Repression of phase-variable cup gene expression
by H-NS-like proteins in Pseudomonas aeruginosa
Isabelle Vallet-Gely*, Katherine E. Donovan*, Rui Fang†, J. Keith Joung†, and Simon L. Dove*‡
*Division of Infectious Diseases, Children’s Hospital, Harvard Medical School, Boston, MA 02115; and†Molecular Pathology Unit, Massachusetts General
Hospital, Charlestown, MA 02129
Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved June 17, 2005 (received for review March 31, 2005)
The cupA gene cluster of Pseudomonas aeruginosa encodes com-
ponents of a putative fimbrial structure that enable this opportu-
nistic human pathogen to form biofilms on abiotic surfaces. In P.
aeruginosa, cupA gene expression is repressed by MvaT, a putative
transcription regulator thought to belong to the H-NS family of
nucleoid-associated proteins that typically function by repressing
variable (ON?OFF) expression of the cupA fimbrial gene cluster.
Using a directed proteomic approach, we show that MvaT associ-
ates with a related protein in P. aeruginosa called MvaU. Analysis
with a bacterial two-hybrid system designed to facilitate the study
of protein dimerization indicates that MvaT and MvaU can form
both heteromeric and homomeric complexes, and that formation
of these complexes is mediated through the N-terminal regions of
MvaT and MvaU, both of which are predicted to adopt a coiled-coil
conformation. We show further that, like MvaT, MvaU can repress
phase-variable expression of the cupA gene cluster. Our findings
suggest that fimbrial genes important for biofilm formation can be
expressed in a phase-variable manner in P. aeruginosa, provide
insight into the molecular mechanism of MvaT-dependent gene
control, and lend further weight to the postulate that MvaT
proteins are H-NS-like in nature.
MvaT ? phase-variation
(CF) patients (1), is a ubiquitous Gram-negative bacterium that
can deploy an impressive array of virulence factors to intoxicate
the human host. In the chronically infected CF lung, the organ-
ism persists as a biofilm, a surface-attached community of
bacteria encased in a polymeric matrix (2). This biofilm mode of
growth augments the resistance of P. aeruginosa to antibiotics
and facilitates evasion of the host immune response (3).
Prominent among those genes that play an important role in
biofilm formation in P. aeruginosa are the cupA genes, which
encode components of a putative fimbrial structure that pre-
sumably facilitates surface attachment (4, 5). Expression of the
cupA gene cluster in P. aeruginosa is repressed by MvaT, a
putative transcription regulator found exclusively in Pseudo-
monads. It has been suggested, based on certain functional
similarities as well as predicted structural similarities, that MvaT
proteins are members of the H-NS family of nucleoid-associated
proteins that typically function to repress transcription (6, 7).
However, there is limited sequence similarity at the primary
amino acid level between members of the MvaT protein family
and those of the H-NS family (6, 8). Indeed, no obvious homolog
of H-NS exists in P. aeruginosa or in any other Pseudomonad (6).
In P. aeruginosa, MvaT was originally described as a global
regulator of virulence gene expression (9). Specifically, inacti-
vation of mvaT was found to up-regulate expression of the lecA
gene encoding the PA-IL lectin and cause overproduction of the
toxic exoproduct pyocyanin (9). Moreover, a microarray-based
transcriptome analysis has revealed that MvaT influences the
expression of ?150 genes in P. aeruginosa, including those
he opportunistic pathogen Pseudomonas aeruginosa, the
leading cause of morbidity and mortality in cystic fibrosis
identified previously together with several others that may be
important for virulence (5).
Here we present evidence for a previously undescribed func-
tion of MvaT, the control of phase-variable gene expression. In
particular, we show that, in the absence of MvaT, expression of
the cupA fimbrial gene cluster phase-varies (i.e., exhibits ON?
OFF expression). Using tandem affinity purification (TAP) (10)
coupled with mass spectrometry, we show further that MvaT is
associated with a related protein in P. aeruginosa called MvaU.
We provide genetic evidence that MvaT associates with MvaU
through a direct protein–protein contact mediated (in whole or
in part) through the N-terminal regions of these two proteins.
