Structure and engineering of Brevibacillus laterosporus Cas9.

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.

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.

Mechanism of Brevibacillus brevis strain TR-4 against leaf disease of Photinia×fraseri Dress.

Background: Colletotrichum species are among the most common pathogens in agriculture and forestry, and their control is urgently needed. Methods: In this study, a total of 68 strains of biocontrol bacteria were isolated and identified from Photinia × fraseri rhizosphere soil. Results: The isolates were identified as Brevibacillus brevis by 16S rRNA. The inhibitory effect of TR-4 on Colletotrichum was confirmed by an in vitro antagonistic experiment. The inhibitory effect of TR-4 was 98% at a concentration of 10 µl/ml bacterial solution, protection of the plant and inhibition of C. siamense was evident. Moreover, the secretion of cellulase and chitosan enzymes in the TR-4 fermentation liquid cultured for three days was 9.07 mol/L and 2.15 µl/mol, respectively. Scanning electron microscopy and transmission electron microscopy confirmed that TR-4 destroyed the cell wall of C. siamense, resulting in leakage of the cell contents, thus weakening the pathogenicity of the bacteria.

Edeine B1 produced by Brevibacillus brevis reduces the virulence of a plant pathogenic fungus by inhibiting mitochondrial respiration.

Plant pathogenic fungi cause serious diseases, which result in the loss of crop yields and reduce the quality of crops worldwide. To counteract the escalating risks of chemical fungicides, interest in biological control agents to manage plant diseases has significantly increased. In this study, we comprehensively screened microbial culture filtrates using a yeast screening system to find microbes exhibiting respiratory inhibition activity. Consequently, we found a soil-borne microbe Brevibacillus brevis HK544 strain exhibiting a respiration inhibitory activity and identified edeine B1 (EB1) from the culture filtrate of HK544 as the active compound of the respiration inhibition activity. Furthermore, against a plant pathogenic fungus Fusarium graminearum, our results showed that EB1 has effects on multiple aspects of respiration with the downregulation of most of the mitochondrial-related genes based on transcriptome analysis, differential EB1-sensitivity from targeted mutagenesis, and the synergistic effects of EB1 with electron transport chain complex inhibitors. With the promising plant disease control efficacy of B. brevis HK544 producing EB1, our results suggest that B. brevis HK544 has potential as a biocontrol agent for Fusarium head blight.IMPORTANCEAs a necrotrophic fungus, Fusarium graminearum is a highly destructive pathogen causing severe diseases in cereal crops and mycotoxin contamination in grains. Although chemical control is considered the primary approach to control plant disease caused by F. graminearum, fungicide-resistant strains have been detected in the field after long-term continuous application of fungicides. Moreover, applying chemical fungicides that trigger mycotoxin biosynthesis is a great concern for many researchers. Biocontrol of Fusarium head blight (FHB) by biological control agents (BCAs) represents an alternative approach and could be used as part of the integrated management of FHB and mycotoxin production. The most extensive studies on bacterial BCAs-fungal communications in agroecosystems have focused on antibiosis. Although many BCAs in agricultural ecology have already been used for fungal disease control, the molecular mechanisms of antibiotics produced by BCAs remain to be elucidated. Here, we found a potential BCA (Brevibacillus brevis HK544) with a strong antifungal activity based on the respiration inhibition activity with its active compound edeine B1 (EB1). Furthermore, our results showed that EB1 secreted by HK544 suppresses the expression of the mitochondria-related genes of F. graminearum, subsequently suppressing fungal development and the virulence of F. graminearum. In addition, EB1 exhibited a synergism with complex I inhibitors such as rotenone and fenazaquin. Our work extends our understanding of how B. brevis HK544 exhibits antifungal activity and suggests that the B. brevis HK544 strain could be a valuable source for developing new crop protectants to control F. graminearum.

High-throughput system for the thermostability analysis of proteins.

Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high-throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate-scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per-plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single-chain variable fragment of nivolumab, harvesting the melting temperature (Tm) ranging from -9.3 up to +10.8°C compared to the wild-type sequence. Our findings will allow for data-driven stabilization in protein design and streamlining the rational approaches.

Mechanism of deltamethrin biodegradation by Brevibacillus parabrevis BCP-09 with proteomic methods.

Ester-containing deltamethrin pesticides are widely used in farmland and have inevitable side effects on the biosphere and human health. Microbia have been used for efficient degradation of deltamethrin, but the related mechanism and enzyme characteristics have not been elucidated. In this study, a species Brevibacillus parabrevis BCP-09 could degrade up to 75 mg L-1 deltamethrin with a degradation efficiency of 95.41%. Proteomic and genomic methods were used to explore its degradation mechanism. Enzymes belonged to hydrolases, oxidases and aromatic compound degrading enzymes were expressed enhanced and might participate in the deltamethrin degradtion. RT-PCR experiment and enzyme activity analysis verified the degradation of deltamethrin by bacterial protein. Additionally, the formation of endospores can help strain BCP-09 resist the toxicity of deltamethrin and enhance its degradation. This study supplies a scientific evidence for the application of Brevibacillus parabrevis BCP-09 in the bioremediation of environmental pollution and enriches the resources of deltamethrin-biodegradable proteins.

