Journal of Bacteriology, August 2002, p . 4374-4383, Vol . 184, No . 16

Cluster II che Genes from Pseudomonas aeruginosa Are Required for an Optimal Chemotactic Response

Abel Ferrández,¹ Andrew C . Hawkins,^1,2 Douglas T . Summerfield,¹ and Caroline S . Harwood^1,2*

Department of Microbiology,¹ Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242-1109²

Received 27 February 2002/ Accepted 14 May 2002

While CheY phosphorylation relies on the activity of the histidine kinase CheA, its dephosphorylation is controlled by CheZ, as well as by an intrinsic dephosphorylation activity . To ensure proper periodic monitoring of the environment, the system is reset by methylation and demethylation of the MCPs . MCP methylation counterbalances the effect of attractant binding and contributes to adaptation by resetting the signaling activity of the receptors, despite the continued presence of stimulus (12) . Two proteins regulate the level of methylation of MCPs . CheR, a methyltransferase, adds methyl groups to conserved cytoplasmic glutamate residues . CheB, a methylesterase, which is active when phosphorylated by CheA-P, removes the methyl groups .

The fundamental characteristics of signal reception and transduction that occur during chemotaxis by E . coli are likely conserved among bacteria and archaea . However, with the recent proliferation of genome sequences, we also now realize that there is much more diversity and complexity in chemotactic signaling pathways in prokaryotes than had been previously anticipated . Most motile bacterial species for which genome sequence information is available have multiple homologues of each of the E . coli che genes; most have many more methyl-accepting chemotaxis genes than the five found in the well-studied enteric species (5) . Bacillus subtilis has chemotaxis genes (cheC and cheD) not found in enterics (22, 31, 45) . The -proteobacteria Rhodobacter sphaeroides, Sinorhizobium meliloti, and Caulobacter crescentus have several copies of genes encoding products homologous to those of E . coli che genes, but each lacks a cheZ homologue (2, 41) .

Pseudomonas aeruginosa is an opportunistic human pathogen and member of the -Proteobacteria . It swims in liquid environments by means of a single polar flagellum, and it can also move on solid surfaces by means of swarming (32) and twitching (49) . P . aeruginosa is chemotactic to most of the organic compounds that it can grow on, and several repellants have been identified (14, 23, 27, 39, 40, 42) . P . aeruginosa has 26 genes that are homologous to E . coli mcp genes . It also has multiple copies of E . coli-like chemotaxis genes arranged in five clusters (Fig . 1) (56) . Two che clusters, cluster I and cluster V, which encode homologues of the six che genes found in E . coli, have previously been shown to be essential for chemotaxis by P . aeruginosa (24, 37) . Cluster IV has been shown to be involved in twitching motility (8, 25) . Here, we investigate the role that cluster II chemotaxis-like proteins may play in P . aeruginosa chemotaxis .

 
Bacterial strains, plasmids, media, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1 . All strains were grown on rich medium, Luria-Bertani (LB) medium (46), at 37°C unless otherwise noted . Antibiotics were used at the following concentrations, where appropriate: carbenicillin, 300 µg per ml; chloramphenicol, 100 µg per ml; gentamicin (Gm), 100 µg per ml; and tetracycline (Tc), 100 µg per ml (P . aeruginosa) and ampicillin (Ap), 100 µg per ml; chloramphenicol, 100 µg per ml; gentamicin, 25 µg per ml; kanamycin, 100 µg per ml; and tetracycline, 25 µg per ml (E . coli) .

