Classification of Trimethoprim‐Resistant Dihydrofolate Reductases Mediated by R‐Plasmids Using Isoelectric Focussing

An analytical isoelectric focussing technique has been developed to distinguish between different groups of R‐plasmid‐mediated trimethoprim‐resistant dihydrofolate reductases. Those mediated by the R‐plasmids R483 and R721, which are type I dihydrofolate reductases have isoelectric point (PI) values of 6.4 whereas those mediated by the R‐plasmids R67bis and R27, which are type I1 enzymes, have PI values of 5.5. There has been some confusion as to the classification of the dihydrofolate reductases mediated by R‐plasmids R388 and R751; however, our studies indicate that the one mediated by R388 is a type I1 enzyme, having a PI value of 5.5, whereas the one mediated by R751 is different from both the type I and type I1 enzymes having a PI value of 7.2. The differences in PI value of these three groups of trimethoprim‐resistant enzymes were confirmed by DEAE‐ cellulose chromatography at pH 8.5 where it was found that the type I enzymes were eluted with a different concentration of NaCl than were the type I1 enzymes including that mediated by R388, whereas the enzyme mediated by R751 did not bind to the DEAE‐cellulose at this pH. A ‘scale‐down’ technique was developed for analytical isoelectric focussing and was used to study the dihydrofolate reductases mediated by ten R‐plasmids present in trimethoprim‐resistant Escherichia coli strains isolated from pigs. All these R‐plasmids coded for type I enzymes and all also coded for resistance to streptomycin, indicating that the trimethoprim‐resistance gene(s) were present on a transposon similar to Tn7.