Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005? 200557616231635Original ArticleTranscriptome of Vibrio cholerae biofilm developmentS.

Moorthy and P. I. Watnick

Molecular Microbiology (2005) 57(6), 1623–1635 doi:10.1111/j.1365-2958.2005.04797.x

Identification of novel stage-specific genetic

requirements through whole genome transcription
profiling of Vibrio cholerae biofilm development

Sudha Moorthy and Paula I. Watnick* development may greatly accelerate the discovery of
Division of Geographic Medicine and Infectious Diseases, novel targets for stage-specific inhibition of biofilm
Tufts-New England Medical Center, Boston, MA 02111, development.
USA.
Introduction
Summary
Most microbes in the natural environment live in surface-
Bacterial biofilm formation has been described as a attached communities called biofilms (Costerton et al.,
developmental process. This process may be divided 1995). Genetic and microscopic studies of biofilm forma-
into three stages: the planktonic stage, the monolayer tion by several Gram-positive and Gram-negative
stage and the biofilm stage. Bacteria in the planktonic organisms have suggested that development of a mature,
stage are not attached to each other or to a surface; three-dimensional biofilm involves the following consecu-
bacteria in the monolayer stage are attached to sur- tive, discrete stages: the planktonic stage, the monolayer
faces as single cells; and bacteria in the biofilm stage stage and, finally, the biofilm stage (O’Toole et al., 2000).
are attached to surfaces as cellular aggregates. In a Free-swimming planktonic cells encountering a surface
study limited to the Vibrio cholerae flaA, mshA and become transiently attached to it. Permanent immobiliza-
vps genes, we previously demonstrated that tran- tion of these cells on the surface results in the formation
scription in monolayer cells is distinct from that in of the monolayer. Induction of extracellular matrix biosyn-
biofilm cells and that the genetic requirements of thesis by cells in the monolayer leads to the development
monolayer formation are distinct from those of biofilm of a multi-layered biofilm through the formation of intercel-
formation. In this work, we sought to identify addi- lular contacts. Biofilm cells have been shown to be phys-
tional stage-specific genetic requirements through iologically and transcriptionally different from planktonic
microarray analysis of the V. cholerae transcriptome cells (Whiteley et al., 2001; Schembri et al., 2003; Stanley
during biofilm development. These studies demon- et al., 2003; Zhu and Mekalanos, 2003; Meibom et al.,
strated unique patterns of transcription in the plank- 2004).
tonic, monolayer and biofilm stages of biofilm Vibrio cholerae is both the agent of the diarrhoeal dis-
development. Based on our microarray results, we ease cholera and a natural inhabitant of aquatic environ-
selected cheY-3 as well as two previously uncharac- ments. The major virulence determinants of V. cholerae
terized genes, bap1 and leuO, for targeted mutation. human infection are cholera toxin (CTX), which is carried
The DcheY-3 mutant displayed a defect in monolayer on the CTX phage, and the toxin co-regulated pilus (TCP)
but not biofilm formation, suggesting that chemotaxis (Mekalanos, 1985; Taylor et al., 1986; 1987; Waldor and
plays a stage-specific role in formation of the Mekalanos, 1996; Tacket et al., 1998). A number of regu-
V. cholerae monolayer. Mutants carrying deletions in latory elements including TcpP and TcpH are responsible
bap1 and leuO formed monolayers that were indistin- for activation of ctx transcription upon entry into the
guishable from those formed by wild-type V. cholerae. human host (Carroll et al., 1997; Hase and Mekalanos,
In contrast, these mutants displayed greatly 1998; Murley et al., 1999).
decreased biofilm accumulation. Our microarray anal- Vibrio cholerae is also found in marine, estuarine and
yses document modulation of the transcriptome of fresh water environments in association with zooplankton,
V. cholerae as it progresses through the stages in phytoplankton, crustaceans, insects and plants (Huq
biofilm development. These studies demonstrate that et al., 1983; 1986; Tamplin et al., 1990; Colwell and Spira,
microarray analysis of the transcriptome of biofilm 1992; Colwell and Huq, 1994; Fotedar, 2001). Surface
attachment is thought to be required for colonization of
Accepted 27 June, 2005. *For correspondence. E-mail pwatnick both the human intestine and the aquatic environment. As
@tufts-nemc.org; Tel. (+1) 617 636 2545; Fax (+1) 617 636 3216. has been observed for other organisms, V. cholerae
© 2005 Blackwell Publishing Ltd
1624 S. Moorthy and P. I. Watnick
passes through the planktonic and monolayer stages prior
to forming a biofilm. Free-swimming planktonic cells are
characterized by the presence of flagella, and the flagellar
genes are actively transcribed in this stage. Transient
interactions with the surface are observed in the plank-
tonic stage, and these are mediated by the mannose-
sensitive haemagglutinin (MSHA), a type IV pilus. The
interaction of MSHA with the surface is blocked by
mannose or by a-methylmannoside (AMM), a non-
metabolizable analogue of mannose. Surface association
leads to repression of flagellar gene transcription, and
this, in turn, leads to permanent attachment of cells to the
surface in a monolayer. Once formed, these permanent
attachments are distinguished from transient attachments
by their resistance to the action of AMM. The flagellar
mutant monolayer is also resistant to the action of AMM.
This supports the hypothesis that flagellar motility must
be absent for permanent attachment to occur (Moorthy
and Watnick, 2004).
Exposure of a wild-type V. cholerae monolayer to
monosaccharides, either by supplementation of the bath-
ing medium or by degradation of a polysaccharide surface
to which the cells are attached, activates transcription of
the vps genes, which are responsible for synthesis of the
VPS exopolysaccharide (Yildiz and Schoolnik, 1999;
Kierek and Watnick, 2003; Moorthy and Watnick, 2004).
The vps synthesis genes, which include vpsA (VC0917)
and vpsL (VC0934), are located within the vps island
encompassing loci VC0916–VC0941. Synthesis of the
VPS exopolysaccharide leads to formation of a mature
biofilm consisting of bacterial pillars attached to a surface
(Watnick and Kolter, 1999; Yildiz and Schoolnik, 1999).
Thus, progression from the planktonic to the biofilm stage
involves changes in gene transcription, extracellular
matrix composition and three-dimensional structure.
Regulation of VPS synthesis has been partially eluci-
dated through the work of several laboratories. Environ-
mental signals such as monosaccharides and
nucleosides have been identified as activators of vps gene
transcription and biofilm formation (Haugo and Watnick,
2002; Kierek and Watnick, 2003), while high cell density
has been identified as an inhibitor of vps gene transcrip-
tion through the action of HapR (Hammer and Bassler,
2003; Vance et al., 2003; Zhu and Mekalanos, 2003; Yildiz
et al., 2004). VpsT, VpsR and VieA are additional regula-
tors of biofilm formation that respond to as yet unidentified
environmental signals (Yildiz et al., 2001; Casper-Lindley
and Yildiz, 2004; Tischler and Camilli, 2004).
A complete understanding of the developmental path-
way leading to formation of the bacterial biofilm requires
exploitation of global whole genome approaches. In this
work, we have used whole genome transcriptional analy-
sis to describe modulation of the V. cholerae transcrip-
tome during passage through the planktonic, monolayer
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635
and biofilm stages of biofilm development. Through these
experiments, we have made the observation that the tran-
scriptomes of the V. cholerae monolayer and biofilm are,
indeed, distinct with the exception of a few similarly reg-
ulated genes. Furthermore, we have demonstrated stage-
specific roles for CheY-3 in monolayer formation and for
two newly identified proteins, Bap1 and LeuO, in formation
of the extracellular biofilm matrix. Genetic requirements
such as these present novel targets for development of
stage-specific biofilm inhibitors.
Results
Distinct modulation of the V. cholerae transcriptome
characterizes entry into the monolayer and biofilm stages
Wild-type V. cholerae forms a monolayer when grown in
minimal medium (MM) alone. When mannose is added to
MM, the monolayer develops into a biofilm. We have pre-
viously used these growth conditions to demonstrate that
transcription levels of genes involved in flagellar and
exopolsaccharide synthesis are different in wild-type
V. cholerae monolayers and biofilms (Moorthy and Wat-
nick, 2004). In the present experiments, our goal was to
use microarray analysis to obtain a genomic perspective
of modulation of gene transcription in response to mono-
layer and biofilm formation. To achieve this, we performed
the following three microarray experiments (Fig. 1). To
determine the effect of monolayer formation on the
V. cholerae transcriptome, we compared levels of gene
transcription in wild-type V. cholerae monolayer cells with
those in planktonic cells grown in MM (monolayer exper-
iment). In order to separate the contributions of mannose
supplementation and surface attachment to modulation of
the V. cholerae transcriptome during biofilm formation in
MM supplemented with mannose, we performed the
microarray analysis of biofilm formation in the following
two steps: (i) a comparison of the transcriptome of plank-
tonic cells grown in MM supplemented with mannose with
that of planktonic cells grown in MM alone (planktonic
experiment) and (ii) a comparison of the transcriptome of
biofilm cells with that of planktonic cells grown in MM
supplemented with mannose (biofilm experiment). Each
comparison was performed in triplicate. The goal of our
microarray analysis was to identify as many stage-specific
genes as possible rather than to accurately measure lev-
els of differential transcription. Thus, we did not base
inclusion in our lists of differentially regulated genes on a
test of statistical significance. Rather, genes that were
induced or repressed by twofold or greater in all three
microarray replicates were included in the lists of differen-
tially regulated genes (see Tables S1–S4).
The monolayer, planktonic and biofilm experiments
identified 150, 128 and 383 differentially regulated genes
 Transcriptome of Vibrio cholerae biofilm development 1625

