Many bacteria live in a liquid environment and explore their surroundings by swimming. When in search of food, bacteria are able to swim toward the highest concentration of food molecules in the environment by a process called chemotaxis. Proteins important for chemotaxis group together in large networks called chemotaxis arrays. In the bacterium Vibrio cholerae chemotaxis arrays are placed at opposite ends (at the “cell poles”) of the bacterium by a protein called ParP. This makes sure that when the bacterium divides, each new cell receives a chemotaxis array and can immediately search for food. In cells that lack ParP, the chemotaxis arrays are no longer placed correctly at the cell poles and the bacteria search for food much less effectively.

To understand how ParP is able to direct chemotaxis arrays to the cell poles in V. cholerae Alvarado et al. searched for partner proteins that could help ParP position the arrays. The search revealed that ParP interacts with other proteins in the chemotaxis arrays. This enables ParP to integrate into the arrays and stimulate new arrays to form. Alvarado et al. also discovered that ParP consists of two separate parts that have different roles. One part directs ParP to the cell pole while the other part integrates ParP into the arrays. By performing both of these roles, ParP links the positioning of the arrays at the cell pole to their formation at this site.

The findings presented by Alvarado et al. open many further questions. For instance, it is not understood how ParP affects how other chemotaxis proteins within the arrays interact with each other. As well as enabling many species of bacteria to spread through their environment, chemotaxis is also important for the disease-causing properties of many human pathogens – like V. cholerae. As a result, learning how chemotaxis is regulated could potentially identify new ways to stop the spread of infectious bacteria and prevent human infections.

Chemotaxis is one of the primary means by which motile bacteria sense, respond, and adapt to changing environmental conditions. This process enables motile bacteria to perceive changes in local concentrations of chemicals; as a result, they can bias their movement away from unfavorable chemical stimuli and towards more favorable compounds (Wadhams and Armitage, 2004; Sourjik and Armitage, 2010). In the best studied model organism Escherichia coli, chemotaxis is mediated by an array of highly organized macromolecular complexes built from core chemotaxis units. The core units are themselves composed of a highly organized set of chemotaxis signaling proteins (Figure 1A–B). In general, the chemotaxis signaling cascade is initiated upon the detection of chemotactic stimuli by methyl-accepting chemotaxis proteins (MCPs). These membrane-spanning receptors then interact with a cytoplasmic histidine kinase, CheA, while the adaptor protein CheW stabilizes this interaction and participates in regulating CheA kinase activity (Ortega et al., 2013; Parkinson et al., 2015). A phosphosignaling cascade is initiated via CheA and its cognate response regulator CheY. Phosphorylated CheY induces a change in flagellar rotation and consequently in the direction of bacterial swimming, which over time results in net movement towards a more favorable environment (Wadhams and Armitage, 2004; Sourjik and Armitage, 2010).

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MCPs usually consist of a variable N-terminal extracellular ligand binding domain, a cytoplasmic HAMP domain, and a well conserved signaling domain (or kinase control domain) with a highly conserved protein interaction tip that directs the assembly and action of receptor signaling complexes (Figure 1A) (Kim et al., 1999; Falke and Hazelbauer, 2001; Alexander and Zhulin, 2007; Hazelbauer et al., 2008). Importantly, the tip contains sites for forming trimers of receptor dimers (Kim et al., 1999; Parkinson et al., 2015), and for binding to CheA and CheW (Miller et al., 2006; Park et al., 2006; Vu et al., 2012; Wang et al., 2012; Li et al., 2013; Piasta et al., 2013; Pedetta et al., 2014; Cassidy et al., 2015) (Figure 1B). The histidine kinase CheA is comprised of five separate domains (P1 to P5) with specific functions (Figure 1B, green). P1 is the phosphotransfer domain and contains the substrate histidine for autophosphorylation; P2 binds CheY for phosphotransfer from P1 (Swanson et al., 1993; Morrison and Parkinson, 1994; Bilwes et al., 1999); P3 is the dimerization domain (Park et al., 2006; Cassidy et al., 2015); P4 is the kinase or ATP binding domain; and P5 is an SH3-like regulatory domain, which binds the signaling tip of MCPs (Borkovich et al., 1989; Gegner et al., 1992; Bilwes et al., 1999; Zhao and Parkinson, 2006). The adaptor protein CheW (Figure 1B, red) consists of a single SH3-like domain, and is structurally similar to P5 of CheA (Griswold et al., 2002; Li et al., 2013; Cassidy et al., 2015).

