CHARACTERIZATION OF A DUAL FUNCTIONING SAM-I RIBOSWITCH AND TRANS ACTING SMALL RNA IN DESULFOVIBRIO VULGARIS HILDENBOROUGH

Author

Megan Leigh Kempher, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bender, Kelly

Abstract

Desulfovibrio vulgaris Hildenborough (DvH) is a sulfate-reducing bacterium that has the potential to be an agent of bioremediation by aiding in the precipitation of heavy metals and radionuclides from contaminated environments via its natural metabolism. Because such environments are commonly associated with a variety of bacterial stressors, it is important to understand how Desulfovibrio regulates its response to changing conditions for successful environmental application. Very few types of regulation have been characterized in Desulfovibrio and many regulatory networks remain unknown. The importance of regulation by both cis and trans acting RNA molecules has become increasingly evident over the last decade. Both small RNAs and riboswitches have been shown in other bacteria to play key roles in sensing cellular conditions and eliciting the appropriate responses. A previous study in this laboratory identified several small regulatory RNAs, but their biological roles have remained largely unknown. Additionally, a few uncharacterized riboswitches have been predicted in DvH based on genome sequence analysis. This dissertation aimed to characterize the most conserved DvH sRNA and to investigate its potential dual role as a S-adenosylmethionine (SAM) sensing riboswitch. Expression of the sRNA, Dv SIC19, was confirmed during normal growth in lactate/sulfate medium via Northern blot analysis. Sequence and expression analyses also indicated that Dv SIC19 is located upstream of a small hypothetical protein DVU1170 and that the two genes are co-transcribed. Molecular techniques and computational analysis were also employed to determine a role for Dv SIC19 and to identify potential targets. Stress analysis using qRT-PCR suggested a potential role for Dv SIC19 under normal growth conditions as opposed to being involved in a specific stress response. While characterization of Dv SIC19 was ongoing, re-annotation of the DvH genome indicated that Dv SIC19 shared both sequence and structural similarity to the SAM-I class of riboswitches. Multiple techniques, both in vitro and in vivo were used to verify the riboswitch activity of Dv SIC19 and its response to SAM. Determining that Dv SIC19 played some role in the methionine biosynthesis pathway lead to the identification of a mRNA target encoding SahR, a predicted transcriptional regulator of methionine biosynthesis genes. Subsequent electrophoretic mobility shift assays confirmed the ability of Dv SIC19 to bind the sahR transcript and qRT-PCR analysis of a Dv SIC19 deletion strain suggested a negative regulatory role. This study presented the first regulatory role for a newly discovered sRNA in Desulfovibrio. Additionally, this study verified that Dv SIC19 acts not only as a trans regulatory molecule, but also as a cis regulatory element in the methionine biosynthesis pathway of DvH.