A B D
Planktonic (–M) Planktonic (–M) DvpsA Planktonic (+M)

Planktonic
Experiment

Monolayer vpsA Monolayer

Experiment Experiment
C Planktonic (+M)

Biofilm
Experiment

Monolayer (–M) Biofilm(+M) DvpsA Monolayer (+M)

Fig. 1. Schematic representation of the state of V. cholerae under each of the conditions in this work.
A. Monolayer experiment. Test: Monolayer (–M)/Reference: Planktonic (–M).
B. Planktonic experiment. Test: Planktonic (+M)/Reference: Planktonic (–M).
C. Biofilm experiment. Test: Biofilm (+M)/Reference: Planktonic (+M).
D. vpsA monolayer experiment. Test: DvpsA mutant monolayer (+M)/Reference: DvpsA planktonic (+M).
+M and –M refer to the presence and absence of mannose in the growth medium respectively.

respectively. Most of these genes were differentially reg-
ulated under the conditions of only one of the three exper-
iments. This result supports our previous hypothesis that
the V. cholerae monolayer and biofilm are distinct devel-
opmental stages.
To obtain an overview of our microarray results, we
classified genes according to the genome annotation and
then tabulated the numbers of differentially regulated
genes in selected functional categories (Table 1; Heidel-
berg et al., 2000). We have previously shown that addi-
tion of mannose to planktonic and monolayer cells
activates vpsL gene transcription and formation of intrac-
ellular contacts (Moorthy and Watnick, 2004). This sug-
gests that synthesis of an extracellular matrix occurs in
the presence of mannose. Construction of an extracellu-
lar matrix requires: (i) transporters to import the neces-
sary substrates for extracellular matrix synthesis; (ii)
enzymes to metabolize these substrates; and (iii) cell
envelope enzymes to catalyse synthesis of the extracel-
lular matrix. In fact, many induced genes were identified
in the categories of transport and binding proteins, carbo-
hydrate metabolism and cell envelope synthesis in the
planktonic and biofilm experiments. As is the case for
many microarray experiments, hypothetical and con-

Table 1. Categories of differentially regulated genes in monolayer, planktonic and biofilm microarrays.

Monolayer Planktonic Biofilm

Tabulation of selected categories Induced Repressed Induced Repressed Induced Repressed

Hypothetical proteins 47 24 22 25 58 85
Transcription regulation 4 7 4 2 9 13
Response regulators 4 3 2 7
Transport and binding proteins 5 1 10 10 17 18
Carbohydrate metabolism 5 2 5 5
Cell envelope synthesis 6 2 6 6
Chemotaxis and Motility 8 1 6 5 6
Total number of differentially regulated genes 91 59 68 60 171 212

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635

1626 S. Moorthy and P. I. Watnick
served hypothetical genes constituted the largest cate-
gory of differentially regulated genes. This supports the yer
er ic ola
widely held view that there is yet much to be learned
lay
ton lm m on
about biofilm development and the bacterial genome, in ono k
an iofi psA
general. M Pl B v

The DvpsA monolayer is similar to the wild-type biofilm

In MM alone, wild-type V. cholerae forms a monolayer on

surfaces. When mannose is added, transcription of the
vps genes is activated, leading to biofilm formation. In
contrast, even in mannose-supplemented medium, a
DvpsA mutant forms a monolayer attributed to the inability
to synthesize exopolysaccharide (Moorthy and Watnick,
2004). This led us to hypothesize that gene transcription
in a DvpsA monolayer formed in the presence of mannose
would be most like that in a wild-type monolayer formed
in the absence of mannose. To evaluate this hypothesis,
we compared gene transcription in DvpsA mutant mono-
layers formed in the presence of mannose with that in
DvpsA mutant planktonic cells grown in MM supplemented
with mannose (vpsA monolayer experiment). We per-
formed a K-means clustering of the expression values of
the monolayer, planktonic, biofilm, and vpsA monolayer
experiments using standard distance as the measure of
similarity (Fig. 2). Our cluster analysis highlighted the
close parallels in modulation of gene transcription as a
result of biofilm formation by wild-type V. cholerae and
monolayer formation by a DvpsA mutant. In fact, 123 sim-
ilarly regulated genes were detected between the wild-
Fig. 2. Cluster analysis of the monolayer, planktonic, biofilm and
type biofilm and vpsA monolayer experiments, while only vpsA monolayer experiments described in this work. The intensity of
10 similarly regulated genes were detected between the red or green represents the extent of gene induction or repression
wild-type monolayer and vpsA monolayer experiments. respectively. Yellow represents no change in gene transcription.
Thus, the DvpsA monolayer is more closely related to the
wild-type biofilm than to the wild-type monolayer.
We identified eight additional genes outside the vps
island that were activated in the planktonic experiment
Extracellular matrix gene transcription is activated by
and repressed in the vpsA experiment (Fig. 3). On
planktonic growth of wild-type V. cholerae in the presence
detailed analysis, all of these genes clustered with one or
of mannose and repressed by DvpsA mutant monolayer
more of the vps genes. We hypothesized that these genes
formation
might also be involved in synthesis of the extracellular
As shown in Fig. 3, most genes in the vps island, including biofilm matrix.
vpsL, were activated in the planktonic experiment and Because our primary goal was to identify stage-specific
repressed in the vpsA monolayer experiment. This obser- genes rather than to accurately quantify stage-specific
vation is in agreement with previous quantitative poly- transcription, we proceeded directly to construction of
merase chain reaction (PCR) experiments demonstrating mutants. Two genes, located at loci VC1888 and VC2485,
that transcription of vpsL is repressed upon DvpsA mutant were selected for deletion. The gene at locus VC1888 is
monolayer formation in the presence of mannose (Moor- one of three encoding haemolysin-related proteins that
thy and Watnick, 2004). This suggests that, when exposed are co-regulated with the vps island. We have named this
to biofilm-activating conditions, surface-associated cells protein Bap1 for biofilm-associated protein 1. It has a
are able to sense a block in extracellular matrix synthesis predicted signal peptide at the N-terminus but no mem-
and, through an unknown negative feedback loop, repress brane-spanning domains, suggesting that it is a secreted
transcription of genes involved in extracellular matrix protein. It also contains four 30-amino-acid repeats that
synthesis. are homologous to FG-GAP domains. These domains are

