Identification and characterization of novel small RNAs in Desulfovibrio vulgaris Hildenborough

Author

Andrew Burns, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2012

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bender, Kelly

Abstract

Desulfovibrio vulgaris strain Hildenborough is a key organism in the bioremediation of environments contaminated with anthropogenic sources of heavy metals and radionuclides. In order for D. vulgaris to be optimized for remediation of such sites in the environment, it is necessary to understand the organism's response to key stressors that are found in conjunction with both heavy metals and radionuclides. Few regulators have been discovered in this bacterium and even less is known about its genetic regulatory response to changing conditions in its environment. This dissertation project aimed to use computational, molecular, and culture-based methods to identify novel, small, regulatory RNAs (sRNAs)--an important class of cellular regulators that have yet to be studied in D. vulgaris--and to determine their effect on cellular processes within the organism. Computational and transcriptomic high-throughput sequencing resulted in over 200 high-quality putative sRNAs with the expression of sixteen of these sRNAs verified by Northern blot analysis. Eight of the confirmed sRNAs were selected and further characterized within this study resulting in three trans--or intergenic--encoded sRNA, three cis--or antisense--encoded sRNAs, and two sRNAs that may encode small proteins. These eight sRNAs included growth phase-specific sRNAs, differentially expressed sRNAs, as well as sRNAs processed into multiple forms. Molecular techniques such as quantitative reverse-transcriptase PCR (qRT-PCR) indicated differential expression of the confirmed sRNAs under a variety of stress conditions. Culture-based methods confirmed growth deficiencies in deletion and over-expression mutants of the sRNAs tested under stressors suggested during qRT-PCR analysis. Both of these tests suggested that the sRNAs discovered in this study participate within the cellular response of the bacterium to environmental stress. Computational methods were employed to reveal likely putative mRNA targets for the confirmed sRNAs in order to determine interactions and function within the regulatory networks. This study presents the first evidence of the existence and expression of sRNAs within D. vulgaris Hildenborough. Furthermore, the information gathered about the sRNAs analyzed in this work indicates that sRNAs are an important part of the regulatory response mechanism of D. vulgaris. Further research into target identification and interaction can close the loop of these regulatory networks and solidify the role of sRNAs in D. vulgaris.