Document Type

Dissertation

Date of Degree

Spring 2014

Degree Name

PhD (Doctor of Philosophy)

Degree In

Microbiology

First Advisor

Timothy L. Yahr

Abstract

Pseudomonas aeruginosa RsmA belongs to the CsrA family of RNA binding proteins. CsrA family members are post-transcriptional regulators of global gene expression and usually function to inhibit translation of target genes, but in some cases can also exert positive regulatory effects. Previous work from our lab determined that RsmA is required for maximal T3SS gene expression in P. aeruginosa strain PA103. Nevertheless, the molecular mechanism underlying the RsmA-mediated control of T3SS gene expression was unknown. Expression of the T3SS is under the direct control of ExsA, a transcriptional activator. Previous microarray analyses showed that exsA transcript levels were reduced two-fold in an rsmA mutant. In chapter II I examine the role of RsmA in regulating ExsA expression. I demonstrate that expression of a ExsA-LacZ translational fusion was reduced two-fold in an rsmA mutant suggesting a specific effect of RsmA on ExsA expression. The effect of RsmA on ExsA expression occurs at a post-transcriptional level and is independent of mRNA and protein stabilization mechanisms. RsmA directly interacts with the exsCEBA transcript at multiple sites. Truncation analyses indicate that the -37 to +85 region (relative to the ATG start codon) is necessary and sufficient for RsmA-dependent control. I identified two binding sites, BS1 (-25 bp) and BS2 (+84), involved in the interaction of RsmA with the exsA transcript using sequence analysis, site-directed mutagenesis, EMSA assays, RNase footprints, and RNaseH cleavage assays. Mutagenesis of both binding sites results in an RsmA-independent phenotype. I further demonstrate that RsmA is able to activate ExsA expression. I propose a model wherein RsmA relieves a block on ExsA translation. Collectively, this work shows that RsmA directly binds and activates ExsA expression at the post-transcriptional level.

Most Pseudomonas species carry at least two homologs of CsrA on the chromosome, but only one copy had been identified in P. aeruginosa. Through the course of other projects in the lab, we observed several phenotypes that could not be accounted for by a single copy of RsmA. In collaboration with the Wolfgang lab, we identified a second CsrA homolog, RsmF in P. aeruginosa. RsmF is dimeric in solution. The structure of RsmF differs substantially from other CsrA homologs by having alpha-helices located between the beta-2 and beta-3 strands. In chapter III I examine the role of RsmF in regulating RsmA-controlled processes associated with acute (T3SS) and chronic (T6SS and biofilm formation) infection. I discovered that while an rsmF mutant alone does not exhibit a phenotype, simultaneous deletion of both rsmA and rsmF significantly accentuates the phenotypes exhibited by an rsmA mutant alone. I show that RsmA directly binds and represses RsmF translation and that the small regulatory RNAs RsmZ and RsmY do not significantly modulate RsmF activity. Site-directed mutagenesis revealed that Arg 62, located in the beta-1 and beta-5 fold, is essential for biological activity in vivo and RNA-binding in vitro suggesting a conserved mechanism of RNA recognition maintained across all CsrA family members. Finally, I show that RsmF binds to only a subset of RsmA targets and is not involved in the regulation of all RsmA-controlled processes. In chapter IV I identified high-affinity RNA ligands from a chemically synthesized oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX) and high-througput sequencing. From preliminary analyses of high-throughput sequencing data, the RsmF-binding consensus was determined as 5'-RUACARGGAC-3', with the ARGGA motif being 95% conserved. Collectively, this work shows that Rsm homologs play important roles in regulating virulence gene expression in P. aeruginosa.

Keywords

CsrA, post-transcriptional regulation, RsmA, RsmF, T3SS

Pages

xi, 147 pages

Bibliography

Includes bibliographical references (pages 131-147).

Copyright

Copyright 2014 Manisha Regina Diaz

Included in

Microbiology Commons

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