Linocin M18 protein from the insect pathogenic bacterium Brevibacillus laterosporus isolates

Abstract

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ~30 kDa. N-terminal sequencing of the ~30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ~30 kDa encapsulating protein of Bl 1821L and Bl 1951during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ~30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N. KEY POINTS: • Gene encoding the 31.4 kDa antibacterial Linocin M18 protein was identified • It defined the autocidal activity of Linocin M18 (encapsulating) protein • Identified the possible killing mechanism of the encapsulins.

Keywords: Antibacterial protein; Brevibacillus laterosporus; Encapsulating protein; Heterologous expression; Insect pathogenic bacterium; Linocin M18 protein.

Fig. 1

Electron micrographs of crude lysates of Bl 1821L (a) and Bl 1951 (b) showing globular or phage capsid-like, hollow sheath-like, contractile phage tail sheath-like, and polysheath-like structures

Fig. 2

SDS-PAGE showing the Bl 1821L and Bl 1951 sucrose density gradient centrifugation purified and 10 kDa MWCO membrane concentrated ~30 kDa putative antibacterial protein (a, c shown with dark blue arrow). Arrows in electron micrographs of purified putative antibacterial protein of Bl 1821L denote globular or phage capsid-like structures (b shown with white arrow) while for Bl 1951, globular or phage capsid-like structures (d shown with white arrow) and polysheath-like structures (d shown with dark blue arrow) are visualised. Scale bar= 100 nm. PM denotes protein marker

Fig. 3

Bl 1821L and Bl 1951 genomic architecture of ~30 kDa N-terminal sequence showing an identified hypothetical protein of 25 kDa (dark blue arrow) and a bacteriocin family protein (encapsulating) of 31.4 kDa (red arrow) residing in Bl 1821L and Bl 1951 genomes along with other proteins of the region with terminology used in GenBank accessions. Bl 1951 genomic region identical to Bl 1821L genome is highlighted with red shaded box region

Fig. 4

Number of viable cells (log₁₀ CFU/mL) of Bl 1821L and Bl 1951 with/without treatment of purified Bl 1821L putative encapsulating protein (~30 kDa) after incubation at 30 °C for various time intervals

Fig. 5

LIVE/DEAD staining of Bl 1821L cells after treatment with the purified Bl 1821L putative encapsulating protein (~30 kDa). Green denotes live cells while red and orange show the cells with compromised membranes. Scale = 10 μm

Fig. 6

LIVE/DEAD stained percentage population proportion of Bl 1821L cells after treatment (left side graph) with the ~30 kDa-purified putative encapsulating protein of Bl 1821L and without treatment (right side graph). Green denotes live cells, and red and orange show the proportion of cells with compromised cell membranes

Fig. 7

Assay test of CFS from Bs WB800N (pHT01-encap, B2) expressing 31.4 kDa putative encapsulating protein against Bl 1821L (a) and Bl 1951 (b) as the host bacterium. Arrows (red) denote the zones of inhibition showing a diameter of ≥11 mm. SDS-PAGE analysis showing a purified ~30 kDa putative encapsulating protein (c shown with red arrow) expressed after 24 h of induction from Bs WB800N (pHT01-encap, B2). PM denotes protein marker

Fig. 8

Assay test of CFS from Bs WB800N (pHT01-hypo.encap) expressing both 25 kDa hypothetical and 31.4 kDa putative encapsulating proteins against Bl 1821L (a) and Bl 1951 (b) as the host bacterium. Arrows (red) denote the zones of inhibition showing a diameter of ≥11 mm. SDS-PAGE analysis showing the purified ~25 kDa hypothetical (shown with dark blue arrow) and ~30 kDa encapsulating proteins (c shown with red arrow) from Bs WB800N (pHT01-hyo.encap). PM denotes protein marker