INTRODUCTION

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Pseudomonas aeruginosa PAO1 is attracted to inorganic phosphate (P_i) (10). P_i taxis is induced when the cells are starved for P_i. Although the chemoreceptor for P_i has never been identified, its specificity for P_i appears to be relatively high. No other phosphorus compounds have been shown to elicit responses similar to those for P_i (10). P_i-starved cells are also attracted to arsenate (AsO₄³ (11). Since P_i competitively inhibits the response to AsO₄³, both P_i and AsO₄³ are likely detected by the same chemoreceptors. The enteric bacterium Enterobacter cloacae, but not Escherichia coli and Salmonella enterica serovar Typhimurium, also exhibits P_i taxis under conditions of P_i limitation (11). Experimental evidence shows that the E. cloacae genes encoding the P_i-specific transport (Pst) system and the PhoU protein are required for P_i taxis.

Previously, we showed that P. aeruginosa mutants PHOB1 (phoB::kan) and PHOR1 (phoR::kan) were not induced by P_i limitation for alkaline phosphatase synthesis but exhibited P_i taxis under conditions of P_i limitation (12). Interestingly, the P. aeruginosa phoU mutant, which was constructed by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis, showed P_i taxis regardless of whether the cells were starved for P_i (constitutive P_i taxis) (12). We also found that P. aeruginosa mutants lacking the Pst complex were constitutive for P_i taxis (17). Based on these results, it has been suggested that the Pst complex, interacting with the PhoU protein, exerts a negative control on P_i taxis, even though it is not positively regulated by the PhoB and PhoR proteins (12). In the present study, we found that P. aeruginosa possesses two chemoreceptors for P_i, designated CtpH and CtpL. CtpH was required for exhibiting P_i taxis at high concentrations of P_i, while CtpL could serve as the major chemoreceptor for P_i at low concentrations. Expression of the gene coding for CtpL (ctpL) was induced by P_i limitation, depending on the PhoB and PhoU proteins. In contrast, the gene coding for CtpH (ctpH) was expressed constitutively.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. E. coli MV1184 and HB101 were used for plasmid construction and DNA manipulation. P. aeruginosa and E. coli were grown at 37°C with shaking in 2× YT medium (19) supplemented with appropriate antibiotics. P. aeruginosa was also grown at 37°C with shaking in T₅ minimal medium (7) containing 5 mM P_i. For P_i limitation, P. aeruginosa cells grown overnight in 2× YT medium were inoculated (a 2.5% inoculum) into T₀ medium, which was prepared by omitting P_i from T₅ medium, and incubated at 37°C with shaking.

Chemotaxis assays. The computer-assisted capillary assays were carried out as described previously (16). Cells were videotaped over the first 3 min. Digital image processing was used to count the number of bacteria accumulating toward the mouth of a capillary containing a known concentration of an attractant plus 1% agarose. The chemotaxis buffer used was T₀ medium supplemented with 2.78 mM glucose. All chemicals used for chemotaxis assays were reagent grade.

DNA manipulation. Standard procedures were used for plasmid DNA manipulations and agarose gel electrophoresis (19). P. aeruginosa chromosomal DNA was prepared as described previously (12). P. aeruginosa was transformed by electroporation (12). TaKaRa Ex Taq DNA polymerase (Takara Shuzo, Shiga, Japan) was used for PCR. The search for DNA sequences corresponding to the highly conserved domain (HCD) of chemotactic transducers was done with the P. aeruginosa genome sequence at the Pseudomonas genome project website (http://www.pseudomonas.com) by using the program TBLASTIN (1). Alignment of amino acid sequences was performed with the program FASTA (18).

Cloning of the ctpH and ctpL genes. The ctpH and ctpL genes were cloned from the PAO1 genome by using PCR. A 3.6-kb DNA fragment, which contained the entire ctpH gene, was amplified with the PCR primers TR1 (5'-TCTGTTTCAGCGTCTGTAGCATCG) and TR2 (5'-ACATCGGTACCAATAGCGAAGTCG). The PCR product was cloned into pGEM-T Easy (Promega) to make pPT10.1. Similarly, a 3.3-kb DNA fragment, which contained the entire ctpL gene, was amplified with the PCR primers TR3 (5'-TCGACGATGTTGTAGTAGACCTCG) and TR4 (5'-GATCATCCTCGACATGTACATGCC). This PCR product was also cloned into pGEM-T Easy to make pPT11.1. For complementation experiments, the 3.6- and 3.3-kb DNA fragments were also cloned into pCP19 (6) to make pPT10.4 and pPT11.4, respectively.

