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 (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 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, 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.
Department of Bacteriology, Medical School, University of Edinburgh.
C J
CJ
Thomson
H K
HK
Young
S G
SG
Amyes
D000595:Amino Acid Sequence; D001322:Autoanalysis; D005075:Biological Evolution; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008727:Methotrexate; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D014296:Trimethoprim Resistance
D004268:DNA-Binding Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate