Pseudomonas chemotaxis

REVIEW ARTICLE

Pseudomonas chemotaxis Inmaculada Sampedro1, Rebecca E. Parales2, Tino Krell3 & Jane E. Hill1 1

Thayer School of Engineering, Dartmouth College, Hanover, NH, USA; 2Department of Microbiology and Molecular Genetics, College of Biological Sciences, University of California, Davis, CA, USA; and 3Department of Environmental Protection, CSIC, Estacion Experimental del Zaidin, Granada, Spain

Correspondence: Jane E. Hill, Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA. Tel.: 1 603 646 8656; fax: 1 603 646 3856; e-mail: [email protected] Received 25 February 2014; revised 5 July 2014; accepted 21 July 2014. DOI: 10.1111/1574-6976.12081 Editor: Alain Filloux

MICROBIOLOGY REVIEWS

Keywords signaling; chemoreceptor; MCP; swimming; twitching; attractant.

Abstract Pseudomonads sense changes in the concentration of chemicals in their environment and exhibit a behavioral response mediated by flagella or pili coupled with a chemosensory system. The two known chemotaxis pathways, a flagella-mediated pathway and a putative pili-mediated system, are described in this review. Pseudomonas shows chemotaxis response toward a wide range of chemicals, and this review includes a summary of them organized by chemical structure. The assays used to measure positive and negative chemotaxis swimming and twitching Pseudomonas as well as improvements to those assays and new assays are also described. This review demonstrates that there is ample research and intellectual space for future investigators to elucidate the role of chemotaxis in important processes such as pathogenesis, bioremediation, and the bioprotection of plants and animals.

Introduction to Pseudomonas chemotaxis Pseudomonas are aerobic, Gram-negative rods belonging to the gammaproteobacterial class of bacteria. Their metabolic diversity is astonishing, as they are able to degrade and/or metabolize many molecules (Harwood et al., 1984; Parales et al., 2000; Lacal et al., 2011b), which likely assists several of them as opportunistic pathogens. Pseudomonads range from 1 to 5 lm in length and can be propelled by polar flagella [monotrichous for Pseudomonas aeruginosa (Leifson, 1951) and lophotrichous for Pseudomonas putida (Lautrop & Jessen, 1964), for example], polar pili [1–25 for P. aeruginosa (Weiss, 1971), for example] or as part of a social response via swarming (Henrichsen, 1972) or sliding motility (P. aeruginosa; Murray & Kazmierczak, 2008). Pseudomonads can sense chemical gradients and respond to them using flagella or pili coupled to a chemosensory system with multiple copies of chemosensory genes. Pseudomonas swim by rotating flagella, generating a force that moves the cell body forward. Flagella-mediated swimming follows a counterclockwise–clockwise–pause– counterclockwise (CCW-CW-P-CCW) flagella rotation pattern (Taylor & Koshland, 1974). When responding FEMS Microbiol Rev && (2014) 1–36

positively to a chemical gradient, the frequency of clockwise rotations deceases. The well-studied two-component chemotaxis signaling system functions by the sensing of molecular species via ligand-binding receptor modules that transduce the signal into a kinase-based pathway, which directs flagellar rotation. A subsequent protein methylation step serves as a molecular memory (Stinson et al., 1977; Craven & Montie, 1983). The specificity of a chemotactic response is determined by the ligand-binding region (LBR), a periplasmic component of methyl-accepting chemotaxis proteins (MCPs), with the exception of aerotaxis, where the LBR is cytosolic (Ferrandez et al., 2002). The cytosolic signaling domains of MCPs show a high degree of sequence conservation, and the presence of a methyl-accepting (MA) domain is the standard criterion for annotation of proteins as MCPs (Alexander & Zhulin, 2007). Pseudomonas species have > 25 MCPs encoded in their genomes, some of which have been functionally characterized (Parales, 2004). For example, 13 of the 26 MCP-like proteins in the genome P. aeruginosa have been functionally characterized (Kato et al., 2008). Recent studies of Pseudomonas provided evidence for cluster II (Lacal et al., 2010a) chemoreceptors with bimodular LBRs containing two sites for the direct recognition of different ligands (Pineda-Molina et al., 2012). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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The mechanism of twitching motility is based on extension–tethering–retraction–extension of type IV pili. When responding to a chemical gradient, it is hypothesized, based on homology to the Escherichia coli pilus structure, that pseudomonads move as a consequence of pilus retraction and the switching of the sites of pilus extension from one pole of the cell to the other (Shi & Sun, 2002). A two-component signaling system analogous in form and function to that used in flagella-mediated chemotaxis is thought to be employed during twitching chemotaxis (Shi & Sun, 2002; Bertrand et al., 2010). Further studies are needed to elucidate the details of the twitching chemotaxis mechanism. Swimming chemotaxis is generally quantified using a modified Adler capillary assay with the agarose-in-plug method and soft agar ‘swim’ plates being used to provide further, qualitative data (Adler, 1966; Yu & Alam, 1997). Twitching chemotaxis is assessed qualitatively by monitoring migration on the surface of an agar plate (Kearns et al., 2001). Alternative assays such as those using microfluidic platforms (Jeong et al., 2010) or fluorescence resonance energy transfer (FRET) (Sourjik et al., 2007) have increased the speed and precision of chemotaxis quantification. Chemoattractants that stimulate flagella-mediated motility include amino acids, aromatic compounds, organic acids, phosphate, chlorinated compounds, and sugars, among others. For example, P. aeruginosa shows positive chemotaxis toward all 20 amino acids (Taguchi et al., 1997), ethylene (Kim et al., 2007), malate (AlvarezOrtega & Harwood, 2007), and chloroform (Kato et al., 2001). In addition to these types of attractants, specific P. putida strains have been shown to be attracted to aromatic hydrocarbons such as toluene and naphthalene (Grimm & Harwood, 1997; Parales et al., 2000; Lacal et al., 2011b). Twitching motility taxis has been primarily measured in response to saturated and unsaturated lipids. For example, P. aeruginosa PA01 can twitch toward dilauroyl-phosphatidylethanolamine (Kearns et al., 2001), and this strain also shows positive chemotaxis toward unsaturated fatty acids such as palmitoleic acid and arachidonic acid (Miller et al., 2008). This review covers the following: (1) The known and putative signaling pathways for swimming and twitching chemotaxis with special emphasis on chemoreceptors. (2) The major assays used to measure positive and negative chemotaxis for swimming and twitching pseudomonads along with suggested improvements. (3) A comprehensive summary of the compounds that induce chemotaxis in strains of Pseudomonas.

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Signaling pathways for swimming and twitching chemotaxis Introduction

There are three known chemotaxis pathways for P. aeruginosa: two flagella-mediated pathways (che and che2) and a putative pili-mediated system (Darzins, 1994; Kato et al., 1999). Parts of the ‘chemotaxis system’ are linked to other phenotypes, such as the gene pilJ and the wsp, which are associated with biofilm formation (Caiazza et al., 2007). The chemotaxis apparatus in P. aeruginosa is remarkably complex. In contrast to the single copy of each signal transduction protein found in E. coli, for example, genome analyses have identified multiple copies of signaling proteins (Hamer et al., 2010) in Pseudomonas and many other species, such as Rhodobacter sphaeroides, which has several paralogues of the cytosolic proteins involved in signal transduction (Porter et al., 2008). The genetically tractable pseudomonads are considered a model organism for the study of complex bacterial chemotaxis systems, particularly P. aeruginosa. Genetic organization of flagella-mediated chemotaxis pathways The two flagella-mediated chemotaxis pathways in P. aeruginosa are formed by two homologous signaling cascades (Fig. 1). The che chemotaxis pathway is essential for chemotaxis, whereas the che2 pathway is required for fine-tuning behavior (Ferrandez et al., 2002). Currently available data suggest that genes encoded in clusters I and V assemble into the che pathway (Masduki et al., 1995; Kato et al., 1999), whereas genes encoded by cluster II assemble into the che2 pathway (Ferrandez et al., 2002; Fig. 1). There are two chemoreceptors encoded in cluster II (McpB and McpA; Guvener et al., 2006), and McpB was found to be essential for the assembly of signaling proteins that form the pathway. It is hypothesized that McpB feeds exclusively into che2, whereas most of the remaining chemoreceptors feed into the che pathway. The cluster III, also called Wsp, is involved in biological functions not related with chemotaxis (Caiazza et al., 2007). Genetic organization of pili-mediated chemotaxis pathway The pathway pil-chp, encoded by cluster IV, controls type IV pilus production and twitching motility in P. aeruginosa (Darzins, 1993, 1994; Whitchurch et al., 2004). Cluster IV contains the pilGHIJK and chpABCDE genes (Fig. 2), which include homologues of all six core genes

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Pseudomonas chemotaxis

Fig. 1. Chemotaxis gene clusters I, II, and V in Pseudomonas aeruginosa. Figure adapted from Guvener et al. (2006).

