Evaluation of probiotic properties of a Brevibacillus laterosporus strain.

Brevibacillus laterosporus is a strain of probiotic bacteria that has been widely used in pest control, cash crop, and other production areas. However, few studies have been conducted on its use as a feed additive in animals. Therefore, the probiotic potential of B. laterosporus PBC01 was evaluated by characterizing hydrophobicity, auto-aggregation activity, bile salt and simulated gastrointestinal fluid tolerance, bienzymatic, and antibacterial activity. Antibiotic susceptibility, hemolysis assays, and supplemental feeding of mice were also performed to evaluate safety features. Our results showed that B. laterosporus PBC01 had moderate hydrophobicity, high auto-agglutination ability. Meanwhile, B. laterosporus PBC01 had good tolerance to bile salt and simulated gastrointestinal fluid. It had the ability to secrete protease, cellulase, and to inhibit various pathogens. In addition, B. laterosporus PBC01 was sensitive to many antibiotics, and did not produce hemolysin. In the safety assessment of mice, it did not cause any deaths, nor did it affect the cell components of blood, antioxidant capacity, and reproductive health. The study indicated the great probiotic characteristics and safety of B. laterosporus PBC01. This may provide a theoretical basis for the clinical application and development of probiotic-based feed additives.

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

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.

Effects of antibacterial peptides from Brevibacillus texasporus on growth performance, meat quality and gut health of cultured largemouth bass (Micropterus salmoides).

The aim of this study was to investigate the effects of antibacterial peptides from Brevibacillus texasporus (BT) on the growth performance, meat quality and gut health of cultured largemouth bass (Micropterus salmoides). Largemouth bass (36.17 ± 1.52 g) were divided into 2 groups and each group was fed with diets supplemented with or without 200 ppm of BT peptides for 130 days. The results showed that BT peptides had no significant influences on growth performance and body indexes, but significantly enhanced total antioxidant capacity and lysozyme content in the serum. Moreover, digestive enzymes activities and intestinal villous height were also prominently increased. From meat quality aspect, no significant differences were found in nutritional components, amino acid composition, fatty acid composition and texture property, except the values of hardness, gumminess and γ-linolenic acid (C18:3n6) were remarkably increased after BT peptides intervention. Finally, the results of gut microbiota and short chain fatty acids revealed that BT peptides significantly decreased the relative abundances of harmful bacteria such as genus Acinetobacter and Pseudomonas, and increased the production of short chain fatty acids. In conclusion, this study confirmed that BT peptides could be used to improve the health of largemouth bass, which provided novel insights into the application of antimicrobial peptides in aquacultures.

The Termite Nest-Associated Bacterium Brevibacillus parabrevis WGTm-23 Contains Unique Biosynthetic Gene Clusters Potentially Coding for Novel Antimicrobial Agents.

Multidrug resistance in clinical pathogens is a significant challenge in healthcare, requiring the development of novel approaches to combat infections. In this study, we report the identification of novel antimicrobial biosynthetic gene clusters from Brevibacillus parabrevis WGTm-23, the bacterial strain isolated from a termitarium. This strain showed an antagonistic effect against drug-resistant clinical pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella paratyphi, Streptococcus gordonii, and enteropathogenic Escherichia coli. The whole genome of this strain was sequenced using the Illumina platform. The genome mining revealed a total of 17 biosynthetic gene clusters (BGCs) responsible for the synthesis of secondary metabolites. The metabolites produced by this strain were predicted by constructing an identity network of the BGCs and performing a comparative analysis with genetically related strains. The genome contains multiple BGCs coding for ribosomally synthesized and post-translationally modified peptides (RiPPs). In the genome of Br. parabrevis WGTm-23, we identified BGCs that code for ulbactin F, ulbactin G, gramicidin, and bacillopaline with the highest identity. We also identified a few BGCs with less than 50% sequence identity to MC-LR/MC-LHty/MC-HphHty/MC-LHph/MC-HphHph, xenocoumacin 1/xenocoumacin II, and tyrocidine. In addition, we found fourteen BGCs that do not resemble or show identity to any entries within the antiSMASH database. Therefore, Br. parabrevis WGTm-23 has the potential to synthesize new classes of antimicrobial compounds.