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.

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.

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.

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.

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.

Enhancing Mechanisms of the Plant Growth-Promoting Bacterial Strain Brevibacillus sp. SR-9 on Cadmium Enrichment in Sweet Sorghum by Metagenomic and Transcriptomic Analysis.

To explore the mechanism by which the plant growth-promoting bacterium Brevibacillus sp. SR-9 improves sweet sorghum tolerance and enriches soil cadmium (Cd) under pot conditions, the effect of strain SR-9 inoculation on the microbial community of sorghum rhizosphere soil was analyzed by metagenomics. Gene expression in sweet sorghum roots was analyzed using transcriptomics. The results showed that strain SR-9 promoted the growth of sweet sorghum and improved the absorption and enrichment of Cd in the plants. Compared with the uninoculated treatment, the aboveground part and root dry weight in strain SR-9 inoculated with sorghum increased by 21.09% and 17.37%, respectively, and the accumulation of Cd increased by 135% and 53.41%, respectively. High-throughput sequencing showed that strain SR-9 inoculation altered the rhizosphere bacterial community, significantly increasing the relative abundance of Actinobacteria and Firmicutes. Metagenomic analysis showed that after inoculation with strain SR-9, the abundance of genes involved in amino acid transport metabolism, energy generation and conversion, and carbohydrate transport metabolism increased. KEGG functional classification showed that inoculation with strain SR-9 increased the abundance of genes involved in soil microbial metabolic pathways in the rhizosphere soil of sweet sorghum and the activity of soil bacteria. Transcriptome analysis identified 198 upregulated differentially expressed genes in sweet sorghum inoculated with strain SR-9, including those involved in genetic information processing, biological system, metabolism, environmental information processing, cellular process, and human disease. Most of the annotated differentially expressed genes were enriched in the metabolic category and were related to pathways such as signal transduction, carbohydrate metabolism, amino acid metabolism, and biosynthesis of other secondary metabolites. This study showed that plant growth-promoting bacteria can alter the rhizosphere bacterial community composition, increasing the activity of soil bacteria and upregulating gene expression in sweet sorghum roots. The findings enhance our understanding of the microbiological and botanical mechanisms by which plant growth-promoting bacterial inoculation improves the remediation of heavy metals by sorghum.

Swarming behavior of a novel strain of Brevibacillus thermoruber.

Brevibacillus thermoruber strain Nabari was isolated from compost and identified based on 16 S rRNA gene sequencing and DNA-DNA hybridization using B. thermoruber DSM 7064 T as the standard, despite some differences in their physiological and structural characteristics. When B. thermoruber Nabari was cultivated on various solid media containing 1.5% agar at 60°C, it rapidly propagated over the entire plate. In particular, on R2A-agar medium, it formed fine dendritic colonies. Macroscopic and microscopic observations of peripheral regions of the colonies indicated that the dendritic patterns were formed by bacterial swarming of some of the cells; large flows of bacterial cell populations were observed in the peripheral regions of the dendritic colonies. The cells were highly flagellated, but no extreme elongation of cells was observed. When B. thermoruber Nabari cells were cultivated at 37°C on R2A-agar plates, most colonies were nonmotile, but some colonies were motile. For example, a wandering colony moved on the plate and split into two, and then they collided to become one again. Additionally, a simple incubation system was devised to record the movement of colonies at high temperatures in this study while protecting the cameras from thermal damage.

Insights into the biodegradation and heavy metal resistance potential of the genus Brevibacillus through comparative genome analyses.

Members of the genus Brevibacillus belonging to the familyPaenibacillaceae are Gram-positive/variable, endospore-forming, and rod-shaped bacteria that dwell in various environmental habitats. Brevibacillus spp. have a wide range of enzyme activities such as degradation of various carbohydrates, plastics, and they possess resistance against heavy metals. These characteristics make them encouraging contenders for biotechnological applications.In this work, we analyzed the reference genomes of 19Brevibacillusspecies, focusing on discovering the biodegradation and heavy metal resistance capabilities of this little studied genus from genomic data. The results indicate that several strain specific traits were identified. For example Brevibacillus halotolerans s-14, and Brevibacillus laterosporus DSM 25 have more glycoside hydrolases (GHs) compared to other carbohydrate-active enzymes, and therefore might be more suitable for biodegradation of carbohydrates. In contrast, strains such as Brevibacillus antibioticus TGS2-1, with a higher number of glycosyltransfereases (GTs) may aid in the biosynthesis of complex carbohydrates. Our results also suggest some correlation between heavy metal resistance and polyurethane degradation, thus indicating that heavy metal resistance strains (e.g. Brevibacillus reuszeri J31TS6) can be a promising source of enzymes for polyurethane degradation. These strain specific features make the members of this bacterial group potential candidates for further investigations with industrial implications. This work also represents the first exhaustive study of Brevibacillus at the genome scale.

