Molecular mechanisms of adaptation in waterborne microbes

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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. **