The Lactococcus lactis triosephosphate isomerase gene, tpi, is monocistronic.
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 (pI 4.0-4.4) was observed to exist as a homodimer (M(r) 57,000) 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 lambda GEM11 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 M(r) of 26,802 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 65 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.
Cancilla
M R
MR
Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Davidson
B E
BE
Hillier
A J
AJ
Nguyen
N Y
NY
Thompson
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D016384:Consensus Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000032:analysis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014158:Transcription, Genetic; D014305:Triose-Phosphate Isomerase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D014305:Triose-Phosphate Isomerase