Fig. 2. Chemotaxis gene cluster IV in Pseudomonas aeruginosa. Figure adapted from Garvis et al. (2009).

for chemosensory pathways (Wuichet & Zhulin, 2010), namely cheA, cheB, cheR, cheW, cheY, and mcp. The chemosensory system that controls twitching motility is encoded predominantly by the pilGHIJK (Darzins, 1994) and chpABC (Whitchurch et al., 2004) genes. The proteins of the pil-chp pathway regulate pilus extension and retraction in P. aeruginosa (Bertrand et al., 2010). Some of these proteins appear to comprise a chemosensory signal transduction pathway similar to, but substantially more complex than, the flagella-mediated chemotaxis system (Burrows, 2012). However, scarce information about the pili-mediated chemotaxis pathway of P. aeruginosa is available. Previous studies indicated that some pilus-deficient strains have mutations in genes encoding homologs of the che chemotaxis proteins that are essential for chemotaxis movements (Darzins, 1994), which suggests crosstalk between pathways involved in twitching and chemotaxis. The following section covers the canonical flagellamediated chemotaxis pathway of Pseudomonas by homology with E. coli, and the putative pili-mediated chemotaxis pathway.

detail in E. coli and serves as a model of chemotaxis signal transduction. By genetic homology with E. coli, the canonical chemotaxis system in Pseudomonas is expected to function similarly although this has yet to be completely determined. After an overview of the canonical pathway based on the E. coli system, this part of the review will focus on the extensive work performed in the area of the MCP chemoreceptors of Pseudomonas species. The chemotaxis pathway in E. coli consists of four modules (see Fig. 3): (1) Sensor module: MCPs (a.k.a., chemoreceptor sensor proteins) that recognize their cognate ligands and are involved in the adaptive response through methylation and demethylation. (2) Transduction module: guarantees modulation of CheA autokinase activity in response to ligand binding. Changes in the CheA phosphorylation state affect the transphosphorylation of the CheY response regulator. (3) Actuator module: alters motor rotation through binding of phosphorylated CheY. (4) Integral feedback module: CheR- and CheB-mediated modulation of the chemoreceptor methylation state, which resets the receptor signaling state.

Flagella-mediated chemotaxis

Chemotaxis is based on the concerted action of excitatory and adaptive mechanisms. As aforementioned, chemosensory cascades are formed by a number of core proteins as well as different auxiliary proteins that may vary among species (Wuichet & Zhulin, 2010). The canonical flagellamediated chemotaxis pathway has been characterized in FEMS Microbiol Rev && (2014) 1–36

Sensor module Methyl-accepting chemotaxis proteins Most of the predicted P. aeruginosa MCPs are transmembrane proteins harboring a periplasmic LBR, a cytosolic HAMP linker, and a MA signaling domain (Ferrandez ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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(a)

(b)

Fig. 3. Swimming and twitching chemotaxis pathways. (a) Swimming chemotaxis pathway. Chemoreceptors recognize their cognate ligands and direct cell behavior by regulating the CheA autokinase activity. CheW couples CheA activity to receptor control. CheA phosphorylates the response regulator CheY. An increase in attractant concentration deactivates CheA autophosphorylation, and the frequency of CW flagella rotations decreases. An increase in repellent concentration (or a decrease in attractant concentration) activates CheA autophosphorylation, and the frequency of CW rotations increases. CW flagellar rotation allows the cell to reorient randomly. CCW rotation of the flagella allows the cells to move in one direction for an extended period of time (run). Dephosphorylation of CheY-P is performed by the CheZ phosphatase. The chemotactic response is also regulated by adaptative modification of the chemoreceptors via methylation and demethylation, catalyzed by CheR and CheB, respectively. CheB-P competes with CheR and removes methyl groups from the MCPs. The integration of the two mechanisms, flagella motor control and sensory adaptation, produces directed motile behavior. Structures and reactions in red are associated with CCW flagellar rotation; those in pink are associated with CW flagellar rotation. Figure adapted from Chevance & Hughes (2008) and Hazelbauer et al. (2008). (b) Putative twitching chemotaxis pathway. By homology with the flagella-mediated chemotaxis pathway, the chemoreceptor may detect changes in the concentration of attractant or repellent and regulate ChpA autokinase activity. Autophosphorylated ChpA phosphorylates PilG and PilH, which interact with the putative type IV pilus motor to control twitching motility. PilG-P mediates pilus extension by interacting with a complex including the ATPases PilB, PilZ, and the diguanylate cyclase FimX. On the other hand, PilH-P mediates pilus retraction by interacting with the ATPases PilT/U. The integration of the excitatory pathway with the adaptation pathway involves the receptor-specific methylesterase ChpB and methyltransferase PilK. Structures and reactions in light blue in the putative twitching pathway are associated with extension and retraction of pili. The putative pili-mediated system is indicated by broken lines.

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et al., 2002). In addition, other receptor topologies exist, like that of the aerotaxis receptors, which contain a pair of transmembrane regions separating the LBR and signaling domain, both of which are located in the cytosol (Hong et al., 2004). Other chemoreceptors, like McpB of P. aeruginosa, lack transmembrane regions and are soluble proteins located in the cytosol (Ferrandez et al., 2002). Polar localization of the chemoreceptor clusters has been reported for several different bacteria (Briegel et al., 2009) and may represent a general feature. Using an in-frame chromosomal MCP–YFP fusion, DeLange et al. (2007) visualized the PilJ chemoreceptor at the poles of P. aeruginosa PAO1 cells. MCP: ligand-binding region Chemoreceptors are classified according to the size of the LBR into cluster I (c. 150 amino acids) or cluster II (c. 250 amino acids) (Lacal et al., 2010a). Receptors with cluster I LBRs (PAS, CACHE, 4_HB) have been extensively studied, which is partly due to the fact that E. coli receptors are all cluster I. However, c. 40% of all chemoreceptors in all bacteria, including Pseudomonas, possess the larger cluster II LBRs (Lacal et al., 2010a). For example, P. putida KT2440 and P. aeruginosa PAO1 possess 14 and 10 cluster II receptors, respectively. Cluster II sensor domains are comprised of two major families, namely the

helical bimodular domain (HBM) and the double PDClike (PhoQ/DcuS/CitA) domain (Lacal et al., 2010a; Zhang & Hendrickson, 2010; Ortega & Krell, 2013; Fig. 4). MCP: HAMP and MA The cytoplasmic portion of the chemoreceptors typically consists of a HAMP linker domain and a MA signaling domain (Ferrandez et al., 2002). In E. coli, HAMP domains modulate the input and output response signals of the chemoreceptors. It was also reported that the cytosolic part of chemoreceptors of E. coli show a high degree of base pair sequence conservation (Alexander & Zhulin, 2007), and therefore, the presence of a MA domain is the standard criterion to annotate a protein as a MCP. Although detailed analyses of the HAMP and MA in Pseudomonas are scarce, the basic mechanism of chemoreceptor signaling is assumed to function similarly to the E. coli system. CheR: receptor methylation The methylation status of the MCP in E. coli is adjusted via competing activities of the constitutive methyltransferase CheR and the methylesterase CheB (Fig. 3a). CheR converts specific glutamyl residues on the cytoplasmic

Fig. 4. Structural diversity of chemoreceptor LBRs in Pseudomonas. Shown are X-ray structures and homology models of different LBRs. Highresolution structures of the Aer-2 PAS [an acronym of the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM)] domain and the McpS HBM domain have been published (Pineda-Molina et al., 2012; Airola et al., 2013). The PAS domain contains bound heme, and the HBM domain is in complex with malate (membrane proximal bundle) and acetate (membrane proximal bundle). Homology models of the PA2652, McpT, and PctA LBRs were generated using pdb entries 2qhk, 3c8c, and 2d4u, respectively, as templates. Image courtesy of T. Krell.

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side of the MCP to glutamyl methyl esters using S-adenosylmethionine as the methyl donor (Springer & Koshland, 1977). The methylation of the glutamyl residues supposes an increase of the activation of CheA autophosphorylation by the chemoreceptors. In response to a reduction in attractant concentration, the chemoreceptors activate CheA autophosphorylation with the consequent increase of CheY-P levels and the tumbling of the bacteria. On the other hand, CheB-P catalyzes demethylation of these glutamyl methyl residues (Anand et al., 1998). The adaptation mediated by CheB-P produces the demethylation of the activate chemoreceptors reducing the activation of CheA with the consequent decrease of CheY-P levels and less tumbling of the bacteria. The adaptation of these two enzymes, CheR and CheB, updates the methylation process with two feedback mechanisms: activation of CheB by CheA-mediated phosphorylation or opposite tendencies for the two receptor conformations. The kinase-on conformation with low-attractant affinity supposes low methylation tendency. However, the kinase-off conformation with high-attractant affinity supposes high methylation tendency. The extent of receptor methylation allows knowing the past levels and current levels of the chemical and make comparisons. By homology, pseudomonads should work similarly. In fact, aromatic compounds that serve as attractants for P. putida stimulated methylation (Harwood et al., 1989b), implying that the pseudomonad adaptation system is more E. coli-like than Bacillus-like.

