Cellular L-arginine pools modulate c-di-GMP turnover and biofilm formation in Pseudomonas putida

The intracellular second messenger cyclic diguanylate (c-di-GMP) is broadly conserved in bacteria, where it influences processes such as virulence, stress resistance and biofilm development. In the plant-beneficial bacterium Pseudomonas putida KT2440, the response regulator with diguanylate cyclase activity CfcR is the main contributor to c-di-GMP levels in the stationary phase of growth. When overexpressed, CfcR increases c-di-GMP levels and gives rise to a pleiotropic phenotype that includes enhanced biofilm formation and crinkly colony morphology. Our group has previously reported that insertion mutants in argG and argH, the genes that encode the last two enzymes in the arginine biosynthesis pathway, do not display the crinkly colony morphology phenotype and show decreased c-di-GMP levels even in the presence of cfcR in multicopy (Ramos-Gonz&#225;lez, M.I. et al. 2016. Front. Microbiol. 7, 1093). Here we present results indicating that L-arginine acts both as an environmental and as a metabolic signal that influences the lifestyles of P. putida through the modulation of c-di-GMP levels and changes in the expression of structural elements of biofilms. Exogenous L-arginine partially restores c-di-GMP levels in arginine biosynthesis mutants, a response that is transduced through CfcR and possibly (an)other diguanylate cyclase(s). At least three periplasmic binding proteins, each forming part of an amino acid transport system, contribute in different ways to the response to external L-arginine. We propose that the turnover of the second messenger c-di-GMP is modulated by the state of global arginine pools in the cell resulting both from anabolism and from uptake.

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Homologous c-di-GMP-Binding Scr Transcription Factors Orchestrate Biofilm Development in Vibrio parahaemolyticus

Journal of Bacteriology ◽

10.1128/jb.00723-19 ◽

2020 ◽

Vol 202 (6) ◽

Author(s):

John H. Kimbrough ◽

J. Thomas Cribbs ◽

Linda L. McCarter

Keyword(s):

Transcription Factors ◽

Biofilm Formation ◽

Vibrio Parahaemolyticus ◽

Matrix Protein ◽

Second Messenger ◽

Minor Role ◽

Binding Motif ◽

Swarming Motility ◽

Content Type ◽

Biofilm Development

ABSTRACT The marine bacterium and human pathogen Vibrio parahaemolyticus rapidly colonizes surfaces by using swarming motility and forming robust biofilms. Entering one of the two colonization programs, swarming motility or sessility, involves differential regulation of many genes, resulting in a dramatic shift in physiology and behavior. V. parahaemolyticus has evolved complex regulation to control these two processes that have opposing outcomes. One mechanism relies on the balance of the second messenger c-di-GMP, where high c-di-GMP favors biofilm formation. V. parahaemolyticus possesses four homologous regulators, the Scr transcription factors, that belong in a Vibrio-specific family of W[F/L/M][T/S]R motif transcriptional regulators, some members of which have been demonstrated to bind c-di-GMP. In this work, we explore the role of these Scr regulators in biofilm development. We show that each protein binds c-di-GMP, that this binding requires a critical R in the binding motif, and that the biofilm-relevant activities of CpsQ, CpsS, and ScrO but not ScrP are dependent upon second messenger binding. ScrO and CpsQ are the primary drivers of biofilm formation, as biofilms are eliminated when both of these regulators are absent. ScrO is most important for capsule expression. CpsQ is most important for RTX-matrix protein expression, although it contributes to capsule expression when c-di-GMP levels are high. Both regulators contribute to O-antigen ligase expression. ScrP works oppositely in a minor role to repress the ligase gene. CpsS plays a regulatory checkpointing role by negatively modulating expression of these biofilm-pertinent genes under fluctuating c-di-GMP conditions. Our work further elucidates the multifactorial network that contributes to biofilm development in V. parahaemolyticus. IMPORTANCE Vibrio parahaemolyticus can inhabit open ocean, chitinous shells, and the human gut. Such varied habitats and the transitions between them require adaptable regulatory networks controlling energetically expensive behaviors, including swarming motility and biofilm formation, which are promoted by low and high concentrations of the signaling molecule c-di-GMP, respectively. Here, we describe four homologous c-di-GMP-binding Scr transcription factors in V. parahaemolyticus. Members of this family of regulators are present in many vibrios, yet their numbers and the natures of their activities differ across species. Our work highlights the distinctive roles that these transcription factors play in dynamically controlling biofilm formation and architecture in V. parahaemolyticus and serves as a powerful example of regulatory network evolution and diversification.

