Antimicrobial peptides produced by <Emphasis Type="Italic">Brevibacillus</Emphasis> spp.: structure,classification and bioactivity: a mini review

Species that are currently listed under the genus Brevibacillus (formerly, Bacillus brevis cluster) have been a rich source of antimicrobial peptides for many decades. The first known peptide antibiotic, gramicidin, is presumed to be produced by a Brevibacillus sp. Members of the genus are widely spread in nature. They can be found in a variety of environments including intestinal tracts of animals, seawater, and soil. Some Brevibacillus strains have been used commercially as probiotics. Bioactive peptides produced by Brevibacillus spp. include antibacterial, antifungal and anti-invertebrate agents. Brevibacillus antimicrobial peptides are synthesized through ribosomal or nonribosomal pathway; these two groups can be further categorized based on specific structural features such as cyclization and presence of lipid chain. Some of the antimicrobial compounds produced by this genus share structural similarities that were overlooked previously. For example, the structural similarity between BT peptide, brevibacillin, and bogorol was revealed only recently. Here we review and classify Brevibacillus antimicrobial peptides and summarize their bioactivities and potential applications.     

Isolation,identification of sludge-lysing strain and its utilization in thermophilic aerobic digestion for waste activated sludge

A strain of sludge-lysing bacteria was isolated from waste activated sludge (WAS) in this study. The result of 16S rRNA gene analysis demonstrated that it was a species of new genus Brevibacillus (named Brevibacillus sp. KH3). The strain could release the protease with molecule weight of about 40 kDa which could enhance the efficiency of sludge thermophilic aerobic digestion. During the sterilized sludge digestion experiment inoculated with Brevibacillus sp. KH3, the maximum protease activity was 0.41 U/ml at pH 8 and 50 °C, and maximum TSS removal ratio achieved 32.8% after 120 h digestion at pH 8 and 50 °C. In the case of un-sterilized sludge digestion inoculated with Brevibacillus sp. KH3, TSS removal ratio in inoculated-group was 54.8%, increasing at 11.86% compared with un-inoculation (46.2%). The result demonstrated that inoculation of Brevibacillus sp. KH3 could help to degrade the EPS and promote the collapse of cells and inhibit the growth of certain kinds of microorganisms. It indicated that Brevibacillus sp. KH3 strain had a high potential to enhance WAS-degradation efficiency in thermophilic aerobic digestion.     

Enrichment,isolation, and characterization of phenol-degrading Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6

The objectives of this research were to isolate pure phenol-degrading strains from enriched mixed cultures, monitoring the variations of species during the enrichment period. Two strains were isolated from the acclimated mixed culture. They were identified as Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6. DGGE indicated that strain P. resinovorans appeared at the beginning, and maintained well during the enrichment period. The second strain, Brevibacillus sp., did not appear in the initial stage, but showed up after 2 weeks of enrichment. The optimum growth temperatures for P. resinovorans and Brevibacillus sp. were 31 and 39 °C, respectively. P. resinovorans could degrade phenol completely within 57.5 h, when the initial phenol concentration was lower than 600 mg l⁻¹. If the initial phenol concentration was lower than 200 mg l⁻¹, Brevibacillus sp. could remove phenol completely within 93.1 h. It was obvious that the phenol-degrading ability of P. resinovorans was much better than that of Brevibacillus sp. The metabolic pathway for P. resinovorans phenol degradation was assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

Due to the worldwide prevalence of multidrug-resistant pathogens and high incidence of diseases such as cancer, there is an urgent need for the discovery and development of new drugs. Nearly half of the FDA-approved drugs are derived from natural products that are produced by living organisms, mainly bacteria, fungi, and plants. Commercial development is often limited by the low yield of the desired compounds expressed by the native producers. In addition, recent advances in whole genome sequencing and bioinformatics have revealed an abundance of cryptic biosynthetic gene clusters within microbial genomes. Genetic manipulation of clusters in the native host is commonly used to awaken poorly expressed or silent gene clusters, however, the lack of feasible genetic manipulation systems in many strains often hinders our ability to engineer the native producers. The transfer of gene clusters into heterologous hosts for expression of partial or entire biosynthetic pathways is an approach that can be used to overcome this limitation. Heterologous expression also facilitates the chimeric fusion of different biosynthetic pathways, leading to the generation of “unnatural” natural products. The genus Streptomyces is especially known to be a prolific source of drugs/antibiotics, its members are often used as heterologous expression hosts. In this review, we summarize recent applications of Streptomyces species, S. coelicolor, S. lividans, S. albus, S. venezuelae and S. avermitilis, as heterologous expression systems.     

