Atay Güneş, Holyavkin Can, Can Hanay, Arslan Mevlüt, Topaloğlu Alican, Trotta Massimo, Çakar Zeynep Petek
Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, İstanbul, Türkiye.
Dr. Orhan Öcalgiray Molecular Biology, Biotechnology and Genetics Research Center (İTÜ-MOBGAM), Istanbul Technical University, İstanbul, Türkiye.
Front Microbiol. 2024 Jun 27;15:1412294. doi: 10.3389/fmicb.2024.1412294. eCollection 2024.
With its versatile metabolism including aerobic and anaerobic respiration, photosynthesis, photo-fermentation and nitrogen fixation, 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 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 CoCl 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 CoCl 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 .
因其具有包括有氧呼吸和无氧呼吸、光合作用、光发酵和固氮在内的多种代谢方式，能够适应各种环境和营养条件，包括存在重金属等各种应激源的情况。因此，它是研究细菌应激反应和抗性分子机制以及用作生物技术应用或生物修复的微生物细胞工厂的重要微生物。在本研究中，通过进化工程，也称为适应性实验室进化（ALE），获得了一种高度耐钴且遗传稳定的菌株，ALE是一种强大的策略，用于改善和表征遗传复杂的所需微生物表型，如抗逆性。为此，在0.1 - 15 mM CoCl的钴应激水平逐渐增加的情况下进行连续批次选择，共64代，且在选择前初始群体未进行任何诱变。从最后一代群体中随机挑选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对铁、镁、镍、铝和NaCl等各种应激源表现出显著的交叉抗性。生长曲线和火焰原子吸收光谱分析结果表明，在4 mM CoCl显著抑制参考菌株生长的情况下，进化菌株的生长不受影响，并且与参考菌株相比，G7细胞中钴离子水平更高。这可能意味着钴离子在G7细胞内或细胞上积累，表明G7具有钴生物修复的潜力。进化菌株的全基因组测序在与转录调节因子、NifB家族 - 铁钼辅因子生物合成、假定的毒力因子、TRAP - T家族转运蛋白、钠/质子反向转运蛋白以及功能未知的基因相关的各种基因中鉴定出23个单核苷酸多态性，这些基因可能在[具体微生物名称未给出]耐钴性中发挥潜在作用。
