Abstract
Rhodobacter sphaeroides has a complex chemosensory system comprising two classic CheAs, two atypical CheAs, and eight response regulators (six CheYs and two CheBs). The classic CheAs, CheA(1) and CheA(2), have similar domain structures to Escherichia coli CheA, whereas the atypical CheAs, CheA(3) and CheA(4), lack some of the domains found in E. coli CheA. CheA(2), CheA(3), and CheA(4) are all essential for chemotaxis. Here we demonstrate that CheA(3) and CheA(4) are both unable to undergo ATP-dependent autophosphorylation, however, CheA(4) is able to phosphorylate CheA(3). The in vitro kinetics of this phosphorylation reaction were consistent with a reaction mechanism in which CheA(3) associates with a CheA(4) dimer forming a complex, CheA(3)A(4). To the best of our knowledge, CheA(3)A(4) is the first characterized histidine protein kinase where the subunits are encoded by distinct genes. Selective phosphotransfer was observed from CheA(3)-P to the response regulators CheY(1), CheY(6), and CheB(2). Using phosphorylation site and kinase domain mutants of CheA we show that phosphosignaling involving CheA(2), CheA(3), and CheA(4) is essential for chemotaxis in R. sphaeroides. Interestingly, CheA(3) was not phosphorylated in vitro by CheA(1) or CheA(2), although CheA(1) and CheA(2) mutants with defective kinase domains were phosphorylated by CheA(4). Because in vivo CheA(3) and CheA(4) localize to the cytoplasmic chemotaxis cluster, while CheA(2) localizes to the polar chemotaxis cluster, it is likely that the physical separation of CheA(2) and CheA(4) prevents unwanted cross-talk between these CheAs.
Highlights
Bacterial chemotaxis uses a two-component phosphotransfer pathway to integrate information from multiple sensory inputs reflecting changes in chemoeffector concentration to produce an appropriate change in swimming behavior
E. coli CheA has five domains: P1 is a histidine phosphotransferase (Hpt)1 domain that contains the conserved phosphorylatable histidine residue [11]; P2 binds the response regulators and, it is not essential for phosphotransfer from the P1 domain to the response regulators, it does accelerate the process [12, 13]; P3 is the dimerization domain; P4 is the kinase domain that binds ATP and phosphorylates the P1 domain; it is characterized by the conserved N, G1, F, and G2 sequence boxes [11]; and P5 is the regulatory domain that interacts with CheW and the chemoreceptors [14]
When CheA1 H49Q was mixed with CheA1 G501K, CheA1 phosphorylation was observed, suggesting that CheA1 H49Q and CheA1 G501K formed a heterodimer in which the kinase domain of CheA1 H49Q was able to phosphorylate CheA1 G501K; similar results were obtained for CheA2 H46Q and CheA2 G472K (Table IV)
Summary
The atypical R. sphaeroides CheAs, CheA3 and CheA4, both lack some of the domains found in classic CheAs. CheA3 contains only the phosphorylatable P1 (Hpt) domain and the regulatory P5 domain; these domains are separated by a 751amino acid sequence that shows no significant similarity to any sequence presently in the TrEMBL and Swiss-Prot databases. We examined the role of CheA phosphorylation in R. sphaeroides chemotaxis and determined whether the classic CheAs could participate in phosphorylation reactions between different CheAs
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