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International Journal of Systematic... Jul 1998Previous DNA relatedness studies showed that strains identified as Bacillus mycoides segregated into two genetically distinct yet phenotypically similar groups, one...
Previous DNA relatedness studies showed that strains identified as Bacillus mycoides segregated into two genetically distinct yet phenotypically similar groups, one being B. mycoides sensu stricto and the other, an unclassified taxon. In the present study, the taxonomic position of this second group was assessed by measuring DNA relatedness and determining phenotypic characteristics of an increased number of B. mycoides strains. Also determined was the second group's 16S RNA gene sequence. The 36 B. mycoides strains studied segregated into two genetically distinct groups showing DNA relatedness of about 30%; 18 strains represented the species proper and 18 the second group with intragroup DNA relatedness for both groups ranging from 70 to 100%. DNA relatedness to the type strains of presently recognized species with G+C contents of approximately 35 mol% (Bacillus alcalophilus, Bacillus cereus, Bacillus circulans, Bacillus lentus, Bacillus megaterium and Bacillus sphaericus) ranged from 22 to 37%. Although shown to be genetically distinct taxa, the two B. mycoides groups exhibited highly similar (98%) 16S RNA sequences. Phylogenetic analyses showed that both B. mycoides and the second group clustered closely with B. cereus. Although not distinguishable by physiological and morphological characteristics, the two B. mycoides groups and B. cereus were clearly separable based on fatty acid composition. The data established that the second B. mycoides group merits recognition as a new species for which the name Bacillus pseudomycoides is proposed. The type stain is NRRL B-617(T).
Topics: Bacillus; Base Sequence; DNA, Bacterial; DNA, Ribosomal; Molecular Sequence Data; Phylogeny; RNA, Ribosomal, 16S
PubMed: 9734060
DOI: 10.1099/00207713-48-3-1031 -
Journal of Molecular Biology Sep 1999The three-dimensional structures of engineered variants of Bacillus lentus subtilisin having increased enzymatic activity, K27R/N87S/V104Y/N123S/T274A (RSYSA) and...
The three-dimensional structures of engineered variants of Bacillus lentus subtilisin having increased enzymatic activity, K27R/N87S/V104Y/N123S/T274A (RSYSA) and N76D/N87S/S103A/V104I (DSAI), were determined by X-ray crystallography. In addition to identifying changes in atomic position we report a method that identifies protein segments having altered flexibility. The method utilizes a statistical analysis of variance to delineate main-chain temperature factors that represent significant departures from the overall variance between equivalent regions seen throughout the structure. This method reveals changes in main-chain mobility in both variants. Residues 125-127 have increased mobility in the RSYSA variant while residues 100-104 have decreased mobility in the DSAI variant. These segments are located at the substrate-binding site and changes in their mobility are believed to relate to the observed changes in proteolytic activity. The effect of altered crystal lattice contacts on segment flexibility becomes apparent when identical variants, determined in two crystal forms, are compared with the native enzyme.
Topics: Bacillus; Binding Sites; Crystallography, X-Ray; Isoenzymes; Models, Molecular; Protein Conformation; Protein Engineering; Regression Analysis; Serine Endopeptidases; Subtilisins; Temperature
PubMed: 10493860
DOI: 10.1006/jmbi.1999.3033 -
Applied and Environmental Microbiology Feb 2000A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy...
A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.
Topics: Bacillus; Cloning, Molecular; Endopeptidases; Hydrogen-Ion Concentration; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Plasmids; Sequence Analysis, DNA
PubMed: 10653758
DOI: 10.1128/AEM.66.2.825-827.2000 -
Bioresource Technology Jan 2011Bacillus lentus BI377, isolated from textile effluent-contaminated soil, was able to degrade 97% and 92% of Reactive Red 120 dye when 1200 and 1500 mg/l, respectively,...