Expression of a novel hydrolase MhpC in Brevibacillus parabrevis BCP-09 and its characteristics for degrading synthetic pyrethroids.

Synthetic pyrethroids are widely used insecticides which may cause chronic diseases in non-target organisms upon long-term exposure. Microbial degradation offers a reliable method to remove them from the environment. This study focused on Brevibacillus parabrevis BCP-09 and its enzymes for degrading pyrethroids. The predicted deltamethrin-degrading genes phnA and mhpC were used to construct recombinant plasmids. These plasmids, introduced into Escherichia coli BL21(DE3) cells and induced with L-arabinose. The results indicated that the intracellular crude enzyme efficiently degraded deltamethrin by 98.8 %, ß-cypermethrin by 94.84 %, and cyfluthrin by 73.52 % within 24 h. The hydrolytic enzyme MhpC possesses a catalytic triad Ser/His/Asp and a typical "Gly-X-Ser-X-Gly" conservative sequence of the esterase family. Co-cultivation of induced E. coli PhnA and E. coli MhpC resulted in degradation rates of 41.44 ± 3.55 % and 60.30 ± 4.55 %, respectively, for deltamethrin after 7 d. This study states that the degrading enzymes from B. parabrevis BCP-09 are an effective method for the degradation of pyrethroids, providing available enzyme resources for food safety and environmental protection.

Unique behavioral patterns of wandering colonies of Brevibacillus thermoruber on agar plates.

Brevibacillus thermoruber strain Nabari cells grow as widely spreading dendritic colonies on reasoner's 2A-agar (1.5%) plates at around 55°C but as small motile colonies at 37°C. Motile colonies can be divided into colonies that move in straight or curved lines over long distances (wandering colonies), and colonies that rotate at a fixed location (rotating colonies). The addition of surfactant to the agar medium greatly increased the frequency of wandering colonies and facilitated the study of such colonies. The morphology of the wandering colonies varied: circular at the tip and pointed at the back, lemon-shaped with pointed ends, crescent-shaped, bullet-shaped, fish-like, and so on. A single colony may split into multiple colonies as it moves, or multiple colonies may merge into a single colony. The most surprising aspect of the movement of wandering colonies was that when a moving colony collides with another colony, it sometimes does not make a U-turn, but instead retreats straight back, as if bouncing back. The migration mechanisms of wandering colonies are discussed based on optical microscopic observations of swimming patterns of single cells in water and scanning electron microscopy of the arrangement of bacterial cells in wandering colonies.

Isolation, Characterization, Genome Annotation, and Evaluation of Hyaluronidase Inhibitory Activity in Secondary Metabolites of Brevibacillus sp. JNUCC 41: A Comprehensive Analysis through Molecular Docking and Molecular Dynamics Simulation.

Brevibacillus sp. JNUCC 41, characterized as a plant-growth-promoting rhizobacterium (PGPR), actively participates in lipid metabolism and biocontrol based on gene analysis. This study aimed to investigate the crucial secondary metabolites in biological metabolism; fermentation, extraction, and isolation were performed, revealing that methyl indole-3-acetate showed the best hyaluronidase (HAase) inhibitory activity (IC50: 343.9 µM). Molecular docking results further revealed that the compound forms hydrogen bonds with the residues Tyr-75 and Tyr-247 of HAase (binding energy: -6.4 kcal/mol). Molecular dynamics (MD) simulations demonstrated that the compound predominantly binds to HAase via hydrogen bonding (MM-PBSA binding energy: -24.9 kcal/mol) and exhibits good stability. The residues Tyr-247 and Tyr-202, pivotal for binding in docking, were also confirmed via MD simulations. This study suggests that methyl indole-3-acetate holds potential applications in anti-inflammatory and anti-aging treatments.

Complete genome sequence analysis and Pks genes identification of Brevibacillus brevis FJAT-0809-GLX with a broad inhibitory spectrum against phytopathogens.

