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Pseudomonas aeruginosa as a model microorganism for investigation of chemotactic behaviors in ecosystem
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 106, No. 1, 1–7. 2008 DOI: 10.1263/jbb.106.1
© 2008, The Society for Biotechnology, Japan
REVIEW Pseudomonas aeruginosa as a Model Microorganism for Investigation of Chemotactic Behaviors in Ecosystem Junichi Kato,1* Hye-Eun Kim,1 Noboru Takiguchi,1 Akio Kuroda,1 and Hisao Ohtake2 Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan1 and Department of Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan2 Received 18 February 2008/Accepted 14 April 2008
Motile bacteria sense changes in the concentration of chemicals in environments and respond in a behavioral manner. This behavioral response is called chemotaxis. Bacterial chemotaxis can be viewed as an important prelude to metabolism, prey–predator relationships, symbiosis, infections, and other ecological interactions in biological communities. Genome analysis reveals that a large number of environmental motile bacteria possess a number of genes involved in chemosensing and chemotatic signal transduction. Pseudomonas aeruginosa has a very complex chemosensory system with more than 20 chemotaxis (che) genes in five distinct clusters and 26 chemoreceptor (methyl-accepting chemotaxis protein [mcp]) genes. Among the 26 MCPs of P. aeruginosa, nine have been identified as MCPs for amino acids, inorganic phosphate, oxygen, ethylene, and volatile chlorinated aliphatic hydrocarbons, whereas 3 MCPs were demonstrated to be involved in biofilm formation and biosynthesis of type IV pilus. Six che genes are essential for chemotactic responses, while genes in Pil-Chp cluster and Wsp cluster are involved in type IV pilus synthesis and twitching motility and biofilm formation, respectively. P. aeruginosa, with its complex chemotaxis system, is a better model microorganism for investigating ecological aspects of chemotaxis in environmental bacteria than Escherichia coli and Salmonella enterica serovar Typhimurium, which possess a relatively simpler chemotaxis system. [Key words: chemotaxis, ecological interactions, Pseudomonas aeruginosa, chemosensing]
The molecular mechanisms that underlie bacterial chemotaxis have been studied extensively in enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium (10). Chemotactic ligands are detected by cell surface chemoreceptors called methyl-accepting chemotaxis proteins (MCPs) (Fig. 1). Upon binding a chemotactic ligand, MCPs generate chemotactic signals that are communicated to the flagellar motor via a series of chemotaxis (Che) proteins. E. coli possesses 6 Che proteins and 5 MCPs. Of these, CheA, a histidine protein kinase autophosphorylates at a specific histidine residue to form CheA-P. The phosphoryl group of CheA-P is transferred to a specific aspartate residue of CheY, which is a response regulator of a two-component regulatory system, to form activated CheY-P that interacts directly with the flagellar motor switch protein to control the direction of flagellar rotation. CheZ is involved in inactivation of CheY-P by dephosphorylation of CheY-P to CheY. MCPs, with help from CheW, modulate the autophosphorylation activity of CheA in response to temporal changes in stimuli intensity. MCPs undergo reversible methylation at several glutamate residues. CheR and CheB are methyltransferase and methyl-
Motile bacteria sense changes in the concentration of chemicals in environments and exhibit a behavioral response (1). This behavioral response is called chemotaxis. It is suggested that most microorganisms in heterogeneous environments are motile (2). The fact that a large number of genes are involved in motility and chemotaxis (3), and that environmental bacteria have retained motility and chemotaxis under natural selection for long periods of time indicate that motility and chemotaxis provide a selective advantage for motile bacteria in natural environments. Bacterial chemotaxis can be viewed as an important prelude to metabolism, prey–predator relationships, symbiosis, infections, and other ecological interactions in biological communities (4). It has been reported that chemotaxis has important roles in colonization of plant roots by plant growth-promoting Pseudomonas fluorescens (5, 6), infections of plants by Pseudomonas syringae and Ralstonia solanacearum (7, 8), and invasive infections of animals by Pseudomonas aeruginosa (9). * Corresponding author. e-mail: [email protected] phone: +81-(0)82-424-7757 fax: +81-(0)82-424-7047 1
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research on chemotaxis and che and mcp genes in P. aeruginosa. CHEMOTACTIC RESPONSES BY P. AERUGINOSA TO VARIOUS CHEMICAL COMPOUNDS AND THEIR MOLECULAR MECHANISMS
FIG. 1. Interactions of MCP, Che proteins, and the flagellar motor in E. coli (A) and the structure of typical MCP (B). HCD denotes highly conserved domain.
