IDENTIFICATION AND CHARACTERIZATION OF THE TKTB GENE ENCODING A 2ND TRANSKETOLASE IN ESCHERICHIA-COLI K-12

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-1 2 which could not grow on MacConkey plates containing D-ribose. Charac terization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutati on was mapped at 63.5 min on the E. coli genetic map, in which the tra nsketolase gene (tkt) had been mapped. A multicopy suppressor gene whi ch complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning a nd sequencing of a 2.7-kb fragment containing the suppressor gene iden tified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the p rotein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced a mino acid sequence showed similarity to sequences of transketolases fr om Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA . Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.