(ACTG)AC-39] is complementary to deduced codons for the amino-terminal sequence YNQDGT
of OmpU from V. anguillarum (Wang et al., 2002) and OmpU-C
[59-GGCACTAGTTCITTITGITC(AG)TC(TC)TG(AG)TC-39] is complementary to V. cholerae ompU codons for amino acids DQDDQNE
(Sperandino et al., 1996). The 560 bp DNA fragment was cloned
into pBluescript (Stratagene), creating pBSOmpU-AC, and sequenced.
The deduced protein sequence of this fragment was 70 % identical
to OmpU of V. cholerae (Sperandino et al., 1996).
The 560 bp fragment was used as a probe to screen a l Zap (Stratagene)
bacteriophage-based V. anguillarum genomic library (Milton et al.,
1992) using previously described methods (Milton et al., 1996). The
pBluescript plasmids containing a chromosomal insert were excised
from positive plaques as described previously (Milton et al., 1992). One
plasmid, pBSOmpU-8, was chosen for sequencing ompU.
wild-type strain under similar conditions. For functional analyses
of the rescue of wild-type phenotypes in the toxR mutant, similar
inducing conditions were used.
Construction of ompU transcriptional gene fusion. A tran-
scriptional gene fusion was constructed between the promoter
region of ompU and the reporter gene lacZ from E. coli. This fusion
was created using previously described methods (Wang et al., 2002).
pDM8-OmpU1 is described in Table 1. However, this plasmid was
very unstable after conjugation into V. anguillarum strains, possibly
due to the high expression of the lacZ gene using the ompU promoter. To obtain a lower copy number, the ompU : : lacZ gene
fusion was subcloned from pDM8-OmpU1 into the suicide vector
pNQ705-1, which requires the pir gene for replication. To create
pDM35-OmpU1, pDM8-OmpU1 was partially digested with
BamHI. The fragment containing the ompU promoter and the lacZ
gene (3400 bp) was purified from an agarose gel and ligated to a
BglII site in the polylinker region of pNQ705-1. pDM35-OmpU1
was then recombined into the chromosome of V. anguillarum
strains. The site of insertion was verified by PCR and the insertion was stable for 30 generations of growth in the absence of
chloramphenicol.
b-Galactosidase assays. Conditions for bacterial growth for
assaying b-galactosidase activity was done as described previously
(Wang et al., 2002). b-Galactosidase assays were performed according
to Miller (1972).
LPS analysis. LPS were prepared from by a Proteinase K digestion
of whole-cell lysates as described by Preston & Penner (1987). LPS
were fractionated by 18 % SDS-PAGE (Laemmli, 1970) and visualized with Silver Stain Plus (Bio-Rad) following the manufacturer’s
instructions.
OMP analyses. OMP preparations were prepared as described pre-
viously (Wang et al., 2002) and separated by 12?5 % SDS-PAGE
(Laemmli, 1970). The gels were fixed and stained with 0?1 %
Coomassie brilliant blue in 40 % methanol/10 % acetic acid and
then destained in 40 % methanol/10 % acetic acid.
Western analysis. Western analysis was done as described pre-
viously (Wang et al., 2002) using a rabbit polyclonal antiserum
raised against OmpU from V. cholerae (a gift from James Kaper,
University of Maryland, School of Medicine, MD, USA).
Construction of an ompU mutant. An ompU in-frame deletion
Flow cell analysis of biofilms. Biofilm formation and analysis
mutation was made in the wild-type strain by allelic exchange as
described previously (Milton et al., 1996). After allelic exchange
using pDMOmpU2, codons for the first 10 amino acids were fused
to the codons encoding the last 49 amino acids. The strain containing this in-frame deletion was called SY12. PCR amplification of
the deleted region from the chromosome and subsequent DNA
sequencing confirmed the ompU in-frame deletion. For complementation analyses, pSup-OmpU, which carries the wild-type ompU and
its promoter, was mobilized into the ompU mutant via conjugation.
using a continuous flow of fresh medium was done using a modification of the previously described protocol of Christensen et al.
(1999). A pre-sterilized three-channel flow cell (Stovall Life
Sciences), which has an acrylic base with a #1 cover slip attached
and a channel size of 464061 mm was used. The flow cell was
filled with biofilm growth medium and the flow rate was stabilized
to 250 ml min21 throughout the experiment. The flow cell was kept
at room temperature. Overnight cultures, grown in biofilm growth
medium, were diluted to an OD600 of 1?0 in fresh biofilm growth
medium. The medium flow was stopped and 0?4 ml was used to
inoculate a channel. The flow cell was inverted with the cover
slip side down for 1 h after which the flow cell was turned right side
up and the flow of medium was initiated again. Biofilms were
allowed to progress for 72 h before staining with acridine orange for
visualization.
