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Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa

Chang Soo Hong , Maiko Shitashiro , Akio Kuroda , Tsukasa Ikeda , Noboru Takiguchi , Hisao Ohtake , Junichi Kato
DOI: http://dx.doi.org/10.1016/S0378-1097(04)00009-6 247-252 First published online: 1 February 2004

Abstract

It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis. In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified. Aerotaxis assays of a number of deletion–insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis. Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis. A double mutant deficient in tlpC and tlpG was negative for aerotaxis. TlpC has 45% amino acid identity with the Escherichia coli aerotactic transducer Aer. The TlpG protein has a predicted C-terminal segment with 89% identity to the highly conserved domain of the E. coli serine chemoreceptor Tsr. A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG. A PAS motif was found in the N-terminal domains of TlpC and TlpG. On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2.

Keywords
  • Aerotaxis
  • Pseudomonas aeruginosa
  • Chemotaxis
  • Behavioral response
  • Transducer

1 Introduction

Pseudomonas aeruginosa is an obligately aerobic bacterium and is capable of swimming by rotating a single polar flagellum. This organism inhabits a wide range of environments, from soil and water to the human host. It is an opportunistic pathogen that is among the most frequently isolated bacteria in nosocomial infections [1]. P. aeruginosa, like most other motile bacteria, has chemotactic responses to a wide range of chemical stimuli. P. aeruginosa is attracted to 20 commonly occurring l-amino acids [2,3], sugars [4], organic acids [5], and inorganic phosphate [6]. P. aeruginosa is repelled by thiocyanic and isothiocyanic esters [7] and volatile chlorinated aliphatic compounds such as trichloroethylene, tetrachloroethylene, trichloroethane, and chloroform [8]. Chemotaxis is a clear indicator of bacterial responses to changing environments. It can be viewed as an important prelude to metabolism, symbiosis, and other ecological interactions in microbial communities [9].

Aerotaxis is the movement of a cell towards or away from oxygen. Bacteria use aerotaxis to swim toward an optimal oxygen concentration for their metabolism. Aerotaxis has been extensively investigated in the facultative anaerobe Escherichia coli. In E. coli, an active respiratory chain and chemotaxis (Che) proteins such as CheA, CheY, and CheW are required and Aer and Tsr function as independent sensor/transducers for aerotaxis [1012]. Aer is a methyl-accepting chemotaxis protein (MCP)-like transducer which contains a PAS (an acronym of the Drosophila period clock protein [PER], vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and Drosophila single-minded protein [SIM]) motif in the N-terminal domain [13]). The PAS motif comprises a binding pocket for a prosthetic group [14] and Bibikov et al. [12] showed that Aer contained high levels of non-covalently associated flavin adenine dinucleotide (FAD). It is postulated that Aer uses FAD to monitor altered redox conditions in the cytoplasm. We recently demonstrated that P. aeruginosa also shows aerotaxis by using the chemotaxis well chamber method [8]. However, the mechanisms of aerotaxis in P. aeruginosa are still poorly understood.

Analysis of the complete genome sequence of P. aeruginosa PAO1 suggested that the P. aeruginosa chemosensory system is very complex, with more than 20 che genes situated in five distinct clusters and 26 mcp-like genes scattered throughout the genome [1517]. We demonstrated that cheY, cheZ, cheA, cheB, and cheW in Che cluster 1 (Fig. 1) and cheR in Che cluster 2 are responsible for chemotactic responses to amino acids and phosphate [18,19]. The pilGHIJ genes in Che cluster 3 are involved in twitching motility [20,21]. Of 26 mcp-like genes, only six genes (pctA, pctB, pctC, ctpH, ctpL, and pilJ) have been characterized to date [2,3,16,21]. The remaining 20 mcp-like genes and Che clusters 4 and 5 are as yet uncharacterized. In this study, we genetically analyzed aerotaxis in P. aeruginosa and found that P. aeruginosa possesses two chemoreceptors for aerotaxis, designated Aer and Aer-2. We also showed that Che cluster 1 and the cheR gene were required for aerotaxis.

