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.

Brevibacillus composti sp. nov., isolated from hyperthermophilic compost.

Two aerobic, Gram-stain-positive, rod-shaped, endospore-forming, thermophilic bacterial strains, designated FJAT-54423T and FJAT-54424, were isolated from hyperthermophilic compost sampled in Shanxi Province, PR China. Growth was observed at 30-60 °C (optimum, 50 °C) and pH 6.0-9.0 (optimum, pH 7.0), with up to 2.0 % (w/v) NaCl (optimum, 0 % NaCl). The 16S rRNA gene sequence similarity between FJAT-54423T and FJAT-54424 was 99.9%, and the maximum similarity to a valid taxon was observed with Brevibacillus borstelensis (98.3%). Further, in phylogenetic and phylogenomic trees, strains FJAT-54423T and FJAT-54424 branched with members of the genus Brevibacillus. The menaquinone was MK-7, and the major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The main polar lipids included phosphatidylmethylethanolamine, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The cell-wall peptidoglycan was found to contain meso-diaminopimelic acid. The DNA G+C content of strains FJAT-54423T and FJAT-54424 were 54.3 and 54.4 mol%, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strain FJAT-54423T and its most closely related reference strain B. borstelensis DSM 6347T were 77.7 and 21.5 %, respectively, which were lower than the recommended species delineation thresholds of ANI (95%) and dDDH (70%). Based on the observed physiological properties, chemotaxonomic characteristics and ANI and dDDH values, FJAT-54423T and FJAT-54424 belong to a novel species of the genus Brevibacillus, for which the name Brevibacillus composti sp. nov. is proposed. The type strain is FJAT-54423T (=GDMCC 1.2054T=KCTC 43273T).

Phylogenetic determinants of toxin gene distribution in genomes of Brevibacillus laterosporus.

Brevibacillus laterosporus is a globally ubiquitous, spore forming bacterium, strains of which have shown toxic activity against invertebrates and microbes and several have been patented due to their commercial potential. Relatively little is known about this bacterium. Here, we examined the genomes of six published and five newly determined genomes of B. laterosporus, with an emphasis on the relationships between known and putative toxin encoding genes, as well as the phylogenetic relationships between strains. Phylogenetically, strain relationships are similar using average nucleotide identity (ANI) values and multi-gene approaches, although PacBio sequencing revealed multiple copies of the 16S rDNA gene which lessened utility at the strain level. Based on ANI values, the New Zealand isolates were distant from other isolates and may represent a new species. While all of the genomes examined shared some putative toxicity or virulence related proteins, many specific genes were only present in a subset of strains.

Brevibacillus migulae sp. nov., isolated from a Yellow River sediment sample.

Strain CFH S0501T, a novel Gram-stain-positive, aerobic, rod-shaped, endospore-forming and motile micro-organism with peritrichous flagella, was isolated from a sediment sample collected from the Yellow River in Henan Province, PR China. Optimum growth was observed at 28 °C, pH 7.0 and without NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain belonged to the genus Brevibacillus and was closely related to Brevibacillus centrosporus DSM 8445T and Brevibacillus ginsengisoli Gsoil 3088T (with 96.8 and 96.7 % sequence similarity, respectively). The predominant menaquinone was MK-7. Major cellular fatty acids were anteiso-C15 : 0 and iso-C15 : 0. Polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, two unidentified phospholipids and an unidentified polar lipid. The cell-wall peptidoglycan was found to contain meso-diaminopimelic acid. The genome size was 5.26 Mbp with a G+C content of 49.7 mol%. The average nucleotide identity (ANI) and in silico DNA-DNAhybridization (DDH) values between CFH S0501T and the other species of the genus Brevibacillus were found to be low (ANIm <86.11 %, ANIb <70.30 % and DDH <25.00 %). Based on physiological properties, chemotaxonomic characteristics and low ANI and DDH results, strain CFH S0501T is considered to represent a novel species, for which the name Brevibacillus migulae sp. nov. is proposed. The type strain is CFH S0501T (=DSM 29940T=BCRC 80809T).

Brevibacillus fortis NRS-1210 produces edeines that inhibit the in vitro growth of conidia and chlamydospores of the onion pathogen Fusarium oxysporum f. sp. cepae.