Bacillus lentus BI377, isolated from textile effluent-contaminated soil, was able to degrade 97% and 92% of Reactive Red 120 dye when 1200 and 1500 mg/l, respectively, of dye was added to nutrient broth (NB) at 35 °C within 12 h. UV-vis spectroscopy, GC-MS, FTIR and 1H NMR revealed the formation of catechol which may be further utilized by the bacterium via the TCA cycle, leading to complete mineralization. Structural analysis of metabolites in conjunction with enzyme activity studies confirmed the involvement of azoreductase, cytochrome P450 monooxygenase and other antioxidant enzymes. Decreases in total organic carbon and in biological and chemical oxygen demand suggest formation of low molecular weight metabolites that could be completely mineralized. These results suggest the potential use of B. lentus BI377 towards online treatment of textile dye effluents by using an appropriate bioreactor over a wide range of pH. This study opens-up a dependable and proficient way to use industrially viable non-pathogenic strains for biotransformation of carcinogenic dyes to ecofriendly compounds.
Topics: Bacillus; Biodegradation, Environmental; Biological Oxygen Demand Analysis; Color; Molecular Sequence Data; Spectrophotometry; Time Factors; Triazines
PubMed: 20864334
DOI: 10.1016/j.biortech.2010.08.094 -
Bioorganic & Medicinal Chemistry Jul 2000Using site directed mutagenesis combined with chemical modification, we have developed a general and versatile method for the glycosylation of proteins which is... (Comparative Study)
Comparative Study
Using site directed mutagenesis combined with chemical modification, we have developed a general and versatile method for the glycosylation of proteins which is virtually unlimited in the scope of proteins and glycans that may be conjugated and in which the site of glycosylation and the nature of the introduced glycan can be carefully controlled. We have demonstrated the applicability of this method through the synthesis of a library of 48 glycosylated forms of the serine protease subtilisin Bacillus lentus (SBL) as single, pure species. As part of our ongoing program to tailor the activity of SBL for use in peptide synthesis, we have screened these enzymes for activity against the esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl. Gratifyingly, 22 enzymes displayed greater than wild type (WT) activity. Glycosylation at positions 62, in the S2 pocket, resulted in five glycosylated forms of SBL that were 1.3- to 1.9-fold more active than WT. At position 217, in the S1' pocket, all glycosylations increased kcat/KM up to a remarkable 8.4-fold greater than WT for the glucosylated enzyme L217C-S-beta-Glc(Ac)3. Furthermore, the ratio of amidase to esterase activity, (kcat/KM)esterase/(kcat/KM)amidase (E/A), is increased relative to wild type for all 48 glycosylated forms of SBL. Again, the most dramatic changes are observed at positions 62 and 217 and L217C-S-beta-Glc(Ac)3 has an E/A that is 17.2-fold greater than WT. The tailored specificity and high activity of this glycoform can be rationalized by molecular modeling analysis, which suggests that the carbohydrate moiety occupies the S1' leaving group pocket and enhances the rate of deacylation of the acyl-enzyme intermediate. These glycosylated enzymes are ideal candidates for use as catalysts in peptide synthesis as they have greatly increased (kcat,KM)esterase and severely reduced (kcat/KM)amidase and will favor the formation of the amide bond over hydrolysis.
Topics: Acetylation; Amidohydrolases; Bacillus; Binding Sites; Combinatorial Chemistry Techniques; Esterases; Glycoproteins; Glycosylation; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Structure-Activity Relationship; Subtilisin
PubMed: 10976502
DOI: 10.1016/s0968-0896(00)00084-5 -
Microbiological Research 2007We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689...
We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Phix174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Phix174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg2+ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 degrees C (pH 8.0) with 100 mM NaCl and 10 mM Mg2+ concentrations.
Topics: Bacillus; Chemical Fractionation; Chromatography, Affinity; Chromatography, Liquid; Coenzymes; DNA Restriction Enzymes; DNA, Bacterial; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Magnesium; Molecular Weight; Osmolar Concentration; Plasmids; Temperature
PubMed: 16644193
DOI: 10.1016/j.micres.2006.01.008 -
Biochemistry Sep 1998Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates...
Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.
Topics: Aspartic Acid; Bacillus; Binding Sites; Catalysis; Computer Simulation; Crystallography, X-Ray; Hydrogen Bonding; Models, Molecular; Structure-Activity Relationship; Subtilisins
PubMed: 9753430
DOI: 10.1021/bi9813983 -
International Journal of Systematic and... Mar 2014A Gram-staining-positive, motile, facultatively anaerobic, endospore-forming and rod-shaped bacterium, designated strain CJ32(T), was isolated from ginseng soil at...
