Download The role of c-di-GMP signaling in an Aeromonas veronii biovar

The role of c-di-GMP signaling in an Aeromonas veronii biovar
sobria strain
Mokhlasur Rahman1,2, Roger Simm1, Abdul Kader1, Eugenie Basseres1, Ute Römling1 & Roland Möllby1
1
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; and 2Department of Natural Science, Södertörns
Högskola University College, Huddinge, Stockholm, Sweden
Correspondence: Mokhlasur Rahman,
Department of Natural Science, Södertörns
högskola University College, Alfred Nobels
alle 7, SE-14189, Huddinge, Stockholm,
Sweden. Tel.: 146 8 6084756; fax: 146 8
6084510; e-mail: [email protected]
Received 2 April 2007; accepted 1 May 2007.
First published online 16 June 2007.
DOI:10.1111/j.1574-6968.2007.00803.x
Editor: Michael Galperin
Keywords
Aeromonas veronii biovar sobria ; c-di-GMP;
GGDEF domain; EAL domain; quorum sensing.
Abstract
Aeromonas is a ubiquitous gram-negative bacterium that persists in the environment. It is shown that all isolates of persistent Aeromonas clones show strong
biofilm formation ability. C-di-GMP regulates biofilm formation in many bacteria.
To investigate the impact of c-di-GMP signaling, we introduced heterologous
GGDEF and EAL domain proteins from Salmonella Typhimurium to an Aeromonas veronii biovar sobria strain. Overexpression of the GGDEF domain protein
AdrA increased c-di-GMP concentration and biofilm formation and reduced
motility. Production of the quorum-sensing signaling molecule C4-homoserine
lactone and adhesion to aquatic plant duckweed and amoeba surfaces were
enhanced. On the other hand, overexpression of the EAL domain protein YhjH
decreased biofilm formation and increased motility.
Introduction
Bacteria make extensive use of the novel secondary messenger cyclic diguanosine monophosphate (c-di-GMP) (D’Argenio & Miller, 2004; Jenal, 2004; Romling et al., 2005). The
molecule c-di-GMP was discovered as an allosteric activator
of the cellulose synthase in Gluconacetobacter xylinus and
Agrobacterium tumefaciens (Amikam & Benziman, 1989).
The cellular level of c-di-GMP was shown to be controlled
through the opposite activities of diguanylate cyclases and
phosphodiesterases, which contained both GGDEF and EAL
domains (Tal et al., 1998). Subsequently, it has been shown
that GGDEF and EAL domain proteins are involved in c-diGMP synthesis and degradation, respectively (Simm et al.,
2004, 2005). Physiologically, c-di-GMP signaling has been
shown to regulate cell surface-associated components distinct from cellulose and multicellular community behaviour
like biofilm formation in Escherichia coli, Salmonella enterica
serovar Typhimurium, Vibrio cholerae, Yersinia pestis and
other bacteria (Kirillina et al., 2004; Simm et al., 2004;
Tischler & Camilli, 2004), swimming, swarming and twitching motility in Pseudomonas aeruginosa (D’Argenio et al.,
2002) and virulence gene expression in Vibrio cholerae
(Tischler & Camilli, 2005).
Aeromonas are a gram-negative facultative anaerobic, opportunistic bacteria mostly associated with child diarrhea and
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
bacteremia in humans and with soft-tissue infections in fish
(Kuhn et al., 1997; Rahman et al., 2002). Aeromonas is a
ubiquitous inhabitant of aquatic ecosystems such as fresh
water, coastal and sewage water; on aquatic plants and animals;
in different water-distribution systems; and in food items
including fish and vegetables (Janda & Duffey, 1988; Albert
et al., 2000). The abundance of Aeromonas in the aquatic
environment is not well characterized, but the ability of
Aeromonas to form biofilms may contribute to the persistence
of the organism in environmental reservoirs. Biofilm production of Aeromonas is only rudimentary characterized; however,
it has been shown to be regulated by the quorum-sensing
signaling molecule C4-HSL (Lynch et al., 2002).
In this study, c-di-GMP signaling in an Aeromonas veronii
biovar sobria strain belonging to a persistent clone (Rahman
et al., 2007) was investigated. It is shown that c-di-GMP signaling influences the multicellular behavior and adherence to
animate and inanimate surfaces as well as the level of the
quorum-sensing molecule C4-HSL in Aeromonas veronii biovar sobria.