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currently unknown whether the modulation of CheA autokinase in Pseudomonas systems is similar to E. coli, similar to B. subtilis, or has a different form of regulation altogether. Response regulator The control of flagellar rotation in the E. coli chemotaxis system is regulated by a specialized single-domain response regulator: CheY. By homology with E. coli (Welch et al., 1993; Toker & Macnab, 1997; McEvoy et al., 1999), it is expected that in Pseudomonas, the autophosphorylation of the central histidine kinase (CheA) phosphorylates CheY (Fig. 3a). CheY-P interacts with the protein FliM, a component of the flagellar switch complex, affecting rotation of the motor and enhancing the probability of CW rotation and thus causing the cell to tumble. Dephosphorylation of CheY-P is carried out by the CheZ phosphatase. Actuator module The chemotaxis signal transduction network modulates the switch between the counterclockwise (CCW) and CW modes. Although nothing is known about the Pseudomonas motor, it is hypothesized that it operates in a manner similar to that in E. coli, that is, in the absence of CheYP, CCW rotation is strongly favored and CheY-P binding to FliM and FliN shifts the equilibrium in favor of CW rotation (Fig. 3a).

Transduction module Integral feedback module Sensor kinase The core of the transduction module in E. coli is a ternary complex composed of MCPs, the CheA histidine kinase, and the CheW coupling protein (Fig. 3a). The molecular stimulus arising from ligand binding in the periplasm is transmitted to the cytoplasmic side of the MCP, where it modulates CheA autophosphorylation activity. Although CheW is apparently required for the regulation of CheA autophosphorylation, its specific role, beyond acting as a scaffold, is unknown. By homology with E. coli (Hess et al., 1988, 1991; Borkovich et al., 1989; Anand et al., 1998), in Pseudomonas, it is expected that two response regulators compete for the CheA phosphoryl group: CheY, which transmits the signal to the flagellar motors, and CheB, which controls the adaptation of the MCPs. In E. coli, attractant binding decreases CheA activity, and repellent binding enhances binding (Borkovich & Simon, 1990). However, in Bacillus subtilis, attractant binding causes the opposite effect, enhancing CheA autophosphorylation (Garrity & Ordal, 1997). It is ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

The chemotaxis pathways each have a kinetically fast as well as slow component, which gives the bacteria a primitive type of memory that is used for temporal comparisons when swimming in a chemical gradient. Changes in the rate of methylation in one part of the signaling cascade occur slower than the phosphorylation–dephosphorylation steps. The timing differential confers the memory required for temporal comparisons of attractant concentrations, an essential mechanism for the chemotaxis system. The purpose of the feedback module is to integrate signals from the fast excitation pathway with those from the slower adaptation pathway when the bacterium is in a gradient that it can sense. The canonical adaptation pathway is comprised of the receptor-specific methylesterase CheB and the receptorspecific methyltransferase CheR (Fig. 3a). In E. coli, CheB possesses a response regulator module that is activated when it is phosphorylated by CheA-P (Anand & Stock, 2002). CheB-P competes with CheR and removes methyl groups from the MCPs (Anand et al., FEMS Microbiol Rev && (2014) 1–36

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1998). As mentioned above for the other modules, the integral feedback module in Pseudomonas is assumed to work in a similar manner to that in E. coli. Although knowledge about flagella-mediated chemotaxis pathway in Pseudomonas is limited, in general, the recognition of cognate ligands by specific chemoreceptors has been well characterized, and some information about the components of the MCPs in Pseudomonas, including the LBR, the HAMP linker domain, and receptor methylation, has been reported recently, as described below. MCP LBRs in Pseudomonas

Pseudomonas sp. have more than 25 MCP-like proteins encoded in their genomes (Parales et al., 2004), and relatively few have been functionally characterized due to the difficulty of identifying their cognate ligands (Kato et al., 2008). For the functional annotation of chemoreceptors, a double approach based on the phenotypic analysis of single mutants and direct-binding ligand-binding studies of signal molecules to the recombinant ligand-binding domains has proven to be successful (Lacal et al., 2010a; Rico-Jimenez et al., 2013). The LBRs of Pseudomonas MCPs studied so far have been found to bind ligands directly, and no evidence has been obtained for the participation of ligand-binding proteins. The size of the LBRs differentiates the chemoreceptors. The cluster I category has c. 150 amino acids, while the cluster II category has c. 250 amino acids (Fig. 4). Note that the LBR clusters are based on the number of amino acids and are specifically associated with the MCPs, that is, they are not part of the genetic clustering organization associated with the different chemotaxis pathways described at the beginning of this section. In addition, chemoreceptor genes have also been identified on a number of plasmids isolated from Pseudomonas strains, such as the hydrocarbon-responsive McpT chemoreceptor from plasmid pGRT1 (P. putida DOT-1E; Molina et al., 2011). This plasmid has also been found in other Pseudomonas species, including Pseudomonas stutzeri AN10 (Brunet-Galmes et al., 2012) and Pseudomonas resinovorans (Maeda et al., 2003). The NahY LBR for naphthalene is also found on a plasmid, in this case, pNAH7 in P. putida G7 (Grimm & Harwood, 1997). MCP LBRs in P. aeruginosa PAO1 The P. aeruginosa PAO1 genome encodes 26 MCPs (Stover et al., 2000; Ramos et al., 2004; Kato et al., 2008). Thirteen of its 26 MCP-like proteins have been functionally characterized, 10 of which have been shown to mediate positive responses to oxygen (Aer and Aer-2), inorganic phosphate (CtpH and CtpL), malate FEMS Microbiol Rev && (2014) 1–36

