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

Abstract

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. Here, we explore the finding that many species of Grampositive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEA DA FG 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 pelDEA DA FG 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.

Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

“…Pel, like GAG, is a partially de-N-acetylated GalNAc-rich polymer involved in biofilm formation in some P. aeruginosa strains [55][56][57][58] . Pel operons have been found in Proteobacteria, including Burkholderia and Moritella species 59 and more recently in a wide range of Gram-positive bacteria 60,61 , suggesting that many bacteria produce a Pel/GAG-like polymer.…”

The Matrix Revisited: Opening Night for the Pel Polysaccharide Across Eubacterial Kingdoms

“…It is unclear what the function of this domain is in the context of Pel biosynthesis, although we hypothesize that it may mediate interactions with membrane-embedded Pel components to keep it associated with active polymer production, similar to the interaction between PelA and PelB in Gram-negatives. 16 Curiously, we noted that while all Gram-positive PelA orthologs lack a glycoside hydrolase domain, many of the associated biosynthetic clusters, including B. cereus, 11 contain a separate gene with predicted similarity to the glycoside hydrolase domain found in P. aeruginosa PelA. 15 Our analyses have confirmed that this protein is an active α-1,4-N-acetyl-galactosaminidase and revealed that deletion of this gene significantly elevated biofilm formation.…”

“…11,18 While this domain is enzymatically inactive, an allosteric product inhibition site retains the ability to bind c-di-GMP, which is required for Pel production in P. aeruginosa and B. cereus. 11,18 We found that in Streptococci the GGDEF domain of PelD is universally absent which, combined with the lack of genomically-encoded c-di-GMP metabolic enzymes, suggests that Pel production is unlikely to be regulated post-translationally by c-di-GMP in this genus (Figure 3). 11 Instead, Streptococci have several additional genes of unknown function directly downstream of pelG that appear to be part of the same operon, which may provide a means to regulate Pel biosynthesis using an as yet undiscovered mechanism.…”

Strain variation inBacillus cereusbiofilms and their susceptibility to extracellular matrix-degrading enzymes

“…On the other hand, a recent study by Whitfield et al [32] suggests that the polysaccharide Pel is essential for the biofilm formation of B. cereus ATCC 10987.…”

“…The cellulase-sensitive properties of strains ATCC 10987 and BC4 suggest that cellulose may play an essential role in their biofilm formation (Fig 2). On the other hand, a recent study by Whitfield et al [32] suggests that the polysaccharide Pel is essential for the biofilm formation of B. cereus ATCC 10987. Considering that Pel is also cellulase-sensitive [33], the degradation of Pel by cellulase may have occurred in strain ATCC 10987, inhibiting its biofilm formation.…”