Cloning, expression and deletion of the cuticle-degrading protease BLG4 from nematophagous bacterium Brevibacillus laterosporus G4

Brevibacillus laterosporus G4, which was isolated from soil sample, kills free-living nematodes (Panagrellus redivius) and plant-parasite nematodes (Bursaphelenchus xylophilus) and degrades their cuticle in previous bioassay. Our works for B. laterosporus G4 had demonstrated that an extracellular alkaline protease BLG4 played a key role as a pathogenic factor in infection against nematode. In this study, the nematicidal activity of BLG4 was further verified by an in vitro assay with purified recombinant BLG4. The encoding gene of BLG4 was cloned and showed high degree of homology with the subtilisin subclass of serine protease gene and another reported cuticle-degrading protease gene from nematophagous bacterium Bacillus sp. B16. Deletion of BLG4 by homologous recombinant had a significant effect on the pathogenicity of B. laterosporus. In infection assays the BLG4-deficient strain (BLG4-6) lost about 50% of its nematocidal activity and in toxicity tests the mortality rate of nematodes decreased with ∼56% in comparison to wild-type strain. This is the first report analyzing the function of a subtilisin enzyme involved in bacterium against nematode at the molecular level, and it is possible to use B. laterosporus as a model to study host-parasite interaction and to gain detailed knowledge of the infection process.     

Direct visualization of bacterial infection process in nematode hosts by an improved immunocytochemical method

Our previous work on Brevibacillus laterosporus G4 had demonstrated that an extracellular protease BLG4 played a key role as a pathogenic factor in bacterial infection of nematodes. In this study, we developed an improved method for the immunofluorescent localization of protease BLG4 to directly visualize the bacterial infection process in a nematode host and demonstrate the involvement of the protease in penetrating nematode cuticles in vivo. This work supplies a new basic knowledge that elucidates the microbial infection processes in nematodes. B.Y. Tian and C.R. Ke have contributed equally to this work.     

Role of an extracellular neutral protease in infection against nematodes by <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> strain G4

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.     

Investigation of protease-mediated cuticle-degradation of nematodes by using an improved immunofluorescence-localization method

In order to facilitate the understanding of the actual process of enzyme-based degradation of nematodes, we visualized the localization of BLG4, a cuticle-degrading protease from the nematophagous bacterium Brevibacillus laterosporus G4, on nematode cuticle by using an improved immuno-labeled fluorescent method. Live nematodes, heat-killed nematodes and extracted nematode cuticles were exposed to the protease, and the localization of the protease and the resulting tissue degradation and destruction were observed microscopically. The bioassay findings showed that live nematodes were significantly more resistant to the protease than the dead nematodes and the extracted cuticles were. The observation of the immuno-labeling fluorescence for BLG4 revealed that the protease localized first in the tail region of the live target; and then spread over the entire target and ultimately destroyed it, including the cuticle. The results indicated the resistance of nematode cuticles to enzymatic attacks and the differences in protease susceptibilities at different regions on the nematode cuticles.     

Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by A. avenae subsp. avenae RS-1

Biological control efficacy of Brevibacillus laterosporus B4 associated with rice rhizosphere was assessed against bacterial brown stripe of rice caused by Acidovorex avenae subsp. avenae. A biochemical bactericide (chitosan) was used as positive control in this experiment. Result of in vitro analysis indicated that B. laterosporus B4 and its culture filtrates (70 %; v/v) exhibited low inhibitory effects than chitosan (5 mg/ml). However, culture suspension of B. laterosporus B4 prepared in 1 % saline solution presented significant ability to control bacterial brown stripe in vivo. Bacterization of rice seeds for 24 h yielded a greater response (71.9 %) for controlling brown stripe in vivo than chitosan (56 %). Studies on mechanisms revealed that B. laterosporus B4 suppressed the biofilm formation and severely disrupted cell membrane integrity of A. avenae subsp. avenae, causing the leakage of intracellular substances. In addition, the expression level of virulence-related genes in pathogen recovered from biocontrol-agent-treated plants showed that the genes responsible for biofilm formation, motility, niche adaptation, membrane functionality and virulence of A. avenae subsp. avenae were down-regulated by B. laterosporus B4 treatment. The biocontrol activity of B. laterosporus B4 was attributed to a substance with protein nature. This protein nature was shown by using ammonium sulfate precipitation and subsequent treatment with protease. The results obtained from this study showed the potential effectiveness of B. laterosporus B4 as biocontrol agent in control of bacterial brown stripe of rice.     

Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement

An efficient method for gene replacement in Bacillus megaterium was developed and used to inactivate the chromosomal neutral protease gene (nprM) from strain DSM319. A temperature-dependent suicide vector was constructed to allow replacement of the normal chromosomal copy with an altered version of the nprM gene. One mutant B. megaterium MS941 was selected for further characterization. Measurement of extracellular protease activity from strain MS941 indicated the existence of an additional minor extracellular protease in B. megaterium. Inhibitor studies revealed that this minor protease, comprising only 1.4% of the wild-type total extracellular protease activities, is a serine-type enzyme.Data presented in this contribution are part of a doctoral thesis of the Naturwissenschaftliche Fakultät Münster, Germany (KDW)     

Characteristics of extracellular proteases produced by Bacillus laterosporus and Flavobacterium sp. isolated from gelatinfactory effluents

Forty bacterial isolates from the effluents of a gelatin factory (Jabalpur, India) were screened for protease activity and the two most potent producers were identified as Bacillus laterosporus and a Flavobacterium sp. The enzymes of both isolates were optimal at pH 8 and 60°C, with maximum activity after 90 min. The enzyme activity of B. laterosporus was suppressed by Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ ions but was enhanced by Ba²⁺ and Ca²⁺. That of Flavobacterium sp. was suppressed by Mg²⁺ and Mn²⁺ ions but enhanced by Ba²⁺, Ca²⁺ and Fe²⁺. The enzyme activity of the former was strongly inhibited by KCN, whereas that of the latter was only slightly inhibited by 8-hydroxyquinoline.     

Enhancement of larvicidal activity of <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> by bioincapsulation in Protozoa <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis> and <Emphasis Type="Italic">Entamoeba moshkovskii</Emphasis>

Conditions for bioincapsulation of crystal-forming strain Brevibacillus laterosporus LAT 006 spores and crystals by using Tetrahymena pyriformis and Entamoeba moshkovskii Protozoa have been developed. Increase in the larvicidal activity of the incapsulated bacteria was demonstrated. Fractions of pure spores and crystals and intact spore-crystal preparation of LAT 006 were shown not to have toxic effect on the protozoa cells.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Electron microscopy of the surfaces of bacillus spores

The surface structures of the spores of Bacillus cereus, Bacillus thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. The diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.     

Gene Cloning and Expression of an Alkaline Serine Protease with Dehairing Function from <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

A new gene (named AP gene) encoding an alkaline serine protease with dehairing function was cloned from Bacillus pumilus UN-31-C-42 and its nucleotide sequence was determined. The expression of AP gene was induced with IPTG in Escherichia coli after the mature protease region was cloned into pET15b and SDS-PAGE showed expressed product clearly, but no alkaline protease activity was detected. In order to express the AP gene in B. subtilis, a recombinant expression plasmid was constructed which contained a promoter Bp53 (also from B. pumilus), the AP gene and an E. coli–B. subtilis shuttle vector pSUGV4. This plasmid was introduced into B. subtilis WB600 and the transformant displayed the hydrolyzed zone on a milk plate. The expressed product can be easily detected with SDS-PAGE and the fermentation fluid of the transformant showed low alkaline protease activity and dehairing activity. This is the first report of a gene cloned from B. pumilus, encoding an alkaline serine protease, which can alone accomplish the whole dehairing process.     

Functional identification of the gene <Emphasis Type="Italic">bace16</Emphasis> from nematophagous bacterium <Emphasis Type="Italic">Bacillus nematocida</Emphasis>

Bacillus nematocida is a Gram-positive bacterium capable of killing nematodes. Our recent studies identified an extracellular serine protease Bace16 in B. nematocida as a candidate of pathogenic factor in the infection against nematodes, which displayed a high similarity with the serine protease family subtilisin BPN’, and the MEROPS ID is S08.034. To further confirm the roles that bace16 played in the mechanism of nematocidal pathogenesis, recombinant mature Bace16 (rm-Bace16) was expressed in Escherichia coli strain BL21 using pET-30 vector system. Bioassay experiments demonstrated that the purified recombinant protease had the ability to degrade nematode cuticles and kill nematodes. In addition, a bace16 knockout mutant of B. nematocida constructed by homologous recombination showed considerably lower proteolytic activity and less than 50% nematocidal activity than the wild-type strain. These results confirmed that Bace16 could serve as an important virulence factor during the infectious process. Qiuhong Niu and Xiaowei Huang contributed equally to this work.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

