Isolation, Purification, and Characterisation of a Phage Tail-Like Bacteriocin from the Insect Pathogenic Bacterium Brevibacillus laterosporus

Abstract

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 the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. 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. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ~48 kD, and 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. Bioinformatic analysis also identified an xkdK homolog 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. Antagonistic activity of the purified ~48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L.

Keywords: PBSX; antibacterial protein; contractile phage tail-sheath; defective phage; insect pathogenic bacterium.

Figure 1

Spectrophotometer reading (OD_{600 nm}) reading of Bl 1821L cultures after treatment with mitomycin C at 1 µg/mL and 3 µg/mL concentrations and control.

Figure 2

SDS-PAGE of sucrose density gradient purified and 10 kD MWCO membrane concentrated lysate showing a protein band of ~48 kD [(A), shown with dark arrow]. Electron micrographs of crude lysate of Bl 1821L showing the structures with hollow sheath, contractile sheath with cores, phage capsid-like, and cog wheel-like [(B,C), shown with white arrow]. Purified lysate showing a hollow tube intermittently joined with knot-like structures [(D), shown with dark arrow), and without knot-like structures [(E) shown, with dark arrow] to form a polysheath. Scale bar = 100 nm.

Figure 3

Number of viable cells (log₁₀ CFU/mL) of Bl 1821L and Bl 1951 with/without treatment of purified Bl 1821L putative phage tail-like protein (~48 kD) after incubation at 30 °C over 24 h.

Figure 4

Dendrogram showing alignment Bl 1821L and Bl 1951 identified (A0A518VEB0) phage-like element PBSX protein XkdK with similar proteins from other Gram-positive bacteria including Aneurinibacillus migulanus (A0A0D1WNL8), Bacillus aerophilus (A0A410KN98), Brevibacillus gelatini (A0A3M8B733), Brevibacillus laterosporus LMG 15441 (A0A075R9L5); uncharacterised protein of Brevibacillus brevis (strain 47/JCM 6285/NBRC 100599), and similar proteins of other Gram-positive bacteria including Bacillus phage PBP180 (R4JQA6), Bacillus sp. ANT_WA51 (A0A5B0B6Z4), Bacillus sp. WR11 (A0A410QZ71), Bacillus subtilis subsp. subtilis str. SMY (A0A6H0H1P2), Bacillus subtilis 168 (P54331), Clostridioides difficile 630 (Q18BN0), and Geomicrobium sp. JCM 19039 (A0A061P351) using the Geneious basic. Key is 0.2 nt substitutions per site.

Figure 5

Amino acid alignment and percentage identity of the identified phage-like element PBSX protein XkdK accession A0A518VEB0 (red arrow) of Bl 1821L and Bl 1951 with the phage tail-sheath proteins of Aneurinibacillus migulanus (A0A0D1WNL8), Bacillus aerophilus (A0A410KN98), Brevibacillus gelatini (A0A3M8B733), Brevibacillus laterosporus LMG 15441 (A0A075R9L5); uncharacterised protein of Brevibacillus brevis (strain 47/JCM 6285/NBRC 100599) (C0Z5G9), and similar proteins of other Gram-positive bacteria, including Bacillus phage PBP180 (R4JQA6), Bacillus sp. ANT_WA51 (A0A5B0B6Z4), Bacillus sp.WR11 (A0A410QZ71), Bacillus subtilis subsp. subtilis str. SMY (A0A6H0H1P2), Bacillus subtilis 168 (P54331), Clostridioides difficile 630 (Q18BN0), and Geomicrobium sp. JCM 19039 (A0A061P351) using the Geneious basic.

Figure 6

Dendrogram showing the alignment of identified putative phage tail-sheath protein (A0A518VEB0) of Bl 1821L and Bl 1951 with the similar protein of different Bl phages including Abouo (S5MUG6), Davies (S5MCF5), Osiris (A0A0K2CNL4), Powder (A0A0K2FLW7), Jimmer1 (S5MNC1), and Jimmer2, (S5MBG7). Key is 0.2 nt substitutions per site.

Figure 7

Genome alignment of the Bl 1821L and Bl 1951 PBSX-like region encoding phage-like element PBSX protein XkdK. Differences between the two strains (shown in red shaded box) with 1700 bp long imperfect repeats of glycine rich proteins residing in the Bl 1951 genome and a corresponding putative phage region in the Bl 1821L genome. On the top bar, green indicates areas of high homology; olive green indicates areas of lower homology. Grey indicates identical sequences on lower bars; black indicates areas of base differences.

Figure 8

Schematic genomic alignment of identified phage-like element PBSX gene xkdK (shown with red arrow) encoding region in the Bl 1821L and Bl 1951 genomes with defective prophage PBSZ, PBSX, and PBP180 of B. subtilis subsp. spizzenii W23, B. subtilis 168, and Bacillus phage PBP180 [36].

Figure 9

Amino acid alignment and percentage identity of identified phage-like element PBSX protein XkdK accession A0A518VEB0 (shown with red arrow) of Bl 1821L and Bl 1951 with similar proteins of defective prophages of Bs W23 (E0U1S9), Bs 168 (P54331), and Bacillus phage PBP180 (R4JQA6) using the Geneious basic.