We demonstrate that both MvaT and MvaU can also form
homomeric complexes and provide evidence that the ability of
MvaT to oligomerize is important for its function. We show
further that, like MvaT, MvaU can repress phase-variable (ON?
OFF) expression of the cupA genes in P. aeruginosa and discuss
the parallels between the MvaT and H-NS proteins that are
highlighted by our findings.
Materials and Methods
Plasmids and Strains. Construction of the plasmids and bacterial
strains used in this study is described in Supporting Text, which
is published as supporting information on the PNAS web site.
Switching-Frequency Calculations. Switching-frequency calcula-
tions were performed essentially as described (11), except that
cells were plated on LB agar plates containing X-Gal and
isopropyl-?-D-thiogalactoside (IPTG) (2 mM) and grown at
TAP. Cells were grown at 37°C with aeration in 200 ml of LB in
1-liter flasks to an OD600of ?2, then harvested by centrifugation
at 4°C. TAP was then performed as described (12).
Western Blots. Purified proteins or cell lysates were separated on
NuPAGE 4–12% gradient gels (Invitrogen), transferred to
poly(vinylidene difluoride) membranes (Invitrogen), then
probed either with antibodies against the Myc-tag (Upstate
Biotechnology, Lake Placid, NY), the calmodulin-binding pep-
tide epitope of the TAP-tag (Open Biosystems, Huntsville, AL),
or the ? subunit of RNA polymerase (RNAP) (Neoclone,
Madison, WI). The TAP tag was detected by using soluble
peroxidase–antiperoxidase complex (Sigma-Aldrich). Proteins
were visualized by chemiluminescent detection by using Super-
Signal West Pico chemiluminescent substrate (Pierce).
Bacterial Two-Hybrid Assays. Cells were grown with aeration at
37°C in LB supplemented with kanamycin (50 ?g?ml), carben-
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: IPTG, isopropyl-?-D-thiogalactoside; TAP, tandem affinity purification;
RNAP, RNA polymerase.
whom correspondenceshouldbe addressed. E-mail: simon.dove@childrens.
© 2005 by The National Academy of Sciences of the USA
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icillin (100 ?g?ml), tetracycline (10 ?g?ml), and IPTG at the
concentration indicated. Cells were permeabilized with SDS-
CHCl3and assayed for ?-galactosidase activity as described (13).
Assays were performed at least three times in duplicate on
separate occasions. Representative data sets are shown. Values
are averages based on one experiment; duplicate measurements
differed by ?10%.
MvaT Represses Phase-Variable Expression of the cupA Genes. MvaT
was recently shown to repress expression of the cupA genes in P.
cupA gene expression, we constructed a strain of PAO1 in which
lacZ was placed downstream of the cupA1 gene (Fig. 1A). When
we introduced an in-frame deletion of the mvaT gene (?mvaT)
into this reporter strain, we were surprised to find that the cupA
genes appeared to be expressed in a phase-variable manner (Fig.
1B). In particular, cells of the ?mvaT mutant strain gave rise to
both blue and white colonies on LB agar plates containing X-Gal
(Fig. 1B). When restreaked on LB X-Gal plates, blue colonies
rise to both blue and white colonies. This apparent ON?OFF
switching of cupA gene expression in the ?mvaT mutant strain
could be complemented with the mvaT gene in trans (Table 1).
Phase-variable expression of the cupA genes could not be
detected in wild-type cells of the reporter strain (Fig. 1B).
Quantification of cupA gene expression in ?mvaT mutant cells
derived from phase-ON (i.e., blue) colonies suggests there is at
least an ?190-fold difference in cupA gene expression between
phase-ON cells and wild-type cells (Fig. 1C). Furthermore,
determination of the frequency with which phase-ON cells
switched to phase-OFF cells, and vice versa, revealed a bias in
favor of the phase-ON to phase-OFF transition (Fig. 1D).
TAP of MvaT. To begin to address the question of whether MvaT
performs its regulatory function in isolation or in association
with other proteins in P. aeruginosa, we took a directed pro-
teomic approach. In particular, to identify those proteins that
associate with MvaT, we adapted the TAP strategy (10) for use
in P. aeruginosa. We constructed a strain of P. aeruginosa PAO1
in which the native chromosomal copy of the mvaT gene had
been altered such that it specified a TAP-tagged form of MvaT.
