Use of a Bifunctional Cosmid for Cloning Large DNA Fragments of B. subtilis

Aubert, Elisabeth; Fargette, F; Fouet, Agnès; Klier, André; Rapoport, Georges

doi:10.1016/b978-0-12-274150-0.50008-4

Cited by 7 publications

(6 citation statements)

References 11 publications

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“…It is possible that extensive deletions occurred in the original cosmid after subculturing in E. coli, even under selective pressure caused by the presence of antibiotics. This situation has already been observed in E. coli for other genes studied in our laboratory, such as sacA, thr, and glyB (3,11). In the case of pBS5A4, vector pQB 79-1 has not been modified, so deletions or rearrangements should have occurred in the cloned B. subtilis sequences.…”

Section: Discussionsupporting

confidence: 57%

“…In this paper, we describe the identification of a recombinant cosmid from a B. subtilis DNA bank established in Escherichia coli (3) harboring the regulatory gene sacU, the isolation of a 2.4-kilobase (kb) fragment containing this locus, and the complementation of sacU-and sacU" mutations in B. subtilis. Expression of the sacU gene was also demonstrated in a minicell-producing E. coli strain.…”

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confidence: 99%

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Cloning and expression in Escherichia coli of the regulatory sacU gene from Bacillus subtilis

Aubert¹,

Klier²,

Rapoport³

1985

J Bacteriol

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The regulatory wild-type locus sacU, which has a pleiotropic effect in Bacillus subtilis, notably on the synthesis of secreted proteins, was obtained from a colony bank of Escherichia coli harboring recombinant cosmids representative of the B. subtilis genome. It was shown that the sacU gene is located on a 2.4-kilobase KpnI-EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. The wild-type phenotype was recovered after transformation of SacU-, SacUh, and SacU-Recstrains with the recombinant cosmid, indicating that the sacU locus has been cloned in totality. The sacU gene was expressed in a minicell-producing E. coli strain, and it was shown that it coded for a 46-kilodalton protein.In addition to the hypersecretion of proteins, SacUh mutants were characterized by the presence of a 46-kilodalton protein in the membrane fraction in higher amounts than were found in the wild-type strain. These mutants were also devoid of a 36-kilodalton polypeptide corresponding to the flagellin subunit.

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Section: Discussionsupporting

confidence: 57%

mentioning

confidence: 99%

Cloning and expression in Escherichia coli of the regulatory sacU gene from Bacillus subtilis

Aubert¹,

Klier²,

Rapoport³

1985

J Bacteriol

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“…This assumption is supported, but not proved, by our failure to identify any clone cross-reacting with GS2 antibody from a constructed gene bank in the cosmid pQB79-1 (data not shown), despite the fact that this cosmid has been used for cloning of DNA carrying other genes (1). This would argue against the expression of GS2 gene from its own promoter in E. coli.…”

Section: Discussionmentioning

confidence: 94%

Molecular cloning of an ornithine-activating fragment of the gramicidin S synthetase 2 gene from Bacillus brevis and its expression in Escherichia coli

Krause¹,

Marahiel²,

Döhren³

et al. 1985

J Bacteriol

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Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 x 103 colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PP, and 2'-dATP-32PPexchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.

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“…Intermolecular rearrangements include Tt'cE-dependent integration of homologous DNA sequences into the B. subtilis chromosome (Aubert et al, 1982). Homology between plasmid and chromosomal sequences results in intermolecular recombination and the integration of plasmid DNA into the chromosome and these events have been exploited to develop a series of integration vectors (see Chapters 4 and 6).…”

Section: Intermolecular Recombinationmentioning

confidence: 99%

Bacillus

1989

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Use of a Bifunctional Cosmid for Cloning Large DNA Fragments of B. subtilis

Cited by 7 publications

References 11 publications

Cloning and expression in Escherichia coli of the regulatory sacU gene from Bacillus subtilis

Cloning and expression in Escherichia coli of the regulatory sacU gene from Bacillus subtilis

Molecular cloning of an ornithine-activating fragment of the gramicidin S synthetase 2 gene from Bacillus brevis and its expression in Escherichia coli

Bacillus

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