Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993). We constructed a mutant allele, ⌬acs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome. Whereas wild-type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (Յ10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (Ն25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested. Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes. Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested. Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence. This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii. The purified E. coli Acs then was used to raise anti-E. coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs. When purified in the presence, but not in the absence, of coenzyme A, the E. coli enzyme activated acetate across a wide range of concentrations in a coenzyme A-dependent manner. On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs.Escherichia coli cells activate acetate to acetyl coenzyme A (acetyl-CoA) by two distinct pathways (Fig. 1) Brown et al. (6) hypothesized that the Acs pathway functions as a catabolite-repressible, acetate-inducible, high-affinity acetate uptake system that scavenges acetate present extracellularly at relatively low concentrations. They also proposed that the Ack-Pta pathway functions primarily in a catabolic role, excreting acetate and generating ATP during mixed-acid fermentation and aerobic growth on excess glucose or other glycolytic intermediates. Finally, they argued that the low-affinity Ack-Pta pathway activates acetate only when that molecule is present extracellularly in large quantity.In addition to their role in acetate metabolism, the acetate activation pathways have been implicated in the regulation of signal transduction by two-component regulatory systems in several bacterial species (reviewed in references 32 and 54; see also references 2, 9, 39, and 55), the regulation of the glucose starvation stimulon of E. coli ...