The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins

The extremely halophilic archaeon Halobacterium salinarum strain ETD5 was previously isolated from the solar saltern of Sfax (Tunisia) and shown to encode and express halocin S8. The Hbt. salinarum ETD5 culture supernatant was shown here to exhibit high antimicrobial activity against several halophilic archaea and bacteria of different genera, showing a cross-domain inhibition. The antimicrobial activity was destroyed by proteases, thus pointing to halocins. A bioguided purification procedure was applied using two chromatography steps and antimicrobial assays directed against Halorubrum chaoviator ETR14. In-gel screening assay showed the presence of two antimicrobial bands of approximately 8 and 14 kDa, for which characterization was investigated by N-terminal sequencing and mass spectrometry. The full-length form of halocin S8 that contains 81 amino acids and differs from the 36 amino acid short-length halocin S8 previously described from an uncharacterized haloarchaeon S8a, was identified in the 8 kDa halocin band. A novel halocin that we termed halocin S14 was found in the 14 kDa band. It exhibits amino acid sequence identities with the N-terminally truncated region of the archaeal Mn-superoxide dismutase. These results show that Hbt. salinarum ETD5 produces multiple halocins, a feature that had not been described until now in the domain Archaea.