Brevibacillus brevis FJAT-0809-GLX has a broad spectrum of antimicrobial activity. Understanding the molecular basis of biocontrol ability of B. brevis will allow us to develop effective microbial agents for sustainable agriculture. In this study, we present the complete and annotated genome sequence of FJAT-0809-GLX. The complete genome size of B. brevis FJAT-0809-GLX was 6,137,019 bp, with 5688 predicted coding sequences (CDS). The average GC content of 47.38%, and there were 44 copies of the rRNAs operon (16S, 23S and 5S RNA), and 127 tRNA genes. A total of 11,162 genes were functionally annotated with the COG, GO, and KEGG databases, and 123 genes belonged to CAZymes. Genomic secondary metabolite analysis indicated 13 clusters encoding potential new antimicrobials. FJAT-0809-GLX was designated as B. brevis according to average nucleotide polymorphism (ANI) and phylogenetic analysis. The pangenome consisted of 7141 homologous genes, and 4469 homologous genes shared by B. brevis FJAT-0809-GLX, B. brevis NBRC100599, B. brevis DSM30, and B. brevis NCTC2611. The number of unique homologous genes of B. brevis FJAT-0809-GLX (419 genes) and B. brevis NBRC100599 (480 genes) were much more than those in B. brevis DSM30 (13 genes), and B. brevis NCTC2611 (6 genes). Nine gene clusters encoding for secondary metabolite biosynthesis were compared in the genome of B. brevis FJAT-0809-GLX with those of B. brevis NBRC100599, B. brevis DSM30 and B. brevis NCTC2611, and the gene clusters encoding for lantipeptide and transatpks-otherks only existed in genome of B. brevis FJAT-0809-GLX. The 11 BbPks genes were included in the B. brevis FJAT-0809-GLX genome, which contained the conserved PS-DH domain. The relative expression of BbPksL, BbPksM2, BbPksM3, BbPksN3, BbPksN4 and BbPksN5 reached a maximum at 120 h and then decreased at 144 h. Our results provided detailed genomic and Pks genes information for the FJAT-0809-GLX strain, and lid a foundation for studying its biocontrol mechanisms.

Simple production of resilin-like protein hydrogels using the Brevibacillus secretory expression system and column-free purification.

Resilin, an insect structural protein, has excellent flexibility, photocrosslinking properties, and temperature responsiveness. Recombinant resilin-like proteins (RLPs) can be fabricated into three-dimensional (3D) structures for use as cell culture substrates and highly elastic materials. A simplified, high-yielding production process for RLPs is required for their widespread application. This study proposes a simple production process combining extracellular expression using Brevibacillus choshinensis (B. choshinensis) and rapid column-free purification. Extracellular production was tested using four representative signal peptides; B. choshinensis was found to efficiently secrete Rec1, an RLP derived from Drosophila melanogaster, regardless of the type of signal peptide. However, it was suggested that Rec1 is altered by an increase in the pH of the culture medium associated with prolonged incubation. Production in a jar fermentor with controllable pH yielded 530 mg Rec1 per liter of culture medium, which is superior to productivity using other hosts. The secreted Rec1 was purified from the culture supernatant via (NH4 )2 SO4 and ethanol precipitations, and the purified Rec1 was applied to ring-shaped 3D hydrogels. These results indicate that the combination of secretory production using B. choshinensis and column-free purification can accelerate the further application of RLPs.

Brevibacillus humidisoli sp. nov., a moderately thermoalkaliphilic and halotolerant species isolated from riverside soil.

