CLONING AND SEQUENCING THE RECA-POLYOXOGENES AND ACETOBACTER-ACETI - CONSTRUCTION OF RECA- MUTANTS OF BY TRANSFORMATION-MEDIATED GENE REPLACEMENT( GENES OF ACETOBACTER)

The recA+ gene of Acetobacter polyoxogenes was cloned as a gene that c onferred methyl methanesulfonate resistance (MMS(R)) on the RecA- Esch erichia coli HB101. The cloned recA+ gene also conferred (i) resistanc e to UV irradiation, (ii) enhanced intrachromosomal recombination, and (iii) permitted prophage phi 80 induction in E. coli recA- lysogens. Nucleotide sequence determination revealed that the recA product consi sts of 348 amino acids (aa) corresponding to 38 kDa, and shows signifi cant similarity to RecA proteins from other Gram- bacteria. Next, a po rtion of recA from Acetobacter aceti was cloned by using polymerase ch ain reaction with oligodeoxyribonucleotide primers design based on the A. polyoxogenes recA sequence. Due to availability of efficient host- vector and transformation systems in A. aceti, recA mutants of A. acet i were obtained by transformation-mediated gene replacement with the c loned A. aceti recA gene which was inactivated by insertion of the kan amycin-resistance-encoding gene from pACYC177. The recA mutants obtain ed in this way showed similar phenotypes to those of E. coli recA stra ins, such as increased sensitivity to MMS and to UV irradiation, and d ecreased homologous recombination.