1

Genetic organization of che clusters of P. aeruginosa PAO1. The locations and orientations of individual ORFs are shown by horizontal arrows. Gene ID numbers used in the P. aeruginosa genome sequencing project (http://www.pseudomonas.com/) are indicated below horizontal arrows. The locations of sequences deleted in deletion–insertion mutants (ΔCHE1, ΔpilHIJK, ΔCHE3, ΔCHE4, and ΔCHE5) are indicated by horizontal lines. Vertical arrows show the insertion sites of the kan and tet gene cassettes in TLPG1 and TLPC1, respectively.

2 Materials and methods

2.1 Bacterial strains and plasmids

The bacterial strains and plasmids used are listed in Table 1. E. coli MV1184 and HB101, which were used for plasmid construction and DNA manipulation, were grown at 37°C with shaking in 2×YT medium [22] supplemented with appropriate antibiotics. This medium was also used for the preparation of P. aeruginosa cells for aerotaxis assays and electroporation.

View this table:
1

Bacterial strains and plasmids used in this study

2.2 Aerotaxis assay

The chemotaxis well chamber method [8] was used to assess aerotaxis. P. aeruginosa strains were transformed by electroporation with plasmid pMRP9-1 [23] which contained the gfp gene under the control of the lac promoter. P. aeruginosa strains harboring pMRP9-1, grown overnight in 2×YT medium with appropriate antibiotics, were inoculated into fresh 2×YT medium (1% inoculum) and incubated at 37°C with shaking for 4 h. Cells were harvested by centrifugation in a 1.5-ml Eppendorf tube for 4 min at 4000×g at room temperature, washed twice with 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer (pH 7.2), and gently resuspended in 1 ml of the same buffer to a concentration of approximately 5×108 cells ml−1. A 1-ml clear acrylic well (Chemotaxicell, Kurabo, Okayama, Japan) was used as an upper well. The bottom of the upper well was sealed by an 8 mm diameter polycarbonate filter with a uniform pore size of 8 μm. The upper well was placed in a 3-ml well of a 24-well microtitration plate (Microplate, Iwaki, Japan). This 3-ml well was used as a lower well. For each assay, 1.5 ml of cell suspension was added to the lower well. The upper well was filled with 0.75 ml of HEPES buffer. The assay was started by inserting the upper well over the lower well allowing bacterial cells to cross the filter in response to an oxygen gradient. Bacterial cells that migrated to the upper well were automatically detected by measuring the green fluorescent protein (GFP) fluorescence intensity using a fluorescence spectrometer (ARVO 1420 Multilabel Counter, Wallac, Turku, Finland) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. All experimental measurements were performed at room temperature.

2.3 DNA manipulation and electroporation

Standard procedures were used for plasmid DNA preparations, restriction enzyme digestions, ligations, transformations, and agarose gel electrophoresis [22]. Polymerase chain reactions (PCR) were carried out using KOD plus DNA polymerase (Toyobo, Tokyo, Japan) according to the manufacturer's instructions. P. aeruginosa was transformed by electroporation as described previously [18].

2.4 Construction of mutant genes/clusters

Deletion–insertion mutations were constructed as detailed below. Genetic organization of Che clusters and the locations of deletion–insertion mutations in P. aeruginosa strains are shown in Fig. 1. Oligonucleotides used for PCR are listed in Table 2.

View this table:
2

Oligonucleotides used for PCR

2.4.1 Che cluster 1

A 2.0-kb ClaI-HpaI fragment containing fleN and fliA from pPT07.1 [19], a 1.3-kb HincII-flanked kan gene cassette (conferring kanamycin resistance [Kmr]) from pUC4K, and a 2.0-kb HincII-HindIII fragment including open reading frames (ORFs) PA1465, PA1466, and PA1467 from pPT07.1 were ligated with the backbone of ClaI/HindIII-digested pHSG396 to obtain pPT07.20.