A Gram-stain-positive, aerobic, rod-shaped, motile, and endospore-forming bacterial strain designated MMS20-4M-10-YT was isolated from riverside soil and subjected to taxonomic characterization. Strain MMS20-4M-10-YT was moderately thermophilic, alkaliphilic and halotolerant, as the strain grew at 25-50 °C (optimum, 45 °C), at pH 7.0-10.0 (optimum, pH 8.0) and in the presence of 0-6 % NaCl (optimum, 0 %). Analysis of 16S rRNA gene sequences indicated that MMS20-4M-10-YT fell into a phylogenetic cluster belonging to the genus Brevibacillus. Strain MMS20-4M-10-YT showed the highest 16S rRNA gene sequence similarity to Brevibacillus marinus SCSIO 07484T (96.7 %). Based on the reults of orthologous average nucleotide identity analysis, MMS20-4M-10-YT was again mostly related to B. marinus SCSIO 07484T with 78.0 % identity, which also shared the highest average nucleotide identity of 68.0 %. In contrast, the digital DNA-DNA relatedness analysis indicated that Aneureibacillus migulanus DSM 2895T was the closest species with 29.5 % similarity. The genome-based analyses indicated that all compared species showed low genomic relatedness with MMS20-4M-10-YT. The major fatty acids of the strain were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0, the major respiratory quinone was MK-7, the diagnostic polar lipids were phosphatidyl-N-methylethanolamine, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, and diagnostic cell-wall diamino acid was meso-diaminopimelic acid, which was consistent with the general chemotaxonomic features of the genus. The total length of the genome was 4.91 Mbp and the DNA G+C content was 51.8 mol%. Based on both phenotypic and phylogenetic evidence, strain MMS20-4M-10-YT should be classified as representing a novel species of the genus Brevibacillus, for which a name Brevibacillus humidisoli sp. nov. (type strain=MMS20-4M-10-YT=KCTC 43333T=LMG 32359T) is proposed.

Brevibacillus ruminantium sp. nov., isolated from cow faeces.

An aerobic, Gram-stain-positive, rod-shaped, endospore-forming bacterial strain, designated BB3-R1T, was isolated from cow faeces sampled in Daejeon, Republic of Korea. Growth was observed at 25-45 °C (optimum, 35-40 °C) and pH 7.0-9.0 (optimum, pH 8.0), with up to 3 % (w/v) NaCl (optimum, 0 % NaCl). blast analysis of 16S rRNA gene sequences revealed the highest sequence similarity of strain BB3-R1T to Brevibacillus borstelensis NRRL NRS-818T (98.8 %) followed by Brevibacillus panacihumi JCM 15085T (97.5 %). According to 16S rRNA gene and whole-genome based phylogenetic trees, strain BB3-R1T clustered with Brevibacillus composti FJAT-54423T and B. borstelensis NRRL NRS-818T. OrthoANI and dDDH values of strain BB3-R1T with the closely related strains were lower than 77.5 and 26.8 %, respectively. The major menaquinones and polar lipids of the strain were MK-7 and phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, respectively. The major fatty acids (>10 %) were C14 : 0 iso, C15 : 0 iso, C15 : 0 anteiso and C16 : 1 ω7c alcohol. The cell-wall peptidoglycan contained cross-linked meso-diaminopimelic acid (type A1 gamma). The phenotypic, chemotaxonomic and genotypic data obtained in this study showed that the strain represents a novel species of the genus Brevibacillus, for which the name Brevibacillus ruminantium sp. nov. (type strain BB3-R1T=KACC 22663T=NBRC 115962T) is proposed.

Brevicillin, a novel lanthipeptide from the genus Brevibacillus with antimicrobial, antifungal, and antiviral activity.

AIM: This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. METHODS AND RESULTS: The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed >30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 µg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 µg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. CONCLUSION: This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

Linocin M18 protein from the insect pathogenic bacterium Brevibacillus laterosporus isolates.

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.

Production of recombinant His-tagged triple-FLAG peptide in Brevibacillus choshinensis and its utilization as an easy-to-remove affinity peptide.

Triple-FLAG (3 × FLAG)-tagged proteins can be affinity purified through binding to an anti-FLAG antibody and competitive elution with excess free 3 × FLAG peptide. To expand the availability of the 3 × FLAG purification system, we produced a recombinant His-tagged 3 × FLAG peptide in Brevibacillus choshinensis. The screening of connecting linkers between His-tag and the 3 × FLAG peptide, culture containers, and culture media showed that the His-tagged 3 × FLAG peptide with an LA linker was most expressed in 2SY medium using a baffled shake flask. The peptide was affinity-purified to give a yield of about 25 mg/L of culture. The peptide was effective for eluting 3 × FLAG-tagged α-amylase from anti-FLAG magnetic beads. Finally, the peptide remaining in the amylase fraction was removed by His-tag affinity purification. These results show that the recombinant His-tagged 3 × FLAG peptide can function as an easy-to-remove affinity peptide in the 3 × FLAG purification system.

Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis Improved the Anti-MRSA Activity of Brevibacillus sp. SPR20.

Brevibacillus sp. SPR20 produced potentially antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). The synthesis of these substances is controlled by their biosynthetic gene clusters. Several mutagenesis methods are used to overcome the restriction of gene regulations when genetic information is absent. Atmospheric and room temperature plasma (ARTP) is a powerful technique to initiate random mutagenesis for microbial strain improvement. This study utilized an argon-based ARTP to conduct the mutations on SPR20. The positive mutants of 40% occurred. The M27 mutant exhibited an increase in anti-MRSA activity when compared to the wild-type strain, with the MIC values of 250-500 and 500 µg/mL, respectively. M27 had genetic stability because it exhibited constant activity throughout fifteen generations. This mutant had similar morphology and antibiotic susceptibility to the wild type. Comparative proteomic analysis identified some specific proteins that were upregulated in M27. These proteins were involved in the metabolism of amino acids, cell structure and movement, and catalytic enzymes. These might result in the enhancement of the anti-MRSA activity of the ARTP-treated SPR20 mutant. This study supports the ARTP technology designed to increase the production of valuable antibacterial agents.

Multiple S-Layer Proteins of Brevibacillus laterosporus as Virulence Factors against Insects.

S-layers are involved in the adaptation of bacteria to the outside environment and in pathogenesis, often representing special virulence factors. Vegetative cells of the entomopathogenic bacterium Brevibacillus laterosporus are characterized by an overproduction of extracellular surface layers that are released in the medium during growth. The purpose of this study was to characterize cell wall proteins of this bacterium and to investigate their involvement in pathogenesis. Electron microscopy observations documented the presence of multiple S-layers, including an outermost (OW) and a middle (MW) layer, in addition to the peptidoglycan layer covering the plasma membrane. After identifying these proteins (OWP and MWP) by mass spectrometry analyses, and determining their gene sequences, the cell wall multilayer-released fraction was successfully isolated and used in insect bioassays alone and in combination with bacterial spores. This study confirmed a central role of spores in bacterial pathogenicity to insects but also detected a significant virulence associated with fractions containing released cell wall multilayer proteins. Taken together, S-layer proteins appear to be part of the toxins and virulence factors complex of this microbial control agent of invertebrate pests.

Comprehensive bacterial-metabolite profiles of Hawaijar, Bekang, and Akhone: a comparative study on traditional fermented soybeans of north-east India.

Preparation of traditionally fermented soybeans varies across ethnicities with distinct tastes, flavour, and nutritional values. The fermented soybean varieties Hawaijar, Bekang, and Akhone of north-east India are associated with diverse ethnic groups from Manipur, Mizoram, and Nagaland, respectively. These varieties differ in substrate and traditional practice that exerts differential bacterial-metabolite profile, which needs an in-depth analysis i. Culture-dependent and independent techniques investigated the bacterial diversity of the fermented soybean varieties. Gas chromatography and mass spectroscopy (GC-MS) studied these varieties' metabolite profiles. The common dominant bacterial genera detected in Hawaijar, Bekang, and Akhone were Bacillus, Ignatzschinaria, and Corynebacterium, with the presence of Brevibacillus and Staphylococcus exclusively in Hawaijar and Oceanobacillus in Bekang and Akhone. The metabolite analysis identified a higher abundance of essential amino acids, amino and nucleotide sugars, and vitamins in Hawaijar, short-chain fatty acids in Bekang, polyunsaturated fatty acids in Akhone and Hawaijar, and prebiotics in Akhone. The bacteria-metabolite correlation analysis predicted four distinct bacterial clusters associated with the differential synthesis of the functional metabolites. While B. subtilis is ubiquitous, cluster-1 comprised B. thermoamylovorans/B. amyloliquefaciens, cluster-2 comprised B. tropicus, cluster-3 comprised B. megaterium/B. borstelensis, and cluster-4 comprised B. rugosus. To the best of our knowledge, this is the first comparative study on traditional fermented soybean varieties of north-east India linking bacterial-metabolite profiles which may help in designing starters for desired functionalities in the future.

