Characterization of bacteriophytochromes from photosynthetic bacteria: histidine kinase signaling triggered by light and redox sensing.
Bacteria detect environmental changes, thanks to two-component signal-transduction systems, composed, in general, of a sensor coupled to a histidine kinase and a DNA binding response regulator. Anoxygenic photosynthetic bacteria like Rhodopseudomonas (Rps.) palustris, possess a highly versatile metabolism and can grow via photosynthesis using light energy or via respiration through oxygen consumption. For photosynthetic bacteria, detecting changes in light quality or quantity, or in oxygen concentration, is therefore of prime importance for adjusting their metabolism for optimal development. A central role is played by bacteriophytochromes for light detection and initiation of regulatory responses. The switch of these chromoproteins between two photointerconvertible forms is the first event in the light-regulated cascade. This chapter describes in vitro and in vivo methods that have been successfully used to investigate the bacteriophytochrome dependent light regulation pathways, in several strains of Rps. palustris and Bradyrhizobium. These approaches range from biochemical and biophysical methods to genetic techniques. Such multiple approaches are indispensable for understanding these complex light-regulated pathway. In a first step, bacteriophytochromes and associated response regulators are overexpressed in Escherichia coli and purified. The spectral and kinetic properties of the two photointerconvertible forms of the purified bacteriophytochromes are then determined by biophysical approaches. Original spectral and kinetic properties found in some of the bacteriophytochromes that we studied necessitated the development of new methods for computing the spectra of the pure forms and the photoconversion yields. In vitro biochemical approaches help to assess the histidine kinase activity of bacteriophytochromes depending on light conditions, the phosphotransfer to response regulators and their affinity to promoter DNA sequences. Finally, gene inactivation tests the importance of specific genes in photosynthesis regulation under particular light and oxygen tension growth conditions. The methods described in this chapter are not restricted to the study of the light-transduction pathways of Rps. palustris and Bradyrhizobium strains but are applicable to the understanding of any bacterial light-regulatory system.
Laboratoire des Symbioses Tropicales et Méditerranéennes, IRD, CIRAD, AGRO-M, INRA, UM2, TA A-82/J, Campus de Baillarguet, Montpellier Cedex 5, France.
Eric
E
Giraud
Jérôme
J
Lavergne
André
A
Verméglio
D001419:Bacteria / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D024202:Electrophoretic Mobility Shift Assay; D000071677:Histidine Kinase; D008027:Light; D010084:Oxidation-Reduction / Q000528:radiation effects; D010766:Phosphorylation; D010788:Photosynthesis / Q000235:genetics / Q000502:physiology*; D010860:Pigments, Biological / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000528:radiation effects
D001426:Bacterial Proteins; D010860:Pigments, Biological; D011494:Protein Kinases; D000071677:Histidine Kinase; D003850:Deoxyribonuclease I