 
Chemotaxis assays. Chemotaxis was examined qualitatively in soft-agar swarm plates . Strains were stab inoculated into the centers of plates that had been solidified with 0.3% agar and that contained one of the following: a defined minimal medium (11) plus 1 mM succinate; diluted LB medium (0.1% [wt/vol] tryptone, 0.05% [wt/vol] yeast extract, and 0.5% [wt/vol] NaCl); or tryptone (0.1% tryptone [wt/vol] and 0.5% NaCl [wt/vol] for P . aeruginosa or 1% tryptone [wt/vol] and 0.5% NaCl [wt/vol] for E . coli) . Wild-type cells and the mcpA and mcpB mutants were screened for chemotaxis in minimal medium swarm plates that contained 1 mM concentrations of 60 different growth substrates . The 60 compounds that were tested as potential chemoattractants included amino acids, sugars, organic acids, and several different kinds of aromatic compounds . To test chemotaxis under anaerobic denitrifying conditions, minimal medium-succinate swarm plates were supplemented with 1 mM KNO₃ and incubated anaerobically in polycarbonate jars to which GasPak hydrogen plus carbon dioxide generator envelopes (BBL Microbiology Systems, Cockeysville, Md.) had been added . Carbon sources were sterilized separately and added to minimal medium after being autoclaved . Soft-agar plates were incubated at 30°C for 18 to 24 h for P . aeruginosa and at 35°C for 5 to 6 h for E . coli . The assays were repeated at least three times .

Construction of a lacZ-Gm^r cassette. A 0.8-kb SmaI fragment containing a Gm^r gene was excised and purified from plasmid pUCGm (48) . This fragment was then ligated into HincII-digested pUC18Not (10), giving pUCGmNot . Plasmid pUTminiTn5lacZ1 (9) was digested with NotI, and the Km^r gene was removed . A NotI fragment that contained the Gm^r gene from pUCGmNot was ligated into pUTminiTn5lacZ1 digested with NotI, yielding pUTlacZ1Gm . A lacZ-gentamicin fragment was purified out of the latter by digesting it with EcoRI . This fragment was blunt ended by treatment with the Klenow fragment of DNA polymerase I (Roche Molecular Biochemicals, Indianapolis, Ind.) and ligated to pUC18Not that was digested with NotI and blunt ended by treatment with Klenow . Finally, the HindIII and PstI restriction sites within the lacZ and the Gm^r genes were removed by partial digestion with HindIII and PstI, Klenow treatment, and then religation . The resulting plasmid, pUClacZGm, is a high-copy-number ori ColE1 vector containing a promoterless lacZ followed by a Gm^r gene . This roughly 4-kb fragment can be excised as either an SmaI or BamHI cassette for the generation of lacZ transcriptional fusions .

Construction of cheB2::lacZ-Gm^r and cheW2::lacZ-Gm^r mutant strains. An in-frame deletion of cheB2 was created by overlap extension PCR as described previously (18, 20, 21) with the following modifications . A region of DNA from the PAO1-Ig chromosome spanning from approximately 1 kb upstream of cheB2 to the 5' end of cheB2 was PCR amplified using primers DcheB2 [1] and DcheB2 [2] . A second region, from approximately 1 kb downstream of cheB2 to the 3' end of cheB2, was PCR amplified using primers DcheB2 [3] (complementary to DcheB2 [2]), and DcheB2 [4] . A mixture of these two DNA fragments (100 ng each) was used as the template in a third PCR amplification including primers DcheB2 [1] and DcheB2 [4] . The product of the third amplification contained a 1,026-bp in-frame deletion in cheB2 including an engineered ScaI site plus approximately 1 kb upstream and 1 kb downstream of cheB2, with engineered HindIII and EcoRI sites on its 5' and 3' ends, respectively . This product was digested with EcoRI and HindIII and was ligated into HindIII/EcoRI-digested pEX19Tc, yielding plasmid pAFB2T . The cheB2-lacZ fusion was generated by ligation of a SmaI lacZ-Gm^r cassette obtained from plasmid pUClacZGm into ScaI-digested pAFB2T, yielding pAFB2TlacZGm . The HindIII/EcoRI insert from pAFB2TlacZGm was then subcloned into pRK415, giving pAFB2TlacZGm2 . This plasmid was then mobilized from E . coli DH5 into PAO1-Ig, using E . coli CC118(pRK600) to provide the transfer functions . The recombinant strain, AFIB2lacZ, was identified by screening for Gm^r and Tc^s colonies and verified by Southern blot analysis (46) . A cheW2::lacZ-Gm^r mutant (strain AFIW2lacZ) was constructed using a similar strategy .

