Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems (https://doi.org/10.1101/769745). Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3,5-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of Bacilli, Clostridia, Streptococci, and Actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.nnAuthor summaryThe Pel polysaccharide is required for biofilm formation in P. aeruginosa and we have previously found that the genes necessary for biosynthesis of this polymer are broadly distributed across Gram-negative bacteria. Herein, we show that many species of Gram-positive bacteria also possess Pel biosynthetic genes and demonstrate that these genes are used Bacillus cereus for biofilm formation. We show that Pel production in B. cereus is regulated by c-di-GMP and have identified two enzymes, a diguanylate cyclase, CdgF, and a phosphodiesterase, CdgE, that control the levels of this bacterial signalling molecule. While Pel production in B. cereus also requires the binding of c-di-GMP to the receptor PelD, the divergence of this protein in Streptococci suggests a c-di-GMP independent mechanism of regulation is used in this species. The discovery of a Pel biosynthetic gene cluster in Gram-positive bacteria and our characterization of the pel operon in B. cereus suggests that Pel is a widespread biofilm component across all bacteria.