Rescue of phenotypes in a toxR mutant by OmpU. To deter-
mine if OmpU can rescue wild-type phenotypes in a toxR mutant, a
PCR fragment (residues 921–2011) containing the ompU gene plus
15 bp upstream of the start codon was amplified and fused to
the inducible Ptac promoter on pMMB208 (Morales et al., 1991),
creating pMMB-OmpU1. The pMMB-OmpU1 plasmid was mobilized into the toxR mutant SY10 via conjugation. To induce ompU
expression in the toxR mutant carrying pMMB-OmpU1, a final
concentration of 1 mM IPTG was added to the medium. After overnight growth under inducing conditions in the toxR mutant, the
amount of OmpU in a whole-cell extract or in an outer-membrane
preparation was approximately 80 % of the OmpU levels in the
http://mic.sgmjournals.org
To stain the biofilm, 200 ml 0?1 % acridine orange in potassium
phosphate buffer (pH 7?4) was injected into the flow cell and allowed
to stain for 20 min after which the biofilm was washed for 60 min
before viewing for images. When using the pMMB208 plasmid
derivatives, 1 mM IPTG was added to the biofilm growth medium to
induce expression of OmpU under the tac promoter.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
1063
S.-Y. Wang and others
Imaging of biofilms in flow cells was done using scanning confocal laser
microscopy (SCLM) (Multiprobe 2001; Molecular Dynamics). The
488 nm laser line of an argon-krypton laser was used to excite the
acridine orange. ImageSpace software (Molecular Dynamics) running
on an O2 workstation (Silicone Graphics) was used to generate vertical
cross sections through the biofilms. Images were further processed for
display using Photoshop software (Adobe). Two sets of images were
obtained from the centre of each flow cell channel for every experiment
and all strains were done in triplicate.
Percentage area coverage of a biofilm. The percentage area
coverage due to biofilm production was determined as described
previously (Wang et al., 2002).
Sequence analysis. Sequence assembly was done using the
Genetics Computer Group Sequence Analysis software (Devereux
et al., 1984) of the Genetics Computer Group, University of
Wisconsin. Database searches were done through the NCBI Web
page using BLAST 2.0.
Fig. 1. Genetic organization of the ompU DNA locus. Each
ORF is indicated by a box and the arrows indicate the direction
of transcription. The dotted line below the ompU gene indicates
the in-frame deletion made in the gene. The homologous V.
cholerae genes from the genomic sequence (Heidelberg et al.,
2000) are indicated below each ORF along with the percentage identity.
Fish infections. Rainbow trout (Oncorhynchus mykiss) with an
approximate weight of 10–15 g were infected with V. anguillarum
either by intraperitoneal injections or by immersion of the fish in
seawater containing V. anguillarum as described previously (Milton
et al., 1996).
RESULTS
Identification and mutagenesis of the ompU
gene
To identify the ompU homologue from V. anguillarum
serotype O1, degenerate PCR primers were designed using
the previously sequenced amino terminus of the major
OMP of V. anguillarum (Wang et al., 2002) and from an
internal sequence within the V. cholerae ompU DNA
sequence (Sperandino et al., 1996). Using these primers, a
560 bp PCR fragment was generated that encoded an amino
acid sequence with 70 % identity to OmpU of V. cholerae
and was used as a probe to screen a V. anguillarum genomic
library for a chromosomal fragment carrying the ompU
homologue. The ompU gene and flanking DNA regions were
sequenced and the genetic organization is shown in Fig. 1.
One complete ORF was identified and named ompU. The
deduced OmpU protein sequence is 330 aa long and is 75 %
identical to OmpU from V. cholerae (Sperandino et al.,
1996), 59 % identical to OmpU of V. fischeri (Aeckersberg
et al., 2001) and 47 % identical to OmpL of Photobacterium
profundum (Welch & Bartlett, 1996).
In addition, two partial ORFs were found (Fig. 1). The
deduced 127 aa sequence of ORF1 is 85 % identical to the V.
cholerae VC0634 gene product from chromosome I,
which encodes the transcription elongation factor GreA
(Heidelberg et al., 2000). The partial deduced protein
sequence of ORF2 is 62 % identical to the V. cholerae
VC0632 gene from chromosome I, which encodes a
D-alanyl-D-alanine carboxypeptidase (DacB) (Heidelberg
et al., 2000). Since the ompU gene corresponds to the V.
cholerae VC0633 gene from chromosome I, the genetic
organization of these three V. anguillarum ORFs is similar to
1064
that from the sequenced V. cholerae strain El Tor N16961
(Heidelberg et al., 2000).
To determine the function of OmpU, an in-frame deletion
mutation was made that fused codons for the first 10 aa to
codons encoding the last 49 aa. Growth of the ompU
mutant (SY12) was similar to that of the wild-type in TSB
(data not shown). For complementation analyses of the
ompU deletion, pSup-OmpU, which carries the wild-type
ompU gene and its promoter, was mobilized into the ompU
mutant.
Major OMP is encoded by ompU
To show that the ompU gene encodes the major OMP in V.
anguillarum serotype O1, OMPs were isolated from overnight broth cultures of the wild-type, the ompU mutant and
the complemented ompU mutant, and separated by 12?5 %
SDS-PAGE. Fig. 2 shows that the major OMP is absent in
the ompU mutant and is present again when the mutation is
complemented with the wild-type ompU gene. In the ompU
mutant, an additional 37 kDa protein can be seen that
migrates with OmpU in the wild-type preparation. To
ensure that this protein is not OmpU, a Western blot
analysis was done using a rabbit polyclonal antiserum
against V. cholerae OmpU. Fig. 2(b) shows that the OmpU
antiserum did not bind to any OMPs from the ompU
mutant, indicating that the 37 kDa OMP is different to
OmpU and that the ompU mutant is lacking the major
OMP.
