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NUTRITIONAL COMPETENCE IN ESCHERICHIA COLI:
THE USE OF DOUBLE-STRANDED DNA AS A NUTRIENT
by
Vyacheslav Palchevskiy
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
August 2007
Copyright 2007 Vyacheslav Palchevskiy
Object Description
| Title | Nutritional competence in Escherichia coli: the use of double-stranded DNA as a nutrient |
| Author | Palchevskiy, Vyacheslav |
| Author email | palche@hotmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Biology |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2007-05-18 |
| Date submitted | 2007 |
| Restricted until | Unrestricted |
| Date published | 2007-06-29 |
| Advisor (committee chair) | Finkel, Steven |
| Advisor (committee member) |
Goodman, Steven Chen, Casey |
| Abstract | Natural genetic competence is the ability of cells to take up extracellular DNA and is an important mechanism for horizontal gene transfer. Another potential benefit of natural competence is that exogenous DNA can serve as a nutrient source for starving bacteria because the ability to "eat" DNA is necessary for competitive survival in environments containing limited nutrients. Given that in "real world" environments extracellular DNA is highly abundant, I was interested in understanding the mechanisms of double-stranded DNA acquisition as a nutrient using E. coli as a model system.; Eight E. coli genes were identified that are homologous to com genes in Haemophilus influenzae and Neisseria gonorrhoeae; however, E. coli is not known to be naturally competent. I am interested in determining the roles of these genes in DNA uptake and metabolism, as well as assessing the effect of these genes on competitive fitness and long-term survival. To begin to identify the functions of com genes in E. coli, null mutants were constructed by replacing each gene with an antibiotic-resistance cassette. Seven mutants showed a stationary phase-specific competition defect (SPCD) phenotype where they were outcompeted by the wild-type cells when grown in co-culture. Additionally, multiplex PCR allowed the establishment of a hierarchy of relative fitness of each mutant with respect to all other mutants when simultaneously co-cultured.; To address the ability of these mutants to consume DNA, a "DNA eating" assay was performed where DNA was provided as the sole source of carbon and energy. Wild-type cells showed a 100-fold higher growth yield as compared to each mutant when cultured in minimal medium containing purified dsDNA. These results indicate that com genes are necessary to use DNA as the sole carbon and energy source and confer a competitive advantage to E. coli during stationary phase incubation. Additionally, ssDNA exonucleases also play a role in the DNA catabolism process. I also determined for the first time the subcellular localization of these proteins and show that homologs of these genes are found throughout the proteobacteria, suggesting that the use of DNA as a nutrient may be a widespread phenomenon. |
| Keyword | nutritional competence; double-stranded DNA; transformation |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Type | texts |
| Legacy record ID | usctheses-m570 |
| Rights | Palchevskiy, Vyacheslav |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Palchevskiy-20070629 |
| Archival file | uscthesesreloadpub_Volume32/etd-Palchevskiy-20070629.pdf |
Description
| Title | Page 1 |
| Full text | NUTRITIONAL COMPETENCE IN ESCHERICHIA COLI: THE USE OF DOUBLE-STRANDED DNA AS A NUTRIENT by Vyacheslav Palchevskiy A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR BIOLOGY) August 2007 Copyright 2007 Vyacheslav Palchevskiy |
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