The pta gene encoding phosphotransacetylase was cloned on a high copy plasmid with or without the ackA gene encoding acetate kinase in Escherichia coli. The acetate kinase and phosphotransacetylase were overproduced in cells harboring the plasmid possessing both genes. Nucleotide sequencing of the pta gene revealed that it is able to produce a polypeptide comprising 714 amino acid residues, which starts at 70 base pairs downstream from the stop codon of the ackA gene. The 77-kDa protein band of overproduced phosphotransacetylase was observed on SDS-polyacrylamide gel electrophoresis, of which the amino terminal sequence corresponds to that of the deduced polypeptide without the amino terminal methionine. Two transcripts of pta of different sizes were found in the cells. A 3,700 nucleotide transcript, which covers the ackA and pta genes, seemed to be produced by the first promoter in the operon and a 2,300 nucleotide transcript, which covers just pta, seemed to be produced by the second promoter. In a synthetic medium containing acetate as the sole carbon source, the growth of an ackA-pta double mutant was greatly impaired. Complementation analyses revealed that both the acetate kinase and phosphotransacetylase were required for the rapid growth in the acetate medium.