Construction of deletion-insertion mutants. Deletion-insertion mutants were constructed by the direct gene replacement technique (12). Plasmid pPT10.1 was digested by XhoI and ligated to a 1.3-kb SalI fragment containing a kan (conferring kanamycin resistance [Km^r]) cassette from pUC4K (Pharmacia) to make pPT10.2. Similarly, pPT11.1 was digested by EcoRI and ligated to a 1.3-kb EcoRI fragment containing a kan cassette from pUC4K to make pPT11.2. Plasmids pPT10.2 and pPT11.2 were individually introduced into PAO1 by electroporation, and Km^r transformants were selected on 2× YT plates containing 1 mg of kanamycin per ml. The resulting ctpH and ctpL single mutants were designated PP1 and PP2, respectively. To construct the ctpH ctpL double mutant, pPT10.1 was digested by XhoI and ligated to a 1.3-kb XhoI fragment containing a tet (conferring tetracycline resistance [Tet^r]) cassette from pUC118Tc. The resulting plasmid, designated pPT10.3, was then introduced into PP2 by electroporation, and Km^r Tc^r transformants were selected on 2× YT plates containing kanamycin (1 mg/ml) and tetracycline (100 µg/ml). The ctpH ctpL double mutant was designated PP3. The deletion-insertions were confirmed by Southern hybridization with a digoxigenin nonradioactive DNA labeling and detection kit (Boehringer Mannheim).

Transcriptional fusion experiments. Transcriptional fusion vectors pKZ27 and pKZ27.1 were derivatives of pKTK40 (9). They contained a multicloning site upstream of lacZ. They differed from each other only in that pKZ27 contained a kan marker, while pKZ27.1 had a carbenicillin resistance (Cb^r) marker. Plasmid pPT10.1 was digested with KpnI, and a 1.8-kb KpnI fragment was cloned in front of the promoterless lacZ gene of pKZ27 and pKZ27.1 to make pPT10.5 and pPT10.6, respectively. Similarly, pPT11.1 was digested with EcoRV and SalI, and a 1.6-kb EcoRV-SalI fragment was inserted upstream of the promoterless lacZ gene of pKZ27 and pKZ27.1 to make pPT11.5 and pPT11.6, respectively. -Galactosidase activities of P. aeruginosa cells were determined as described by Miller (15), with the modification that the enzymatic reaction was carried out at 28°C.

Nucleotide accession numbers. The sequences of the P. aeruginosa PAO1 ctpH and ctpL genes are deposited in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases under accession numbers AB039333 and AB039332, respectively.

	RESULTS AND DISCUSSION

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Cloning of the ctpH and ctpL genes. We initially identified the ctpH and ctpL genes as open reading frames (ORFs) discovered in the P. aeruginosa genome sequencing project. The methyl-accepting chemotaxis proteins (MCPs) from phylogenetically diverse bacteria have been shown to possess the HCD which is likely to be important for intracellular chemotactic signaling (21). Based on the conserved amino acid sequence (IADQTNILALNAAIEAARAGDQGRGFAVVADEVR KLA),computer analysis of the PAO1 genome sequence predicted that PAO1 possesses 26 ORFs which likely encode proteins containing the HCD (Table 2). Among them were the known genes such as pctA (13), pctB, pctC (22), and pilJ (5). Thirteen randomly chosen ORFs were individually amplified by PCR using the sequence-specific primers and cloned into the vector plasmid pGEM-T Easy (Promega). Individual genes were then disrupted by inserting a kan cassette into the wild-type genes in the PAO1 genome, and Km^r mutants and PAO1 were examined for the ability to exhibit P_i taxis by using the computer-assisted capillary assay technique (16).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Chemotactic responses to Pi by Pi-starved (A) and Pi-sufficient (B) cells of P. aeruginosa wild-type strain PAO1 and Pi-starved cells of ctpH single mutant PP1 (C), ctpL single mutant PP2 (D), and ctpH ctpL double mutant PP3 (E). Digital image processing was used to count the number of bacteria accumulating around the mouth of the capillary containing a known concentration of Pi plus 1% agarose. One videotape frame was analyzed at each time point. The chemotactic response is presented at the number of bacteria per videotape frame as described previously (16). Pi concentrations (millimolar) in the capillary: , 0.01; , 0.1; , 5; , 10.

