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.

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).

Degradation of Poly(ε-caprolactone) by a Thermophilic Community and Brevibacillus thermoruber Strain 7 Isolated from Bulgarian Hot Spring.

The continual plastic accumulation in the environment and the hazardous consequences determine the interest in thermophiles as possible effective plastic degraders, due to their unique metabolic mechanisms and change of plastic properties at elevated temperatures. PCL is one of major biodegradable plastics with promising application to replace existing non-biodegradable polymers. Metagenomic analysis of the phylogenetic diversity in plastic contaminated area of Marikostinovo hot spring, Bulgaria revealed a higher number taxonomic groups (11) in the sample enriched without plastic (Marikostinovo community, control sample, MKC-C) than in that enriched in the presence of poly-ε-caprolactone (PCL) (MKC-P), (7). A strong domination of the phylum Proteobacteria was observed for MKC-C, while the dominant phyla in MKC-P were Deinococcus-Thermus and Firmicutes. Among the strains isolated from MKC-P, the highest esterase activity was registered for Brevibacillus thermoruber strain 7 at 55 °C. Its co-cultivation with another isolate resulted in ~10% increase in enzyme activity. During a 28-day biodegradation process, a decrease in PCL molecular weight and weight loss were established resulting in 100% degradation by MKC-P and 63.6% by strain 7. PCL degradation intermediate profiles for MKC-P and pure strain were similar. Broken plastic pieces from PCL surface and formation of a biofilm by MKC-P were observed by SEM, while the pure strain caused significant deformation of PCL probes without biofilm formation.

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).

Siderophore Production by Rhizosphere Biological Control Bacteria Brevibacillus brevis GZDF3 of Pinellia ternata and Its Antifungal Effects on Candida albicans.

Brevibacillus brevis GZDF3 is a gram-positive, plant growth-promoting rhizosphere bacterium (PGPR) isolated from the rhizosphere soil of Pinellia ternata (an important herb in traditional Chinese medicine). The GZDF3 strain produces certain active compounds, such as siderophores, which are the final metabolite products of non-ribosomal peptide synthetase (NRPS) and independent non-ribosomal peptide synthetase (NIS) activity. With the present study, we attempted to investigate the siderophore production characteristics and conditions of Bacillus sp. GZDF3. The antibacterial activity of the siderophores on pathogenic fungi was also investigated. Optimal conditions for the synthesis of siderophores were determined by single factor method, using sucrose 15 g/l, asparagine 2 g/l, 32°C, and 48 h. The optimized sucrose asparagine medium significantly increased the production of siderophores, from 27.09% to 54.99%. Moreover, the effects of different kinds of metal ions on siderophore production were explored here. We found that Fe3+ and Cu2+ significantly inhibited the synthesis of siderophores. The preliminary separation and purification of siderophores by immobilized-metal affinity chromatography (IMAC) provides strong antibacterial activity against Candida albicans. The synergistic effect of siderophores and amphotericin B was also demonstrated. Our results have shown that the GZDF3 strain could produce a large amount of siderophores with strong antagonistic activity, which is helpful in the development of new biological control agents.

Know your enemy: Unexpected, pervasive and persistent viral and bacterial contamination of primary cell cultures.

In biomedical research, cell culture contamination is one of the main culprits of experimental failure. Contamination sources and concomitant remedies are numerous and challenging to manage. We herein describe two cases of uncommon contamination of cell cultures that we encountered, and the successful determination and eradication strategies. The first case describes the infection with human adenovirus C that originated from pharyngeal tonsils used for isolation of primary tonsillar epithelial cells. It is known that viral contamination of in vitro cell cultures can occur symptomless and is therefore difficult to identify. The contamination was pervasive and persistent, as it was widely spread in flow cabinets and apparatus, and has caused a serious delay to our research projects and the inevitable loss of valuable (patient-derived) cell sources. Eradication was successful by formalin gas sterilization of the flow cabinet and elimination of all infected cell lines from our biobank after PCR-guided determination. Secondly, we encountered a spore-forming bacterium, namely Brevibacillus brevis, in our cell culture facility. This bacterium originated from contaminated tap water pipes and spread via regular aseptic culture techniques due to survival of the bacterial spores in 70% ethanol. B brevis overgrew the cultures within a few days after seeding of the primary cells. Chlorine solution effectively killed this spore-forming bacterium. Both cases of contamination were identified using DNA sequencing which enabled the deployment of targeted aseptic techniques for the elimination of the persistent contamination.

A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (Kcat/Km (s-1 M-1) = 8.80 × 1011), which is significantly higher than that of AacCas12b (Kcat/Km (s-1 M-1) = 7.56 × 108) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis.

Insights of organic fertilizer micro flora of bovine manure and their useful potentials in sustainable agriculture.