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635

 Transcriptome of Vibrio cholerae biofilm development 1627

9 8 8 5 6 7 8 37
9 28 929 930 931 932 933 934 935 936 937 938 939 940 941 048 163 188 248 248 248 248 A08
0 V
0
C0V
0
C00VC00VC00VC00VC00VC00VC00VC00VC00VC00VC00VC00VC0 C
V0 V0 C V0 C V0 C 0VC 0VC0VC 0VC

Planktonic

V V V V V V V V V V V V V V V V V V V V V V
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

DvpsA
monolayer
V V V V V V V V V V V V V V V V V V V V V V

Gene ID Product Function

VC0489 Haemolysin, putative Pathogenesis

VC1638 DNA binding response regulator Regulatory
VC1888 Haemolysin-related protein
VC2485 Transcriptional regulator, LysR family Regulatory
VC2486 Hypothetical protein
VC2487 Conserved hypothetical protein
VC2488 Conserved hypothetical protein
VCA0837 Haemolysin, putative Pathogenesis

Fig. 3. Modulation of transcription of vps genes and similarly regulated genes in the planktonic and vpsA experiments. The intensity of red or
blue represents the extent of gene induction or repression respectively. Yellow represents no change in gene transcription. Putative gene products
and functions are listed in a table below.

also found in eukaryotic membrane-associated integrins, comparable areas of the substratum (Fig. 4A). In LB, both
which mediate adhesion of eukaryotic cells to proteins in the Dbap1 and DleuO mutants showed reduced biofilm
the extracellular matrix such as fibrinogen and fibronectin accumulation, demonstrating that these mutants are
(Baneres et al., 2000). The protein encoded at locus defective for biofilm formation (Fig. 4B). Interestingly, both
VC2485 is homologous to transcriptional regulators of the these mutants demonstrated greater surface accumula-
LysR family, with 50% identity and 75% similarity to LeuO, tion in LB than a DvpsA mutant, suggesting that VPS
a positive regulator of the leu operon in Escherichia coli. synthesis does occur in these mutants and that synthesis
No better match to LeuO is encoded in the V. cholerae of VPS contributes to surface attachment.
genome. Furthermore, the V. cholerae leuABCD operon Because decreased biofilm formation by V. cholerae
is located near VC2485 at loci VC2490–VC2493. This mutants has previously been correlated with decreased
arrangement of the leu genes is similar to that found in transcription of the vps genes, we measured vpsL tran-
the E. coli genome. Thus, we have named this protein scription in LB by wild-type V. cholerae, a Dbap1 mutant
LeuO. and a DleuO mutant carrying chromosomal vpsL–lacZ
In order to determine if Bap1 and LeuO play a stage- reporter fusions. The levels of vpsL transcription in LB
specific role in biofilm development, we quantified surface broth were comparable for wild-type and mutant strains
association in the monolayer and biofilm stages by wild- (Fig. 4C). This indicates that decreased biofilm formation
type V. cholerae as well as Dbap1, DleuO and DvpsA by the mutants is not the result of decreased vpsL gene
mutants. Because biofilm accumulation is sparse in mini- transcription.
mal medium and therefore difficult to quantify by macro-
scopic methods, we chose to study Dbap1 and DleuO
The monolayer and biofilm stages have 12 commonly
biofilms formed in LB, a medium in which the predominant
regulated gene clusters
surface-associated state is the vps-dependent biofilm
(Kierek and Watnick, 2003). As shown in Fig. 4A, the We identified 27 genes that were similarly regulated in the
monolayers made by wild-type V. cholerae, the Dbap1 monolayer and biofilm experiments. In contrast, only four
mutant, the DleuO mutant and a DvpsA mutant covered differentially regulated genes were common to the plank-
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635
1628 S. Moorthy and P. I. Watnick
tonic and monolayer experiments, and only three were
A
Total surface area covered (µm2) common to the planktonic and biofilm experiments. This
suggested to us that a small subset of differentially regu-
350 lated genes might be used to describe the footprint of the
V. cholerae surface-associated state. To define this foot-
300
print more precisely, we searched for genes that were
250 similarly regulated in the monolayer, biofilm and vpsA
monolayer experiments as well as similarly regulated
200 genes that either were in common operons, were in spatial
proximity, or were related in function (Table 2). Twelve
150 clusters of similarly regulated genes were identified.
Although the transcription patterns of these gene clusters
100
were not confirmed by quantitative PCR, in all cases they
50 were observed in more than one microarray experiment
comparing transcription in a surface-associated state to
0 transcription in the corresponding planktonic state. The
WT Dbap1 DleuO DvpsA function of most of these gene clusters remains to be
B elucidated. However, two interesting themes emerged
from this tabulation. First of all, the gene cluster including
0.4 tcpP and tcpH, which is involved in virulence gene regu-
Absorbance (655 nm)

lation, was repressed in all three surface-associated

states. Futhermore, the CTXF genes rstR, ctxA and ctxB
0.3 were repressed in the wild-type and vpsA monolayer
experiments. These data suggest that, under the condi-
0.2 tions of these experiments, surface association may
repress virulence gene transcription.
Secondly, the large gene clusters within VC1514–
0.1 VC1519 and VCA0676–VCA0685 include proteins con-
taining iron-sulphur clusters as well as those involved in
formate dehydrogenation and nitrate reduction
0 respectively. These types of proteins are involved in
WT Dbap1 DleuO DvpsA anaerobic respiration, and transcriptional activation of
C these gene clusters suggests that surface-attached cells
0.2 may induce proteins involved in anaerobic respiration.