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A universal stress protein in Mycobacterium smegmatis sequesters the cAMP-regulated lysine acyltransferase and is essential for biofilm formation

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011373 ◽

2019 ◽

Vol 295 (6) ◽

pp. 1500-1516 ◽

Cited By ~ 2

Author(s):

Sintu Samanta ◽

Priyanka Biswas ◽

Arka Banerjee ◽

Avipsa Bose ◽

Nida Siddiqui ◽

...

Keyword(s):

Stress Responses ◽

Mycobacterium Smegmatis ◽

Biofilm Formation ◽

Stress Proteins ◽

Response Regulator ◽

Stress Protein ◽

Colony Morphology ◽

Regulator Gene ◽

Its Gene

Universal stress proteins (USPs) are present in many bacteria, and their expression is enhanced under various environmental stresses. We have previously identified a USP in Mycobacterium smegmatis that is a product of the msmeg_4207 gene and is a substrate for a cAMP-regulated protein lysine acyltransferase (KATms; MSMEG_5458). Here, we explored the role of this USP (USP4207) in M. smegmatis and found that its gene is present in an operon that also contains genes predicted to encode a putative tripartite tricarboxylate transporter (TTT). Transcription of the TTT-usp4207 operon was induced in the presence of citrate and tartrate, perhaps by the activity of a divergent histidine kinase-response regulator gene pair. A usp4207-deleted strain had rough colony morphology and reduced biofilm formation compared with the WT strain; however, both normal colony morphology and biofilm formation were restored in a Δusp4207Δkatms strain. We identified several proteins whose acetylation was lost in the Δkatms strain, and whose transcript levels increased in M. smegmatis biofilms along with that of USP4207, suggesting that USP4207 insulates KATms from its other substrates in the cell. We propose that USP4207 sequesters KATms from diverse substrates whose activities are down-regulated by acylation but are required for biofilm formation, thus providing a defined role for this USP in mycobacterial physiology and stress responses.

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Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽

10.1128/mbio.00242-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 69

Author(s):

Varisa Huangyutitham ◽

Zehra Tüzün Güvener ◽

Caroline S. Harwood

Keyword(s):

Signal Transduction ◽

Biofilm Formation ◽

Response Regulator ◽

Cluster Formation ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Regulator Protein ◽

Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

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The cyclic di-GMP network is a global regulator of phase-transition and attachment-dependent host colonization in Erwinia amylovora

10.1101/2021.02.01.429191 ◽

2021 ◽

Author(s):

Roshni R. Kharadi ◽

Kayla Selbmann ◽

George W. Sundin

Keyword(s):

Signal Transduction ◽

Biofilm Formation ◽

Erwinia Amylovora ◽

Initial Surface ◽

Diguanylate Cyclase ◽

Wide Range ◽

Biofilm Development ◽

Bacterial Systems

AbstractCyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189Δ12. Comparisons between the representative transcriptomic profiles of Ea1189Δ12 and the combinatorial edc gene knockout mutant (Ea1189Δ5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189Δ12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189Δ12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. Additionally, complementing individual eliminated genes back into Ea1189Δ12, and the collective evaluation of several virulence factors, enabled the correlative clustering of the functional effect rendered by each Edc and Pde enzyme in the system.SignificanceCyclic-di-GMP dependent regulation, in the context of biofilm formation, has been studied in several bacterial systems. However, the comprehensiveness of the studies exploring the role of individual genetic components related to cyclic-di-GMP is affected by the often large number of diguanylate cyclase and phosphodiesterase enzymes present within individual bacterial systems. To explore the evolutionary dependencies related to cyclic-di-GMP in E. amylovora, we used a collective elimination approach, whereby all of the enzymes involved in cyclic-di-GMP metabolism were eliminated from the system. This approach enabled us to highlight the critical importance of cyclic-di-GMP in plant xylem colonization due to its effect on surface attachment. Additionally, we highlight the global transcriptomic effect of cyclic-di-GMP dependent signaling at various stages of biofilm development. Our approach is aimed at exploring the regulatory role of individual cyclic-di-GMP related enzymes in a background that is free from any redundancy-based feedback.

