Year of Award

2015

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Department or School/College

Division of Biological Sciences

Committee Chair

Michael F. Minnick

Commitee Members

Stephen J. Lodmell, D. Scott Samuels, Scott Miller, Keith Parker

Abstract

Coxiella burnetii is an obligate intracellular bacterial pathogen that undergoes a biphasic developmental cycle, alternating between a small cell variant (SCV) and a large cell variant (LCV). Despite the remarkable niche and life cycle of C. burnetii, little is known about its modes of regulation and the roles that non-coding RNAs play in its growth and development. One such element is the intervening sequence (IVS); a 444-nt RNA element that is inserted within helix 45 of CoxiellaÆs precursor 23S rRNA. C. burnetii may have acquired IVS through horizontal transfer, and it has been subsequently maintained by all strains of C. burnetii through vertical transfer. The IVS of C. burnetii contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). However, our data show that the S23p-encoding ORF is probably undergoing reductive evolution and therefore not expressed in vivo. Additionally, we observed that following RNase III-mediated excision, IVS RNA is degraded and levels of the resulting fragments of 23S rRNA differ significantly from each other and the 16S rRNA. Since the fragment of 23S rRNA that is lowest in quantity may dictate the number of mature ribosomes that are ultimately formed, we hypothesize that the biological role of IVS is to moderate CoxiellaÆs growth by fragmentation of its 23S rRNA thereby fostering CoxiellaÆs tendency towards slow growth and chronic infection. Further, we identified fifteen novel Coxiella burnetii sRNAs (CbSRs) using RNA-seq, which were verified using Northern analyses. Additionally, some of these CbSRs were upregulated in LCVs or during intracellular growth, suggesting adaptive roles in those contexts. Furthermore, we also identified and characterized the 6S RNA of C. burnetii and found that it accumulated during the SCV phase of the bacterium. The location of ssrS gene and the secondary structure of 6S RNA were similar to those of other eubacteria, indicating functionality. We also demonstrated that the 6S RNA of C. burnetii interacts specifically with RNA polymerase (RNAP). Finally, 6S RNA was highly expressed during intracellular growth of C. burnetii indicating that it probably regulates stress response by interacting with RNAP during transcription.

Download

Share

COinS
 

© Copyright 2015 Indu Ramesh Warrier