Year of Award

2013

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Other Degree Name/Area of Focus

Integrative Microbiology and Biochemistry

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

D. Scott Samuels

Commitee Members

Mike Minnick, Stephen Lodmell, Michele McGuirl, Kent Sugden

Keywords

Borrelia burgdorferi, gene regulation, ribosomal RNA, ribosome biogenesis, spirochete

Abstract

Here we demonstrate the first characterization of an RNase III enzyme from a spirochete and its role in processing rRNA transcripts from the unusual rRNA gene operons of Borrelia burgdorferi. In most bacteria, the three rRNA transcripts (16S, 23S, and 5S rRNAs) that form the ribosome are produced as a single transcript from an operon with minimal spacing between genes. In the B. burgdorferi genome, however, a single 16S rRNA gene is encoded more than 3 kb from the bicistronic 23S-5S rRNA operons. The 23S-5S operons are tandemly duplicated, yielding an uneven number of rRNA genes, a feature unique to Lyme disease Borrelia. Additionally, the 16S and tandem 23S-5S operons appear to be synthesized as two separate transcripts. Our data show that B. burgdorferi RNase III processes the 3´ end of the 16S, 23S, but not the 5S, rRNA transcripts, as in other bacteria. However, 16S rRNA 5´ end processing proceeds by an as yet unidentified mechanism, which is an unprecedented finding. We hypothesize that this deviation from the canonical 16S rRNA processing pathway is likely an adaptation of B. burgdorferi to rRNA gene rearrangement during genome reduction and transition to a host-restricted lifestyle. In agreement with this finding, the 16S rRNA gene is transcribed as part of a larger operon containing unrelated genes, suggesting alternative regulation of the rRNA transcripts. Additionally, we show that the 23S rRNA is transcribed from identical promoters present in front of both tandem 23S rRNA genes and that this creates our observed 2.5 to 3-fold excess of 23S rRNA compared to 16S rRNA. Finally, single deletion mutants in each of the 23S rRNA genes were constructed. Surprisingly, deletion of the first 23S rRNA gene produces a severe growth phenotype and increased erythromycin susceptibility in vitro and a strain that is non-infectious in vivo. A mutant with a deletion in the second 23S rRNA gene shows no phenotype. The 23S rRNA genes have begun to acquire single nucleotide polymorphisms. However, their pattern currently indicates that they are the products of genetic drift. We conclude that the mechanism of rRNA transcription is unique in B. burgdorferi.

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© Copyright 2013 Melissa Lynn Hargreaves