2.4.2 Che cluster 2

The isolation of the cheR mutant PC4 has been described previously [19].

2.4.3 Che cluster 3

The deletion of the pilHIJK genes has been described previously [24]. A 0.7-kb DNA fragment containing an N- and C-terminally truncated copy of pilJ was amplified with the PCR primers pilJf and pilJr and the PCR product was cloned into pUC118 to make pPT07.21. A 0.9-kb DNA fragment containing the 3′ end of chpB amplified with the primers chpBf and chpBr was cloned into pUC118 to obtain pPT07.22. A 0.7-kb XbaI-EcoRI fragment from pPT07.21, a 1.3-kb EcoRI-flanked kan gene cassette from pUC4K, and a 0.9-kb EcoRI-SphI fragment from pPT07.22 were ligated with the backbone of the XbaI/SphI-digested pUC118 to obtain pPT07.23.

2.4.4 Che cluster 4

A 1.7-kb DNA fragment, which contained the entire tlpF gene and the 5′ end of cheY2, was amplified with the PCR primers tlpFf and cheY2r. The PCR product was cloned into pBluescript II KS+ to make pPT07.24. A 1.0-kb SalI-BamHI chromosomal DNA fragment containing the 3′ end of cheB2 was cloned into pUC118 to obtain pPT07.25. A 1.7-kb XbaI-XhoI fragment from pPT07.24, a 1.3-kb SalI-flanked kan cassette from pUC4K, and a 1.0-kb SalI-BamHI fragment from pPT07.25 were ligated with the backbone of the XbaI/BamHI-digested pUC118 to obtain pPT07.26.

2.4.5 Che cluster 5

A 1.1-kb DNA fragment containing the 3′ end of tlpM and the 5′ end of cheW3 was amplified with the primers tlpMf and cheW3r and the PCR product was cloned into pUC118 to make pPT07.27. A 1.4-kb DNA fragment containing the 3′ end of cheB3 and the 5′ end of cheY3 was amplified with the primers cheB3f and cheY3r and the PCR product was cloned into pUC118 to obtain pPT07.28. A 1.1-kb KpnI-HincII fragment from pPT07.27, a 1.3-kb HincII fragment containing kan, and a 1.3-kb EcoRV-BamHI fragment from pPT07.28 were ligated with the backbone of the KpnI/BamHI-digested pUC118 to obtain pPT07.29.

2.4.6 tlpC (aer)

PCR was used to generate a fragment containing tlpC. The PCR product was cloned into pUC118 to give pTLPC01. pTLPC01 was partially digested with PstI and ligated with a 1.3-kb PstI-flanked tet gene cassette (conferring tetracycline resistance [Tcr]) from pUC118Tc to obtain pTLPC01.1.

2.4.7 tlpG (aer-2)

A 2.1-kb SalI fragment containing tlpG was cloned into pUC118 to make pTLPG01. The tlpG gene was insertionally inactivated by cloning a 1.3-kb SalI flanked kan gene cassette into the XhoI site in the tlpG ORF on pTLPG01 to give pTLPG01.1.

The mutations were transferred into the chromosome of PAO1 by allelic exchange as described previously [19]. The deletion–insertions were confirmed by Southern hybridization with a digoxigenin non-radioactive DNA labeling and detection kit (Roche Diagnostics).

