Show simple item record

dc.contributor.advisorMarshall, Craig
dc.contributor.advisorGerth, Monica Leigh
dc.contributor.authorEhrhardt, Maximilian Karl Gregor
dc.date.available2019-11-06T01:34:05Z
dc.date.copyright2019
dc.identifier.citationEhrhardt, M. K. G. (2019). Engineering of bacterial chemoreceptors (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/9749en
dc.identifier.urihttp://hdl.handle.net/10523/9749
dc.description.abstractMany motile bacteria are able to swim towards certain chemicals and are repelled by others. This process is called chemotaxis and is intimately linked with growth and survival, as it directs bacteria towards favorable environmental conditions. The chemotactic process is mediated by bacterial chemoreceptor proteins, which fulfill two important functions: (i) detection of chemical cues (ligands), by their ligand binding domains (LBDs) and (ii) initiating an intracellular signaling cascade, which controls the swimming behavior. Ultimately, the repertoire of expressed bacterial chemoreceptors and their individual specificities control the chemotactic response of a bacterium. Thousands of bacterial chemoreceptor proteins exist within bacterial genomes, but despite their physiological importance, only a few have been characterized in detail and little is known about the underlying structure-function relationships, which determine their affinity and specificity. Therefore, the main goal of this work was to further our understanding of the affinity and specificity determining structure-function relationships within bacterial chemoreceptors. In addition, the modulation of chemoreceptor binding affinity by rational mutagenesis and directed evolution was explored. In Chapter 3 chemoreceptors with high degrees of amino acid sequence identity, but varying ligand binding capabilities were studied. Two of the chemoreceptors, PscC and McpC, had similar affinities for one ligand (L-proline), but markedly different binding affinities for another (GABA). Rational mutagenesis of the PscC chemoreceptor was carried out to identify residues critical for GABA binding. In part, the mutagenesis approach proved successful and a variant with ~ 7 fold reduced affinity for GABA was obtained. However, none of the rationally designed variants could completely explain the observed differences in ligand binding between PscC and McpC. In Chapter 4 a high-throughput screening platform for the directed evolution of bacterial chemoreceptor LBDs was developed. This novel screening platform relies on chimeric fusion proteins and a fluorescent reporter strain. Coupling of two high-throughput screens allows for the identification of promising variants and exclusion of constitutively-active variants. In addition, the use of fluorescence as reporter signal allows for the quantification of signal strength and the use of fluorescence activated cell sorting (FACS) to achieve a high-throughput screen. The screening platform was validated by screening an error-prone PCR library of PscH chemoreceptor variants and identifying a promising single point mutant (Ser99Gly) with improved malate binding affinity. In Chapter 5 the newly established high-throughput screening platform was exploited to screen additional PscH variants and identify further promising mutations with effect on the malate binding affinity. An in silico analysis strategy was developed and employed to identify six promising single point mutations. Promising variants were examined using isothermal titration calorimetry (ITC). The best variant had a ~ 6 times lower dissociation constant for malate in comparison to the wild-type PscH LBD. In this work, for the first time, both rational mutagenesis and random mutagenesis were explored side to side as techniques to alter the binding capabilities of bacterial chemoreceptors. In conclusion, the findings from both mutagenesis approaches suggest that the affinity of bacterial chemoreceptors is influenced by residues apart from the immediate environment of the ligand binding site. As these residues are hard to predict rationally, the random mutagenesis of chemoreceptors ultimately has the greater potential. In the future, the established high-throughput screening platform holds great promise for the mutagenesis of other chemoreceptor LBDs and the development of new sensory elements based on bacterial chemoreceptor proteins.
dc.format.mimetypeapplication/pdf
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.subjectBacterial chemoreceptors
dc.subjectDirected evolution
dc.subjectRational mutagenesis
dc.subjectLigand binding domain
dc.subjectMalate
dc.subjectPseudomonas syringae pv. actinidiae
dc.subjectFluorescence
dc.subjectFACS
dc.titleEngineering of bacterial chemoreceptors
dc.typeThesis
dc.date.updated2019-11-05T23:52:59Z
dc.language.rfc3066en
thesis.degree.disciplineBiochemistry
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
 Find in your library

Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record