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(PA2652), amino acids and gamma aminobutyrate (GABA) (PctABC), ethylene (TlpQ), and chloroethylenes (McpA; Taguchi et al., 1997; Wu et al., 2000; Hong et al., 2004; Shitashiro et al., 2005; Kim et al., 2006, 2007; Alvarez-Ortega & Harwood, 2007; Rico-Jimenez et al., 2013). The receptors Aer and Aer-2 mediate aerotaxis/energy taxis in P. aeruginosa (Hong et al., 2004). Aer is a cluster I LBR that has 45% amino acid homology to the E. coli Aer and contains the PAS [an acronym of the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM)) motif]. The function of Aer-2 (also called McpB), a cytoplasmic protein, which contains a cluster I LBR (PAS domain, Fig. 4), is uncertain, as other authors have found that mutation of mcpB does not affect aerotaxis (Guvener et al., 2006). Interestingly, when introduced into E. coli, recombinant McpB was found to elicit a repellent response to oxygen, carbon monoxide, and nitric oxide (Watts et al., 2011). The MCPs responsible for detecting the 20 natural L-amino acids are the paralogous PctA, PctB, and PctC receptors (Kuroda et al., 1995; Taguchi et al., 1997). The LBRs of PctA, B, and C are cluster II receptors containing a double PhoQ/DcuS/CitA (PDC-like) fold. The phylogenetically distant organism B. subtilis has similar amino acid receptors (i.e. both are cluster II LBRs with the double PDC fold), which are different than those of E. coli which possess 4-helix bundle (4_HB) LBRs (Glekas et al., 2010, 2012; Rico-Jimenez et al., 2013; Fig. 4). PctA appears to recognize and mediate the response to most of the natural L-amino acids, while PctB is primarily involved in mediating taxis to Gln, and PctC to His, Pro, and the human neurotransmitter c-aminobutyric acid (GABA; Rico-Jimenez et al., 2013). PctA is also the major MCP for trichloroethylene. The affinities of 17 different L-amino acids for the ligand-binding domain of the PctA chemoreceptor were found to differ largely. L-Thr was identified as the tightest binding amino acid (KD = 0.28 lM), whereas the weakest binding amino acid was L-Leu with a KD of 116 lM. The three Pct paralogues also appear to be responsible for a repellent response toward chloroform and methylthiocyanate (Shitashiro et al., 2005). Interestingly, chloroform and methylthiocyanate do not bind to recombinant LBRs of PctA, B, or C; therefore, they may be recognized indirectly via a complex with periplasmic ligand-binding proteins (Rico-Jimenez et al., 2013). TlpQ, a cluster I LBR, is the chemoreceptor responsible for positive chemotaxis to ethylene (Kim et al., 2007). The amino acid sequences of TlpQ and McpA are 73% and 70% identical, respectively, to the highly conserved domain of the E. coli chemotaxis transducer Tsr (Kim et al., 2006, 2007). Other P. aeruginosa strains including ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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PA7, PA14, and 2192 possess TlpQ homologs. The sequence of the TlpQ-LBR is 29–57% identical to the N-terminal regions of MCPs from other pseudomonads, like P. putida strains KT2440 and F1, or Pseudomonas fluorescens Pf-5 and PfO-1. Pseudomonas putida KT2440 and F1 chemotaxis toward ethylene. While some strains of Pseudomonas fluorescens chemotaxis toward ethylene, it is unknown whether Pf-5 and PfO-1 are capable of this response. The phosphate receptor CtpH (Wu et al., 2000) and the malate receptor PA2652 (Alvarez-Ortega & Harwood, 2007) are cluster I receptors that adopt a 4-helix bundle fold (like the LBRs of E. coli receptors). The receptor McpA, a cluster I LBR, mediates taxis toward trichlorethylene, tetrachloroethylene, and dichloroethylene (Kim et al., 2006). Additional cluster II LBRs in P. aeruginosa PA01 include CtpL, which responds to phosphate (Wu et al., 2000) and 4-chloroaniline (Vangnai et al., 2013). MCP LBRs in P. putida Pseudomonas putida and P. aeruginosa have approximately the same number of MCP-like genes, although there is relatively low amino acid sequence similarity between MCPs in the two species. The most conserved MCPs in P. putida F1 and P. aeruginosa PAO1 are Aer and PilJ, which share 77% amino acid sequence identity (Liu et al., 2009). The MCP Aer, a cluster I LBR, is the energy taxis receptor in P. putida PRS2000 (Nichols & Harwood, 1999), while Sarand et al. (2008) reported that the response of P. putida KT2440 toward oxygen, phenols, and methylphenols is mediated by Aer-2. This energy taxis receptor was shown to mediate responses to oxygen and phenylacetic acid in P. putida F1 (Luu et al., 2013). McpS, a cluster II LBR in P. putida KT2440, is the chemoreceptor for TCA cycle intermediates (e.g. succinate, fumarate, oxaloacetate, citrate, and isocitrate), as well as acetate and butyrate (Lacal et al., 2010b, 2011a; PinedaMolina et al., 2012). The ligand-binding domain of the McpS chemoreceptor was found to bind TCA cycle intermediates and butyrate with affinities ranging from 8 to 337 lM. The strongest responses were mediated by malate, fumarate, and succinate. It appears that binding an elevated binding affinity is a necessary requisite but not the only determinant for the responses. This is illustrated by succinate that caused strong responses whereas its binding affinity to McpS-LBD was only slightly higher than that of citrate and butyrate which caused very weak responses. The three-dimensional structure of this MCP has been determined by Pineda-Molina et al. (2012) for P. putida KT2440 (Fig. 4). This LBR is composed of two helical modules to which chemoattractants can be ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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bound. Interestingly, ligand binding to either of the two modules induced a chemotactic response. The authors hypothesized that the recognition of multiple signals by different modules of a single chemoreceptor LBR may enable fine-tuning of the chemotaxis response. Ortega & Krell (2013) defined a family of McpS-LBR-like domains that is termed the HBM. This domain is found in chemoreceptors and sensor kinases in both Archaea and Bacteria (Ortega & Krell, 2013). PcaK is required for 4-hydroxybenzoate (4-HBA) chemotaxis in P. putida (Ditty & Nichols, 1996). This protein is not a classical chemoreceptor, but a member of the major facilitator superfamily of transporters (Ditty & Harwood, 2002). The MCP cluster I LBR NahY, which is encoded on the NAH7 catabolic plasmid, is the receptor for naphthalene in P. putida G7 (Grimm & Harwood, 1999). McpT, also encoded on a plasmid, is the receptor in P. putida DOT-T1E for hydrocarbons such as benzene, p-nitrotoluene, and naphthalene (Lacal et al., 2011b). Both NahY and McpT are cluster I LBRs that possess 4-helix bundle domains. Positive chemotaxis toward cysteine has been linked to the product of the mcpC gene in P. putida F1 (Liu et al., 2009). PctApp of P. putida F1 (which is 69% identical in amino acid sequence to PctA from P. aeruginosa) is involved in negative chemotaxis to trichloroethylene (Oku et al., 2010). Parales et al. (2013) presented evidence that orthologous MCPs in closely related strains may not have identical functions. The MCP McfS in P. putida F1 is an orthologue of McpS from P. putida KT2440. The authors demonstrated that a deletion of the gene encoding McfS in P. putida F1 did not result in a loss of the response to the organic acids detected by McpS in P. putida KT2440, indicating that other receptors are involved in the response (McfR and McfQ; Parales et al., 2013). MCP LBRs in other pseudomonads There are 49 putative MCPs in Pseudomonas syringae DC3000 (Parales, 2004) and 32 putative MCPs in Pseudomonas entomophila (Munoz-Martinez et al., 2012); however, there is little known about their MCPs. What is known is that the P. fluorescens proteins CtaA, CtaB, and CtaC (homologues of P. aeruginosa PctABC) are the major chemosensory proteins for P. fluorescens Pf0-1 chemotaxis toward amino acids (Oku et al., 2012). Additionally, NbaY, a cluster I LBR, is the chemoreceptor for 2-nitrobenzoate in P. fluorescens KU-7 (Iwaki et al., 2007). Liu & Parales (2009) demonstrated that pyrimidines and s-triazines are detected by a single chemoreceptor in Pseudomonas sp. strain ADP, which has yet not been identified. FEMS Microbiol Rev && (2014) 1–36

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Although there is now some focus on understanding the poorly characterized LBR cluster II family, there is still much work to be carried out to fully elucidate the chemotaxis mechanisms for pseudomonads. MCP: HAMP domain and receptor methylation in Pseudomonas

The function and mechanism of HAMP has been extensively studied in E. coli (Parkinson, 2010); however, the information available for this domain in Pseudomonas is scarce. The HAMP domain in E. coli is characterized by four-helix bundle architecture previously described, for example, for the aerotaxis receptor Aer and Tar (Swain & Falke, 2007; Watts et al., 2008). The only published information on Pseudomonas is on P. aeruginosa PA01. PA01 has HAMP domains with the four-helix bundle architecture (Airola et al., 2010). In addition, PA01 houses an atypical HAMP domain structure on the N-terminal fragment of the receptor Aer-2 (McpB). Interestingly, this structure has five HAMP domains (each of them adopt a four-helix bundle; Airola et al., 2010). The adaptation mechanism, in which CheR mediates receptor methylation, has also been extensively studied in E. coli. In terms of receptor methylation, by homology, the CheR in pseudomonads should work similarly to E. coli. For example, P. aeruginosa and P. putida harbor methyltransferases (four and three gene copies, respectively) that are homologous to E. coli CheR. A comparative analysis of the three CheR paralogues of P. putida KT2440 was reported recently (Garcia-Fontana et al., 2013). The three proteins were found to bind the product of the methylation reaction, S-adenosylhomocysteine, with significantly higher affinity compared to the substrate, S-adenosylmethionine, which is indicative of a product feedback inhibition mechanism, like that found in E. coli (Springer & Koshland, 1977; Garcia-Fontana et al., 2013). Mutations in CheR2 abolished chemotaxis. Interestingly, CheR2 was the only paralogue that methylated the McpS and McpT chemoreceptors, demonstrating high specificity in CheR chemoreceptor recognition (Garcia-Fontana et al., 2013). Recent studies show evidence of a secondary CheR binding site in P. aeruginosa. Many bacteria, including Pseudomonas species, have an elevated number of chemoreceptors and multiple copies of paralogous signaling proteins that are thought to assemble specifically into different signaling pathways that carry out different functions. The E. coli Tar and Tsr receptors contain a C-terminal extension of c. 30 amino acids that ends in a conserved pentapeptide, which serves as a secondary FEMS Microbiol Rev && (2014) 1–36

CheR binding site (Djordjevic & Stock, 1998; Li & Hazelbauer, 2006). The physiological relevance of this secondary CheR binding site in species like E. coli that harbor a single CheR is not fully understood, but it has been proposed that it increases the local concentration of CheR (Wu et al., 1996). This phenomenon has now been assessed using P. aeruginosa, which has four CheR paralogues and three receptors (McpA, Aer2/McpB, and PA0411) with a C-terminal extension (Garcia-Fontana et al., 2014). The authors showed that of the four paralogues, only CheR2 bound to the C-terminal extension on the Aer2/McpB chemoreceptor. The function of this binding has yet to be fully determined. Removal of the pentapeptide from McpB abolished CheR2 binding and methylation. Sequence analysis shows that the pentapeptidedependent and pentapeptide-independent methyltransferases are members of two different protein families. It can therefore be concluded that the presence of the C-terminal extension on McpB permits the specific targeting of CheR2 to McpB, which may be one of the mechanisms that permits the specific association of paralogous proteins to form distinct chemosensory pathways. Pili-mediated chemotaxis

