An official website of the United States government.
Acetobacter aceti is a Gram-negative alpha-proteobacterium that converts ethanol to acetic acid, each of which is a potent food preservative and microbial poison. Acetic acid bacteria, most prominently A. aceti, are plant-associated organisms that have been used for millenia in the production of vinegar from wine. Vigorous aeration is a key requirement for both ethanol oxidation and bacterial survival at high acetic acid levels. Vintners suppress acetic acid bacteria by excluding O2 and adding SO2 (forming sulfites). Despite extensive experience manipulating acetic acid bacteria in food production, we have little understanding of their essential biochemistry, including how these organisms survive acetic acid and ethanol levels sufficient to kill essentially all other microbes. Acetic acid resistance is broadly relevant to food contamination by pathogenic bacteria that have gained the ability to resist killing by acetic acid treatments.
<P>
Our studies of a "professional" acidophile should shed light on how the "amateurs" defend themselves. We study adaptations in enzyme structure and metabolic function that confer the required resistance traits in a particularly acetic acid-resistant strain isolated in a vinegar factory. We have shown that A. aceti strain 1023 acetic acid resistance gene aarC encodes succinyl-coenzyme A (CoA):acetate CoA-transferase. We proposed that AarC supports acetic acid resistance by oxidation of internal acetic acid to CO2, using a modified version of the citric acid cycle and O2 as the terminal oxidant. Of five acyl-CoA transferases we identified in a draft genome sequence, AarC is the only one with a known function. Catabolism of other carboxylic acids may depend on the others (UctA-D). An operon containing UctB and UctC resembles an E. coli acid-resistance cluster, suggesting they function as resistance factors. UctB and UctC are clustered together with an oxalyl-CoA decarboxylase homologue, suggesting one or both recognizes oxalate.
<P> This proposal seeks support for the following Specific Aims: <P> AIM 1. Assembly and annotation of the Acetobacter aceti 1023 genome. Inferred open reading frames (ORFs) will then be assigned to candidate functions. <P> AIM 2. Succinyl-CoA:acetate CoA-transferase (AarC) expression analysis will be correlated with stressor levels. <P> AIM 3. Identifying acyl-CoA and carboxylic acid substrates for the orphan CoA-transferases UctB and UctC, to learn which acid(s) are processed by these potential acid resistance genes.
NON-TECHNICAL SUMMARY: The use of vinegar as a preservative is based on its ability to kill a broad range of microorganisms, with the notable exception of the vinegar-producing bacterium Acetobacter aceti. Our objective is to study acetic acid resistance by completing the A. aceti genome sequence, monitoring its production of an acid-defense enzyme, and determining if related enzymes protect it from other toxic carboxylic acids. These findings may also indicate how several pathogenic bacteria have gained the ability to survive acetic acid treatments intended to decontaminate food.
<P>
APPROACH: The following approaches will be used to address each Specific Aim: <P>AIM 1. Assembly and annotation of the Acetobacter aceti 1023 genome. Available Sanger and 454 whole-genome shotgun sequence information will be combined with physical mapping data to "close" and thereby complete the genome sequence. A key challenge is the proper handling of numerous identical or near-identical repeat sequences that can only be distinguished by flanking sequence context. Inferred ORFs will be annotated using information from sequence searches and new information from this project. <P>AIM 2. Succinyl-CoA:acetate CoA-transferase (AarC) expression analysis. Expression of the acetic acid resistance factor AarC will be assessed by semi-quantitative Western blotting and enzyme activity measurements, then correlated with acetic acid, ethanol, pH and oxygen levels in bacterial cultures. We also will collaborate with the group of Prof. A. Okamoto-Kainuma to do global transcriptional profiling of Acetobacter strains. <P>AIM 3. Identifying substrates for the orphan CoA-transferases UctB and UctC. An analysis of a similar E. coli gene cluster involved in acid resistance suggests that UctB or UctC process oxalate and oxalyl-CoA. Both proteins are available in purified form. We will chemically synthesize candidate acyl-CoA substrates by CoA alkylation and pair them with one or more candidate carboxylic acid substrates. Liquid chromatography will be used to detect formation of new acyl-CoAs.