Cloning and characterization of the beta-amylase gene from Bacillus polymyxa.

The gene for beta-amylase was isolated from Bacillus polymyxa by molecular cloning in B. subtilis. B. subtilis cells containing this gene express and secrete an amylase which resembles the B. polymyxa beta-amylase and barley beta-amylase in terms of the products it generates during carbohydrate hydrolysis. Starch hydrolysis with this beta-amylase produces maltose, not glucose, whereas maltotriose and cycloheptaose are resistant to the action of this beta-amylase. The enzyme has a molecular weight of approximately 68,000. Restriction endonuclease mapping demonstrated that the DNA inserted in pBD64 and containing the gene is approximately 3 kilobases in length.     

Cloning and sequencing of Schizosaccharomyces pombe DNA topoisomerase I gene, and effect of gene disruption.

We cloned the structural gene topl+ for Schizosaccharomyces pombe DNA topoisomerase I (topo I) by hybridization. An eight-fold increase of topo I relaxing activity was obtained in S. pombe cells transformed with multicopy plasmid with topl+ insert. Nucleotide sequence determination showed a hypothetical coding frame interrupted by two short introns, encoding a 812 residue polypeptide (M.W. 94,000), 43 residues longer than and 47% homologous to Saccharomyces cerevisiae topo I. We show that the topl (null) strain made by gene disruption is viable, although its generation time is 20% longer than that of wild type. The topl locus is mapped in the long arm of chromosome II, using the Leu+ marker integrated with the cloned topl+ sequence. We constructed a double mutant topl (null) top2 (ts) and found its defective phenotype similar to that of previously obtained topl (heat sensitive) top2 (ts). The other double mutant topl (null) top2 (cs), however, was lethal. Our results suggest that topl+ gene of S. pombe is dispensable only if topo II activity is abundant.     

Cloning and sequencing of a new holin-encoding gene of Bacillus licheniformis

A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec). Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptide of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively). Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1–G2, respectively, exhibited higher extracellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398. The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin-encoding gene. Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of β-galactosidase into the extracellular fraction.     

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.     

Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans.

The gene coding for proline iminopeptidase in Bacillus coagulans was cloned and expressed in Escherichia coli. Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein. The calculated molecular weight of the product was 32,415. The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence. The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme. By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved. The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold. The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36%. The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column. The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.     

Cloning and sequencing of the gerD gene of Bacillus subtilis

A Tn917 insertion in the same region of the chromosome as gerD gave rise to a mutant (ger-97) with a germination phenotype similar to that of two gerD mutants which germinate abnormally in a range of germinants. The insertion and two gerD mutations were cotransformed with ribosomal protein genes rpoB, rpsE and rpsI. DNA cloned from one side of the insertion carried the 16S end of the ribosomal RNA operon rrnI. These data were consistent with the order rpoB-rpsE-rpsI-gerD/ger-97::Tn917-rrnI. Insertion into the wild-type chromosome of a plasmid carrying DNA adjacent to the insertion permitted the recovery of a 1.8 kb fragment of DNA which complemented ger-97::Tn917 and the gerD mutations. The DNA nucleotide sequence of the region of this fragment at which Tn917 had inserted revealed a 555 bp open reading frame, preceded by a ribosome-binding site and potential sigma E and sigma A promoter regions and encoding a predicted polypeptide of 21,117 Da. This polypeptide was largely hydrophilic but contained a hydrophobic region at the N-terminus resembling a signal peptide.     

Cloning and sequencing of the hrcA gene of Bacillus stearothermophilus

We report on cloning and sequencing of a 2.0-kb PCR fragment of chromosomal DNA from thermophilic Bacillus stearothermophilus carrying the complete hrcA gene. In addition, this amplicon contains the 3' end of an open reading frame exhibiting significant homology to the hemN gene of Bacillus subtilis (Bs) and other bacterial species. The hrcA gene could complement an Bs hrcA deletion mutant by repressing expression of class I heat shock (HS) genes. Furthermore, we could show that the HrcA protein derived from the thermophilic microorganism responds to HS in a similar way as reported for the Bs HrcA protein.     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

炭疽杆菌水肿因子基因的克隆与序列测定

采用聚合酶链反应（ＰＣＲ）从炭疽芽孢杆菌减毒株ＹＢ１中扩增其水肿因子（ＥＦ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２ ３０１ ｂｐ,编码７６７个氨基酸,与已报道的Ｓｔｅｒｎｅ标准株的ＥＦ基因完全一致。     

炭疽杆菌保护性抗原基因的克隆与序列测定

采用聚合酶链反应从炭疽芽孢杆菌减毒株ＹＢ１中扩增其保护性抗原（ＰＡ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２２０５ｂｐ,编码７３５个氨基酸残基,与献报道的标准菌株Ｓｔｅｒｎｅ株的ＰＡ序列只有４个碱基的差异。     

Cloning and sequencing of the beta-fructofuranosidase gene from Bacillus sp. V230.

