Bacillus pumilus plasmid pPL10: properties and insertion into Bacillus subtilis 168 by transformation

Abstract

B. pumilus ATCC 12140 harbors .gtoreq. 10 copies/chromosome of each of 2 small plasmids. Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent. Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled DNA. Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; MW .apprx.4.4 .times. 106; .apprx.20 copies/chromosome; .rho. [buoyant density] = 1.698). Resulting Klll+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10. Transfer of pPL10 from strain L10 to B. pumilus sttrain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype. Strain NRS 576 naturally harbors .apprx.2 copies/chromosome of a 28 .times. 106-dalton plasmid, pPL576. In Kill+ transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of .apprx.11 molecules of pPL10 to 1 molecule of pPL576. Therefore, pPL576 and pPL10 are compatible plasmids. B. subtilis 168 is naturally resistant to pPL10-determined killing activity. Plasmid pPL10 was therefore inserted into B. subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient. The plasmid is stably maintained at an estimated 10 copies/chromosome in the spore- recipient and in spore+ transformants. pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoR1.