0.16
Chemotaxis plays a role in monolayer formation
A415/A655

0.12 The V. cholerae genome contains 43 homologues of

methyl-accepting chemotaxis proteins (MCPs). Eight of
0.08 these MCPs were activated in the monolayer experiment
but none were repressed (Table 3). In the planktonic
0.04 experiment, only one MCP displayed increased transcrip-
tion. In the biofilm experiment, six MCPs were differen-
0
WT Dbap1 DleuO tially transcribed. Three of these displayed increased
transcription, and three displayed decreased transcription.
Fig. 4. Monolayer formation, biofilm formation and vpsL transcription Based on these observations, we questioned whether
by wild-type V. cholerae, a Dbap1 mutant, a DleuO mutant and a chemotaxis might play a role in V. cholerae biofilm devel-
DvpsA mutant.
A. Total surface area covered by wild-type and mutant monolayers opment. In E. coli, CheY plays a central role in chemot-
formed over 3 h. axis, interacting directly with the flagellar motor to change
B. Quantification of wild-type and mutant biofilm accumulation in LB. the direction of rotation. V. cholerae possesses multiple
C. Quantification of vpsL transcription in wild-type and mutant
V. cholerae harbouring a chromosomal fusion of the vpsL promoter to CheY homologues. Only CheY-3, however, has been
the lacZ gene. shown to play a role in classical flagellum-mediated
chemotaxis (Butler and Camilli, 2004). To investigate the
role of chemotaxis in surface association, we constructed

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635

 Transcriptome of Vibrio cholerae biofilm development 1629
Table 2. Similarly regulated gene clusters in the monolayer, biofilm and vpsA monolayer experiments.

Gene ID Product Monolayer Biofilm vpsA Monolayer

Upregulated
VC1075 Conserved hypothetical protein 3.80 8.23
VC1077 Hypothetical protein 9.02
VC1330 Hypothetical protein 7.58
VC1332 Conserved hypothetical protein 83.90 26.08
VC1333 Hypothetical protein 8.37 80.21 20.61
VC1334 Conserved hypothetical protein 5.46 124.97 3.10
VC1335 Transcriptional regulator, GntR family 6.46 29.63 12.09
VC1336 Carboxyphosphonoenolpyruvate phosphonomutase 8.45 21.70 27.76
VC1338 Aconitate hydratase 1 2.34
VC1339 Conserved hypothetical protein 3.12
VC1514 Hypothetical protein 5.58 5.03
VC1515 Chaperone, formate dehydrogenase-specific, putative 8.58 7.82
VC1516 Iron-sulphur cluster-binding protein 3.37 3.63
VC1517 Hypothetical protein 3.98
VC1518 Hypothetical protein 3.32
VC1519 Formate dehydrogenase accessory protein 5.51
VC2705 Sodium/solute symporter, putative 3.95 21.33
VC2706 Conserved hypothetical protein 5.96
VC2708 Guanylate kinase 8.74
VC2709 DNA-directed RNA polymerase, omega subunit 13.85
VC2711 ATP-dependent DNA helicase RecG 4.03 4.75
VCA0676 Iron-sulphur cluster-binding protein NapF 9.93 3.51 4.13
VCA0677 napD protein 6.19
VCA0678 Periplasmic nitrate reductase 5.77
VCA0680 Periplasmic nitrate reductase, cytochrome c-type protein 3.18
VCA0682 Transcriptional regulator UhpA 28.09
VCA0684 Regulatory protein UhpC 8.86 5.86
VCA0685 Iron(III) ABC transporter, periplasmic iron-compound-binding protein 6.67
Downregulated
VC0826 Toxin co-regulated pilus biosynthesis protein P 0.31a 0.29a
VC0827 Toxin co-regulated pilus biosynthesis protein H 0.23 0.26a 0.35a
VC0940 Conserved hypothetical protein 0.10 0.12
VC0943 Lipoic acid synthetase 0.30
VC0944 Lipoate-protein ligase B 0.33
VC1443 Hypothetical protein 0.24
VC1444 Hypothetical protein 0.33
VC1445 Sensor histidine kinase/response regulator 0.32 0.38
VC1455 Transcriptional repressor RstR 0.42
VC1456 Cholera enterotoxin, B subunit, ctxB 0.21
VC1457 Cholera enterotoxin, A subunit, ctxA 0.25a 0.34a
VC2698 Aspartate ammonia-lyase 0.12 0.29
VC2699 C4-dicarboxylate transporter, anaerobic 0.40
VCA0817 Hypothetical protein 0.11 0.30
VCA0818 Magnesium transporter MgtE, putative 0.37
VCA0820 Chaperonin, 60 Kd subunit 0.29
VCA1045 PTS system, mannitol-specific IIABC component 0.39 0.29 0.20
VCA1046 Mannitol-1-phosphate 5-dehydrogenase 0.16 0.38 0.28

a. Denotes that the expression value is an average of two replicates.

a V. cholerae DcheY-3 mutant and tested its ability to form
a monolayer and a biofilm. As illustrated in Fig. 5A and
quantified in Fig. 5B, after 24 h, the substratum surface
area covered by the Dche Y-3 mutant monolayer was only
slightly less than that covered by the wild-type V. cholerae
monolayer. We have previously shown that incubation with
AMM may be used to differentiate between permanently
and transiently attached cells. To determine the numbers
of permanently attached cells in the wild-type and Dche
Y-3 mutant monolayers, monolayers formed over 24 h
were treated with AMM. More than 50% of the cells in the
Dche Y-3 mutant monolayer were removed by AMM treat-
ment (Fig. 5A and B), while only a small proportion of wild-
type cells were removed by this treatment. This suggests
that the transition from transient to permanent attachment
is retarded in the Dche Y-3 mutant. Interestingly, the
DcheY-3 mutant displayed no impairment in biofilm forma-
tion in LB (Fig. 5C). Thus, the surface-attachment defect
of the DcheY-3 mutant is monolayer-specific.
Discussion
In this work, we have used microarray analysis to study
the transcriptome of V. cholerae during each stage of bio-

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635

1630 S. Moorthy and P. I. Watnick
Table 3. Differentially regulated methyl-accepting chemotaxis genes film development. Our results indicate that the wild-type
in monolayer, planktonic and biofilm microarrays.
V. cholerae monolayer and biofilm stages are transcrip-
Induced Repressed
tionally distinct at the whole genome level and, thus, sup-
port previous assertions that the monolayer and biofilm
Average Average are distinct stages in biofilm development. Based on our
expression expression
Gene ID value Gene ID value microarray analysis, we have identified three proteins,
namely CheY-3, LeuO and Bap1, which are required in a
Monolayer VC1248 4.5
VC1394 4.5 specific stage of biofilm development.
VC1405 5.0 In E. coli, the bacterial chemotaxis machinery directs
VC1868 3.3 cells towards favourable stimuli and away from noxious
VCA0008 7.4
VCA0031 7.8 stimuli by altering the frequency of flagellar pauses and
VCA0906 4.1 reversals of direction (Lapidus et al., 1988; Eisenbach
VCA0988 3.7
et al., 1990). CheY, which is at the centre of the chemot-
Planktonic VCA0663 6.7 VC1406 0.3
actic signal transduction system, is phosphorylated by the
Biofilm VC0216 6.6 VC1535 0.1
VC0282 8.7 VCA0268 0.3
kinase CheA (Szurmant and Ordal, 2004). This process
VC1898 9.6 VCA0663 0.3 is modulated by MCPs, which serve as the sensors of
environmental stimuli. In the absence of CheY-P, the fla-

Fig. 5. Effect of AMM treatment on wild-type

A V. cholerae and DcheY-3 mutant monolayers
formed over 24 h.
A. Phase contrast micrographs of monolayers
before (Monolayer) and after
(Monolayer + 0.1% AMM) treatment with AMM.
B. Total surface area covered by monolayer
cells before and after treatment with AMM.
WT C. Quantification of wild-type V. cholerae and
DcheY-3 mutant biofilm accumulation in LB.

