具有多种代谢功能,包括有氧和无氧呼吸,光合作用,光发酵和固氮,球形红细菌可以适应不同的环境和营养条件,包括重金属等各种压力源的存在。因此,是研究细菌应激反应和抗性分子机制的重要微生物,并用作生物技术应用或生物修复的微生物细胞工厂。在这项研究中,通过进化工程获得了一种高度耐钴且遗传稳定的沙氏菌菌株,也被称为适应性实验室进化(ALE),改善和表征基因复杂性的强大策略,所需的微生物表型,如抗压力。为此,在0.1-15mMCoCl2之间逐渐增加的钴胁迫水平存在下进行连续批次选择,传代64次,并且在选择之前没有对初始种群进行任何诱变。从最后一个群体中随机选择突变个体并进行详细分析。其中,一种名为G7的高度耐钴且遗传稳定的进化菌株显示出对各种应激源(如铁)的显着交叉抗性,镁,镍,铝,和NaCl。生长曲线和火焰原子吸收光谱分析结果表明,在存在显着抑制参考菌株生长的4mMCoCl2时,进化菌株的生长不受影响,与参考菌株相比,更高水平的钴离子与G7细胞相关。这可能意味着钴离子在G7电池中或上积累,表明G7用于钴生物修复的潜力。进化菌株的全基因组测序鉴定了与转录调节因子相关的各种基因中的23个单核苷酸多态性。NifB家族-FeMo辅因子生物合成,假定的毒力因子,TRAP-T家族转运蛋白,钠/质子反转运蛋白,以及功能未知的基因,这可能对R.spaeroides的钴抗性有潜在的作用。
With its versatile metabolism including aerobic and anaerobic respiration, photosynthesis, photo-fermentation and nitrogen fixation, Rhodobacter sphaeroides can adapt to diverse environmental and nutritional conditions, including the presence of various stressors such as heavy metals. Thus, it is an important microorganism to study the molecular mechanisms of bacterial stress response and resistance, and to be used as a microbial cell factory for biotechnological applications or bioremediation. In this study, a highly cobalt-resistant and genetically stable R. sphaeroides strain was obtained by evolutionary engineering, also known as adaptive laboratory evolution (ALE), a powerful strategy to improve and characterize genetically complex, desired microbial phenotypes, such as stress resistance. For this purpose, successive batch selection was performed in the presence of gradually increased cobalt stress levels between 0.1-15 mM CoCl2 for 64 passages and without any mutagenesis of the initial population prior to selection. The mutant individuals were randomly chosen from the last population and analyzed in detail. Among these, a highly cobalt-resistant and genetically stable evolved strain called G7 showed significant cross-resistance against various stressors such as iron, magnesium, nickel, aluminum, and NaCl. Growth profiles and flame atomic absorption spectrometry analysis results revealed that in the presence of 4 mM CoCl2 that significantly inhibited growth of the reference strain, the growth of the evolved strain was unaffected, and higher levels of cobalt ions were associated with G7 cells than the reference strain. This may imply that cobalt ions accumulated in or on G7 cells, indicating the potential of G7 for cobalt bioremediation. Whole genome sequencing of the evolved strain identified 23 single nucleotide polymorphisms in various genes that are associated with transcriptional regulators, NifB family-FeMo cofactor biosynthesis, putative virulence factors, TRAP-T family transporter, sodium/proton antiporter, and also in genes with unknown functions, which may have a potential role in the cobalt resistance of R. sphaeroides.