Isolation, purification, and characterisation of a phage tail-like bacteriocin from the insect pathogenic bacterium Brevibacillus laterosporus

The gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to often restricted growth of these endemic isolates, production can be an issue. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages tail-like structures. PEG 8000 precipitated lysate harbouring the putative phage tail-like particles demonstrated broad-spectrum activity against several gram-positive bacteria. SDS-PAGE of purified and concentrated lysate showed a prominent protein band of ∼48 kD from where transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ∼48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. An xkdK homolog was also identified in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-L-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ∼48 kD sequenced protein of Bl 1821L. Assays of purified ∼48 kD protein of Bl 1821L caused a decrease of 34.2% in the number of viable cells of Bl 1951, 18 hours after treatment as compared to the control.