Onions can be damaged by Fusarium basal rot caused by the soilborne fungus Fusarium oxysporum f. sp. cepae (FOC). Control of this pathogen is challenging since there is limited genetic resistance in onion. The identification of molecules that inhibit this pathogen is needed. Antagonism screening showed Brevibacillus fortis NRS-1210 secreted antifungal compounds into growth medium. The spent growth medium, diluted 1:1, inhibited growth of FOC conidia after seven hours and killed 67-91% of conidia after 11 h. The spent medium also inhibited growth of propagules from F. graminearum, F. proliferatum, F. verticillioides and Galactomyces citri-aurantii. Full strength spent growth medium did not effectively kill FOC conidia and chlamydospores inoculated into a sand cornmeal mixture. In silico analysis of the B. fortis NRS-1210 genome indicated the biosynthetic clusters of several antibiotics. Fractionation of spent medium followed by reverse-phase liquid chromatography with tandem mass spectrometry analysis found that fractions with the most antifungal activity contained a combination of edeines A, B and F and no other recognized antibiotics. 1H NMR signals of the active fraction corresponded to edeine, a pentapeptide with broad spectrum antimicrobial activity which blocks translation in both prokaryotes and eukaryotes. Comparative genomics of Brevibacillus genomes shows edeine producers form a clade which consists of: Brevibacillus brevis, Brevibacillus formosus, 'Brevibacillus antibioticus', Brevibacillus schisleri, Brevibacillus fortis, and Brevibacillus porteri. This observation suggests edeine played an important role in the evolution and speciation of the Brevibacillus genus.

Phylogenomic analysis of the Brevibacillus brevis clade: a proposal for three new Brevibacillus species, Brevibacillus fortis sp. nov., Brevibacillus porteri sp. nov. and Brevibacillus schisleri sp. nov.

During a screen for antifungal activity of Brevibacillus strains in the Northern Regional Research Laboratory collection we identified two strains with strong activity. Subsequent genomic sequencing and phylogenomic analysis revealed that these strains (NRRL NRS-1210T and NRRL B-41110T) are likely novel species. To confirm their taxonomic placement, we conducted a 16S rRNA phylogenetic analysis and subsequently sequenced the genomes of 10 Brevibacillus type strains with a 16S homology > 97%. Phylogenomic analysis of these type strains and of representative Brevibacillus strains deposited in GenBank also identified several novel clades that should be recognised as novel species. For one of these novel clades, we were able to obtain a publicly available isolate (ATCC 35690T) that could serve as a type strain. The three new species were subjected to a polyphasic characterisation to confirm their taxonomic status. Cells of strains NRRL NRS-1210T, NRRL B-41110T and ATCC 35690T are Gram-staining positive, motile and form tan colonies. All three strains are obligate aerobic mesophiles with a broad pH range for growth. The two most prominent fatty acids of the three strains were identified as iso-C15:0 and anteiso-C15:0. The DNA G+C contents of strains NRRL NRS-1210T, NRRL B-41110T and ATCC 35690T are 47.2 mol%, 47.1 mol% and 47.3 mol%, respectively. Based on these characteristics, three novel species are proposed: Brevibacillus fortis sp. nov. (NRRL NRS-1210T = DSM 9886T = ATCC 51666T), Brevibacillus porteri sp. nov. (NRRL B-41110T = KACC 19693T) and Brevibacillus schisleri sp. nov. (ATCC 35690T = LMG 17055T).

A PCR-Based Method for Distinguishing between Two Common Beehive Bacteria, Paenibacillus larvae and Brevibacillus laterosporus.

