Purification, Gene Cloning And Biological Characterization Of Halocin C8, A Novel Peptide Antibiotic From Halobacterium Strain AS7092

Halocins are bacteriocin-like proteins or peptides produced by many species of the family Halobacteriaceae and are externalized into the environment, where they kill or inhibit other haloarchaeons. Halocin is a useful model for studying the gene expression regulation and protein structure/ function in the haloarchaea.In order to explore halocin diversity at both the protein and gene expression levels, 22 haloarchaeal strains were used to study antagonistic interactions among them. The results of antagonistic interactions showed that all the tested strains produced halocins except Halobacterium sp. QD3. Among them, six halocins (Hal C6, Hal C8, Hal C9, Hal C16, Hal Cl7 and Hal C22) displayed wide inhibitory spectra and could inhibit the growth of most (>10) haloarchaeal strains tested. Stability analysis of the six halocins indicated that halocin C8 was the most stable one, so halocin C8 was chosen for competitive study in this work.Halocin C8 is a single 6.3-kDa polypeptide with an isoelectric point of 4.4, which is sensitive to proteinase K but not to trypsin. Halocin C8 is quite stable, as it can be desalted, boiled, frozen, subjected to organic solvents, and stored in culturesupernatant at 4C or in dH2O at -20C for more than one year without losing activity. The purification of this halocin was achieved by combination of tangential flow filtration (TFF), Sephadex G50 and DEAE-Sepharose chromatography. The N-terminal amino acid sequence was also determined by Edman degradation. Halocin C8 appeared to have a very wide activity spectrum, including mosthaloarchaea and even some haloalkaliphilic rods, but it showed no inhibitory activity against bacterial strains tested. When a sensitive strain of Halorubrum saccharovorum ATCC29252 was exposed to halocin C8, the treated cells swelled at the initial stage, the cell wall appeared to be nicked and the cytoplasm was extruded out afterwards, and the whole cell was eventually completely lysed. These results revealed that halocin C8 is cytocidal and its primary target might be located in the cell wall of the sensitive cells.Two degenerate oligoxynucleotide primers (C8-2, C8-4) were designed according to the N-terminal amino acid sequence of halocin C8. Using these primers, partial sequence of the halC8 gene was amplified by PCR. This PCR product was cloned into a T-vector and resulted in a plasmid pLY11, which was used as the template to generate a RNA probe of halocin C8 for screening the halC8 gene in the AS7092 genome by Southern blot analysis. According to the Southern blot results, a 6.5 kb BamH I restriction fragment containing the gene coding for Hal C8 was cloned and sequenced. The entire halCS gene is located on a megaplasmid and contains a 849-bp open reading frame that encodes a polypeptide of 283 amino acids. The promoter is typically haloarchaeal, but the start site of transcription is only seven bases from the 5'end of the initiator AUG codon, making the halCS transcript another example of a "leadless transcript" in the haloarchaea.