DcheY

Monolayer Monolayer
+0.1% AMM
B C
WT 0.25
DcheY
Total area covered (mm2)

Absorbance (655nm)

3500
0.2
3000
2500 0.15
2000
1500 0.1

1000
0.05
500
0 0
24 h monolayer 24 h monolayer WT DcheY
+ 0.1% AMM
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635
 Transcriptome of Vibrio cholerae biofilm development 1631
gellum undergoes counterclockwise rotation, leading to a
straight swimming phenotype. When CheY-P interacts
with the flagellar motor, clockwise rotation is induced, and
flagellar pauses and reversals of direction increase in
frequency. Recent studies suggest that a similar mecha-
nism is at work in V. cholerae chemotaxis (Gosink et al.,
2002; Butler and Camilli, 2004).
The role of chemotaxis in biofilm development varies
with the bacterial species under study. Pratt and Kolter
(1998) have previously shown that chemotaxis is dispens-
able for initiation of E. coli biofilm formation. In contrast,
Aeromonas caviae chemotaxis mutants display a defect
in biofilm formation (Kirov et al., 2004). V. cholerae has
multiple homologues of CheY and CheA. However, only
CheY-3 and CheA-2 have been shown to be involved in
flagellar-based chemotaxis (Gosink et al., 2002; Butler
and Camilli, 2004).
In this work, we have found the first evidence that
V. cholerae monolayer formation is facilitated by the bac-
terial chemotactic apparatus. We have previously pre-
sented evidence that the absence of flagellar motility is
required for permanent attachment (Moorthy and Watnick,
2004). One possibility is that flagellar pauses, which are
observed in the presence of a functional chemotactic
apparatus, facilitate initiation of the genetic changes
required for permanent attachment. Alternatively, CheY-3
may enhance permanent attachment through a signalling
pathway that is independent of the flagellar motor.
Transcriptional activation of several genes encoding
MCPs was observed upon monolayer formation. The
types of ligands bound by these MCPs have not yet been
studied, and the observed transcriptional activation of
these MCPs does not necessarily imply a role in mono-
layer formation, maintenance, or dissolution under the
experimental conditions utilized here. However, this pat-
tern of transcription suggests to us that the chemotactic
apparatus of monolayer cells remains prepared for a rapid
response to environmental stimuli and, hence, that mono-
layer cells are not fully committed to a surface-associated
existence.
The transition to the biofilm stage is initiated by various
environmental signals through the action of specific tran-
scription factors (Yildiz et al., 2001; Haugo and Watnick,
2002; Hammer and Bassler, 2003; Kierek and Watnick,
2003; Zhu and Mekalanos, 2003; Casper-Lindley and
Yildiz, 2004). To date, all identified transcription factors
alter biofilm formation by modulation of vps gene tran-
scription. We have identified two biofilm-specific proteins
that increase V. cholerae biofilm accumulation without
increasing transcription of the vps genes. Because there
is little similarity between Bap1 and the V. cholerae
haemolysin HlyA, it seems unlikely that Bap1 functions as
a haemolysin. Rather we favour the hypothesis that, like
VPS, it is transported into the extracellular milieu during
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635
biofilm formation and plays a direct role in stabilization of
the extracellular matrix. However, we do not exclude the
possibility that Bap1 may use a post-transcriptional mech-
anism to alter exopolysaccharide synthesis and transport.
LeuO has been implicated in post-transcriptional repres-
sion of rpoS in E. coli and activation of virulence genes
and porins in Salmonella (Repoila and Gottesman, 2001;
Tenor et al., 2004). Thus, it seems likely that LeuO is a
global regulator. As has been observed in E. coli, LeuO
may also utilize a post-transcriptional mechanism to
increase exopolysaccharide synthesis. Alternatively, it
may directly regulate transcription of genes encoding ele-
ments of the extracellular matrix other than exopolysac-
charide. Experiments are underway in the laboratory to
determine the roles of Bap1 and LeuO in V. cholerae
biofilm formation.
Biofilm development proceeds in three stages: (i) the
planktonic stage, (ii) the monolayer stage and (iii) the
biofilm stage. In this work, we have used microarray anal-
ysis to obtain a comprehensive description of modulation
of the V. cholerae transcriptome in each stage of biofilm
development. Using mutational analysis, we have identi-
fied three proteins that are uniquely required at one stage
in this process. Thus, microarray analysis is a powerful
tool for dissecting the transcriptome of biofilm develop-
ment and for identifying stage-specific proteins that may
then be used to develop stage-specific inhibitors of biofilm
development.
Experimental procedures
Bacterial strains, plasmids and media
The bacterial strains and plasmids used in this study are
listed in Table 4. All experiments were done in either LB broth
or MM alone or supplemented with 0.2% mannose (Sigma;
Moorthy and Watnick, 2004). Where specified, 0.1 M phos-
phate buffered saline (PBS; pH 7.0) was used to rinse the
cells and AMM was added to a final concentration of 0.1%.
Construction of the deletion mutants
The DleuO mutant was constructed by amplification of a
447 bp fragment at the 5¢ end and a 447 bp fragment at the
3¢ end of VC2485 using primer pairs listed in Table 4. Internal
primers included a complementary 15 bp sequence at their
3¢ and 5¢ ends, respectively, as previously described (Haugo
and Watnick, 2002). These two fragments were joined using
the gene splicing by overlap extension technique (Horton
et al., 1990; Lefebvre et al., 1995). This resulted in the con-
struction of a fragment including only the first 66 bp and last
36 bp of leuO with a deletion of 866 bp within the coding
region. The fragment containing the deletion was purified and
ligated into pCR2.1TOPO. This fragment was then removed
from pCR2.1TOPO by digestion with SpeI and XhoI and
ligated into pWM91 to create the suicide plasmid
pWM91DleuO. This plasmid was used to create leuO dele-
1632 S. Moorthy and P. I. Watnick
Table 4. Bacterial strains, plasmids and primers.

Strain/plasmid/primers Genotype/sequence Source or reference

E. coli
SM10lpir thi thr leu tonA lacy supE Miller and Mekalanos (1988)
recA::RP4-2-Tc::MulpirR6K; Kmr
PW614 SM10lpir (pCVD443lac::DcheY-3) Butler and Camilli (2004)
PW659 SM10lpir (pWM91Dbap1) This study
PW680 SM10lpir (pWM91DleuO) This study
V. cholerae
PW249 MO10 O139, Smr Waldor and Mekalanos (1994)
PW357 MO10lacZ::vpsLpÆlacZ, Smr Haugo and Watnick (2002)
PW396 MO10 DvpsA, Smr Kierek and Watnick (2003)
PW631 MO10 DcheY-3 This study
PW662 MO10Dbap1 lacZ::vpsLpÆlacZ; Smr This study
PW662 MO10DleuO lacZ::vpsLpÆlacZ; Smr This study
Plasmids
pWM91 oriR6KmobRP4 lacI pTac tnp miniTn10Km; Kmr, Apr Metcalf et al. (1996)
pWM91Dbap1 pWM91 carrying a fragment of bap1 harbouring an This study
internal, unmarked deletion
pWM91DleuO pWM91 carrying a fragment of leuO harbouring an This study
internal, unmarked deletion
Primers
P301 5¢-cca acc cgc tgt atg aac tt-3¢ Primers used for construction of bap1 deletion
P302 5¢-tta cga gcg gcc gca gct tat cgc ggt caa cgt tt-3¢
P303 5¢-tgc ggc cgc tcg taa agc tgg tta acc cac aac ag-3¢
P304 5¢-gca gtg agt gct tcc tta cca-3¢
P358 5¢-ttg gca aaa agt cga ttt cc-3¢ Primers used for construction of leuO deletion
P359 5¢-tta cga gcg gcc gca gct ttc cat gcg gta ac-3¢
P360 5¢-tgc ggc cgc tcg taa atg gtg att tgt ggc gaa gt-3¢
P361 5¢-caa aat cag caa tcg tcc aa-3¢

