N-terminal amino-acid sequence and subunit structure of the type IV trimethoprim-resistant plasmid-encoded dihydrofolate reductase

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Abstract
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 (pUK1123), and from E. coli C600 (pUK1150) containing a 2.6-kb HindIII fragment of pUK1123 cloned into plasmid pBR322. 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 seuqence 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 harboring the original clinical plasmid, pUK1123, 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 DHFR of Mr 33 000 coupled to a DNA-binding protein.

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