Molecular cloning of structural and immunity genes for megacins A-216 and A-19213 in Bacillus megaterium.

A host-vector system was developed for molecular cloning in Bacillus megaterium and used to clone the structural and immunity genes for megacins A-216 and A-19213. Recombinant clones that expressed immunity only or both immunity to and production of each megacin were obtained. Restriction mapping of native megacinogenic plasmids and recombinant clones was used to construct physical and genetic maps of megacinogenic plasmids pBM309 and pBM113. Limited sequence homology between pBM309 and pBM113 was detected by Southern blot hybridization and was mapped to, at most, a 6.4-kilobase-pair region of pBM309 and a 6.1-kilobase-pair region of pBM113.     

Bacteriocin from Bacillus megaterium ATCC 19213: comparative studies with megacin A-216

A bacteriocin produced by Bacillus megaterium ATCC 19213 was identified, purified, and compared with megacin A from B. megaterium 216. The ATCC 19213 bacteriocin was inducible with mitomycin C and showed phospholipase A activity. Both megacin A-216 and megacin A-19213 contained two dissimilar polypeptide subunits. Megacin A-216 contains a 30,000-dalton alpha subunit and a 15,000-dalton beta subunit. Megacin A-19213 is composed of an alpha subunit 18,000 daltons in mass and a beta subunit about 7,500 daltons in mass. No sequence similarities between alpha and beta subunits of either megacin were detected. The two megacins were further distinguished by quantitative differences in activity spectra and by immunodiffusion analyses.     

A new bacteriocinogenic activity: Megacin BII encoded by plasmid pSE 203 in strains of Bacillus megaterium

Mesophilic strains producing a new bacteriocin: Megacin BII, have been isolated from strains of Bacillus megaterium. Facultatively thermophilic strains producing Megacin BI were less sensitive to this new activity than nonproducing mesophiles and strains producing Megacin BII were also also more resistant to Megacin BI. Strains producing Megacin BII contained a large plasmid of 36·10⁶:pSE 203. This plasmid was introduced into non-megacinogenic acceptor strains by protoplast transformation, they then became megacin producers and immune to Megacin BII. Plasmid pSE 203 has been mapped with endonucleases. No similarity to the Megacin A plasmids pBM 309 [Rostás et al. (1980) and pBM 113 (von Tersch and Carlton (1983b)] was evident.Abbreviations CFU colony forming units - CCO covalently closed circular - OC open circular - LIN linear - EB ethidium bromide - Meg megacin - Tris tris (hydroxyl methyl) amino methane - Ot obligately thermophilic - Ft facultatively thermophilic - Sm streptomycin - Trim trimethoprim - Rif rifampicin - Thy thymine - Tc tetracycline     

Cloning and characterization of the polC region of Bacillus subtilis.

The polC gene of Bacillus subtilis is defined by five temperature-sensitive mutations and the 6-(p-hydroxyphenylazo)-uracil (HPUra) resistance mutation azp-12. Biochemical evidence suggests that polC codes for the 160-kilodalton DNA polymerase III. A recombinant plasmid, p154t, was isolated and found to contain the azp-12 marker and one end of the polC gene (N. C. Brown and M. H. Barnes, J. Cell. Biochem. 78 [Suppl.]: 116, 1983). The azp-12 marker was localized to a 1-kilobase DNA segment which was used as a probe to isolate recombinant lambda phages containing polC region sequences. A complete polC gene was constructed by in vitro ligation of DNA segments derived from two of the recombinant phages. The resulting plasmid, pRO10, directed the synthesis of four proteins of 160, 76, 39, and 32 kilodaltons in Escherichia coli maxicells. Recombination-deficient (recE) B. subtilis PSL1 containing pRO10 produced an HPUra-resistant polymerase III activity which was lost when the strain was cured of pRO10. In vivo, the HPUra resistance of the plasmid-encoded polymerase III appeared to be recessive to the resident HPUra-sensitive polymerase III enzyme.     

The purification and characterization of a DNA nicking-closing enzyme from Bacillus megaterium.

Although several eucaryote DNA nicking--closing (N--C) enzymes have been characterized, only the Escherichia coli enzyme has been extensively studied amongst procaryotes. The latter enzyme is distinctly different from the eucaryotic enzymes and we have therefore purified the N--C enzyme from Bacillus megaterium to determine if procaryotes form a distinctive class of N--C enzymes. The purified B. megaterium N--C enzyme has a molecular weight of 120,000, only partly relaxes negative supercoils, does not affect positive supercoils, requires Mg2+, and is inhibited by 0.2 M KCl. The enzyme is also inhibited by 1 mM nalidixic or oxolinic acids but unaffected by novobiocin. A crude N--C enzyme preparation from Micrococcus luteus shows very similar properties.     

Isolation and characterization of forespores from Bacillus megaterium

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine. Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension.     

Photoabsorption study of Bacillus megaterium, DNA and related biological materials in the phosphorus K-shell edge region

We measured the X-ray transmission spectra of several biologically related samples in the phosphorus K-shell edge absorption region. These include red phosphorus, hydrated sodium phosphate (Na(3)PO(4).12 H(2)O), deoxyribonucleic acid (DNA), adenosine triphosphate (ATP), diolylphosphatidyl choline (DOPC), and Bacillus megaterium spores. Red phosphorus essentially displays an edge-jump. All other spectra are similar in form and energy position: Each is dominated by a narrower, more intense first peak and a broader but less intense second peak. The corresponding K-shell edge absorption thresholds are shifted toward higher energy relative to that for red phosphorus, as expected for increasing degrees of phosphorus oxidation. The B. megaterium spectrum has aspects common to both the phosphate and DNA spectra and is therefore interpreted as a composite of spectra arising from DNA, ribonucleic acid (RNA) and phosphates within the spore. The B. megaterium spore spectrum provides information for resonant radiation damage studies in the phosphorus K-shell edge absorption region by identifying candidate photoexcitations. In addition, the absorption spectra will be useful in X-ray microscopy and macromolecular crystallography studies at the phosphorus K-shell edge.     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9 Kb的基因片段.核酸序列分析结果表明,该片段全长1 984 bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9Kb的基因片段.核酸序列分析结果表明,该片段全长1984bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.     

Isolation and some properties of the site-specific endonuclease and methylase Bme2161 from Bacillus megaterium 216

The site-specific endonuclease Bme2161 was isolated as a homogeneous preparation by chromatography on phosphocellulose, hydroxyapatite and heparin-agarose. The molecular mass of the enzyme, determined by gel filtration and by electrophoresis under denaturing conditions, was found to be 60 kDa and 30 kDa respectively. These data indicate that the native enzyme consists of two identical subunits. The enzyme recognized the decreases pentanucleotide sequence 5'-GGACC-3' X 3'-CCTGG-5' and cleaves the sequence as indicated by arrows. The increases optimal concentration for endonuclease reaction is 6-7 mM Mg2+. The endonuclease relaxes its specificity in the presence of glycerol or dimethyl sulfoxide at low Mg2+ concentration (1-3 mM). Methylase Bme2161, which protects DNA against endonuclease Bme2161 action by DNA methylation, was isolated from the same bacterial strain.     

Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium

A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His(6)-tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry.     

Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Due to its chemical structure, (D-O-Leu-D-Ala-L-O-Val-L-Val)(3), cereulide might be synthesized nonribosomally. Therefore, degenerate PCR primers targeted to conserved sequence motifs of known nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a cereulide-producing B. cereus strain. Sequence analysis of one of the amplicons revealed a DNA fragment whose putative gene product showed significant homology to valine activation NRPS modules. The sequences of the flanking regions of this DNA fragment revealed a complete module that is predicted to activate valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene. Disruption of the peptide synthetase gene by insertion of a kanamycin cassette through homologous recombination produced cereulide-deficient mutants. The valine-activating module was highly conserved when sequences from nine emetic B. cereus strains isolated from diverse geographical locations were compared. Primers were designed based on the NRPS sequence, and the resulting PCR assay, targeting the ces gene, was tested by using a panel of 143 B. cereus group strains and 40 strains of other bacterial species showing PCR bands specific for only the cereulide-producing B. cereus strains.     

Proline iminopeptidase from Bacillus megaterium: purification and characterization

Proline iminopeptidase [EC 3.4.11.5] was purified about 1,700-fold from cell free extract of Bacillus megaterium by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose and hydroxyapatite, and gel filtration on Toyopearl FW-55. The purified enzyme still contained a minor contaminant as judged by disc gel electrophoresis. The enzyme was most active at pH 7.0 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminal. The enzyme was completely inactivated by p-chloromercuribenzoate (PCMB), but was not inhibited by metal chelators, diisopropylphosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by adding 2-mercaptoethanol. From this result and the chromatographic profile on PCMB-T-Sepharose, the enzyme seems to be a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0. The molecular weight of the enzyme was estimated to be 58,000 by gel filtration on Toyopearl and 60,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is a monomer.     

Cloning in Escherichia coli of a mitochondrial DNA fragment from Acremonium chrysogenum containing a region responsible for DNA replication

A bireplicone plasmid pSU901,4.6 kb in length, was constructed on the basis of plasmid pUC19 and the pstIB fragment, 1.9 kb in length, from mitochondrial DNA of A. chrysogenum. Based on the hybrid plasmid pSU901 and kanamycin resistance determinant, an autonomically replicating vector for A. chrysogenum, a culture producing cephalosporin C, is being constructed.     

A sucrose-inducible promoter system for the intra- and extracellular protein production in Bacillus megaterium

A sucrose-inducible promoter system (P(sacB)) from Bacillus megaterium was identified using a secretome approach. It was successfully employed for the extracellular production of the homologous levansucrase SacB (4252.4 U l(-1)) and the heterologous green fluorescent protein GFP (7.9 mg g(CDW)(-1)). Mutational analysis of B. megaterium P(sacB) allowed the identification of important promoter elements. The sucrose-inducible promoter provides a useful alternative to the established xylose-inducible promoter system (P(xylA)) for recombinant gene expression in B. megaterium.     

Cloning and characterization of a region responsible for the maintenance of megaplasmid pTAV3 of Paracoccus versutus UW1

Using cointegrate formation, we constructed a basic replicon of the megaplasmid/mini-chromosome pTAV3 of Paracoccus versutus UW1. It is composed of two adjacent modules, responsible for plasmid replication (rep) and partitioning (par). Functional analysis of the par region identified a determinant of incompatibility (inc2), whose presence is crucial for proper partitioning (the partitioning site). Database searches revealed that the only known replicon with significant homology to that of pTAV3 is encoded by the chromosome cII of Rhodobacter sphaeroides 2.4.1. Incompatibility studies showed that closely related basic replicons are also encoded by megaplasmids (above 400 kb) harbored by four strains of P. pantotrophus. Basic replicons of the pTAV3-type are able to maintain large bacterial genomes, therefore they appear to be good candidates for the construction of vectors specific for Alphaproteobacteria.