( 1991 )
Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.
PMID : 1744041 : DOI : 10.1128/jb.173.24.7848-7855.1991 PMC : PMC212576
A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.
( 2007 )
Role of an extracellular neutral protease in infection against nematodes by Brevibacillus laterosporus strain G4.
PMID : 17109172 : DOI : 10.1007/s00253-006-0690-1
Proteases have been proposed as virulence factors in microbial pathogenicity against nematodes. However, what kinds of extracellular proteases from these pathogens and how they contribute to the pathogenesis of infections against nematode in vivo remain largely unknown. A previous analysis using a strain with a deletion in an extracellular alkaline protease BLG4 gene from Brevibacillus laterosporus demonstrated that BLG4 was responsible for the majority of nematicidal activity by destroying host's cuticle. In recent studies, a neutral protease NPE-4, purified from the mutant BLG4-6, was found to be responsible for the majority of the remaining EDTA-inhibited protease activity. However, the purified NPE-4 and recombinant NPE-4 in a related species Bacillus subtilis showed little nematicidal activity in vitro and were unable to degrade the intact cuticle of the host. It is interesting to note that the addition of NPE-4 improved the pathogenicity of crude enzyme extract from wild-type B. laterosporus but had no effect on the BLG4-deficient mutant. This result suggests that NPE-4 functions in the presence of protease BLG4. Moreover, NPE-4 could degrade proteins from the inner layer of purified cuticles from nematode Panagrellus redivivus in vitro. These results indicated that the two different bacterial extracellular proteases might play differential roles at different stages of infection or a synthetic role in penetration of nematode cuticle in B. laterosporus. This is among the first reports to systematically evaluate and define the roles of different bacterial extracellular proteases in infection against nematodes.
( N/A )
An extracellular protease from Brevibacillus laterosporus G4 without parasporal crystals can serve as a pathogenic factor in infection of nematodes.
PMID : 15950127 : DOI : 10.1016/j.resmic.2005.02.006
Brevibacillus laterosporus is an aerobic spore-forming bacterium with the ability to produce canoe-shaped lamellar parasporal inclusions adjacent to spores. An isolate named G4 was identified as a B. laterosporus which does not produce parasporal crystals and shows significant toxic activity toward nematodes. Crude extracellular protein extract from culture supernatant of B. laterosporus G4 killed the nematodes within 12 h and finally destroyed the targets within 24 h, which suggested possible proteinaceous pathogeny. A homogeneous extracellular protease with nematicidal activities, purified by chromatography, confirmed the hypothesis that it might serve as a pathogenic factor during infection of the G4 strain. Characterization of the purified protease revealed a molecular mass of 30 kDa and optimum activity at pH 10 and 50 degrees C. The protease hydrolyzed relatively broad substrates including collagen and the cuticle of nematodes, and histopathological observations demonstrated the resulting destroyed nematode cuticle upon treatment by purified protease. Our present study reveals that extracellular protease, but not previously reported parasporal crystals, can be employed in infection against invertebrates by the B. laterosporus G4 strain.
( 2017 )
Spore surface proteins of Brevibacillus laterosporus are involved in insect pathogenesis.
PMID : 28256631 : DOI : 10.1038/srep43805 PMC : PMC5335551
Outer spore envelope proteins of pathogenic bacteria often present specific virulence factors and tools to evade the defence system of their hosts. Brevibacillus laterosporus, a pathogen of invertebrates and an antimicrobial-producing species, is characterised by a unique spore coat and canoe-shaped parasporal body (SC-CSPB) complex surrounding the core spore. In the present study, we identified and characterised major proteins of the SC-CSPB complex of B. laterosporus, and we investigated their entomopathogenic role. Employing a proteomic approach and a B. laterosporus-house fly study model, we found four highly conserved proteins (ExsC, CHRD, CpbA and CpbB) that function as insect virulence factors. CpbA was associated with a significantly higher mortality of flies and greater relative gene expression levels during sporulation, compared to the other SC-CSPB proteins. Taken together, we suggest that spore surface proteins are a part of a complex set of toxins and virulence factors that B. laterosporus employs in its pathogenicity against flies.
( 2015 )
PeBL1, a novel protein elicitor from Brevibacillus laterosporus strain A60, activates defense responses and systemic resistance in Nicotiana benthamiana.
PMID : 25662975 : DOI : 10.1128/AEM.03586-14 PMC : PMC4375336
We report the identification, characterization, and gene cloning of a novel protein elicitor (PeBL1) secreted from Brevibacillus laterosporus strain A60. Through a purification process consisting of ion-exchange chromatography and high-performance liquid chromatography (HPLC), we isolated a protein that was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry (ESI-Q-TOF-MS-MS). The 351-bp PeBL1 gene produces a 12,833-Da protein with 116 amino acids that contains a 30-residue signal peptide. The PeBL1 protein was expressed in Escherichia coli. The recombinant protein can induce a typical hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana, like the endogenous protein. PeBL1-treated N. benthamiana exhibited strong resistance to the infection of tobacco mosaic virus-green fluorescent protein (TMV-GFP) and Pseudomonas syringae pv. tabaci compared to control N. benthamiana. In addition, PeBL1 triggered a cascade of events that resulted in defense responses in plants, including reactive oxygen species (ROS) production, extracellular-medium alkalization, phenolic-compound deposition, and expression of several defense-related genes. Real-time quantitative-PCR analysis indicated that the known defense-related genes PR-1, PR-5, PDF1.2, NPR1, and PAL were upregulated to varying degrees by PeBL1. This research not only provides insights into the mechanism by which beneficial bacteria activate plant systemic resistance, but also sheds new light on a novel strategy for biocontrol using strain A60.
( 2012 )
Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60.
PMID : 22244810 : DOI : 10.1016/j.peptides.2012.01.001
A novel antimicrobial peptide, with molecular mass of 1602.0469Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC(50) values of 7.89, 0.60 and 21.96 �gg ml(-1), respectively.
( 2018 )
Survey of Brevibacillus laterosporus insecticidal protein genes and virulence factors.
PMID : 29753714 : DOI : 10.1016/j.jip.2018.05.002
The pathogenic action of the bacterium Brevibacillus laterosporus against invertebrates involves a toxin-mediated mechanism. Several studies, conducted with specific strains against diverse targets, suggested the implication of different toxins. Recent genome sequencing and annotation of some insecticidal strains revealed several putative virulence factors highly conserved in this species. After determining the pathogenicity of strain UNISS 18 against different Lepidopteran and Dipteran larvae, in this study we have investigated the actual expression of genes encoding for enzymes (i.e., chitinases, proteases), toxins, and other virulence factors, either in vitro and in vivo at the transcriptional level. Selected genes encode for two chitinases, a collagenase-like protease, a GlcNAc-binding protein, two protective antigen proteins, a bacillolysin, a thermophilic serine proteinase, two spore surface proteins, an insecticidal toxin homologous to Cry75Aa. All target genes were well expressed in pure bacterial cultures with significant differences between bacterial growth phases. Their expression level was generally enhanced in the bacterial population developing in the insect body cavity, compared with pure culture. The expression of certain genes increased substantially over time after insect inoculation. These results support a complex mechanism of action leveraging a variety of available virulence factors, and can also explain the ability of this bacterial species to act against diverse invertebrate targets.