PROJECT SUMMARY / ABSTRACT
Post-transcriptional regulation of gene expression has significant effects on the physiology of the bacterial cells.
While much has been uncovered about post-transcriptional regulation of gene expression in bacteria, there are
still some key unknowns that are hindering our ability to understand how these processes work and differ
throughout the prokaryotic domain of life. In this project, our overall goal is to expand our understanding of the
diversity of these processes in gram-negative model organisms, firmicutes, and mycobacteria. Our propsosed
research program has two sub-projects. Project #1 is entitled: Epitranscriptomic regulation of gene expression
throughout the prokaryotic domain. In sub-project 1, we will expand our understanding of RNA modifications
within a wide range of bacterial species. RNA modifications include an isomerization or chemical additions to
canonical nucleotides within RNA. Generally found in tRNA molecules, RNA modifications are critical in
promoting translational fidelity by promoting ribosome mRNA decoding errors. There are several key knowledge
gaps driving our experimental approach. The regulatory role of RNA modifications, cis-acting signals that may
promoting regulatory targeting of RNA modifications, and the effect of GC content on the ability of codon usage
to drive targeting of genes by modified tRNAs. We will use a multi-omics approach to address these key
knowledge gaps using several bacterial model organisms: Escherichia coli, Staphylococcus aureus,
Clostridioides difficile, and Mycobacterium smegmatis. Project #2 is entitled: Post-transcriptional gene regulation
via novel proteins and sRNAs in firmicutes. In sub-project 2, we will expand our understanding of the regulatory
role of novel proteins and sRNAs in the regulation of the RNome in firmicutes. Much our understanding of sRNA
in bacteria has come from extensive studies in model organisms. This has assisted in our understanding sRNA
regulation of bacterial physiology in general. However, recent studies have uncovered hundreds of species-
specific sRNA in S. aureus. The vast majority of these are uncharacterized. Since we lack a full picture of sRNA
regulons in S. aureus, our understanding of bacterial sRNA biology is incomplete. In addition, many model
organisms utilize the RNA chaperone Hfq as a sRNA co-factor in some cases to promote sRNA-mRNA-RNase
interactions. However, Hfq is dispensable for growth and sRNA function. It is unknown if there are other protein
factors that may act as specific-specific chaperones in S. aureus or other firmicutes. We will have preliminary
data to suggest that novel regulatory proteins may exist, in S. aureus and C. difficile, in the mRNA regulation
and may also modulate levels of sRNAs. We will execute RNA-immunoprecipitation sequencing of this candidate
protein in S. aureus and C. difficile to identify its RNA-interactome. We have also identified predicted sRNA
interactions with several important proteins in S. aureus and execute experiments to test this interaction.
