Construction and properties of chimeric plasmids in Bacillus subtilis.

Abstract

Antibiotic resistance chimeric plasmids have been constructed by in vitro enzymatic manipulation and introduced into Bacillus subtilis by transformation. The parental plasmids used had been introduced into B. subtiis from Staphylococcus aureus by transformation. Of the seven recombinant plasmids constructed using restriction endonucleases, one was made using EcoRI, another using Hpa II, and five with Xba I (from Xanthomonas badrii), demonstrating the utility of the latter enzyme for molecular cloning experiments. Although all of the recombinant plasmids we have made replicate and express their antibiotic resistance characters, three of them have suffered a loss of DNA, either in vitro or, more likely, in vivo. The deletion event in all cases involved-one of the two termini used to join the parental plasmids. The plasmid chimeras reported in this paper should prove useful for the study of plasmid replication, incompatibility, and recombination. In addition, the utility of the B. subtifis system for molecular cloning has been clearly illustrated.The ability to carry out molecular cloning in Bacillus subtilis would be useful for a variety of studies on sporulation, transformation, and gene expression. In addition, such a capability might be industrially significant, because Bacillus species are of considerable commercial importance. Ehrlich (1) has shown that several chloramphenicol and tetracycline resistance plasmids isolated from Staphylococcus aureus can be introduced by transformation into B. subtilis. This raised the possibility that S. aureus plasmids might be useful as vectors for molecular cloning in R. subtilis. We have transferred additional S. aureus plasmids to competent B. subtilis strains and have initiated a study of the molecular biology of these plasmids. This paper reports the construction of several plasmid chimeras by molecular cloning, thus demonstrating the utility of the B. subtilis system for recombinant DNA experiments and providing a collection of new plasmids for studies on replication, incompatibility, and transformation as well as for the engineering of better cloning vectors. MATERIALS AND METHODSThe B. subtilis host strain used in this study was BD170 (trpC2 thr-5). The plasmids used were originally isolated from S. aureus and were introduced into B. subtilis by transformation. All plasmid DNA preparations used in this study were isolated from B. subtilis. The plasmids are listed in Table 1 together with some relevant restriction endonuclease sites and molecular weights. The documentation of this data will be published elsewhere.'The isolation of covalently closed circular plasmid DNA was carried out by the sodium dodecyl sulfate/NaCl method of Guerry et al. (5) followed by dye-buoyant density centrifugation (6). The preparation of bacterial DNA and competent cells and the transformation procedure was as described previously (7), except that 1 mM ethylene glycol bis(#-aminoethyl ether)-N,N'-tetraacetic acid was added to the plasmid transformation mixtures, because this raise...

Cloning Vectors Derived from Plasmids and Phage of Bacillus

“…In some cases ifplasmid DNA from B. subtilis cells transformed with this plasmid is isolated immediately after transformation, plasmids indistinguishable in size from pJK50l are found, while after subcultivation large deletions are again observed. This clearly demonstrates that in this case the deletion event occurs after the uptake of the plasmid into the cell, as has also been proposed by others (Gryczan and Dubnau 1978).…”

“…coli hybrid plasmids of this type (pJID10, pJID12, pJID21, pJK521, and pJK523) (Goebel et al 1979; Kreft and Goebel, manuscript in preparation). Two of them, pJK310 (pUBllO + pBR325) (Gryczan et al 1978;Bolivar 1978) and pJK523 (p021 + pBR322) (Novick 1976;Bolivar et al 1977), express resistance to two antibiotics in B. subtilis and carry single restriction sites in these markers.…”

Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis

“…Preparation of competent cells (12) and PBS1 transduction (15) were performed as previously described. Measurement of ␤-galactosidase activity was done as described in the accompanying paper, and the activity was expressed as specific activity per milligram of protein (27).…”

Mutations in pts cause catabolite-resistant sporulation and altered regulation of spo0H in Bacillus subtilis

“…For TSS media in which the only nitrogen source was glutamine or proline, NH4Cl was omitted from the TSS salts and glutamine or proline was added to the medium at a final concentration of 0.1%. The doubling times listed in Table 2 were obtained by monitoring the growth curves of liquid cultures and were calculated by the formula (18). The uses of SPI in specialized transduction and its propagation have been described previously (63).…”