- Title
- Development of novel genetic tools for molecular investigations of pathogenic bacteria
- Creator
- Mende, Linda
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The prevalence of multi-drug resistance in pathogenic bacteria is globally on the rise, and represents an immense threat to the effective treatment of infectious diseases. Alarming reports about bacterial strains that are resistant to all clinically available antibiotics have emerged. The urgent demand for newly developed antibiotics entering the market requires both the identification of potential target sites for antimicrobial action as well as a clear understanding of respective bacterial pathogenesis. This project aimed to develop novel genetic tools for molecular investigations of pathogenic bacteria, with a particular focus on one member of the ESKAPE pathogens: Acinetobacter. A rapidly increasing number of non-treatable infections, mainly caused by one member of the genus – A. baumannii, makes fundamental studies of this group of human pathogens highly relevant. Two different Tn5-based transposon systems, denoted as pTnInt and pTnTEV, have been constructed that could provide two novel methods for inducible protein knock-out in living cells. Such knock-out strains enable not only the identification of essential genes in various bacteria but also the characterisation of their function if unknown through real time phenotypic studies. Both systems could provide a useful tool for studies of particular genes of interest. Whilst with one of the systems (pTnInt) the expression of a certain gene of interest is controllable (knock-out induction on gene transcript level), the other system (pTnTEV) allows for a controlled depletion of a certain gene product of interest (knock-out induction on protein level). Independent of the knock-out strategy, consequences on cell function can be observed in real time. Validation studies have successfully demonstrated the ability of both systems to result in viable transposition events in E. coli. Unfortunately, the utility of the systems to achieve conditional gene knockouts could not be confirmed due to unexpected complications and delays during the process of construct generation. Apart from the development of the two different transposon tools pTnInt and pTnTEV, genomic sequence analysis has revealed the first-time identification and characterisation of the thus far unknown chromosomal origin of replication, oriC, of the human pathogen A. baumannii strain ATCC19606. Essential structures for bacterial replication origins were identified, i.e. 13 putative DnaA boxes with a proposed A. baumannii ATCC19606 consensus DnaA box sequence TTATNCACA as well as clustered AT-rich repeats as potential DNA unwinding elements. Through systematic truncation, 223 bp of the oriC region including 8 of the proposed DnaA boxes were defined as being sufficient to function as a replication origin. Moreover, an E. coli – Acinetobacter shuttle vector, using the newly identified oriC as its replication origin, was constructed and successfully utilised to complement a gene deletion in A. baylyi ADP1. This vector complements the limited repertoire of genetic tools for future studies in Acinetobacter species. The laboratory stock of A. baumannii ATCC19606 (denoted as IG645) harbours two plasmids, the previously described pMAC as well as a second, thus far uncharacterised, plasmid. Isolation and partial sequencing of these extrachromosomal elements revealed the existence of genes that may provide physiological advantages to the host as well as genes that may be involved in essential plasmid functions like replication. Sequence identities to genetic determinants involved in conjugative mobilisation as well as organic peroxide resistance may promote horizontal gene transfer. A better understanding of fundamental gene functions and/or gene essentiality has clinical significance as it leads to a greater insight of pathogenesis of organisms like A. baumannii, but also provides possible future target sites for antimicrobial compounds.
- Subject
- A. baumannii; novel genetic tools; pathogenic bacteria; infectious diseases; antibiotics
- Identifier
- http://hdl.handle.net/1959.13/1332726
- Identifier
- uon:26926
- Rights
- Copyright 2016 Linda Mende
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 5 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 297 KB | Adobe Acrobat PDF | View Details Download |