3 Results and discussion

3.1 Che cluster 1 and cheR are required for aerotaxis

The aerotactic responses of P. aeruginosa were assessed with the chemotaxis well chamber method [8]. When both the upper and lower wells contained HEPES buffer alone, P. aeruginosa PAO1(pMRP9-1) moved from the lower to the upper well through the filter, responding to the gradient of oxygen. After the gfp-tagged P. aeruginosa PAO1 cells were introduced into the lower well, the GFP fluorescence intensity in the upper well continuously increased (Fig. 2A). The gfp-tagged ΔpilHIJK (ΔpilH pilI pilJ pilK), ΔCHE3 (ΔpilJ pilK pilL chpA chpB), and ΔCHE5 (ΔcheW3 cheR3 cheW4 cheA3 cheB3) cells exhibited normal aerotactic responses, however, ΔCHE1 (ΔcheY cheZ cheA cheB motA2 motB2 cheW) and PC4 (cheR mutant) had an impaired ability to respond to oxygen. Microscopic analysis showed that ΔCHE1 and PC4 were fully motile. ΔCHE1 and PC4 were also defective in chemotaxis toward peptone and phosphate (data not shown). pPT07.1 (carrying the Che cluster 1) restored the ability of ΔCHE1 to respond to oxygen (Fig. 2A). These results suggest that the mutation phenotypes were not due to polar effects of the cassette insertion and the Che cluster 1 was required for aerotaxis in P. aeruginosa PAO1. The cheR gene was shown to be essential for aerotaxis, suggesting that aerotaxis is the MCP-dependent chemotaxis [25].

2

Aerotactic responses by wild-type and mutant strains of P. aeruginosa. The changes in the GFP fluorescence intensity of the upper well were measured by a fluorescence spectrometer. (A) (○), PAO1 (wild-type); ?, ΔCHE1 (ΔcheY cheZ cheA cheB motA2 motB2 cheW); △, ΔCHE3 (ΔpilJ pilK pilL chpA chpB); ◻, Δ pilHIJK (ΔpilH pilI pilJ pilK); ?, ΔCHE1(pPT07.1); ?, ΔCHE5 (ΔcheW3 cheR3 cheW4 cheA3 cheB3); ♦, PC4 (cheR mutant). (B) ?, ΔCHE4 (ΔcheY2 cheA2 cheW2 tlpG cheR2 cheB2); ◻, ΔCHE4(pTLPG01.2).

3.2 TlpG is a chemotactic transducer for aerotaxis

ΔCHE4 (ΔcheY2 cheA2 cheW2 tlpG cheR2 cheB2) showed decreased aerotaxis (Fig. 2B). ΔCHE4 was fully motile. In contrast to ΔCHE1, ΔCHE4 exhibited positive chemotactic responses to peptone and phosphate (data not shown) and the Che cluster 4 contains the mcp-like gene, tlpG. These findings suggest the possibility that TlpG is a chemotactic transducer for aerotaxis and the mutation phenotype of ΔCHE4 is due to the deletion of the tlpG gene. To investigate this possibility, pTLPG01.2 (containing tlpG) was introduced into ΔCHE4 and the transformant was examined for aerotaxis. The introduction of pTLPG01.2 restored the ability of ΔCHE4 to respond to oxygen (Fig. 2B). To confirm that TlpG is a chemotactic transducer for aerotaxis, we inactivated the tlpG gene by inserting a kan cassette into the wild-type gene in the PAO1 genome. The single tlpG mutant, designated TLPG1, showed decreased aerotaxis (Fig. 3A). pTLPG01.2 complemented the mutation of TLPG1, indicating the absence of polar effects. ΔCHE4(pTLPG01.2) and TLPG1(pTLPG01.2) showed stronger aerotactic responses than PAO1 (Figs. 2B and 3A). PAO1(pTLPG01.2) also showed stronger aerotaxis than PAO1 (Fig. 3A). These results indicate that the level of TlpG protein in the cell determines the strength of its positive aerotactic response.

3

Aerotactic responses by P. aeruginosa strains. (A) ○, PAO1; ?, TLGP1 (ΔtlpG); ◻, TLPG1(pTLPG01.2); △, PAO1(pTLPG01.2). (B) ▲, TLPC1 (ΔtlpC); ○, TLPC1(pTLPC01.2); △, PAO1(pTLPC01.2). (C) ?, TLPCG1 (ΔtlpC tlpG); △, TLPCG1(pTLPG01.2); ◻, TLPCG1(pTLPC01.2).

