Date of Award


Degree Type


Degree Name

Master of Science




Biology (MS)

First Advisor/Chairperson

Josh Sharp


Pseudomonas aeruginosa is an opportunistic bacterial pathogen notable for its ability to colonize the lungs of cystic fibrosis patients. Once the bacterium infects and colonizes humans, it is extremely difficult to eradicate. This leads to long-term infections that significantly damage the lungs and other tissues. P. aeruginosa infections are challenging to treat due to the bacterium’s natural antibiotic resistance and the rise of multidrug resistant strains. Development of novel drug treatments are a necessity.

In all organisms, the regulation of gene expression is a highly controlled process. Remarkably, in P. aeruginosa bioinformatics studies showed that 20% of its genome is dedicated to regulating transcription, the first stage of gene expression. However, the vast majority of proteins that regulate transcription in P. aeruginosa are poorly understood. Understanding gene regulation is a promising strategy for discovery of novel drug targets. RNA polymerase (RNAP) is an essential enzyme controlling gene regulation, and its activity is modulated through a plethora of transcription factors or other proteins. The regulation of gene expression has been best studied in E. coli.

The aim of this work was to develop a platform to study RNAP-interacting proteins in P. aeruginosa. To do this, we took advantage of an E. coli RNAP “coreome” library. The RNAP “coreome” was a term developed to describe 38 different plasmids each expressing surface exposed regions of RNAP inputted into a bacterial two-hybrid assay. In general, RNAP is a highly conserved enzyme among bacteria. Alignments of the amino acids of each piece of the E. coli RNAP coreome to specific domains of P. aeruginosa RNAP have shown a high degree of similarity (between 82-100%). The coreome was constructed based on the high-resolution structure of the bacteria Thermus aquaticus RNAP suggesting that E. coli β RNAP can be parsed into 10 sub-domains. Analysis of the E. coli β’ subunit of RNAP suggested that it could be parsed into 18 sub-domains that cover the entire gene (Nickels, 2009).

A bacterial two-hybrid assay can be utilized to determine if a specific target protein interacts with domains of RNAP found within the RNAP coreome. Data from the IntAct Molecular Interaction Database identified 147 E. coli proteins that directly or indirectly interact with RNAP. We performed a bioinformatic analyses to identify P. aeruginosa homologs to these E. coli proteins. Initially, sixteen P. aeruginosa proteins were screened against the E. coli RNAP coreome in the bacterial two-hybrid assay.

In this work five P. aeruginosa proteins, AlgQ, NusG, ClpA, DppF, and Tig were shown to interact with particular fragments of the E. coli RNAP coreome. Specifically, we show that AlgQ interacted with σ 528-613, NusG interacted with β’249-328 and β’264-308, ClpA interacted with β 829-930 and β 831-1059, DppF interacted with β' 114-190 and β 1137-1226, and Tig interacted with β' 735-790 and β 450-530. These results indicate the E. coli RNAP coreome can be utilized to uncover RNAP-interacting proteins in P. aeruginosa and to discover the precise domains they contact. In the future if the RNAP-binding determinants or proteins that control expression of virulence factors are identified using the RNAP coreome, it may be possible to design novel drugs that either disrupt the function of those proteins or their interaction with RNAP. Ultimately, this could lead to improved treatment options for P. aeruginosa infections.

Access Type

Open Access

Included in

Bacteria Commons