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Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose.

ABSTRACT: In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of "local" c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo. DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist.

SUBMITTER: Richter AM

PROVIDER: S-EPMC7397504 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A major target of c-di-GMP signaling is the production of biofilm-associated extracellular polymeric substances (EPS), which in Escherichia coli K-12 include amyloid curli fibers, phosphoethanolamine-modified cellulose, and poly-N-acetylglucosamine. However, the characterized c-di-GMP-binding effector systems are largely outnumbered by the 12 diguanylate cyclases (DGCs) and 13 phosphodiesterases (PDEs), which synthetize and degrade c-di-GMP, respectively. E. coli possesses a single protein with a potentially c-di-GMP-binding MshEN domain, NfrB, which-together with the outer membrane protein NfrA-is known to serve as a receptor system for phage N4. Here, we show that NfrB not only binds c-di-GMP with high affinity but, as a novel c-di-GMP-controlled glycosyltransferase, synthesizes a secreted EPS, which can impede motility and is required as an initial receptor for phage N4 infection. In addition, a systematic screening of the 12 DGCs of E. coli K-12 revealed that specifically DgcJ is required for the infection with phage N4 and interacts directly with NfrB. This is in line with local signaling models, where specific DGCs and/or PDEs form protein complexes with particular c-di-GMP effector/target systems. Our findings thus provide further evidence that intracellular signaling pathways, which all use the same diffusible second messenger, can act in parallel in a highly specific manner. IMPORTANCE Key findings in model organisms led to the concept of "local" signaling, challenging the dogma of a gradually increasing global intracellular c-di-GMP concentration driving the motile-sessile transition in bacteria. In our current model, bacteria dynamically combine both global and local signaling modes, in which specific DGCs and/or PDEs team up with effector/target systems in multiprotein complexes. The present study highlights a novel example of how specificity in c-di-GMP signaling can be achieved by showing NfrB as a novel c-di-GMP binding effector in E. coli, which is controlled in a local manner specifically by DgcJ. We further show that NfrB (which was initially found as a part of a receptor system for phage N4) is involved in the production of a novel exopolysaccharide. Finally, our data shine new light on host interaction of phage N4, which uses this exopolysaccharide as an initial receptor for adsorption.

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Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose.

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