Effects of the phoU and phoB mutations on P_i taxis. We previously showed that chromosomal phoU mutant APC1, which had been selected after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, was constitutive for P_i taxis (12). To further investigate the effect of phoU on P_i taxis, we inactivated the ctpH and ctpL genes individually by inserting a kan cassette into the wild-type genes in the APC1 genome. The ctpH phoU and ctpL phoU double mutants were designated PP4 and PP5, respectively. The computer-assisted capillary assays revealed that PP4 failed to exhibit P_i taxis at any concentration ranging from 0.01 to 10 mM even under conditions of P_i limitation, whereas PP5, like APC1, was constitutive for P_i taxis at P_i concentrations higher than 5 mM (Fig. 2). As expected, the ctpL phoU double mutant PP5 was unable to respond to P_i at 0.01 mM. We also previously showed that a chromosomal phoB mutant PHOB1 (phoB::kan) exhibited chemotactic responses toward 10 mM P_i under conditions of P_i limitation (12). Interestingly, it was now found that the phoB single mutant could not respond to 0.01 mM P_i (Fig. 3). To further investigate the effect of phoB on P_i taxis, we also disrupted the ctpH and ctpL genes individually in the genome of PHOB1 by insertional mutagenesis. The ctpH phoB double mutant, designated PP6, did not show P_i taxis at any concentration ranging from 0.01 to 10 mM, even when the cells were starved for P_i limitation (Fig. 3). Like PHOB1, the ctpL phoB double mutant, designated PP7, exhibited strong chemotactic responses toward P_i at concentrations higher than 5 mM when the cells were starved for P_i. However, both PHOB1 and PP7 failed to exhibit P_i taxis under conditions of P_i excess.

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Chemotactic responses to Pi by P. aeruginosa phoU single mutant APC1 (A), ctpH phoU double mutant PP4 (B), and ctpL phoU double mutant PP5 (C). P. aeruginosa cells were grown with either Pi excess (dotted lines) or Pi limitation (solid lines). Pi concentrations (millimolar) in the capillary: , 0.01; , 0.1; , 10.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Chemotactic responses to Pi by Pi-starved (A) and Pi-sufficient (B) cells of P. aeruginosa phoB single mutant PHOB1, Pi-starved cells of ctpH phoB double mutant PP6 (C), and Pi-starved (D) and Pi-sufficient (E) cells of ctpL phoB double mutant PP7. Pi concentrations (millimolar) in the capillary: , 0.01; , 0.1; , 5; , 10.

Expression of ctpH::lacZ and ctpL::lacZ. The nucleotide sequences upstream of ctpH and ctpL were scanned for the presence of a pho box, the consensus sequence shared by the pho promoters, using the consensus sequence published previously (20). A putative pho box was found in the sequence upstream of ctpL (data not shown). There was a 13/18-bp match with the consensus pho box sequence (21). On the other hand, no pho box sequence was found with the promoter region of ctpH. To investigate the ctpH and ctpL promoter activities, the promoter regions of ctpH and ctpL were inserted individually upstream from the promoterless lacZ gene in transcriptional fusion vectors pKZ27 and pKZ27.1. The control strain PAO1 and phoU single mutant APC1 were transformed with either pPT10.5 (Km^r; carrying ctpH::lacZ) or pPT11.5 (Km^r; carrying ctpL::lacZ). Since the phoB single mutant PHOB1 had a Km^r marker, this strain was transformed by either pPT10.6 (Cb^r; carrying ctpH::lacZ) or pPT11.6 (Cb^r; carrying ctpL::lacZ). -Galactosidase activities were then measured in the wild-type and transformant strains of P. aeruginosa under conditions of P_i excess and P_i limitation (Table 3).

View this table: [in this window] [in a new window]   TABLE 3.   -Galactosidase activities in P. aeruginosa strains

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Model for Pi taxis in P. aeruginosa. P. aeruginosa possesses two Pi chemoreceptors, CtpH and CtpL. CtpH is required for exhibiting Pi taxis at high concentrations of Pi, while CtpL serves as the major chemoreceptor for Pi at low concentrations. The Pst complex, together with PhoU, is likely to exert a negative control on Pi detection by CtpH at high concentrations of Pi at the posttranscriptional level. In contrast, these proteins are required for Pi detection by CtpL at low concentrations of Pi. A putative pho box sequence exists in the promoter region of ctpL. The two-component regulatory proteins, PhoR and PhoB, activate its transcription under conditions of Pi limitation. The Pst complex, together with PhoU, also causes the repression of CtpL synthesis under conditions of Pi excess. A plus sign signifies gene activation, while a minus sign means inhibition or repression.