To do this, we made use of an integration vector we specifically
designed for this purpose (see Fig. 2A). The resulting strain
(PAO1 MvaT-TAP) thus synthesizes MvaT with a TAP-tag
fused to its C terminus at a level that likely reflects that of the
wild-type protein. As a control, we constructed a strain that
synthesized AceF (a subunit of the pyruvate dehdrogenase
complex) with a TAP-tag fused to its C terminus (PAO1
AceF-TAP). Lysates were made from wild-type PAO1 cells, cells
of the PAO1 MvaT-TAP strain, and cells of the PAO1 AceF-
TAP strain. Proteins were then purified by TAP (10, 12),
separated by SDS?PAGE, and stained with silver. A protein with
an apparent molecular weight of ?14 kDa was found that
specifically copurified with MvaT (Fig. 2B). Nanoelectrospray
tandem MS was used to identity the protein as MvaU (PA2667),
a putative transcription regulator from P. aeruginosa that shares
47% identity and 65% similarity with MvaT (5).
MvaT Copurifies with MvaU-TAP. We reasoned that if MvaT and
MvaU were associated with one another in P. aeruginosa, then
MvaT would be expected to copurify with TAP-tagged MvaU.
To test this prediction, we constructed two strains. One of these
MvaU-TAP). The other strain was identical to the first, except
that it harbored an in-frame deletion of the mvaT gene (PAO1
?mvaT MvaU-TAP) and therefore no longer synthesized MvaT.
Fig. 3A shows the results following TAP of MvaU from these two
strains. In support of the hypothesis that MvaT and MvaU
associate with one another, a protein with the expected molec-
ular weight of MvaT was found to copurify with MvaU only in
the wild-type strain (Fig. 3A).
To test explicitly the hypothesis that MvaT copurifies with
MvaU-TAP, we constructed two additional strains. Each of
to its C terminus (MvaT-Myc5). Whereas one strain was engi-
neered to synthesize MvaU-TAP, the other was engineered to
synthesize AceF-TAP. Immunoblot analyses of the TAP mate-
rial from these two strains revealed that, although equal amounts
the absence of MvaT. (A) Schematic of the cupA gene cluster in the cupA lacZ
reporter strain. (B) Phenotypes of strains containing the cupA lacZ construct
when plated on LB agar containing X-Gal and grown overnight at 37°C. The
colonies. (C) Quantification of cupA lacZ expression in cultures of the wild-
type reporter strain and in cultures derived from phase-ON or phase-OFF
colonies of the ?mvaT mutant derivative. (D) Frequencies of switching from
phase-ON to phase-OFF and from phase-OFF to phase-ON in ?mvaT mutants.
The cupA fimbrial gene cluster exhibits phase-variable expression in
Table 1. Switching frequencies
(per cell per
? 5.5 ? 0.1 ? 10?6
21.4 ? 3.6 ? 10?3
13.5 ? 1.3 ? 10?3
? 1.3 ? 0.4 ? 10?5
? 2.8 ? 2.0 ? 10?5
33.8 ? 11.7 ? 10?3
1.9 ? 0.7 ? 10?3
48.3 ? 3.2 ? 10?3
1.1 ? 0.7 ? 10?4
Vallet-Gely et al. PNAS ?
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of MvaT-Myc5 were made by each strain (data not shown),
MvaT-Myc5 specifically copurified with MvaU (Fig. 3B). From
these findings, we conclude that MvaT and MvaU are associated
with one another in P. aeruginosa.