Exploration of diverse environmental samples for plant growth-promoting microbes to fulfill the increasing demand for sustainable agriculture resulted in increased use of bacterial biofertilizer. We aimed for the isolation of plant growth-promoting as well as antibiotic sensitive bacteria from bovine manure samples. The basic theme of our study is to highlight potentials of bacteria in manure and the unchecked risk associated with the application of manure i.e. introducing antibiotic-resistant microbial flora, as fertilizer. Fifty-two, morphologically distinct isolates; from eight different manure samples, were subjected to plant growth-promoting parametric tests along with antibiotic resistance. Thirteen antibiotic sensitive bacterial strains with potentials of plant growth promotion further characterized by 16S rRNA ribotyping and the identified genera were Stenotrophomonas, Achromobacter, Pseudomonas, and Brevibacillus. Successful radish seeds germination under sterile in-vitro conditions showed the potential of selected bacterial isolates as plant growth-promoting bacteria. The results of this study confirmed plant growth-promoting characteristics of bovine manures' bacterial strains along with an alarming antibiotic resistance load which comprises 75% of bacterial isolated population. Our study showed distinct results of un-explored manure bacterial isolates for plant growth promotion and flagged ways associated with unchecked manure application in agriculture soil through high load of antibiotic resistant bacteria.

Brevibacillus antibioticus sp. nov., with a broad range of antibacterial activity, isolated from soil in the Nakdong River.

A Gram-stain-positive, aerobic, motile, and rod-shaped bacterial strain designated TGS2-1T was isolated from sediment soil in the Nakdong River, Republic of Korea. The optimal growth of strain TGS2-1T was observed at 28°C and pH 7.0 without NaCl supplementation. Strain TGS2-1T revealed antibiosis against various bacteria, including Staphylococcus aureus KCCM 4051, CCARM 3089 (methicillin resistant strains), Enterococcus faecalis KCCM 11814, Escherichia coli KCTC 2443, Candida albicans KACC 7270, and Filobasidium neoformans KCTC 7902. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that strain TGS2-1T belonged to the genus Brevibacillus and shared 93.8-99.7% sequence similarity with Brevibacillus species. Whole-genome sequencing of strain TGS2-1T revealed a genome size of 6.2 Mbp and DNA G + C content of 47.0 mol%. The TGS2-1T genome shared an average nucleotide identity and digital DNA-DNA hybridization of 74.6-93.3% and 18.6-67.1%, respectively, with six related Brevibacillus genomes. The major fatty acid constituents of strain TGS2-1T were anteiso-C15:0 (62.3%) and anteiso-C17:0 (10.8%). Cells of strain TGS2-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, seven unidentified aminophospholipids, and five unidentified lipids. The isoprenoid quinone detected in the strain was menaquinone-7 (MK-7). Based on data obtained from this polyphasic taxonomic study, strain TGS2-1T represents a novel species belonging to genus Brevibacillus, for which the name B. antibioticus sp. nov. is proposed. The type strain is TGS2-1T (= KCCM 90326T = NBRC 113840T = FBCC-B2501).

Inhibition of Paenibacillus larvae by an extracellular protein fraction from a honeybee-borne Brevibacillus laterosporus strain.

The inhibitory action that a Brevibacillus laterosporus strain isolated from the honeybee body causes against the American Foulbrood (AFB) etiological agent Paenibacillus larvae was studied by in-vitro experiments. A protein fraction isolated from B. laterosporus culture supernatant was involved in the observed inhibition of P. larvae vegetative growth and spore germination. As a result of LC-MS/MS proteomic analyses, the bacteriocin laterosporulin was found to be the major component of this fraction, followed by other antimicrobial proteins and substances including lectins, chaperonins, various enzymes and a number of putative uncharacterized proteins. The results obtained in this study highlight the potential of B. laterosporus as a biological control agent for preserving and improving honeybee health.

Rapid polymerase chain reaction assays for Brevibacillus laterosporus detection.

The identification of the ubiquitous spore-forming bacterium Brevibacillus laterosporus, whose interest in pharma, agriculture, and other industrial sectors is raising, mostly relies on 16S ribosomal RNA gene sequence analysis. However, due to bacterial gene homology, this method appears insufficient for a proper discrimination of this species, so that the availability of other target genes is necessary. Leveraging the morphological and genetic feature uniqueness of B. laterosporus, a sensitive and reliable detection and quantification method based on polymerase chain reaction (PCR) and quantitative PCR assays, respectively, was developed. Targeting a highly conserved spore surface protein-related gene, B. laterosporus could be easily found in different matrices including soil, food, and insect body. Primer set selectivity was confirmed to be very specific and no false positives or negatives were observed using DNA of different bacterial species as a template. The method developed is also suitable for the rapid identification of newly isolated B. laterosporus strains.

