Isolation and characterization of Bacillus sp. BP-6 LipA,a ubiquitous lipase among mesophilic Bacillus species

AIMS: The aim of this study was to perform the isolation, cloning and characterization of a lipase from Bacillus sp. BP-6 bearing the features of a biotechnologically important group of enzymes. METHODS AND RESULTS: Strain Bacillus sp. BP-6, showing activity on tributyrin plates, was used for isolation of lipase-coding gene lipA by means of inverse and direct PCR. The complete 633 nucleotide ORF isolated was cloned in Escherichia coli for further characterization. The amino acid sequence of the cloned protein was 98% identical to B. subtilis and B. megaterium lipases, the enzyme also showing similar molecular and biochemical features. CONCLUSIONS: The gene coding for Bacillus sp. BP-6 LipA was found in all mesophilic Bacillus species assayed, indicating its ubiquity in the genus. The cloned enzyme displayed the same properties as those of homologous lipases. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall profile of Bacillus sp. BP-6 LipA was found to be that of a ubiquitous and highly conserved subfamily I.4 bacterial lipase. Previously described lipases within this family have shown to be well suited for biotechnological applications, suggesting that the cloned enzyme could be used accordingly.     

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315?±?16 IU/mL acidic xylanase, 290?±?20 IU/mL alkaline xylanase, and 88?±?9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10?mM MgSO₄, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60?hr at 200?rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3?hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6?hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.     

Structural requirements of the unique disulphide bond and the proline-rich motif within the alpha4-alpha5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Both the disulphide bond (Cys192-Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the alpha4-alpha5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the flexibility of the alpha4-alpha5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha4-alpha5 loop of the Cry4Aa toxin.     

Co-existence of clpB and clpC in the Bacillaceae

The gene encoding ClpC in Bacillus anthracis was amplified from the chromosome by polymerase chain reaction using degenerate oligonucleotide primers. These primers also amplified a second DNA fragment identified as a clpB homolog. Both genes were suggested to be functional. Contrary to Bacillus subtilis which possesses clpC but not clpB, many Bacillus species were found to harbor both clpC and clpB. We also found that Clostridium strains could possess clpB, clpC, or both. All the Gram-negative strains tested had clpB only.     

Activation process of the mosquitocidal delta-endotoxin Cry39A produced by Bacillus thuringiensis subsp. aizawai BUN1-14 and binding property to Anopheles stephensi BBMV

Most delta-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a delta-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg(61) and Gly(62). In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction.     

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

Genome-scale modeling for Bacillus coagulans to understand the metabolic characteristics

Lactic acid is widely used in many industries, especially in the production of poly-lactic acid. Bacillus coagulans is a promising lactic acid producer in industrial fermentation due to its thermophilic property. In this study, we developed the first genome-scale metabolic model (GEM) of B. coagulans iBag597, together with an enzyme-constrained model ec-iBag597. We measured strain-specific biomass composition and integrated the data into a biomass equation. Then, we validated iBag597 against experimental data generated in this study, including amino acid requirements and carbon source utilization, showing that simulations were generally consistent with the experimental results. Subsequently, we carried out chemostats to investigate the effects of specific growth rate and culture pH on metabolism of B. coagulans. Meanwhile, we used iBag597 to estimate the intracellular metabolic fluxes for those conditions. The results showed that B. coagulans was capable of generating ATP via multiple pathways, and switched among them in response to various conditions. With ec-iBag597, we estimated the protein cost and protein efficiency for each ATP-producing pathway to investigate the switches. Our models pave the way for systems biology of B. coagulans, and our findings suggest that maintaining a proper growth rate and selecting an optimal pH are beneficial for lactate fermentation.     

Comparative characterization of the conjugation capacity and large plasmids in native strains of Bacillus subtilis from different regions of the East European Plain

The properties of large plasmids harbored by Bacillus subtilis strains isolated from soils of Moscow and Moscow oblast and from different regions of the Republic of Belarus have been studied. All large plasmids in the collection of strains from Belarus were capable of conjugative mobilization of the small plasmid pUB110 and were similar in size and other properties. Most of the tested plasmids harbored by strains isolated from Moscow soils had no mobilization ability; they were of different sizes and showed no homology with the replication region of plasmids from the Belarussian collection. The uniformity of the plasmids present in strains from Belarussian soils may be due to their active horizontal transfer under natural conditions.     

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

The twin-arginine translocation (Tat) pathway can transport folded and co-factor-containing cargo proteins over bacterial cytoplasmic membranes. Functional Tat machinery components, a folded state of the cargo protein and correct co-factor insertion in the cargo protein are generally considered as prerequisites for successful translocation. The present studies were aimed at a dissection of these requirements with regard to the Rieske iron-sulfur protein QcrA of Bacillus subtilis. Notably, QcrA is a component of the cytochrome bc₁ complex, which is conserved from bacteria to man. Single amino acid substitutions were introduced into the Rieske domain of QcrA to prevent either co-factor binding or disulfide bond formation. Both types of mutations precluded QcrA translocation. Importantly, a proofreading hierarchy was uncovered, where a QcrA mutant defective in disulfide bonding was quickly degraded, whereas mutant QcrA proteins defective in co-factor binding accumulated in the cytoplasm and membrane. Altogether, these are the first studies on Tat-dependent protein translocation where both oxidative folding and co-factor attachment have been addressed in a single native molecule.