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    Carotenoid regulation in Myxococcus xanthus

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    The Gram-negative soil dwelling bacterium Myxococcus xanthus synthesises carotenoids on exposure to UV illumination. These pigments give the colonies a distinctive orange/red colour, as well as affording them valuable protection from high energy species, generated by chemical reactions fuelled by light. Genetic dissection of the regulation of carotenogenesis has allowed elucidation of transduction of the light signal to the carotenogenic machinery within the cell. The key element in the carotenogenic regulon is the genetic switch manifested by CarR and CarQ, CarR is an integral membrane protein (anti-sigma factor) which sequesters CarQ, the sigma factor, to the membrane in the dark. When cells are illuminated the photosensitiser protoporphyrin IX becomes excited, being unstable it transfers its excitation energy to molecular oxygen. This generates the high energy species singlet oxygen, which is capable of causing severe cellular damage. Singlet oxygen interacts with CarR possibly through the mediation of CarF, the net result is the destruction of CarR and the release of the sigma factor CarQ. CarQ then mediates transcription from the carQRS promoter and the crtl promoter. Transcription from the carQRS promoter leads to the generation of more CarQ and CarS, CarS causes de-repression of the crtEBDC operon. The carotenogenic enzymes encoded by crtl and the crtEBDC cluster catalyse the production of carotenoids, which quench the initial signalling molecules singlet oxygen and protoporphyrin IX. CarR levels accumulate and CarQ is once again secured in an inactive state. This provides a nice example of negative feedback. This work investigates the interaction of CarQ with its cognate promoter at carQRS through in vivo and in vitro molecular and genetic techniques. Site directed mutations were assessed in vivo through the use of lacZ transcriptional fusions, this allowed the identification of important regions in the carQRS promoter. The interaction between the carQRS promoter and the divergent gufA promoter was also assessed. In vitro experiments were used to attempt to further characterise individual mutations. The negative feedback loop was assessed in a crtl mutant to define whether crtl was subject to autoregulation. Previously identified genes downstream of crtl were mutated to allow phenotypic analysis and identification of putative roles in carotenogenesis
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