Characterisation of the Brochothrix thermosphacta sortase A enzyme

Gram-positive bacteria use sortase A enzymes to anchor surface proteins, which influence their interactions with the environment and contribute to physiological and virulence traits. While extensively studied in pathogens like *Staphylococcus* and *Streptococcus*, the role of sortase A in spoilage bacteria remains less understood. The spoilage bacterium *Brochothrix thermosphacta* has not been well characterized in this context. Prior research has shown that sortase A recognizes the LPXTG motif and catalyzes transpeptidation reactions to anchor proteins to the cell wall. However, the function of sortase A in *B. thermosphacta* has remained unclear. This gap motivated the current investigation into the enzyme’s activity and potential substrates. The study aimed to explore how this poorly characterized spoilage bacterium might use sortase A to mediate environmental interactions. No prior work had resolved the specific activity or substrate specificity of *B. thermosphacta* sortase A. This uncertainty drove the investigation into the enzyme’s function and its role in surface protein anchoring. The study sought to determine whether this enzyme could facilitate adhesion to meat surfaces, a key factor in spoilage. The findings could contribute to understanding spoilage mechanisms in food systems.

The study aimed to characterize the sortase A enzyme from *Brochothrix thermosphacta* to better understand its role in surface protein anchoring and environmental interactions. Researchers sought to determine whether this enzyme could recognize and process typical LPXTG-motif-containing substrates. The goal was to investigate the enzyme’s catalytic activity and its potential role in bacterial adhesion. The study also aimed to identify possible substrates encoded in the *B. thermosphacta* genome. By expressing and purifying a His-tagged variant of the enzyme, the researchers could test its functional properties. The purpose was to assess the enzyme’s ability to form thioacyl intermediates and mediate protein conjugation. The study aimed to determine if the enzyme could facilitate adhesion to meat surfaces, a spoilage-related trait. This information could help clarify the molecular mechanisms behind *B. thermosphacta* colonization of meat.

Researchers identified a putative sortase A gene in the *B. thermosphacta* genome and cloned it for expression. An N-terminal truncated, His-tagged variant of the enzyme (His6-BtSrtA) was produced and purified. The catalytic activity of the recombinant enzyme was tested using LPXTG-tagged model substrates. The formation of thioacyl intermediates was assessed using tri-glycine as a nucleophile. The genome was analyzed for potential substrates of the sortase A enzyme. Bioinformatics tools were used to identify proteins containing LPXTG motifs and adherence-related domains. The enzyme’s ability to mediate protein conjugation was evaluated through reaction efficiency measurements. The study combined biochemical assays with genomic analysis to determine the enzyme’s functional role.

The recombinant His6-BtSrtA enzyme formed intermediate complexes with LPXTG-tagged proteins, indicating recognition of the sorting motif. Thioacyl intermediates were generated, though the reaction was inefficient. Tri-glycine nucleophilic attack occurred, suggesting the enzyme could mediate protein conjugation. The *B. thermosphacta* genome contained 11 potential sortase A substrates. Two of these substrates contained domains associated with adherence to extracellular matrix proteins. These findings suggest the enzyme may play a role in bacterial attachment to surfaces. The low-efficiency reaction indicates that the enzyme may function under specific conditions. The presence of adherence-related domains in substrates supports a possible role in meat colonization.

The study suggests that the *B. thermosphacta* sortase A enzyme can recognize LPXTG motifs and mediate protein conjugation. The formation of thioacyl intermediates indicates functional activity, though the reaction was inefficient. The presence of adherence-related domains in potential substrates implies a possible role in surface attachment. The findings support the idea that the enzyme may facilitate bacterial adhesion to meat surfaces. The low-efficiency reaction suggests that the enzyme may function under specific environmental conditions. The study provides a foundation for further investigation into the enzyme’s role in spoilage mechanisms. The identification of 11 potential substrates opens avenues for future research into their functional roles. The results highlight the need for additional studies to clarify the enzyme’s contribution to *B. thermosphacta* colonization.