Identification, purification and characterization of laterosporulin, a novel bacteriocin produced by Brevibacillus sp. strain GI-9

Background

Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology.

Methodology/Findings

The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database.

Conclusions

We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.     

Lipases from the genus Rhizopus: Characteristics,expression, protein engineering and application

Lipases are versatile catalysts that hydrolyze ester bonds of water-insoluble glycerides or carry out reversible reactions at the water/lipid interface. The remarkable characteristics of lipases from the genus Rhizopus are their high sn-1,3-positional specificity, enantioselectivity and activity in nonaqueous media, which make them one of the most desirable enzymes for many applications, including lipid modification and biodiesel and chiral organic compound synthesis. sn-1,3-Position-specific Rhizopus lipases are particularly useful for the production of structured triacylglycerols. Significant progress has been made regarding lipases from the genus Rhizopus, including gene sequencing, elucidation of the protein structure and catalytic function, heterologous expression and redesigning Rhizopus lipases for valuable properties, which is receiving increasing academic and industrial attention. In this review, we present a comprehensive overview of Rhizopus lipases, focusing on (a) the characteristics of Rhizopus lipases, (b) Rhizopus lipase genes and structural features, (c) strategies for heterologous expression of Rhizopus lipase genes in yeast system, (d) progress in protein engineering for the improvement of the properties of Rhizopus lipases, and (e) development of biotechnological applications.     

Metabolites produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. enable a Gram-positive bacterium to achieve extracellular electron transfer

Previous studies revealed the abundance of Pseudomonas sp. in the microbial community of a microbial fuel cell (MFC). These bacteria can transfer electrons to the electrode via self-produced phenazine-based mediators. A MFC fed with acetate where several Pseudomonas sp. were present was found to be rich in a Gram-positive bacterium, identified as Brevibacillus sp. PTH1. Remarkably, MFCs operated with only the Brevibacillus strain in their anodes had poor electricity generation. Upon replacement of the anodic aqueous part of Brevibacillus containing MFCs with the cell-free anodic supernatants of MFCs operated with Pseudomonas sp. CMR12a, a strain producing considerable amounts of phenazine-1-carboxamide (PCN) and biosurfactants, the electricity generation was improved significantly. Supernatants of Pseudomonas sp. CMR12a_Reg, a regulatory mutant lacking the ability to produce PCN, had no similar improvement effect. Purified PCN, together with rhamnolipids as biosurfactants (1 mg L⁻¹), could clearly improve electricity generation by Brevibacillus sp. PTH1, as well as enable this bacterium to oxidize acetate with concomitant reduction of ferric iron, supplied as goethite (FeOOH). When added alone, PCN had no observable effects on Brevibacillus’ electron transfer. This work demonstrates that metabolites produced by Pseudomonas sp. enable Gram-positive bacteria to achieve extracellular electron transfer. Possibly, this bacterial interaction is a key process in the anodic electron transfer of a MFC, enabling Brevibacillus sp. PTH1 to achieve its dominance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner

Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649–658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The “Brevibacillus in vivo cloning” method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.     

Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.     

Isolation and Characterization of Marine Brevibacillus sp. S-1 Collected from South China Sea and a Novel Antitumor Peptide Produced by the Strain

A Gram-positive, rod-shaped bacterium, designated as S-1, was isolated from a marine sediment sample collected from South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence showed that S-1 belongs to the genus Brevibacillus. A novel cytotoxic peptide was isolated from the fermentation broth of the marine-derived bacterium Brevibacillus sp. S-1, using ion-exchange chromatography and reverse-phase HPLC chromatography. The molecular weight of this peptide was determined as 1570 Da by MALDI-TOF mass spectrometry, and its structure was proposed as a cyclic peptide elucidated by MALDI-TOF/TOF mass spectrometry and de novo sequencing. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that this peptide exhibited cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, U251 human glioma cells and MCF-7 human breast carcinoma cells. Additionally, SBP exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells. The result suggested that the cytotoxic effect of the peptide is specific to tumor cells.     

A Brevibacillus sp. antagonistic to mycotoxigenic Fusarium spp.