Construction of P . aeruginosa cheB, cheZ, cheY, cheW, cheA, and cheA2 in-frame deletion mutants. An in-frame deletion in cheB was constructed by overlap extension PCR, as described above, using primers DcheB [1]-DcheB [2] and DcheB [3]-DcheB [4] . The product of the third amplification contained a 1,065-bp in-frame deletion in cheB including an engineered ScaI site plus approximately 1 kb upstream and 1 kb downstream of cheB, with engineered HindIII and EcoRI sites on its 5' and 3' ends, respectively . This 2,125-bp fragment was cloned as an EcoRI/HindIII cassette into pEX19Gm to give plasmid pAFBG . pAFBG was mobilized from E . coli S17-1 into PAO1-Ig by conjugation . A single recombination event was selected by growth on selective medium containing gentamicin . The double recombinant was selected by growth on LB medium plus 5% sucrose . Correct colonies were identified by screening for Gm^s and the ability to grow on 5% sucrose . cheB mutations were screened by PCR and further confirmed by Southern blot analysis, yielding the nonpolar, in-frame deletion mutant AFIB . Similar cloning strategies and methods were used to construct in-frame deletion mutations in cheZ, cheY, cheW, cheA, and cheA2 genes in P . aeruginosa strain PAO1-Ig .

Construction of cluster II mcpB::Gm^r and mcpA::Gm^r mutants. mcpB was PCR amplified from PAO1-Ig chromosomal DNA using the primers RPA04672F (which includes an engineered EcoRI site) and RPA04672R (which includes an engineered HindIII site) . The product was cloned into pUC18 digested with EcoRI/HindIII, and the resulting plasmid was named pAF27 . pAF27 was digested with AvaI, removing a 234-bp fragment from the center of mcpB; treated with Klenow and shrimp alkaline phosphatase (Roche Molecular Biochemicals), and ligated to the SmaI fragment containing a Gm^r cassette from pUCGm . The resulting plasmid was named pAF28 . The EcoRI/HindIII fragment from pAF28 was cloned into pRK415, giving pAF29 . This plasmid was transferred from E . coli DH5 to P . aeruginosa PAO1-Ig by conjugation, using E . coli CC118(pRK600) to provide the transfer functions . A double recombinant was selected as described above to give strain AFD4672 . The mutation was verified by Southern blotting and PCR . An mcpA::Gm^r mutant (AFD3216) was constructed by a similar strategy .

Construction of plasmids for expression of cluster II genes. The coding sequences of cheA2, cheB2, cheW2, cheY2, mcpA, and mcpB were PCR amplified from PAO1-Ig chromosomal DNA using the appropriate primer pairs . The upstream primers for each PCR contained an additional sequence (CCGAATTCTGATTAACTTTATAAGGAGGAAAAACATATG...) containing an engineered EcoRI site (underlined), an optimized Shine-Delgarno sequence (in boldface) (38), a translational enhancer from gene 10 of phage 7 (in italics) (38), and the start codon of the gene being amplified (double underline) . The products from PCR amplifications were digested with EcoRI/HindIII and cloned into the EcoRI/HindIII sites of pEX1.8 . These new plasmids were designated pEXA2, pEXB2, pEXW2, pEXY2, pHAH128, and pAF27B and were used to express CheA2, CheB2, CheW2, CheY2, McpA, and McpB, respectively . Each plasmid was then introduced into either P . aeruginosa competent cells (58) or E . coli competent cells (The NEB Transcript, vol . 6, p . 7, New England Biolabs, 1994) and selected for by incorporation of carbenicillin (P . aeruginosa) or ampicillin (E . coli) into the growth medium . Transformants were verified by agarose gel electrophoresis of plasmid DNA isolated by alkaline lysis (3) . Proteins were expressed under the control of the P_tac promoter and induced by the addition of 10, 100, or 1,000 µM isopropyl-ß-D-thiogalactopyranoside (IPTG) (Research Product International Corp., Mt . Prospect, Ill.) to the growth medium .

