Summary: 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.

Escherichia coli can commonly be found, either as a commensal, probiotic or a pathogen, in the human gastrointestinal (GI) tract. Biofilm formation and its regulation is surprisingly variable, although distinct regulatory pattern of red, dry and rough (rdar) biofilm formation arise in certain pathovars and even clones. In the GI tract, environmental conditions, signals from the host and from commensal bacteria contribute to shape E. coli biofilm formation within the multi-faceted multicellular communities in a complex and integrated fashion. Although some major regulatory networks, adhesion factors and extracellular matrix components constituting E. coli biofilms have been recognized, these processes have mainly been characterized in vitro and in the context of interaction of E. coli strains with intestinal epithelial cells. However, direct observation of E. coli cells in situ, and the vast number of genes encoding surface appendages on the core or accessory genome of E. coli suggests the complexity of the biofilm process to be far from being fully understood. In this review, we summarize biofilm formation mechanisms of commensal, probiotic and pathogenic E. coli in the context of the gastrointestinal tract. Graphical Abstract

Cellulose, a 1,4 beta-glucan polysaccharide, is produced by a variety of organisms including bacteria. Although the production of cellulose has a high biological, ecological and economical impact, regulatory mechanisms of cellulose biosynthesis are mostly unknown. Family eight cellulases are regularly associated with cellulose biosynthesis operons in bacteria; however, their function is poorly characterized. In this study, we analysed the role of the cellulase BcsZ encoded by the bcsABZC cellulose biosynthesis operon of Salmonella enterica serovar Typhimurium (S. Typhimurium) in biofilm related behavior. We also investigated the involvement of BcsZ in pathogenesis of S. Typhimurium including a murine typhoid fever infection model. In S. Typhimurium, cellulase BcsZ with a putative periplasmic location negatively regulates cellulose biosynthesis. Moreover, as assessed with a non-polar mutant, BcsZ affects cellulose-associated phenotypes such as the rdar biofilm morphotype, cell clumping, biofilm formation, pellicle formation and flagella-dependent motility. Strikingly, although upregulation of cellulose biosynthesis was not observed on agar plate medium at 37 °C, BcsZ is required for efficient pathogen-host interaction. Key virulence phenotypes of S. Typhimurium such as invasion of epithelial cells and proliferation in macrophages were positively regulated by BcsZ. Further on, a bcsZ mutant was outcompeted by the wild type in organ colonization in the murine typhoid fever infection model. Selected phenotypes were relieved upon deletion of the cellulose synthase BcsA and/or the central biofilm activator CsgD. Although the protein scaffold has an additional physiological role, our findings indicate that the catalytic activity of BcsZ effectively downregulates CsgD activated cellulose biosynthesis. Repression of cellulose production by BcsZ subsequently enables Salmonella to efficiently colonize the host.

The secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium. The GGDEF/EAL domain proteins regulate the c-di-GMP turnover. There are twenty- two GGDEF/EAL domain proteins in the genome of S. typhimurium. In this study, we dissect the role of individual GGDEF/EAL proteins for csgD expression and rdar biofilm development. Among twelve GGDEF domains, two proteins upregulate and among fifteen EAL domains, four proteins down regulate csgD expression. We identified two additional GGDEF proteins required to promote optimal csgD expression. With the exception of the EAL domain of STM1703, solely, diguanylate cyclase and phosphodiesterase activities are required to regulate csgD mediated rdar biofilm formation. Identification of corresponding phosphodiesterases and diguanylate cyclases interacting in the csgD regulatory network indicates various levels of regulation by c-di-GMP. The phosphodiesterase STM1703 represses transcription of csgD via a distinct promoter upstream region. The enzymatic activity and the protein scaffold of GGDEF/EAL domain proteins regulate csgD expression. Thereby, c-di-GMP adjusts csgD expression at multiple levels presumably using a multitude of input signals.