RT Journal Article SR Electronic(1) A1 Cancilla, Michael R. A1 Davidson, Barrie E. A1 Hillier, Alan J. A1 Nguyen, Nga Y. A1 Thompson, JohnYR 1995 T1 The Lactococcus lactis triosephosphate isomerase gene, tpi, is monocistronic JF Microbiology, VO 141 IS 1 SP 229 OP 238 DO https://doi.org/10.1099/00221287-141-1-229 PB Microbiology Society, SN 1465-2080, AB SUMMARY: 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-1) was obtained from 70 g (wet wt) cells. In solution, triosephosphate isomerase (pl 4·0-4·4) was observed to exist as a homodimer (Mr 57000) of noncovalently linked subunits. The sequence of the first 37 amino acid residues from the NH2-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 λGEM11 library of L. lactis LM0230 DNA. The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH2-terminal sequence as that determined for the purified enzyme and a subunit Mr of 26802 after removal of the NH2-terminal methionine. Escherichia coli cells harbouring a plasmid containing tpi had 15-fold 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 DL11 chromosome map was determined to be between map coordinates 1·818 and 1·978., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-141-1-229