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GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.

ABSTRACT: In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli?ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S.?enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain containing glycosyltransferase BcsA.

SUBMITTER: Fang X

PROVIDER: S-EPMC4116459 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.

Fang Xin X Ahmad Irfan I Blanka Andrea A Schottkowski Marco M Cimdins Annika A Galperin Michael Y MY Römling Ute U Gomelsky Mark M

Molecular microbiology 20140701 3

In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. Th ...[more]

PMID: 24942809

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Project description:Bis-(3'-5')-cyclic-dimeric GMP (c-di-GMP) is an important bacterial regulatory signaling molecule affecting biofilm formation, toxin production, motility, and virulence. The genome of Bacillus anthracis, the causative agent of anthrax, is predicted to encode ten putative GGDEF/EAL/HD-GYP-domain containing proteins. Heterologous expression in Bacillus subtilis hosts indicated that there are five active GGDEF domain-containing proteins and four active EAL or HD-GYP domain-containing proteins. Using an mCherry gene fusion-Western blotting approach, the expression of the c-di-GMP-associated proteins was observed throughout the in vitro life cycle. Of the six c-di-GMP-associated proteins found to be present in sporulating cells, four (CdgA, CdgB, CdgD, and CdgG) contain active GGDEF domains. The six proteins expressed in sporulating cells are retained in spores in a CotE-independent manner and thus are not likely to be localized to the exosporium layer of the spores. Individual deletion mutations involving the nine GGDEF/EAL protein-encoding genes and one HD-GYP protein-encoding gene did not affect sporulation efficiency, the attachment of the exosporium glycoprotein BclA, or biofilm production. Notably, expression of anthrax toxin was not affected by deletion of any of the cdg determinants. Three determinants encoding proteins with active GGDEF domains were found to affect germination kinetics. This study reveals a spore association of cyclic-di-GMP regulatory proteins and a likely role for these proteins in the biology of the B. anthracis spore. IMPORTANCE The genus Bacillus is composed of Gram-positive, rod shaped, soil-dwelling bacteria. As a mechanism for survival in the harsh conditions in soil, the organisms undergo sporulation, and the resulting spores permit the organisms to survive harsh environmental conditions. Although most species are saprophytes, Bacillus cereus and Bacillus anthracis are human pathogens and Bacillus thuringiensis is an insect pathogen. The bacterial c-di-GMP regulatory system is an important control system affecting motility, biofilm formation, and toxin production. The role of c-di-GMP has been studied in the spore-forming bacilli Bacillus subtilis, Bacillus amyloliquefaciens, B. cereus, and B. thuringiensis. However, this regulatory system has not heretofore been examined in the high-consequence zoonotic pathogen of this genus, B. anthracis.

Project description:Most bacteria respond to surfaces by biogenesis of intracellular c-di-GMP, which inhibits motility and induces secretion of biofilm-promoting adherence factors. Bacterial cellulose is a widespread biofilm component whose secretion in Gram-negative species requires an inner membrane, c-di-GMP-dependent synthase tandem (BcsAB), an outer membrane porin (BcsC), and various accessory subunits that regulate synthase assembly and function as well as the exopolysaccharide's chemical composition and mechanical properties. We recently showed that in Escherichia coli, most Bcs proteins form a megadalton-sized secretory nanomachine, but the role and structure of individual regulatory components remained enigmatic. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other's folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner BcsF. Crystallographic and solution-based data show that BcsE's predicted GIL domain is a degenerate receiver-GGDEF domain tandem (BcsEREC*-GGDEF*), where the divergent diguanylate cyclase module binds both dimeric c-di-GMP and BcsQ through mutually independent interfaces. In addition, we reveal that a third N-terminal domain (BcsENTD) determines the protein's homooligomerization and targeting of BcsERQ to the membrane as well as previously unreported interactions with transcription antitermination complex components. Together, the data suggest that BcsE acts on multiple levels to fine-tune bacterial cellulose secretion, from the early stages of secretion system assembly to the maintenance of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation.IMPORTANCE Bacterial cellulose is a widespread biofilm component that can modulate microbial fitness and virulence both in the environment and infected hosts. Whereas its secretion generally involves an inner membrane c-di-GMP-dependent synthase tandem (BcsAB) across the bacterial domain of life, enterobacteria feature sophisticated Escherichia coli-like Bcs secretion systems, where multiple additional subunits are either required for secretion or contribute to the maximal production of the polysaccharide in vivo. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other's folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner, BcsF. Crystallographic and functional data reveal that BcsE features unexpected domain architecture and likely acts on multiple levels to fine-tune bacterial cellulose production, from the early stages of secretion system assembly to the maintenence of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation.

Dataset Information

GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.

Publications

GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.

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