tions in the relevant strains by double homologous recombi-
nation and sucrose selection as previously described (Haugo
and Watnick, 2002). The Dbap1 mutant was constructed sim-
ilarly. The bap1 deletion included 33 bp at the 5¢ end and
57 bp at the 3¢ end of the coding region, resulting in a
1986 bp in frame deletion. The strain SM10lpir
(pCVD443lac::DcheY-3) was used to create the DcheY-3
deletion mutant (Butler and Camilli, 2004).
Phase contrast microscopy and quantitative analysis of
monolayer structures
Cells were grown in sterile 24-well polystyrene microtiter
dishes. Wells were filled with 300 ml of MM and inoculated
with overnight cultures of the relevant strain to yield a starting
A655 of 0.001. The dishes were incubated at 27∞C with gentle
shaking for the indicated length of time. After incubation, the
planktonic cells were removed. PBS was added to wells
followed by vigorous agitation for 5 min, removal of medium
and replacement with fresh PBS. This procedure was
repeated three times. After removal of planktonic cells, mono-
layer development at the bottom surface of the well was
visualized with an Eclipse TE-200 microscope (Nikon)
equipped with an Orca digital CCD camera (Hamamatsu).
For treatment with AMM, monolayers were formed as
described above and visualized by phase-contrast micros-
copy. They were then rinsed, and PBS supplemented with
AMM was added to the wells. The cells were incubated for
three additional hours at 27∞C, rinsed, and visualized again.
A computer equipped with Metamorph Imaging software
(Universal Imaging) was used for image acquisition,
processing and quantification. This software quantifies the
total surface area covered by cells. All measurements were
performed in triplicate and repeated several times. Monolayer
accumulation was reported as a mean measurement. Error
bars represent the standard deviation.
Quantitative analyis of biofilms formed in LB
The strains to be tested were grown overnight in LB broth,
pH 7.4 at 27∞C with shaking. The following morning, 6 ml of
these cultures was used to inoculate borosilicate tubes filled
with 300 ml of fresh LB broth. These cultures were incubated
statically for 24 h at 27∞C. At the end of the incubation,
planktonic cells were removed, and the OD655 of this cell
suspension was measured. Fresh LB and an 100 ml volume
of 1 mm glass beads were added to each tube. This mixture
was vortexed to disperse biofilm-associated cells, and the
OD655 of the resulting suspension was measured. All mea-
surements were performed in triplicate and repeated several
times. Biofilm accumulation was reported as a mean mea-
surement. Error bars represent the standard deviation.
b-Galactosidase measurements of vpsL transcription
For analysis of vpsL transcription, the strains to be tested
were grown to stationary phase in LB broth, pH 7.4 at 27∞C
with shaking. Twenty microlitres of these cultures was used
to inoculate tubes filled with 2 ml of LB broth, pH 7.4. These
cultures were incubated overnight at 27∞C with shaking. The

