| The peptide antibiotic, halocin R1 (HalR1) is produced by Halobacterium salinarum GN101 and appears in culture supernatants as the culture makes the transition into stationary phase. The onset of HalR1 activity is cell-density dependent and cannot be induced by amino-acid limitation, nitrate starvation, phosphate starvation or oxygen limitation.;HalR1 is was purified from culture supernatants by using tangential flow filtration (TFF) with filters of successively smaller nominal molecular weight cut-offs. Ninety percent of HalR1 activity was retained by a 30 kDa filter. This 30 kDa retentate was subjected to heat, acetone precipitation, gel filtration chromatography and RP-HPLC against base and acid.;Amino acid sequencing by Edman degradation revealed that halocin R1 is a 38 amino acid peptide with a calculated molecular weight of 4.4 kDa, making it the second microhalocin to be purified. Gel filtration chromatography suggests that it may be associated with a ∼29 kDa "carrier protein" that can be eliminated under denaturing conditions such as heat and exposure to organic solvents. The microhalocin is resistant to acid, base, 100% (v/v) acetonitrile and 50% (v/v) acetone. HalR1 activity is unaffected by incubation at 60°C for 2 hours, but half of its activity is lost after incubation at 98°C for 10 minutes. It is hydrophobic, eluting from reversed phase columns at 62% (v/v) ACN. HalR1 is 63% identical and 71% similar to HalS8 and contains no charged residues. It appears to have no sequence similarity to bacteriocins or to eucaryocins and BLAST studies reveal homology to only one other protein, HalS8. Like HalS8, it is missing the N-terminal methionine, indicating that it is also the product of post-translational processing. The sequence similarities to HalS8 suggest that HalR1 may be cleaved from the middle of a larger pro-protein.;Attempts to clone the halR1 gene using oligodeoxynucleotide probes designed from the HalR1 amino acid sequence or random primer probes generated from a PCR amplification product of the halS8 gene revealed the presence of a cryptic halS8 gene. This gene was presumably inactive due to its association with an upstream IS240-type insertion element. |
