Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein repA.

In vitro mutagenesis with hydroxylamine of a ParD- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174. Some of these mutants had a thermosensitive phenotype. The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42 degrees C. The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced. Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3' end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene. These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.