The Lactococcus lactis t r iose p hosp hate isomerase gene , tpi , is rnonocistronic
نویسنده
چکیده
Triosephosphate isomerase (EC 5.3.1.1) from Lactococcus lactis was purified to electrophoretic homogeneity. Approximately 3 mg purified enzyme (specific activity 3300 U mg-l) was obtained from 70 g (wet wt) cells. In solution, triosephosphate isomerase (pl4*044) was observed to exist as a homodimer (M, 57000) of noncovalently linked subunits. The sequence of the first 37 amino acid residues from the NH,-terminus were determined by step-wise Edman degradation. This sequence, and that of a region conserved in all known bacterial triosephosphate isomerases, was used to design oligonucleotide primers for the synthesis of a lactococcal tpi probe by PCR. The probe was used to isolate a molecular clone of tpi from a RGEMII library of L. lactis LM0230 DNA. The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH,-terminal sequence as that determined for the purified enzyme and a subunit M, of 26802 after removal of the NH,-terminal methionine. Escherichia coli cells harbouring a plasmid containing tpi had 15fold higher triosephosphate isomerase activity than isogenic plasmid-free cells, confirming the identity of the cloned gene. Northern analysis of L. lactis LM0230 RNA showed that a 900 base transcript hybridized with tpi. The 5‘ end of the transcript was determined by primer extension analysis to be a G located 64 bp upstream from the tpi start codon. These transcript analyses indicated that in L. lactis, tpi is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to tpi did not encode another Embden-Meyerhoff-Parnas pathway enzyme. The location of tpi on the L. lactis DLll chromosome map was determined to be between map coordinates 1-818 and 1.978.
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