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The Journal of Biological Chemistry May 1987In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome...
Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts.
In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique. The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B. megaterium enzyme and contains the same N-terminal amino acid sequence. The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase. In E. coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital. However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B. megaterium via an E. coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital. This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA. The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B. megaterium but is absent in the E. coli clone.
Topics: Bacillus megaterium; Cloning, Molecular; Cytochrome P-450 Enzyme System; Enzyme Induction; Escherichia coli; Gene Expression Regulation; Genes; Genes, Bacterial; Genetic Vectors; Oxygenases; Phenobarbital; Plasmids
PubMed: 3106359
DOI: No ID Found -
Journal of Applied Microbiology Dec 2011This work was aimed to isolate, purify and characterize an extracellular polysaccharide (EPS) produced by a freshwater dynamic sediment-attached micro-organism, Bacillus...
Composition analysis and material characterization of an emulsifying extracellular polysaccharide (EPS) produced by Bacillus megaterium RB-05: a hydrodynamic sediment-attached isolate of freshwater origin.
AIMS
This work was aimed to isolate, purify and characterize an extracellular polysaccharide (EPS) produced by a freshwater dynamic sediment-attached micro-organism, Bacillus megaterium RB-05, and study its emulsifying potential in different hydrocarbon media.
METHODS AND RESULTS
Bacillus megaterium RB-05 was found to produce EPSs in glucose mineral salts medium, and maximum yield (0.864 g l(-1) ) was achieved after 24-h incubation. The recovery rates of the polysaccharide material by ion-exchange and gel filtration chromatography were around 67 and 93%, respectively. As evident from HPLC and FT-IR analyses, the polysaccharide was found to be a heteropolymer-containing glucose, galactose, mannose, arabinose, fucose and N-acetyl glucosamine. Different oligosaccharide combinations namely hexose(3), hexose(4), hexose(5) deoxyhexose(1) and hexose(5) deoxyhexose(1) pentose(3) were obtained after partial hydrolysis of the polymer using MALDI-ToF-MS. The polysaccharide with an average molecular weight of 170 kDa and thermal stability up to 180°C showed pseudoplastic rheology and significant emulsifying activity in hydrocarbon media.
CONCLUSIONS
Isolated polysaccharide was found to be of high molecular weight and thermally stable. The purified EPS fraction was composed of hexose, pentose and deoxyhexose sugar residues, which is a rare combination for bacterial polysaccharides. Emulsifying property was either better or comparable to that of other commercially available natural gums and polysaccharides.
SIGNIFICANCE AND IMPACT OF THE STUDY
This is probably one of the few reports about characterizing an emulsifying EPS produced by a freshwater sediment-attached bacterium. The results of this study contribute to understand the influence of chemical composition and material properties of a new microbial polysaccharide on its application in industrial biotechnology. Furthermore, this work reconfirms freshwater dynamic sediment as a potential habitat for bioprospecting extracellular polymer-producing bacteria. This study will improve our knowledge on the exploitation of a nonconventional renewable resource, which also seems to be ecologically significant.
Topics: Bacillus megaterium; Culture Media; Emulsifying Agents; Extracellular Space; Fresh Water; Geologic Sediments; Glucose; Molecular Weight; Polysaccharides, Bacterial; Rheology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Water Microbiology
PubMed: 21973188
DOI: 10.1111/j.1365-2672.2011.05162.x -
Journal of Bacteriology May 1975Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells,... (Comparative Study)
Comparative Study
Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.
Topics: Albumins; Anilides; Azo Compounds; Bacillus megaterium; Bacterial Proteins; Carbon Radioisotopes; Caseins; Cell-Free System; Dipeptides; Endopeptidases; Hemoglobins; Hydrolysis; Oligopeptides; Peptide Hydrolases; Spores, Bacterial
PubMed: 805126
DOI: 10.1128/jb.122.2.642-649.1975 -
The Journal of General and Applied... Jul 2019An extracellular L-asparaginase was isolated and purified from Bacillus megaterium MG1 to apparent homogeneity. The purification procedure involved a combination of...
Isolation, purification and characterization of an extracellular L-asparaginase produced by a newly isolated Bacillus megaterium strain MG1 from the water bodies of Moraghat forest, Jalpaiguri, India.
An extracellular L-asparaginase was isolated and purified from Bacillus megaterium MG1 to apparent homogeneity. The purification procedure involved a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration techniques, resulting in a purification factor of 31.52 fold with a specific activity of 215 U mg. The molecular mass of the purified enzyme was approximately 47 kDa on SDS-PAGE and 185 kDa on native PAGE gel as well as in gel filtration column chromatography, revealing that the enzyme was a homotetramer. The K and V values of the purified enzyme were calculated to be 2.0 ⅹ 10 M and 1.198 mM s. Maximum enzyme activity was observed over a wide range of temperature and pH values with an optimum temperature of 37°C and pH 8.5. SDS and metal ions such as Fe, Cu, Mg, Co, Mn, and Ca decreased the enzyme activity remarkably, whereas the addition of Na and K led to an increase in activity. The insensitivity of the protein in the presence of EDTA suggested that the enzyme might not essentially be a metalloprotein. Its marked stability and activity in organic solvents and reducing agents suggest that this asparaginase is highly suitable as a biotechnological tool with industrial applications.
Topics: Asparaginase; Asparagine; Bacillus megaterium; Bacterial Proteins; Enzyme Activation; Enzyme Stability; Extracellular Space; Forests; Hydrogen-Ion Concentration; India; Kinetics; Molecular Weight; Phylogeny; Substrate Specificity; Temperature; Water Microbiology
PubMed: 30568045
DOI: 10.2323/jgam.2018.07.004 -
Acta Crystallographica. Section F,... Aug 2013Alanine dehydrogenase (L-AlaDH) from Bacillus megaterium WSH-002 catalyses the NAD⁺-dependent interconversion of L-alanine and pyruvate. The enzyme was expressed in...
Alanine dehydrogenase (L-AlaDH) from Bacillus megaterium WSH-002 catalyses the NAD⁺-dependent interconversion of L-alanine and pyruvate. The enzyme was expressed in Escherichia coli BL21 (DE3) cells and purified with a His6 tag by Ni²⁺-chelating affinity chromatography for X-ray crystallographic analysis. Crystals were grown in a solution consisting of 0.1 M HEPES pH 8.0, 12%(w/v) polyethylene glycol 8000, 8%(v/v) ethylene glycol at a concentration of 15 mg ml⁻¹ purified protein. The crystal diffracted to 2.35 Å resolution and belonged to the trigonal space group R32, with unit-cell parameters a = b = 125.918, c = 144.698 Å.
Topics: Alanine Dehydrogenase; Bacillus megaterium; Bacterial Proteins; Crystallization; Crystallography, X-Ray
PubMed: 23908047
DOI: 10.1107/S1744309113019672 -
Journal of Bacteriology May 1964Hancock, R. (Harvard Medical School, Boston, Mass.), and P. C. Fitz-James. Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus...
Hancock, R. (Harvard Medical School, Boston, Mass.), and P. C. Fitz-James. Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium. J. Bacteriol. 87:1044-1050. 1964.-Penicillin and cycloserine do not inhibit the growth of protoplasts of Bacillus megaterium, indicating that inhibition of cell-wall synthesis is the only significant process by which they inhibit growth of bacteria. In contrast, bacitracin and vancomycin inhibit growth of protoplasts and bacteria at similar concentrations, indicating that they have important sites of action other than their known inhibition of cell-wall synthesis. At concentrations which inhibit mucopeptide synthesis, penicillin, bacitracin, and vancomycin each cause an increased rate of efflux of K ions from growing bacteria. This effect of penicillin is prevented by chloramphenicol or hypertonic sucrose, whereas the effects of bacitracin and vancomycin are unchanged under these conditions. It is concluded that bacitracin and vancomycin have direct effects on the cytoplasmic membrane, and it is proposed that their inhibition of cell-wall synthesis could be a consequence of these effects. Bacitracin and vancomycin do not compete with penicillin for binding to cells of B. megaterium, a further indication that they have a different primary site of action.
