Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi - 3 - T in Escherichia coli ( DNA cloning / plasmid / transformation / hybridization )

The thymidylate synthetase gene of B. subtifis bacteriophage Phi-3-T, when cloned in plasmids pSC101 or pMB9, is expressed in E. colt. The promoter of the cloned tene is likely to originate in Phi4-T. Rearrangements of hybrid plasmid sequences during the cloning have been noted. B. subtifis strains can be transformed with hybrid DNAs. The transformants contain sequences of Phi-3T, but not those of plasmid vectors. Phi-3-T is a Bacillus subtilis temperate bacteriophage capable of lysogenic conversion of thyB. subtilis clones to prototrophy (1): we describe here the cloning of the synthetase gene carried by Phi-3-T on the Escherichia coli plasmids pSC101 (2) and pMB9. The gene complements thymine-deficiency mutation in E. coli which indicates its correct transcription and translation in this new host. The promoter of the gene, cloned in pSC101, is likely to be contained within the inserted segment. Hybrid plasmid DNA transforms B. subtilis, although about 10fold less efficiently than the intact phage DNA. B. subtilis clones transformed with the hybrid plasmid, DNA do not contain detectable pSC101 sequences, but do show sequences homologous with part of the Phi-3-T genome. MATERIALS AND METHODS Bacterial Strains. E. coli strains used were W5443 thr-1 leu-6 thi-1 supE44 lacYl rmthystr (tonB trypdelta) (SB2 of D. Finnegan), W5469 thyhsargA metB leuxyllacY strA polAts214 from D. Helinski, C600(pSCIOI) from S. Cohen, HB129 (pMB9) endo" rB+ mB+ gallacstrepr leuprothifrom H. Boyer, and C600 (ColElAmp) (3) from V. Hershfield. B. subtilis strains included SB168 trypC2, SB591 thy-, SB1158 thyA (thyB ilvD6 delta) from this laboratory and SB168(Phi-3-T) from D. Dean. DNAs and Enzymes. Phage, purified by differential centrifugation followed by two CsCl density bandings, was used to prepare Phi-3-T DNA by phenol extraction. Plasmid DNAs were prepared by the clear lysis procedure (4). EcoRI nuclease was prepared according to an unpublished procedure (T. Landers, personal communication). T4 ligase was prepared and used as described (5). EcoRI cleavage was done as described (6). RNA polymerase was a gift of D. Brutlag. 32P-Labeled complementary RNA [cRNA (1)] was synthesized on DNA templates with RNA polymerase as described (7). Transformation Procedures. Competence induction and transformation of E. coli and B. subtilis strains was done according to published procedures (8, 9). Cautions appropriate for a P2 level of containment were adopted for the relevant parts of this investigation. Electrophoresis and Electron Microscopy. Separations were done on horizontal agarose slab gels essentially as described (6). Heteroduplex analysis was done according to ref 10. Hybridization. E. cohand B. subtilis colonies on Millipore filters were prepared for in situ hybridization according to unpublished procedure of J. Lis and L. Prestidge (personal communication). Hybridization of cRNA treated clones or to DNA segments after electrophoretic separation was as described (11, 12). Removal of pSClOl-thy Hybrid Plasmid from E. coli. About 1000 cells were inoculated per ml of L-broth containing 120 sg/ml of ethidium bromide and grown overnight. A 0.1 ml aliquot of the culture was diluted into 5 ml of L-broth. After 1 hr at 370, 20,gg/ml of tetracycline was added, and after another hour 50 /Ag/ml of ampicillin was added. Two hours later, cells were spun down, resuspended in L-broth, and plated on rich medium. Colonies appearing overnight were replica plated on medium containing tetracycline: one out of eight experiments yielded Tcs clones free of plasmid.