Refined Description of a Gene Locus in Escherichia coli Encoding for Bile Salt Resistance and Sensitivity

1. Refined Description of a Gene Locus in Escherichia coli Encoding for Bile Salt Resistance and Sensitivity Eden Bernstein, Anna Graves, Jonna Whetsel, and James BidlackDepartment of Biology, University of Central Oklahoma, Edmond, OK 73034 Abstract Methods Cultures: Five E. coli strains were utilized in this experiment: BW25113, JW1271, JW1272, JC3272F, and JC3272I. They were stored in a -80˚ freezer and streaked on LB agar plates (Figure 1). DNA Extraction: Using an inoculating loop, randomly selected colonies were placed into microcentrifuge tubes containing 100 µL of deionized H2O. PCR mixture: PCR mixture included: DI H2O, Mg2+, Primers, Extracted DNA, Taq polymerase, and dNTP’s. Thermal Cycler: A pre-set program was used on the Thermo PCR Sprint Thermal Cycler to amplify the targeted locus (Figure 2). SDS Polyacrylamide Electrophoresis: A gel was prepared using a mixture of 1.5 g agarose and 150 mL of TE Buffer. This mixture was heated until clear, with 5 µL of EtBr then added. Carolina blue loading dye was added to the amplified PCR samples for electrophoresis. The gel and amplified PCR mixtures were ran on a FisherBiotech Model H5 Horizontal Gel Electrophoresis System. Gel Imaging: The Kodak Gel Logic 100 gel imager was used to identify and document the results from electrophoresis. Bile salt plate: 15 grams of bile salt were mixed with 40 grams of LB agar and 1000 mL DI H2O to form bile salt plates. 2 µL of various dilutions of the colonies were plated onto respective sections. This project is a continued investigation of a chromosomal mutation that causes bile salt sensitivity in Escherichia coli. Previous attempts to narrow down the location of the mutation suggest that it affects the yciS and yciM genes. Our new focus is to conclusively demonstrate that the mutation confers bile salt sensitivity using several strains of E. coli, including the wild type and mutant strains of the bacterium as well as two genetically-engineered strains that lack the yciS and yciM genes. Primers for the yciS and yciM genes have been used to amplify DNA segments from the wild type and mutant strains of E. coli and we are now in the process of cloning and sequencing target DNA. Once cloned, genes for bile salt resistance and sensitivity will be inserted into counterpart bacterial strains to determine if resistant strains can be made sensitive to bile salts and if sensitive strains can be made resistant to bile salts. Results will provide details for a new gene locus description that includes the location, sequence, and phenotype for bile salt resistance and sensitivity in E. coli. Figure 5: E. coli growth on LB agar Figure 6: E. coli growth on 1.5% bile salt LB plates Results and Discussion The PCR reactions must result in an abundant quantity of DNA products such that the amplified DNA from a single isolated colony is sufficient for several procedures. These include gel electrophoresis for verification of the desired 1.8 Kbp length, sequencing, and cloning through pUC19 (plasmid). Thus, PCR techniques are currently being refined to allow for optimal results. Previous trials (Figure 3) have not yielded the desired quantity of products. We are planning to run bulk PCR reactions involving several reactions from each individual colony to circumvent this issue. Other members of the group are investigating the growth of the strains in LB agar plates and 1.5% bile salt LB agar plates (Figures 5 and 6). The appropriate plasmid and restriction enzymes have already been designated and ordered, such that the PCR products can be cloned once the new PCR procedures are implemented and the PCR products are verified. Future research could investigate the specific mechanism which confers the mutated strain sensitivity to bile salt. Furthermore, a continuation of this project could consider alternative phenotypic properties of the mutation, such as results of exposure to other substrates, including antibiotics. Introduction Multi-drug resistant bacteria are a prominent concern among the scientific and medical community. Drug resistance in bacteria is known to proliferate through horizontal gene transfer. Genes conferring resistance through a variety of mechanisms (including efflux pumps among others) may be encoded in plasmids and transferred among cells through conjugation mediated by a pillus. In 2002, a strain of Escherichia coli displayed impaired growth on a bile salt agar plate, suggesting a bile salt sensitive phenotype distinct from other Gram-negative bacteria. Bile salt sensitivity in E. coli (Strain JC3272I) was initially thought to be associated with a type IV secretion system coded by the F plasmid, conferring sensitivity to F+ strains (Bidlack and Silverman, 2004). It was later discovered that the sensitivity was the result of a chromosomal mutation involving the genes yciS and yciM, and not coded by the F plasmid (unpublished results). yciS is a conserved inner membrane protein and yciM is a putative heat shock protein. The mechanism of sensitivity is not yet known. Knock out experiments of these genes have demonstrated a correlation with not only bile salt, but antibiotic sensitivity (Baba et al., 2004). Hence, the biomedical implications of investigating the phenotypic properties of the mutation through gene transfer include possible insight to methods of antibiotic resistance. This preliminary study will expectantly serve as the foundation for future clinical trials that will apply these concepts to the betterment of human lives. Literature Cited Figure 1: Anna streaking E. coli on LB agar Figure 3: Gel amplification at the desired gene locus Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. Wanner, and H. Mori. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molec. Syst. Biol. 2:2006.0008. Bidlack, J.E., and P.M. Silverman. 2004. An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts. Journal of Bacteriology 186:5202-5209. Acknowledgements Funding for this project was provided by the University of Central Oklahoma CURE-STEM program and Office of Research & Grants. We thank Dr. Philip Silverman of the Oklahoma Medical Research Foundation for his research and guidance necessary for the current progress of this experiment. We also thank our Research Group for much needed assistance and support (Figure 4). Figure 2: Jonna loading samples into thermal cycler Figure 4: Research group