Second messenger signalling governs Escherichia coli biofilm induction upon ribosomal stress

Abstract

SummaryBiofilms are communities of surface-attached, matrixembedded microbial cells that can resist antimicrobial chemotherapy and contribute to persistent infections. Using an Escherichia coli biofilm model we found that exposure of bacteria to subinhibitory concentrations of ribosome-targeting antibiotics leads to strong biofilm induction. We present evidence that this effect is elicited by the ribosome in response to translational stress. Biofilm induction involves upregulation of the polysaccharide adhesin poly-b-1,6-N-acetyl-glucosamine (poly-GlcNAc) and two components of the poly-GlcNAc biosynthesis machinery, PgaA and PgaD. Poly-GlcNAc control depends on the bacterial signalling molecules guanosine-bis 3Ј, 5Ј(diphosphate) (ppGpp) and bis-(3Ј-5Ј)-cyclic di-GMP (c-di-GMP). Treatment with translation inhibitors causes a ppGpp hydrolase (SpoT)-mediated reduction of ppGpp levels, resulting in specific derepression of PgaA. Maximal induction of PgaD and poly-GlcNAc synthesis requires the production of c-di-GMP by the dedicated diguanylate cyclase YdeH. Our results identify a novel regulatory mechanism that relies on ppGpp signalling to relay information about ribosomal performance to the Pga machinery, thereby inducing adhesin production and biofilm formation. Based on the important synergistic roles of ppGpp and c-di-GMP in this process, we suggest that interference with bacterial second messenger signalling might represent an effective means for biofilm control during chronic infections.

The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine

“…PgaD is a small integral inner membrane protein required for PNAG biosynthesis (17). The exact function of PgaD is unknown, but it has been predicted to assist PgaC in polymerization and transport of PNAG across the inner membrane (17) and in the regulation of PNAG production, because levels of PgaD are controlled post-transcriptionally by cellular concentrations of the bacterial second messenger bis-(3Ј-5Ј)-cyclic dimeric GMP (18). PgaB is a twodomain outer membrane protein that contains a putative lipidation site and is responsible for the partial de-N-acetylation of PNAG (17).…”

The diguanylate cyclase YddV controls production of the exopolysaccharide poly-N-acetylglucosamine (PNAG) through regulation of the PNAG biosynthetic pgaABCD operon

“…In contrast, our results suggest that the YddV protein promotes PNAG production by activating the expression of the PNAG biosynthetic operon pgaABCD (Figs 4 and 5), possibly via interaction with a c-di-GMPresponsive regulatory protein. In addition to YddV, PNAG production is controlled by another DGC, YdeH, which positively affects PgaD protein stability via a yet unknown mechanism (Boehm et al, 2009). Similarly, cellulose biosynthesis is regulated by DGC proteins at both the gene expression and the protein activity levels: the YdaM protein positively regulates csgDEFG transcription (Weber Thus, it appears that DGCdependent control at multiple levels is a common mechanism for EPS biosynthesis regulation in E. coli.…”

“…Regulation of EPS production by DGCs can take place at different levels: cellulose production is stimulated by AdrA through allosteric activation of the cellulose synthase protein machinery (Zogaj et al, 2001;Simm et al, 2004); the YdeH protein affects PNAG production through stabilization of the PgaD protein (Boehm et al, 2009); and finally, the YdaM protein activates curli and cellulose production via upregulation of csgDEFG transcription (Weber et al, 2006). We tested the possibility that the YddV protein regulates PNAG production by affecting transcription of the pgaABCD operon, encoding the proteins involved in PNAG biosynthesis.…”

N-Acetylglucosamine-dependent biofilm formation in Pectobacterium atrosepticum is cryptic and activated by elevated c-di-GMP levels

“…In E. coli, the second messengers c-di-GMP and ppGpp regulate biofilm induction upon ribosomal stress. Biofilm induction involves the upregulation of at least two components of the E. coli PGA biosynthesis machinery (PgaA and PgaD; Boehm et al, 2009). The molecular mechanism through which these two secondary messengers regulate PGA production remains to be elucidated.…”

“…CsrA has also been associated with c-di-GMP metabolism in E. coli, in which this protein regulates the expression of several GGDEF proteins at the post-transcriptional level (Jonas et al, 2008). The regulation of PGA by c-di-GMP is not well defined, and recently c-di-GMP has been proposed to control expression of the E. coli pgaABCD operon at posttranscriptional and post-translational levels (Boehm et al, 2009;Tagliabue et al, 2010). A role for c-di-GMP in the regulation of PGA synthesis by the hmsHFRS operon of the animal pathogen Yersinia pestis has also been described (Bobrov et al, 2008(Bobrov et al, , 2011Kirillina et al, 2004).…”