ToxR positively regulates ompU
To determine if ToxR regulates the expression from ompU, a
transcriptional gene fusion was made to the E. coli lacZ gene
and carried on the suicide plasmid pDM35-OmpU1. This
plasmid was recombined into the wild-type, the toxR
mutant and the complemented toxR mutant at the ompU
promoter region. Since the promoter region was duplicated
on the chromosome downstream of the plasmid insertion,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
Microbiology 149
OmpU affects bile resistance and biofilm production
Fig. 2. OMP analyses. OMPs were isolated from the wild-type,
the ompU mutant, the ompU mutant carrying a plasmidencoded wild-type ompU gene (pSup-OmpU), the opaque (Op)
variant of the ompU mutant and the translucent (Tr) variant of
the ompU mutant. In some cases, the strains were grown in
the presence of 0?04 % bile. OMPs were applied to a 12?5 %
SDS-PAGE gel, separated and stained with (a) Coomassie
brilliant blue or (b) transferred to nitrocellulose followed by
Western blotting using an antiserum raised against OmpU from
V. cholerae. Lanes 1–7 are labelled in (a) and are the same for
both (a) and (b). Molecular mass markers are included in the
left-most lane and sizes are indicated.
an ompU mutation was not made and the expression level of
OmpU was still that of the wild-type (data not shown).
Fig. 3 shows that the expression of ompU : : lacZ in the toxR
mutant is 50- to 100-fold less than in the wild-type
throughout growth, indicating that ToxR positively regulates expression from the ompU promoter. When the toxR
defect was complemented with the wild-type toxR gene,
LacZ activity returned almost to that of the wild-type.
Fig. 3. The effect of a toxR mutation on the b-galactosidase
activity of ompU : : lacZ transcriptional gene fusion inserted into
the chromosome using pDM35-OmpU1. Overnight cultures
were diluted into fresh TSB medium to an OD600 of 0?05 and
then incubated with shaking at 24 ˚C. Samples were taken at
various times and analysed for growth (OD600; dotted lines)
and b-galactosidase expression (Miller units; solid lines).
Squares, wild-type; circles, DtoxR; triangles, DtoxR/pToxR.
OmpU enhances growth in the presence of bile
To test if OmpU from V. anguillarum plays a role in
resistance to bile, equal amounts of bacterial cells were
spread onto TSA plates containing various concentrations of
bile salts (Difco). After 3 days, the number of c.f.u. was
counted. Results are presented in Table 2. The wild-type was
resistant up to 2 % bile salts, after which the c.f.u. began to
decrease. However, the ompU mutant did not grow in the
presence of 0?2 % bile and even at the lowest concentration
of bile salts (0?04 %), the c.f.u. was decreased. For all
concentrations of bile, the colony size of the ompU mutant
was much smaller than the wild-type on a similar medium.
When the ompU mutation was complemented with the
wild-type gene and its promoter, growth in the presence of
all concentrations of bile was regained.
Table 2. Effect of bile salts on the survival of V. anguillarum and motility analysis
Genotype
Wild-type
DompU
DompU/pSup-OmpU
DtoxR
DtoxR/pMMB-OmpU13
Motility (%)
100
71±1?1
92±2?3
84±2?7
81±2?1
c.f.u. with indicated bile salts concentration (%) in TSA
0
0?04
0?08
0?2
0?4
0?8
2
4
115
107
81
100
264
120
45*
52
57*
243
115
10*
60
13*
235
100
0
41
0
158
107
0
29
0
139
115
0
35
0
108
102
0
35
0
42
90
0
34
0
49
*Colony size was greatly reduced compared to wild-type.
3IPTG (1 mM) was added to the medium to induce ompU expression.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
1065
S.-Y. Wang and others
ensure that a spontaneous mutation that affects virulence
did not occur. Equal numbers of cells were spotted onto a
TSB/0?25 % agar plate. Migration of cells from the centre
spot was measured and compared to the wild-type
(Table 2). The ompU mutant was only 71 % as motile as
the wild-type and when the ompU mutation was complemented with the wild-type gene, motility returned to that of
the wild-type.
Biofilm formation
Fig. 4. The effect of bile on the transcription of ompU.
b-Galactosidase activity of an ompU : : lacZ transcriptional gene
fusion (pDM35-OmpU1), which was inserted into the chromosome of the wild-type (NB10), was measured in the presence
[0?04 (circles) and 0?4 % (triangles)] or absence (squares) of
bile. Overnight cultures were diluted into fresh TSB medium to
an OD600 of 0?05 and then incubated with shaking at 24 ˚C.
Samples were taken at various times and analysed for growth
(OD600; dotted lines) and b-galactosidase expression (Miller
units; solid lines).
In addition, LacZ activity was measured from the ompU : :
lacZ transcriptional fusion in the wild-type strain in the
presence (0?04 and 0?4 %) or absence of bile. In Fig. 4, a 1?5fold increase in LacZ expression occurred at the onset of
stationary phase in the presence of 0?4 % bile, but not in the
presence of 0?04 % bile. Taken together, these data suggest
that OmpU is required for optimal growth of V. anguillarum
in the presence of bile and that induction of ompU by bile
occurs at the onset of stationary phase.