Paenibacillus larvae and Brevibacillus laterosporus are two bacteria that are members of the Paenibacillaceae family. Both are commonly found in beehives and have historically been difficult to distinguish from each other due to related genetic and phenotypic characteristics and a shared ecological niche. Here, we discuss the likely mischaracterization of three 16S rRNA sequences previously published as P. larvae and provide the phylogenetic evidence that supported the GenBank reassignment of the sequences as B. laterosporus We explore the issues that arise by using only 16S rRNA or other single-gene analyses to distinguish between these bacteria. We also present three sets of molecular markers, two sets that distinguish P. larvae from B. laterosporus and other closely related species within the Paenibacillus genus and a third set that distinguishes B. laterosporus from P. larvae and other closely related species within the Brevibacillus genus. These molecular markers provide a tool for proper identification of these oft-mistaken species.IMPORTANCE 16S rRNA gene sequencing in bacteria has long been held as the gold standard for typing bacteria and, for the most part, is an excellent method of taxonomically identifying different bacterial species. However, the high level of 16S rRNA sequence similarity of some published strains of P. larvae and B. laterosporus, as well as possible horizontal gene transfer events within their shared ecological niche, complicates the use of 16S rRNA sequence as an effective molecular marker for differentiating these two species. Additionally, shared characteristics of these bacteria limit the effectiveness of using traditional phenotypic identification assays, such as the catalase test. The results from this study provide PCR methods to quickly differentiate between these two genera and will be useful when studying Brevibacillus, Paenibacillus, and other disease-relevant bacteria commonly found in beehives.

Isolation and Characteristics of Biotechnologically Important Antagonistic Thermophilic Bacteria from Rhizosphere of Haloxylon salicornicum.

Rhizobacteria are an active part of microbial population in the rhizosphere of plants. In this study, twenty rhizobacteria were isolated from the rhizosphere of a perennial grass, Haloxylon salicornicum, found in Cholistan desert, an arid landmass near Bahawalpur Pakistan, in one set of experimental conditions. Colony characteristics, biochemical and molecular analyses of these isolates were performed. All isolates were bacilli, gram positive with off-white colonies and exhibited typical bacilli colony morphology. None of the isolates was gelatinase, urease, indole, H2S and catalase producer. Eleven isolates were amylase producers and 8 isolates were acid producers. All isolates fermented glucose, 3 fermented lactose and 19 fermented fructose. Molecular data revealed that out of twenty isolates, 14 isolates showed 91-99% identity with Brevibacillus borstelensis, 4 with Bacillus subtilis (97-98%) and 2 with Bacillus licheniformis (94-99%) through BLAST analysis. All identified bacterial isolates cladded with their respective groups in the phylogenetic tree. Many (11-15 out of 20) of the isolates were more effective in inhibiting growth of the tested bacterial strains as compared to the positive control (Ampicillin 50 µg/disc). We conclude that bacilli are the predominant form populating rhizosphere of this desert grass. Among the isolated bacteria Brevibacillus borstelensis, Bacillus subtilis and Bacillus licheniformis are the most predominant species.

Brevibacillus parabrevis MTCC 12105: a potential bacterium for pulp and paper effluent degradation.

A gram positive, rod shaped, bacterium was isolated from pulp and paper mill sludge and characterized as Brevibacillus parabrevis (MTCC 12105) by biochemical tests and 16S rRNA gene sequencing. CD and EOP stage wastewater, collected from a leading pulp and paper mill situated in North India, was used for degradation study. Wastewater degradation efficiency of the bacterial isolate was evaluated by Flask study in batch mode and Reactor study in semi continuous mode. The isolated bacterium showed a considerable reduction of the colour (59%), chemical oxygen demand (62%) and lignin (53.8%) content of Kraft paper mill effluent at 37 °C after 5 days. During reactor study it reduced 42.6% Lignin, 51.6% Colour and 60.3% COD (chemical oxygen demand) of the wastewater at 48 h RT (retention time). The isolate was deposited at MTCC Chandigarh, India with an accession no. MTCC 12105.

Brevibacillus halotolerans sp. nov., isolated from saline soil of a paddy field.

