- Title
- Transcription factors and complementation strategies in bacteria
- Creator
- Jordan, Matthew James
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosopy (PhD)
- Description
- The threat to health posed by antibiotic resistant bacterial pathogens is one of the great challenges facing humanity in the 21st century. As current antibiotics lose their efficacy, it is imperative that new drugs be developed. Bacterial transcription offers an attractive and hitherto underutilised target for the development of novel therapeutic agents. The first step in the rational design of targeted antimicrobial drugs is an understanding of the drug target, and its importance during bacterial growth or pathogenesis. The work presented here examines three bacterial proteins; the well-characterised transcription factors NusA and ρ (Rho factor), and the novel virulence associated factor, AtfA. By using phage display to screen a library of random peptide sequences, a 7-residue peptide showing affinity to the N-terminal domain of Bacillus subtilis NusA was isolated. This peptide was capable of titrating NusA away from RNAP at concentrations as low as 70 nM, but did not show affinity for the correspondingprotein fragment. Modelling of the enriched peptide sequence revealed no similarity to the NusA binding site on the β-flap tip region of RNAP, indicating that this peptide sequence represents a novel NusA binding motif. The transcription termination factor ρ has roles in transcriptional control and genome maintenance. The importance of ρ varies among bacteria, and it is annotated as essential in most Gram-negative species. An exception is the model organism Acinetobacter baylyi, in which the rho gene is currently considered dispensable. The work presented here suggests that GFP tagging of ρ results in a temperature sensitive strain, with consequences for growth rate, cell morphology, and chromosome shape. Furthermore, despite repeated attempts to delete rho using a targeted gene deletion cassette, a null strain could not be produced, nor could a strain featuring a second wild type copy of the rho gene. This data leads to the conclusion that rho is, in fact, essential in A. baylyi, and redefines this protein as a potential target for antimicrobials in this species. A new class of highly antibiotic resistant Gram-negative pathogens has recently emerged, which thrive in the hospital environment, and among these, A. baumannii and Pseudomonas aeruginosa are of particular concern. The persistence and pathogenicity of these species is related to the expression of a series of virulence factors, which enable persistence in the hospital environment, and potentiate human disease. The novel transcription factor AtfA has been implicated in a range of these processes, including biofilm formation, ethanol metabolism, pilus assembly, and iron acquisition. Phylogenetic analysis revealed that AtfA is a diverse protein, confined to the γ-proteobacteria, including several important pathogens. Deletion of atfA in Acinetobacter results in increased cell size, decreased ethanol metabolism, and increased pathogenicity. A P. aeruginosa null strain showed reduced biofilm formation in minimal medium, but was unaffected for cell size and ethanol metabolism. This work contributes to an understanding of the role of this transcription factor in virulence related pathways, and indicates that the role of AtfA varies among even closely related organisms. Reinsertion of a gene into a deletion strain (complementation) is an important tool in understanding the role of that gene product in the pathobiology of an organism, but for poorly studied organisms such as Acinetobacter spp., genetic tools are currently lacking. Using atfA as subject for genetic complementation, currently available methods for gene complementation were evaluated. Replicative plasmid-based options demonstrated prohibitive cell toxicity, and were ineffective in rescuing deletion phenotypes. Reinsertion of atfA into the A. baylyi chromosome via single crossover resulted in a cell size phenotype distinct from both the wild type and deletion strains, an intermediate ethanol metabolism phenotype, and quantification of protein production showed an ~9 fold increase in AtfA when the gene was expressed from the site of insertion (the lip1 lipase gene) when compared with the native locus. Reintroduction of atfA into an A. baumannii deletion strain via double crossover rescued the cell size phenotype, but not ethanol metabolism. Taken together these results contribute to an understanding of the tools currently available for gene complementation, and the challenges faced when working with these important bacteria.
- Subject
- antibiotic; complementation; transcription factor; AtfA; rho; NusA; acinetobacter; pseudomonas; virulence
- Identifier
- http://hdl.handle.net/1959.13/1342445
- Identifier
- uon:28967
- Rights
- Copyright 2017 Matthew James Jordan
- Language
- eng
- Full Text
- Hits: 1900
- Visitors: 2675
- Downloads: 627
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 5 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 223 KB | Adobe Acrobat PDF | View Details Download |