Virulence analyses
V. anguillarum has been shown to utilize the intestines as a
site of attachment, colonization and proliferation (Horne &
Baxendale, 1983; Olsson et al., 1996, 1998). Since OmpU
enhances growth in the presence of bile, the ompU mutant
may be defective in virulence. Virulence of the ompU mutant
was assayed via both the immersion and intraperitoneal
infection routes. For the intraperitoneal route, the LD50
values for the ompU mutant and the wild-type were 69±14
and 107±80 bacteria, respectively. For the immersion
route, the LD50 values for the ompU mutant and the wildtype were 16104±96103 and 76103±36103 bacteria
(ml seawater)21, respectively. These results indicate that
ompU is not essential for V. anguillarum to cause disease
in fish.
Motility analysis
Since motility aids the entry of V. anguillarum into the
fish host (Milton et al., 1996; O’Toole et al., 1996), this
phenotype is routinely checked when mutants are made to
1066
Since OmpU does not appear to have an essential role in
virulence, maybe it is important for survival of V.
anguillarum outside of the fish host. OmpU is located in
the outer membrane and could affect surface structures or
surface charges that may be important for biofilm
formation. The percentage area coverage of a biofilm on a
glass surface was determined for 2–14 h of growth for the
wild-type, ompU mutant and the complemented ompU
strains (Fig. 5). All strains showed the same percentage area
coverage through 8 h of growth. After 8 h, the percentage
area coverage for the ompU mutant increased above that of
the wild-type and reached a 10-fold greater percentage area
coverage by 14 h. The surface area coverage for the wildtype, on the other hand, did not change much after 8 h. In
some cases, the level of biofilm coverage by the wild-type
often dropped after 12 h. When the ompU mutation was
complemented, near wild-type levels were obtained during
the later hours of growth.
In addition to the percentage area coverage determination,
flow cell analysis was done to analyse the thickness of the
biofilm for the wild-type, the ompU mutant and the
complemented ompU mutant. Using SCLM, images of a
vertical cross-section through a 3-day-old biofilm for each
strain were taken. As shown in Fig. 6, the ompU mutant
produced a biofilm that was thicker than that of the wildtype and complementation of the mutation with the wildtype gene resulted in a near wild-type biofilm. These data
may suggest that the ompU mutant either was more
proficient than the wild-type at forming a biofilm or was
unable to detach from the glass surface as well as the wildtype, thus enhancing surface biofilm coverage and biofilm
thickness.
ompU expression complements some
phenotypes of a toxR mutation
For the ompU mutant, alterations in biofilm formation, bile
resistance and motility were similar to the same phenotypes
for the toxR mutant (Wang et al., 2002). To test if these three
toxR mutant phenotypes were due to the loss of OmpU or to
other ToxR-regulated proteins, ompU was fused to the tac
promoter carried on pMMB208, creating pMMB-OmpU1
and this plasmid was mobilized into the toxR deletion
mutant SY10. This fusion places ompU under the control of
a ToxR-independent promoter. To determine if ompU was
expressed when 1 mM IPTG was added to the growth
medium, OMP preparations from induced and uninduced
cultures of toxR carrying pMMB-OmpU1 were compared to
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
Microbiology 149
OmpU affects bile resistance and biofilm production
Fig. 5. Percentage area coverage of a biofilm on a glass surface. (a) Typical image of
biofilm formation on a glass slide for each
strain at 14 h. Slides were stained with acridine orange and viewed with a fluorescent
microscope. Bars, 20 mm. (b) Biofilm formation was quantified by determining the percentage area coverage on a glass slide. For
each strain, five images were taken at each
time point and saved as a computer image.
These images were converted into black
(background) and white (biofilm) pixels and
the percentage of white pixels is given as
the percentage of biofilm covering the glass
surface. The data for 2, 4 and 6 h were collected from separate experiments to those of
8, 10, 12 and 14 h but included in the
same figure for easy comparison.
those of the wild-type and toxR mutant (data not shown). In
the toxR/pMMB-OmpU1 strain, ompU is expressed to
approximately 80 % of the wild-type levels in the presence of
IPTG. Western analysis confirmed that this induced protein
was OmpU.
A second OMP involved in bile resistance
Moreover, the ompU mutant was made a second time and a
similar phenotypic alteration occurred. Thus, the alteration
did not appear to be a spontaneous mutation unrelated to
ompU. Interestingly, bacteria from the translucent colony
could grow in the presence of 2 % bile like the wild-type,
whereas the opaque colony maintained the ompU mutant
phenotype and was unable to grow in the presence of 0?2 %
bile (data not shown). OMP preparations were made from
the opaque and translucent colonies in the presence and
absence of bile. Fig. 2 shows that the translucent colony
contains an abundant 37 kDa OMP that is induced by a low
concentration of bile and that does not bind OmpU
antibodies, suggesting that there may be more than one
OMP that plays a role in bile resistance. In the OMP
preparations from the opaque colony, the amount of a
50 kDa protein was increased, but was not induced by bile.
When doing c.f.u. counts using the ompU mutant, about
2 % of the colonies had an increased translucent colony
morphology compared to other colonies. PCR analysis
indicated that both colony types carried the ompU deletion.
