Roles of Small Rnas and Paralogous Proteins In Bacillus Anthracis Virulence Gene Regulation

<p>Bacteria have evolved a myriad of regulatory mechanisms to control gene expression. One of the most common mechanisms is post-transcriptional control through the function of small regulatory RNAs (sRNAs). Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. In this dissertation, I investigated two sRNAs encoded within the virulence plasmid pXO1 of <em>Bacillus anthracis</em>, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for p­XO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of <em>B. anthracis</em>. sRNA levels are highest during culture conditions that promote AtxA expression and activity. Stability of the sRNAs is unaffected in <em>hfq</em>-null mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. <em>In silico</em> analysis revealed complementarity between XrrA and the 5’ UTR of seven mRNA transcripts whose expression is affected by the sRNA, suggesting base-pairing interactions. A translational fusion of one of these targets, the secreted metalloprotease <em>inhA1</em>, to GFP suggests XrrA-mediated regulation of protease translation. XrrA appears to mediate base-pairing interactions with the<em> inhA1 </em>RBS via a C-rich motif typical of other Gram-positive sRNAs. In a mouse model for systemic anthrax, the lungs, livers, and brains of animals infected with <em>xrrA</em>-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in <em>B. anthracis</em> pathogenesis. Deletion of the XrrA-regulated branched chain amino acid (BCAA) biosynthesis (<em>ilvD</em>) and transport (<em>brnQ3</em>) genes led to severe attenuation in the same model. XrrA and XrrB are the first reported sRNAs of <em>B. anthracis</em>. Future work should focus on the molecular basis for sRNA function, including investigations of potential RNA and/or protein interacting partners of XrrA and XrrB.</p> <p>Another common mechanism of bacterial gene regulation is transcriptional control through the function of regulatory proteins that bind DNA. The <em>pagA</em> gene, encoding the protective antigen component of the anthrax toxin of <em>B. anthracis</em>, is part of a bicistronic operon that codes for its own repressor, the dimeric protein PagR1. PagR1 also regulates <em>sap</em> and <em>eag</em>, two genes encoding components of the surface layer (S-layer), a protein layer found between the cell wall and capsule which serves as a mounting structure for key surface-associated proteins. Genomic and transcriptomic studies identified a 70% identical paralog of PagR1, termed PagR2, found in the pXO2 plasmid. In this work I examined PagR1 and PagR2 for functional similarity. Recombinant PagR1 and PagR2 expressed individually in a ∆<em>pagR1</em> pXO1⁺/pXO2^- background comparably repressed expression of <em>pagA</em>, <em>atxA</em>, <em>sap</em>, and <em>eag</em>, suggesting functional redundancy. PagR1 readily bound promoters of these genes, while PagR2 weakly bound only the <em>pagA</em> and <em>atxA </em>promoters <em>in vitro</em>. The unequal DNA binding capacity of these two highly identical proteins is partially explained by one key amino acid difference between them. The eighty-first amino acid, which in PagR1 is a surface-exposed tyrosine (Y), is a buried serine (S) residue in PagR2. Recombinant PagR2 S81Y bound the S-layer<em> </em>promoters with binding affinity comparable to that of PagR1. Furthermore, while PagR1 forms readily detectable dimers, PagR2 dimers can only be visualized using a crosslinking agent. Mutation of S81 to Y allows PagR2 to form dimers detected without crosslinking. Interaction between native PagR1 and PagR2 S81Y suggests a role for the tyrosine residue in mediating protein:protein interactions. Future studies should focus on assessing <em>in vivo</em> interactions between the native proteins and expanding our knowledge of the PagR1 and PagR2 regulons.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2022

Authors dc:creator

• Corsi, Ileana
• <p>http://www.orcid.org/0000-0002-5422-9105</p>

Contributors dc:contributor

• Theresa M. Koehler
• Ambro van Hoof
• Danielle Garsin

• Bacillus anthracis
• plasmid
• pXO1
• sRNA
• RNA-seq
• transcription
• translation
• paralogs
• toxin
• Bacteriology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/1157

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-2222