There has not been elucidated the conclusive mechanism of high resistance mutant on R factor. In this studies, we have examined that the demonstration of R factor mutant capable of hypersynthesis of penicillinase and genetic studies of the mutant.
When the strains of E. coli W3630 harboring RM201 factor (TC.CM.SM.SA.APC) were inoculated on plates containing various concentration of APC, mutants carrying various levels of APC resistance were due to a quantitative increase in the formation of penicillinase. By conjugation experiments and transduction analysis, the mutation was found to affect the gene (amp) governing APC resistance on the R factor. The R-factor mutnats carrying high and stable APC resistance were conjugally transferred at the same frequency as their parent R factors, and the level of their resistance to drugs other than APC was not distinct from that conferred by their parents. Such R-factor mutants could easily be obtained from wild-type R factors carrying low APC resistance. The hypersynthesis of penicillinase by such R-factor mutants was considered to be due to the replication of the amp gene on the R factor at hyper-rates and the integration of multiple copies of the amp gene (amp-hyper) in the R-factor genome.
The physical characteristics of a mutant RM201-2 capable of conferring high and stable APC-resistance was analysed. The RM201-2 and its parent R factor DNA could be isolated as an extrachromosomal and covalently closed circular form, their buoyant densities both 1.712 g/cm3 and the molecular weight were about 8.2× 107 and 6.4 × 107 daltons respectively when measured by CsCl and sucrose density gradient analyses. The contour lengths under electromicroscope were 35.9±0.6μm and 31±0.6μm, respectively. Using the extracted R factor DNA, the mutant and parent characters were transformable to another E. coli strain, respectively. The mutant R factor showed an increased amount of DNA even after conjugaltransfer to Protues. Increase in the size of R factor DNA was thus considered to be the cause for the high level of APC resistance.