Cloning of Bacillus subtilis leucine A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli

Nagahari, Kenji; Sakaguchi, Kenji

doi:10.1007/bf00267197

Cited by 48 publications

(13 citation statements)

References 25 publications

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“…The LPA carried by pBRE2H4cI was inserted into the leuB region in the BEST6225 genome as follows. Plasmid pBR322Cm (12), which harbors a chloramphenicol resistance gene (cat) cassette in the EcoRI site of pBR322, was linearized at a unique PvuII site and then inserted into a unique BamHI site in the leuB gene of plasmid RSF2124.B.leuB (25) by using a T4 DNA polymerase-based blunting kit. The resulting plasmid, pBMAP103-322CA, was used to transform BEST6225, generating BEST6234, which has pBR322Cm inserted in the genomic leuB region (Fig.…”

Section: Methodsmentioning

confidence: 99%

Horizontal Transfer of Iturin A Operon, itu , to Bacillus subtilis 168 and Conversion into an Iturin A Producer

Tsuge

Inoue

Ano

et al. 2005

Antimicrob Agents Chemother

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Iturin A and its derivatives are lipopeptide antibiotics produced by Bacillus subtilis and several closely related bacteria. Three iturin group operons (i.e., iturin A, mycosubtilin, and bacillomycin D) of those antibioticproducing strains have been cloned and sequenced thus far, strongly implying the horizontal transfer of these operons. To examine the nature of such horizontal transfer in terms of antibiotic production, a 42-kb region of the B. subtilis RB14 genome, which contains a complete 38-kb iturin A operon, was transferred via competent cell transformation to the genome of a non-iturin A producer, B. subtilis 168, using a method based on double-crossover homologous recombination with two short landing pad sequences (LPSs) in the genome. The recombinant was positively selected by confirming the elimination of the cI repressor gene, which was localized between the two LPSs and substituted by the transferred segment. The iturin A operon-transferred strain 168 was then converted into an iturin A producer by the introduction of an sfp gene, which encodes 4-phosphopantetheinyl transferase and is mutated in strain 168. By inserting the pleiotropic regulator degQ, the productivity of iturin A increased sevenfold and was restored to about half that of the donor strain RB14, without the transfer of additional genes, such as regulatory or self-resistance genes.

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Section: Methodsmentioning

confidence: 99%

Horizontal Transfer of Iturin A Operon, itu , to Bacillus subtilis 168 and Conversion into an Iturin A Producer

Tsuge

Inoue

Ano

et al. 2005

Antimicrob Agents Chemother

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“…subtilis were cloned into the ampicillin (Ap) or the tetracycline (Tc') determinants, respectively, of cloning vector pJH101. Plasmid construction resulted in the following integrable derivatives of pJH101: (i) pGR102 containing a 1.2-kilobase (kb) PstI-EcoRI fragment from pMS102-B7 (24,30), (ii) pWR103 containing a 1.0-kb PstI-EcoRI fragment from pBC194 (33), and (iii) pER102 containing a 3.2-kb EcoRI-BamHI fragment from RSF2124-leu of B. subtilis (22).…”