Together, MCPs, CheA, and CheW form stable core signaling complexes. As shown in Figure 1B, one CheA dimer joins two MCP trimer-of-dimers and two CheW proteins. The helix formed by the dimerization of the P3 domains of CheA positions itself between the two MCP dimer-of-trimers (Briegel et al., 2011; Li and Hazelbauer, 2011; Briegel et al., 2012; Liu et al., 2012, 2013; Briegel et al., 2014a) and each P5 domain of a CheA dimer binds to one CheW. Therefore, a single core unit is arranged in a hexagonal structure held together by contacts between: (i) CheA-MCPs, (ii) CheW-MCPs and (iii) CheA-CheW. According to the current model, further hexagonal core units then join to form a super-lattice structure, commonly known as the chemosensory array (Briegel et al., 2009, 2012; Liu et al., 2012, 2013; Briegel et al., 2014a, 2014b; Piasta and Falke, 2014) (Figure 1B). In vivo and in vitro observations indicate that CheA-CheW interactions bridge the two receptor trimers of every core and give the array its characteristic stability and high sensitivity (Zhao and Parkinson, 2006; Hazelbauer et al., 2008; Erbse and Falke, 2009; Briegel et al., 2009; Li and Hazelbauer, 2011; Briegel et al., 2012; Slivka and Falke, 2012; Sourjik and Wingreen, 2012; Liu et al., 2012; Briegel et al., 2014a; Piasta and Falke, 2014). However, while there is much knowledge of array structure, the mechanisms that underlie the formation and localization of these elaborate structures are incompletely understood, especially in systems other than E. coli.

As mentioned above, chemotaxis has been extensively studied in E. coli, a peritrichously flagellated bacterium. Here, array formation is thought to be a stochastic process in which individual receptors are inserted randomly in the membrane, and subsequently diffuse freely until they either join existing arrays or nucleate new ones (Thiem and Sourjik, 2008). This process results in a non-uniform distribution of signaling arrays at cell poles and randomly along the cell length (Sourjik and Berg, 2000), and likely ensures that sensory arrays are in close proximity to the lateral flagella. In organisms such as Caulobacter crescentus, Pseudomonas aeruginosa, and Rhodobacter sphaeroides, and several Vibrio species, chemosensory arrays are actively localized to the cell poles (Alley et al., 1992; Maddock and Shapiro, 1993; Wadhams et al., 2003; Bardy and Maddock, 2005; Ringgaard et al., 2011; Ringgaard et al., 2014). In the polarly flagellated pathogens Vibrio cholerae and Vibrio parahaemolyticus, we recently reported that chemosensory arrays are exclusively localized at one or both cell poles by a mechanism that depends on the partner proteins ParC and ParP, both of which are encoded within the chemotaxis operon (Ringgaard et al., 2011; Yamaichi et al., 2012; Ringgaard et al., 2014). For V. cholerae, chemotaxis proteins encoded by chemotaxis operon II, e.g. CheA1 and CheW1, are directed to the cell pole by ParC and ParP (Ringgaard et al., 2015; Briegel et al., 2016), and from here on, CheA and CheW will be used instead of CheA1 and CheW1, respectively. In newborn Vibrio cells, these signaling arrays are exclusively localized to the old flagellated cell pole, then recruited to the new cell pole as cells enlarge, resulting in a bi-polar localization pattern. Thus, at cell division each daughter cell inherits a signaling array positioned at its old pole (Ringgaard et al., 2011, 2014). In the absence of either ParC or ParP, the chemotaxis arrays are no longer properly recruited to the cell poles. Instead, signaling arrays form and localize randomly along the cell length, and bi-polar localization is not established prior to cell division. Therefore, daughter cells do not faithfully inherit a signaling array at their old poles, resulting in altered motility and decreased chemotaxis (Ringgaard et al., 2011, 2014).