The beta-fructofuranosidase gene (bff) from Bacillus sp. V230 has been cloned in Escherichia coli and its nucleotide sequence has been analyzed. The product of bff consists of a signal sequence of 32 amino acid (a.a.) residues for secretion and 455 a.a. residues of the extracellular beta-fructofuranosidase. The a.a. sequence of the bff product has similarities with those of the Bacillus subtilis levanscrase (63.7% identity), the Streptococcus mutans fructosyltransferase (33.7%), and the Zymomonas mobilis levanscrase and beta-fructofuranosidase (15%).     

Cloning and characterization of an exoinulinase from Bacillus polymyxa

A gene encoding an exoinulinase (inu) from Bacillus polymyxa MGL21 was cloned and sequenced. It is composed of 1455 nucleotides, encoding a protein (485 amino acids) with a molecular mass of 55522 Da. Inu was expressed in Escherichia coli and the His-tagged exoinulinase was purified. The purified enzyme hydrolyzed sucrose, levan and raffinose, in addition to inulin, with a sucrose/inulin ratio of 2. Inulinase activity was optimal at 35°C and pH 7, was completely inactivated by 1 mM Ag⁺ or Hg²⁺. The K _m and V _max values for inulin hydrolysis were 0.7 mM and 2500 M min^–1 mg^–1 protein. The enzyme acted on inulin via an exo-attack to produce fructose mainly.     

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1.

A cosmid library was prepared from a partial BamHI digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons Tn501 and Tn21. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADP-ribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.     

Cloning and nucleotide sequencing of phenylalanine dehydrogenase gene of Bacillus sphaericus

The gene coding for phenylalanine dehydrogenase [PDH; L-phenylalanine: NAD+ oxidoreductase (deaminating); EC 1.4.1.-] from Bacillus sphaericus SCRC-79a was cloned onto plasmid pUC9, and the nucleotide sequence of the 2-kb DNA region of the insert was determined. A 1143-bp open reading frame consisting of 381 codons was identified as a pdh gene coding for PDH.     

Cloning and nucleotide sequence of the gene coding for enzymatically active fragments of the Bacillus polymyxa beta-amylase.

The gene encoding beta-amylase was cloned from Bacillus polymyxa 72 into Escherichia coli HB101 by inserting HindIII-generated DNA fragments into the HindIII site of pBR322. The 4.8-kilobase insert was shown to direct the synthesis of beta-amylase. A 1.8-kilobase AccI-AccI fragment of the donor strain DNA was sufficient for the beta-amylase synthesis. Homologous DNA was found by Southern blot analysis to be present only in B. polymyxa 72 and not in other bacteria such as E. coli or B. subtilis. B. polymyxa, as well as E. coli harboring the cloned DNA, was found to produce enzymatically active fragments of beta-amylases (70,000, 56,000, or 58,000, and 42,000 daltons), which were detected in situ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nucleotide sequence analysis of the cloned 3.1-kilobase DNA revealed that it contains one open reading frame of 2,808 nucleotides without a translational stop codon. The deduced amino acid sequence for these 2,808 nucleotides encoding a secretory precursor of the beta-amylase protein is 936 amino acids including a signal peptide of 33 or 35 residues at its amino-terminal end. The existence of a beta-amylase of larger than 100,000 daltons, which was predicted on the basis of the results of nucleotide sequence analysis of the gene, was confirmed by examining culture supernatants after various cultivation periods. It existed only transiently during cultivation, but the multiform beta-amylases described above existed for a long time. The large beta-amylase (approximately 160,000 daltons) existed for longer in the presence of a protease inhibitor such as chymostatin, suggesting that proteolytic cleavage is the cause of the formation of multiform beta-amylases.     

Cloning,sequencing, and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene

Bacillus sp. B-0618 produces both creatinase (Cre; creatine amidinohydrolase; EC 3.5.3.3) and sarcosine oxidase (Sox; EC 1.5.3.1) enzymes when grown in the presence of an inducer, choline chloride. A genomic library of Bacillus sp. B-0618, prepared in the plasmid vector pACYC184, was screened to obtain a gene (sox) encoding Sox by a convenient colorimetric assay. A plasmid, pOXI101, isolated from a sox-positive clone, contained a 14.2-kb insert of Bacillus DNA. The nucleotide sequence of a 1.7-kb segment containing the sox gene was determined, and it was found that an open reading frame encoding a protein consisting of 390 amino acids was located upstream from the cre structural gene cloned previously. When a 1.6-kb XhoI-BglII fragment of pOXI101 was inserted into the pUC118 vector and introduced into Escherichia coli, transformants cultured in the absence of the inducer produced Sox about 50-fold more than Bacillus sp. B-0618 cultured in the presence of the inducer. The Bacillus Sox had the -¹¹Gly-X-¹³Gly-X-X-¹⁶Gly- sequence motif that is highly conserved in flavoproteins. We created an FAD-free Sox by changing ¹³Gly to Asp in the motif of the parental Sox by oligodeoxynucleotide-directed mutagenesis. The mutant protein no longer expressed the Sox activity, even on the addition of FAD.