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
August 2, 2005 ?
vol. 102 ?
no. 31 www.pnas.org?cgi?doi?10.1073?pnas.0502663102
Parallels Between MvaT and H-NS. Our findings with MvaT and
MvaU strengthen the claim that, despite the lack of sequence
similarity, members of the MvaT protein family functionally
resemble H-NS (6). Like H-NS, we found that MvaT and MvaU
contain protein–protein interaction motifs in their N-terminal
regions that can mediate the formation of both heteromeric and
homomeric complexes. Moreover, we found that overproduction
of the N-terminal portions of either MvaT or MvaU can result
in dominant negative effects on gene expression in P. aeruginosa;
similar observations have been made with truncated versions of
H-NS in E. coli (19, 20). Our findings are consistent with the idea
that MvaT exerts its negative effect on cupA gene expression in
protein called StpA (reviewed in ref. 17). StpA shares 58%
identity with H-NS at the primary amino acid level and can
evidently form both homodimers and heterodimers (with H-NS)
through a predicted coiled-coil motif present in its N-terminal
domain (reviewed in ref. 16). We suggest that the interaction of
MvaT with MvaU in P. aeruginosa is formally analogous to the
interaction of H-NS with StpA in E. coli (see ref. 17). It is also
striking that the reciprocal regulatory interaction we observe
repress the gene encoding the other protein (17, 26, 27).
Although MvaT is known to repress expression of mvaU (5), it
remains to be determined whether the effect of MvaU on MvaT
protein levels (Fig. 5) is mediated at the level of mvaT gene
Phase-variable expression of the cupA fimbrial genes in the
absence of MvaT provides yet another parallel between MvaT
and H-NS. In E. coli, H-NS represses both the phase-variable
expression of genes encoding type 1 fimbriae (28, 29), and the
phase-variable expression of genes encoding the Pap pili (30).
However, we do not yet know the mechanism governing the
apparent phase-variable expression of the cupA fimbrial genes
that occurs in the absence of MvaT, nor do we know whether
MvaT mediates its effects on cupA gene expression directly or
Association Between MvaT and MvaU. Our findings with MvaT and
MvaU in P. aeruginosa may be relevant to several observations
made regarding MvaT homologs from other Pseudomonads.
Specifically, MvaT in Pseudomonas mevalonii was originally
described as a heteromeric transcription regulator composed of
two subunits, P16 and P15, with molecular weights of 16 and 15
kDa, respectively (31). The P16 subunit of MvaT from P.
mevalonii is 82% similar to MvaT from P. aeruginosa (5).
Although the identity of the P15 subunit of P. mevalonii MvaT
was not determined (31), based on our findings, we speculate
that P15 is the equivalent of P. aeruginosa MvaU. More recently,
in Pseudomonas putida, a homolog of MvaT called TurA was
identified that repressed transcription from the TOL plasmid Pu
promoter (7). TurA was found to copurify with a smaller related
protein called TurB (7). Although an alternative explanation was
proposed (7), we suggest that TurA and TurB in P. putida, like
MvaT and MvaU in P. aeruginosa, may copurify, because they
physically interact with one another.
and that MvaU can complement one of the phenotypes of an
mvaT mutant, raise the possibility that MvaU may influence the
expression of a subset of the genes that are controlled by MvaT
in P. aeruginosa. Indeed, expression of the lecA gene, which
encodes the PA-IL lectin, appears to be repressed by both MvaT
and MvaU (5). It will be interesting to determine the extent to
which those genes that belong to the MvaT regulon overlap with
those of the putative MvaU regulon. Because MvaT appears to
be more abundant in the cell than MvaU (Fig. 5), we speculate
that the majority of MvaU will be complexed with MvaT.
Therefore, although MvaU homomers may form, we expect that
in the cell. In E. coli, heterodimers of the nucleoid-associated
protein HU (composed of HU? and HU? subunits) have been
shown to have functions distinct from those of the corresponding
homodimeric species (reviewed in ref. 17). It will be important
to determine whether MvaT-MvaU heteromers have properties
distinct from those of either MvaT or MvaU homomers.
We thank Arne Rietsch (Harvard Medical School, Boston) and Julia
Ko ¨hler (Children’s Hospital, Boston) for plasmids and strains, Ross
Tomano (Harvard Medical School, Boston) for tandem MS analysis,
Renate Hellmiss for artwork, and Ann Hochschild and Arne Rietsch for
comments on the manuscript. This work was supported in part by a Child
Health Research Grant from the Charles H. Hood Foundation (to
S.L.D.) and by a grant from the Cystic Fibrosis Foundation (to S.L.D.).