Broad-spectrum cytotoxicity to cancer cells of Brevilaterin C from Brevibacillus laterosporus and its specific mechanism on human epidermal cancer cells.

Antimicrobial peptides (AMP) from Brevibacillus laterosporus have good prospects as clinical treatments for cancer. Nevertheless, details about their anticancer spectrum and mode of cytotoxicity remain poorly understood. A newly found AMP (named Brevilaterin C) secreted by B. laterosporus S62-9 exhibited strong inhibition on almost cancer cell lines examined at a concentration of 8 µg/ml but was relatively safe for normal cells. We further systematically examined its cytotoxicity and mechanism toward human epidermal cancer cell A431. A dosage of 3 µg/ml of Brevilaterin C could significantly increase lactate dehydrogenase release of tumor cells. Moreover, it could remarkably increase the ratio of apoptosis and reactive oxygen species generation of A431, indicating effective induction of apoptosis. Moreover, the formation of JC-1 aggregates was effectively prevented by a low concentration of Brevilaterin C, indicating its effective induction of A431's apoptosis. Brevilaterin C exhibited broad-spectrum cytotoxicity to cancer cells, indicating a good potential prospect in the medical field.

Isolation, purification, and characterization of a novel exopolysaccharide isolated from marine bacteria Brevibacillus borstelensis M42.

Marine microbes produce polysaccharides with unique physicochemical and functional properties that help them survive in harsh marine environments. However, only a handful of marine exopolysaccharides (EPSs) have been reported to date. The present study explored the seashore of Visakhapatnam, India, to report a novel exopolysaccharide designated as Br42 produced by Brevibacillus borstelensis M42. The isolate was identified through morphological, biochemical, phylogenetic, and genome sequencing analysis. The studies on fermentation kinetics revealed that EPS Br42 was a primary metabolite with a maximum production of 1.88 ± 0.02 g/L after 60 h when production broth was fortified with 2% glucose. Additionally, EPS Br42 was found to be a heteropolysaccharide consisting of glucose and galacturonic acid with a molecular weight of about 286 kDa. Interestingly, this molecule possesses industrially relevant functional properties such as water-holding (510 ± 0.35%), oil-holding (374 ± 0.12% for coconut oil and 384 ± 0.35% for olive oil), and swelling capacities (146.6 ± 5.75%). EPS Br42 could form an emulsion that was stable at a wide pH range for about 72 h and, in fact, performed better as compared to Span 20, a commercially used synthetic emulsifier. Moreover, this EPS was also found to be heat stable and exhibited non-Newtonian pseudoplastic behavior. These physicochemical and functional properties of polysaccharides suggest that the EPS Br42 has potential for multifarious industrial applications as an emulsifier, stabilizer, viscosifier, and binding agent.

Addition of arginine hydrochloride and proline to the culture medium enhances recombinant protein expression in Brevibacillus choshinensis: The case of RBD of SARS-CoV-2 spike protein and its antibody.

Brevibacillus choshinensis is a gram-positive bacterium that is known to efficiently secrete recombinant proteins. However, the expression of these proteins is often difficult depending upon the expressed protein. In this study, we demonstrated that the addition of arginine hydrochloride and proline to the culture medium dramatically increased protein expression. By culturing bacterial cells in 96-well plates, we were able to rapidly examine the expression conditions and easily scale up to 96 mL of culture for production. Although functional expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein without any solubility-enhancing tag in bacterial strains (including Escherichia coli) has not been reported to date, we succeeded in efficiently producing RBD which showed a similar CD spectrum to that of RBD produced by eukaryotic cell expression systems. Furthermore, RBD from the omicron variant (B.1.1.529) was also produced. Physicochemical analyses indicated that omicron RBD exhibited markedly increased instability compared to the wild-type. We also revealed that the Fab format of the anti-SARS-CoV-2 antibody C121 can be produced in large quantities using the same expression system. The obtained C121 Fab bound to wild-type RBD but not to omicron RBD. These results strongly suggest that the Brevibacillus expression system is useful for facilitating the efficient expression of proteins that are difficult to fold and will thus contribute to the rapid physicochemical evaluation of functional proteins.