SummaryComponents of bacterial chemosensory pathways which sense via transmembrane receptors have been shown to localize to the cell poles. Many species, however, have operons encoding multiple putative chemosensory pathways, some including putative cytoplasmic receptors. In-genome fusions to single or multiple genes encoding components of two chemosensory pathways in Rhodobacter sphaeroides , che Op 2 and che Op 3 , revealed that while sensory transducing proteins associated with transmembrane receptors and encoded on che Op 2 were targeted to the cell poles, the proteins associated with putative cytoplasmic receptors and encoded on che Op 3 were all targeted to a cytoplasmic cluster. No proteins were localized to both sites. These data show that bacteria target components of related pathways to different sites in the cell, presumably preventing direct crosstalk between the different pathways, but allowing a balanced response between extracellular and cytoplasmic signals. It also indicates that there is intracellular organization in bacterial cells, with specific proteins targeted and localized to cytoplasmic regions.

SummaryThe purple photosynthetic bacterium Rhodobacter sphaeroides has three loci encoding multiple homologues of the bacterial chemosensory proteins: 13 putative chemoreceptors, four CheW, four CheA, six CheY, two CheB and three CheR. Previously, studies have shown that, although deletion of che Op 1 led to only minor changes in behaviour, deletion of che Op 2 led to a loss of taxis. The third locus encodes two CheA, one CheR, one CheB, one CheW, one CheY, a putative cytoplasmic chemoreceptor (TlpT) and a protein showing homology to the chromosomal partitioning factor Soj (designated Slp). Here, we show that every protein encoded by this locus is essential for normal chemotaxis. Phototaxis is also dependent upon all the components of this locus, except CheB 2 and Slp. The two putative CheA proteins encoded in this locus are unusual. CheA 3 has only the P1 domain and the P5 regulatory domain linked by a large internal domain, whereas CheA 4 lacks the P1 and P2 domains required for phosphorylation and response regulator binding. These data indicate that the minimal set of proteins required for normal chemotaxis in R. sphaeroides is all the proteins encoded by che Op 2 and the third chemotaxis locus, and that the multiple chemosensory protein homologues found in R. sphaeroides are not redundant.

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SummaryMany proteins have recently been shown to localize to different regions of the bacterial cell. This is most striking in the case of the Escherichia coli chemotaxis pathway in which the components localize at the cell poles. Rhodobacter sphaeroides has a more complex chemotaxis system with two complete pathways, each localizing to different positions, one pathway at the pole and one at a discrete cluster within the cytoplasm of the bacterium. Using genomic replacement of the wild-type chemotaxis genes in R. sphaeroides with their corresponding fluorescent protein fusions in conjunction with in frame deletions of other chemotaxis genes, we have investigated which proteins are required for the formation of the polar and cytoplasmic chemotaxis protein clusters. As in E. coli , the polarly targeted CheA and CheW homologues are required for the formation of the polar cluster. However, the formation of the cytoplasmic cluster requires the cytoplasmic chemoreceptors and CheW but not the CheAs. Interestingly, even when deletion of a component resulted in the chemotaxis proteins of one pathway becoming delocalized and diffuse in the cytoplasm, in no case were any chemotaxis proteins seen to localize to the other signalling cluster.