Antagonistic microbes were isolated from soils to control mycotoxin contamination of cereals by limiting the growth of mycotoxigenic Fusarium species. In total, 341 bacterial isolates were examined for antifungal activity against eight mycotoxigenic Fusarium species using dual culture assays. The screening identified 11 isolates that inhibited mycelial growth of all Fusarium species tested. The culture filtrates of 2 of the 11 isolates completely inhibited germination of conidia up to 21 days of incubation. These two isolates exhibited identical activity toward the fungi tested and were identified as Brevibacillus spp. based on 16S rRNA sequence homology. The most closely related species based on phylogenetic analysis was Brevibacillus reuszeri. Additional dual culturing using further fungal species showed that the antagonistic Brevibacillus inhibited the growth of most Fusarium species tested (39 of 46 species), two Epicoccum spp., one Alternaria sp., three Aspergillus spp. (3 of 11), and three Penicillium spp. (3 of 8). The in vivo assay was performed to test the efficacy of antagonistic Brevibacillus isolates on maize ears and revealed that the application of microbes suppressed ear rot (ANOVA, p = 0.0020). This Brevibacillus sp. may be an antagonist of the majority of Fusarium species, including mycotoxigenic species.     

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

Current biotechnological applications of the genus Amycolatopsis

Recently there has been increasing interest in possible biotechnological applications of the bacterial genus Amycolatopsis. This genus originally attracted attention for its antibiotic producing capabilities; although it is actually a multifaceted genus and a more diverse range of studies involving biotechnological processes have now been undertaken. Several works have demonstrated that the versatility shown by these bacteria is valuable in industrial applications. Here, we provide a condensed overview of the most important biotechnological applications such as bioremediation, biodegradation and bioconversion, as well as aspects that need to be explored further in order to gain a fuller insight into this genus, including its possible potential in the production of biofuel. Antibiotic production is not discussed since this is well covered by the latest edition of Bergey’s Manual of Systematic Bacteriology. To our knowledge this is the first report highlighting the versatility and biotechnological potential of the genus Amycolatopsis.     

Description of a methanotrophic strain BOH1, isolated from Al-Bohyriya well,Al-Ahsa City,Saudi Arabia

Methanotrophic bacteria have a unique ability to utilize methane as their carbon and energy sources. Therefore, methanotrophs play a key role in suppressing methane emissions from different ecosystems and hence in alleviating the global climate change. Despite methanotrophs having many ecological, economical and biotechnological applications, little is known about this group of bacteria in Al-Ahsa. Therefore, the main objective of the current work was to expand our understanding of methane oxidizing bacteria in Al-Ahsa region. The specific aim was to describe a methanotrophic strain isolated from Al-Bohyriya well, Al-Ahsa using phenotypic, genotypic (such as 16S rRNA and pmoA gene sequencing) and phylogenetic characterization. The results indicated that the strain belongs to the genus Methylomonas that belongs to Gammaproteobacteria as revealed by the comparative sequence analysis of the 16S rRNA and pmoA genes. There is a general agreement in the profile of the phylogenetic trees based on the sequences of 16srRNA and pmoA genes of the strain BOH1 indicating that both genes are efficient taxonomic marker in methanotrophic phylogeny. The strain possesses the particulate but not the soluble methane monooxygenase as a key enzyme for methane metabolism. Further investigation such as DNA:DNA hybridization is needed to assign the strain as a novel species of the genus Methyomonas and this will open the door to explore the talents of the strain for its potential role in alleviating global warming and biotechnological applications in Saudi Arabia such as bioremediation of toxic by-products released in oil industry. In addition, the strain enhances our knowledge of methanotrophic bacteria and their adaptation to desert ecosystems.     

Genome mining of the Streptomyces avermitilis genome and development of genome-minimized hosts for heterologous expression of biosynthetic gene clusters

To date, several actinomycete genomes have been completed and annotated. Among them, Streptomyces microorganisms are of major pharmaceutical interest because they are a rich source of numerous secondary metabolites. S. avermitilis is an industrial microorganism used for the production of an anthelmintic agent, avermectin, which is a commercially important antiparasitic agent in human and veterinary medicine, and agricultural pesticides. Genome analysis of S. avermitilis provides significant information for not only industrial applications but also understanding the features of this genus. On genome mining of S. avermitilis, the microorganism has been found to harbor at least 38 secondary metabolic gene clusters and 46 insertion sequence (IS)-like sequences on the genome, which have not been searched so far. A significant use of the genome data of Streptomyces microorganisms is the construction of a versatile host for heterologous expression of exogenous biosynthetic gene clusters by genetic engineering. Since S. avermitilis is used as an industrial microorganism, the microorganism is already optimized for the efficient supply of primary metabolic precursors and biochemical energy to support multistep biosynthesis. The feasibility of large-deletion mutants of S. avermitilis has been confirmed by heterologous expression of more than 20 exogenous biosynthetic gene clusters.     

