Author ORCID Identifier

http://www.orcid.org/0000-0002-5422-9105

Date of Graduation

5-2022

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Theresa M. Koehler

Committee Member

Ambro van Hoof

Committee Member

Danielle Garsin

Committee Member

Jeffrey Frost

Committee Member

Nicholas De Lay

Abstract

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 Bacillus anthracis, 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 B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity. Stability of the sRNAs is unaffected in hfq-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. In silico 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 inhA1, to GFP suggests XrrA-mediated regulation of protease translation. XrrA appears to mediate base-pairing interactions with the inhA1 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 xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis. Deletion of the XrrA-regulated branched chain amino acid (BCAA) biosynthesis (ilvD) and transport (brnQ3) genes led to severe attenuation in the same model. XrrA and XrrB are the first reported sRNAs of B. anthracis. 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.

Another common mechanism of bacterial gene regulation is transcriptional control through the function of regulatory proteins that bind DNA. The pagA gene, encoding the protective antigen component of the anthrax toxin of B. anthracis, is part of a bicistronic operon that codes for its own repressor, the dimeric protein PagR1. PagR1 also regulates sap and eag, 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 ∆pagR1 pXO1+/pXO2- background comparably repressed expression of pagA, atxA, sap, and eag, suggesting functional redundancy. PagR1 readily bound promoters of these genes, while PagR2 weakly bound only the pagA and atxA promoters in vitro. 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 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 in vivo interactions between the native proteins and expanding our knowledge of the PagR1 and PagR2 regulons.

Keywords

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

Available for download on Tuesday, April 11, 2023

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Bacteriology Commons

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