Molecular mechanisms of adaptation in waterborne microbes

我的研究计划的长期目标是了解细菌在环境中的分子适应机制。我将在我的研究中使用大肠杆菌作为模型，因为(I)E.coliK-12是研究得最好的模式菌；(Ii)E.coli自然分离株在各种来源都可以找到；(Iii)我的实验室拥有构建突变和基因表达所需的所有遗传工具。*天然大肠杆菌菌株的基因组不仅高度分化，而且在胁迫适应和抗生素耐药性方面也有不同的表型。人们对环境菌株中大量基因的功能知之甚少。我推测，这些环境基因编码了细胞适应特定宿主或环境条件的重要功能。唯一的环境是处理设施中的城市污水，该设施从城市居民那里收集单个肠道微生物以及抗生素和其他药物。在处理过程中，微生物受到包括渗透休克、氧化应激和紫外线暴露在内的应激条件的挑战。可以想象，这些不利的条件为可能编码特定酶或调节剂的基因提供了强有力的选择，以减轻压力/抗生素毒性。*我的具体目标是：(1)鉴定具有增强的应激和抗生素耐药性的大肠杆菌分离株；(2)利用基因组测序和功能基因组学方法鉴定抗压和抗生素所需的基因。我的实验室已经建立了一个从卡尔加里污水处理厂分离出来的大型大肠杆菌收集(&gt；4000个分离物)。利用这一大肠杆菌收集，我们将筛选出对压力条件和抗生素耐药性高度耐受的菌株。我们假设这些选定的菌株已经获得了编码特定酶或调节因子的适应特异性基因，以缓解压力和抗生素暴露。我们将使用全基因组测序和转座子突变来确定适应所需的基因。我们还将使用包括亲和下拉、免疫共沉淀和染色质免疫沉淀在内的生化分析来表征基因功能和潜在的适应机制。*这项研究将为城市污水中微生物的适应策略提供重要的机械见解，城市污水是最复杂的环境之一。**

The long-term objective of my research program is to understand the molecular adaptation mechanisms of bacteria in the environment. I will use Escherichia coli as a model in my study because (i) E. coli K-12 is the best studied model bacterium; (ii) E. coli natural isolates can be found in various sources; (iii) my lab has all the required genetic tools for mutant construction and gene expression.*** Not only are the genomes of natural E. coli strains highly divergent but also they differ phenotypically in stress adaption and antibiotic resistance. Little is known about the functions of a vast number of genes in environmental strains. I hypothesize that these environmental genes code for important functions for cells to adapt to specific host or environmental conditions. A uniqe environment is municipal wastewater at the treatment facility that collects the individual gut microbiomes, as well as antibiotics and other pharmaceutical drugs, from the city residents. During treatment process, microbes are challenged with stress conditions including osmotic shock, oxidative stress, and UV exposure. Those hostile conditions conceivably pose a strong selection for genes that may encode specific enzymes or regulators to alleviate stress/antibiotic toxicity. *** My specific objectives are: (1) to identify E. coli isolates with enhanced stress and antibiotic resistance; (2) to characterize genes required for resistance to stress and antibiotics using genome sequencing and functional genomics approaches. My lab has established a large E. coli collection (>4000 isolates) isolated from the Calgary Wastewater Treatment Plants. Using this E. coli collection, we will screen for isolates that are highly resistant to stress conditions and antibiotic resistance. We hypothesize that these selected isolates have acquired adaptation specific genes encoding specific enzymes or regulatory factors to alleviate the stress and antibiotic exposure. We will use whole genome sequencing and transposon mutagenesis to identify genes required for the adaptation. We will also use biochemical assays including affinity-pull down, co-immunoprecipitation, and chromatin-immunoprecipitation to characterize the gene functions and underlying mechanisms of adaptation. *** This study will provide important mechanistic insights regarding the adaption strategy of microbes in municipal wastewater, one of the most complex environments. **