Bacterial Two-Hybrid Analysis of Interactions Involving MvaT and
MvaU. Our findings with TAP-tagged forms of MvaT and MvaU
suggest these proteins may interact with one another directly to
form a heteromeric complex. Because MvaT and MvaU are
similar to one another at the primary amino acid level, we sought
to determine whether MvaT and?or MvaU could also form
homomeric complexes. To test for both heteromeric and homo-
meric complexes, we modified the design of a previously devel-
oped bacterial two-hybrid assay (13, 14) to permit the detection
of both dimeric and higher-order complexes. This two-hybrid
assay is based on the finding that any sufficiently strong inter-
action between two proteins can activate transcription in Esch-
erichia coli provided one of the interacting proteins is tethered
to the DNA by a DNA-binding protein, and the other is tethered
to a subunit of E. coli RNAP (13, 14). In the modified version
to the ? subunit of E. coli RNAP and another protein fused to
a zinc-finger DNA-binding protein (referred to as Zif) activates
transcription of a lacZ reporter gene (Fig. 4A) (13, 15). Because
Zif (the Zinc-finger DNA-binding domain from the murine
Zif268 protein) binds its cognate recognition site as a monomer,
and because the ? subunit is monomeric in the RNAP holoen-
zyme complex, this modified configuration of the assay is ideally
suited to detecting interactions between two protein monomers
(i.e., dimer formation).
Using this newly configured two-hybrid assay (Fig. 4A), we
first sought to detect an interaction between MvaT and MvaU
from P. aeruginosa. Accordingly, we fused full length MvaU
(residues 1–117) to the N terminus of Zif, and we fused full
length MvaT (residues 1–124) to the N terminus of ?. We then
determined whether the MvaU-Zif fusion protein could activate
transcription from a suitable test promoter in cells containing
the MvaT-? fusion protein. Plasmids expressing MvaU-Zif and
MvaT-? chimeras were introduced into E. coli strain KDZif1?Z,
which harbors the test promoter depicted in Fig. 4A linked to
lacZ on an F? episome. (KDZif1?Z also bears a deletion of the
chromosomal rpoZ gene encoding ?.) In support of the idea that
MvaU and MvaT interact with one another directly, the MvaU-
Zif fusion protein activated transcription strongly (up to ?18-
fold) in cells containing the MvaT-? chimera, whereas Zif did
not (Fig. 4B). An additional control revealed that MvaU-Zif did
not activate transcription from the test promoter in the presence
of the unrelated Gal11P-? chimera (Fig. 4B).
Having demonstrated the utility of the assay to detect a direct
protein–protein interaction between MvaT and MvaU, we next
asked whether either MvaT or MvaU (or both) could undergo
homotypic interactions. Accordingly, we made two additional
fusion proteins, one in which full length MvaU (residues 1–117)
MvaT (residues 1–124) was fused to the N terminus of Zif.
Results depicted in Fig. 4B show that MvaU-Zif activated
transcription from the test promoter by a factor of up to ?11 in
cells containing the MvaU-? chimera. Similarly, MvaT-Zif ac-
tivated transcription by a factor of up to ?9 in cells containing
the MvaT-? chimera (Fig. 4B). Control assays indicated that
TAP-tag integration vector and its use to make MvaT-TAP. The calmodulin-
binding peptide (CBP), protein A moieties (ProtA), and TEV cleavage site that
constitute the TAP-tag are shown (10). (B) SDS?PAGE analysis of proteins that
were tandem affinity purified, electrophoresed on a 4–12% Bis-Tris NuPAGE
gel and stained with silver. Lane 1, proteins purified from the nontagged
PAO1 wild-type strain. Lane 2, proteins purified from strain PAO1 MvaT-TAP.
Lane 3, proteins purified from strain PAO1 AceF-TAP. Molecular weights are
indicated on the left.
TAP of MvaT from P. aeruginosa. (A) Schematic representation of
associated with MvaU-TAP in a wild type (lane 1) or in a ?mvaT mutant
on a 4–12% Bis-Tris NuPAGE gel, and stained with silver. Lane 1, proteins
purified from strain PAO1 MvaU-TAP. Lane 2, proteins purified from strain
PAO1 ?mvaT MvaU-TAP. (B) Western blot analysis of proteins associated with
tandem affinity-purified MvaU-TAP (lane 1), and AceF-TAP (lane 2). Both
strains (that containing MvaU-TAP, and that containing AceF-TAP) produce
Myc-tagged MvaT (MvaT-Myc5). (Upper) Immunoblot probed with an anti-
Myc antibody demonstrates MvaT-Myc5 is only detected among the MvaU-
TAP associated proteins. (Lower) Immunoblot probed with an anti-
calmodulin-binding peptide (CBP) antibody shows comparable amounts of
tandem affinity-purified material were loaded in each lane. Molecular
weights are indicated on the left.