Section: Methodsmentioning

confidence: 99%

Mapping of rRNA genes with integrable plasmids in Bacillus subtilis

LaFauci¹,

Widom²,

Eisner³

et al. 1986

J Bacteriol

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Integrable plasmids pGR102 and pWR103 containing ribosomal sequences from within the transcriptional units for 16S and 23S were used to transform Bacillus subtilis. To date, these plasmids integrated into 7 of 10 known rrn operons. Two such events occurred at unassigned operons, revealing a close linkage of the CAT gene of the plasmid to pha-1 situated between dal-) and purB33 for rrnE and to thiA78 situated between glyB133 and tre-12 for rrnD. All seven integration events that led to the loss of unique ribosomal BclI fragments can now be assigned to known rrn operons.In the genome of Bacillus subtilis there are as many as 10 rRNA gene sets clustered in three groups (5). A major group is composed of rrnO and rrnA located at the replication origin (24,26,35) and the closely situated (5, 26) repeats (rrnI, rrnH, rrnG) found near the attachment site of phage SP02 (5). Minor clusters containing rrnB and rrnC have been mapped in the region between thr-S and aroG (5), and one cistron is believed to be located at the ilvBC-leu region (7,11,31). Strains of B. subtilis have been shown to contain only nine operons because of a deletion (11,18) or to exhibit rearrangements involving ribosomal sequences (3,11). Each transcriptional unit is arranged in the order 16S, 23S, and 5S rRNA genes, with two to three operons (including rrnO and rrnA) containing tRNAAla and tRNAlie genes in the abutment region between the 16S and 23S rDNA sequences (18, 33). We employed the integrative mapping method of Haldenwang et al. (13) to ascertain whether integrations can occur in operons, to locate various unmapped rrn genes on the chromosome, and to determine the physiological effects on growth and sporulation of such events in B. subtilis. In this study, the bifunctional plasmid pJH101 was used (10). The plasmid cannot replicate in B. subtilis because it does not contain an origin of replication for B. subtilis. If a region of homology with the B. subtilis chromosome is inserted in pJH101, the resultant plasmid gains the ability to transform competent cells and also, on occasion, integrates by Campbell-like recombination (10). We subcloned two similar but not identical ribosomal fragments from within the transcriptional unit for 16S and 23S which originated from the operons rrnO and rrnI. The resultant plasmids pGR102 and pWR103 can theoretically integrate into any of the 10 ribosomal operons by a Campbell-like insertion, leaving the entire plasmid with its antibiotic marker adjacent to the homologous region. We report here seven different integration events by these plasmids, two of which occur at unassigned operons (rrnD and rrnE), increasing the number from seven to nine known genomic locations in B. subtilis. containing integrated plasmids were constructed by competent cell transformation, verified for integration by hybridization, and checked for site of integration by PBS1 transduction mapping. We designated the various transformants as the B. subtilis strain Ql plasmids, with fl denoting plasmid integration. The plasmids with and wi...

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“…The leuABCD gene cluster, responsible for leucine biosynthesis, has been previously mapped and cloned in B. szlbtili~ (Nagahari & Sekaguchi, 1978). The sequence of leaB (formerly lez/C) has been described previously (Imai et a/., 1987).…”

Section: Homology Search and Properties Of Newly Identified Orfsmentioning

confidence: 99%

The dnaB-pheA (256 -240 ) region of the Bacillus subtilis chromosome containing genes responsible for stress responses, the utilization of plant cell walls and primary metabolism

Wipat

Carter²,

Brignell³

et al. 1996

Microbiology

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Within the framework of the international programme to sequence the genome of Baci//us subti/is strain 168, we were allocated the region between dna8 (256") and pheA (240"). The sequencing of this region is now complete and we report our primary analysis of the 114 kb region containing 114 ORFs. In addition to previously characterized genes, we have identified genes involved in the utilization of plant cell wall pOlySaCCharideS, stress responses and the metabolism of amino acids, cell walls, DNA and fatty acids. We also discuss various structural and physical features, including the orientation of genes with respect to replication, putative start and stop codons, ribosome binding sites and pindependent transcription terminators. I I

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Cloning of Bacillus subtilis leucine A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli

Cited by 48 publications

References 25 publications

Horizontal Transfer of Iturin A Operon, itu , to Bacillus subtilis 168 and Conversion into an Iturin A Producer

Horizontal Transfer of Iturin A Operon, itu , to Bacillus subtilis 168 and Conversion into an Iturin A Producer

Mapping of rRNA genes with integrable plasmids in Bacillus subtilis

The dnaB-pheA (256 -240 ) region of the Bacillus subtilis chromosome containing genes responsible for stress responses, the utilization of plant cell walls and primary metabolism

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