A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae

McKee, Robert W.; Kariisa, Ankunda; Mudrak, Benjamin; Whitaker, Courtney; Tamayo, Rita

doi:10.1186/s12866-014-0272-9

Cited by 22 publications

(29 citation statements)

References 67 publications

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“…This phenotype is not observed in the El Tor biotype, as the transcription of vieSAB is subject to H-NS silencing (39,40). We also did not observe a phenotype for the ΔVC1348 or ΔVCA0210 strain; however, this was also consistent with previously published findings, which demonstrated a biofilm phenotype only when these proteins were overexpressed (32). Interestingly, we observed a 47-fold decrease in vpsL expression in the ΔvarA (VC1213) strain, which is contradictory to results from previously published work (31).…”

Section: Discussionsupporting

confidence: 92%

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The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

2017

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Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium Vibrio cholerae is no exception and harbors 52 RR genes. Using in-frame deletion mutants of each RR gene, we performed a systematic analysis of their role in V. cholerae biofilm formation. We determined that 7 RRs impacted the expression of an essential biofilm gene and found that the recently characterized RR, VxrB, regulates the expression of key structural and regulatory biofilm genes in V. cholerae. vxrB is part of a 5-gene operon, which contains the cognate HK vxrA and three genes of unknown function. Strains carrying ΔvxrA and ΔvxrB mutations are deficient in biofilm formation, while the ΔvxrC mutation enhances biofilm formation. The overexpression of VxrB led to a decrease in motility. We also observed a small but reproducible effect of the absence of VxrB on the levels of cyclic di-GMP (c-di-GMP). Our work reveals a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels.IMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, providing protection from environmental stresses and contributing to the transmission of V. cholerae to the human host. V. cholerae can utilize two-component systems (TCS), composed of a histidine kinase (HK) and a response regulator (RR), to regulate biofilm formation in response to external cues. We performed a systematic analysis of V. cholerae RRs and identified a new regulator of biofilm formation, VxrB. We demonstrated that the VxrAB TCS is essential for robust biofilm formation and that this system may regulate biofilm formation via its regulation of key biofilm regulators and cyclic di-GMP levels. This research furthers our understanding of the role that TCSs play in the regulation of V. cholerae biofilm formation.KEYWORDS Vibrio cholerae, VxrAB, biofilms, c-di-GMP, motility V ibrio cholerae is the causative agent of the gastrointestinal disease cholera, responsible for approximately 3 to 5 million cases of severe diarrhea and 120,000 deaths annually (1, 2). A resident of aquatic reservoirs, V. cholerae can be found as freeswimming planktonic cells or in matrix-protected cellular aggregates, known as biofilms (2-4). Evidence suggests that biofilms form during the aquatic and intestinal phases of the V. cholerae life cycle and play an important role in environmental survival, as well as in the intestinal and transmission stages of infection (5-9). V. cholerae biofilm formation requires the production and secretion of an extracellular matrix composed of matrix proteins, nucleic acids, and Vibrio polysaccharide (VPS), a glycoconjugate that is essential for the formation of three-dimensional biofilm structures (10-16). A complex regulatory network governs this process,...

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Section: Discussionsupporting

confidence: 92%

“…VC1348 and VCA0210 are RRs that contain HD-GYP domains with predicted cyclic di-GMP (c-di-GMP) phosphodiesterase activity. Overexpression of these RRs led to a significant decrease in biofilm formation (32). To date, there is no systematic analysis reporting on the contribution of each TCS to biofilm formation in Vibrio cholerae.…”

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confidence: 99%

The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

2017

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“…Detailed kinetic analyses indicate that PA4781 has low enzymatic activity but hydrolyzes 5=-pGpG more effectively than cyclic di-GMP (81). Although similar kinetic experiments on other HD-GYP domain proteins have not been reported, the available evidence suggests that differences in the relative activity against 5=-pGpG compared to cyclic di-GMP do occur (66,69,71,76,77,83,84).…”

