Evidence for two putative holin-like peptides encoding genes of Bacillus pumilus strain WAPB4.

An open reading frame encoding a 71-amino acid BhlA bacteriocin-related holin-like peptide was present upstream of 86-amino acid holin-like peptide, xhlB, encoding gene in the genome of Bacillus pumilus strain WAPB4. Analysis of BhlA using TMHMM server suggested one putative transmembrane domain at the N-terminal part and a number of highly charged amino acid residues at the C-terminal part. XhlB of B. pumilus strain WAPB4 composed of two putative transmembrane domains separated by a β-turn, and numerous charged residues in the C-terminus. The dual start motifs were found in both BhlA and XhlB. Structural analysis of their sequence revealed features characteristic for holin. To analyze the effect of BhlA on bacteria cell, its ORF was cloned and expressed in Escherichia coli BL21(DE3). Expression of holin-like peptide, BhlA, was found to be toxic to the host cell. The site of action of BhlA is on the cell membrane and caused bacterial death by cell membrane disruption as clearly demonstrated by transmission electron microscopy or TEM.