γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes

Abstract

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

The structure of PghL hydrolase bound to its substrate poly‐γ‐glutamate

“…The residues which coordinate the zinc ion are strongly conserved between the structures of PghP and other non‐γ‐PGA‐specific carboxypeptidases such as carboxypeptidase A, if not in their sequential position, then in their three‐dimensional location. PghL residues His41 and His102, and the two carboxyl oxygens of Glu46 in a bidentate mode overlap well, for instance, with His40, His103 and Glu45 of PghP and are conserved in other homologues . The zinc atom sits precisely in the same position as observed in PghP with which it also shares a similar environment (Fig.…”

“…Recently, four unannotated B . subtilis gene products, YjqB, YmaC, YndL, and YoqZ, were found to share with PghP high sequence similarity (27–37% identity and 41–54% homology) . The authors also demonstrated that recombinant YndL and YoqZ were efficient at γ‐DL‐PGA degradation thus highlighting their functional homology to PghP.…”

“…With the aim of understanding the activity and stereoselectivity of γ‐PGA hydrolases ultimately aimed at exploring their potential use as therapeutics for the treatment of recalcitrant infections, we have solved the structure of the recombinant PghL hydrolase from B. subtilis . We had previously demonstrated that the recombinant protein is fully enzymatically active and able to efficiently cut γ‐PGA . We now characterised further the cleavage properties of this enzyme and crystallised PghL both in isolation and in the presence of γ‐PGA and solved the structures of a zinc‐free, zinc‐loaded and γ‐PGA‐enzyme complex.…”

“…Genes for γ‐PGA biosynthesis are abundantly present in soil microorganisms, particularly, but not exclusively, of the genera Streptomyces , Staphylococcus and Bacillus . However, a much larger number of organisms, including archaea and a few eukaryotes, produce and exploit the polymer .…”

“…These proteins were thus renamed by homology PghB, PghC, PghL and PghZ respectively. Their genes are likely derived from integrated prophages, as judged by their localisation in prophagic regions of the B. subtilis genome .…”