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Second messenger c-di-GMP modulates exopolysaccharide Pea-dependent phenotypes via regulating eppA expression in Pseudomonas putida

Applied and Environmental Microbiology ◽

10.1128/aem.02270-21 ◽

2022 ◽

Author(s):

Yujie Xiao ◽

Qingyuan Liang ◽

Meina He ◽

Nianqi Wu ◽

Liang Nie ◽

...

Keyword(s):

Pseudomonas Putida ◽

Structure Prediction ◽

Direct Interaction ◽

Second Messenger ◽

Genetically Engineered ◽

Colony Morphology ◽

Base Pairs ◽

Pellicle Formation ◽

Unknown Protein ◽

Relationship Of

Exopolysaccharides (EPSs) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida . The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through unknown mechanism(s). Herein, we found that c-di-GMP modulated wrinkly colony morphology via regulating expression of eppA ( PP_5586 ), a small individually transcribed gene with 177 base pairs, and this gene was adjacent to the upstream of pea cluster. Phenotype observation revealed that eppA was essential for Pea-dependent phenotypes. The deletion of eppA led to smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with the loss of the entire pea operon. EppA expression was positively regulated by c-di-GMP via the transcriptional effector FleQ, and eppA was essential for the c-di-GMP-induced wrinkly colony morphology. Structure prediction results implied that EppA had two transmembrane regions, and Western blot revealed that EppA was located on cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on transcriptomic profile of P. putida . Bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in pea cluster and adjacent operons. Overall, these findings reveal that EppA is essential for Pea-dependent phenotypes, and that c-di-GMP modulates Pea-dependent phenotypes via regulating eppA expression in P. putida . IMPORTANCE Microbe-secreted EPSs are high molecular weight polysaccharides that have the potential to be used as industrially important biomaterials. The EPS Pea in P. putida is essential for wrinkly colony morphology and pellicle formation. Here, we identified a function-unknown protein EppA, which was also essential for Pea-dependent wrinkly colony morphology and pellicle formation, and EppA was probably involved in Pea secretion. Meanwhile, our results indicated that the second messenger c-di-GMP positively regulated the expression of EppA, resulting in Pea-dependent wrinkly colony morphology. Our results reveal the relationship of c-di-GMP, EppA, and Pea-dependent phenotypes, and provide possible pathway to construct genetically engineered strain for high Pea production.

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A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

mBio ◽

10.1128/mbio.00156-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 28

Author(s):

Nathan Feirer ◽

Jing Xu ◽

Kylie D. Allen ◽

Benjamin J. Koestler ◽

Eric L. Bruger ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Signaling Pathway ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Dominant Role ◽

Second Messenger ◽

Dual Function ◽

Diguanylate Cyclase ◽

Cyclic Diguanylate ◽

Content Type

ABSTRACTThe motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogenAgrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP inA. tumefaciensare controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins.IMPORTANCEPathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have characterized a novel signaling pathway that plays a dominant role in the regulation of biofilm formation in the model pathogenAgrobacterium tumefaciens. This control pathway involves small metabolites called pterins, well studied in eukaryotes, but this is the first example of pterin-dependent signaling in bacteria. The described pathway controls levels of an important intracellular second messenger (cyclic diguanylate monophosphate) that regulates key bacterial processes such as biofilm formation, motility, and virulence. Pterins control the balance of activity for an enzyme that both synthesizes and degrades the second messenger. These findings reveal a complex, multistep pathway that modulates this enzyme, possibly identifying new targets for antibacterial intervention.

