Haloferax mediterranei Halolysin R4 Confers Antagonistic and Defensive Capabilities

Halolysins, which are subtilisin-like serine proteases of haloarchaea, are usually secreted into the extracellular matrix via the twin-arginine translocation pathway. A small number of activated molecules can greatly affect cell growth owing to their proteolytic activity. It is, however, unclear as to whether this proteolysis-based growth inhibition by halolysins conveys antagonistic or defensive effects against other resident and potentially competitive microorganisms. Here, we report that halolysin R4 (HlyR4), encoded by the hlyR4 gene, is the key enzyme in the initial steps of extracellular protein utilization in Haloferax mediterranei (Hfx. mediterrand). HlyR4 shows significant antagonistic activity against other haloarchaeal strains. Deletion of hlyR4 completely halts the inhibition activity of Hfx. mediterranei toward other haloarchaea, while correspondingly, complementation of hlyR4 almost completely restores the inhibition activity. Furthermore, Hfx. mediterranei strains containing hlyR4 showed a certain amount of resistance to halocins and halolysins in a hypersaline environment, and this function of hlyR4 is reproducible in Haloarcula hispanica. The versatility of HlyR4 enables its encoding organism to outcompete other haloarchaea living in the same hypersaline environment. Intriguingly, unlike the growth phase-dependent halolysins SptA and Nep, it is likely that HlyR4 may be secreted independently of growth phase. This study provides a new peptide antibiotic candidate in haloarchaea, as well as new insight into a better understanding of the ecological roles of halolysins. IMPORTANCE This study shows that halolysin R4 from Haloferax mediterranei provides its host antagonistic and defensive activities against other haloarchaea, which expands our knowledge of the traditional function of haloarchaeal extracellular proteases. Haloarchaeal extracellular serine proteases have been previously discussed as growth phase-dependent proteins, whereas our study reports constitutive expression of halolysin R4. This work also clearly reveals a hidden diversity of extracellular proteases from haloarchaea. Studies on multifunctional halolysins reveal that they play an important ecological role in shaping microbial community composition and provide a new perspective toward understanding the intricate interactions between haloarchaeal cells in hypersaline environments. HlyR4 can lyse competing cells hying in the same environment, and the cell debris may probably be utilized as nutrients, which may constitute an important part of nutrient cycling in extremely hypersaline environments.

Automated summary

Halolysin R4 from Haloferax mediterranei provides its host antagonistic and defensive activities against other haloarchaea, expanding our knowledge of the traditional function of haloarchaeal extracellular proteases. This protein may be secreted independently of growth phase, lysing competing cells in the same environment and utilizing cell debris as nutrients, potentially playing a crucial role in nutrient cycling in extremely hypersaline environments.