Experimental procedures
Aeromonas strains and construction
In total 1315 presumptive Aeromonas isolates from different
sites of a duckweed aquaculture-based hospital sewage water
FEMS Microbiol Lett 273 (2007) 172–179
173
C-di-GMP in Aeromonas
treatment plant, from hospitalized children suffering from
diarrhea, from environmental-control ponds and from
healthy humans were collected over a period of 3 years. The
isolates were biochemically typed by the PhenePlate rapid
screening system (PhP-AE), which is based on the kinetics of
fermentation of 11 reagents especially selected to discriminate between individual Aeromonas strains (Möllby et al.,
1993; Kuhn et al., 1997). The isolates were distributed over a
large number of common (192) and single PhP types.
Eighteen PhP types [representing 841 of the 1315 studies
Aeromonas isolates (64%)] isolated from multiple sites and/
or during several occasions were referred to as major types
(MTs) (Rahman, 2005; Rahman et al., 2007). Several isolates
belonging to each MT, in total 92 isolates (at least four from
each of the 18 MTs, representing different sampling sites
or sampling occasions), were investigated for biofilm
formation.
Aeromonas veronii biovar sobria strain AEW43 (MT-G
clonal group), isolated from a child suffering from diarrhea,
was chosen to study c-di-GMP signaling. The GGDEF
domain protein AdrA and the EAL domain protein YhjH
were cloned in broad host range vector pLAFR3 (Simm
et al., 2004) and were introduced into Aeromonas veronii
biovar sobria AEW43 by conjugation.
Detection of c-di-GMP
Detection of c-di-GMP from Aeromonas veronii biovar
sobria AEW43 overexpressing GGDEF and EAL domain
proteins was carried out as described (Simm et al., 2004).
Phenotypic assays
Aeromonas veronii biovar sobria AEW43 containing GGDEF
and EAL domain proteins was grown on Congo Red (CR)
agar plates (Römling et al., 2000) for 20 h at 37 1C. For
phenotypic assessment, biofilm formation was observed as
adherence to the wall of the glass tube. Overnight cultures of
Aeromonas were inoculated in Luria–Bertani (LB) medium
for 16 h with shaking at 150 r.p.m. and adherent bacteria
stained with crystal violet were dissolved in dimethylsulfoxide to measure OD540 nm. Swimming motility was observed using 0.3% LB agar plates inoculated with a constant
cell number from overnight cultures.
Adherence to duckweed and amoeba surfaces
Single colonies of Aeromonas veronii biovar sobria AEW43
from overnight cultures were resuspended in LB broth
supplemented with appropriate antibiotics and arabinose
(0.01%) or isopropyl-b-D-thiogalactopyranoside. The cells
were grown for 6 h at 37 1C and adjusted to 107 CFU mL 1.
The bacterial suspension and the duckweed (gentamicin
washed) was incubated for 1 h with shaking (145 r.p.m) at
FEMS Microbiol Lett 273 (2007) 172–179
room temperature. To eliminate nonattached bacteria, the
duckweed was washed five times with phosphate-buffered
saline (PBS). For quantification, the duckweed and attached
bacteria were incubated in sterile water containing 0.1%
Triton for 10 min. The ability of the bacteria to adhere to the
duckweed was determined by spreading the cell suspension
onto nutrient agar plates and counting the number of
colonies after incubation overnight at 37 1C.
Acanthamoeba castellanii (ATCC 30234) cells were grown
at 30 1C in ATCC medium no 712 (ATCC) and adjusted to
105 cells mL 1 before use. Bacteria and amoeba were incubated in a Falcon tissue culture flask at room temperature
for 1 h with shaking (145 r.p.m.) and washed five times with
PBS in order to eliminate nonattached bacteria. After
washing, attachment of Aeromonas to the amoeba cells was
investigated by light microscopy (Leica Microscopy Systems,
Switzerland). From each sample, a certain culture volume
was placed on coverslips, fixed in methanol, stained with
giemsa for 1–2 min and thereafter examined.
Detection of quorum-sensing molecule
N-acyl homoserine lactoneg molecules were extracted as
described (Lynch et al., 2002). To detect HSL, plasmid
pSB536, which preferentially detects N-(butanoyl)-L-homoserine lactone (C4-HSL), was used (Swift et al., 1997;
Winson et al., 1998). To estimate the amount of HSL in the
culture, a standard curve of relative light units as a function
of C4-HSL concentration was constructed using E. coli
JM109 (pSB536) as the biosensor (Swift et al., 1997). The
mean value of at least three independent experiments was
considered.
