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

Bacteria synthesize and export adhesive macromolecules to enable biofilm formation. These macromolecules, collectively called the biofilm matrix, are structurally varied and often unique to specific bacterial species or subspecies. This heterogeneity in matrix utilization makes it difficult to facilitate direct comparison between biofilm formation mechanisms of different bacterial species. Despite this, some matrix components, in particular the polysaccharides poly-β-1,6--acetyl-glucosamine (PNAG) and bacterial cellulose, are utilized by many Gram-negative species for biofilm formation. However, there is a very narrow distribution of these components across Gram-positive organisms, whose biofilm matrix determinants remain largely undiscovered. We found that a genetic locus required for the production of a biofilm matrix component of , the Pel polysaccharide, is widespread in Gram-negative bacteria and that there is a variant form of this cluster present in many Gram-positive bacterial species. We demonstrated that this locus is required for biofilm formation by ATCC 10987, produces a polysaccharide that is similar to Pel, and is post-translationally regulated by cyclic-3',5'-dimeric-guanosine monophosphate (c-di-GMP) in a manner identical to . However, while the proposed mechanism for Pel production appears remarkably similar between and , we identified several key differences between Gram-negative and Gram-positive Pel biosynthetic components in other monoderms. In particular, 4 different architectural subtypes of the c-di-GMP-binding component PelD were identified, including 1 found only in Streptococci that has entirely lost the c-di-GMP recognition domain. These observations highlight how existing multi-component bacterial machines can be subtly tweaked to adapt to the unique physiology and regulatory mechanisms of Gram-positive organisms. Collectively, our analyses suggest that the Pel biosynthetic locus is one of the most phylogenetically widespread biofilm matrix determinants in bacteria, and that its mechanism of production and regulation is extraordinarily conserved across the majority of organisms that possess it.