esterase enzymes, respectively, responsible for methylation modification of MCPs. CheB is another response regulator, which is phosphorylated by CheA-P to form CheB-P. CheB-P exhibits higher methylesterase activity than CheB and is not affected by environmental stimuli. CheR continually adds methyl groups to MCPs. The methylation level of MCPs is controlled in response to environmental stimuli and affects their conformation. This reversible methylation of MCPs is required for temporal sensing of chemical gradients. The best characterized of the chemotactic systems in environmental bacteria is that of P. aeruginosa PAO1. P. aeruginosa is an obligately aerobic bacterium and is capable of locomotion by rotating a single polar flagellum. It inhabits a wide range of environmental niches, including soil, water, and human hosts. Sequence analysis of the P. aeruginosa PAO1 genome suggested that the P. aeruginosa chemotaxis system is very complex, with more than 20 che genes in five distinct clusters and 26 mcp-like genes scattered throughout the genome (Fig. 2) (11–13). Whole genome sequences of several environmental motile bacteria have been determined. Sequence analysis reveals that several environmental bacteria possess a number of che and MCP genes, like P. aeruginosa. Therefore, P. aeruginosa is thought to be a better model microorganism for investigation of ecological aspects of chemotaxis in environmental bacteria than E. coli, which has a relatively simple chemotactic system. In this paper, we provide an overview of molecular biological
MCPs are major chemosensory proteins in bacterial chemotaxis. They are membrane-spanning homo dimers and exhibit typical structural features (14) (Fig. 1) including a positively charged N terminus followed by a hydrophobic membrane-spanning region, a hydrophilic periplasmic domain, a second hydrophobic membrane-spanning region, and a hydrophilic cytoplasmic domain. Periplasmic domains contain variable sequences to which chemotactic ligands bind. In contrast, cytoplasmic domains are relatively conserved. Particularly, a 44-amino acid domain between two reversible methylation sites is highly conserved. MCPs from phylogenetically diverse bacteria have been shown to possess this highly conserved domain (HCD) (15), which is involved in the interaction between MCPs and CheW as well as CheA (16). Putative mcp genes are easily found within genomic sequences by similarity search using HCD as a query sequence. However, a limited number of MCPs have been characterized due to difficulty in identification of their cognate ligands. In P. aeruginosa, 12 of 26 MCPs have been characterized (17–22). Here, we describe chemotactic responses by P. aeruginosa PAO1 of which MCPs have been identified and characterized. Amino acids P. aeruginosa is attracted by twenty commonly occurring amino acids (17). Kuroda et al. isolated a P. aeruginosa mutant defective in chemotaxis toward L-serine but responsive to peptone from N-methyl-N′-nitro-N-nitrosoguanidine-mutagenized cells and cloned the pctA gene (AAG07697 [protein ID in the DDBJ/EMBL/NCBI databases]) encoding a MCP for L-serine by phenotypic complementation of the mutant. DNA sequence analysis of regions upstream and downstream of pctA showed two mcp genes (pctB [AAG07698] and pctC [AAG07695]) that were highly homologous to pctA (18). Chemotaxis assays of pctA pctB pctC triple mutant containing each one of these MCP genes
FIG. 2. Putative mcp genes and Che gene clusters on the genome of P. aeruginosa PAO1 (A) and genetic organization of Che gene clusters (B). (A) The putative mcp genes are shown outside of the circle. The mcp genes which have been characterized are underlined. (B) A, B, R. W, and Y in parentheses denote homologues of cheA, cheB, cheR, cheW, and cheY, respectively.