3.3 Identification of TlpC as a second aerotaxis transducer

Although tlpG-deficient cells showed decreased aerotactic responses, aerotaxis was not abolished in tlpG-deficient cells (Fig. 3A). This result suggests the existence of an additional transducer for aerotaxis. To identify an additional transducer for aerotaxis, a series of mutants that have deletion–insertion mutations in individual mcp-like genes in the PAO1 genome were constructed. PAO1 possesses 26 mcp-like genes [1517]. We amplified 25 mcp-like genes other than tlpG by PCR using the sequence-specific primers, cloned into the vector plasmid pUC118, and disrupted individual genes by inserting a tet cassette into the wild-type genes in the PAO1 genome. After the introduction of pMRP9-1, each mutant was tested for aerotaxis. Aerotaxis assays revealed that the tlpC (PA1561) mutant TLPC1 showed decreased aerotaxis (Fig. 3B). pTLPC01.2 carrying the tlpC gene complemented the mutation of TLPC1. TlpC overproduction also increased the aerotactic response (PAO1[pTLPC01.2], Fig. 3B). We further constructed the double mutant TLPCG1 by inserting a tet cassette into the wild-type tlpC gene in the TLPG1 genome. Aerotaxis was abolished in the tlpC tlpG double mutant TLPCG1 (Fig. 3C). Aerotaxis was partially restored in the tlpC tlpG double mutant when TlpC or TlpG was expressed from a plasmid. These results suggest that P. aeruginosa PAO1 possesses two aerotaxis transducers, TlpC and TlpG.

3.4 Sequence analysis of the tlpC and tlpG genes

The potential product of tlpC was 521-amino acid TlpC. The tlpC gene product is 78 and 45%, respectively, identical to the Pseudomonas putida and E. coli Aer proteins, transducers involved in aerotaxis [11,12,26]. The N-terminal domain of the tlpC gene product (residues 21–106) contains a PAS motif, which is known to comprise a binding pocket for a prosthetic group [14]. Only one hydrophobic sequence (residues 146–191) was predicted in the TlpC protein. This hydrophobic sequence may serve to anchor TlpC to the cytoplasmic side of the inner membrane. TlpC residues 354–397 are 84% identical to the 44-amino acid highly conserved domain (HCD) of the E. coli chemotaxis transducer Tsr. MCPs from phylogenetically diverse bacteria have been shown to possess the HCD [27], which is likely to be important for the interaction between MCPs and CheW as well as CheA [28]. Thus the TlpC protein closely resembled the P. putida and E. coli Aer proteins and it was experimentally demonstrated that the tlpC gene is involved in aerotaxis. On this basis, the tlpC gene was renamed aer.

The putative product of the tlpG gene was a 679-residue protein (72.6 kDa) that had a predicted C-terminal segment (residues 481–524) with 89% identity to the HCD of the E. coli Tsr protein. In the C-terminal domain of TlpG, there are two potential methylation regions (residues 412–434 and 600–624) that have 87 and 72% identity to the K1 and R1 regions of E. coli Tsr [29], respectively. Thus, the C-terminal domain of TlpG has typical structural features of MCPs. A hydropathy plot of TlpG indicated that two hydrophobic membrane-spanning regions in the N-terminal domain of typical MCPs are missing in TlpG. TlpG may be a cytoplasmic protein and sense intracellular stimuli. Reverse position-specific Blast search [30] against the database of the National Center for Biotechnology Information predicted the presence of a PAS motif in the N-terminal domain of TlpG (residues 174–216). TlpC and TlpG had 18% identity in the PAS domain. Since genetic analysis demonstrated the involvement of the tlpG gene in aerotaxis, tlpG was renamed aer-2. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2.

Acknowledgements

We thank Matthew R. Parsek for providing the pMRP9-1 plasmid containing the gfp gene. This work was supported by the Showa Shell Sekiyu Foundation for Promotion of Environmental Research and the Kurita Research Foundation.

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