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This chapter describes both the in vivo and in vitro methods that have been successfully used to analyze the chemotaxis pathways of R. sphaeroides, showing that two operons each encode a complete chemosensory pathway with each forming into independent signaling clusters. The methods used range from in vitro analysis of the chemotaxis phosphorylation reactions to protein localization experiments. In vitro analysis using purified proteins shows a complex pattern of phosphotransfer. However, protein localization studies show that the R. sphaeroides chemotaxis proteins are organized into two distinct sensory clusters -- one containing transmembrane receptors located at the cell poles and the other containing soluble chemoreceptors located in the cytoplasm. Signal outputs from both clusters are essential for chemotaxis. Each cluster has a dedicated chemotaxis histidine protein kinase (HPK), CheA. There are a total of eight chemotaxis response regulators in R. sphaeroides, six CheYs and two CheBs, and each CheA shows a different pattern of phosphotransfer to these response regulators. The spatial separation of homologous proteins may mean that reactions that happen in vitro do not occur in vivo, suggesting great care should be taken when extrapolating from purely in vitro data to cell physiology. The methods described in this chapter are not confined to the study of R. sphaeroides chemotaxis but are applicable to the study of complex two-component systems in general.

Original publication

DOI

10.1016/S0076-6879(07)23018-6

Type

Journal article

Journal

Methods Enzymol

Publication Date

2007

Volume

423

Pages

392 - 413

Keywords

Bacterial Proteins, Biochemistry, Chemotaxis, Cluster Analysis, Gene Expression Regulation, Bacterial, Genome, Bacterial, Green Fluorescent Proteins, Image Processing, Computer-Assisted, In Vitro Techniques, Models, Biological, Phosphoric Monoester Hydrolases, Phosphorylation, Recombinant Fusion Proteins, Rhodobacter sphaeroides, Signal Transduction, Time Factors