Kataoka Naoya, Matsutani Minenosuke, Matsumoto Nami, Oda Misuzu, Mizumachi Yuki, Ito Kohei, Tanaka Shuhei, Kanesaki Yu, Yakushi Toshiharu, Matsushita Kazunobu
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, Japan.
Front Microbiol. 2022 May 12;13:802010. doi: 10.3389/fmicb.2022.802010. eCollection 2022.
An acetic acid bacterium, NBRC 3288, was adapted to higher growth temperatures through an experimental evolution approach in acetic acid fermentation conditions, in which the cells grew under high concentrations of ethanol and acetic acid. The thermally adapted strains were shown to exhibit significantly increased growth and fermentation ability, compared to the wild strain, at higher temperatures. Although the wild cells were largely elongated and exhibited a rough cell surface, the adapted strains repressed the elongation and exhibited a smaller cell size and a smoother cell surface than the wild strain. Among the adapted strains, the ITO-1 strain isolated during the initial rounds of adaptation was shown to have three indel mutations in the genes , , and . Among these, two dispensable genes, and , were further examined in this study. Rough cell surface morphology related to mutation suggested that membrane vesicle-like structures were increased on the cell surface of the wild-type strain but repressed in the ITO-1 strain under high-temperature acetic acid fermentation conditions. The Δ strain could not grow at higher temperatures and accumulated a large amount of membrane vesicles in the culture supernatant when grown even at 30°C, suggesting that the mutation is involved in cell surface stability. As the gene of ITO-1 lost a 3'-end of 424 bp, which includes one (Act-4) of the possible two regulatory domains (TGS and Act-4), two mutant strains were created: one (ΔTGSAct) with a drug cassette in between the 5'-half catalytic domain and 3'-half regulatory domains of the gene, and the other (ΔAct-4) in between TGS and Act-4 domains of the regulatory domain. These mutants exhibited different growth responses; ΔTGSAct grew better in both the fermentation and non-fermentation conditions, whereas ΔAct-4 did only under fermentation conditions, such as ITO-1 at higher temperatures. We suggest that cell elongation and/or cell size are largely related to these mutations, which may be involved in fermentation stress and thermotolerance.
一株醋酸菌，NBRC 3288，通过实验进化方法在醋酸发酵条件下适应了更高的生长温度，在该条件下细胞在高浓度乙醇和醋酸中生长。与野生菌株相比，热适应菌株在较高温度下显示出显著提高的生长和发酵能力。尽管野生细胞大多伸长且细胞表面粗糙，但适应菌株抑制了伸长，并且与野生菌株相比细胞尺寸更小且细胞表面更光滑。在适应菌株中，在适应初期分离出的ITO - 1菌株在基因、和中显示有三个插入缺失突变。其中，本研究进一步检测了两个非必需基因和。与突变相关的粗糙细胞表面形态表明，在高温醋酸发酵条件下，野生型菌株细胞表面的膜泡样结构增加，但在ITO - 1菌株中受到抑制。Δ菌株在较高温度下无法生长，即使在30°C生长时，培养上清液中也积累了大量膜泡，这表明突变与细胞表面稳定性有关。由于ITO - 1的基因失去了424 bp的3'末端，其中包括可能的两个调控域（TGS和Act - 4）之一（Act - 4），构建了两个突变菌株：一个（ΔTGSAct）在基因的5'半催化域和3'半调控域之间带有一个药物盒，另一个（ΔAct - 4）在调控域的TGS和Act - 4域之间。这些突变体表现出不同的生长反应；ΔTGSAct在发酵和非发酵条件下生长都更好，而ΔAct - 4仅在发酵条件下生长良好，如ITO - 1在较高温度下的情况。我们认为细胞伸长和/或细胞大小在很大程度上与这些突变有关，这些突变可能与发酵应激和耐热性有关。