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FIG. 3. Amino acids detected by PctA, PctB, and PctC of P. aeruginosa PAO1.
revealed that PctA, PctB, and PctC detected 18 amino acids, 6 amino acids, and 2 amino acids, respectively (Fig. 3). Blast search revealed that Pseudomonas species, Sinorhizobium meliloti, Agrobacterium tumefaciens, and Chromobacterium violaceum also possessed 1 to 3 PctA homologues. Many of these bacteria are known to interact with plants, and amino acids are one of the major constituents in plant exudates. Therefore, it would be interesting to investigate the involvement of PctA homologues in interaction between these bacteria and plants. Inorganic phosphate Phosphorus compounds are essential constituents in organisms. In nature, inorganic phosphate (Pi) is often found to be a growth-limiting factor for organisms. Therefore, it is not surprising that motile bacteria show chemotactic responses to Pi. Such responses were first reported in 1992 (23), even though the first reports on bacterial chemotaxis emerged at the end of the 19th century (24, 25). This is because researchers most commonly analyzed chemotactic responses by Adler’s capillary method (26) where Pi buffer is recommended as the cell suspension buffer, and E. coli does not have the ability to respond chemotactically to Pi (27). Pi-starved cells of P. aeruginosa are attracted to Pi, whereas no positive response to Pi is observed with P. aeruginosa cells grown under Pi-sufficient conditions (23). A series of mutants that have deletion-insertion mutations in individual mcp-like genes in the P. aeruginosa PAO1 genome were constructed to identify the MCP for Pi. Chemotaxis assays of the mcp mutant library identified CtpH (AAG05949) and CtpL (AAG08229) as MCPs for Pi (12). CtpH and CtpL are functional at different Pi concentrations. CtpH serves as the major chemoreceptor for Pi at high concentrations, while CtpL is required for exhibiting Pi chemotaxis at low Pi concentrations (Fig. 4). Pho regulon is known to be the major regulatory system responding to Pi limitation in many bacteria (28) including P. aeruginosa. It is a two-component regulatory system comprising His protein kinase PhoR and response regulator PhoB (29). It increases autophosphorylation activity of PhoR; the phosphoryl group of PhoR-P is transferred to PhoB. PhoB-P activates transcription of the Pho regulon genes upon binding to a specific sequence called Pho box in their promoter regions. Sufficient Pi stimulates dephosphorylation
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FIG. 4. Model for Pi taxis in P. aeruginosa PAO1. CtpH and CtpL are MCPs responsible for detection of high concentrations and low concentrations of Pi, respectively. Pi chemotaxis in P. aeruginosa is induced by Pi limitation. PhoB and PhoR, which are a response regulator and a sensor kinase of Pho regulon, respectively, positively control the transcription of ctpL, while Pi-specific transport (Pst) system and PhoU are involved in the negative regulation of ctpL transcription. Pst system and PhoU are essential for Pi detection by CtpL. Pst-PhoU complex negatively regulates CtpH expression at the posttranscriptional level.
