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Analysis of Genetic Regulatory Mechanisms that Control Ethanolamine Utilization in Enterococcus faecalis

dc.contributor.advisorWinkler, Wade Cen_US
dc.contributor.authorGebbie, Margo Pageen_US
dc.date.accessioned2017-09-14T05:38:21Z
dc.date.available2017-09-14T05:38:21Z
dc.date.issued2017en_US
dc.identifierhttps://doi.org/10.13016/M2V40K04R
dc.identifier.urihttp://hdl.handle.net/1903/19917
dc.description.abstractIn this project, we studied the genetic regulatory mechanisms that affect utilization of ethanolamine, an abundant compound in the gastrointestinal environment. In Enterococcus faecalis, the ethanolamine utilization (eut) gene cluster encodes for a two-component regulatory system (TCS), comprised of a histidine kinase, EutW, which autophosphorylates upon sensing EA, and a cognate response regulator, EutV, which dimerizes upon receiving the phosphoryl group from EutW and binds the nascent transcript to prevent premature transcription termination. This TCS is responsible for coupling sensing of ethanolamine to production of eut transcripts. However, clues from other organisms had previously suggested that adenosylcobalamin (AdoCbl) might also be an important genetic regulatory signal for the E. faecalis eut genes. Indeed, we discovered a novel trans-acting noncoding RNA (EutX) that contained an AdoCbl-responsive riboswitch. Our data demonstrated that the riboswitch promotes a shortened form of EutX when cellular AdoCbl levels are replete. In contrast, a longer form is synthesized when AdoCbl levels are depleted. We demonstrated that structural motifs contained in the longer form of EutX act to sequester the EutV protein, preventing it from promoting transcription elongation of eut transcripts. These unexpected data revealed an important new type of regulatory mechanism for riboswitch RNAs. In support of this overall genetic regulatory model, we recapitulated the full genetic circuitry in a heterologous host. Using this system, we employed extensive site-directed mutagenesis to examine the functional importance of highly conserved EutV residues. This led to the identification of a cluster of positively charged residues, which we speculated are important determinants for RNA-binding activity. Consistent with this hypothesis, mutations of these residues resulted in loss of RNA-binding activity. Furthermore, we also explored whether the eut gene cluster was affected by additional genetic regulatory mechanisms. From these efforts, we concluded that oxygen is not a genetic regulatory feature of eut genes, in contrast to previously published speculation. However, we did find that it is likely to be repressed under conditions of high glucose. Therefore, these aggregate studies revealed new mechanisms of post-initiation genetic regulation, and showed how E. faecalis specifically controls expression of ethanolamine catabolism genes.en_US
dc.language.isoenen_US
dc.titleAnalysis of Genetic Regulatory Mechanisms that Control Ethanolamine Utilization in Enterococcus faecalisen_US
dc.typeDissertationen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.contributor.departmentBiochemistryen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pquncontrolledEnterococcus faecalisen_US
dc.subject.pquncontrolledEthanolamineen_US
dc.subject.pquncontrolledPost-transcriptional regulationen_US
dc.subject.pquncontrolledRiboswitchen_US
dc.subject.pquncontrolledRNAen_US
dc.subject.pquncontrolledtwo-component regulatory systemen_US


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