Two novel aerobic bacteria, designated strains LAM0312T and LAM0313, were isolated from saline soil samples collected from a paddy field in Dezhou city, Shandong Province, China. Cells of these strains were Gram-stain-positive, sporogenous, rod-shaped and motile with peritrichous flagella. The optimal growth temperature and pH were 30 °C and pH 7.0-8.0. Strain LAM0312T was able to grow in the presence of 12 % (w/v) NaCl. The major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The dominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, glycolipids, five unidentitied lipids and phosphatidylmonomethylethanolamine. The cell-wall peptidoglycan was found to contain meso-diaminopimelic acid. The predominant menaquinone was identified as menaquinone-7. The G+C content of the genomic DNA of strains LAM0312T and LAM0313 was 45.0 and 46.0 mol%, respectively, as determined by the Tm method. 16S rRNA gene sequence similarity analysis indicated that the strains were closely related to Brevibacillus laterosporus DSM 25T and Brevibacillus fluminis JCM 15716T with 98.5 and 96.4 % sequence similarity, respectively. The DNA-DNA hybridization value between strain LAM0312T and LAM0313 was 92±0.6 % (reciprocal 90±0.2 %) and the value between strain LAM0312T and Brevibacillus laterosporus DSM 25T was 48±0.5 % (reciprocal 40±0.4 %). On the basis of the phenotypic, phylogenetic and chemotaxonomic characteristics, the two strains are proposed to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus halotolerans sp. nov. is proposed. The type strain is LAM0312T (=ACCC 06527T=JCM 30849T).

Classification of Bacillus and Brevibacillus species using rapid analysis of lipids by mass spectrometry.

Bacillus are aerobic spore-forming bacteria that are known to lead to specific diseases, such as anthrax and food poisoning. This study focuses on the characterization of these bacteria by the detection of lipids extracted from 33 well-characterized strains from the Bacillus and Brevibacillus genera, with the aim to discriminate between the different species. For the purpose of analysing the lipids extracted from these bacterial samples, two rapid physicochemical techniques were used: matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography in conjunction with mass spectrometry (LC-MS). The findings of this investigation confirmed that MALDI-TOF-MS could be used to identify different bacterial lipids and, in combination with appropriate chemometrics, allowed for the discrimination between these different bacterial species, which was supported by LC-MS. The average correct classification rates for the seven species of bacteria were 62.23 and 77.03 % based on MALDI-TOF-MS and LC-MS data, respectively. The Procrustes distance for the two datasets was 0.0699, indicating that the results from the two techniques were very similar. In addition, we also compared these bacterial lipid MALDI-TOF-MS profiles to protein profiles also collected by MALDI-TOF-MS on the same bacteria (Procrustes distance, 0.1006). The level of discrimination between lipids and proteins was equivalent, and this further indicated the potential of MALDI-TOF-MS analysis as a rapid, robust and reliable method for the classification of bacteria based on different bacterial chemical components. Graphical abstract MALDI-MS has been successfully developed for the characterization of bacteria at the subspecies level using lipids and benchmarked against HPLC.

The status of the species Shewanella irciniae Lee et al. 2006. Request for an opinion.

During a taxonomic study in describing a novel species of the genus Shewanella from a mangrove ecosystem, it was found that the type strain Shewanella irciniae was not available from the Japan Collection of Microorganisms (JCM) and the type strain received from the Agricultural Research Service (ARS) culture collection and from the author who described this species were found to be members of the genus Brevibacillus based on 16S rRNA gene sequence analysis. Therefore, this species cannot be included in any further scientific studies. It is therefore proposed that the Judicial Commission of the International Committee of Systematics of Prokaryotes place the name Shewanella irciniae on the list of rejected names, if a suitable replacement for the type strain is not found or a neotype strain is not proposed within two years following the publication of this Request for an Opinion.

Determination of cypermethrin degradation potential of soil bacteria along with plant growth-promoting characteristics.

The pyrethroid insecticide cypermethrin is in extensive use since 1980s for insect control. However, its toxicity toward aquatic animals and humans requires its complete removal from contaminated areas that can be done using indigenous microbes through bioremediation. In this study, three bacterial strains isolated from agricultural soil and identified as Acinetobacter calcoaceticus MCm5, Brevibacillus parabrevis FCm9, and Sphingomonas sp. RCm6 were found highly efficient in degrading cypermethrin and other pyrethroids. These bacterial strains were able to degrade more than 85 % of cypermethrin (100 mg L(-1)) within 10 days. Degradation kinetics of cypermethrin (200 mg kg(-1)) in soils inoculated with isolates MCm5, FCm9, and RCm6 suggested time-dependent disappearance of cypermethrin with rate constants of 0.0406, 0.0722, and 0.0483 d(-1) following first-order rate kinetics. Enzyme assays for Carboxylesterase, 3-PBA dioxygenase, Phenol hydroxylase, and Catechol-1,2 dioxygenase showed higher activities with cypermethrin treated cell-free extracts compared to non-treated cell-free extracts. Meanwhile, SDS-PAGE analysis showed upregulation of some bands in cypermethrin-treated cells. This might suggest that cypermethrin degradation in these strains involves inducible enzymes. Besides, the isolates displayed substantial plant growth-promoting traits such as phosphate solubilization, Indole acetic acid production, and ammonia production. Implying the efficient biodegradation potential along with multiple biological properties, these isolates can be valuable candidates for the development of bioremediation strategies.