MvaT copurifies with MvaU-TAP. (A) SDS?PAGE analysis of proteins
www.pnas.org?cgi?doi?10.1073?pnas.0502663102Vallet-Gely et al.
neither Zif nor MvaT-Zif activated transcription from the test
promoter in the presence of the MvaU-? chimera, or the
Gal11P-? chimera, respectively (Fig. 4B). These findings suggest
that both MvaT and MvaU can form homomeric complexes.
N-Terminal Regions of MvaT and MvaU Mediate Protein–Protein
Interactions. H-NS-like proteins typically contain coiled-coil mo-
tifs in their N-terminal regions that can mediate the formation
of both homodimeric and heterodimeric complexes (16, 17). An
MvaT homolog from Pseudomonas strain Y1000, which has been
described as an H-NS-like protein, is predicted to contain a
coiled-coil in its N-terminal region (6). Moreover, analysis of P.
aeruginosa MvaT and MvaU using COILS (18) predicts that
residues 1–39 of MvaT and residues 1–35 of MvaU are likely to
adopt coiled-coil conformations (not shown). We therefore
asked whether the N-terminal regions of MvaT and MvaU
harboring these putative coiled-coil motifs could mediate both
the heteromeric and homomeric interactions of these proteins.
To do this, we fused the N-terminal regions of MvaT (residues
1–62) and MvaU (residues 1–62) to Zif and then assayed the
ability of the resulting fusion proteins to interact with full length
MvaT and MvaU. In reporter strain KDZif1?Z, the MvaU
(1–62)-Zif fusion protein activated transcription strongly in cells
containing either the MvaT-? chimera (up to ?32-fold) or the
(1–62)-Zif fusion protein strongly stimulated reporter gene
expression in cells containing either the MvaT-? or the MvaU-?
chimera by factors of up to ?29 and ?33, respectively (Fig. 4C).
Control assays revealed that both MvaU (1–62)-Zif and MvaT
(1–62)-Zif failed to activate transcription in the presence of the
Gal11P-? chimera (Fig. 4C).
We then wished to determine whether the N-terminal regions of
both MvaT and MvaU could suffice to mediate both the hetero-
meric and homomeric interactions of MvaT and MvaU. Accord-
ingly, we made two additional chimeras in which either MvaT
(residues 1–62) or MvaU (residues 1–62) were fused to ?. We then
assayed the ability of each of these chimeras to interact with the
MvaU (1–62)-Zif and MvaT (1–62)-Zif fusion proteins in reporter
strain KDZif1?Z. Fig. 4D shows that MvaT (1–62)-Zif strongly
activated transcription from the test promoter in cells containing
the MvaT (1–62)-? chimera (by a factor of up to ?18). Moreover,
whereas MvaU (1–62)-Zif activated transcription by factors of up
to ?16 and ?10 in cells containing the MvaT (1–62)-? and MvaU
(1–62)-? chimeras, respectively, Zif did not (Fig. 4D). We can
therefore conclude that the N-terminal regions of both MvaT and
MvaU are sufficient to mediate the formation of the respective
heteromeric and homomeric complexes.
Dominant-Negative Effect of Overproducing N-Terminal Regions of
MvaT and MvaU. We have shown, using a bacterial two-hybrid
assay, that the N-terminal regions of both MvaT and MvaU can
interact with both full length MvaT and full length MvaU (Fig.