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635

 Transcriptome of Vibrio cholerae biofilm development 1633
following morning, 100 ml of this culture was removed, and
an OD655, representing the final cell density of each culture,
was measured. Of each culture 1.5 ml was placed in an
eppendorf tube, spun for 5 min at 3000 r.p.m. to pellet cells,
washed with 1 ml of Z-buffer, and then resuspended in 100 ml
of Z-buffer (Miller, 1992). After three freeze-thaw cycles, 17 ml
of a 4 mg ml-1 solution of o-nitrophenyl-b-D-galactopyrono-
side (Sigma) was added to the lysed cells, and this
preparation was incubated at 37∞C for 23 h. Cell debris was
then removed by centrifugation, and the OD415 of the super-
natant was measured. All b-galactosidase measurements are
reported as the OD415 of the supernatant divided by the
OD655, reflecting the final cell density of the respective culture.
b-Galactosidase measurements were performed in triplicate
and reported as a mean measurement. Error bars represent
the standard deviation.
Array printing
Each microarray consisted of ~4000 DNA oligonucleotides
spotted onto UltraGAPS coated slides (Corning) using a
Biorobotics MicroGrid II. Oligonucleotides were 70-mers
designed to correspond to unique sequences within
V. cholerae open reading frames (Illumina) and additional
control sequences.
RNA extraction
All the strains were grown in MM or MM supplemented with
mannose in sterile 90 mm Petri dishes. Briefly, for preparation
of planktonic cell RNA, 10 ml aliquots of the cultures were
pelleted by centrifugation. Two millilitres of TRIzol (Invitrogen)
were added to each pellet, and cells were lysed by repetitive
pipetting. After removal of planktonic cells, the remaining
surface-associated cells were rinsed with PBS three times
and then lysed in situ with 7 ml of TRIzol. The lysates of both
planktonic and surface-associated cells were incubated at
room temperature for 10 min. Chloroform was then added to
the samples at a ratio of 0.2 ml for every millilitre of TRIzol.
Samples were shaken vigorously for 15 min and then incu-
bated at room temperature for 3 min. The samples were
centrifuged at 4500 g at 4∞C for 10 min to separate the aque-
ous layer, which was then transferred to a microcentrifuge
tube. The RNA, along with 10 mg of carrier t-RNA, was incu-
bated with isopropyl alcohol in a ratio of 0.5 ml for every
millilitre of TRIzol to allow precipitation of RNA. Precipitated
RNA was pelleted by centrifugation at 12 000 g for 30 min at
4∞C, washed with 75% ethanol, dried, and then dissolved in
100 ml of RNase-free water. To remove contaminating DNA,
the total RNA was incubated with RNase-free DNAse I
(Ambion) for 30 min at 37∞C. Finally, the RNeasy Mini Kit
(Qiagen) was used to clean up the RNA after DNase I diges-
tion. The RNA samples were checked for presence of resid-
ual genomic DNA by standard PCR. A measurement of
absorbance at 260 nm was used to quantify RNA concentra-
tion, and RNA was subsequently stored at -80∞C.
cDNA synthesis
Ten micrograms of total RNA was reverse transcribed in a
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635
final volume of 30 ml as follows. A pool of random hexamers,
at a concentration of 5 mM, was added to the RNA prior to
denaturation at 70∞C for 10 min. To this mixture was then
added (i) the first strand buffer [75 mM KCl, 50 mM Tris-HCl
(pH 8.3), 3 mM MgCl2]; (ii) a mixture containing 0.5 mM dATP,
0.5 mM dCTP, 0.5 mM dGTP, 0.2 mM dTTP and 0.3 mM ami-
noallyl dUTP; (iii) 10 mM DTT; and (iv) 400 units of Super-
script II RNase H-Reverse Transcriptase (Invitrogen). The
reaction mixture was incubated at 42∞C for 2 h. The RNA was
hydrolysed by incubation with 0.1 M EDTA and 0.2 M NaOH
at 65∞C for 15 min and then neutralized with 0.1 M HCl before
purification of the cDNA with a Minelute column (Qiagen).
The purified cDNA was quantified by measuring absorbance
at 260 nm.
Labelling of the cDNA probes
The cDNAs were coupled to monoreactive Cy3 and Cy5
(Amersham) in the presence of 0.05 M NaHCO3, for 60 min
at room temperature. The reaction was quenched with 1.2 M
Hydroxylamine and the labelled cDNAs were purified with
Qiaquick columns (Qiagen). The efficiency of incorporation
was determined by measuring the absorbance of the
labelled cDNA at 260 nm and 550 nm (for Cy3 incorpora-
tion) and 260 nm and 650 nm (for Cy5 incorporation). Cy3-
and Cy5-labelled corresponding reference and test cDNAs
(see Fig. 1) were mixed in ratios such that the incorporated
dyes were equal, and then dried down and resuspended in
the hybridization buffer (25% formamide, 5¥ SSC, 0.1%
SDS, 1 mg ml-1 salmon sperm DNA, 2 mg ml-1 yeast tRNA).
Probes were denatured at 95∞C for 3 min before
hybridization.
Hybridization
Prior to hybridization, printed slides were cross-linked in a
UV Stratalinker after brief hydration over boiling dH2O. Slides
were prehybridized at 42∞C in 5¥ SSC, 0.1% SDS, 2% BSA
and then briefly rinsed with water and isopropanol. Each
array was hybridized simultaneously to differentially labelled
cDNA probes corresponding to sample and control RNAs
prepared previously. After hybridization for 36 h at 42∞C,
slides were washed with (i) 2¥ SSC, 0.2% SDS; (ii) 0.1¥ SSC,
0.2% SDS; and (iii) 0.1¥ SSC. Hybridized, washed slides
were scanned for Cy5 and Cy3 fluorescence intensities using
a Packard Scanarray 4000. Laser power and/or PMT were
adjusted such that the fluorescence from Cy3- and Cy5-
labelled probes was balanced.
Image quantification and data analysis
For each of the four experiments (Fig. 1), data were compiled
from at least three microarray replicates. Scans were analy-
sed with Imagene version 5.6.1 (BioDiscovery) to calculate
spot intensity and background. Gene expression levels for all
replicates were calculated from the background-subtracted
fluorescent intensities of the sample and control channels.
These data were further analysed on GeneSpring version 6.1
(Silicon Genetics). The data were normalized using intensity-
dependent Lowess normalization per spot per chip. Genes
1634 S. Moorthy and P. I. Watnick
were scored as differentially regulated if the ratio of
test:reference transcripts was either >2.0 or <0.5 in all tech-
nical replicates of the experiment.
Acknowledgements
We thank Susan Butler and Andrew Camilli for generous
sharing of the DcheY-3 mutant constructs. We thank Dr Anne
Kane of the Tufts-NEMC GRASP Center and her staff for their
expert preparation of many reagents. We thank Lan Wei of
the Tufts Microarray Core Facility for help with the printing
and scanning of microarray slides, Dagmar Kapfhammer for
optimization of microarray experiments, and Emily Lyettefi for
assistance with analysis of microarray data. This work was
supported by the Tufts-NEMC GRASP Center NIH/NIDDK,
P30 DK34928 and NIH R01 AI50032 to P.I.W.
References
Baneres, J.L., Roquet, F., Martin, A., and Parello, J. (2000)
A minimized human integrin alpha(5)beta(1) that retains
ligand recognition. J Biol Chem 275: 5888–5903.
Butler, S.M., and Camilli, A. (2004) Both chemotaxis and net
motility greatly influence the infectivity of Vibrio cholerae.
Proc Natl Acad Sci USA 101: 5018–5023.
Carroll, P.A., Tashima, K.T., Rogers, M.B., DiRita, V.J., and
Calderwood, S.B. (1997) Phase variation in tcpH modu-
lates expression of the ToxR regulon in Vibrio cholerae. Mol
Microbiol 25: 1099–1111.
Casper-Lindley, C., and Yildiz, F.H. (2004) VpsT is a tran-
scriptional regulator required for expression of vps biosyn-
thesis genes and the development of rugose colonial
morphology in Vibrio cholerae O1 El Tor. J Bacteriol 186:
1574–1578.
Colwell, R.R., and Huq, A. (1994) Environmental reservoir of
Vibrio cholerae. The causative agent of cholera. Ann N Y
Acad Sci 740: 44–54.
Colwell, R.R., and Spira, W.M. (1992) The ecology of Vibrio
cholerae. In Cholera. Barua, D., and Greenough, W.B.I.
(eds). New York: Plenum, pp. 107–127.
Costerton, J.W., Lewandowski, Z., Caldwell, D.E., Korber,
D.R., and Lappin-Scott, H.M. (1995) Microbial biofilms.
Annu Rev Microbiol 49: 711–745.
