Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon
Haloferax mediterranei
ATCC 33500. Despite attempts to express
halH4
heterologously in
Escherichia coli
and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by
Hfx. mediterranei
ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4’s antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species’ cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea.
IMPORTANCE
The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species (
Haloarchaeobius
spp.,
Haloarcula
spp.,
Haloferax
spp.,
Halorubellus
spp., and
Halorubrum
spp.), as well as selected bacterial species (
Aliifodinibius
spp. and
Salicola
spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4,
halH4
, from the model halophilic archaeon
Haloferax mediterranei
ATCC 33500 can be expressed in
Escherichia coli
without codon optimization.