Statistical analysis
Statistical significance was assessed using Student’s t-test.
Results
A total of 92 Aeromonas isolates, representing 18 common
PhP clonal types, were tested for biofilm formation by
assessing adherence to the wall of a glass tube. A biofilm
formation score was assigned to each strain. Thereby, 25
isolates (27%) showed strong biofilm formation, 41 (44%)
showed moderate biofilm formation and the remaining 26
(29%) did not show any biofilm formation (data not
shown). Biofilm formation varied within the members
of a PhP type except for two PhP clonal types (MT-F and
MT-G), where all members showed strong biofilm formation (Fig. 1). MT-F persisted in different environmental
niches (hospital sewage water, lagoon where duckweed is
grown for sewage water purification, fish culture pond
where sewage-grown duckweed was used as fish feed and
control environmental pond). MT-G was not only detected
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
174
M. Rahman et al.
from all different sites of the treatment plant but also found
to be associated with diarrhea in hospitalized children. This
may indicate that transmission by the fish cultured for
human use through the sewage water treatment system has
been demonstrated (Rahman et al., 2007). In conclusion,
these data suggest that biofilm formation contributes to
persistence and to successive transmission of certain Aeromonas clonal types.
GGDEF and EAL domain proteins regulate c-diGMP concentration in Aeromonas veronii biovar
sobria
C-di-GMP signaling controls biofilm formation in many
bacteria (Romling et al., 2005; Romling & Amikam, 2006).
To investigate c-di-GMP signaling in Aeromonas, the
GGDEF domain protein AdrA and the EAL domain protein
YhjH from S. Typhimurium were introduced into Aeromonas veronii biovar sobria AEW43. To obtain an insight into
whether AdrA and YhjH are functional in Aeromonas veronii
biovar sobria AEW43, c-di-GMP production was measured
(Fig. 2). When AdrA was overexpressed, the c-di-GMP
Arbitrary biofilm formation
6
5
4
3
2
1
0
MT-G
MT-H
MT-F
MT-C
MT-B
Fig. 1. Biofilm formation capacities of the members of different clonal
PhP groups of Aeromonas. Within 18 PhP clonal groups, biofilm formation scores are shown for six groups.
concentration was 200-fold higher than in the vector control. When YhjH was overexpressed, the c-di-GMP level was
1.2-fold higher than in the vector control (Table 1). Slightly
enhanced c-di-GMP concentrations have also been observed
when YhjH was overexpressed in S. Typhimurium in the
wild-type background, although YhjH was demonstrated to
be a phosphodiesterase in vitro (unpublished data).
GGDEF and EAL domain proteins regulate the
multicellular behavior of Aeromonas veronii
biovar sobria AEW43
The effect of c-di-GMP on multicellular behavior was
assessed. Bacteria can express multicellular behavior in
various ways. On agar plates, the appearance of a wrinkled
colony morphology in Pseudomonas aeruginosa, the rdar
colony morphology of S. Typhimurium and the rugose
colony morphology of V. cholerae is indicative of the
expression of adhesive extracellular matrix components
such as exopolysaccharides and fimbriae (D’Argenio &
Miller, 2004). When AdrA was overexpressed in Aeromonas
veronii biovar sobria AEW43, a CR binding reddish colony
was formed (Fig. 3a). YhjH overexpression showed a slightly
enhanced reddish colony morphology. Furthermore, when
Aeromonas veronii biovar sobria AEW43 overexpressing
AdrA was lifted with a tooth pick, the cells stuck to each
other indicating the production of extracellular matrix
components. On the other hand, neither the YhjH-expressing strain nor the vector control showed such behavior.
Furthermore, the effect of c-di-GMP was assessed by
testing the adherence to the wall of a glass tube. When AdrA
was overexpressed in Aeromonas veronii biovar sobria
AEW43 biofilm formation was induced, while YhjH decreased biofilm formation (Fig. 4).
The effect of c-di-GMP on swimming motility was also
tested. Figure 3b shows that the swimming motility increased when YhjH was overexpressed and decreased when
AdrA was overexpressed.
Fig. 2. Detection of c-di-GMP in nucleotide
extracts from Aeromonas veronii biovar sobria
AEW43 by HPLC analysis. The peak at 32 min
(arrow) represents c-di-GMP, as compared with
the retention time of synthetic c-di-GMP and
MALDI-TOF analysis (data not shown).
