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dc.contributor.advisorRonson, Clive
dc.contributor.advisorRamsay, Joshua
dc.contributor.advisorSullivan, John
dc.contributor.authorMorris, Calum Ross Pettigrew
dc.date.available2021-07-07T22:06:52Z
dc.date.copyright2021
dc.identifier.citationMorris, C. R. P. (2021). Decoding the dual target antiactivator QseM (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/12092en
dc.identifier.urihttp://hdl.handle.net/10523/12092
dc.description.abstractRegulation of transfer of the symbiosis island of Mesorhizobium japonicum strain R7A, ICEMlSymR7A, is tightly controlled by a complex multi-component regulatory system. The master antiactivator of this system is QseM, which robustly inactivates two proteins, FseA and TraR, with both FseA and QseM containing a DUF2285 domain. Through these interactions QseM blocks both quorum sensing and Integrative and Conjugative Element (ICE) transfer, effectively quashing biological noise that could otherwise unleash the transfer mechanism population-wide with deleterious effects. Quorum sensing (QS) systems are widely utilised by bacteria to time critical changes in cellular physiology and have been increasingly implemented into the development of synthetic circuits to take advantage of this timed population-wide response to improve circuit function. However, these QS systems are prone to inappropriate activation by biological noise and crosstalk, leading to inefficient operation and signal collapse. There is a lack of components that can be utilised in combination to control cross-talk and enable complex circuit construction, especially in the cases where integration of multiple quorum sensing pathways is desired. Identification and adaption of naturally evolved components is one way in which this lack of components can be overcome. Some QS systems have evolved antiactivators, which dampen the effects of biological noise and improve the function of natural QS circuits. The role of QseM in regulating ICEMlSymR7A and its imperviousness to biological noise makes it an ideal candidate for use in synthetic applications. However, the mechanisms QseM utilises to exert antiactivation are unknown. The aims of this work were to identify critical features in QseM that are involved in the interaction and inactivation of FseA and TraR with the view of adapting it to control biological noise and crosstalk in synthetic circuits. To identify critical residues and motifs within QseM, alanine scanning mutagenesis was performed in combination with previous work to create 33 QseM mutants. These mutants were assessed for function against FseA and TraR and two critical regions in QseM were identified, one in the first predicted α-helix and one shared between the end of the third, and fourth α- helices. The majority of the residues critical for FseA inactivation were also critical for TraR inactivation; however, a four-residue motif, RLLD, was specific for FseA with mutation of an arginine at position 28 abolishing inactivation but not interaction in Bacterial-two-Hybrid assays. The NMR structure of QseM except for its N- and C-terminal regions was determined by our collaborators and the structure of the whole QseM protein was reinforced by the development of co-evolution models of QseM (this work) and FseA (William Jowsey, this lab, unpublished data). Overlay of mutations critical for FseA inactivation on the structure showed that these clustered on one side of QseM, with helix one and the end of helix three to four forming a broad interaction interface on the surface of the protein. The structure of QseM identified it as an uncharacterised tetra-helical HTH domain protein with the typical DNA- binding HTH features, but the mutational analysis showed that critical DNA-binding HTH residues were not important for QseM function. The conservation of the HTH structure may be due to its suggested evolutionary history as a duplication of the DUF2285 domain of FseA. FseA inactivation is at the core of QseM function with homologues down to 43% identity able to completely inactivate FseA despite their own FseA homologues showing as little as 25% identify. However only one of five QseM homologues tested, found on an ICE in the marine bacterium Stappia indica, was able to inactivate ICEMlSymR7A TraR. Substitution of regions within QseM with a homologue that was unable to inactivate TraR indicated that the C-terminus of QseM is involved in TraR inactivation. Incorporation of TraR into the regulatory system of QseM appears to a be a recent evolutionary event, with very few homologues outside of mesorhizobia containing linked qseM and traR genes that were close in the genome. The mutational and structural analyses provided evidence that QseM acts as a competitive inhibitor of FseA dimerisation, obscuring the FseA dimerisation domain in a cleft between the RLLD and GY motifs of QseM, similar to the mechanism that FseA may use to dimerise. QseM also interacts with the AHL-binding domain of TraR, blocking its function likely through the formation of inactive heterodimers. Finally, a chimeric LuxR family regulator was created using TraR and LasR. The 170 amino acid AHL-binding domain of TraR was fused to the DNA-binding domain of LasR as a ‘QseM-tag’. This chimera responded to the native TraR AHL 3-oxo-C6-HSL and had a similar AHL response profile despite having reduced function. Upon addition of AHL, the chimera activated transcription of the LasR target promoter, lasIp indicating that a successful fusion had been made. Importantly, it was shown that the chimeric protein was susceptible to QseM- mediated inactivation while the donor LasR was not. The construction of this chimera highlighted the modular chimeric QS regulators can be developed to improve specificity and regulation.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectSynthetic Biology
dc.subjectQuorum Sensing
dc.subjectMicrobial Genetics
dc.subjectMolecular Biology
dc.subjectMesorhizobium
dc.subjectLuxR
dc.subjectAHL
dc.subjectAntiactivator
dc.subjectChimera
dc.subjectChimeric LuxR Regulator
dc.subjectQseM
dc.subjectDUF2285
dc.titleDecoding the dual target antiactivator QseM
dc.typeThesis
dc.date.updated2021-07-06T06:53:54Z
dc.language.rfc3066en
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanno
otago.openaccessAbstract Only
otago.evidence.presentYes
otago.abstractonly.term26w
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