Identification of Bacillus species: Implication on the quality of probiotic formulations.

Spores of several Bacillus species have long history of consumption and safe use as probiotics and a variety of formulations containing these organisms are available in the global market. Considering the difficulties in the identification of Bacillus species and the poor microbiological quality of many probiotic formulations, we used three up-to-date methodological approaches for analyzing the content of ten formulations marketed in Italy and labeled to contain Bacillus spores. We compared the performance of biochemical tests based on the BCL Vitek2 card and MALDI-TOF mass spectrometry, using 16S rDNA sequencing as the reference technique. The BCL card performed well in identifying all Bacillus probiotic strains as well as the Bruker's MALDI Biotyper. Nevertheless, the MALDI score values were sometimes lower than those indicated by the manufacturer for correct species identification. Contaminant bacteria (Lysinibacillus fusiformis, Acinetobacter baumannii, Bacillus cereus, Brevibacillus choshinensis, Bacillus licheniformis, Bacillus badius) were detected in some formulations. Characterization of the B. cereus contaminant showed the potential pathogenicity of this strain. Microbial enumeration performed by the plate count method revealed that the number of viable cells contained in many of the analyzed products differed from the labeled amount. Overall, our data show that only two of the ten analyzed formulations qualitatively and quantitatively respect what is on the label. Since probiotic properties are most often strain specific, molecular typing of isolates of the two most common Bacillus species, B. clausii and B. coagulans, was also performed. In conclusion, the majority of the analyzed products do not comply with quality requirements, most likely leading to reduced/absent efficacy of the preparation and representing a potential infective risk for consumers.

Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials.

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.

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.

Screening of a beta-cypermethrin-degrading bacterial strain Brevibacillus parabrevis BCP-09 and its biochemical degradation pathway.

A novel beta-cypermethrin (Beta-CP)-degrading strain isolated from activated sludge was identified as Brevibacillus parabrevis BCP-09 based on its morphological and physio-biochemical characteristics, and 16S rRNA gene analysis. Strain BCP-09 could effectively degrade Beta-CP at pH 5.0-9.0, 20-40 °C, and 10-500 mg L-1 Beta-CP. Under optimal conditions (pH 7.41, 38.9 °C, 30.9 mg L-1 Beta-CP), 75.87% Beta-CP was degraded within 3 days. Beta-CP degradation (half-life, 33.45 h) and strain BCP-09 growth were respectively described using first-order-kinetic and logistic-kinetic models. Seven metabolites were detected by high-performance liquid chromatography and gas chromatography-mass spectrometry- methyl salicylate, catechol, phthalic acid, salicylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzaldehyde, and 3-phenoxybenzoic acid (3-PBA). The major Beta-CP metabolite, 3-PBA was further degraded into phenol, benzoic acid, and 4-methylhexanoic acid. BCP-09 also degraded aromatic compounds such as phenol, catechol, and protocatechuic acid. Beta-CP appears to be mainly degraded into 3-PBA, which is continuously degraded into smaller benzene or chain compounds. Thus, strain BCP-09 could form a complete degradation system for Beta-CP and might be considered a promising strain for application in the bioremediation of environments and agricultural products polluted by Beta-CP.

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.

Subsurface Endospore-Forming Bacteria Possess Bio-Sealant Properties.

Concrete is a strong and fairly inexpensive building substance, but has several disadvantages like cracking that allows corrosion, thus reducing its lifespan. To mitigate these complications, long-lasting microbial self-healing cement is an alternative that is eco-friendly and also actively repairs cracks. The present paper describes the detailed experimental investigation on compressive strength of cement mortars, mixed with six alkaliphilic bacteria, isolated from subsurface mica mines of high alkalinity. The experiments showed that the addition of alkaliphilic isolates at different cell concentrations (104 and 106 cells/ml) enhanced the compressive strength of cement mortar, because the rapid growth of bacteria at high alkalinity precipitates calcite crystals that lead to filling of pores and densifying the concrete mix. Thus, Bacillus subtilis (SVUNM4) showed the highest compressive strength (28.61%) of cement mortar at 104 cells/ml compared to those of other five alkaliphilic isolates (Brevibacillus sp., SVUNM15-22.1%; P. dendritiformis, SVUNM11-19.9%; B. methylotrophicus, SVUNM9-16%; B. licheniformis, SVUNM14-12.7% and S. maltophilia, SVUNM13-9.6%) and controlled cement mortar as well. This method resulted in the filling of cracks in concrete with calcite (CaCO3), which was observed by scanning electron microscopy (SEM). Our results showed that the alkaliphilic bacterial isolates used in the study are effective in self-healing and repair of concrete cracks.