Aeromonas veronii biovar sobria AEW43
overexpressing AdrA (solid line); vector control,
dashed line.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Microbiol Lett 273 (2007) 172–179
175
C-di-GMP in Aeromonas
Table 1. C-di-GMP concentration, C4-HSL concentration and adherence to amoebal cells of Aeromonas veronii biovar sobria AEW43 containing
plasmids encoding the GGDEF domain protein AdrA and the EAL domain protein YhjH
Strains
Aeromonas veronii biovar sobria
AEW 43 overexpressing AdrA
Aeromonas veronii biovar sobria
AEW 43 overexpressing YhjH
Aeromonas veronii biovar sobria
AEW 43 vector control
C-di-GMP
(pmol mg 1 cells, mean SD)
AHL concentration
(nmol mL 1, mean SE)
% adherence
(mean SD)
185.5 60.5
2.03 0.16
27.4 3.35
1.14 0.14
1.06 0.06
7.2 2.6
0.92 0.68
0.93 0.05
5.1 1.4
Value shown is the mean SD of the number of amoeba cells positive for adherence. The samples were regarded as positive adherent when more than
20 bacteria were attached to an amoeba cell. The results derive from three independent experiments and at least 100 amoeba cells were examined in
each experiment.
(b)
(a)
AdrA
AdrA
YhjH
YhjH
Vector
Vector
(c)
(d)
(e)
Fig. 3. Phenotypes of Aeromonas veronii biovar sobria AEW43 influenced by c-di-GMP signaling. (a) Colony morphology of Aeromonas veronii biovar
sobria AEW43 by AdrA and YhjH overexpression on CR plates. Reddish colonies indicate the production of extracellular matrix components. (b)
Swimming behavior of Aeromonas veronii biovar sobria AEW43 was repressed by the GGDEF domain protein AdrA and enhanced by the EAL domain
protein YhjH. (c–e) Adherence to the cell surface of Acanthamoeba castellanii. High numbers of Aeromonas veronii biovar sobria AEW43 were found to
adhere to the amoeba cell surface when the GGDEF domain protein AdrA was overexpressed (c) when compared with YhjH overexpression (d) and
vector control (e) was overexpressed, respectively. Bar = 5 mm.
FEMS Microbiol Lett 273 (2007) 172–179
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
176
(a)
M. Rahman et al.
(b)
Absorbance at 540 nm
5
4
3
Fig. 4. Adherence of Aeromonas veronii biovar
sobria AEW43 to glass test tubes; (a) Crystal
violet staining of adherent bacteria (I, Vector
control; II, AdrA; III, YhjH), (b) Quantification of
adherence. Data shown are the average of
three independent experiments with error bars
indicating the SEs. Biofilm is enhanced by the
GGDEF domain protein AdrA and decreased
by EAL domain protein YhjH
2
1
0
Vector
Consequently, results of this study showed that GGDEF
and EAL domain proteins had an effect on the multicellular
behavior and motility of Aeromonas.
C-di-GMP concentrations influence Aeromonas
veronii biovar sobria AEW43 adhesion to plant
and animal surfaces
Microorganisms are typically attached to biotic and abiotic
surfaces in the aquatic environment (Davey & O’Toole,
2000). Aquatic plants harbour more Aeromonas compared
with water and sediment samples (Parveen et al., 1995). It
has been have shown that high numbers of Aeromonas are
attached to aquatic plant duckweed, whereby the duckweed
is used as a vehicle for transmission of Aeromonas (Rahman
et al., 2007).
To investigate the role of c-di-GMP signaling on
Aeromonas attachment to duckweed, adhesion experiments
were carried out. Aeromonas veronii biovar sobria AEW43
overexpressing AdrA showed significantly enhanced ability
(P o 0.001) to adhere to duckweed as compared with the
vector control or the strain overexpressing YhjH (Fig. 5).
The YhjH overexpressing strain adhered more than the
vector control, but with no statistical significance.
Bacteria can persist in the environment in free-living
amoeba, a protective eukaryotic host (Greub & Raoult,
2004; Abd et al., 2005; Philippe et al., 2006). To investigate
the role of c-di-GMP signaling on the attachment of
Aeromonas to free-living amoeba, Acanthamoeba castellanii
was incubated with Aeromonas veronii biovar sobria AEW43
overexpressing AdrA, YhjH or the vector control. A significantly higher number of bacterial cells attached to the
amoeba cell surface, when Aeromonas expressed AdrA in
comparison with Aeromonas expressing the EAL domain
protein YhjH or the vector control (Table 1 and Fig. 3c–e).
