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.

Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development

“…Increased c‐di‐GMP in ∆ rmdA and ∆ rmdB is not surprising, but is unexpected for ∆ cdgB and ∆ cdgC and shows that levels of c‐di‐GMP do not correlate with the opposing sporulation phenotypes of the PDE and DGC mutants. In E. coli , global c‐di‐GMP levels do not correlate with biofilm formation phenotype since local c‐di‐GMP‐signaling mechanisms control the synthesis of curli fibers and of pEtN‐cellulose (Sarenko et al ., 2017) (Richter et al ., 2020). Thus, it is likely that locally acting c‐di‐GMP is also involved in regulation of Streptomyces sporulation.…”

Common plant flavonoids prevent the assembly of amyloid curli fibres and can interfere with bacterial biofilm formation

“…2), we tested the expression of the relevant genes using single‐copy lacZ reporter fusions to csgB (the first gene in the curli biosynthesis csgBAC operon) and dgcC . DgcC is a diguanylate cyclase required to specifically activate cellulose synthase BcsA, which is allosterically controlled by c‐di‐GMP binding to its PilZ domain (García et al ., 2004; Morgan et al ., 2014; Richter et al ., 2020). Transcription of both the csgBAC operon and dgcC requires the biofilm regulator CsgD (Römling et al ., 2000; Brombacher et al ., 2003), whose expression in turn depends on the stationary phase sigma factor RpoS and additional complex c‐di‐GMP input (Lindenberg et al ., 2013; Sarenko et al ., 2017; Pfiffer et al ., 2019).…”

“…allosterically controlled by c-di-GMP binding to its PilZ domain (García et al, 2004;Morgan et al, 2014;Richter et al, 2020). Transcription of both the csgBAC operon and dgcC requires the biofilm regulator CsgD (Römling et al, 2000;Brombacher et al, 2003), whose expression in turn depends on the stationary phase sigma factor RpoS and additional complex c-di-GMP input (Lindenberg et al, 2013;Sarenko et al, 2017;Pfiffer et al, 2019).…”

Architecture and regulation of an enterobacterial cellulose secretion system

“…The altered BcsA:BcsB ratio is also particularly interesting, as it demonstrates a biosynthetically expensive stoichiometry for a complex that features a single catalytic subunit. Possible roles for the BcsB polymeric crown might include local perturbations of the IM for facilitated BcsA membrane sorting or function, a ratchetlike organization of the BcsB crown lumen for more efficient cellulose extrusion, a supramolecular assembly for recruitment of regulatory enzymes [e.g., the hydrolase BcsZ, the pEtN transferase BcsG, or BcsB-interacting c-di-GMP metabolizing enzymes PdeK and DgcC (1,9,29)], periplasmic peptidoglycan rearrangements for facilitated guidance toward the outer membrane export channel, or introduction of overall secretion system asymmetry related to the role of the essential SIMIBI subunit BcsQ (see below). Furthermore, the link between BcsB oligomerization and membrane deformation could also explain preferential secretion system assembly at the cell pole (11), where the native membrane curvature is inherently highest.…”