 
A cheB2 mutant is defective in chemotaxis. We constructed mutations in cluster II cheB2, cheW2, and cheA2 genes and found that the cheW2 and cheA2 mutants behaved like the wild-type parent in swarm plate assays . The cheB2 mutant, however, formed swarm rings that were smaller than those of the wild type (Fig . 3A) . This general defect was observed in minimal swarm media as well as in rich medium . The cheB2 mutant grew at the same rate as its wild-type parent in liquid medium . cheB2 mutant cells did not have an obvious defect in swimming behavior when observed under the microscope . The cheB2 mutant phenotype was complemented by the addition of cheB2 in trans (Fig . 3A) .

 
CheB2 restores chemotaxis to the nonchemotactic cheB mutant. To determine whether cluster I chemotaxis proteins can interact with cluster II proteins, four cluster II proteins, CheA2, CheB2, CheW2, and CheY2, were expressed in strains that contained a defect in the homologous cluster I gene . Of these, only CheB2 could complement a defect in the cluster I paralog, cheB . CheB2 partially restored chemotaxis to the nonchemotactic, cluster I, cheB-deficient strain AFIB (Fig . 3B) .

mcpA and mcpB mutants have a general chemotaxis defect. Cluster II contains two genes predicted to encode MCP-like proteins: mcpA and mcpB . Each gene was mutated by replacing part of the gene with a Gm^r cassette . The mcpA and mcpB mutants were then screened for chemotaxis to 60 carbon sources in minimal medium using the soft-agar plate assay (data not shown) . Carbon sources that were tested as chemoattractants included amino acids, sugars, organic acids, and aromatic compounds . Wild-type cells formed well-defined chemotactic swarm rings in swarm plates that included any of the 60 carbon sources . The mcpA mutant strain, AFD3216, behaved like the wild type in minimal-medium soft-agar plates containing any of the organic chemoattractants that we tested (not shown), but it had a general chemotaxis defect in tryptone soft-agar plates (Fig . 4A) . This phenotype was not due to a defect in the growth rate in tryptone media (data not shown) . This led us to examine compositional differences between tryptone and our minimal medium . We determined that the presence of magnesium in the minimal medium allowed the mcpA mutant to overcome the mutant phenotype (Fig . 4B) . Magnesium, in concentrations as low as 100 µM, added to the tryptone soft-agar plates as either MgCl₂ or MgSO₄, restored wild-type chemotaxis to the mcpA mutant . Overexpression of McpA in P . aeruginosa led to a nonchemotactic phenotype in soft-agar plates (Fig . 4C) . The same phenotype was observed when McpA was overexpressed in E . coli K-12 (Fig . 4D) . Individual P . aeruginosa or E . coli cells overexpressing McpA had a smooth-swimming phenotype .

 
The mcpB mutant strain, AFD4672, grew at wild-type rates (data not shown) but had a general chemotaxis defect on LB medium, tryptone, and minimal-medium soft-agar swarm plates containing any of 60 different attractants . This defect was observed when plates were incubated either aerobically or anaerobically under denitrifying conditions (Fig . 5A) . The defect was complemented when mcpB was expressed in trans (Fig . 5A) . Overexpression of McpB in P . aeruginosa led to a nonchemotactic phenotype on soft-agar plates (not shown) . The same phenotype was observed when McpB was overexpressed in E . coli (Fig . 5B) . Individual P . aeruginosa cells overexpressing McpB had a constantly smooth-swimming phenotype when observed microscopically . The same was observed when McpB was overexpressed in E . coli .