ParC mediates polar localization of ParP, which in turn interacts with a specific domain of CheA that is only present in CheA proteins with an associated ParC/ParP-system (CheA-LID) (Ringgaard et al., 2014). ParP prevents dissociation of CheA from chemotaxis arrays and disruption of either ParP-ParC or ParP-CheA interactions results in defective recruitment of chemotaxis arrays to the cell poles, leading to their random instead of polar localization (Ringgaard et al., 2011, 2014). However, the molecular mechanisms by which this protein interaction network governs the dynamic localization of chemotactic signaling arrays remain to be elucidated. Notably, there is little knowledge of how factors promoting array positioning are able to access and guide localization of chemotaxis proteins. In particular, it is not clear how such factors are integrated within the widely conserved structure of signaling arrays.

Here, using V. cholerae as a model organism, we analyze how ParP is able to gain access to and interact with chemotaxis proteins positioned within the highly ordered structure of signaling arrays and how it mediates their intracellular localization. We identify MCP proteins as a new interaction partner for ParP. Via interactions with MCPs and CheA, ParP is a part of the chemotaxis core unit and integrates into the chemotactic signaling arrays. Importantly, ParP integrates into arrays and promotes their formation via a C-terminal Array Integration and Formation (AIF) domain, which is linked to ParP’s N-terminal ParC interaction domain. Linkage of these domains within ParP couples array formation and localization and results in localized formation of arrays at the cell poles and thus promotes cell pole maturation.

The highly ordered structure and distribution of signaling arrays within the cell is essential for proper chemotactic responses and bacterial competitiveness. However, it is not well understood how factors responsible for array positioning are able to access chemotaxis proteins within arrays to mediate localization. In addition to interacting with ParC and CheA, we found that ParP also interacts with MCP proteins. ParP interacts with the MCP interaction tip domain in a manner analogous to CheA and CheW proteins and promotes array formation. Thus, ParP, like CheA and CheW, appears to be a component of the chemotaxis core unit. Mapping of ParP’s interaction interfaces revealed that its C-terminal SH3-like AIF domain includes distinct surfaces that enable interaction with MCPs and CheA. Furthermore, its N-terminal ParC interaction domain is responsible for recruitment of ParP and signaling arrays to the cell pole. The linkage of these domains within ParP couples array formation and localization and results in localized formation of arrays at the cell poles. By stimulating polar array formation, ParP also promotes its own and ParC’s polar localization. Collectively, by defining ParP’s interaction network and interfaces, we uncovered how this protein couples array formation and polar localization.

We identified MCP proteins as essential interaction partners for ParP. The screen identified 15 distinct MCPs, and all but two MCPs of V. cholerae possess the motif within the conserved protein interaction tip that mediates ParP-MCP interaction. The screen likely only identified 15 of the 45 predicted MCPs encoded by V. cholerae because only ~50,000 colonies were screened, and thus the screen was not comprehensive. Furthermore, the screen only identifies ParP interaction partners that were fused in frame with the t18 gene during library generation. These factors likely explain why a subset of MCPs, as well as known interaction partners ParC and CheA were not identified in the screen.