4C). In E. coli, overproduction of the N-terminal portion of
H-NS results in dominant-negative effects on gene expression
because this portion of H-NS sequesters the full length protein
in an inactive heterodimer that is incapable of binding DNA (19,
20). We therefore wished to determine whether overproduction
of the N-terminal regions of MvaT and MvaU would result in a
dominant-negative effect on cupA gene expression in P. aerugi-
nosa. To do this, we constructed two vectors, pM-MvaT (1–61)
and pM-MvaU (1–61), carrying genes encoding MvaT (1–61)
and MvaU (1–61), respectively, under the control of the IPTG-
inducible tac promoter. Each vector, as well as the empty parent
vector (pMMB67EH), was then introduced into the cupA lacZ
reporter strain. Overproduction of either MvaT (1–61) or MvaU
(1–61) resulted in phase-variable expression of the cupA genes
(Table 1). Furthermore, overproduction of MvaT (1–61) in cells
of a ?mvaU mutant resulted in phase-variable expression of the
cupA genes (data not shown), demonstrating that MvaU itself is
genes. Therefore, akin to the situation with truncated versions of
H-NS in E. coli (19, 20), overproduction of the N-terminal
portions of either MvaT or MvaU can result in dominant-
ing MvaT and MvaU. (A) Schematic representation of the two-hybrid system.
expression of lacZ. The diagram depicts test promoter placZif1–61, which
bears a Zif-binding site centered 61 bp upstream of the transcription start site
of the lac core promoter. In E. coli strain KDZif1?Z, this test promoter is linked
to lacZ on an F? episome. (B–D) KDZif1?Z cells harboring compatible plasmids
directing the synthesis of the indicated proteins were grown in the presence
of different concentrations of IPTG and assayed for ?-galactosidase activity.
(B) Transcription activation by MvaU-Zif in the presence of the MvaT-? or
MvaU-? chimeras and by MvaT-Zif in the presence of the MvaT-? chimera. (C)
of the MvaT-? or MvaU-? chimeras. (D) Transcription activation by MvaU
(1–62)-Zif in the presence of the MvaT (1–62)-? or MvaU (1–62)-? chimeras
and by MvaT (1–62)-Zif in the presence of the MvaT (1–62)-? chimera.
Bacterial two-hybrid analysis of protein–protein interactions involv-
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negative effects on gene expression in P. aeruginosa. These
findings are consistent with a model in which MvaT exerts its
negative effect on cupA gene expression in oligomeric form.
MvaU Can Substitute Functionally for MvaT in the Control of cupA
Gene Expression. Because MvaU is similar to MvaT, we next
wished to determine whether MvaU could functionally substi-
tute for MvaT with respect to its role in cupA gene expression.
For this purpose, we constructed a vector, pM-MvaU, in which
expression of the mvaU gene is under the control of the
IPTG-inducible tac promoter. We found that when overex-
pressed, mvaU could complement the ?mvaT mutant cells and
repress phase-variable expression of the cupA genes (Table 1).
Deletion of mvaU Increases the Amount of MvaT in the Cell.Although
MvaU associates with MvaT in P. aeruginosa (Figs. 2 and 3), and
although MvaU can repress phase-variable expression of the
cupA genes (Table 1), deletion of mvaU evidently has no effect
on cupA gene expression (ref. 5 and data not shown). We
wondered why this was the case. Because deletion of mvaT
results in an increase in expression of the mvaU gene (5), we
wished to determine whether the converse situation might be
true; if deletion of mvaU were to result in an increase in the
amount of MvaT in the cell, this compensatory change might be
sufficient to mask any role MvaU might normally play in
repression of the cupA genes.
To determine whether deletion of mvaU influences the
amount of MvaT in the cell, we introduced an in-frame deletion
of mvaU into our strain synthesizing TAP-tagged MvaT (PAO1
MvaT-TAP) to create strain PAO1 ?mvaU MvaT-TAP. Immu-
noblot analysis of the amount of MvaT-TAP in these two strains,
at different points of the growth curve, revealed that deletion of
mvaU results in a modest increase in the intracellular concen-
tration of MvaT-TAP (Fig. 5). In addition, using our PAO1
MvaU-TAP strain together with a ?mvaT mutant derivative, we
also found that deletion of mvaT results in an increase in the
amount of MvaU-TAP in the cell (Fig. 5). This finding is
consistent with the earlier observation that deletion of mvaT
results in increased expression of the mvaU gene (5). Our
findings with the ?mvaU mutant suggest that one unanticipated
consequence of deleting mvaU is that the intracellular concen-
tration of MvaT increases. This might confound the interpreta-
tion of phenotypes (or lack thereof) of ?mvaU mutants.