LPS profiles
LPS, which constitute the majority of the outer surface of
the outer membrane, have been shown to play a role in bile
Bile resistance, motility and biofilm formation were assayed
under inducing conditions (1 mM IPTG) for the toxR
mutant carrying pMMB-OmpU1. Figs 5 and 6 and Table 2
show that the thickness and percentage area coverage of
biofilm formation and bile resistance returned to approximate wild-type levels in the toxR mutant expressing OmpU;
however, motility (Table 2) was unaffected. Thus, OmpU is
a major player from the ToxR regulon for biofilm formation
and bile resistance, but not for motility.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
1067
S.-Y. Wang and others
Fig. 7. Silver-stained LPS profiles of V. anguillarum mutant
strains. LPS were isolated from equal numbers of bacterial cells
as described in Methods and separated on an 18 % SDSPAGE gel.
DISCUSSION
Fig. 6. SCLM images of biofilms formed in a flow cell. The
images represent vertical cross-sections through 72 h biofilms
formed on a glass surface after continuous flow of fresh
medium. The biofilms were stained with acridine orange and
SCLM was used to take images of the biofilm. Bars, 20 mm.
resistance (Gunn, 2000). We wondered if the loss or gain of
these major OMPs had any affect on the LPS in the outer
membrane. LPS were isolated from the various strains and
separated in an 18 % SDS-PAGE gel. The wild-type LPS
profile in Fig. 7 shows that the O1 serotype produced a
smooth LPS with a tight O-antigen modal length distribution as shown by Boesen et al. (1999). Compared to the wildtype, the toxR, ompU and ompU (opaque) mutants have
an increased amount of the medium molecular mass
O-antigen, and show an increase in the modal length of
the repeating O-antigen units (lanes 2, 4 and 6, respectively),
which when complemented decreased to the wild-type levels
(lanes 3 and 5). Interestingly, the translucent ompU mutant
(lane 7) contains predominately the medium molecular
mass O-antigen and very little of the lipid A core. This LPS
profile is similar to a translucent variant of a wild-type V.
cholerae strain (Finkelstein et al., 1997). These data suggest
that changes in the OMP content can alter the amount and
length of the smooth LPS in the outer leaflet of the cell.
1068
Bacterial outer membranes form an adaptive barrier to the
external environment, protecting the bacterial cell contents
from damaging substances while allowing the selective
uptake of nutrients (for reviews see Achouak et al., 2001;
Koebnik et al., 2000; Nikaido, 1996). At the basis of this
selective uptake are proteins called porins, which form
hydrophilic channels in the outer membrane that permit
selective transport of molecules into the bacterial cell. Porins
may be either specific or non-specific in their transport
properties. Regulation of the synthesis of porins is often
dependent on signals found in the environment, such as
osmolarity, nutrient limitation, temperature and phosphate.
Thus, turning the expression of various porins on and off
alters outer-membrane permeability.
The V. anguillarum major OMP from serotypes O1 and O2
has been shown to be a general diffusion porin for which a
trimeric form can be isolated similar to that of OmpF
and OmpC from E. coli and OmpU from V. cholerae
(Chakrabarti et al., 1996; Davey et al., 1998; Simón et al.,
1996). The predominant major OMPs from 10 V.
anguillarum serotypes are antigenically similar to each
other and for serotypes O1 and O2, they are also
antigenically similar to the major OMP in other Vibrio
species, including OmpU from V. cholerae (Davey et al.,
1998; Simón et al., 1996; Suzuki et al., 1996; Wang et al.,
2002). In this study, the ompU gene, encoding the major
OMP from V. anguillarum serotype O1 was cloned. OmpU
is 75 % identical to OmpU from V. cholerae (Sperandino
et al., 1996) and the N-terminal sequence of this protein is
100 % identical to the Omp35La from at least four different
serotypes of V. anguillarum (Suzuki et al., 1996). This
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
Microbiology 149
OmpU affects bile resistance and biofilm production
suggests that the major OMP of V. anguillarum serotypes is
likely to be a homologue to the V. cholerae OmpU porin.
Porins are the most abundant OMPs in the outer membrane
and have been suggested to play an important role in bile
resistance. Porins exist as trimers in the outer membrane
that form pores of variable size and charge, resulting in
variable permeability to bile salts (for reviews see Gunn,
2000; Lin et al., 2002). By regulating specific porins, bacteria
can alter their membrane permeability and thus modulate
bile resistance. In E. coli, OmpC is more important for bile
resistance than OmpF as it has a smaller pore size than
OmpF (Thanassi et al., 1997). In V. cholerae, the positive and
negative regulation of OmpU and OmpT, respectively, is
ToxR-dependent and is critical for bile resistance. OmpU,
like OmpC in E. coli, makes a smaller pore that is more
cation-selective than OmpT and is less permeable to bile
salts (Provenzano & Klose, 2000). A similar mechanism of
bile resistance is likely to be used by V. anguillarum since
OmpU exhibits weak cationic selectivity and a moderate
surface charge (Simón et al., 1996) and is critical for bile
resistance. Moreover, the translucent and opaque variants of
the ompU mutant clearly showed an induction of several
additional OMPs in the absence of OmpU. The opaque
variant was still bile-sensitive; however, for the translucent
variant, the induction of one major OMP and several minor
OMPs resulted in a gain of bile resistance. The translucent
variant represents a small population of cells that have
managed to adapt to high levels of bile. Bile resistance
has previously been described as an adaptive response
that involves a global alteration in protein production
(Gunn, 2000).