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
YhjH
104 CFU per duckweed (c. 0.01 g)
AdrA
4
3
2
1
0
AdrA
YhjH
Vector
Fig. 5. Adherence of Aeromonas veronii biovar sobria AEW43 on duckweed surfaces. Adherence was significantly enhanced by GGDEF domain
protein AdrA, but only slightly by the EAL domain protein YhjH. Data
shown are the average of three independent experiments with SDs.
GGDEF-type regulator influences the level of
quorum sensing molecules in Aeromonas
veronii biovar sobria
Quorum sensing is a mechanism of controling gene expression in response to bacterial cell density (Swift et al., 2001;
Winzer & Williams, 2001; Withers et al., 2001). Previous
studies have shown that biofilm formation of Aeromonas
hydrophila is regulated by the C4-HSL quorum-sensing
molecules (Lynch et al., 2002; Kirke et al., 2004). To study
the influence of c-di-GMP on the production of quorumsensing molecule, the concentration of C4-AHL was measured in Aeromonas veronii biovar sobria AEW43 overexpressing AdrA or YhjH. Aeromonas veronii biovar sobria
AEW43 overexpressing the GGDEF domain protein AdrA
produced significant higher amounts of C4-HSL than Aeromonas veronii biovar sobria AEW43 overexpressing the EAL
domain protein YhjH or the vector control (P o 0.001)
(Table 1). In summary, a link between c-di-GMP signaling
FEMS Microbiol Lett 273 (2007) 172–179
177
C-di-GMP in Aeromonas
and the production of the quorum-sensing C4-HSL molecule was demonstrated.
Discussion
Aeromonas is a ubiquitous inhabitant of aquatic ecosystems,
which has been found to persist for a prolonged period in a
drinking water distribution system or in a water microcosm
(Kuhn et al., 1997; Messi et al., 2002). The factors that
determine the ubiquity of Aeromonas in the environment are
mainly unknown. Biofilm formation is thought to contribute to the persistence of the bacteria in the environment.
For example, it has been shown that expression of multicellular behavior in S. Typhimurium enhances the long-term
survival in response to desiccation stress (White et al., 2006).
In a previous study, the authors have shown that certain
clonal types of A. veronii biovar sobria have the ability
to persist in different niches and/or in the environment
(Rahman, 2005; Rahman et al., 2007). This study demonstrates that all members of this clonal type of Aeromonas
veronii biover sobria have the ability to produce strong
biofilm, which may successively lead to colonization of
different niches and persistence in the environment.
The recently published Aeromonas hydrophila (ATCC
7966) whole-genome sequence (Seshadri et al., 2006) suggests 32 proteins with a GGDEF, nine proteins with an EAL
domain and 13 proteins with both domains. Thus the c-diGMP signaling network is present in Aeromonas species.
However, when this study was initiated the Aeromonas
genome sequence was not available. The GGDEF domain
protein AdrA and the EAL domain protein YhjH from
S. Typhimurium were used, which are well characterized
with respect to c-di-GMP synthesis and degradation and
shown to be functional in a variety of species (Simm et al.,
2004; Thormann et al., 2006). Subsequently, it was shown
that the c-di-GMP regulatory network is also functional in
Aeromonas veronii biovar sobria AEW43. Previous studies
have shown that c-di-GMP signaling reciprocally influences
multicellular behavior and motility (Simm et al., 2004). The
achievement of high and low c-di-GMP concentrations by
overexpression of the diguanylate cyclase AdrA and the
phosphodiesterase YhjH in Aeromonas veronii biovar sobria
showed that adherence to the wall of a glass tube and
motility are inversely regulated by c-di-GMP concentrations. However, although colony development, adherence
to duckweed and amoeba, and production of the quorumsensing molecule C4-HSL were stimulated by high c-diGMP concentrations, expression of YhjH had no or even a
slight adverse effect. It might be that the c-di-GMP concentration is already low under the environmental conditions
tested, so that no change in phenotype can be observed.
Alternatively, YhjH might be nonfunctional under certain
conditions in Aeromonas veronii biovar sobria.
