Stable Positional Cloning of Long Continuous DNA in the Bacillus subtilis Genome Vector

Itaya, Mitsuhiro; Fujita, Kyoko; Ikeuchi, Masahiko; Koizumi, Maki; Tsuge, Kenji

doi:10.1093/jb/mvg168

Cited by 30 publications

(25 citation statements)

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“…Km (500 g/ml), Hm (100 g/ml), and/or Bm (20 g/ml) were added to the medium when needed. B. subtilis strain BEST7003, in which a linearized pBR322 sequence was inserted into the proB gene, has been described previously (19). This genomic pBR322 sequence is called GpBR.…”

Section: Methodsmentioning

confidence: 99%

An Extreme Thermophile,Thermus thermophilus, Is a Polyploid Bacterium

2010

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An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology.

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Section: Methodsmentioning

confidence: 99%

An Extreme Thermophile,Thermus thermophilus, Is a Polyploid Bacterium

2010

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“…To investigate the nature of the horizontal transfer of an antibiotic producer gene in terms of antibiotic production, we transferred the iturin A operon of RB14 to the non-iturinproducing strain 168, using a positive-selection system that employs the cI repressor gene of lambda phage as a reverse marker (14,15,16). In this study, although sfp is necessary, we show that the iturin A operon is essentially the only operon required for conversion of strain 168 into an iturin A producer.…”

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confidence: 90%

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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“…3) The cI cassettes in pCISP310B (cI-spc) 5,6) and pBR322CI-BS (cI-bsr) 8) were directly integrated to these genomic pBR by selection with the linked markers, as shown in Fig. 1.…”

Section: Notementioning

confidence: 99%

“…This method, using a neomycin resistance gene with Prpromoter (Pr-neo) regulated by presence or absence of the CI repressor binding protein, enabled rapid selection for the unmarked DNA integration process. [5][6][7] But careful screening must be carried out to discriminate spontaneous neomycin resistant colonies that appear at frequencies ranging from 10 À5 to 10 À6 due to mutation of the cI gene in the B. subtilis genome (as described in ref. 6 and evident in our unpublished data).…”

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confidence: 99%

“…The two cI gene cassettes, one linked with the spectinomycin resistance gene (cI-spc), and the other with the blasticidin S resistance gene (cI-bsr), were prepared from pBR322-derivative plasmids, pCISP310B (cI-spc) 5,6) and pBR322CI-BS (cI-bsr) 8) respectively. These two cI genes were used in this study to construct mutants for genes relevant to myo-inositol utilization.…”

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