The putative pili-mediated chemotaxis pathway in P. aeruginosa utilizes pil-chp genes found in gene cluster IV (Fig. 2; Darzins, 1993, 1994; Whitchurch et al., 2004). Some of the proteins encoded by these genes appear to comprise a chemosensory signal transduction pathway very similar (by gene homology) to the flagella-mediated chemotaxis system (Burrows, 2012). Although direct information about this chemotaxis system is scarce, a hypothetical pili-mediated chemotaxis pathway has been proposed (Mattick, 2002; Whitchurch et al., 2004; Bertrand et al., 2010). This hypothetical pathway consists of the four analogous modules described previously for swimming chemotaxis (Fig. 3): Sensor module Methyl-accepting chemotaxis protein Darzins (1994) provided genetic evidence, via sequence analysis, suggesting that pilJ from cluster IV in P. aeruginosa encodes a MCP. The chemoreceptor PilJ is localized at both poles in P. aeruginosa PAO1 and is required for type IV pili retraction/extension (DeLange et al., 2007). By homology with the chemoreceptors of the flagellamediated chemotaxis pathway, PilJ is expected to detect changes in the concentration of attractant or repellent and signal through the chemosensory signaling cascades to control twitching motility (Fig. 3b). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Receptor methylation The P. aeruginosa pilK gene encodes a homolog of a chemotactic methyltransferase CheR (Darzins, 1995). The methylation status of the MCP in the pili-mediated chemotaxis system could be adjusted via the activity of the CheR-like protein PilK and the methylesterase CheB-like protein ChpB (Bertrand et al., 2010; Fig. 3b). Transduction module Sensor kinase In a manner analogous to the sensor kinase for the flagella-mediated chemotaxis pathway, the molecular stimulus generated by the sensor module is expected to be transmitted via the signaling domain that forms a complex with a CheA-like histidine kinase called ChpA and two CheW homologues called PilI and ChpC (Fig. 3b). The histidine kinase ChpA senses changes through the chemoreceptor PilJ, which induces the modulation of ChpA autophosphorylation activity. The 2476 amino acid ChpA of P. aeruginosa PAO1 is composed of at least 12 domains that contain nine potential phosphorylation sites (Whitchurch et al., 2004). There are only five domains in the CheA of E. coli. The reason for such a complex domain arrangement in Pseudomonas is not understood. Response regulator The control of pili movement in the chemotaxis twitching system is likely regulated by two CheY homologues, PilG, and PilH (Fig. 3b). The central histidine kinase (ChpA) phosphorylates PilG and PilH, each of which interacts with the putative type IV pilus motor to control twitching motility (Bertrand et al., 2010). The Pil-Chp system lacks a homologue of the CheZ phosphatase. Actuator module Chemotaxis signal transduction modulates the phosphorylation of PilG and PilH (CheY-like response regulator proteins). It is expected that PilG-P mediates pilus extension by interaction with a complex including the ATPases PilB and PilZ as well as the diguanylate cyclase FimX. On the other hand, PilH-P mediates pilus retraction by the interaction with the ATPases PilT/U (Fig. 3b). Whether the two response regulators PilG and PilH compete for phosphorylation is unclear and remains to be determined. Residual activity of both ATPases PilB and PilT has been demonstrated in the absence of signaling input from components of the Pil-Chp system (Bertrand et al., 2010).

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Integral feedback module The concerted integration of the excitation and adaptation stimuli is likely achieved by the receptor-specific methylesterase ChpB and the receptor-specific methyltransferase PilK (Fig. 3b). By analogy with the flagella-mediated chemotaxis pathway, phosphorylation of the response regulator module of ChpB may result in an activation of this enzyme. It is plausible that pathway adaptation is achieved by modulating the ratios of the opposing ChpB and PilK activities. To date, only genetic studies provide information about the putative pili-mediated chemotaxis pathway, which reveal striking parallels with the flagella-mediated chemotaxis pathway. Future biochemical analyses are necessary to increase our understanding of this chemotaxis system.

Chemotaxis assays Pseudomonads can sense and respond to certain chemical gradients. Cells may be attracted or repelled by these compounds in what is known as positive or negative chemotaxis, respectively. In the late 1800s, Engelman’s interest in bacterial chemotaxis centered on the observation of what was likely a mixture of bacteria containing Bacterium termo toward oxygen (Engelmann, 1881). Around the same time, Pfeffer (Pfeffer, 1885, 1904) reported observations of bacterial aerotaxis. Sherris et al. (1957) appear to have been the first to observe the chemotaxis behavior of a Pseudomonas (P. viscosa) toward air and arginine in a flat capillary tube. Adler (1966) was the first to develop quantitative methods to measure E. coli chemotaxis, and then, others modified these methods to monitor Pseudomonas chemotaxis (e.g. Harwood et al., 1989b; Parales, 2004; Liu et al., 2009). Measurement of twitching chemotaxis is a recent development with many areas for improvement, including the development of a method to quantify rather than just observe the twitching response. This section of the review covers the most frequently used swimming and twitching chemotaxis assays (capillary and agarose based) for Pseudomonas, including an historical overview, method optimization, standard chemical and biological controls, constraints and considerations, adaptations to respond to new questions, and emerging techniques. The following section covers flagella-mediated chemotaxis methods, and covers the pilimediated chemotaxis method. Methods to assess flagella-mediated chemotaxis

This subsection focuses on the evolution of the capillary assay, including its use in competition and negative

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chemotaxis as well as two agarose-based methods for qualitatively visualizing chemotaxis responses. Quantitative chemoattraction capillary assays Adler’s first capillary assay method centered on the generation of a chemical gradient by bacteria metabolizing nutrients in situ (Adler, 1966). In this assay, chemotaxis is monitored by observing the speed of moving bands of bacteria in a capillary tube (see Fig. 5a) over a period of hours. Quantification (Fig. 5b) is achieved by removing fractions from the tube with a Pasteur pipet and plating. Quantification of the bacteria by light scattering (Dahlquist et al., 1972) improved the utility of the assay. While having the advantage of being able to see the bands of bacteria by eye, this assay cannot distinguish between metabolism-linked chemotaxis and the chemical-specific response of the bacteria. Julius Adler transformed the bacterial chemotaxis field by modifying the capillary assay into a true reflection of (a)

the chemotaxis phenomenon. By placing a capillary containing a solution of a compound into a suspension of motile bacteria (Fig. 6a), it became possible to measure the qualitative (Fig. 6b) and quantitative response of the bacteria to a chemical gradient (Adler, 1969, 1973) over a period of about 30–60 min. The qualitative assay is quick and easy and can be used to process many samples (different attractants strains, mutants, etc.). The quantitative assay, while rather tedious and time-consuming, provides a direct quantitative measurement of the response of a population of bacterial cells to a gradient of attractant, which is what cells would encounter in their natural environment. The chemotaxis apparatus consists of a U-shaped chamber (typically bent from a 5-cm-length melting point capillary tube sealed at both ends) between a microscope slide and a cover slip. Pregrowth of Pseudomonas in a specific medium (e.g. Moulton & Montie, 1979; Liu & Parales, 2009; Liu et al., 2009) can be used to determine whether the chemotactic response under study is inducible. After microscopic verification of motility, exponentially grown cells are harvested, washed, and suspended in chemotaxis buffer (pH 7; Harwood et al., 1984) to a final density of c. 6 9 107 cells mL 1 and placed in the chamber.

(a)

(b)

(b)

Fig. 5. Response of Escherichia coli to galactose. (a) Photograph and diagram of a capillary tube filled with galactose dissolved in chemotaxis buffer, inoculated at one end with E. coli, and then closed at the ends with agar and a clay plug. The tubes are incubated in a horizontal position at 37 °C. (b) To quantify, cell fractions are removed from end B with a Pasteur pipette. Figures reproduced from Adler (1966) with permission.

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Fig. 6. Modified capillary assay. (a) Apparatus showing suspended cells in a U-shaped pool with a 1-lL capillary containing the attractant. Quantification is achieved by expelling cells from the capillary after a 30- to 60-min incubation and conducting plate counts. (b) The chemotactic response at the mouth of the capillary can be visualized using dark-field microscopy at 409 magnification. Figure (a) reproduced from Parales & Harwood (2002), with permission.

