CheR- and CheB-Dependent Chemosensory Adaptation System of Rhodobacter sphaeroides

Abstract

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in three major operons and other, unlinked, loci. These include cheA 1 and cheR 1 (che Op 1 ) and cheA 2 , cheR 2 , and cheB 1 (che Op 2 ). In-frame deletions of these cheR and cheB homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR 2 and CheB 1 were essential for normal chemotaxis, whereas CheR 1 was not. cheR 2 and cheB 1 , but not cheR 1 , were also able to complement the equivalent E. coli mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in R. sphaeroides. In E. coli, CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high-and low-abundance receptors. This motif is not contained on any R. sphaeroides chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR 2 either interacts with the NWETF motif of E. coli methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that R. sphaeroides has an adaptation system that is different from that of Bacillus subtilis and E. coli, with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA 2 , but not CheA 1 , is able to phosphorylate CheB 1 , suggesting that signaling through CheA 1 cannot initiate feedback receptor adaptation via CheB 1 -P.

The CheYs of Rhodobacter sphaeroides

“…Protein Purification-His-tagged CheA and CheY proteins were overexpressed and purified as described previously (22,23,29). Mutant CheY proteins were overexpressed and purified in the same way as the wild-type versions of the proteins.…”

Localization of MreB inRhodobacter sphaeroidesunder Conditions Causing Changes in Cell Shape and Membrane Structure

“…His-tagged MreB was expressed in E. coli M15 pREP4 cells containing pMLP1. Purification was performed as described previously (14). A rabbit antibody was raised against purified MreB (Eurogentec).…”

Chemotaxis in Rhodobacter sphaeroides Requires an Atypical Histidine Protein Kinase

“…Cloning CheAs into Expression Vectors-The construction of the pQEA1, pQEA2, and pQEA4 expression plasmids has been described previously (15,22); the kinase domain and phosphorylation site mutants of cheA 1 , cheA 2 , and cheA 4 were cloned into the expression plasmid pQE30 in the same way. The coding sequences of cheA 3 and cheA 3 H51Q (excluding the stop codons) were amplified by PCR from genomic DNA of R. sphaeroides strains WS8N and JPA1210, respectively.…”

“…Purification of Chemotaxis Proteins-His-tagged CheA 1 , CheA 2 , CheY 1 , CheY 2 , CheY 3 , CheY 4 , CheY 5 , CheY 6 , CheB 1 , and CheB 2 were overexpressed and purified as described (22,28). CheA 3 and CheA 4 were overexpressed from His-tagging expression vectors and purified in the same way as CheA 1 and CheA 2 , except that for CheA 3 the His tag was C-terminal instead of N-terminal.…”

“…The proteins of immediate relevance to this study are: CheA 1 , CheY 1 , CheY 2 , and CheY 5 encoded by cheOp 1 (18,20); CheA 2 , CheB 1 , and CheY 3 encoded by cheOp 2 (19); CheA 3 , CheA 4 , CheB 2 , and CheY 6 encoded by cheOp 3 (15); CheY 4 encoded in a separate genetic locus with the chemoreceptor McpG (21). Genetic studies have shown that, although CheA 2 , CheA 3 , CheA 4 , CheB 1 , CheB 2 , and CheY 6 are essential for chemotaxis (15,22,23), all of the proteins encoded by cheOp 1 have no detectable role in chemotaxis (18). Chemotaxis also requires either of CheY 3 The chemosensory proteins of R. sphaeroides are found in two distinct clusters, the polar chemotaxis cluster and the cytoplasmic chemotaxis cluster, each containing a specific subset of chemosensory proteins.…”