of PhoB-P, likely by PhoR (28, 30, 31), forming unphosphorylated or inactive form of PhoB. Phosphate-specific transport (Pst) system is required for negative regulation of Pho regulon (32–34). Pst system is an ATP-binding cassette (ABC) membrane transporter that transports Pi into the cell. It is composed of four proteins (PstS, PstC, PstA, and PstB) encoded by the pstSCAB-phoU operon (34). The PhoU protein is predicted to be a cytoplasmic protein and is involved in negative control of Pho regulon (33). Although it is speculated that Pst system and PhoU negatively regulate Pho regulon through stimulating phosphatase activity of PhoR (28, 29), its molecular mechanism has not been elucidated yet. The ctpL gene belongs to Pho regulon and its inducible transcription is dependent on PhoB/PhoR, while transcription of ctpH is not dependent on PhoB/PhoR and ctpH is constitutively transcribed (12). The ctpL gene is constitutively transcribed in pst and phoU mutants of P. aeruginosa, but these mutant strains fail to show chemotactic responses to low concentrations of Pi, suggesting that the Pst complex and PhoU are required for Pi detection by CtpL. Mutations of pst and phoU do not affect transcription of ctpH. However, CtpH-dependent Pi chemotaxis is constitutive in pst and phoU mutants. Thus, the Pst complex and PhoU are likely to exert a negative control on CtpH expression at the posttranscriptional level. Blast search reveals that Pseudomonas species, including P. putida, P. entomophila, P. stutzeri, P. fluorescens, P. mendocina, and P. syringae, have homologues of ctpH and ctpL. Among them, P. putida was experimentally demonstrated to exhibit Pi taxis (35). It was reported that Enterobacter cloacae is also attracted by Pi and that the Pst system is essential for Pi taxis, although a MCP for Pi has not been identified (36). Thus, Pi chemotaxis seems to be a prevalent trait in environmental motile bacteria. Aerotaxis Aerotaxis is the movement of a cell toward
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or away from oxygen. P. aeruginosa is an aerobic bacterium. Therefore, it is not surprising that this bacterium shows positive aerotaxis. P. aeruginosa possesses two MCPs for aerotaxis, Aer (AAG04950) and Aer-2 (AAG03566) (19). P. aeruginosa Aer is 45% identical to E. coli Aer (37, 38), and has only one hydrophobic sequence, which may serve to anchor Aer to the cytoplasmic side of the inner membrane. We observed that the Aer-GFP fusion protein mainly localized to the cell pole in P. aeruginosa (Kato and Hong, unpublished data). The N-terminal domain of Aer 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, which is known to comprise a binding pocket for a prosthetic group. It was shown that E. coli Aer contains high levels of non-covalently associated flavin adenine dinucleotide (FAD) (37, 39). It is postulated that E. coli Aer uses FAD to monitor altered redox conditions in the cytoplasm. Aer-2 lacks transmembrane domains and it is thought to be a soluble MCP. However, Güvener et al. demonstrated that an Aer-2-YFP fusion protein localized to the cell pole (40). Aer-2 may associate with the inner membrane via other membrane bound proteins. Aer-2 also contains a PAS motif in the N-terminal region, suggesting this MCP may monitor changes of redox potential via the PAS motif. Expressions of aer and aer-2 are controlled by different regulators. The aer promoter contains two FNR/ANR boxes, to which the anaerobic regulator ANR binds; transcription of aer is induced under oxygen-limited conditions by ANR (41). The cheY2, cheA2, cheW2, and aer-2 genes are expressed as a single transcript (42, 43). This transcription is dependent on alternative sigma factor RpoS and is induced in the stationary phase. Chloroethylenes Chloroethylenes, including trichloroethylene (TCE), tetrachloroethylene (PCE), and dichloroethylene (DCE), are xenobiotics and the most frequently detected groundwater contaminants (44). P. aeruginosa is repelled by chloroethylenes (45). This reaction is thought to occur because of toxic compounds. Screening of the mcp mutant library revealed that MCPs for amino acids, PctA, PctB, and PctC, are chemosensory proteins of negative chemotaxis to chloroethylenes (20). Among the three MCPs, PctA is the major MCP for chloroethylenes. TCE-degrading bacterium P. putida F1 exhibits chemotactic responses to TCE. In contrast to P. aeruginosa, P. putida F1 is attracted by TCE (46). The response by P. putida F1 to TCE is induced by toluene in the presence of TodS and TodT, which are regulatory proteins of toluene dioxygenase operon. Chemotactic responses by P. aeruginosa to chloroethylenes are complex. Kim et al. investigated chemotactic responses by the pctA pctB pctC triple mutant of P. aeruginosa to TCE (21). As expected, the mutant showed weaker repelled responses to high concentrations of TCE than the wild-type strain. However, unexpectedly, the mutant was attracted by low concentrations of TCE (Fig. 5). This result suggests that P. aeruginosa has the ability to both negatively and positively respond to TCE and that the positive responses are detected only after disruption of the pctABC genes encoding the MCPs for negative chemotaxis to TCE. CttP
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FIG. 5. Chemotactic responses of P. aeruginosa and P. putida strains to TCE in agarose plug assays. Photographs were taken 10 min after addition of bacterial suspensions to solidified agarose plugs containing 1 mM and 3.8 mM TCE. (A) Responses of the wild-type P. aeruginosa PAO1 and the pctA pctB pctC triple mutant of PAO1. (B) Responses of the wild-type P. putida F1 and the F1 transformant harboring the P. aeruginosa cttP gene. P. putida strains were grown in the absence of toluene.