FEMS Microbiol Lett 273 (2007) 172–179
Biofilm formation aids attachment to biotic surfaces and
virulence. For example, the infection phenotype of Yersinia
is caused by biofilm formation on the head of Caenorhabditis elegans, which blocks feeding of the nematode (Joshua
et al., 2003). Salmonella Typhimurium forms biofilms on
epithelial cell surface layers, which is thought to aid colonization and infection (Ledeboer et al., 2006). This study
showed that enhanced biofilm formation aided by overexpression of the GGDEF domain protein AdrA enhanced
bacterial adhesion to the plant and amoebal surface. C-diGMP-mediated biofilm formation might aid in the transmission of Aeromonas between different ecological niches,
from the hospital sewage setting to the fish pond via the
duckweed, which is grown in the sewage setting. On the
other hand, c-di-GMP-mediated biofilm formation might
also aid persistence of Aeromonas. Adherence of Aeromonas
to ameba might be followed by invasion and intracellular
persistence in this protozoal organism, which is a survival
strategy of many environmental and pathogenic bacteria
(Marciano-Cabral & Cabral, 2003). Thus in this study
results suggest that c-di-GMP signaling may play a conserved ecological role in bacterial adhesion to plant and
animal surfaces.
However, the intracellular c-di-GMP signaling is not the
only signal controlling biofilm formation. Extracellular
quorum-sensing molecules are also involved in the cell
population density-dependent regulation of virulence factor
expression, multicellular behavior, metabolic production
and biofilm formation in many different bacteria (Camara
et al., 2002). Previous studies have shown that Aeromonas
hydrophila produced C4-HSL. This work showed that Aeromonas veronii biovar sobria produced C4-HSL while the
strain overexpressing AdrA produced higher C4-HSL levels
than the strain overexpressing YhjH and the vector control.
The relationship between the intracellular signaling molecule c-di-GMP and the extracellular quorum-sensing signaling is unknown (Camilli & Bassler, 2006). But one simple
explanation could be that increased aggregation and biofilm
formation lead to increased C4-HSL production. However, a
connection between c-di-GMP signaling and quorum sensing has been demonstrated before, whereby knockout of the
YciR GGDEF-EAL domain protein, which has a homolog in
S. Typhimurium, has been shown to decrease production of
quorum-sensing molecules in Burkholderia cepacia (Huber
et al., 2002). It is not known whether quorum sensing
signaling also influences the c-di-GMP concentrations in
the cell.
Conclusions and perspectives
In this study, the GGDEF domain protein AdrA altered the
c-di-GMP level in Aeromonas veronii biovar sobria AEW43,
thus regulating the multicellular behavior, biofilm
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
178
formation and adherence to aquatic weed (duckweed) and
free living animal (amoeba) surfaces. It also influenced the
level of the C4-HSL quorum-sensing molecule in Aeromonas
veronii biovar sobria AEW43. The control of biofilm formation is of significant interest to the industrial, public health
and medical sectors. Because the intracellular concentration
of c-di-GMP and the extracellular concentration of quorum
sensing play a critical role in biofilm formation, a new focus
for investigating the molecular mechanisms of biofilm
development could be to elucidate the connection between
extracellular quorum-sensing signaling and intracellular
c-di-GMP signaling.
Acknowledgements
This work was supported by SIDA/SAREC grant 1999–255
for PhD fellowship for M.R. and the Karolinska Institutet
fund. Research in the laboratory of U.R. was funded by the
Karolinska Institutet (elitforskartjänst) and Vetenskapsrådet. Prof. Paul Williams is thanked for providing
plasmids pSB536 and C4-HSL. M.R. acknowledges Dr
Dinesh Diraviam Sriramalu for supporting the quorumsensing molecule detection protocols, and Dr Gunnar
Sandstrom and Dr Hadi Abd for supplying the Amoeba cell
culture.
References
Abd H, Weintraub A & Sandstrom G (2005) Intracellular survival
and replication of Vibrio cholerae O139 in aquatic free-living
amoebae. Environ Microbiol 7: 1003–1008.
Albert MJ, Ansaruzzaman M, Talukder KA, Chopra AK, Kuhn I,
Rahman M, Faruque AS, Islam MS, Sack RB & Mollby R
(2000) Prevalence of enterotoxin genes in Aeromonas spp.
isolated from children with diarrhea, healthy controls, and the
environment. J Clin Microbiol 38: 3785–3790.
Amikam D & Benziman M (1989) Cyclic Diguanylic Acid and
Cellulose Synthesis in Agrobacterium tumefaciens. J Bacteriol
171: 6649–6655.
