c-di-GMP-mediated regulation of virulence and biofilm formation
Peggy A Cotter1and Scott Stibitz2
It is now apparent that the signaling molecule 30,50-cyclic
diguanylic acid (c-di-GMP) is a central regulator of the
prokaryote biofilm lifestyle and recent evidence also links this
molecule to virulence. Environmentally responsive signal
transduction systems that control expression and/or activity of
are responsible for synthesis and degradation of c-di-GMP
have recently been identified. Members of the phosphorelay
family feature prominently amongst these systems, which
include several with hybrid polydomain sensors and one that is
similar to well-characterized chemotaxis-controlling pathways.
These findings support the hypothesis that c-di-GMP levels are
tightly controlled in response to a broad range, in terms of both
diversity and intensity, of extracellular signals. Insight into how
c-di-GMP affects changes in gene expression and/or protein
activity has come from the demonstration that proteins
containing the PilZ domain can bind c-di-GMP and control
phenotypes involved in biofilm formation and virulence. These
recent developments should pave the way for researchers to
answer the important question of how a vast array of
extracellular signals that are sensed by multiple sensory
transduction pathways which all lead to the production or
destruction of c-di-GMP are coordinated such that the
appropriate phenotypic response is produced.
1Department of Molecular, Cellular, and Developmental Biology,
University of California, Santa Barbara, Santa Barbara, CA 93106-9610,
2Division of Bacterial, Parasitic and Allergenic Products, Center for
Biologics Evaluation and Research, Food and Drug Administration, 8800
Rockville Pike, Bethesda, MD 20892, USA
Corresponding author: Cotter, Peggy A (email@example.com)
Current Opinion in Microbiology 2007, 10:17–23
This review comes from a themed issue on
Host-microbe interactions: bacteria
Edited by Pamela Small and Gisou van der Goot
Available online 8th January 2007
1369-5274/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
Most, if not all, bacteria are able to live either as inde-
pendent planktonic cells or as members of organized
surface-anchored communities called biofilms . As
both lifestyles confer specific advantages and liabilities,
choosing the right one in any particular environment is
crucial for bacterial survival. For pathogens, it appears
that this choice is often tied to virulence. Vibrio cholerae,
for example, is thought to form biofilms on the chitinous
exoskeleton of zooplankton and phytoplankton [2,3], but
switches to a planktonic form upon arrival in the mam-
malian gut where it causes the profusely diarrhoeal dis-
ease cholera [4–6]. For the phytopathogen Xanthomonas
campestrispathovar campestris, biofilm formation isimport-
is important for vascular disease . The ubiquitous
environmental bacterium Pseudomonas aeruginosa is
thought to exist in biofilms in ex vivo environments,
and factors required for this lifestyle are expressed reci-
procally with those required to cause acute infections in
burns and other wounds . By contrast, colonization of
pacemakers and other indwelling devices by Staphylococci
and chronic lung infection of cystic fibrosis patients by
responsible for the remarkable resistance these infections
display [8,9]. Whereas much has been learned regarding
nothing was known about how the crucial switch between
biofilm and planktonic lifestyles is regulated.
c-di-GMP is a key regulator of biofilm versus
Pioneering work from a handful of laboratories over the
past few years has revealed the core of an evolutionarily
conserved regulatory mechanism for controlling pheno-
types associated with the biofilm lifestyle. The central
feature of these systems is the second messenger 30,50-
diguanylate cyclase (DGC) and degraded by phosphodi-
esterase A (PDEA; see [10?,11,12] for recent excellent
reviews on this topic). The basic system was actually
discovered in the 1980s through the work of Benzimen
and colleagues [13,14] who found that c-di-GMP was a
positive allosteric activator of the cellulose synthase
enzyme used by Gluconacetobacter xylinus to produce an
extracellular cellulose matrix. The current explosion of
interest in this signaling mechanism is fueled, in part, by
share sequence similarity and are called GGDEF proteins
members of the EAL domain family, similarly named,
although recently a type II c-di-GMP-specific phosphodi-
PDEA activity . Many GGDEF and EAL proteins
contain both types of domain, yet in all cases examined,
these proteins possess only one enzymatic activity, with
the enzymatically inactive domain potentially serving a
regulatory function [16–18].
Current Opinion in Microbiology 2007, 10:17–23
31. Camilli A, Mekalanos JJ: Use of recombinase gene fusions to
identify Vibrio cholerae genes induced during infection.
Mol Microbiol 1995, 18:671-683.
32. Lee SH, Angelichio MJ, Mekalanos JJ, Camilli A: Nucleotide
sequence and spatiotemporal expression of the Vibrio
cholerae vieSAB genes during infection. J Bacteriol 1998,
This paper demonstrates a role for c-di-GMP in control of cholera toxin
gene expression in V. cholerae.
Tischler AD, Camilli A: Cyclic diguanylate regulates Vibrio
cholerae virulence gene expression. Infect Immun 2005,
a cyclic diguanylate phosphodiesterase.