(AAG03570) was identified as an MCP for positive chemotaxis to TCE through screening of the mcp mutant library. CttP detects PCE and three isomers of DCE as well as TCE. P. aeruginosa cannot utilize chloroethylenes as carbon and energy sources. Although P. putida F1 is capable of degrading TCE in the presence of toluene, it cannot use TCE as a growth substrate. These findings lead one to a question as to why these bacteria possess MCPs for positive chemotaxis to chloroethylenes. Parales et al. speculated that chemotaxis towards chloroethylenes, which are not growth substrates, is a fortuitous consequence of a broad-substrate-specificity chemoreceptor (46). P. putida F1 harboring the P. aeruginosa cttP gene showed positive chemotaxis to TCE even when the cells were grown in the absence of toluene (Fig. 5). The migration of environmental pollutant-degrading bacteria toward environmental pollutants might speed up the biodegradation process because it would bring the cells into contact with environmental pollutants (47). The cttP gene may be used for improvement of the ability of TCE-degrading bacteria to migrate to TCE. Blast search did not find cttP homologues in the bacterial genomes other than those of P. aeruginosa strains. Ethylene Based on the chemotactic responses by P. aeruginosa and P. putida to chlorinated ethylenes, questions were raised about the ability of environmental motile bacteria to respond to ethylene. Ethylene is one of plant hormones and is essentially produced by all plant tissues. Therefore, chemotaxis to ethylene may provide an advantage for initiation of microbe-plant interactions. Kim et al. found that P. aeruginosa was attracted by ethylene and that CttP was not involved in ethylene chemotaxis (22). They identi-
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fied TlpQ (AAG06402) as a MCP for ethylene. Plant-growth promoting bacteria P. fluorescens and P. putida and plant pathogen P. syringae contain tlpQ homologues in their genomes. Chemotaxis assays revealed that these Pseudomonas strains are also attracted by ethylene. Further studies may elucidate a role of ethylene chemotaxis in microbe-plant interactions. FUNCTIONAL ANALYSIS OF che GENE CLUSTERS IN P. AERUGINOSA P. aeruginosa possesses more than 20 che genes in 5 gene clusters (Che, Che2, Pil-Chp, Wsp, and cheVR clusters) in its genome (Fig. 2). Genetic analysis revealed that each of 5 che genes (cheY [AAG04845], cheZ [AAG04846], cheA [AAG04847], cheB [AAG04848], and cheW [AAG04853]) in the Che cluster and cheR (AAG06736) are essential for chemotaxis in P. aeruginosa (48, 49). Deletion of other che genes does not affect chemotactic responses (19). Among Che proteins in P. aeruginosa, gene products of cheY2 (AAG03569), cheA2 (AAG03568), cheW2 (AAG03567), cheB2 (AAG03565), and cheW2 (AAG03563) in the Che2 cluster show the highest similarities to E. coli counterparts. Overexpression of cheB2 complemented cheB mutation in P. aeruginosa (50), suggesting the possibility that this gene product is involved in chemotaxis under certain conditions. Overexpression of cheA2 and cheW2 inhibited chemotaxis in E. coli, suggesting that these gene products compete with endogenous E. coli chemotaxis proteins. Pil-Chp and Wsp clusters contain cheA, cheB, cheR, cheW, and cheY homologues and one mcp gene (Fig. 2). Recent researches reported implications of Pil-Chp and Wsp clusters in biological functions other than chemotaxis. Pil-Chp cluster is involved in biosynthesis of type IV pilus (51, 52) and control of twitching motility (53), while Wsp cluster is involved in biofilm formation (54). WspR (AAG07089) and WspF (AAG07090) are a cyclic diguanylate (c-diGMP)synthesizing enzyme and a negative regulator of WspR, respectively, which