ParP integrates into signaling arrays through its interactions via its AIF domain with the conserved protein interaction tip of MCP proteins and with CheA. Through these complex interactions, ParP promotes array formation rather than compromising array structure. ParP-AIF’s similarity to CheW and CheA-P5 in regions that mediate interaction with MCP suggests that ParP might compete with CheW and CheA-P5 for MCP binding, and thereby to become part of the chemotactic core unit. Previous studies indicate that other proteins with SH3-like structures compete to become part of the array in a comparable manner (Levit et al., 2002; Asinas and Weis, 2006; Erbse and Falke, 2009); e.g., CheV can replace CheW (Alexander et al., 2010) and CheA-P5 (Briegel et al., 2012) within signaling arrays. Although CheW, CheA-P5 and ParP-AIF all appear to have the capacity to recognize and bind the MCP interaction tip within the chemotactic core unit, all have distinct functions and interestingly form their own distinct clades of SH3-like domains. Thus, evolution has exploited the ability of the SH3 domain to interact with MCPs within the core unit of arrays for diverse functions including signal transduction, array formation and the intracellular localization of signaling arrays.

Transactions between ParP and its array partners - MCPs and the LID domain of CheA - likely reflect the balancing of the requirement for an additional array component mediating array localization (ParP) with preservation of array structure and function. Arrays can still form if one of ParP or CheA is absent, due to the presence of the other. owever, in the absence of both proteins, the chemotaxis core unit is no longer assembled and signaling arrays are barely able to form. Arrays form at almost wild-type levels in the absence of CheA, suggesting that ParP is able to fully replace CheA within the core unit. However, we cannot rule out the possibility that ParP also is able to compete with CheW for integration into the core unit. In the prevailing model of array structure, two CheA proteins are present within a core unit, dimerized through their P3 domain (Figure 9, type #1) – an interaction that contributes to array stability and signal transduction (Briegel et al., 2011; Li and Hazelbauer, 2011; Briegel et al., 2012; Li et al., 2013; Briegel et al., 2014b). If ParP-AIF replaces a CheA protein within the arrays, CheA dimerization, and its associated array stabilization, would be lost; however, it was shown that the ParP-CheA interaction (via CheA-LID) reduces dissociation of CheA from arrays (Ringgaard et al., 2014) and thereby provides an alternate means of stabilization. Thus, our data are consistent with a model where ParP-AIF replaces some CheA-P5 in binding the MCP interaction tip within the chemotaxis core unit, and is tethered there through binding to the LID domain of the remaining CheA of the core unit. Presumably this AIF-LID interaction is able to substitute for the absence of CheA-P3 dimerization within the core unit and thus maintains the stability of the array structure (Figure 9, type #2). Since ParP is able to dimerize via its N-terminus (Ringgaard et al., 2014), it is also possible that a ParP dimer is able to replace the CheA dimer within the core unit of wild-type cells, resulting in a core unit comprised of two CheWs and a ParP dimer (Figure 9, type #3). Core units consisting only of CheW, ParP, and MCPs presumably constitute the arrays observed within a CheA-deficient strain, which form at close to wild-type levels. Additional studies will be required to elucidate whether interactions between ParP/CheW and ParP/CheA-P5 occur and the factors that modulate the MCP interaction tip’s accessibility to different partners. In a wild-type background arrays might consist of all three types of core units (Figure 9—figure supplement 1), whereas in the ΔparP and ΔcheA deletion backgrounds, arrays consist of type #1 and type #3 core units only, respectively (Figure 9—figure supplement 1). Furthermore, it is also a possibility that the flexible dimerization domain of ParP could link ParP proteins from neighboring core units, and in this way augment retention of ParP itself and chemotaxis proteins within the array, thereby contributing to array stability and ultimately their sequestration at the cell pole via ParP’s ParC interaction domain.