In addition to OMPs, the outer membrane of Gramnegative bacteria also consists of LPS and phospholipids (for
a review see Nikaido, 1996). LPS aid in the formation of an
outer membrane that has low permeability and low fluidity
and LPS are required for trimeric porin formation. Loss of
the O-antigen creates a rough LPS, which results in the loss
of porins and an increase in phospholipids in the outer
membrane. An increase in phospholipid patches increases
permeability of the outer membrane and hence increases
sensitivity to bile (Gunn, 2000). Loss of the major OMP in V.
anguillarum did lead to alterations in the LPS profile and
thus may result in an increase in phospholipids in the outer
membrane. In addition to the possible selective permeability
of porins, the LPS and phospholipid composition of the
outer membrane may also play a role in bile resistance in
V. anguillarum.
Resistance to bile should be critical for V. anguillarum to
colonize the intestines of the fish host (Horne & Baxendale,
1983; Olsson et al., 1996, 1998). Therefore, it is puzzling why
the ompU mutant was not decreased in virulence. An
alternate OMP that also functions in bile resistance, such as
the 37 kDa OMP, could be one explanation. The 37 kDa
OMP may be preferentially expressed to OmpU in the
intestinal environment. The expression of the 37 kDa OMP
was increased at least threefold in 0?04 % bile, whereas
http://mic.sgmjournals.org
OmpU expression was not induced in 0?04 % bile and was
only mildly induced using 0?4 % bile. Hence, V. anguillarum
may have two general porins critical for bile resistance
expressed differentially as OmpC and OmpF are in E. coli
(for a review see Nikaido, 1996). OmpC expression is
induced with high osmolarity (0?15 M NaCl) and high
temperature (37 ˚C), conditions found in human body
fluids. On the other hand, OmpF expression is induced
under environmental conditions found outside of the
human body, such as low osmolarity and low temperature.
However, preliminary studies have shown that a higher
proportion of translucent colonies was not observed when
the ompU mutant was isolated from infected fish kidney
tissue, indicating a more complex regulation than that
initially predicted.
Under the conditions assayed, ompU is likely to be the most
critical of the genes regulated by ToxR that play a role in
biofilm formation and bile resistance. However, OmpU did
not rescue the wild-type motility in a toxR mutant. In other
vibrios, the OMP profile is altered in a toxR mutant,
suggesting that ToxR modulates the expression of more
than one OMP (Provenzano et al., 2000). Consequently, the
expression of ompU from a ToxR-independent promoter in
a toxR mutant may not create a wild-type outer membrane.
The loss of other OMPs may alter the amount of LPS or
phospholipid. The polar flagella of vibrios are covered in a
sheath, a possible extension of the cell membrane (for a
review see McCarter, 2001). The sheath may be involved in
flagellar assembly, which may have implications for flagellar
morphogenesis and gene expression. Variations in the
content of the outer membrane may affect sheath assembly
and thus flagellar assembly. On the other hand, LPS mutants
of E. coli are reduced in motility at varying levels, suggesting
that the levels of LPS may affect flagellar functions
(Genevaux et al., 1999).
Survival for an aquatic bacterium in seawater outside the
fish host may depend on biofilm formation. Bacteria that are
part of the normal microflora of seawater, such as V.
anguillarum (Muroga et al., 1986; West et al., 1983), are
normally found attached to surfaces, as opposed to a freeswimming form, which is likely to provide an adaptive or
survival advantage for bacteria in the aquatic environment
(Costerton et al., 1987, 1995). The cellular location of
OmpU is ideal for playing a role in bacterial cell–cell or cell–
surface interactions needed for biofilm formation. In this
study, a new role is proposed for the OmpU porin in biofilm
formation. OmpU may limit or prohibit biofilm formation,
or it may have a role in detachment from the biofilm
possibly in environmental niches that are less advantageous
for the bacterium. On the other hand, an ompU mutant may
just be better at forming a biofilm than the wild-type.
The modulation of expression and the variation of the cellsurface-exposed domains of outer-membrane porins in
Gram-negative bacteria is a remarkable means by which
bacteria adapt and respond to environmental changes.
Porins affect various different physiological phenotypes,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
1069
S.-Y. Wang and others
allowing the bacterium to adapt quickly to aid survival of the
cell. The V. anguillarum porin OmpU was shown to affect
two important physiological functions, bile resistance and
biofilm formation. These physiological functions are likely
to be critical for the adaptation and survival of this
bacterium to particular environmental niches, such as the
fish host and seawater.
Genevaux, P., Bauda, P., DuBow, M. S. & Oudega, B. (1999).
ACKNOWLEDGEMENTS
Horne, M. T. & Baxendale, A. (1983). The adhesion of Vibrio
Identification of Tn10 insertions in the rfaG, rfaP, and galU
genes involved in lipopolysaccharide core biosynthesis that affect
Escherichia coli adhesion. Arch Microbiol 172, 1–8.
Gunn, J. S. (2000). Mechanisms of bacterial resistance and response
to bile. Microbes Infect 2, 907–913.
Heidelberg, J. F., Eisen, J. A., Nelson, W. C. & 29 other authors
(2000). DNA sequence of both chromosomes of the cholera
pathogen Vibrio cholerae. Nature 406, 477–484.