control intracellular levels of c-diGMP (54). Deletion of wspF resulted in increased levels of intracellular c-diGMP, which led to enhancement of biofilm formation. BdlA (AGG04812), one of MCPs in P. aeruginosa, also affects biofilm dispersion via modulation of intracellular levels of c-diGMP (55). However, there is no clear evidence of any communication between BdlA and Wsp proteins. FUTURE PERSPECTIVES MCPs for amino acids, Pi, oxygen, ethylene, and volatile chlorinated aliphatic hydrocarbons have been identified, and 12 of 26 MCPs have been characterized in P. aeruginosa. It is shown that P. aeruginosa exhibits chemotactic responses to dicarboxylic acids such as succinate, citrate, and malate (56, 57), sugars (56, 58), nitrate and nitrite (59), oligopeptides (60), and aromatic compounds such as benzoate, p-hydroxybenzoate, vanillate, vanillin, benzene, toluene, phenol, and catechol (Kim and Kato, unpublished data). Some of these compounds are major constituents of plant exudates and secretions of animal tissues. Chemotaxis to these com-
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pounds may contribute to initiation of interactions of environmental bacteria with their hosts. It is expected that MCPs for these compounds will be identified in P. aeruginosa in the near future using the mcp mutant library. The symbiotic bacterium Sinorhizobium meliloti is attracted by the plant flavone luteolin which acts as an inducer of its nodulation genes (61), and the plant pathogenic bacterium Agrobacterium tumefaciens shows positive chemotaxis to opines which are produced by crown galls of dicotyledonous plants (62). Therefore, it is interesting to see whether P. aerguinosa responds to these compounds. Previous researches indicated that chemotaxis contributes to colonization of plant roots, infection of plant tissues, and invasive infections of animal tissues by motile bacteria (6– 10). Some compounds have been suggested as chemotactic ligands involved in these interactions. However, direct evidence has not been found. This is primarily because of a lack of information about MCPs in these bacteria and a difficulty in obtaining mutants deficient in chemotaxis to a specific compound. We believe that molecular biological information regarding chemotaxis in P. aeruginosa is of benefit for the investigation of ecological aspects of chemotaxis in environmental bacteria. REFERENCES 1. Adler, J.: Chemotaxis in bacteria. Science, 153, 708–716 (1966). 2. Fenchel, T.: Microbial behavior in a heterogeneous world. Science, 296, 1068–1071 (2002). 3. Macnab, R. M.: Flagella and motility, p. 123–145. In Cutiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Rezinikoff, W. S., Riley, M., Schaechter M., and Umbarger, H. E. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C. (1996). 4. Chet, I. and Mitchell, R.: Ecological aspects of microbial chemotactic behavior. Annu. Rev. Microbiol., 30, 221–239 (1979). 5. De Weger, L. A., van der Vlugt, C. I., Wijfjes, A. H., Bakker, P. A., Schippers, B., and Lugtenberg, B.: Flagella of a plant-growth-stimulating Pseudomonas fluorescens strain are required for colonization of potato roots. J. Bacteriol., 169, 2769–2773 (1987). 6. de Weert, S., Vermeiren, H., Mulders, I. H., Kuiper, I., Hendrickx, N., Bloemberg, G. V., Vanderleyden, J., De Mot, R., and Lugtenberg, B. J.: Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens. Mol. Plant Microbe Interact., 15, 1173–1180 (2002). 7. Panopoulos, N. J. and Schroth, M. N.: Role of flagellar motility in the invasion of bean leaves by Pseudomonas phaseolicola. Phytopathology, 64, 1389–1397 (1974). 8. Yao, J. and Allen, C.: Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum. J. Bacteriol., 188, 3697–3708 (2006). 9. Drake, D. and Montie, T. C.: Flagella, motility and invasive virulence of Pseudomonas aeruginosa. J. Gen. Microbiol., 134, 43–52 (1988). 10. Stock, J. B. and Surette, M. G.: Chemotaxis, p. 1103–1129. In Cutiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Rezinikoff, W. S., Riley, M., Schaechter, M., and Umbarger, H. E. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for
