Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Abstract

Background: Bacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics.

Results: Using a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature.

Conclusion: This study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Keywords: Brevibacillus laterosporus; Antibacterial proteins; Biochemical characterisation; High molecular-weight; Insect pathogenic isolates; Production kinetics; Spontaneous induction.

Fig. 1

Transmission electron microscope images of the putative phage structural parts observed in the mitomycin C- induced flitrates of Bl 1821L (1a, 1b) and Bl 1951 (1c, 1d). Structures resembling to phage head (capsid) and incomplete phage particles with an empty contracted sheath, contractile tail sheath, and contractile sheath with cores were seen (Fig. 1a- 1d). Scale bar = Fig. 1a = 100 nm, Fig. 1b- 1d = 50 nm

Fig. 2

Mean values of Bl 1821L cells growth (log₁₀ CFU/mL) at various time intervals are shown with the standard error. Mean values are averaged from the pooled data of three experiments

Fig. 3

Mean values of Bl 1951 cells growth (log₁₀ CFU/mL) at various time intervals are shown with the standard error. Mean values are averaged from the pooled data of three experiments

Fig. 4

SDS-PAGE analysis of spontaneously induced putative antibacterial proteins of Bl 1821L across various time intervals. (a) shows the spontaneously induced putative antibacterial proteins after 3, 6, 12, 18, 24, 36, 48, 60, 72 h and (b) shows the protein bands of spontaneously induced putative antibacterial proteins after 96, 120, 144, 168, 192, 216, and 240 h of Bl 1821L cultivation at 30 °C and 250 rpm. Black arrows denote the mitomycin C- induced putative antibacterial proteins of ~ 30 kDa and ~ 48 kDa. PM and MMC stands for protein marker and mitomycin-C respectively

Fig. 5

SDS-PAGE analysis of spontaneously induced putative antibacterial proteins of Bl 1951 across various time intervals. (a) shows the spontaneously induced putative antibacterial proteins after 3, 6, 12, 18, 24, 36, 48, 60, 72 h and (b) shows the protein bands of spontaneously induced putative antibacterial proteins after 96, 120, 144, 168, 192, 216, and 240 h of Bl 1951 cultivation at 30 °C and 250 rpm. Black arrows denote the mitomycin C- induced putative antibacterial proteins of ~ 30 kDa and ~ 48 kDa. PM and MMC stands for protein marker and mitomycin-C respectively