Azorhizobium caulinodans Chemotaxis Is Controlled by an Unusual Phosphorelay Network

Abstract

Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms root nodules on its host legume, Sesbania rostrata . This agriculturally significant symbiotic relationship is important in lowland rice cultivation, and allows for nitrogen fixation under flood conditions. Chemotaxis plays an important role in bacterial colonization of the rhizosphere. Plant roots release chemical compounds that are sensed by bacteria, triggering chemotaxis along a concentration gradient toward the roots. This gives motile bacteria a significant competitive advantage during root surface colonization. Although plant-associated bacterial genomes often encode multiple chemotaxis systems, A. caulinodans appears to encode only one. The che cluster on the A. caulinodans genome contains cheA , cheW , cheY2 , cheB , and cheR . Two other chemotaxis genes, cheY1 and cheZ , are located independently from the che operon. Both CheY1 and CheY2 are involved in chemotaxis, with CheY1 being the predominant signaling protein. A. caulinodans CheA contains an unusual set of C-terminal domains: a CheW-like/Receiver pair (termed W2-Rec), follows the more common single CheW-like domain. W2-Rec impacts both chemotaxis and CheA function. We found a preference for transfer of phosphoryl groups from CheA to CheY2, rather than to W2-Rec or CheY1, which appears to be involved in flagellar motor binding. Furthermore, we observed increased phosphoryl group stabilities on CheY1 compared to CheY2 or W2-Rec. Finally, CheZ enhanced dephosphorylation of CheY2 substantially more than CheY1, but had no effect on the dephosphorylation rate of W2-Rec. This network of phosphotransfer reactions highlights a previously uncharacterized scheme for regulation of chemotactic responses. IMPORTANCE Chemotaxis allows bacteria to move towards nutrients and away from toxins in their environment. Chemotactic movement provides a competitive advantage over non-specific motion. CheY is an essential mediator of the chemotactic response with phosphorylated and unphosphorylated forms of CheY differentially interacting with the flagellar motor to change swimming behavior. Previously established schemes of CheY dephosphorylation include action of a phosphatase and/or transfer of the phosphoryl group to another receiver domain that acts as a sink. Here, we propose A. caulinodans uses a concerted mechanism in which the Hpt domain of CheA, CheY2, and CheZ function together as a dual sink system to rapidly reset chemotactic signaling. To the best of our knowledge, this mechanism is unlike any that have previously been evaluated. Chemotaxis systems that utilize both receiver and Hpt domains as phosphate sinks likely occur in other bacterial species.

Generalizable strategy to analyze domains in the context of parent protein architecture: A CheW case study

“…However, the only experimental investigation of a Class 5 CheW-like domain of which we are aware was in A. caulinodans CheA, where removal of the C-terminal CheW-like domain diminishes chemotaxis but does not reveal the functional role of the Class 5 domain. 51 Class 5 CheW-like domains were substantially different from experimentally characterized CheW-like domains. Therefore, because investigation of Class 5 is especially likely to reveal new information, Class 5 arguably represents the highest priority for future research on CheW-like domains.…”

“…In each case, the C-terminal receiver domain is functionally important. However, the only experimental investigation of a Class 5 CheW-like domain of which we are aware was in A. caulinodans CheA, where removal of the C-terminal CheW-like domain diminishes chemotaxis but does not reveal the functional role of the Class 5 domain 50. Class 5 CheW-like domains were substantially different from experimentally characterized CheW-like domains.…”

“…Perhaps the receiver domain must be held in a particular orientation to be accessible to multiple Hpt domains.Class 5 was the C-terminal CheW-like domain in CheA proteins with dual CheWlike domains. A. caulinodans CheA,50 Azospirillum brasilense CheA1,54 and Rhodospirillum centenum CheA1 55 have been explored experimentally and all belong to the CheA.V Architecture with two CheW-like domains. In each case, the C-terminal receiver domain is functionally important.…”

Generalizable strategy to analyze domains in the context of parent protein architecture: A CheW case study

“…In each case, the C‐terminal receiver domain is functionally important. However, the only experimental investigation of a Class 5 CheW‐like domain of which we are aware was in A. caulinodans CheA, where removal of the C‐terminal CheW‐like domain diminishes chemotaxis but does not reveal the functional role of the Class 5 domain 51 . Class 5 CheW‐like domains were substantially different from experimentally characterized CheW‐like domains.…”

“…However, the fact that CheW-like domains in CheV and CheA proteins were in different Classes (1 vs. 3, 4, or 5) strongly suggests that adjacent CheW-like and receiver domains in CheA proteins do not act as CheV analogs. Consistent with this prediction, the adjacent CheW-like domain did not affect autodephosphorylation of the receiver domain from Azorhizobium caulinodans CheA,51 in contrast to B. subtilis CheV 49. Third, as far as we know, obvious questions about CheW-lineage proteins with multiple domains have not been explored experimentally.…”