Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase

The Corynebacterium glutamicum ack and pta genes encoding the acetate-activ ating enzymes acetate kinase and phosphotransacetylase were isolated, subcl oned on a plasmid and re-introduced into Corynebacterium glutamicum. Relati ve to the wild-type, the recombinant strains showed about tenfold higher sp ecific activities of both enzymes. Sequence analysis of a 3657 bp DNA fragm ent revealed that the ack and pta genes are contiguous in the corynebacteri al chromosome, with pta upstream and the last nucleotide of the pta stop co don (TAA) overlapping the first of the ack start codon (ATC). The predicted gene product of pta consists of 329 amino acids (M-r 35 242), that of ack consists of 397 amino acids (M-r 43 098) and the amino acid sequences of th e two polypeptides show up to 60% (phosphotransacetylase) and 53 % (acetate kinase) identity in comparison with respective enzymes from other organism s. Northern (RNA) blot hybridizations using pta- and ack-specific probes an d transcriptional cat fusion experiments revealed that the two genes are tr anscribed as a 2.5 kb bicistronic mRNA and that the expression of this oper on is induced when Corynebacterium glutamicum grows on acetate instead of g lucose as a carbon source. Directed inactivation of the chromosomal pta and ack genes led to the absence of detectable phosphotransacetylase and aceta te kinase activity in the respective mutants and to their inability to grow on acetate. These data indicate that no isoenzymes of acetate kinase and p hosphotransacetylase are present in Corynebacterium glutamicum and that a f unctional acetate kinase/phosphotransacetylase pathway is essential for gro wth of this organism on acetate.