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Note that assay reproducibility was shown to suffer considerably for E. coli at lower bacterial concentrations (Adler, 1973), although this has not been studied for Pseudomonas. The chemotaxis buffer should contain a chelating agent, such as EDTA, to remove traces of heavy metal ions, which inhibit motility in E. coli (Moulton & Montie, 1979), although the presence of magnesium ions seems potentially important in E. coli (Moench & Konetzka, 1978). The basic formulation of this buffer is used in Pseudomonas studies. To prolong motility, an energy source such as glycerol is sometimes added to the chemotaxis buffer (Parales et al., 2000; Liu et al., 2009; Lacal et al., 2011b). For studies of chemotaxis toward phosphate, a phosphate-free HEPESbased buffer is used (Kato et al., 1992). The capillary tubes containing the potential attractant are 1-lL disposable micropipettes with one end sealed via flame. After incubation in the pool of cells for 30–60 min at room temperature (Lynch, 1980), the capillary is removed, the exterior is rinsed with sterile water, and the contents of the capillaries are transferred to tubes of chemotaxis buffer. Dilution in sterile minimal medium or saline and then plating on appropriate plates to determine CFU mL 1 allows for quantitation of the chemotactic response. Experimental controls should include a chemoattractant blank (buffer only) and a positive control (typically 0.1% casamino acids), and, if available, mutants lacking the cognate chemoreceptor; for example, P. aeruginosa amino acid chemotaxis studies would use pctA, B, and/ or C mutants (Rico-Jimenez et al., 2013). A range of attractant concentrations can be tested to determine the peak attractant concentration and the threshold of detection. To reduce the number of assays that are necessary, it is recommended that a quick qualitative method (see below) be used first to screen for potential attractants and gauge the relevant concentrations. Constraints and considerations of the adler-based chemoattraction capillary assay Although the capillary assay is relatively simple and quite sensitive, this method has constraints. The cells must be very motile for reproducible results to be obtained; checking the motility of the washed cells is critical, and only proceed with the assay if > 60% of the cells in the population appear to be motile. Several biological and technical replicates should be carried out to obtain statistically significant results. Perhaps, the biggest issue associated with this assay is with working with the capillaries, as expulsion of the contents can be tricky. To address this problem, after rinsing the capillary, one can break it open and place it in a tube and expel the contents via centrifugation. Some researchers have had difficulty generating U-shaped tubes to supª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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port the coverslip, and as a result, Lacal et al. (2011b) modified the set-up to overcome this issue. Their method can be tedious; however, use of the high-throughput capillary assay (96-well-microtiter plate format; see below) eliminates some of the time-consuming steps. Finally, while diffusion is the key to generating the chemical gradient to which one is testing the chemotactic response, each molecule being tested has not typically had its diffusion constant calculated. Thus, quantifying the rate of chemotaxis is necessarily a little weak, though not substantially so. Modifications to chemoattraction capillary assay: quantitative 96-well plate format Liu et al. (2009) adapted the traditional quantitative capillary assay into a high-throughput capillary assay for Pseudomonas using a 96-well-microtiter plate format. This assay is based on the method of Bainer et al. (2003) for E. coli and allows one to carry out multiple, simultaneous chemotaxis assays (Fig. 7). Capillaries sealed with 3% agar are filled under vacuum with chemotaxis buffer or an attractant dissolved in chemotaxis buffer. The plate is removed, and the capillaries are inserted into the wells of a second plate prefilled with 300 lL of a motile bacterial cell suspension (each well) with an empty sterile pipette tip tray inserted between the plates as a spacer. After incubation at room temperature for 30–60 min, the plate with the capillaries is removed. The capillaries are rinsed with chemotaxis buffer, and the contents of each capillary are collected, diluted in a sterile 96-well plate, and enumerated as CFUs by plate counts on an appropriate solid medium. Modifications to chemoattraction capillary assay: darkfield microscopy for qualitative and quantitative assessment Qualitative assessment of chemotaxis by dark-field microscopy has been carried out using a modification of

Fig. 7. High-throughput 96-well plate capillary reproduced from Liu et al. (2009), with permission.

assay.

Figure

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the classical capillary assay. In this assay, the cells accumulate at the mouth of a 1-lL capillary containing a known concentration of an attractant plus 1% agarose (Nikata et al., 1992), 1.5% agarose (Kato et al., 1992), or 2% agarose (Parales et al., 2013). The presence of the agarose prevents cells from swimming into the capillary, so they appear as a bright cloud that accumulates over time. This accumulation can be photographed through the microscope with the focus maintained at the capillary mouth (Fig. 6b). Depending on the strain and attractant, responses can be observed in 1–30 min. This qualitative capillary assay is very useful for screening many potential attractants, different concentrations of attractants, or the responses of different strains. It is also useful for determining whether chemotactic responses are inducible by pregrowing cells in specific growth media. Kato et al. (1992) quantified the Pseudomonas response using microscopy (209) to calculate the number of bacterial cells accumulating near the mouth of a capillary containing attractant (e.g. Fig. 8).

eliminate the ability of the cells to respond to the test attractant. Adaptation: measuring negative chemotaxis using a capillary assay Smith & Doetsch (1969) were the first to semi-quantitatively measure chemorepulsion in Pseudomonas (specifically, P. fluorescens) by tracking the migration of cells into a flat glass capillary. More robustly, an adapted Adler capillary method can be employed for the quantitative measurement of negative chemotaxis. In this situation, the putative repellent is present in the bacterial suspension, that is, not the capillary (Fig. 9a), and the number of bacteria that enter the capillary for ‘refuge’ is used as a measure of chemorepulsion; for example, see Fig. 9b (Tso & Adler, 1974).

(a)

Adaptation: measuring relative chemoattraction (competition) using a capillary assay This assay is carried out by including a competing attractant at its peak response concentration in the cell suspension and in the capillary (Mesibov & Adler, 1972; Liu & Parales, 2009). The competing ‘test’ attractant is placed in the capillary. With this assay, it is possible to evaluate whether the same or different chemoreceptors are used to detect the two attractants. If both are detected by the same chemoreceptor, the presence of the competing attractant in the cell suspension will reduce or

Fig. 8. Concentration–response curve for Pseudomonas aeruginosa to phosphate. Cells were imaged at 209 magnification at 30-s intervals at the mouth of a capillary containing attractant. Standard deviations for each measurement are shown. Figure reproduced from Kato et al. (1992), with permission.

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(b)

Fig. 9. Modified capillary assay for measurement of negative chemotaxis. (a) Apparatus showing suspended cells and the repellent in U-shaped pool. Quantification of bacteria that enter the capillary ‘for refuge’ is achieved by expelling cells from the capillary after a 30- to 60-min incubation and conducting plate counts. (b) Concentration–response curve of chemorepulsion for Escherichia coli to acetate. Figure (b) reproduced from Tso & Adler (1974), with permission.

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Qualitative and quantitative soft agar ‘swim’ plates for chemoattraction In this assay, soft agar (0.3%) swim plates containing the attractant are inoculated with bacteria picked from a colony (Fig. 10). The low-viscosity agar allows the bacteria to swim freely. The chemical gradient necessary for chemotaxis is set up by degradation of the attractant by the bacteria, which creates a gradient that can activate the chemotactic response. Therefore, this assay requires that the attractant is a growth substrate. Migrating bands of bacteria demonstrate positive chemotaxis. The diameter of the ring is monitored or measured over a period of several hours [10 h (Craven & Montie, 1981); 16 h (Guvener et al., 2006)] (Fig. 11). The chemotactic response can be photographed using backlighting (Parkinson, 2007). By first growing liquid cultures, and harvesting, washing, and resuspending cells in buffer or minimal medium to equal densities, one can inoculate equal numbers of cells to make the assay quantitative. Subtle but significantly reduced responses can be detected by comparing the relative responses of wild-type and mutant strains inoculated on the same plate (several replicate plates are necessary), and measuring the diameters of the colonies. This method is quite useful for screening for chemoreceptor mutants (Parales et al., 2013). Constraints and considerations of the soft agar ‘swim’ plate assay This method requires that the attractant is the growth substrate, and the size of the colony is due to a combination of growth and chemotaxis. Therefore, it is important to carry out growth studies in liquid medium to determine whether wild-type and mutant strains grow on the

Fig. 11. Diameter of the ring of Pseudomonas aeruginosa in soft agar ‘swim’ plates. Plate containing tryptone, yeast extract, and sodium chloride and was incubated over a period of 10 h at 37 °C. Figure reproduced from Craven & Montie (1981) with permission.

potential attractant at the same rate. If not, then differences in colony size could be due to differences in growth rate rather than a chemotaxis defect. If this is the case, an additional assay that does not involve growth should be used. The use of appropriate chemotaxis mutants like ΔcheYZABW or for energy taxis Δaer2 (Luu et al., 2013) as well as a positive (e.g. 0.2% casamino acids) control allows for an initial decoupling of energy and standard chemotaxis behavior. Where known or suspected, chemoreceptor mutant strain controls, for example, in P. aeruginosa amino acid chemotaxis studies, pctA, B, or C mutants can be used (Kuroda et al., 1995; Taguchi et al., 1997). Note: This assay was previously called a ‘swarm’ plate, but it is more accurately described as a ‘swim’ plate. Swimming is the individual movement of bacteria in agar concentrations at or below 0.3%, which requires flagella; in contrast, swarming is the multicellular movement of bacteria over a surface at agar concentrations between 0.3% and 1%, which requires both flagella and pili and is not linked to the chemotaxis phenomenon in Pseudomonas. Qualitative agar/agarose plug assays for chemoattraction and chemorepulsion

Fig. 10. Pseudomonas putida F1 cheA mutant (generally nonchemotactic; top colony) and wild-type (bottom three colonies) chemotaxis response to 1 mM succinate in the swim plate assay. Plate was incubated at 30 °C for 20 h. Image courtesy of R. Parales.