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Interestingly, the intracellular localization of cytosolic chemotaxis arrays in R. sphaeroides may have functional similarities to the Vibrio ParP/ParC system. In R. sphaeroides, a ParA-like protein, PpfA, (ParC is also a ParA-like protein) is thought to ensure proper segregation and positioning of the cytosolic chemotaxis arrays over the bacterial nucleoid by means similar to that used by ParA proteins involved in plasmid segregation (Thompson et al., 2006; Ringgaard et al., 2011; Roberts et al., 2012). PpfA mediates array localization in concert with a predicted cytoplasmic chemoreceptor TlpT, which, in combination with a CheW, is required for array formation. TlpT interacts with PpfA via its N-terminus, thereby likely stimulating PpfA ATPase activity – an action that is required for PpfA function (Wadhams et al., 2005; Thompson et al., 2006; Roberts et al., 2012). It is possible that TlpT integrates into the cytoplasmic arrays, bridging chemotaxis proteins and PpfA to promote array formation and localization in a manner similar to that of ParP.

Importantly, ParP’s interactions with the chemotaxis proteins in the core unit (CheA and MCPs) are also important for ParC-mediated sequestration of ParP at the poles and for the polar localization of ParC itself. Disruption of ParP’s interactions with MCPs and CheA resulted in a much higher percentage of non-polar (cytosolic) ParC and ParP. Thus, although ParC is still able to recruit ParP to the cell poles, sequestration of ParC and ParP at this site is diminished when ParP interactions with MCPs and CheA are disrupted. As seen with ParC (Ringgaard et al., 2011), here we show that there is a continuous exchange of ParP between the cell pole and the cytoplasm. Our photobleaching-based comparisons of ParP and ParP2PM suggest that ParP’s capacity to integrate into signaling arrays does not influence its recruitment to the cell pole. In contrast, the capacity of ParP to bind MCPs and CheA and integrate into signaling arrays had a significant impact on its release from the pole into the cytoplasm. Particularly, that integration of ParP into signaling arrays prevents the release of ParP molecules from the cell pole and consequently promotes its retention at this site. Thus, ParP’s integration into arrays modifies its own and likely in turn ParC’s subcellular localization dynamics, promoting their polar retention.

We have shown that ParP is a protein of high network connectivity and functions as an important nexus that facilitates chemotaxis array formation, array localization, and regulates the localization dynamics of its network elements. ParP retains partial function as long as one of its network connections to the core unit exists (i.e. to either CheA or MCPs). Only loss of both ParP’s connections to the core unit results in a non-functional ParP variant. In contrast, when either of its connections to the core unit are disrupted, the retention of ParP, and ParC, at the cell pole is compromised to the same extend as when both connections simultaneously are disrupted. This suggests that when a ParP loses a network connection to the core unit, ParP is still able to function partially in mediating polar array localization due to the other connection, however, the ability of ParP to mediate retention of its network constituents at the cell pole is lost – ultimately resulting in its partial loss of function. This further emphasizes the importance of ParP’s interconnectivity within the chemotaxis protein interaction network in regulating the polar retention of itself and its network constituents and in mediating the proper polar localization of chemotactic signaling arrays.

Taken together our findings show that ParP’s high connectivity allows it to serve as a critical nexus that regulates the temporal dynamics of its network constituents and stabilizes the polar localization of the cell-pole anchor ParC and itself. Furthermore, it facilitates the localized assembly and inheritance of signaling arrays at the pole, hereby ensuring proper cell pole development.

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Author details

1. Alejandra Alvarado

   Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   Formal analysis, Investigation, Writing—review and editing, Data interpretation and generation of working model

   Competing interests

   No competing interests declared

2. Andreas Kjær

   Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Wen Yang

   Institute of Biology, Leiden University, Leiden, Netherlands

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Petra Mann

   Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Ariane Briegel

   Institute of Biology, Leiden University, Leiden, Netherlands

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Matthew K Waldor

   1. Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, United States
   2. Howard Hughes Medical Institute, Harvard Medical School, Boston, United States
   3. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States

   Contribution

   Conceptualization, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1843-7000

7. Simon Ringgaard

   Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing, Data interpretation and generation of working model

   For correspondence

   simon.ringgaard@mpi-marburg.mpg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4980-5964

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