Antifungal activity of Brevibacillus laterosporus JX-5 and characterization of its antifungal components.

The establishment of safe and effective methods for controlling fungal disease is an urgent issue in agriculture and forestry. Microbiological control of plant disease is expected to achieve better results than use of chemically derived fungicides. This study aimed to establish Brevibacillus laterosporus JX-5 as a potential microbiological control agent of poplar canker. The bacterium was isolated from the poplar rhizosphere and demonstrated significant growth inhibition of several pathogenic fungi in vitro. The antifungal components of Br. laterosporus JX-5 were isolated and identified. The fermentation broth of Br. laterosporus JX-5 and its main antifungal component, designated as component B, reduced Botryosphaeria dothidea associated canker of the excised poplar branch by 70 and 90%, respectively. Component B is considerably heat-stable, adaptable to a broad pH range, and UV-resistant. It could inhibit Bo. dothidea by permeating the fungal membrane, fracturing the nuclei, damaging the cell wall, and eventually killing the pathogenic fungus. The antifungal activity exhibited by Br. laterosporus JX-5 and its bioactive metabolic products indicate its feasibility as a potential biocontrol agent for plant diseases.

Brevibacillus fulvus sp. nov., isolated from a compost pile.

Two strains, designated K2814(T) and K282, were isolated from a compost pile in Japan. These strains were Gram-stain-variable, aerobic, motile and endospore-forming rods. The strains produced a characteristic brown non-diffusible pigment. The 16S rRNA gene sequences of the strains were 100% identical and had high similarity to that of Brevibacillus levickii LMG 22481(T) (97.3%). Phylogenetic analyses based on 16S rRNA gene sequences revealed that these strains belong to the genus Brevibacillus. Strains K2814(T) and K282 contained meso-diaminopimelic acid in their cell walls. Strains K2814(T) and K282 contained MK-7 (96.0 and 97.2%, respectively) and MK-8 (4.0 and 2.8%, respectively) as the major and minor menaquinones, respectively. Their major cellular fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(15 : 0) and iso-C(17 : 0). The DNA G+C contents of strains K2814(T) and K282 were 48.8 and 49.8 mol%, respectively. Polar lipids of strain K2814(T) were composed of phosphatidyl-N-methylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, three unidentified polar lipids, an unidentified aminophospholipid and an unidentified aminolipid. The level of DNA-DNA relatedness between strains K2814(T) and K282 was 99 or 100%, and levels between strain K2814(T) and the type strains of seven related species of the genus Brevibacillus, including Brevibacillus levickii LMG 22481(T), were below 59%. From the chemotaxonomic and physiological data and the levels of DNA-DNA relatedness, these two strains should be classified as representing a novel species of the genus Brevibacillus, for which the name Brevibacillus fulvus sp. nov. (type strain K2814(T) = JCM 18162(T) = ATCC BAA-2417(T) = DSM 25523(T)) is proposed.

Brevibacillus themoruber: a promising microbial cell factory for exopolysaccharide production.

AIMS: This study aims to identify a high level exopolysaccharide (EPS) producer thermophile that in turn could be used as a model organism to study the biological mechanisms and whole genome organization of EPS-producing thermophilic bacteria. METHODS AND RESULTS: Thermophilic isolates were screened, and then growth and EPS production of the best producer Brevibacillus thermoruber strain 423 were investigated under different carbon and nitrogen sources, temperature, pH and agitation rates. Rheological characterization revealed that the EPS behaved like a typical Newtonian fluid and viscosity of the EPS solution increased with increasing Ca(2+) ion concentration. Chemical characterization by TLC, GC-MS, FT-IR and NMR suggested a heteropolymer structure with glucose as major monomer unit. High biocompatibility of pure EPS fractions suggested their potential use in biomedical applications. CONCLUSIONS: This study reports on the comprehensive description of microbial production conditions as well as chemical, rheological and biological characterization of the EPS produced by B. thermoruber strain 423. The bioreactor cultures were found to reach two times higher yields and three times higher productivities when compared with literature. SIGNIFICANCE AND IMPACT OF THE STUDY: Brevibacillus thermoruber strain 423 combined the advantages of its nonpathogenicity with the advantages of fast productivity and hence proved to be a very promising model organism and cell factory for microbial EPS production.

