Limited Diversity of Thermal Adaptation to a Critical Temperature in Zymomonas mobilis: Evidence from Multiple-Parallel Laboratory Evolution Experiments

Laboratory evolution is an effective means of understanding microbial adaptation to the environment. We previously isolated four thermoadapted <i>Zymomonas mobilis</i> mutants, which showed a 2 °C rise in the critical high temperature (CHT), by performing multiple-parallel adaptation experiments. In the present study, the individual mutations in these mutants were intensively analyzed. Two mutations in each adapted mutant were found to primarily contribute to the increase in the upper temperature limit. RNA sequencing (RNA-seq) analysis revealed that the two mutations led to the upregulation of 79-185 genes and the downregulation of 242-311 genes. The findings from transcriptomic and physiological experiments suggest two common and primary mechanisms for thermal resistance: a decrease in the activity of diacylglycerol kinase, which may change the structure of lipopolysaccharide (LPS) probably to strengthen the membrane structure, and an increase in the expression of genes for GroEL/GroES or cell wall hydrolase to repair the protein or membrane damage that occurs at such critical temperatures. Additionally, transporters including efflux pumps may contribute to intracellular homeostasis by expelling toxic compounds such as ethanol and acetate or by maintaining the K⁺ concentration. The results of this study on four independently thermoadapted mutants led to the conclusion that the mutants have almost the same thermal adaptation strategies and thus their molecular diversity is limited.