Figure X: Insolubility of AarCH6-C357Y

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By Elwood A. Mullins1, T. Joseph Kappock2

1. Purdue University and Washington University in St. Louis 2. Purdue University

Supplementary to Mullins E.A. and Kappock T.J. (2013) "Functional analysis of the acetic acid resistance (aar) gene cluster in Acetobacter aceti strain 1023." Acetic Acid Bacteria 2: e3. (DOI: 10.4081/aab.2013.s1.e3).

Version 1.0 - published on 22 Oct 2013 doi:10.4231/D31834278 - cite this Archived on 25 Oct 2016

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Vinegar production requires acetic acid bacteria that produce, tolerate, and conserve high levels of acetic acid. When ethanol is depleted, aerobic acetate “overoxidation” to carbon dioxide ensues. The resulting diauxic growth pattern has two logarithmic growth phases, the first associated with ethanol oxidation and the second associated with acetate overoxidation. The vinegar factory isolate ''Acetobacter aceti'' strain 1023 has a long intermediate stationary phase that persists at elevated acetic acid levels. Strain 1023 conserves acetic acid despite possessing a complete set of citric acid cycle (CAC) enzymes, including succinyl-!CoA:acetate !CoA-transferase (SCACT), the product of the acetic acid resistance (''aar) gene aarC''. In this study, cell growth and acid production were correlated with the functional expression of aar genes using RT-PCR, Western blotting, and enzyme activity assays. Citrate synthase (!AarA) and SCACT (!AarC) were abundant in ''A. aceti'' strain 1023 during both log phases, suggesting the transition to acetate overoxidation was not a simple consequence of CAC enzyme induction. A mutagenized derivative of strain 1023 lacking functional !AarC readily oxidized ethanol but was unable to overoxidize acetate, indicating that the CAC is required for acetate overoxidation but not ethanol oxidation. The primary role of the ''aar'' genes in the metabolically streamlined industrial strain ''A. aceti ''1023 appears to be to harvest energy via acetate overoxidation in otherwise depleted medium.

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Previously unreleased Figure X. Caption: “Figure X. Insolubility of !AarCH6-C357Y. SDS-PAGE was used to assess the solubility of heterologously overproduced !AarCH6-C357Y in E. coli C41(DE3). While a large proportion of the total protein (tot) was !AarCH6-C357Y, no detectable !AarCH6-C357Y remained in the soluble fraction (sol) after the lysate was clarified. Lane sol contains 5 µg protein. Size standard positions are indicated.” Figure X supports: Mullins, E. A. and Kappock, T. J. (2013) "Functional analysis of the acetic acid resistance (''aar'') gene cluster in ''Acetobacter aceti'' strain 1023." ''Acetic Acid Bacteria'' 2, e3 (DOI:

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