Whitworth, David E. (1999) Feedback and molecular interactions in the process of light-induced carotenogenesis in Myxococcus xanthus. PhD thesis, University of Warwick.

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Abstract

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.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH426 Genetics
Library of Congress Subject Headings (LCSH):	Myxococcus xanthus, Carotenoids, Molecular genetics
Official Date:	March 1999
Dates:	Date Event March 1999 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Hodgson, D. A. (David A.)
Extent:	227 leaves
Language:	eng

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