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Tso & Adler (1974) employed an agar plug-in Petri dish assay to monitor negative chemotaxis. The advantage of this assay is that it is easy to set up, and, using a simple lighting system, a response can usually be seen by eye in about 30 min. This subsection covers the standard agar FEMS Microbiol Rev && (2014) 1–36

Pseudomonas chemotaxis

plug-in Petri dish assay for chemoattraction, a modification of this assay (gradient swim plate assay) and the agarose-in-plug slide assay adaptation. In the original assay used for E. coli (Tso & Adler, 1974), a plug of agar (2%) containing a repellent is placed on top of a suspension of cells in 0.25% agar. A repellant response will result in the development of a clear area around the plug within c. 30 min (Fig. 12). While ostensibly qualitative, measurement of the distance from the plug to the ring can provide information for the generation of a concentration–response curve. Luu et al. (2013) employed adaptations made to the original assay (Storch et al., 1999) to measure positive chemotaxis in a small Petri dish (e.g. 35 9 10 mm) with an agar plug containing an attractant. After incubation at room temperature, a positive chemotactic response is indicated by the formation of a ring of cell density around the agar plug (Fig. 13). The chemotactic response can be photographed after 1 h using backlighting (Parkinson, 2007). As with other chemotaxis assays, experimental controls should include a chemoattractant blank and a positive control (2% casamino acids), and if available, a nonmotile mutant control (i.e. flagella mutants (such as flgK), a generally nonchemotactic mutant control (such as cheA), and, where known or suspected, mutants lacking the cognate chemoreceptor). Constraints and considerations of the agar plug assays This assay is particularly useful for testing responses to compounds that do not serve as growth substrates as the response does not involve growth on the attractant. It is also useful for determining whether a response is inducible, as the culture can be pregrown under different conditions prior to setting up the assay. However,

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Fig. 13. Pseudomonas putida F1 responding to 0.2% casamino acids in the agarose-in-plug Petri dish assay. Photograph was taken after 1 h. Image courtesy of R. Pareles.

because this method can detect both positive and negative responses, a second type of assay that can distinguish between chemoattraction and chemorepulsion must be carried out. Adaptation of the qualitative agar plug-in-petri dish: gradient swim plate assay This variation of the swim plate assay allows the detection of responses to attractants that are not metabolized by the test organism (Pham & Parkinson, 2011; Luu et al., 2013). The attractant diffuses from an agar plug placed on the surface of a soft agar plate (0.25% agar) that has been poured at least 12 h earlier. The medium contains a carbon and energy source, preferably a weak or nonattractant for the bacteria. Cells (OD660 nm 0.2) are inoculated 2 cm from the attractant plug. A chemotactic response is recorded after 20–24 h using backlighting (Parkinson, 2007). An oblong colony migrating toward the plug indicates a positive response (Fig. 14). This variation of the swim plate assay allows one to test nonmetabolizable analogs as attractants as well as the responses of catabolic mutants, as the assay does not depend on growth on the test compound. Adaptation: qualitative agarose plug slide assays for chemoattraction

Fig. 12. Escherichia coli chemorepulsion to acetate in the agarose-inplug Petri dish assay. Photograph was taken after 30 min. Figure reproduced from Tso & Adler (1974) with permission.

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Yu & Alam (1997) adapted the traditional agar plugin-Petri dish to assess chemotaxis in Halobacterium salinarum. A few years later, a slight modification of this assay was used to study the chemotactic response of P. putida F1 (Parales et al., 2000). A chamber is formed ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Constraints and considerations of the agarose plug assays

Fig. 14. Pseudomonas putida F1 (top) and an energy taxis (aer2) mutant (bottom) responding to 5 mM phenylacetic acid (in the plugin center) in the gradient plate assay. Plate contained 1 mM glycerol and was incubated at 30 °C for 25 h. Image courtesy of R. Parales.

by placing two plastic strips on a microscope slide and placing an agarose plug made with c. 10 lL of preheated 2% low-melting temperature agarose (NuSieve GTG Agarose) prepared in chemotaxis buffer, which is placed in the middle of the two plastic strips and topped with a glass coverslip (Parales et al., 2000). A few crystals of Coomassie blue help provide contrast and to improve the visualization of the process. Bacterial cells are harvested in the exponential phase and resuspended in chemotaxis buffer to an OD660 nm of c. 0.7. The cell suspension is inserted between the microscope slide and the glass coverslip, and a ring of chemotactic cells around the agarose plug can be seen in 5–30 min (Fig. 15). The chemotactic response is observed directly, with a 409 phase contrast objective, or by dark-field microscopy with a 109 objective.

This assay is quick and easy to set up, the response is obvious to the naked eye, and it can be easily documented. Because it is so quick and easy, it is particularly useful for screening many isolates or mutant strains for responses, or for screening responses to several different chemicals. This assay can be used to determine whether a chemotactic response is inducible by pregrowing the cells in appropriate media. In addition, this assay can detect both positive and negative responses, as the band of cells usually forms at some distance from the plug, depending on the chemical, its concentration, and whether it is an attractant or repellant. Therefore, it is important to confirm results with another type of assay that can distinguish attractant and repellant responses. One key factor contributing to the success of this assay is the choice of agarose. Some types of agarose contain contaminating compounds that are sensed by the bacteria as attractants. Most studies have reported the use of NuSieve GTG Agarose or Omnipur agarose. Therefore, the grade of agarose is important and negative controls are absolutely essential. Alternative and emerging flagella-mediated chemotaxis assays Several additional methods that have been developed to monitor flagella-based chemotaxis are briefly described in Table 1. Methods to assess pili-mediated chemotaxis

This subsection covers the qualitative twitching assay applied to Pseudomonas. Chemotaxis twitching plate assay

Fig. 15. Pseudomonas putida F1 responding to 2% casamino acids in the agarose-in-plug slide assay. Photograph was taken after 5 min. Image courtesy of R. Parales.

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Kearns & Shimkets (2001) adapted an assay employed to determine the chemotactic response of Myxococcus xanthus to chemical gradients (Dworkin & Eide, 1983) to visualize, monitor, and measure the motile response of P. aeruginosa PA01 to a chemical gradient when cellular movement is dominated by type IV pili contraction/extension (Fig. 16). A chemical gradient in twitching assay plates is established by placing the attractant onto the agar surface followed by 30 °C incubation for 24 h to allow gradient development. The attractant location is labeled with a marker on the Petri dish. In parallel and to test the expected chemical gradient generated after incubation, a fluorescent dye is spotted onto an equivalent twitching plate and diffusion is quantified using a spectrometer (Kearns & Shimkets, FEMS Microbiol Rev && (2014) 1–36

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In this assay, the swimming trajectories of the cells after addition of attractant are imaged and the time required for one-half of the cell population to return to pre-stimulus behavior is determined

Temporal quantitative assay

Flagella are sheared off, and the cells are attached to a microscope slide via the single remaining flagellum using a flagellin-specific antibody. Cells rotate CW/CCW in response to an attractant and are visualized under a 409 objective

This assay analyzes the swimming paths of individual cells, and behavior is assessed quantitatively in terms of the average number of changes of swimming direction per second for a population of cells

Computer-assisted motion analysis

Tethered cell analysis

Definition

Chemotaxis is measured by adding the bacterial suspension to the lower cell of the chemotaxis chamber and by the quantification of the number of bacteria passing through the membrane

Method

Chemotaxis chamber

Table 1. Alternative and emerging flagella-mediated chemotaxis assays Diagram

References

Silverman & Simon (1974), Qian et al. (2013)

Shioi et al. (1987), Parales (2004)

Harwood et al. (1989b, 1990), Schmidt et al. (2011)

Armitage et al. (1977), Armitage & Evans (1983), Shitashiro et al. (2003)

Advantage (A)/disadvantage (D)

A: This assay can accurately separate CCW and CW rotations and measure rotational speed. The dynamic chemotaxis properties of tethered bacterial cells are analyzed

A: This assay monitors adaptation of the bacteria to the attractant

A: Allows differentiation between random swimming behavior (above 0.4 changes of direction per second) and chemotactic stimulation (below 0.3 changes of direction per second)

