- Title
- A study into DNA recombination proteins and novel plasmid recombination sites from Acinetobacter baumannii
- Creator
- Ahammed, Sadia Khaleda
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2022
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The discovery of multiple, inverted, dif-like recombination sites (pdif) flanking antibiotic resistance genes (dif modules), has prompted great interest in the area. Many of these recombination sites are located on plasmids originating from clinical isolates of the serious pathogen Acinetobacter baumannii. The study hypothesied inverted pdif sites on either side of a gene could function as a novel gene transfer and / or gene shuffling system via Xer site-specific recombination. Xer site-specific recombination is a process where the recombination proteins XerC and XerD bind to dif sites to catalyse two rounds of DNA strand cleavage, exchange and ligation. The recombination of dif sites on a plasmid can lead to the excision or inversion of the internal DNA, depending on the orientation of the recombination sites. Understanding this process could uncover mechanisms by which antibiotic resistance genes can mobilise and disseminate throughout bacterial populations. The current study evaluated the binding interaction between two pdif sites and the A. baumannii recombinases XerC and XerD. A series of electrophoretic mobility shift assays (EMSA) demonstrated XerC and XerD cooperatively bind to the pdif sites, a crucial step preceding the catalysis of Xer site-specific recombination. The study then predicted potential cross-species binding interactions between A. baumannii FtsKɣ (FtsKɣ_AB) and E. coli XerD (XerDEC), an interaction necessary to activate catalysis of Xer site-specific recombination. A combination of predictive structural software, and hydrophobic and electrostatic protein profiles, revealed the possibility of a cross-species binding interaction. Subsequent in vivo recombination assays of the A. baumannii chromosome dif site, involving FtsKɣ_AB and XerDEC resulted in weak levels of recombination, a likely indication FtsKɣ_AB and XerDEC do interact to allow recombination to proceed. Other in vivo recombination assays involved the inverted pdif sites, which were able to undergo recombination to generate inversion products. The recombination assay was performed within a host E. coli cell, which demonstrated the ability of E. coli Xer proteins to catalyse recombination events at variant dif sites. The ability of non-native Xer recombinases to recognise and catalyse variant dif sites could be prevalent in other bacterial species, which could aid the mobilisation of dif modules harbouring antibiotic resistance genes. The study also investigated the A. baumannii DNA translocase FtsK (FtsKAB), which could serve as a future therapeutic target. FtsKAB was confirmed to be a strong DNA-dependent ATPase. However, the identification of an FtsK orientating polar sequence (KOPS) on the A. baumannii chromosome remains elusive. The study suggested several octomers that could potentially function as the KOPS motif on the A. baumannii chromosome. Yet, none of the listed octomers were as highly skewed or over-represented as the well-established KOPS motif GGGNAGGG on the E. coli chromosome. Nevertheless, it is likely the KOPS motif on the A. baumannii chromosome differs from that on the E. coli chromosome. The difference in motif sequence could indicate key FtsK proteins folds responsible for recognising the substrate DNA differs between the two species, which could be beneficial in developing new antibiotics specifically targeting FtsK within pathogenic strains of Acinetobacter. Overall, the study demonstrated the novel pdif sites can be recognised and cooperatively bound by the A. baumannii Xer recombinases. Additionally, the pdif sites can undergo recombination within E. coli, which serves as an early indication that the novel pdif sites could function as a gene transfer and / or gene shuffling system, in support of the hypothesis.
- Subject
- molecular microbiology; Acinetobacter; DNA recombination; antibiotic resistance
- Identifier
- http://hdl.handle.net/1959.13/1449363
- Identifier
- uon:43648
- Rights
- Copyright 2022 Sadia Khaleda Ahammed
- Language
- eng
- Full Text
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