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In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes.

ABSTRACT: BACKGROUND:The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences. RESULTS:The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5T, A. lipoferum 4B, Azospirillum sp. B510, A. thiophilum BV-ST, A. halopraeferens DSM3675, A. oryzae A2P, and A. brasilense Sp7, Sp245 and Az39 encode for 29 to 41 of these predicted proteins. Notably, only 15 proteins were conserved in all nine genomes: eight GGDEF, three EAL and four GGDEF-EAL hybrid domain proteins, all of which corresponded to core genes in the genomes. The predicted proteins exhibited variable lengths, architectures and sensor domains. In addition, the predicted cellular localizations showed that some of the proteins to contain transmembrane domains, suggesting that these proteins are anchored to the membrane. Therefore, as reported in other soil bacteria, the Azospirillum genomes encode a large number of proteins that are likely involved in c-di-GMP metabolism. In addition, the data obtained here strongly suggest host specificity and environment specific adaptation. CONCLUSIONS:Bacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to colonize and benefit their host plant. Gaining information on the structures of proteins involved in c-di-GMP metabolism in Azospirillum appears to be an important step in determining the c-di-GMP signaling pathways, involved in the transition of a motile cell towards a biofilm life-style, as an example of microbial genome plasticity under diverse in situ environments.

SUBMITTER: Mata AR

PROVIDER: S-EPMC5845226 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes.

Mata Alberto Ramírez AR Pacheco César Millán CM Cruz Pérez José F JF Sáenz Martha Minjárez MM Baca Beatriz E BE

BMC microbiology 20180309 1

<h4>Background</h4>The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promot ...[more]

PMID: 29523074

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Project description:Vibrio parahaemolyticus rapidly colonizes surfaces using swarming motility. Surface contact induces the surface-sensing regulon, including lateral flagellar genes, spurring dramatic shifts in physiology and behavior. The bacterium can also adopt a sessile, surface-associated lifestyle and form robust biofilms. These alternate colonization strategies are influenced reciprocally by the second messenger c-di-GMP. Although V. parahaemolyticus possesses 43 predicted proteins with the c-di-GMP-forming GGDEF domain, none have been previously been identified as contributors to surface colonization. We sought to explore this knowledge gap by using a suppressor transposon screen to restore the swarming motility of a nonswarming, high-c-di-GMP strain. Two diguanylate cyclases, ScrJ and ScrL, each containing tetratricopeptide repeat-coupled GGDEF domains, were demonstrated to contribute additively to swarming gene repression. Both proteins required an intact catalytic motif to regulate. Another suppressor mapped in lafV, the last gene in a lateral flagellar operon. Containing a degenerate phosphodiesterase (EAL) domain, LafV repressed transcription of multiple genes in the surface sensing regulon; its repressive activity required LafK, the primary swarming regulator. Mutation of the signature EAL motif had little effect on LafV's repressive activity, suggesting that LafV belongs to the subclass of EAL-type proteins that are regulatory but not enzymatic. Consistent with these activities and their predicted effects on c-di-GMP, scrJ and scrL but not lafV, mutants affected the transcription of the c-di-GMP-responsive biofilm reporter cpsA::lacZ Our results expand the knowledge of the V. parahaemolyticus GGDEF/EAL repertoire and its roles in this surface colonization regulatory network.IMPORTANCE A key survival decision, in the environment or the host, is whether to emigrate or aggregate. In bacteria, c-di-GMP signaling almost universally influences solutions to this dilemma. In V. parahaemolyticus, c-di-GMP reciprocally regulates swarming and sticking (i.e., biofilm formation) programs of surface colonization. Key c-di-GMP-degrading phosphodiesterases responsive to quorum and nutritional signals have been previously identified. c-di-GMP binding transcription factors programming biofilm development have been studied. Here, we further develop the blueprint of the c-di-GMP network by identifying new participants involved in dictating the complex decision of whether to swarm or stay. These include diguanylate cyclases with tetratricopeptide domains and a degenerate EAL protein that, analogously to the negative flagellar regulator RflP/YdiV of enteric bacteria, serves to regulate swarming.

