AN OPERON ENCODING 3 GLYCOLYTIC-ENZYMES IN LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS - GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, PHOSPHOGLYCERATE KINASE AND TRIOSEPHOSPHATE ISOMERASE

The structural genes gap, pgk and tpi encoding three glycolytic enzyme s, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerat e kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulga ricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplifi cation of chromosomal DNA with degenerate primers corresponding to ami no acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequ encing revealed that the three genes were organized in the order gap-p gk-tpi, The translation start codons of the three genes were identifie d by alignment of the N-terminal sequences. These genes predicted poly peptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, a nd by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed noticeab ly from that in other chromosomal genes. The site of transcriptional i nitiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon, Northern hybridization of tota l RNA with specific probes showed two transcripts, an mRNA of 1.4 kb c orresponding to the gap gene, and a less abundant mRNA of 3.4 kb corre sponding to the gap-pgk-tpi cluster. The absence of a visible terminat or in the 3'-end of the shorter transcript and the location of this 3' -end inside the pgk gene indicated that this shorter transcript was pr oduced by degradation of the longer one, rather than by an early termi nation of transcription after the gap gene.