Effect of in vitro DNA Rearrangement in the NH2-terminal Region of the Penicillinase Gene from Bacillus licheniformis on the Mode of Expression in Bacillus subtilis

Imanaka, Tadayuki; Himeno, Takeshi; Aiba, Setsuya

doi:10.1099/00221287-131-7-1753

Cited by 21 publications

(19 citation statements)

References 16 publications

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“…Cloning of the mini-Tn10-disrupted gene was carried out as follows. To construct a B. subtilis MI113 plasmid library of the RB14 chromosome, chromosomal DNA of the transposoncontaining mutant was digested with HindIII, ligated at the HindIII site of plasmid pTB522 which could be replicated in B. subtilis (9), and then transformed into B. subtilis MI113 competent cells. The plasmid of the chloramphenicol-resistant transformant resulting from cloning of a chromosomal fragment containing mini-Tn10 was obtained from the library and designated pTB1006.…”

Section: Methodsmentioning

confidence: 99%

Cloning, Sequencing, and Characterization of the Iturin A Operon

2001

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Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609-and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser3L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn3L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.Many Bacillus subtilis strains produce a small peptide(s) with a long fatty moiety, the so-called lipopeptide antibiotics. The peptide portions of these compounds contain ␣-amino acids with a D configuration and are produced nonribosomally with templates of the multifunctional peptide synthetases. As in the synthesis of the peptide antibiotic gramicidin S, the peptide chain grows in a defined sequence by moving on the template of the multifunctional peptide synthetase (18,35,44). On the basis of the structural relationships, the lipopeptides that have been identified in B. subtilis are generally classified into three groups: the surfactin group (27), the plipastatin-fengycin group (16,41,42), and the iturin group (17). The members of the surfactin and plipastatin-fengycin groups are ...

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Section: Methodsmentioning

confidence: 99%

Cloning, Sequencing, and Characterization of the Iturin A Operon

2001

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“…Transformation of Escherichia coli with plasmid DNA was done as described earlier (14). Transformants were selected on L agar (10 g analysis, and isolation were all performed as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

“…We have independently cloned penP and the repressor gene penI from both the wild-type and constitutive strains of B. licheniformis 9945A (14). By use of the penP signal sequence, many protein secretion vectors have been constructed in Bacillus subtilis (3,10,13).…”

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confidence: 99%

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Himeno¹,

Imanaka²,

Aiba³

1986

J Bacteriol

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Bacillus licheniformis penicillinase genes, penP and penl, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penl(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48°C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB6OE24), the coding region was concluded to be the pen! gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penl promoter. The regulation of penP and pen! by the repressor is discussed.

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“…We constructed a system to analyze the dynamics of noncognate genomic DNA horizontal transfer by using a multicopy plasmid, pTB522, from a thermophilic Bacillus strain that shows -type replication in B. subtilis (10,11). There are two advantages to the use of this multicopy plasmid.…”

mentioning

confidence: 99%

“…Plasmid pTB522 (11) was linearized at the unique HindIII site by HindIII digestion and blunted by T4 DNA polymerase. The blunted pTB522 was ligated with an Escherichia coli plasmid, pBR322, which had been linearized by PvuII and dephosphorylated with alkaline phosphatase.…”

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confidence: 99%

Recombinational Transfer of 100-Kilobase Genomic DNA to Plasmid in Bacillus subtilis 168

Tsuge

Itaya

2001

J Bacteriol

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Transformation of Bacillus subtilis by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic B. subtilis recombinational repair pathway. The vector, pGETS103, a derivative of the -type replicating plasmid pTB19 of thermophilic Bacillus, had the full length of Escherichia coli plasmid pBR322. A multimeric form of pGETS103 yielded tetracycline-resistant transformants of B. subtilis. In contrast, linearized pGETS103 gave tetracycline-resistant transformants only when the recipient strain had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.

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Effect of in vitro DNA Rearrangement in the NH2-terminal Region of the Penicillinase Gene from Bacillus licheniformis on the Mode of Expression in Bacillus subtilis

Cited by 21 publications

References 16 publications

Cloning, Sequencing, and Characterization of the Iturin A Operon

Cloning, Sequencing, and Characterization of the Iturin A Operon

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Recombinational Transfer of 100-Kilobase Genomic DNA to Plasmid in Bacillus subtilis 168

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