A Gram-staining-positive, motile, facultatively anaerobic, endospore-forming and rod-shaped bacterium, designated strain CJ32(T), was isolated from ginseng soil at Geumsan in Korea. The isolate grew optimally at 30 °C, 2% (w/v) NaCl and pH 7.0. Colonies of strain CJ32(T) were beige and circular with an entire margin on LB agar plates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CJ32(T) was associated with the genus Bacillus and was most closely related to Bacillus graminis YC6957(T) (97.3% similarity) and Bacillus lentus IAM 12466(T) (97.1%). DNA-DNA hybridization with closely related strains was below 31.3%. The major respiratory isoprenoid quinone was MK-7. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The polar lipid profile of strain CJ32(T) consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several unidentified lipids, including phospholipids, aminolipids and aminophospholipids. The predominant fatty acids of strain CJ32(T) were iso-C15:0 and anteiso-C15:0. The G+C content of the genomic DNA was 35.1 mol%. Based on phenotypic, genotypic and phylogenetic data, strain CJ32(T) should be classified within a novel species of the genus Bacillus, for which the name Bacillus panacisoli sp. nov. is proposed. The type strain is strain CJ32(T) ( = KACC 17503(T) = JCM 19226(T)).
Topics: Bacillus; Bacterial Typing Techniques; Base Composition; Cell Wall; DNA, Bacterial; Diaminopimelic Acid; Fatty Acids; Molecular Sequence Data; Nucleic Acid Hybridization; Panax; Peptidoglycan; Phospholipids; Phylogeny; RNA, Ribosomal, 16S; Republic of Korea; Sequence Analysis, DNA; Soil Microbiology; Vitamin K 2
PubMed: 24277860
DOI: 10.1099/ijs.0.054320-0 -
Bioorganic & Medicinal Chemistry Nov 1999The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually...
The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have recently adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). We now describe the use of this strategy to introduce multiple positive charges. A series of mono-, di- and triammonium methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutants (CMM) enzymes were determined at pH 8.6. The presence of up to three positive charges in the S1, S1' and S2 subsites of SBL resulted in up to 77-fold lowered activity, possibly due to interference with the histidinium ion formed in the transition state of the hydrolytic reactions catalyzed.
Topics: Amino Acids; Bacillus; Catalysis; Electrochemistry; Kinetics; Mutagenesis, Site-Directed; Quaternary Ammonium Compounds; Substrate Specificity; Subtilisin; Sulfhydryl Reagents; Thiosulfonic Acids
PubMed: 10632040
DOI: 10.1016/s0968-0896(99)00168-6 -
Bioresource Technology Feb 2013Bacillus lentus BI377 (B. lentus BI377) an alkaliphilic strain has accomplished the discriminate color removal strategy for Reactive Red sulfonated azoic recalcitrant...
Bacillus lentus BI377 (B. lentus BI377) an alkaliphilic strain has accomplished the discriminate color removal strategy for Reactive Red sulfonated azoic recalcitrant irrespective of their molecular structure. During the decolorization experiment, it was observed that the diazo dye first followed chromophoric cleavage by azoreductase via typical azoreduction whereas, in case of monoazo dye, cleavage took place by peroxidase via successive electron transfers to oxide surface resulting in the asymmetric cleavage of the azo bond. Dismutation of oxidative stress by reactive metabolites has confirmed by superoxide dismutase activity. Carbon monoxide (CO) binding spectra, the content of cytochrome P450 and spectroscopy analysis by GCMS, FTIR and (1)H NMR of intermediate metabolites indicated the differentiate pattern of diazo and monoazo dye decolorization fuse to central metabolic pathway. Declined percentage of TOC and the cytotoxicity (MTT) study confirmed that environmentally benign intermediates may lead to mineralization.
Topics: Animals; Bacillus; Cell Line; Coloring Agents; Mice; Naphthalenesulfonates; Spectrophotometry; Tetrazolium Salts; Thiazoles; Triazines; Water Pollutants, Chemical
PubMed: 23313681
DOI: 10.1016/j.biortech.2012.12.019