Our lab created ionizing radiation (IR) resistant populations of E. coli MG1655 through twenty cycles of exposure to IR and outgrowth. To identify the genetic alterations responsible for increased IR tolerance, a total of seven isolates from the evolved final population were re-sequenced. However, the individual isolates sequence analysis revealed wide variety of unique genomic alterations with little overlap between the seven isolates and therefore, did not reveal the clear picture of the mutations responsible for IR resistance. Our current research focused on distinguishing the relevant mutations from the non-relevant mutations that contribute to IR resistance by using two methods: population sequencing and TaqMAMA with the assumption that beneficial mutations evolve and accumulate in the final population. A 255X deep coverage sequencing of the genomic DNA from the IR resistant final population determined mutant allelic percentage in this population for each polymorphism detected. TaqMAMA assay acted as a tool for verifying the sequencing results. This assay also revealed two evolutionary phenomena: clonal interference and hitchhiking suggesting that not all polymorphisms seen in the final population could contribute to IR resistance. The analysis of population sequencing results along with the phylogenetic analysis reduced the number of potential candidates responsible of IR resistance in these isolates. These possible contributors enable at least three unique mechanisms in the seven isolates and the survival data revealed that the related isolates have same levels of IR resistance possibly from the same mechanism. The identified candidates were transferred into the parent strain alone or in combination and the resulting strains were tested for survivability against IR. This experiment identified the possible reasons of IR resistance in these isolates. In fact, a RecA modification has been determined to be a lone contributor for IR resistance in the four isolates. The methods used here, enabled us to separate relevant mutations that accumulated during evolution from non-relevant mutations that do not and thus helped to reduce the number of alleles possibly that could contribute to IR resistance. However, genetic characterization is required to confirm their role in IR resistance due to complexity that exists in allelic selection during evolution.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-03272013-195756 |
| Date | 05 April 2013 |
| Creators | Selvam, Kathiresan |
| Contributors | Pettis, Gregg S., Lee, Yong-Hwan, Ding, Huangen, Eades, Susan C., Battista, John R. |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-03272013-195756/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0101 seconds