MvaT influences the expression of a large number of genes in P.
aeruginosa, including several that are important for virulence (5,
9). We present evidence that a cluster of genes involved in
biofilm formation exhibits phase-variable expression in the
absence of MvaT. Using TAP (10) in combination with tandem
MS, we have found that in P. aeruginosa MvaT interacts with a
related protein called MvaU. This finding potentially implicates
MvaU in the control of target gene expression by MvaT.
Furthermore, using a bacterial two-hybrid system specifically
designed to study protein dimerization, we have shown that not
homo- or heterooligomeric species have distinct functions in the
Phase-Variable Expression of the cupA Fimbrial Genes. Of the 150 or
so genes in P. aeruginosa whose expression is altered as a result
of deleting mvaT, the cupA genes are among those whose
expression is most strongly affected (5). The cupA genes con-
stitute a putative fimbrial operon whose products are involved in
the early stages of biofilm formation (4). In a previous study (5),
it was found that MvaT represses expression of the cupA genes,
either directly or indirectly. We present evidence that, in the
absence of MvaT, the cupA genes are expressed in a phase-
variable manner. Thus, within a population of mvaT mutant
cells, some cells will express the cupA fimbrial genes, whereas
others will not. Although phenotypic variation is known to occur
in P. aeruginosa (21–23), to our knowledge this is the first explicit
demonstration of phase-variable gene expression in P. aerugi-
nosa. In addition, although MvaU has not previously been
implicated in the control of cupA gene expression (5), we have
shown that, when overexpressed, mvaU can complement an
mvaT deletion and repress phase-variable expression of the cupA
genes (Table 1). Because deletion of mvaU results in an increase
in the amount of MvaT in the cell, this compensatory change
might be sufficient to mask any role MvaU might normally play
in repression of the cupA genes. Thus, we have yet to establish
whether MvaU contributes to the repression of cupA gene
expression. We note that phase-variable expression of the cupA
(5); possibly the difference between our observations and the
previous observations lies in the fact that the cupA1 promoter
region was present on a multicopy plasmid in the prior study.
Many examples exist in bacteria of surface structures such as
fimbriae being produced in a phase-variable manner (reviewed
in ref. 24), and some of these are involved in biofilm formation
(see, for example, ref. 25). We speculate that phase-variable
(ON?OFF) expression of the cupA genes might contribute to the
fitness of the cell population as a whole. For example, in the host,
expression of the cupA genes (in phase-ON cells) might facilitate
initial biofilm formation, whereas any subsequent switch to the
phase-OFF expression state might better enable cells to persist,
because they no longer produce a potentially immunogenic
surface structure. Although we have been unable to detect
phase-variable expression of the cupA genes in the presence of
MvaT (i.e., in wild-type cells), it is tempting to speculate that
there may be specific environmental cues that promote phase-
variable expression of the cupA genes. Our demonstration that
overproduction of the N-terminal region of MvaT or MvaU had
a dominant negative effect on cupA gene expression establishes
MvaT sequestration as a potential mechanism that could be used
to promote phase-variable expression of the cupA genes in
respectively, on the intracellular concentrations of MvaT or MvaU. The
curve of a wild-type strain (lanes 1–4) and a ?mvaU mutant derivative (lanes
the growth curve of a wild-type strain (lanes 5–8) and a ?mvaT mutant
derivative (lanes 13–16). Samples were taken at mid-log (lanes 1, 5, 9, and 13),
late-log (lanes 2, 6, 10, and 14), early stationary (lanes 3, 7, 11, and 15), and
stationary (lanes 4, 8, 12, and 16) phases of growth. The OD600at which each
sample was taken is indicated, and the corresponding growth curves are
site. Equivalent amounts of cell lysates were loaded for each sample. (Upper)
Immunoblot probed with anti-TAP. (Lower) Immunoblot probed with anti-
body against the ? subunit of RNAP serves as a control for sample loading.
Western blot analysis of the effects of deleting mvaU or mvaT,
www.pnas.org?cgi?doi?10.1073?pnas.0502663102Vallet-Gely et al.