Antagonistic interactions and production of halocin antimicrobial peptides among extremely halophilic prokaryotes isolated from the solar saltern of Sfax, Tunisia.

Thirty-five extremely halophilic microbial strains isolated from crystallizer (TS18) and non-crystallizer (M1) ponds in the Sfax solar saltern in Tunisia were examined for their ability to exert antimicrobial activity. Antagonistic assays resulted in the selection of eleven strains that displayed such antimicrobial activity and they were further characterized. Three cases of cross-domain inhibition (archaea/bacteria or bacteria/archaea) were observed. Four archaeal strains exerted antimicrobial activity against several other strains. Three strains, for which several lines of evidence suggested the antimicrobial activity was, at least in part, due to peptide/protein agents (Halobacterium salinarum ETD5, Hbt. salinarum ETD8, and Haloterrigena thermotolerans SS1R12), were studied further. Optimal culture conditions for growth and antimicrobial production were determined. Using DNA amplification with specific primers, sequencing and RT-PCR analysis, Hbt. salinarum ETD5 and Hbt. salinarum ETD8 were shown to encode and express halocin S8, a hydrophobic antimicrobial peptide targeting halophilic archaea. Although the gene encoding halocin H4 was amplified from the genome of Htg. thermotolerans SS1R12, no transcript could be detected and the antimicrobial activity was most likely due to multiple antimicrobial compounds. This is also the first report that points to four different strains isolated from different geographical locations with the capacity to produce identical halocin S8 proteins.

Characterization of halo-alkaline and thermostable protease from Halorubrum ezzemoulense strain ETR14 isolated from Sfax solar saltern in Tunisia.

A total of 54 halophilic strains were isolated from crystallizer TS18 (26 strains) and non-crystallizer M1 (28 strains) ponds and screened for their ability to produce protease, amylase, and lipase activities. Enzymatic assays allowed the selection of thirty-two active strains, among them, the ETR14 strain from TS18 showed maximum protease production yields and therefore, selected for further analysis. The results from 16S rRNA gene sequence analysis revealed that the strain belonged to Halorubrum ezzemoulense (Hrr. ezzemoulense) species. Further results indicated that optimum growth and protease production yields were obtained with 10-15% NaCl concentrations in the DSC-97 medium. The enzyme was also able to maintain high levels of protease activity at salt concentrations of up to 25%. While readily available carbon sources were noted to significantly reduce protease production, the combination between yeast extract and peptone enhanced protease excretion, which reached a maximum of 284 U ml(-1) at the end of the exponential growth phase. The enzyme exhibited optimum activity at pH 9 and 60 °C. The halophilic protease retained 87% of its initial activity after 1 h incubation at 70 °C and showed high stability over a wide range of pH, ranging from 7 to 10. This protease exhibited good temperature, pH, and salinity tolerance, which distinguishes it from other proteases previously described from other members of the holoarchaea genera and makes it a promising candidate for application in various industries.

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.