Topics: Bacillus megaterium; Bacitracin; Cell Membrane Permeability; Cell Wall; Cycloserine; Mucoproteins; Novobiocin; Penicillin G; Potassium; Potassium Isotopes; Protoplasts; Vancomycin
PubMed: 4959792
DOI: 10.1128/jb.87.5.1044-1050.1964 -
Journal of Bacteriology Nov 1968The techniques of Nomarski interference contrast microscopy and phase-contrast microscopy were compared for their utility in monitoring sporulation and germination in...
The techniques of Nomarski interference contrast microscopy and phase-contrast microscopy were compared for their utility in monitoring sporulation and germination in Bacillus megaterium. The Nomarski technique permitted rapid and easy delineation of septation and engulfment during sporulation, whereas with phase contrast microscopy these stages were not detected at all. The later stages of sporulation were easily seen by either technique. Thus, of the seven stages of sporulation as recognized by the electron microscopy of thin sections, five can now be routinely detected quantitatively by optical microscopy: septation (stage II), engulfment (stage III), phase-dark forespore (corresponding to cortex formation, stage IV), phase-bright spore in a sporangium (corresponding to coat formation, stage V), and the free spore (stage VII). This means that now only stage I (axial filament) and stage VI (maturation of the refractile spore) require electron microscopy for routine detection. There was no advantage in using Nomarski optics for germination studies.
Topics: Bacillus megaterium; Microscopy, Interference; Microscopy, Phase-Contrast; Spores
PubMed: 4973131
DOI: 10.1128/jb.96.5.1811-1817.1968 -
Proceedings of the National Academy of... Jun 2019Germination of spores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore's inner membrane. GRs typically...
Germination of spores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore's inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of the GerK GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substrate-binding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination.
Topics: Bacillus megaterium; Bacterial Proteins; Molecular Docking Simulation; Protein Domains; Protein Subunits; Spores, Bacterial
PubMed: 31113879
DOI: 10.1073/pnas.1903675116 -
Acta Crystallographica. Section D,... Mar 2014The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four...
Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution.
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.
Topics: Amino Acid Sequence; Bacillus megaterium; Crystallization; Crystallography, X-Ray; Hydroxymethylbilane Synthase; Molecular Sequence Data; Oxidation-Reduction; Porphobilinogen
PubMed: 24598743
DOI: 10.1107/S139900471303294X -
The Journal of Biological Chemistry Sep 1980Pulse labeling of cells of Bacillus megaterium followed by cell disruption and immunoprecipitation has shown that the spore-specific Proteins A and C are synthetized...
Pulse labeling of cells of Bacillus megaterium followed by cell disruption and immunoprecipitation has shown that the spore-specific Proteins A and C are synthetized only during a discrete time period in sporulation. At its maximum, the synthesis of the A- and C-proteins accounted for 5% of the protein being synthesized in vivo, but the mRNA for the A- and C-proteins had a lifetime no longer than that of other mRNAs translated at that time. No evidence was found for synthesis of Proteins A or C in high molecular weight precursor form, and essentially all of the newly synthesized A- and C-protein was found in the forespore. Isolation of total RNA from cells in various stages of growth and sporulation, translation of this RNA in a cell-free system from vegetative cells, and immunoprecipitation showed that the ability of cellular RNA to promote A- and C-protein synthesis in vitro was directly proportional to the rate at which the cells had been synthesizing Proteins A and C in vivo. These data indicate that synthesis of Proteins A and C during sporulation in B. megaterium is primarily under transcriptional control. The identity of the immunoprecipitated labeled material material synthesized in vitro with the A- and C-proteins was established by: 1) their co-migration on sodium dodecyl sulfate-polyacrylamide gels; 2) co-migration on high performance liquid chromatography of tryptic peptides from an [35S]methionine-labeled immunoprecipitate with the methionine-containing tryptic peptides of the A- and C-proteins; and 3) digestion of the labeled immunoprecipitate with a protease specific for the A- and C-proteins.
Topics: Antigens, Bacterial; Bacillus megaterium; Bacterial Proteins; Cell-Free System; Kinetics; Protoplasts; Sigma Factor; Spores, Bacterial; Transcription Factors
PubMed: 6773942
DOI: No ID Found