Antiserum against OmpU of V. cholerae was kindly provided by James
Kaper. We thank Roland Rosqvist for technical assistance with the
confocal microscope. This work was supported by a grant from the
Swedish Council for Environment, Agricultural Sciences and Spatial
Planning and by a grant from the Carl Tryggers Foundation, Sweden,
which are gratefully acknowledged.
REFERENCES
Achouak, W., Heulin, T. & Pagès, J.-M. (2001). Multiple facets of
bacterial porins. FEMS Microbiol Lett 199, 1–7.
Actis, L. A., Tolmasky, M. E. & Crosa, J. H. (1999). Vibriosis. In Fish
Diseases and Disorders, Vol. 3: Viral, Bacterial and Fungal Infections,
pp. 523–557. Edited by P. T. K. Woo & E. W. Bruno. Wallingford:
CABI.
Aeckersberg, F., Lupp, C., Feliciano, B. & Ruby, E. G. (2001). Vibrio
fischeri outer membrane protein OmpU plays a role in normal
symbiotic colonization. J Bacteriol 183, 6590–6597.
Austin, B. & Austin, D. A. (1999). Bacterial Fish Pathogens: Disease of
Farmed and Wild Fish. Chichester: Springer-Praxis.
Boesen, H. T., Pedersen, K., Larsen, J. L., Koch, C. & Ellis, A. E.
(1999). Vibrio anguillarum resistance to rainbow trout
(Oncorhynchus mykiss) serum: role of O-antigen structure of
lipopolysaccharide. Infect Immun 67, 294–301.
Chakrabarti, S. R., Chaudhuri, K., Sen, K. & Das, J. (1996). Porins of
anguillarum to host tissues and its role in pathogenesis. J Fish Dis 6,
461–471.
Koebnik, R., Locher, K. P. & Van Gelder, P. (2000). Structure and
function of bacterial outer membrane proteins: barrels in a nutshell.
Mol Microbiol 37, 239–253.
Laemmli, U. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lin, J., Shouxiong, H. & Zhang, Q. (2002). Outer membrane
proteins: key players for bacterial adaptation in host niches. Microbes
Infect 4, 325–331.
McCarter, L. L. (2001). Polar flagellar motility of the Vibrionaceae.
Microbiol Mol Biol Rev 65, 445–462.
McGee, K., Hörstedt, P. & Milton, D. L. (1996). Identification and
characterization of additional flagellin genes from Vibrio anguillarum. J Bacteriol 178, 5188–5198.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory.
Miller, V. L. & Mekalanos, J. J. (1988). A novel suicide vector and its
use in construction of insertion mutations: osmoregulation of outer
membrane proteins and virulence determinants in Vibrio cholerae
requires toxR. J Bacteriol 170, 2575–2583.
Milton, D. L., Norqvist, A. & Wolf-Watz, H. (1992). Cloning of a
metalloprotease gene involved in the virulence mechanism of Vibrio
anguillarum. J Bacteriol 174, 7235–7244.
Milton, D. L., O’Toole, R., Hörstedt, P. & Wolf-Watz, H. (1996).
Davey, M., Hancock, R. E. W. & Mutharia, L. M. (1998). Influence of
Flagellin A is essential for the virulence of Vibrio anguillarum.
J Bacteriol 178, 1310–1319.
Morales, V. M., Bäckman, A. & Bagdasarian, M. (1991). A series of
wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97, 39–47.
Muroga, K., Iida, M., Matsumoto, H. & Nakai, T. (1986). Detection of
Vibrio anguillarum from waters. Bull Jpn Soc Sci Fish 52, 641–647.
Nakasone, N. & Iwanaga, M. (1998). Characterization of outer
membrane protein OmpU of Vibrio cholerae O1. Infect Immun 66,
4726–4728.
Nikaido, H. (1996). Outer membrane. In Escherichia coli and
Salmonella, Cellular and Molecular Biology, pp. 29–47. Edited by
F. C. Neidhardt and others. Washington, DC: American Society for
Microbiology.
Norqvist, A., Hagström, Å. & Wolf-Watz, H. (1989). Protection of
rainbow trout against vibriosis and furunculosis by the use of
attenuated strains of Vibrio anguillarum. Appl Environ Microbiol 55,
1400–1405.
culture conditions on expression of the 40-kilodalton porin protein
of Vibrio anguillarum serotype O2. App Environ Microbiol 64, 138–146.
Okuda, J., Nakai, T., Chang, P. S., Oh, T., Nishino, T., Koitabashi, T.
& Nishibuchi, M. (2001). The toxR gene of Vibrio (Listonella)
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive
set of sequence analysis programs for the VAX. Nucleic Acids Res 12,
387–395.
anguillarum controls expression of the major outer membrane
proteins but not virulence in a natural host model. Infect Immun 69,
6091–6101.
Finkelstein, R. A., Boesman-Finkelstein, M., Sengupta, D. K., Page,
W. J., Stanley, C. M. & Phillips, T. E. (1997). Colonial
Olsson, J. C., Jöborn, A., Westerdahl, A., Blomberg, L., Kjelleberg, S.
& Conway, P. L. (1996). Is turbot, Scophthalmus maximus (L.),
opacity variations among the choleragenic vibrios. Microbiol 143,
23–34.
intestine a portal of entry for the fish pathogen Vibrio anguillarum.