Project description:Bacterial lifestyles are dictated by the conditions encountered during colonization of a specific ecological niche or host. A wide range of environmental stimuli triggers sensory modules, that modify activity of proteins, or/and initiate a transfer of information in complex regulatory networks, which modulate gene expression, and therefore adaptation. The transition between planktonic and biofilm growth is dependent on the homeostasis of the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclase (DGC) activity favor biofilm while low levels maintained by phosphodiesterase (PDE) enzymes encourage planktonic style or biofilm dispersal. In Pseudomonas aeruginosa over 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual enzymes possessing both canonical DGC and PDE motifs, i.e. GGDEF and EAL, respectively. It has been previously reported that the deletion of the PDE/DGC dual enzyme PA0285, one of 5 c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms and causes elevated c-di-GMP levels in P. aeruginosa. Here we confirm that this role is conserved in various P. aeruginosa (PAO1, PA14 and PAK) and Pseudomonas putida strains. Deletion of PA0285 has the highest impact during the early stage of surface colonization, and RNA-seq analysis in biofilm context indicates that it may be associated with upregulation of cupA fimbrial genes. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds the respective GTP (GGDEF) and c-di-GMP (EAL) substrates but only exhibits PDE activity in vitro. Both GGDEF and EAL domains are important for PA0285 PDE activity in vivo as suggested by site-directed mutants in each of the key motifs. Complementation of the PA0285 mutant strain with the C-terminal GGDEF/EAL portion in trans is not as effective as with the full-length gene suggesting that the transmembrane region and PAS domains contained in PA0285’s N-terminal portion influence its PDE activity in vivo. We speculate that the transmembrane portion or/and PAS domains of PA0285 impede its PDE activity in vivo through modulating the conformation of the enzyme, as shown for P. aeruginosa RbdA, in response to an unknown stimulus.

Project description:The second messenger cyclic dimeric GMP (c-di-GMP) is almost ubiquitous among bacteria as are the c-di-GMP turnover proteins, which mediate the transition between motility and sessility. EAL domain proteins have been characterized as c-di-GMP-specific phosphodiesterases. While most EAL domain proteins contain additional, usually N-terminal, domains, there is a distinct family of proteins with stand-alone EAL domains, exemplified by Salmonella enterica serovar Typhimurium proteins STM3611 (YhjH/PdeH), a c-di-GMP-specific phosphodiesterase, and the enzymatically inactive STM1344 (YdiV/CdgR) and STM1697, which regulate bacterial motility through interaction with the flagellar master regulator, FlhDC. We have analyzed the phylogenetic distribution of EAL-only proteins and their potential functions. Genes encoding EAL-only proteins were found in various bacterial phyla, although most of them were seen in proteobacteria, particularly enterobacteria. Based on the conservation of the active site residues, nearly all stand-alone EAL domains encoded by genomes from phyla other than proteobacteria appear to represent functional phosphodiesterases. Within enterobacteria, EAL-only proteins were found to cluster either with YhjH or with one of the subfamilies of YdiV-related proteins. EAL-only proteins from Shigella flexneri, Klebsiella pneumoniae, and Yersinia enterocolitica were tested for their ability to regulate swimming and swarming motility and formation of the red, dry, and rough (rdar) biofilm morphotype. In these tests, YhjH-related proteins S4210, KPN_01159, KPN_03274, and YE4063 displayed properties typical of enzymatically active phosphodiesterases, whereas S1641 and YE1324 behaved like members of the YdiV/STM1697 subfamily, with Yersinia enterocolitica protein YE1324 shown to downregulate motility in its native host. Of two closely related EAL-only proteins, YE2225 is an active phosphodiesterase, while YE1324 appears to interact with FlhD. These results suggest that in FlhDC-harboring beta- and gammaproteobacteria, some EAL-only proteins evolved to become catalytically inactive and regulate motility and biofilm formation by interacting with FlhDC.IMPORTANCE The EAL domain superfamily consists mainly of proteins with cyclic dimeric GMP-specific phosphodiesterase activity, but individual domains have been classified in three classes according to their functions and conserved amino acid signatures. Proteins that consist solely of stand-alone EAL domains cannot rely on other domains to form catalytically active dimers, and most of them fall into one of two distinct classes: catalytically active phosphodiesterases with well-conserved residues of the active site and the dimerization loop, and catalytically inactive YdiV/CdgR-like proteins that regulate bacterial motility by binding to the flagellar master regulator, FlhDC, and are found primarily in enterobacteria. The presence of apparently inactive EAL-only proteins in the bacteria that do not express FlhD suggests the existence of additional EAL interaction partners.

Dataset Information

In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes.

Publications

In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes.

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