Section: Diversity In Substrate Binding and Catalysismentioning

confidence: 72%

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

2017

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Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins.KEYWORDS cyclic dinucleotide second messenger, GGDEF domain, EAL domain, HD-GYP domain, DHH/DHHA1 domain, cyclic GAMP, cyclic di-AMP, cyclic di-GMP, second messenger T he dinucleotide cyclic di-GMP is the most abundant second messenger in bacteria. It promotes the environmental lifestyle switch between sessility and motility, as well as the host-related lifestyle switch between acute and chronic/benign infection. A hallmark of the cyclic di-GMP signaling network is an apparent redundancy of cyclic di-GMP turnover proteins encoded in one genome. However, many of these proteins have distinct N-terminal sensing and signaling domains, suggesting that their activities in cyclic di-GMP turnover respond posttranslationally to various (and different) intraand extracellular signals. In gross terms, the number of cyclic di-GMP turnover proteins is linearly correlated with genome size within the different bacterial phyla, with Thermotogae having one of the highest cyclic di-GMP-related "IQs," the density of enzymes per megabase pair, with some species harboring over 100 cyclic di-GMP turnover proteins (http://www.ncbi.nlm.nih.gov/Complete_Genomes/c-di-GMP.html). As in other domain superfamilies, extensive sequence diversity exists. Here, we review the knowledge on the translation of sequence diversity of cyclic di-GMP turnover proteins into functional diversity. We conclude by discussing whether and how a unified nomenclature for cyclic di-GMP turnover proteins can be established.

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“…For example, it is not known whether HD-GYPs are functional duplicates of EAL enzymes emerging from convergent evolution or whether they have a different role within more complex regulatory pathways. If this is the case, since HD-GYPs are not ubiquitous in bacteria, the presence of genes encoding these domains may well be considered a hallmark of bacterial species with more sophisticated biofilm-mediated survival strategies, such as Pseudomonas aeruginosa (9,10) or Vibrio cholerae (11). Indeed, HD-GYPs have been shown to interact with GGDEF domains within multien-zyme complexes, while EAL domains seem to interact with GGDEF domains only in hybrid proteins (i.e., when they are fused within the same polypeptide chain) (12,13).…”

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confidence: 99%

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

et al. 2015

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The intracellular level of the bacterial secondary messenger cyclic di-3=,5=-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously characterized in vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site. IMPORTANCEIn bacteria, the capability to form biofilms, responsible for increased pathogenicity and antibiotic resistance, is almost universally stimulated by the second messenger cyclic di-GMP (c-di-GMP). To design successful strategies for targeting biofilm formation, a detailed characterization of the enzymes involved in c-di-GMP metabolism is crucial. We solved the structure of the HD-GYP domain of PA4781 from Pseudomonas aeruginosa, involved in c-di-GMP degradation. This is the third structure of this class of phosphodiesterases to be solved, and with respect to its homologues, it shows significant differences both in the nature and in the binding mode of the coordinated metals, indicating that HD-GYP proteins are able to fine-tune their function, thereby increasing the chances of the microorganism to adapt to different environmental needs. The dinucleotide cyclic di-GMP (c-di-GMP) serves as a core signal in the coordinated lifestyle switch of most bacteria in response to different environmental stimuli. In particular, high levels of c-di-GMP generally flag hostile conditions and result in reduced motility, attachment, and biofilm formation, while low levels of c-di-GMP indicate that it is time to go back to virulence and cell division (1-5). The intracellular levels of c-di-GMP are fine-tuned by the opposite action of dedicated enzymes: diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) (6). The first class, characterized by a GGDEF domain, synthesize c-di-GMP from two GTP molecules, while the second one hydrolyzes it. PDEs are further divided into two unrelated superfamilies of hydrolases: the more abundant EAL proteins, which degrade c-di-GMP to the linear dinucleotide pGpG, and the HD-GYP proteins, which are able to hydrolyze c-di-GMP into two molecules of GMP. Bacteria display a wide variety of genes encoding these three enzymatic domains,...

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A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae

Cited by 22 publications

References 67 publications

The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

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