ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor

Bacterial chemotaxis proteins are organized into ordered arrays. In peritrichous organisms, such as Escherichia coli, stochastic assembly processes are thought to account for the placement of chemotaxis arrays, which are nonuniformly distributed. In contrast, we previously found that chemotactic signaling arrays in polarly flagellated vibrios are uniformly polar and that array localization is dependent on the ParA-like ATPase ParC. However, the processes that enable ParC to facilitate array localization have not been described. Here, we show that a previously uncharacterized protein, ParP, interacts with ParC and that ParP is integral to array localization in Vibrio parahaemolyticus. ParC’s principal contribution to chemotaxis appears to be via positioning of ParP. Once recruited to the pole by ParC, ParP sequesters arrays at this site by capturing and preventing the dissociation of chemotactic signaling protein (CheA). Notably, ParP also stabilizes chemotactic protein complexes in the absence of ParC, indicating that some of its activity is independent of this interaction partner. ParP recruits CheA via CheA’s localization and inheritance domain, a region found only in polarly flagellated organisms that encode ParP, ParC, and CheA. Thus, a tripartite (ParC–ParP–CheA) interaction network enables the polar localization and sequestration of chemotaxis arrays in polarly flagellated organisms. Localization and sequestration of chemotaxis clusters adjacent to the flagella—to which the chemotactic signal is transmitted—facilitates proper chemotaxis as well as accurate inheritance of these macromolecular machines.Motile bacteria use chemotaxis to survey their environments and navigate in response to them. In particular, chemotactic sensing and response systems enable bacteria to recognize chemical gradients in their surroundings and to direct themselves toward favorable environments and away from detrimental ones. Chemotactic behavior is mediated by two-component signal-transduction pathways that detect such gradients and transmit this information via a phosphorelay to the bacterial flagella. Unfavorable gradients, i.e., a decrease in attractants or an increase in repellants, generate a signal to change flagellar rotation, which results in a change in swimming direction and a net movement toward more favorable conditions. This ability to react to changes in the external milieu can be essential for viability and competitiveness (reviewed in ref. 1).Chemoeffectors typically are detected by transmembrane chemosensory proteins termed “methyl-accepting chemotaxis proteins” (MCPs). These receptors interact with a histidine kinase, CheA, whose interaction with MCPs is stabilized by an adaptor protein, CheW. Upon recognition of a decrease in attractant or increase in repellant, the MCPs induce CheA autophosphorylation. Phosphorylated CheA transfers its phosphate group to the response regulator CheY, which diffuses freely in the cytoplasm. Accumulation and binding of phosphorylated CheY to the flagellar switch protein FliM increases the probability of a change in flagellar motor rotation, resulting in movement toward more favorable conditions (Fig. S1A) (1).Chemosensory proteins are found universally in large macromolecular clusters known as “chemotactic signaling arrays” (2–5). MCPs, together with CheA and CheW, form the sensory core of these clusters. Interactions between the cytoplasmic signaling domains of the receptors are sufficient for the formation of receptor arrays (6), although the formation is enhanced in the presence of CheW or CheA (6–8). Recent studies have suggested that array formation in Escherichia coli is a stochastic process in which individual receptors are inserted randomly in the membrane, where they diffuse freely and either join existing arrays or nucleate new ones (9). This process results in a nonuniform distribution of signaling arrays at cell poles and randomly along the cell length (10). In other organisms chemosensory arrays are localized only to the cell poles (11–14); however, the mechanisms behind polar localization are incompletely understood.We recently reported that the principal chemotaxis proteins in Vibrio cholerae, the Gram-negative bacterium responsible for the diarrheal disease cholera, display a markedly different distribution from that observed in E. coli (15). Newborn cells contain a single focus of chemotaxis proteins at their old pole. Subsequently, as cells mature, a second focus develops at the new pole, resulting in bipolar localization. Uni-and bipolar targeting of these chemotaxis proteins is dependent on ParC, a representative of a distinct family of ParA-like ATPases encoded within chemotaxis operons of many polar-flagellated bacteria. Like the chemotaxis signaling proteins, ParC exhibits a cell cycle-dependent unipolar/bipolar distribution (15). The mechanisms that enable ParC to control the localization of chemotactic signaling arrays have not been described. Targeting of ParC to the pole is dependent on the polar determinant, HubP, although HubP and ParC do not interact directly (16).ParA-like ATPases are responsible for the spatiotemporal positioning of several additional intracellular processes, including positioning of chromosomes and plasmids and identification of the cell division plane (reviewed in refs. 17 and 18). ParA-like proteins modulate the localization of cytosolic chemotaxis clusters and carboxysomes in Rhodobacter sphaeroides (19) and Synechococcus elongatus (20), respectively. The localization and activity of ParA-like proteins typically is controlled by ATP binding and hydrolysis, because ATP binding governs their capacity to oligomerize and to interact with other proteins (reviewed in refs. 17 and 18). Notably, many ParA-like proteins are encoded adjacent to genes encoding interacting proteins that also are required for their function (e.g., ParB). Like ParA proteins, these partners typically display a restricted and dynamic distribution within the cell. In general, the role of the partner protein is to act as a bridge between ParA and its target and to regulate the enzyme’s ATPase activity.Here, we investigated the function of an unannotated open reading frame, now designated parP, which is encoded downstream of parC. ParP proved to be a ParC partner protein, and, like ParC, ParP colocalizes with polar chemotactic signaling arrays in Vibrio parahaemolyticus. Positioning of ParP is dependent on ParC, and localization of ParP appears to be ParC’s principal function. ParP promotes array stability as well as positioning and retention at the pole. Following its recruitment to the pole by ParC, ParP sequesters arrays at this site by capturing and preventing the dissociation of CheA. Notably, in contrast to many other ParA-associated proteins, we found that ParP has activity that is independent of its ParA-like partner. Even in the absence of ParC, ParP prevents the dissociation of CheA from clusters of chemotaxis proteins. ParP maintains clusters of CheA by interacting with a localization and inheritance domain (LID) that is present only in CheA proteins with coresident ParP and ParC and also can be bound by ParC. Thus, our data indicate that a tripartite ParC–ParP–CheA interaction network promotes proper polar localization, sequestration, and inheritance of the macromolecular machine that is responsible for chemotactic signaling.