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。我将使用大肠杆菌作为我的研究模型，因为（i）E. coliK-12是目前研究最好的模式菌;大肠杆菌天然分离物可以在各种来源中找到;（iii）我的实验室拥有突变体构建和基因表达所需的所有遗传工具。 不仅天然E.大肠杆菌菌株高度分化，但它们在胁迫适应和抗生素抗性方面也存在表型差异。对于环境菌株中大量基因的功能知之甚少。我假设这些环境基因编码细胞适应特定宿主或环境条件的重要功能。一个独特的环境是处理设施的城市废水，该设施收集城市居民的单个肠道微生物组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压休克、氧化应激和UV暴露在内的应激条件的挑战。可以想象，这些不利的条件对可能编码特定酶或调节剂以减轻压力/抗生素毒性的基因构成了强烈的选择。*** 具体目标是：（1）鉴定E.（2）利用基因组测序和功能基因组学方法，对胁迫抗性和抗生素抗性所需的基因进行鉴定。我的实验室已经建立了一个大的E。从卡尔加里污水处理厂分离的大肠杆菌集合（>4000个分离株）。使用这个E。大肠杆菌的收集，我们将筛选分离株，是高度耐应力条件和抗生素耐药性。我们假设这些选择的分离株已经获得了编码特定酶或调节因子的适应特异性基因，以减轻压力和抗生素暴露。我们将使用全基因组测序和转座子诱变来鉴定适应所需的基因。我们还将使用生物化学测定，包括亲和下拉，免疫共沉淀和染色质免疫沉淀，以表征基因功能和适应的潜在机制。*** 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制见解，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. **