Camara M, Williams P & Hardman A (2002) Controlling
infection by tuning in and turning down the volume of
bacterial small-talk. Lancet Infect Dis 2: 667–676.
Camilli A & Bassler BL (2006) Bacterial small-molecule signaling
pathways. Science 311: 1113–1116.
D’Argenio DA & Miller SI (2004) Cyclic di-GMP as a bacterial
second messenger. Microbiology 150: 2497–2502.
D’Argenio DA, Calfee MW, Rainey PB & Pesci EC (2002)
Autolysis and autoaggregation in Pseudomonas aeruginosa
colony morphology mutants. J Bacteriol 184: 6481–6489.
Davey ME & O’Toole GA (2000) Microbial biofilms: from
ecology to molecular genetics. Microbiol Mol Biol Rev 64:
847–867.
Greub G & Raoult D (2004) Microorganisms resistant to
free-living amoebae. Clin Microbiol Rev 17: 413–433.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
M. Rahman et al.
Huber B, Riedel K, Kothe M, Givskov M, Molin S & Eberl L
(2002) Genetic analysis of functions involved in the late stages
of biofilm development in Burkholderia cepacia H111. Mol
Microbiol 46: 411–426.
Janda JM & Duffey PS (1988) Mesophilic aeromonads in human
disease: current taxonomy, laboratory identification, and
infectious disease spectrum. Rev Infect Dis 10: 980–997.
Jenal U (2004) Cyclic di-guanosine-monophosphate comes of
age: a novel secondary messenger involved in modulating cell
surface structures in bacteria? Curr Opin Microbiol 7: 185–191.
Joshua GWP, Karlyshev AV, Smith MP, Isherwood KE, Titball RW
& Wren BW (2003) A Caenorhabditis elegans model of Yersinia
infection: biofilm formation on a biotic surface. Microbiology
149: 3221–3229.
Kirillina O, Fetherston JD, Bobrov AG, Abney J & Perry RD
(2004) HmsP, a putative phosphodiesterase, and HmsT, a
putative diguanylate cyclase, control Hms-dependent biofilm
formation in Yersinia pestis. Mol Microbiol 54: 75–88.
Kirke DF, Swift S, Lynch MJ & Williams P (2004) The Aeromonas
hydrophila LuxR homologue AhyR regulates the N-acyl
homoserine lactone synthase, AhyI positively and negatively in
a growth phase-dependent manner. FEMS Microbiol Lett 241:
109–117.
Kuhn I, Albert MJ, Ansaruzzaman M et al. (1997)
Characterization of Aeromonas spp. isolated from humans
with diarrhea, from healthy controls, and from surface water in
Bangladesh. J Clin Microbiol 35: 369–373.
Ledeboer NA, Frye JG, McClelland M & Jones BD (2006)
Salmonella enterica serovar Typhimurium requires the Lpf,
Pef, and Tafi fimbriae for biofilm formation on HEp-2 tissue
culture cells and chicken intestinal epithelium. Infect Immun
74: 3156–3169.
Lynch MJ, Swift S, Kirke DF, Keevil CW, Dodd CE & Williams P
(2002) The regulation of biofilm development by quorum
sensing in Aeromonas hydrophila. Environ Microbiol 4: 18–28.
Marciano-Cabral F & Cabral G (2003) Acanthamoeba spp. as
agents of disease in humans. Clin Microbiol Rev 16: 273–307.
Messi P, Guerrieri E & Bondi M (2002) Survival of an Aeromonas
hydrophila in an artificial mineral water microcosm. Water Res
36: 3410–3415.
Möllby R, Kühn I & Katouli M (1993) Computerized biochemical
fingerprinting. A new tool for typing of bacteria. Rev of Med
Microbiol 4: 231–241.
Parveen S, Islam MS & Huq A (1995) Abundance of Aeromonas
spp. in river and lake waters in and around Dhaka, Bangladesh.
J Diarrhoeal Dis Res 13: 183–186.
Philippe C, Blech MF & Hartemann P (2006) Intra-amoebal
development of Legionella pneumophila and the potential role
of amoebae in the transmission of Legionnaires’ disease. Med
Mal Infect 36: 196–200.
Rahman M (2005) Health hazards associated with dissemination
of bacterial strains in waste water recycling. Microbiology and
Tumor Biology Center, 66pp. PhD thesis, Karolinska Institutet,
Stockholm.
