Feedback and molecular interactions in the process of light-induced carotenogenesis in Myxococcus xanthus
Myxococcus xanthus is a soil-dwelling bacterium which produces carotenoids upon irradiation with blue light. Genetic analysis has allowed elucidation of transduction of the light signal to the carotenogenic machinery within the cell. The primary element within the carotenogenic regulon is the genetic switch manifested by CarR and CarQ. CarR is an integral membrane protein which binds to the sigma factor CarQ and holds it in an inactive state at the cell membrane. Illumination of the cell with blue light excites the photosensitiser protoporphyrin IX (PPIX) within the bacterial membrane, which then excites molecular oxygen to the excited singlet state. Both singlet oxygen and excited triplet state PPIX can cause large amounts of cellular damage. Carotenoids prevent this damage by absorbing the excess energy from these excited species and dissipating it harmlessly as heat. The presence of singlet oxygen within the bacterial membrane causes the inactivation/degradation of CarR. Removal of CarR releases CarQ from the membrane enabling it to mediate transcription from various promoters. CarQ causes transcription of the crtI gene and of the carQRS operon which produces further CarQ and CarS. CarS causes de-repression of the crtEBDC cluster. The carotenogenic enzymes encoded by crtI and the crtEBDC cluster catalyse the production of carotenoids which quench the initial signalling molecules, singlet oxygen and triplet PPIX. This causes down-regulation of the regulon as a whole as CarR is no longer degraded and once again carries nascent CarQ to the membrane in an inactive state. The negative feedback loop described above is an important consideration when assessing mutants which produce carotenoids either constitutively (Car c phenotype), or under no conditions (Car- phenotype). This work investigates the consequences of Carc and Car- mutations on the activity of promoters within the Car regulon in order to clarify the roles of various genetic loci. It is demonstrated that CarA has no regulatory role in expression of crtI or carQRS and that the expression of crtI has no regulatory consequences. Sequencing downstream of crtI revealed a novel gene gufB (gene of unknown function B) which has homologues of no known function. The critical event in the activation of the carotenogenic system is expression of the carQRS operon allowed by the release of CarQ from its complex with CarR at the membrane. Attempts were made to extract information about the interaction of CarQ with its cognate promoter at carQRS through a variety of in vivo and in vitro molecular and genetic techniques. Site-directed mutations within pcarQRS were assessed in vivo through the use of lacZ transcriptional fusions, enabling identification of important regions within the carQRS promoter. In vitro experiments provided information about the possibility of using molecular methods to assess interactions between CarQ and the pcarQRS promoter.