A: This high-throughput assay allows the evaluation of the aerotactic response of bacteria by the use of fluorescently labeled bacteria on a plate reader

Pseudomonas chemotaxis

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Definition

This device consists of a main detection channel and of a gradient generator. The chemoeffector solution or buffer blank is injected into inlets 1 and 3 and the fluorescently labeled bacteria into inlet 2. Fluorescent images are collected at 0.1-s intervals

A chemoattractant gradient is established along the channel containing glass beads with an average diameter of 100 lm and is maintained for several hours. Replacing the chemoattractant with fluorescent rhodamine B allows the determination of the concentration gradient. Bacterial swimming behavior is recorded using a confocal microscope in a fixed focal plane

This device generates multiple spatial chemical gradients inside a microfluidic chamber in a 2-D plane

This assay quantifies the fluorescence resonance energy transfer (FRET) signal arising from the ligand induced change in the binding of fluorescence labelled forms of CheY and CheZ. The FRET signal linearly correlates with taxis

Method

Microfluidic assay

Microfluidic assay l-Slide

Microfluidic palette

Fluorescence resonance energy transfer assay (FRET assay)

Table 1. Continued Diagram

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Sourjik et al. (2007), Neumann et al. (2010, 2012), Bi et al. (2013)

Atencia et al. (2009), Wu et al. (2013)

Chen & Jin (2011)

Jeong et al. (2010)

References

A: This assay is more precise than standard assays, allowing quantitative noninvasive measurements in real time

A: These assays allow the quantification of cell migration under controlled gradient conditions D: Flow can influence negative swimming dynamics (Hill et al., 2007)

Advantage (A)/disadvantage (D)

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Pseudomonas chemotaxis

(a)

(b)

(c)

(d)

(a)

(b)

Fig. 16. Twitching chemotaxis response of Pseudomonas aeruginosa PA01 (a, b) and a pilJ mutant (c, d) toward chloroform (a, c) and phosphatidylethanolamine (b, d). Figure reproduced from Kearns et al. (2001) with permission.

Fig. 17. Twitching chemotaxis response of Pseudomonas aeruginosa PA01 toward palmitic acid and palmitoleic acid. Observation of cell migration was taken after c. 16–24 h using a light microscope at a magnification of 1009. Figure reproduced from Miller et al. (2008) with permission.

2001). To inoculate, late exponential phase Pseudomonas cells are suspended to 5 9 109 CFU mL 1 in LB-India ink-MOPS buffer (pH 7.6). The India ink allows for the visualization of the original inoculum location. The bacteria are spotted 5 mm from the center of the attractant drop. The plate is then incubated at the optimal growth temperature for the strain of Pseudomonas until the cells have migrated 1 mm from the India ink origin in the negative controls (c. 16–24 h). Observation of cell migration is typically conducted using a light microscope at a magnification of 1009 (Fig. 17). Preferential migration, that is, chemoattraction, is grossly calculated by dividing the distance traveled up gradient by the distance traveled down gradient. FEMS Microbiol Rev && (2014) 1–36

Considerations and constraints of the chemotaxis twitching plate assay The key consideration for monitoring twitching chemotaxis is the need for high magnification (e.g. 1009) to image the phenomenon. The key constraint is that the assay is not quantitative. There is a need for the development of a quantitative twitching chemotaxis assay. A note on energy taxis

To test whether the response is metabolism dependent, the investigator needs to use catabolic mutants as well as a mutant with a defective energy taxis receptor (identifiª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Table 2. Compounds that induce flagella-mediated chemotaxis in Pseudomonas

(a)

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(b)

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(c)

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(c)

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(c)

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(d)

(e)

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(e)

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(f)

(g)

(h)

(i)

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Table 3. Compounds that induce pili-mediated chemotaxis in Pseudomonas

cation based on the PAS domain). Confounding this issue, however, is that pseudomonads have more than one PAS domain containing receptor. Two different Aer homologs are responsible for energy taxis in two different P. putida strains (Nichols & Harwood, 2000; Luu et al., 2013). Reviews covering methodology and a summary of known phenomena are available (Taylor et al., 2007; Schweinitzer & Josenhans, 2010).

Compounds that induce chemotaxis in Pseudomonas Pseudomonads sense a wide range of chemoeffectors. Tables 2 and 3 summarize the compounds that induce flagella-mediated chemotaxis and pili-mediated chemotaxis in Pseudomonas, respectively. The compounds that induce flagella-mediated chemotaxis in Pseudomonas are classified into several groups corresponding to their general chemical structures and their derivative compounds, as follows: (a) carboxylic acids, (b) amino acids, (c) aromatic compounds, (d) biphenyl, (e) ethylene, (f) furan, (g) dichloromethane, (h) pyrimidines, FEMS Microbiol Rev && (2014) 1–36

and (i) triazines (see Table 2). All but a few of the compounds are chemoattractants. Those Pseudomonas strains that exhibit chemorepellent behavior are noted. In contrast to flagella-mediated chemotaxis, few studies have measured twitching motility taxis and this literature review unearthed only six compounds, which are classified as phospholipids or unsaturated long-chain fatty acids (Table 3). The Pseudomonas strains in which chemoreceptors for specific compounds have been identified are noted. The tables do not include studies using extracts, exudates, or otherwise uncharacterized mixtures, even if chemotaxis has been observed.

Concluding remarks and future perspectives Pseudomonads can sense and respond to chemical gradients using flagella or pili coupled with a chemosensory system. This review describes the two known chemotaxis pathways: a flagella-mediated pathway and a putative pili-mediated system. The mechanism of the canonical flagella-mediated chemotaxis in Pseudomonas is quite well ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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understood, as many pieces of the pathway have homology with the well-described E. coli system. The specificity of the canonical flagella-mediated chemotactic response is determined by the > 25 MCPs present in pseudomonads. While cluster I LBRs have been well studied, little is known about cluster II LBRs, which have a unique, bimodular architecture. Pseudomonas respond positively to a wide range of chemicals using the canonical pathway, but far less is known about the detection of chemorepellants or the interesting dichotomy of concentration-dependent attraction and repulsion (Kato et al., 2008). This situation is necessarily limited by the development and use of highthroughput assays. Improvement of flagella-mediated chemotaxis measurements via high-throughput capillary assays as well as the development and use of microfluidic devices should allow for faster assessment of chemotaxis in pseudomonads. Although not high throughput, the fluorescence resonance energy transfer method recently employed in E. coli studies captures the chemotaxis process in real time via the direct monitoring of Che-Y phosphorylation and so is also a useful tool. Unfortunately, little is known about twitching chemotaxis in Pseudomonas. Biochemical testing as well as the development of quantitative chemotaxis assays to elucidate the mechanisms and evaluation of various potential attractants and repellents is needed to gain greater insight into this interesting phenomenon. Genetic homology with E. coli suggests that these studies are achievable. Intriguingly, there could be a third, noncanonical chemotaxis mechanism in Pseudomonas as has been reported in E. coli (Pham & Parkinson, 2011; Neumann et al., 2012; Yuan et al., 2012). It remains to be established whether this mechanism also exists in pseudomonads. Interestingly, chemotaxis proteins have been associated with nonchemotaxis functions. For example, PilJ, a MCP from chemotaxis gene cluster IV, inversely regulates biofilm formation and swarming promoted by SadB (surface attachment defective B; Caiazza et al., 2007). A deletion of PilJ results in a biofilm-defective phenotype and hyperswarming. Alternative functions of the many proteins in the chemotaxis machinery have not been explored. There are over 200 species of Pseudomonas and countless strains; however, only seven Pseudomonas species have had their chemotaxis abilities screened and published: P. aeruginosa, P. fluorescens, Pseudomonas pseudoalcaligenes, P. stutzeri, P. syringae, P. entomophila, and P. putida. The study of more clinical isolates and environmental strains would also help elucidate the role of chemotaxis in important processes such as pathogenesis, bioremediation, and the bioprotection of plants and aniª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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mals. There is ample research and intellectual space for future investigators.

Acknowledgements Figure 3 credit: Borja Andres Sampedro Medina. We thank Daniel B. Kearns for helpful comments on the manuscript. Research in the Parales Lab is supported by the National Science Foundation (MCB0919930). Research in the Krell lab is supported by FEDER Funds and Fondo Social Europeo from the Junta de Andalucia (CVI-7335).

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Graphical Abstract The contents of this page will be used as part of the graphical abstract of html only. It will not be published as part of main article.

(a)

(b)

This review covers the known and putative signaling pathways for swimming and twitching chemotaxis with special emphasis on chemoreceptors, the major assays used to measure positive and negative chemotaxis for swimming and twitching, and a comprehensive summary of the compounds that induce chemotaxis in strains of Pseudomonas.