N-TERMINAL AMINO-ACID-SEQUENCE AND SUBUNIT STRUCTURE OF THE TYPE-IV TRIMETHOPRIM-RESISTANT PLASMID-ENCODED DIHYDROFOLATE-REDUCTASE

The type IV plasmid-mediated dihydrofolate reductase (DHFR), from a clinical strain of Escherichia coli isolated in South India, was prepared from a transconjugant containing the original clinical plasmid, E. coli J62-2 (pUK 1123), and from E. coli C600 (pUK 1150) containing a 2.6-kb HindIII fragment of pUK 1123 cloned into plasmid pBR 322. Both preparations were purified by methotrexate affinity chromatography. Automatic amino-acid sequencing of the N-terminal of the purified type IV enzyme from both sources gave an identical sequence which was clearly distinct from other plasmid-mediated trimethoprim-resistant DHFRs. The type IV DHFR showed most homology with the endogenous, chromosomally-encoded E. coli enzyme. Amino-acid sequence analysis also showed that the type IV enzyme preparation from E. coli J62-2 harbouring the original clinical plasmid, pUK 1123, also contained the E. coli DNA-binding protein NS1. Analysis by polyacrylamide gel electrophoresis suggested that the type IV enzyme, in its native form, consists of a DHER of M(r) 33 000 coupled to a DNA-binding protein.