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Bacterial cyclic diguanylate signaling networks sense temperature

Nature Communications ◽

10.1038/s41467-021-22176-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Henrik Almblad ◽

Trevor E. Randall ◽

Fanny Liu ◽

Katherine Leblanc ◽

Ryan A. Groves ◽

...

Keyword(s):

Second Messenger ◽

Opportunistic Pathogen ◽

Signaling Networks ◽

Pas Domain ◽

Diguanylate Cyclase ◽

Temperature Dependent ◽

Cyclic Diguanylate ◽

Biofilm Production ◽

Thermal Sensing ◽

Biofilm Development

AbstractMany bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

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Bordetella bronchisepticaDiguanylate Cyclase BdcA Regulates Motility and Is Important for the Establishment of Respiratory Infection in Mice

Journal of Bacteriology ◽

10.1128/jb.00011-19 ◽

2019 ◽

Vol 201 (17) ◽

Cited By ~ 1

Author(s):

Keila Belhart ◽

María de la Paz Gutierrez ◽

Federico Zacca ◽

Nicolás Ambrosis ◽

Monica Cartelle Gestal ◽

...

Keyword(s):

Respiratory Tract ◽

Biofilm Formation ◽

Abiotic Factors ◽

Second Messenger ◽

Bordetella Bronchiseptica ◽

Diguanylate Cyclase ◽

Content Type ◽

Motility Regulation ◽

Tract Infections ◽

Motility Inhibition

ABSTRACTBacteria can be motile and planktonic or, alternatively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, includingBordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation inB. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of thebdcAgene,B. bronchisepticashowed enhanced motility, strengthening the hypothesis that BdcA regulates motility inB. bronchiseptica. We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of thebdcAgene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation inB. bronchiseptica, as well as its impact on pathogenesis.IMPORTANCEPathogenesis ofBordetellaspp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. InBordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized aB. bronchisepticadiguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability ofB. bronchisepticato colonize the murine respiratory tract. These results provide us with a better understanding of howB. bronchisepticacan infect a host.

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Vibrio cholerae O1 Strain TSI-4 Produces the Exopolysaccharide Materials That Determine Colony Morphology, Stress Resistance, and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.64.10.3648-3655.1998 ◽

1998 ◽

Vol 64 (10) ◽

pp. 3648-3655 ◽

Cited By ~ 166

Author(s):

Sun Nyunt Wai ◽

Yoshimitsu Mizunoe ◽

Akemi Takade ◽

Shun-Ichiro Kawabata ◽

Shin-Ichi Yoshida

Keyword(s):

Vibrio Cholerae ◽

Stress Resistance ◽

Biofilm Formation ◽

Acquired Resistance ◽

Electron Microscopic Examination ◽

Culture Conditions ◽

Colony Morphology ◽

Electron Microscopic ◽

Biofilm Development ◽

El Tor

ABSTRACT Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced byV. cholerae TSI-4/R was found to have a composition ofN-acetyl-d-glucosamine,d-mannose, 6-deoxy-d-galactose, andd-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.

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A Staphylococcal GGDEF Domain Protein Regulates Biofilm Formation Independently of Cyclic Dimeric GMP

Journal of Bacteriology ◽

10.1128/jb.00375-08 ◽

2008 ◽

Vol 190 (15) ◽

pp. 5178-5189 ◽

Cited By ~ 74

Author(s):

Linda M. Holland ◽

Sinéad T. O'Donnell ◽

Dmitri A. Ryjenkov ◽

Larissa Gomelsky ◽

Shawn R. Slater ◽

...

Keyword(s):

Biofilm Formation ◽

Cyclase Activity ◽

Mrna Levels ◽

Diguanylate Cyclase ◽

Diguanylate Cyclases ◽

Exopolysaccharide Biosynthesis ◽

Ggdef Domain ◽

Domain Protein ◽

Biofilm Development

ABSTRACT Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

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