J Fish Dis 19, 225–234.
Vibrio cholerae: purification and characterization of OmpU.
J Bacteriol 178, 524–530.
Christensen, B. B., Sternberg, C., Andersen, J. B., Palmer, R. J., Jr,
Nielsen, A. T., Givskov, M. & Molin, S. (1999). Molecular tools for
study of biofilm physiology. Methods Enzymol 310, 20–42.
Costerton, J. W., Cheng, K.-J., Geesy, G. G., Ladd, T. I., Nickel, J. C.,
Dasgupta, M. & Marrie, T. J. (1987). Bacterial biofilms in nature and
disease. Annu Rev Microbiol 41, 435–464.
Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R. &
Lappin-Scott, H. M. (1995). Microbial biofilms. Annu Rev Microbiol
49, 711–745.
Croxatto, A., Chalker, V. J., Lauritz, J., Jass, J., Hardman, A.,
Williams, P., Cámara, M. & Milton, D. L. (2002). VanT, a homologue
of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment,
and biofilm production in Vibrio anguillarum. J Bacteriol 184,
1617–1629.
1070
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
Microbiology 149
OmpU affects bile resistance and biofilm production
Olsson, J. C., Jöborn, A., Westerdahl, A., Blomberg, L., Kjelleberg, S.
& Conway, P. L. (1998). Survival, persistence, and proliferation of
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range
Vibrio anguillarum in juvenile turbot, Scophthalmus maximus (L.),
intestine and faeces. J Fish Dis 21, 1–10.
mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1,
787–796.
Oppenheimer, C. H. (1962). Marine fish diseases. In Fish as Food,
Sperandino, V., Giron, J. A., Silveira, W. D. & Kaper, J. B. (1995). The
Vol. 2., pp. 541–572. Edited by Georg Borgström. New York:
Academic Press.
OmpU outer membrane protein, a potential adherence factor of
Vibrio cholerae. Infect Immun 63, 4433–4438.
O’Toole, R., Milton, D. L. & Wolf-Watz, H. (1996). Chemotactic
Sperandino, V., Bailey, C., Giron, J. A., DiRita, V. J., Silveirak, W. D.,
Vettore, A. L. & Kaper, J. B. (1996). Cloning and characterization of
motility is required for invasion of the host by the fish pathogen
Vibrio anguillarum. Mol Microbiol 19, 625–637.
Preston, M. A. & Penner, J. L. (1987). Structural and antigenic
properties of lipopolysaccharides from serotype reference strains of
Campylobacter jejuni. Infect Immun 55, 1806–1812.
Provanzano, D. & Klose, K. E. (2000). Altered expression of the
ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae
bile resistance, virulence factor expression, and intestinal colonization. Proc Natl Acad Sci U S A 97, 10220–10224.
Provenzano, D., Schuhmacher, D. A., Barker, J. L. & Klose, K. E.
(2000). The virulence regulatory protein ToxR mediates enhanced
bile resistance in Vibrio cholerae and other pathogenic Vibrio species.
Infect Immun 68, 1491–1497.
Provenzano, D., Lauriano, C. M. & Klose, K. E. (2001).
Characterization of the role of the ToxR-modulated outer membrane
porins OmpU and OmpT in Vibrio cholerae virulence. J Bacteriol
183, 3652–3662.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:
a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.
Silhavy, T., Berman, M. & Enquist, L. (1984). Experiments with Gene
Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Simón, M., Mathes, A., Blanch, A. & Engelhardt, H. (1996).
Characterization of a porin from the outer membrane of Vibrio
anguillarum. J Bacteriol 178, 4182–4188.
http://mic.sgmjournals.org
the gene encoding the OmpU outer membrane protein of Vibrio
cholerae. Infect Immun 64, 5406–5409.
Suzuki, S., Kuroe, K., Yasue, K. & Kusuda, R. (1996). Antigenicity
and N-terminal amino acid sequence of a 35 kDa porin-like protein
of Listonella (Vibrio) anguillarum: comparison among different
serotypes and other bacterial species. Lett Appl Microbiol 23,
303–306.
Thanassi, D. G., Cheng, L. W. & Nikaido, J. (1997). Active efflux of
bile salts by Escherichia coli. J Bacteriol 179, 2512–2518.
Wang, S.-Y., Lauritz, J., Jass, J. & Milton, D. L. (2002). A ToxR
homolog from Vibrio anguillarum serotype O1 regulates its own
production, bile resistance, and biofilm formation. J Bacteriol 184,
1630–1639.
Welch, T. J. & Bartlett, D. H. (1996). Isolation and characterization of
the structural gene for OmpL, a pressure-regulated porin-like protein
from the deep-sea bacterium Photobacterium species strain SS9.
J Bacteriol 178, 5027–5031.
West, P. A., Lee, J. V. & Bryant, T. N. (1983). A numerical taxonomic
study of species of Vibrio isolated from the aquatic environment and
birds in Kent, England. J Appl Bacteriol 55, 263–282.
Wibbenmeyer, J. A., Provenzano, D., Landry, C. F., Klose, K. E. &
Delcour, A. H. (2002). Vibrio cholerae OmpU and OmpT porins are
differentially affected by bile. Infect Immun 70, 121–126.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 21:42:56
1071