Transductional selection of cloned bacteriophage phi 105 and SP02 deoxyribonucleic acids in Bacillus subtilis

Abstract

The Bacillus subtilis temperate bacteriophages phi 105 and SP02 are incapable of transduction of the small, multicopy drug resistance plasmids pUB110 and pCM194. Cloning endonuclease-generated fragments of phi 105 or SP02 DNA into each of the plasmids renders the chimeric derivatives susceptible to transduction specifically by the phage whose deoxyribonucleic acid is present in the chimera. The majority of phage deoxyribonucleic acid fragments identified that render plasmids transducible by phi 105 or SP02 appear to be internal fragments, not fragments containing the cohesive ends. However, the highest overall transduction frequency was observed in SP02-mediated transduction of a derivative of pUB110 containing a 1.6-megadalton EcoRI fragment that likely contains the SP02 cohesive ends (plasmid pPL1010). The transducing activity present in a phi 105 transducing lysate had a buoyant density slightly greater than infectious particles, whereas the majority of transducing particles in an SP02(pPL1010) transducing lysate had a buoyant density slightly less than infectious particles. Although no detectable change in plasmid structure resulted from transduction by phi 105 or SP02, deoxyribonucleic acid isolated from a purified SP02(pPL1010) transducing lysate contained no detectable monomeric pPL1010, but did contain a form of pPL1010 of higher molecular weight than the monomer.

Bacteriophage SPO2-mediated plasmid transduction in transpositional mutagenesis within the genus Bacillus

“…The importance of SP02 sequences near the cos site to bacteriophage DNA replication and packaging may be elucidated by future studies employing Tn917-directed mutagenesis of a plasmid-borne SP02 cos region. A 1.2-kb EcoRI-HindIII fragment of pPL1017 is derived from the 3.2-kb EcoRI fragment F of the bacteriophage +105 genome and confers immunity to 4105 infectious activity (4,5,13,15). The approximately 0.5-kb region between the inserts of plasmids pLK250 and pLK253 (Fig.…”

“…The prototrophic strain B. amyloliquefaciens DSM7 was obtained from Bayer A.G. (15). Media used included tryptose blood agar base (TBAB; Difco Laboratories), antibiotic medium 3 (Penassay broth [PB]; Difco), and M medium prepared for broth and solid-base cultures as previously described (13,17). Resistance to antibiotics was determined at concentrations of 5 ,ug/ml for chloramphenicol (Cam9 and 10 ,ug/ml for neomycin sulfate (Neo9.…”

“…Strains of B. subtilis were brought to competence and transformed by the method of Boylan et al (2). Bacteriophages were propagated and assays for infectious and transducing activities were performed as previously described (13). Lysogenization of sensitive strains of B. subtilis with bacteriophages SP02 and 4105 and the tests for immunity to superinfection by these two bacteriophages have been described (12,14).…”

“…Cesium chloride-ethidium bromide buoyant density gradient purification of plasmid DNA was performed by the method of Lovett and Keggins (10). Rapid plasmid isolations were performed by using cleared lysates of small-volume cell suspensions as previously described (13). Restriction endonuclease analysis of plasmid DNA was done with enzymes obtained from Bethesda Research Laboratories and used to the specifications supplied by the manufacturer.…”

“…Derivatives of staphylococcal antibiotic resistance plasmids containing the cohesive end site (cos) of bacteriophage SP02 are transducible by SP02 at high frequency among strains of B. subtilis 168 (11,13). Recombination-proficient strains lysogenic for SP02 and harboring SPO2 cos plasmids can be induced to yield lysates with transducing frequencies ranging from 0.5 x 10-2 to 2.0 x 10-2 transductants per PFU for plasmid-borne markers (11).…”