Author's School

Graduate School of Arts & Sciences

Author's Department/Program



English (en)

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

T Kappock


The characteristic ability of acetic acid bacteria to aerobically oxidize ethanol to acetic acid has been harnessed for millennia to produce vinegar. Acetic acid permeates cell membranes at low pH and generally inhibits bacterial growth at low millimolar concentrations. The strains used for vinegar production, however, thrive in near-molar concentrations. This remarkable resistance results from the combined contributions of several molecular mechanisms. This study examines the inherent acid stability of proteins from the industrial vinegar-production strain Acetobacter aceti 1023 and the process by which cytoplasmic acetic acid is "overoxidized" to carbon dioxide. Acetate overoxidation in A. aceti strain 1023 is catalyzed by a variant citric acid cycle: CAC) that lacks succinyl-coenzyme A: CoA) synthetase. The acetic-acid-resistance protein succinyl-CoA:acetate CoA-transferase: SCACT, AarC) circumvents this deficiency and bypasses substrate-level phosphorylation and/or adenylation of acetate. Continuous acetate dissimilation by this specialized CAC is dependent upon only favorable oxidation of reduced cofactors. Biphasic growth of A. aceti strain 1023 in yeast extract-peptone-dextrose-ethanol medium is accompanied by distinct stages of acetate production, conservation, and depletion. Acetate is initially accumulated as ethanol is oxidized in the first log phase, transiently maintained in the first stationary phase, and ultimately consumed in the second log phase. The high levels of AarC and SCACT activity present prior to the acetate depletion phase suggest that regulation of CAC enzyme levels is not the only mechanism by which premature acetate overoxidation is avoided. Acetate catabolism may be further minimized during ethanol oxidation by the maintenance of a low cytoplasmic acetate concentration. A. aceti employs multiple means of active acetic acid efflux which may be driven by the energetically favorable oxidation of ethanol. Selective pressure to function in a primary metabolic role increased the specificity and catalytic rapidity of AarC relative to other class I CoA-transferases. Catalysis relies upon a novel oxyanion hole configuration composed partly of the distal amide nitrogen of CoA to stabilize tetrahedral oxyanion intermediates. Structural alignments suggest this mechanism is employed by all class I enzymes. Favorable hydrogen-bonding and electrostatic interactions between the protein and the diphosphate moiety of CoA induce a protein conformational change that was previously predicted to accelerate CoA transfer. This motion is influenced by an auxiliary binding site that preconcentrates carboxylate substrates.


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