Establishment of a Simple Lactobacillus plantarum Cell Consortium for Cellulase-Xylanase Synergistic Interactions

Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into L. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by L. plantarum at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD₆₀₀) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of L. plantarum for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.     

Cysteine-rich low molecular weight antimicrobial peptides from <Emphasis Type="Italic">Brevibacillus</Emphasis> and related genera for biotechnological applications

The production of natural antimicrobial peptides (AMPs) is an innate immunity trait of all life forms including eukaryotes and prokaryotes. While these AMPs are usually called as defensins in eukaryotes, they are known as bacteriocins in prokaryotes. Bacteriocins are more diverse AMPs considering their varied composition and posttranslational modifications. Accordingly, this review is focused on cysteine-rich AMPs resembling eukaryotic defensins such as laterosporulin from Brevibacillus spp. and associated peptides secreted by the members of related genera. In fact, structural studies of laterosporulin showed the pattern typically observed in human defensins and therefore, should be considered as bacterial defensin. Although the biosynthesis mechanism of bacterial defensins displayed high similarities, variations in amino acid composition and structure provided the molecular basis for a better understanding of their properties. They are reported to inhibit Gram-positive, Gram-negative, non-multiplying and human pathogenic bacteria. The extreme stability is due to the presence of intra-molecular disulfide bonds in prokaryotic defensins and reveals their potential clinical and food preservation applications. Notably, they are also reported to have potential anticancer properties. Therefore, this review is focused on multitude of diverse applications of bacterial defensins, exploring the possible correlations between their structural, functional and possible biotechnological applications.     

Comparative genome characterization of Achromobacter members reveals potential genetic determinants facilitating the adaptation to a pathogenic lifestyle

Members of the Achromobacter genus are Gram-negative bacteria including both environmental and clinical isolates, which are increasingly recovered from patients with cystic fibrosis (CF) as emerging pathogens. To better understand the features of the genus and its potential pathogenic mechanisms, six available Achromobacter genomes were compared in this study. The results revealed that: (1) Achromobacter had a pan-genome size of 10,750 genes with 3,398 core genes and a similar global classification of protein functions; (2) the Achromobacter genomes underwent a relatively low recombination that introduced nearly twice nucleotide substitutions less than the point mutation in genome evolution; (3) phylogenomic analysis based on 436 conserved proteins and average nucleotide identity both indicated that the Achromobacter genus had the closest relationship to the human/animal pathogen Bordetella rather than to Alcaligenes. The entire group of Achromobacter clustered with Bordetella in phylogeny, strongly suggesting a common origin, which therefore highlighted the potentially pathogenic nature of Achromobacter from the phylogenetic perspective, and (4) the CF clinical isolate possessed markedly unique genomic features discriminated from the environmental isolate and was equipped with numerous factors that facilitate its adaptation to a pathogenic lifestyle, such as a type III secretion system, a “polysaccharide island” (36.0 kb) of capsular/cellulose synthesis, adhesion-related proteins, alcaligin biogenesis, and several putative toxins. This study provided the first comprehensive genomic comparative analysis for Achromobacter, revealed information to better understand this far less-known genus on the genomic scale, and, importantly, identified potential virulence factors of the Achromobacter pathogen.     

FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System

Regulation and assembly of the flagellar type III secretion system is one of the most investigated and best understood regulational cascades in molecular biology. Depending on the host organism, flagellar morphogenesis requires the interplay of more than 50 genes. Direct secretion of heterologous proteins to the supernatant is appealing due to protection against cellular proteases and simplified downstream processing. As Escherichia coli currently remains the predominant host organism used for recombinant prokaryotic protein expression, the generation of a strain that exhibits inducible flagellar secretion would be highly desirable for biotechnological applications.Here, we report the first engineered Escherichia coli mutant strain featuring flagellar morphogenesis upon addition of an external inducer. Using FlgM as a sensor for direct secretion in combination with this novel strain may represent a potent tool for significant improvements in future engineering of an inducible type III secretion for heterologous proteins.