Mutagenesis and functional characterisation of toxin HicA from the HicBA TA system in Burkholderia pseudomallei.
Bare, Harriet Leah
Thesis or dissertation
University of Exeter
This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement.
Reason for embargo
To allow time for me to publish a paper from my thesis.
Four type II toxin-antitoxin (TA) systems were previously identified in Burkholderia pseudomallei K96243. Type II TA toxins are able to induce cell growth arrest or death by interfering with key processes within the organism. BPSS0390-0391 is one of the TA systems previously identified and has homology to hicBA system in Acinetobacter baumannii. B. pseudomallei HicA is able to cause a reduction in the number of culturable cells after expression in E. coli. This study aimed to characterise B. pseudomallei HicA in three ways: by inducing expression of HicA in bacterial species other than E. coli, by identifying amino acids in HicA involved in toxicity and neutralisation by the antitoxin HicB and by examining the interaction of HicA with other TA antitoxins identified within B. pseudomallei genome. A broad host range plasmid encoding BPSS0390 was transformed into a range of Gram negative bacteria including Yersinia pseudotuberculosis IP32953, Vibrio vulnificus E64MW, Salmonella enterica serovar Typhimurium SL1344 and Burkholderia thailandensis E264. Expression of BPSS0390 was toxic in all bacterial species tested, despite the presence of antitoxin BPSS0391 homologues in some species. Unregulated expression in E. coli resulted in the appearance of escape mutants encoding non-toxic variants of HicA. An alanine scanning mutagenesis study of HicA identified 20 mutants where toxicity was abolished despite high levels of expression, but identified no mutants that affected TA complex formation. Finally an existing co-expression assay was modified to examine interactions between HicA and other type II TA antitoxins in B. pseudomallei. The assay revealed no interaction between HicA and non-cognate antitoxins and clarified the role of IPTG as an inhibitor of PBAD promoter on the arabinose operon.
University or Exeter and DSTL.
Doctoral thesis studying the TA toxin HicA from Burkholderia pseudomallei. Firstly, toxin HicA was evaluated as a potential antimicrobial compound, through ectopic expression in a range of bacterial species. Secondly, residues or regions of the toxin involved in toxicity or TA complex formation were identified using a scanning alanine mutagenesis study. Thirdly, interactions between non-cognate TA pairs were examined to determine if type II TA systems are able to form co-operative networks in B. pseudomallei.
PhD in Biological Sciences