Brevibacillus as a biological tool: a short review.

The significance of Brevibacillus has been documented scientifically in the published literature and commercially in heterologous recombinant protein catalogs. Brevibacillus is one of the most widespread genera of Gram-positive bacteria, recorded from the diverse environmental habitats. The high growth rate, better transformation efficiency by electroporation, availability of shuttle vectors, production of negligible amount of extracellular protease, and the constitutive expression of heterologous proteins make some strains of this genus excellent laboratory models. Regarding biotechnological applications, this genus continues to be a source of various enzymes of great biotechnological interest due to their ability to biodegrade low density polyethylene, ability to act as a candidate bio-control agent, and more recently acknowledged as a tool for the overexpression. This article reviews the properties of Brevibacillus spp. as better biological tools with varied applications.

Sequence polymorphism of GroEL gene in natural population of Bacillus and Brevibacillus spp. that showed variation in thermal tolerance capacity and mRNA expression.

GroEL, a class I chaperonin, plays an important role in the thermal adaptation of the cell and helps to maintain the viability of the cell under heat shock condition. Function of groEL in vivo depends on the maintenance of proper structure of the protein which in turn depends on the nucleotide and amino acid sequence of the gene. In this study, we investigated the changes in nucleotide and amino acid sequences of the partial groEL gene that may affect the thermotolerance capacity as well as mRNA expression of bacterial isolates. Sequences among the same species having differences in the amino acid level were identified as different alleles. The effect of allelic variation on the groEL gene expression was analyzed by comparison and relative quantification in each allele under thermal shock condition by RT-PCR. Evaluation of K a/K s ratio among the strains of same species showed that the groEL gene of all the species had undergone similar functional constrain during evolution. The strains showing similar thermotolerance capacity was found to carry same allele of groEL gene. The isolates carrying allele having amino acid substitution inside the highly ATP/ADP or Mg(2+)-binding region could not tolerate thermal stress and showed lower expression of the groEL gene. Our results indicate that during evolution of these bacterial species the groEL gene has undergone the process of natural selection, and the isolates have evolved with the groEL allelic sequences that help them to withstand the thermal stress during their interaction with the environment.

Transformation of Brevibacillus, a soil microbe to an uropathogen with hemagglutination trait.

An urinary tract infection (UTI) with Brevibacillus agri, an aerobic soil bacteria is discussed. The preliminary urine analysis tested negative for UTI, while the patient was diagnosed with focal pyelonephritis. The urine sample was analyzed for the presence of possible micro-organisms. The isolated micro-organism was phenotypically characterized and compared with a standard B. agri strain and an established uropathogen Eschericia coli, CFT073. Each experimental mouse was trans-urethrally infected using 2.5 × 10(8) c.f.u. for the generation of an UTI model. The kidney tissues were stored in buffered 10 % formaldehyde solution for histopathological analysis. The destruction of the glomerular and tubular morphology with prominent hemagglutination in the tubular region in the mouse kidneys were confirmed by light microscopic examination of the H&E stained sections. Hence, the identity of pathogen was confirmed using the 16S rRNA gene sequencing. The phylogenetic tree constructed using the 16S rRNA sequence obtained from the isolated microbial strain showed 99 % similarity with the strain, B. agri AB112716. Finally this study concludes based on the phenotypic characteristics, production of lipopolysaccharide, the ability to aggregate in the presence of ammonium sulphate, agglutinate erythrocytes in the presence of mannose, and the potential to resist the interactions of serum, the possibility that the soil microbe could have undergone genotypic modification to cause UTI. However, further detailed and in-depth genetic analysis are required to point out exactly how the soil bacterium has adapted itself to cause infection in a human subject.

Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers.

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.