A Ferric Dicitrate Uptake System Is Required for the Full Virulence of Bacillus cereus

Bacillus cereus is an opportunistic human pathogen of increasing prevalence. Analysis of the Bacillus cereus genome sequence identified a potential ferric dicitrate uptake system. The three-gene operon was confirmed to be negatively regulated by the ferric uptake repressor (Fur). The Fec operon was genetically silenced using the integration suicide vector pMUTIN4. The mutant strain displayed no growth defect under iron-limited conditions but was unable to grow on ferric citrate as a sole iron source. The virulence of the mutant strain was attenuated in a lepidopteran infection model, highlighting the importance of iron uptake systems to the virulence of B. cereus and the potential of these systems to act as targets for novel antimicrobial agents.     

Extracellular production of cycloisomaltooligosaccharide glucanotransferase and cyclodextran by a protease-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> host–vector system

A cycloisomaltooligosaccharide (CI; cyclodextran) production system was developed using a Bacillus subtilis expression system for the cycloisomaltooligosaccharide glucanotransferase (CITase) gene. The CITase gene of Bacillus circulans T-3040, along with the α-amylase promoter (PamyQ) and amyQ signal sequence of Bacillus amyloliquefaciens, was cloned into the Bacillus expression vector pUB110 and subsequently expressed in B. subtilis strain 168 and its alkaline (aprE) and neutral (nprE) protease-deficient strains. The recombinant CITase produced by the protease-deficient strains reached 1 U/mL in the culture supernatant within 48 h of cultivation, which was approximately 7.5 times more than that produced by the industrial CITase-producing strain B. circulans G22-10 derived from B. circulans T-3040. When aprE- and nprE-deficient B. subtilis 168 harboring the CITase gene was cultured with 10% dextran 40 for 48 h, 17% of the dextran in the culture was converted to CIs (CI-7 to CI-12), which was approximately three times more than that converted by B. circulans G22-10 under the same dextran concentration. The B. subtilis host–vector system enabled us to produce CIs by direct fermentation of dextran along with high CITase production, which was not possible in B. circulans G22-10 due to growth inhibition by dextran at high concentrations and limited production of CITase.     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

Larvicidal activity of crystal-forming strains of <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis>

The optimum conditions for growth, sporulation, and crystal-formation in four isolated crystal-forming strains of Bacillus laterosporus were determined. It was shown that culture broth and pellets of bacterial culture liquid possess larvicidal activity against larvae of mosquitoes A. stephensi and A. aegypti. The protein nature of crystal was shown. Crystals are monocomponent containing a protein with MM of 68 or 130 kDa. Purified protein crystals demonstrated larvicidal activity. Specific larvicidal activity of crystals of various strains essentially differed. High larvicidal activity of B. laterosporus strains allows for them to be recommended as producers of antimosquito biological preparations.     

A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis

Incorporation of starch or casein into protoplast-regeneration medium facilitated shotgun cloning of α-amylase and neutral protease genes from an unidentified Bacillus sp. in Bacillus subtilis by polyethylene glycol-induced protoplast transformation. This modification and the use of the plasmid vector pPL603b enabled us to simultaneously select for promoter-bearing recombinant plasmids that expressed amylase or protease activity. The inserts were found to be 4 and 4.6 kb, respectively. Although protease activity directed by the cloned gene was only 2- to 4-fold higher than for the donor strain, that of α-amylase was 28-fold higher.     

Insecticidal Activity of Bacillus laterosporus

The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The larvicidal activity of B. laterosporus was associated with spores and crystalline inclusions. Purified B. laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.     

Isolation,Biological Properties,and Spatial Structure of Antibiotic Loloatin A

A peptide antibiotic with cyanolytic activity was isolated from the IGM52 strain of the Brevibacillus laterosporus Gram-positive spore-forming bacteria. By ¹H NMR spectroscopy, this antibiotic was identified as loloatin A, a cyclic decapeptide cyclo(-Asn-Asp-Tyr-Val-Orn-Leu-D-Tyr-Pro-Phe-D-Phe-). The spatial structure of loloatin A in solution was determined.