FEMS Microbiol Lett 273 (2007) 172–179
179
C-di-GMP in Aeromonas
Rahman M, Colque-Navarro P, Kuhn I, Huys G, Swings J &
Mollby R (2002) Identification and characterization of
pathogenic Aeromonas veronii biovar sobria associated with
epizootic ulcerative syndrome in fish in Bangladesh. Appl
Environ Microbiol 68: 650–655.
Rahman M, Huys G, Rahman M, Albert MJ, Kuhn I & Mollby R
(2007) Persistence, transmission and virulence characteristics
of Aeromonas strains in a duckweed aquaculture-based
hospital sewage water recycling plant in Bangladesh. Appl
Environ Microbiol 73: 144–151.
Romling U & Amikam D (2006) Cyclic di-GMP as a second
messenger. Curr Opin Microbiol 9: 218–228.
Römling U, Rohde M, Olsén A, Normark S & Reinköster J (2000)
AgfD, the checkpoint of multicellular and aggregative
behaviour in Salmonella typhimurium regulates at least two
independent pathways. Mol Microbiol 36: 10–23.
Romling U, Gomelsky M & Galperin MY (2005) C-di-GMP: the
dawning of a novel bacterial signalling system. Mol Microbiol
57: 629–639.
Seshadri R, Joseph SW, Chopra AK et al. (2006) Genome
sequence of Aeromonas hydrophila ATCC 7966T: jack of all
trades. J Bacteriol 188: 8272–8282.
Simm R, Morr M, Kader A, Nimtz M & Romling U (2004)
GGDEF and EAL domains inversely regulate cyclic di-GMP
levels and transition from sessility to motility. Mol Microbiol
53: 1123–1134.
Simm R, Fetherston JD, Kader A, Romling U & Perry RD (2005)
Phenotypic convergence mediated by GGDEF-domaincontaining proteins. J Bacteriol 187: 6816–6823.
Swift S, Karlyshev AV, Fish L, Durant EL, Winson MK, Chhabra
SR, Williams P, Macintyre S & Stewart GS (1997) Quorum
sensing in Aeromonas hydrophila and Aeromonas salmonicida:
identification of the LuxRI homologs AhyRI and AsaRI and
their cognate N-acylhomoserine lactone signal molecules.
J Bacteriol 179: 5271–5281.
FEMS Microbiol Lett 273 (2007) 172–179
Swift S, Downie JA, Whitehead NA, Barnard AM, Salmond GP &
Williams P (2001) Quorum sensing as a population-densitydependent determinant of bacterial physiology. Adv Microb
Physiol 45: 199–270.
Tal R, Wong HC, Calhoon R et al. (1998) Three cdg operons
control cellular turnover of cyclic di-GMP in Acetobacter
xylinum: genetic organization and occurrence of conserved
domains in isoenzymes. J Bacteriol 180: 4416–4425.
Thormann KM, Duttler S, Saville RM, Hyodo M, Shukla S,
Hayakawa Y & Spormann AM (2006) Control of
formation and cellular detachment from Shewanella
oneidensis MR-1 biofilms by cyclic di-GMP. J Bacteriol 188:
2681–2691.
Tischler AD & Camilli A (2004) Cyclic diguanylate (c-di-GMP)
regulates Vibrio cholerae biofilm formation. Mol Microbiol 53:
857–869.
Tischler AD & Camilli A (2005) Cyclic diguanylate regulates
Vibrio cholerae virulence gene expression. Infect Immun 73:
5873–5882.
White AP, Gibson DL, Kim W, Kay WW & Surette MG (2006)
Thin aggregative fimbriae and cellulose enhance long-term
survival and persistence of Salmonella. J Bacteriol 188:
3219–3227.
Winson MK, Swift S, Fish L, Throup JP, Jorgensen F, Chhabra SR,
Bycroft BW, Williams P & Stewart GS (1998) Construction
and analysis of luxCDABE-based plasmid sensors for
investigating N-acyl homoserine lactone-mediated quorum
sensing. FEMS Microbiol Lett 163: 185–192.
Winzer K & Williams P (2001) Quorum sensing and the
regulation of virulence gene expression in pathogenic bacteria.
Int J Med Microbiol 291: 131–143.
Withers H, Swift S & Williams P (2001) Quorum sensing
as an integral component of gene regulatory networks
in Gram-negative bacteria. Curr Opin Microbiol 4:
186–193.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c