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Title: A systematic analysis of two-component systems of Streptomyces spp.
Author: Lo, Rebecca
ISNI:       0000 0004 7962 7825
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2019
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Found in all domains of life, two-component systems (TCSs) are signal transduction pathways which are canonically composed of a membrane bound sensor kinase (SK) and a cognate response regulator (RR). Upon stimulation, the SK relays the signal to the RR through transfer of a phosphate group to a conserved aspartate residue. The active RR~P is then able to modulate gene expression in response to the initial stimulus. This output could be the regulation of metabolite production, development or movement for example. Streptomyces species encode a high number of TCSs which reflects the multitude of environmental challenges they must face. Streptomyces species are prolific producers of bioactive natural products (NPs) and account for over half of the clinically used antibiotics. Many TCSs have been shown to regulate antibiotic biosynthesis including the global regulators MtrAB and AfsQ1/Q2. The newly emerging model organism S. venezuelae possesses 59 TCSs and a predicted 30 NP biosynthetic gene clusters. In this work, a library of TCS operon deletion mutants were generated through PCR targeting and CRISPR/Cas9 gene editing. High throughput screening of this library as well as a more targeted approach through analysis of potential regulons has been utilised in an effort to characterise these mutants. In this study, through analysis of adjacent genes, a TCS was identified to regulate tunicamycin resistance (TunRS; Sven15_3170/71). Another TCS which mediates antibiotic resistance is VanRS, in response to vancomycin. Vancomycin is a glycopeptide antibiotic widely used in clinics to treat infections caused by Gram-positive bacteria such as Staphylococcus aureus and Clostridium difficile, for example. With the emergence and spread of vancomycin resistance, it is important to understand not just the mechanism of reistance but also the mechanism of recognition. This is with the aim to develop a means of sequestering recognition and resistance. It is currently unclear whether vancomycin binds directly to the SK (VanS) or first binding and forming a complex with another cell component. Here, work has been presented on the purification of the membrane protein VanS with the aim of elucidating the mechanism of vancomycin and VanS interaction. The study of TCSs shows us how bacteria link their external surroundings with adaptive responses. The study of TCSs allows us to better understand not just how bacteria perceive their surroundings but could be used as a means to activate biosynthetic gene clusters of desirable products such as bioactives. With many TCSs in the genus Streptomyces uncharacterised, TCSs were rewired in an effort to activate antibiotic production through the RR AfsQ1 via the non-cognate SK VanS, which is involved in vancomycin resistance. The in vitro and in vivo analyses carried out in this investigation to test the effects of these chimeras, have produced results which are inconclusive in determining whether the use of vancomycin is able to activate antibiotic production through an AfsQ1-dependent pathway.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available