Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: Its use as cloning vehicle in B. subtilis 168

Sequence Analysis of Replication Origin of Plasmid pLS11 of Bacillus subtilis IFO 3022

“…The source of the dihydrofolate reductase-coding gene was. a Tmpr strain, TTK24, of B. subtilis 168 17 ) and has been cloned in the pBR322 plasmid of Escherichia coli. DNAs from pTL 12, carrying the Tmpr dihydrofolate reductase gene, which was constructed by Tanaka and Kawano, 16) and pBR322 were both treated with EcoRI and HindUI, mixed and ligated by T4 ligase, then pATEl was constructed.…”

Designed Gene Amplification on the Bacillus subtilis Chromosome

“…Tmr transformants were selected on nutrient agar containing 10 pg tunicamycin ml-l. Ten Tmr transformants were obtained, four of which (designated NMM 13-NMM 16) showed a-amylase hyperproductivity efficiency, we transformed strain APT139 (see Table 1) with 5 pg of intact pKH81 in the presence of 40 m~-MgCl,, using the modified transformation method of Tanaka & Sakaguchi (1978) and selected on plates containing 5 pg or 10 pg tunicamycin ml-l. As a result, 68 and 15 Tmr transformants were obtained, respectively, and 65 and 10 of them showed a-amylase hyperproductivity. Restriction patterns of the latter 10 chromosomal DNAs were analysed; they were identical to those of NMMl3 and NMM14 (data not shown).…”

Cloning Vectors Derived from Plasmids and Phage of Bacillus

“…Insertion of foreign DNA into the single BamHI site inactivates leuA but not leuC, which can thus be used as a marker (Tanaka and Sakaguchi 1978). A derivative of pLS103 termed pLLIQ, has only one EcoRI site and complements leuA and leuB but not, in contrast to pLS103, leuC.…”

“…Plasmids can of course recombine with each other or with the chromosome if they contain homologous segments (Keggins et al 1978;Tanaka and Sakaguchi 1978), the recE4 mutation (Dubnau and Cirigliano 1974) preventing this recombination process. Intramolecular recombination, on the other hand, can occur without involvement of the recE4 function (Tanaka 1979b).…”