Eisenbach, M., Wolf, A., Welch, M., Caplan, S.R., Lapidus,
I.R., Macnab, R.M., et al. (1990) Pausing, switching and
speed fluctuation of the bacterial flagellar motor and their
relation to motility and chemotaxis. J Mol Biol 211: 551–563.
Fotedar, R. (2001) Vector potential of houseflies (Musca
domestica) in the transmission of Vibrio cholerae in India.
Acta Trop 78: 31–34.
Gosink, K.K., Kobayashi, R., Kawagishi, I., and Hase, C.C.
(2002) Analyses of the roles of the three cheA homologs
in chemotaxis of Vibrio cholerae. J Bacteriol 184: 1767–
1771.
Hammer, B.K., and Bassler, B.L. (2003) Quorum sensing
controls biofilm formation in Vibrio cholerae. Mol Microbiol
50: 101–104.
Hase, C.C., and Mekalanos, J.J. (1998) TcpP protein is a
positive regulator of virulence gene expression in Vibrio
cholerae. Proc Natl Acad Sci USA 95: 730–734.
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635
Haugo, A.J., and Watnick, P.I. (2002) Vibrio cholerae CytR
is a repressor of biofilm development. Mol Microbiol 45:
471–483.
Heidelberg, J.F., Eisen, J.A., Nelson, W.C., Clayton, R.A.,
Gwinn, M.L., Dodson, R.J., et al. (2000) DNA sequence of
both chromosomes of the cholera pathogen Vibrio chol-
erae. Nature 406: 477–483.
Horton, R.M., Cai, Z.L., Ho, S.N., and Pease, L.R. (1990)
Gene splicing by overlap extension: tailor-made genes
using the polymerase chain reaction. Biotechniques 8:
528–535.
Huq, A., Small, E.B., West, P.A., Huq, M.I., Rahman, R., and
Colwell, R.R. (1983) Ecological relationships between
Vibrio cholerae and planktonic crustacean copepods. Appl
Environ Microbiol 45: 275–283.
Huq, A., Huq, S.A., Grimes, D.J., O’Brien, M., Chu, K.H.,
Capuzzo, J.M., and Colwell, R.R. (1986) Colonization of
the gut of the blue crab (Callinectes sapidus) by Vibrio
cholerae. Appl Environ Microbiol 52: 586–588.
Kierek, K., and Watnick, P.I. (2003) Environmental determi-
nants of Vibrio cholerae biofilm development. Appl Environ
Microbiol 69: 5079–5088.
Kirov, S.M., Castrisios, M., and Shaw, J.G. (2004) Aeromo-
nas flagella (polar and lateral) are enterocyte adhesins that
contribute to biofilm formation on surfaces. Infect Immun
72: 1939–1945.
Lapidus, I.R., Welch, M., and Eisenbach, M. (1988) Pausing
of flagellar rotation is a component of bacterial motility and
chemotaxis. J Bacteriol 170: 3627–3632.
Lefebvre, B., Formstecher, P., and Lefebvre, P. (1995)
Improvement of the gene splicing overlap (SOE) method.
Biotechniques 19: 186–188.
Meibom, K.L., Li, X.B., Nielsen, A.T., Wu, C.Y., Roseman,
S., and Schoolnik, G.K. (2004) The Vibrio cholerae chitin
utilization program. Proc Natl Acad Sci USA 101: 2524–
2529.
Mekalanos, J.J. (1985) Cholera toxin: genetic analysis, reg-
ulation, and role in pathogenesis. Curr Top Microbiol Immu-
nol 118: 97–118.
Metcalf, W.W., Jiang, W., Daniels, L.L., Kim, S.K., Haldimann,
A., and Wanner, B.L. (1996) Conditionally replicative and
conjugative plasmids carrying lacZ a for cloning, mutagen-
esis, and allele replacement in bacteria. Plasmid 35: 1–13.
Miller, J.H. (1992) A Short Course in Bacterial Genetics.
Plainview: Cold Spring Harbor Laboratory Press.
Miller, V.L., and Mekalanos, J.J. (1988) A novel suicide vector
and its use in construction of insertion mutations: osmo-
regulation of outer membrane proteins and virulence deter-
minants in Vibrio cholerae requires toxR. J Bacteriol 170:
2575–2583.
Moorthy, S., and Watnick, P.I. (2004) Genetic evidence that
the Vibrio cholerae monolayer is a distinct stage in biofilm
development. Mol Microbiol 52: 573–587.
Murley, Y.M., Carroll, P.A., Skorupski, K., Taylor, R.K., and
Calderwood, S.B. (1999) Differential transcription of the
tcpPH operon confers biotype-specific control of the Vibrio
cholerae ToxR virulence regulon. Infect Immun 67: 5117–
5123.
O’Toole, G., Kaplan, H.B., and Kolter, R. (2000) Biofilm for-
mation as microbial development. Annu Rev Microbiol 54:
49–79.
 Transcriptome of Vibrio cholerae biofilm development 1635
Pratt, L.A., and Kolter, R. (1998) Genetic analysis of Escher-
ichia coli biofilm formation: roles of flagella, motility, chemo-
taxis and type I pili. Mol Microbiol 30: 285–293.
Repoila, F., and Gottesman, S. (2001) Signal transduction
cascade for regulation of RpoS: temperature regulation of
DsrA. J Bacteriol 183: 4012–4023.
Schembri, M.A., Kjaergaard, K., and Klemm, P. (2003) Glo-
bal gene expression in Escherichia coli biofilms. Mol Micro-
biol 48: 253–267.
Stanley, N.R., Britton, R.A., Grossman, A.D., and Lazazzera,
B.A. (2003) Identification of catabolite repression as a
physiological regulator of biofilm formation by Bacillus sub-
tilis by use of DNA microarrays. J Bacteriol 185: 1951–
1957.
Szurmant, H., and Ordal, G.W. (2004) Diversity in chemot-
axis mechanisms among the bacteria and archaea. Micro-
biol Mol Biol Rev 68: 301–319.
Tacket, C.O., Taylor, R.K., Losonsky, G., Lim, Y., Nataro,
J.P., Kaper, J.B., and Levine, M.M. (1998) Investigation of
the roles of toxin-coregulated pili and mannose-sensitive
hemagglutinin pili in the pathogenesis of Vibrio cholerae
O139 infection. Infect Immun 66: 692–695.
Tamplin, M.L., Gauzens, A.L., Huq, A., Sack, D.A., and Col-
well, R.R. (1990) Attachment of Vibrio cholerae serogroup
O1 to zooplankton and phytoplankton of Bangladesh
waters. Appl Environ Microbiol 56: 1977–1980.
Taylor, R.K., Miller, V.L., Furlong, D.B., and Mekalanos, J.J.
(1986) Identification of a pilus colonization factor that is
coordinately regulated with cholera toxin. Ann Sclavo Col-
lana Monogr 3: 51–61.
Taylor, R.K., Miller, V.L., Furlong, D.B., and Mekalanos, J.J.
(1987) Use of phoA gene fusions to identify a pilus coloni-
zation factor coordinately regulated with cholera toxin. Proc
Natl Acad Sci USA 84: 2833–2837.
Tenor, J.L., McCormick, B.A., Ausubel, F.M., and Aballay, A.
(2004) Caenorhabditis elegans-based screen identifies
Salmonella virulence factors required for conserved host–
pathogen interactions. Curr Biol 14: 1018–1024.
Tischler, A.D., and Camilli, A. (2004) Cyclic diguanylate (c-
di-GMP) regulates Vibrio cholerae biofilm formation. Mol
Microbiol 53: 857–869.
Vance, R.E., Zhu, J., and Mekalanos, J.J. (2003) A constitu-
tively active variant of the quorum-sensing regulator LuxO
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1623–1635
affects protease production and biofilm formation in Vibrio
cholerae. Infect Immun 71: 2571–2576.
Waldor, M.K., and Mekalanos, J.J. (1994) Emergence of a
new cholera pandemic: molecular analysis of virulence
determinants in Vibrio cholerae O139 and development of
a live vaccine prototype. J Infect Dis 170: 278–283.
Waldor, M.K., and Mekalanos, J.J. (1996) Lysogenic conver-
sion by a filamentous phage encoding cholera toxin. Sci-
ence 272: 1910–1914.
Watnick, P.I., and Kolter, R. (1999) Steps in the develop-
ment of a Vibrio cholerae biofilm. Mol Microbiol 34: 586–
595.
Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R.,
Teitzel, G.M., Lory, S., and Greenberg, E.P. (2001) Gene
expression in Pseudomonas aeruginosa biofilms. Nature
413: 860–864.
Yildiz, F.H., and Schoolnik, G.K. (1999) Vibrio cholerae O1
El Tor: identification of a gene cluster required for the
rugose colony type, exopolysaccharide production, chlo-
rine resistance, and biofilm formation. Proc Natl Acad Sci
USA 96: 4028–4033.
Yildiz, F.H., Dolganov, N.A., and Schoolnik, G.K. (2001)
VpsR, a member of the response regulators of the two-
component regulatory systems, is required for expression
of vps biosynthesis genes and EPS(ETr)-associated phe-
notypes in Vibrio cholerae O1 El Tor. J Bacteriol 183:
1716–1726.
Yildiz, F.H., Liu, X.S., Heydorn, A., and Schoolnik, G.K.
(2004) Molecular analysis of rugosity in a Vibrio cholerae
O1 El Tor phase variant. Mol Microbiol 53: 497–515.
Zhu, J., and Mekalanos, J.J. (2003) Quorum sensing-depen-
dent biofilms enhance colonization in Vibrio cholerae. Dev
Cell 5: 647–656.
Supplementary material
The following supplementary material is available for this
article online:
Table S1. Monolayer experiment.
Table S2. Planktonic experiment.
Table S3. Biofilm experiment.
Table S4. vpsA monolayer experiment.