 
Cluster II proteins CheA2, CheB2, and CheW2 disrupt chemotaxis when expressed in E . coli K-12, but not when expressed in P . aeruginosa PAO1-Ig. CheA2, CheB2, and CheW2 were expressed in E . coli K-12 from a high-copy-number ori ColE1 plasmid (pEX1.8) . Each of these proteins disrupted normal chemotaxis in 1% (wt/vol) tryptone soft-agar plates (Fig . 6) . Overexpression of CheY2 had no effect on E . coli chemotaxis . Each strain was motile when a sample was removed from the plate and observed microscopically . Thus, the effect was not due to a disruption of motility . Overexpression of CheA2, CheB2, CheW2, and CheY2 in wild-type P . aeruginosa cells had no effect on chemotaxis as assayed in soft-agar swarm plates . Expression was induced by IPTG concentrations up to1,000 µM (data not shown) .

 
The P . aeruginosa cluster I CheA, CheB, CheW, CheY, and CheZ proteins have 32, 36, 30, 58, and 32% amino acid identity with the corresponding E . coli Che proteins . CheR proteins from P . aeruginosa (encoded in cluster V) and E . coli are 30% identical . Given that the cluster I che gene mutants are completely defective in chemotaxis, it was surprising to find, on inspection of the P . aeruginosa genome sequence, an additional cluster of che genes (cluster II) whose predicted protein products, with the exception of cheY2, had even higher overall sequence identities to the orthologous E . coli chemotaxis proteins (Table 2) . Key amino acid residues shown to be important for E . coli chemotaxis protein function are conserved in both the cluster I and the cluster II Che proteins from P . aeruginosa .

 
Our data suggest that P . aeruginosa cluster II che genes participate in chemotaxis . A cheB2 mutant is impaired in chemotaxis . Also, CheB2, when overexpressed, can complement a P . aeruginosa cheB mutant and restore it to a wild-type pattern of motile behavior and chemotactic response . Although the cheA2 and cheW2 mutants did not have a discernible chemotaxis defect, CheA2 and CheW2 proteins that were expressed in an E . coli K-12 background disrupted E . coli chemotaxis . This suggests that CheA2 and CheW2 can compete with endogenous E . coli chemotaxis proteins and interfere with the normal chemotactic response . There are a number of ways in which cluster II Che proteins might function in chemotaxis . They may contribute to an optimal chemotactic response by coexisting and participating in a major signal transduction pathway that is dominated by cluster I proteins . Alternatively, cluster II Che proteins may form complexes that are physically and functionally distinct from cluster I signaling complexes . The observation that overexpression of cluster II proteins interferes with E . coli, but not P . aeruginosa, chemotaxis favors the second possibility .

A distinct cluster II chemotaxis-signaling complex may be important for sensing some general parameter of cellular physiology through associated McpA and McpB proteins . Such a role is consistent with the observation that mcpA and mcpB mutants have general chemotaxis defects and unusual molecular architectures . Most mcp mutants that have been described are defective in chemotaxis to a subset of the chemoattractants that a particular bacterial species can detect . For example, mutants in the P . aeruginosa mcp genes ctpH and ctpL that are specifically nonchemotactic to inorganic phosphate have been described (59) . Similarly, the mcp mutant pctA is defective in chemotaxis to L-serine but attracted to other amino acids (33) . Most of the 26 predicted P . aeruginosa MCPs have a typical E . coli-like MCP architecture that consists of two transmembrane regions in the N-terminal half of the protein and a domain, called the highly conserved domain, that is involved in sensory signaling (Fig . 7) . Analysis of the amino acid sequence of McpA using the Simple Modular Architecture Research Tool (SMART) (35, 47) indicates that McpA has a highly conserved domain (34) but only one predicted transmembrane domain (Fig . 7) . McpB (35, 47) has a highly conserved domain (34), a PAS (for Per, ARNT, Sim) (57) domain, and no predicted transmembrane domains (Fig . 7) . PAS domains are typically involved in sensing redox potential, oxygen, or light (57) . McpA and McpB are the only P . aeruginosa MCPs that have C-terminal pentapeptides (EVELF in the case of McpA and GWEEF in the case of McpB) related to that found on the high-abundance receptors of E . coli (NWETF) (43, 60) . This reinforces the concept that McpA and McpB may play a more general and central role in chemotactic signal transduction than do typical MCPs .