Tamayo R, Tischler AD, Camilli A: The EAL domain protein VieA
is a cyclic diguanylate phosphodiesterase. J Biol Chem 2005,
Hickman JW, Tifrea DF, Harwood CS: A chemosensory
system that regulates biofilm formation through modulation
of cyclic diguanylate levels. Proc Natl Acad Sci USA 2005,
Here, Hickman et al. show that the P. aeruginosa Wsp system, which is
analogous to chemotaxis-controlling systems, controls biofilm formation
by controlling c-di-GMP levels with the EAL domain protein WspR.
Kulasekara HD, Ventre I, Kulasekara BR, Lazdunski A, Filloux A,
Lory S: A novel two-component system controls the
expression of Pseudomonas aeruginosa fimbrial cup genes.
Mol Microbiol 2005, 55:368-380.
See annotation for [37??]
Kuchma SL, Connolly JP, O’Toole GA: A three-component
regulatory system regulates biofilm maturation and type III
secretion in Pseudomonas aeruginosa. J Bacteriol 2005,
These papers [36??,37??] identify the RocS1RA1/SadSRA regulatory sys-
tem and demonstrate its role in controlling the regulation of genes
involved in virulence and biofilm formation.
de Vroom E, Fidder A, de Paus P, Sliedregt LA et al.: The cyclic
diguanylic acid regulatory system of cellulose synthesis in
Acetobacter xylinum. Chemical synthesis and biological
activity of cyclic nucleotide dimer, trimer, and phosphothioate
derivatives. J Biol Chem 1990, 265:18933-18943.
39. Mayer R, Ross P, Weinhouse H, Amikam D, Volman G, Ohana P,
Calhoon RD, Wong HC, Emerick AW, Benziman M: Polypeptide
composition of bacterial cyclic diguanylic acid-dependent
cellulose synthase and the occurrence of immunologically
crossreacting proteins in higher plants. Proc Natl Acad Sci USA
Amikam D, Galperin MY: PilZ domain is part of the bacterial
c-di-GMP binding protein. Bioinformatics 2006, 22:3-6.
Thisbioinformatic analysis providedcompellingevidenceforthepotential
c-di-GMP-binding ability of the PilZ domain.
Ryjenkov DA, Simm R, Romling U, Gomelsky M: The PilZ domain
is a receptor for the second messenger c-di-GMP. The PilZ
domain protein YcgR controls motility in enterobacteria.
J Biol Chem 2006, 281:30310-30314.
that PilZ domain proteins, and their c-di-GMP binding ability, are impor-
tant for controlling biofilm and virulence phenotypes.
42. Goodman AL, Kulasekara B, Rietsch A, Boyd D, Smith RS, Lory S:
A signaling network reciprocally regulates genes associated
with acute infection and chronic persistence in Pseudomonas
aeruginosa. Dev Cell 2004, 7:745-754.
43. Laskowski MA, Osborn E, Kazmierczak BI: A novel sensor
kinase-response regulator hybrid regulates type III secretion
and is required for virulence in Pseudomonas aeruginosa.
Mol Microbiol 2004, 54:1090-1103.
44. Ventre I, Goodman AL, Vallet-Gely I, Vasseur P, Soscia C, Molin S,
Bleves S, Lazdunski A, Lory S, Filloux A: Multiple sensors control
reciprocal expression of Pseudomonas aeruginosa regulatory
RNA and virulence genes. Proc Natl Acad Sci USA 2006,
dispersal in Xanthomonas campestris is controlled by
cell-cell signaling and is required for full virulence to plants.
Proc Natl Acad Sci USA 2003, 100:10995-11000.
46. Cotter PA, Jones AM: Phosphorelay control of virulence gene
expression in Bordetella. Trends Microbiol 2003, 11:367-373.
47. Akerley BJ, Cotter PA, Miller JF: Ectopic expression of the
flagellar regulon alters development of the Bordetella-host
interaction. Cell 1995, 80:611-620.
48. Cotter PA, Miller JF: BvgAS-mediated signal transduction:
analysis of phase-locked regulatory mutants of Bordetella
bronchiseptica in a rabbit model. Infect Immun 1994,
49. Martinez de Tejada G, Cotter PA, Heininger U, Camilli A,
Akerley BJ, Mekalanos JJ, Miller JF: Neither the Bvg- phase nor
the vrg6 locus of Bordetella pertussis is required for
respiratory infection in mice. Infect Immun 1998, 66:2762-2768.
50. Merkel TJ, Stibitz S, Keith JM, Leef M, Shahin R: Contribution of
regulation by the bvg locus to respiratory infection of mice by
Bordetella pertussis. Infect Immun 1998, 66:4367-4373.
51. Veal-Carr WL, Stibitz S: Demonstration of differential virulence
gene promoter activation in vivo in Bordetella pertussis using
RIVET. Mol Microbiol 2005, 55:788-798.
52. Merkel TJ, Barros C, Stibitz S: Characterization of the bvgR
locus of Bordetella pertussis. J Bacteriol 1998, 180:1682-1690.
c-di-GMP-mediated regulation of virulence and biofilm formation Cotter and Stibitz 23
Current Opinion in Microbiology 2007, 10:17–23