Implication of amino acid metabolism and cell surface integrity for the thermotolerance mechanism in the thermally adapted acetic acid bacterium Acetobacter pasteurianus TH-3

Abstract

ABSTRACT An acetic acid bacterium, Acetobacter pasteurianus SKU1108, was adapted to higher growth temperatures through an experimental evolution approach under acetic acid fermentation conditions. The thermally adapted strain, TH-3, exhibited significantly increased growth and fermentation ability compared with the wild-type strain at higher temperatures (M. Matsutani, M. Nishikura, N. Saichana, T. Hatano, et al., J Biotechnol 165:109–119, 2013, https://doi.org/10.1016/j.jbiotec.2013.03.006 ). A previous study showed that the TH-3 strain has a total of 11 mutations in the genome, of which marR has been shown to be involved in a higher acetic acid fermentation ability, but mutations related to thermotolerance have not yet been elucidated. In this study, we identified almost all of the mutated genes and found that mutation of three genes, ans (asparagine permease), dct (dicarboxylate transporter), and glnD (uridylyltransferase PII), with ans and dct becoming dysfunctional but glnD seemingly functionally modified, was sufficient to reproduce the increased thermotolerance of the TH-3 strain. In addition, these mutations induced two phenotypic changes in TH-3: altered intracellular amino acid pool and cell size reduction. We further observed cell surface modification in TH-3, including increased phospholipid and lipopolysaccharide contents, as well as increased respiratory activities with reduced intracellular reactive oxygen species generation. These results suggest that mutation of the three genes enabled the TH-3 strain to be more thermotolerant by increasing the cell surface integrity and energy generation, which could be caused by increased membrane components and altered membrane protein synthesis via changes in the intracellular amino acid pool, together with the cell size reduction, which may enhance nutrient or oxygen availability. IMPORTANCE Acetobacter pasteurianus , an industrial vinegar-producing strain, is suffered by fermentation stress such as fermentation heat and/or high concentrations of acetic acid. By an experimental evolution approach, we have obtained a stress-tolerant strain, exhibiting significantly increased growth and acetic acid fermentation ability at higher temperatures. In this study, we report that only the three gene mutations of ones accumulated during the adaptation process, ansP , dctD , and glnD , were sufficient to reproduce the increased thermotolerance of A. pasteurianus . These mutations resulted in cell envelope modification, including increased phospholipid and lipopolysaccharide synthesis, increased respiratory activity, and cell size reduction. The phenotypic changes may cooperatively work to make the adapted cell thermotolerant by enhancing cell surface integrity, nutrient or oxygen availability, and energy generation.