1. Govan, J. R. & Deretic, V. (1996) Microbiol. Rev. 60, 539–574.
2. Singh, P. K., Schaefer, A. L., Parsek, M. R., Moninger, T. O., Welsh, M. J. &
Greenberg, E. P. (2000) Nature 407, 762–764.
3. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. (1999) Science 284,
4. Vallet, I., Olson, J. W., Lory, S., Lazdunski, A. & Filloux, A. (2001) Proc. Natl.
Acad. Sci. USA 98, 6911–6916.
5. Vallet, I., Diggle, S. P., Stacey, R. E., Camara, M., Ventre, I., Lory, S.,
Lazdunski, A., Williams, P. & Filloux, A. (2004) J. Bacteriol. 186, 2880–2890.
6. Tendeng, C., Soutourina, O. A., Danchin, A. & Bertin, P. N. (2003) Microbi-
ology 149, 3047–3050.
7. Rescalli, E., Saini, S., Bartocci, C., Rychlewski, L., de Lorenzo, V. & Bertoni,
G. (2004) J. Biol. Chem. 279, 7777–7784.
8. Tendeng, C. & Bertin, P. N. (2003) Trends Microbiol. 11, 511–518.
9. Diggle, S. P., Winzer, K., Lazdunski, A., Williams, P. & Camara, M. (2002) J.
Bacteriol. 184, 2576–2586.
10. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. & Seraphin, B.
(1999) Nat. Biotechnol. 17, 1030–1032.
11. Blyn, L. B., Braaten, B. A., White-Ziegler, C. A., Rolfson, D. H. & Low, D. A.
(1989) EMBO J. 8, 613–620.
12. Rietsch, A., Vallet-Gely, I., Dove, S. L. & Mekalanos, J. J. (2005) Proc. Natl.
Acad. Sci. USA 102, 8006–8011.
13. Dove, S. L. & Hochschild, A. (1998) Genes Dev. 12, 745–754.
14. Dove, S. L., Joung, J. K. & Hochschild, A. (1997) Nature 386, 627–630.
15. Joung, J. K., Ramm, E. I. & Pabo, C. O. (2000) Proc. Natl. Acad. Sci. USA 97,
16. Dorman, C. J., Hinton, J. C. D. & Free, A. (1999) Trends Microbiol. 7, 124–128.
17. Dorman, C. J. (2004) Nat. Rev. Microbiol. 2, 391–400.
18. Lupas, A. (1996) Methods Enzymol. 266, 513–525.
19. Williams, R. M., Rimsky, S. & Buc, H. (1996) J. Bacteriol. 178, 4335–4343.
20. Free, A., Porter, M., Deighan, P. & Dorman, C. J. (2001) Mol. Microbiol. 42,
21. Deziel, E., Comeau, Y. & Villemur, R. (2001) J. Bacteriol. 183, 1195–1204.
22. Drenkard, E. & Ausubel, F. M. (2002) Nature 416, 740–743.
23. Webb, J. S., Lau, M. & Kjelleberg, S. (2004) J. Bacteriol. 186, 8066–8073.
24. van der Woude, M. W. & Baumler, A. J. (2004) Clin. Microbiol. Rev. 17,
25. Pratt, L. A. & Kolter, R. (1998) Mol. Microbiol. 30, 285–293.
26. Sonden, B. & Uhlin, B. E. (1996) EMBO J. 15, 4970–4980.
27. Zhang, A., Rimsky, S., Reaban, M. E., Buc, H. & Belfort, M. (1996) EMBO J.
28. Higgins, C. F., Dorman, C. J., Stirling, D. A., Waddell, L., Booth, I. R., May,
G. & Bremer, E. (1988) Cell 52, 569–584.
29. Kawula, T. H. & Orndorff, P. E. (1991) J. Bacteriol. 173, 4116–4123.
30. White-Ziegler, C. A., Hill, M. L. A., Braaten, B. A., van der Woude, M. W. &
Low, D. A. (1998) Mol. Microbiol. 28, 1121–1137.
31. Rosenthal, R. S. & Rodwell, V. W. (1998) Protein Sci. 7, 178–184.
Vallet-Gely et al. PNAS ?
August 2, 2005 ?
vol. 102 ?
no. 31 ?