 
Cluster II has previously been referred to as the -subgroup-like cluster of che genes (53) . However, genetic organization and physiological observations suggest that -proteobacteria and P . aeruginosa have different chemotaxis systems . None of the multiple clusters of chemotaxis genes identified in the genomes of the sequenced -proteobacteria R . sphaeroides, S . meliloti, and C . crescentus resembles P . aeruginosa cluster II in gene organization (7, 15, 36) . Furthermore, the proteins predicted to be encoded by cluster II genes, with the exception of CheY2, have the highest degree of sequence identity to E . coli rather than to -proteobacteria orthologs (Table 2) (56) . The best-described chemotaxis system in a bacterium belonging to the subgroup of proteobacteria is that of R . sphaeroides (50) . A model for R . sphaeroides chemotaxis has been proposed in which two chemotaxis-signaling complexes, one encoded by operon 1 and a second encoded by operon 2, contribute to an optimal chemotactic response (50) . In the absence of operon 2, a repellent response to the attractant propionate was seen (50) . An operon 1 deletion mutant had no obvious chemotaxis phenotype (50) . Significantly, motor bias phenotypes (in the case of R . sphaeroides, either smooth swimming or stopped) were seen only in mutants that had defects in both operon 1 and operon 2 (50) . P . aeruginosa differs from R . sphaeroides and other -proteobacteria in that a single set of chemotaxis genes clearly dominates the chemotactic response, as evidenced by the observation that cluster I mutants have profound effects on the rotational bias of the flagellar motor . This is likely to facilitate studies of the contribution of cluster II genes to the ability of P . aeruginosa to sense and respond to its environment .

One cannot, at this point, exclude the possibility that the major output from a cluster II signaling complex may be something other than a chemotactic response . Such a hypothetical output would likely be processed through CheY2 . Overexpression and complementation data offer no support for the idea that CheY2 interacts with the flagellar motor . Moreover, CheY2 did not complement a P . aeruginosa cheZ mutant . These data suggest that CheY2 may not act as a phosphate sink, as has been reported for R . sphaeroides and S . meliloti CheY proteins (53) .

The -proteobacteria Pseudomonas syringae pv . tomato, Shewanella oneidensis MR-1, and Vibrio cholerae each have a set of cluster II-like chemotaxis genes (Table 2 and Fig . 8) . Pseudomonas putida (The Institute for Genomic Research [TIGR] unfinished genomes BLAST search [http://tigrblast.tigr.org/ufmg/]) and Pseudomonas fluorescens (Pseudomonas fluorescens Genome Project [http://www.jgi.doe.gov/JGI_microbial/html/pseudomonas/pseudo_homepage.html]), species that are closely related to P . aeruginosa, do not have cluster II chemotaxis genes . They do, however, have sets of cluster I, III, IV, and V chemotaxis genes with high amino acid sequence identity (on the order of 70 to 80% identity) to the orthologous P . aeruginosa che genes . The function of a cluster II signal transduction pathway could be associated with physiological capabilities that cluster II bacteria have in common . However, it is not immediately clear what these commonalities may be . V . cholerae, S . oneidensis, and P . aeruginosa are able to grow anaerobically by denitrification . Yet P . syringae is an obligate aerobe and P . fluorescens, which lacks cluster II genes, can denitrify . P . syringae, V . cholerae, and P . aeruginosa are each either an animal or plant pathogen . S . oneidensis strain MR-1 (formerly Shewanella putrefaciens MR-1) is not known to be pathogenic, but some Shewanella species are opportunistic human pathogens (29) . This suggests that it might be worth exploring a possible connection between the activity of a cluster II signal transduction pathway and environmental stress conditions of the type that pathogens are known to experience .

We thank Steve Lory and Barbara Iglewski for providing P . aeruginosa strains . We also gratefully acknowledge Rebecca Parales for critical review of the manuscript .

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