Whole-genome analysis and secondary metabolites production of a new strain Brevibacillus halotolerans 7WMA2: A potential biocontrol agent against fungal pathogens.

The extensive usage of synthetic fungicides against fungal diseases has caused adverse impacts on both human and agricultural crops. Therefore, the current study aims to establish a new bacterium 7WMA2, as a biocontrol agent to achieve better antifungal results. The strain 7WMA2 was isolated from marine sediment, displayed a broad spectrum of several fungi that includes Alternaria alternata, Cladosporium sp., Candida albicans, Fusarium oxysporum, Trichosporon pullulans, and Trichophyton rubrum. The 16S rRNA phylogeny inferred that strain 7WMA2 was a member of Brevibacillus. The phylogenetic and biochemical analyses revealed that the strain 7WMA2 belongs to the species of Brevibacillus halotolerans. The complete genome sequence of Brevibacillus halotolerans 7WMA2 consists of a circular chromosome of 5,351,077 bp length with a GC content of 41.39 mol %, including 4433 CDS, 111 tRNA genes, and 36 rRNA genes. The genomic analysis showed 23 putative biosynthetic secondary metabolite gene clusters responsible for non-ribosomal peptides, polyketides and siderophores. The antifungal compounds concentrated from cell-free fermentation broth demonstrated strong inhibition of fungi, and the compounds are considerably thermal stable and adaptable to pH range 2-12. This complete genome sequence has provided insight for further exploration of antagonistic ability and its secondary metabolite compounds indicated feasibility as biological control agents against fungal infections.

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.

Brevibacillus dissolubilis sp. nov., Isolated from Fresh Water.

A Gram-positive-staining, strictly aerobic, motile, ellipsoidal endospore-forming bacterial strain, designated CHY01T, was isolated from the Chishui river in a section of Maotai Town, Guizhou Province, Southwest China. Strain CHY01T was found to grow optimally at pH 8.0 and 28 °C. The 16S rRNA gene sequence analysis indicated that strain CHY01T belonged to the genus Brevibacillus and clustered with the type strain of Brevibacillus panacihumi, with which it exhibited 16S rRNA gene sequence similarity values of 97.8%. The predominant respiratory quinone was MK-7, and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major fatty acids were C14:0, iso-C15:0, anteiso-C15:0, C16:0, C15:1iso-H and/or C13:0 3-OH, and C16:1ω7c and/or C16:1ω6c. Genome sequencing revealed a genome size of 6.1 Mbp and a G + C content of 50.6%. The results of physiological and biochemical tests allowed strain CHY01T to be distinguished genotypically and phenotypically from Brevibacillus species with validly published names. Pairwise determined whole-genome average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values suggested that strain CHY01T represents a new species, for which we propose the name Brevibacillus dissolubilis sp. nov. with the type strain CHY01T (= CGMCC 1.15916 T = KCTC 33863 T).

Characterization of biosorption potential of Brevibacillus biomass isolated from contaminated water resources for removal of Pb (II) ions.

Various activities of different industries are found to be the main reason for water pollution with heavy metals. Use of microorganisms that are tolerant even of a high concentration of metal ions could be a valuable tool for remediation of contaminated water resources. In the present study, microorganisms that showed high resistance to lead ions were isolated and evaluated for biosorption efficiency for removal of lead ions from waste water. Biochemical identification and 16S rRNA gene sequence analysis indicated that the isolated strain was Brevibacillus. The conditions of pH, biomass concentration, temperature, time, agitation and Initial concentration of metal for biosorption of Pb (II) were optimized. Based on induction coupled plasma optical emission spectroscopy (ICP-OES) analysis, the biosorption efficiency of Brevibacillus at optimized conditions of initial metal concentration of 150 µg/mL, 1 g/L of biomass dose, pH 6.0, 40 °C, for 12 h at 80 rpm was 78.58% and the biosorption capacity (qe) is 128.58 mg/g of the biosorbent. Of the three isotherm models investigated, the Freundlich isotherm model was identified as a good fit with high correlation coefficient, while kinetic data followed the pseudo first order model as best fit. Surface characterization by scanning electron microscopy (SEM) analysis revealed morphological changes with a bulged rod-shape cell having metal depositions and rough texture. The presence of lead within the cell was detected by transmission emission microscopy (TEM). The key functional groups that participate in biosorption were analyzed by Fourier transform infrared (FTIR) spectroscopy and were found to be carboxyl, hydroxyl, amino and phosphate groups. From the real-time study, it proves that the biomass of Brevibacillus can be used as a promising biosorbent for removal of metals including lead from waste water.

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.

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.

BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).