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phosphate regulon, p. 1357–1381. In Cutiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Rezinikoff, W. S., Riley, M., Schaechter, M., and Umbarger, H. E. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C. (1996). Siehnel, R. J., Worobec, E. A., and Hancock, R. E.: Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coli. Mol. Microbiol., 2, 347–352 (1988). Wanner, B. L. and Wilmes-Riesenberg, M. R.: Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli. J. Bacteriol., 174, 2124–2130 (1992). Carmany, D. O., Hollingworth, K., and McCleary, W. R.: Genetic and biochemical studies of phosphatase activity of PhoR. J. Bacteriol., 185, 1112–1115 (2003). Poole, K. and Hancock, R. E.: Phosphate transport in Pseudomonas aeruginosa. Involvement of a periplasmic phosphatebinding protein. Eur. J. Biochem., 144, 607–612 (1984). Kato, J., Sakai, Y., Nikata, T., and Ohtake, H.: Cloning and characterization of a Pseudomonas aeruginosa gene involved in the negative regulation of phosphate taxis. J. Bacteriol., 176, 5874–5877 (1994). Nikata, T., Sakai, Y., Shibata, K., Kato, J., Kuroda, A., and Ohtake, H.: Molecular analysis of the phosphate-specific transport (pst) operon of Pseudomonas aeruginosa. Mol. Gen. Genet., 250, 692–698 (1996). Wu, H., Kosaka, H., Kato, J., Kuroda, A., Ikeda, T., Takiguchi, N., and Ohtake, H.: Cloning and characterization of Pseudomonas putida genes encoding the phosphatespecific transport system. J. Biosci. Bioeng., 87, 273–279 (1999). Kusaka, K., Shibata, K., Kuroda, A., Kato, J., and Ohtake, H.: Isolation and characterization of Enterobacter cloacae mutants which are defective in chemotaxis toward inorganic phosphate. J. Bacteriol., 179, 6192–6195 (1997). Bibikov, S. I., Biran, R., Rudd, K. E., and Parkinson, J. S.: A signal transducer for aerotaxis in Escherichia coli. J. Bacteriol., 179, 4075–4079 (1997). Rebbapragada, A., Johnson, M. S., Harding, G. P., Zuccarelli, A. J., Fletcher, H. M., Zhulin, I. B., and Taylor, B. L.: The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior. Proc. Natl. Acad. Sci. USA, 94, 10541–10546 (1997). Taylor, B. L. and Zhulin, I. B.: PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol. Mol. Biol. Rev., 63, 479–506 (1999). Güvener, Z. T., Tifrea, D. F., and Harwood, C. S.: Two different Pseudomonas aeruginosa chemosensory signal transduction complexes localize to cell poles and form and remould in stationary phase. Mol. Microbiol., 61, 106–118 (2006). Hong, C. S., Kuroda, A., Ikeda, T., Takiguchi, N., Ohtake, H., and Kato, J.: The aerotaxis transducer gene aer, but not aer-2, is transcriptionally regulated by the anaerobic regulator ANR in Pseudomonas aeruginosa. J. Biosci. Bioeng., 97, 184–190 (2004). Schuster, M., Hawkins, A. C., Harwood, C. S., and Greenberg, E. P.: The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol. Microbiol., 51, 973–985 (2004). Hong, C. S., Kuroda, A., Takiguchi, N., Ohtake, H., and Kato, J.: Expression of Pseudomonas aeruginosa aer-2, one of two aerotaxis transducer genes, is controlled by RpoS. J. Bacteriol., 187, 1533–1535 (2005). Scott, C. S. and Cogliano, V. J.: Trichloroethylene health risks—state of the science. Environ. Health Perspect., 108
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(suppl. 2), 159–160 (2000). 45. Shitashiro, M., Kato, J., Fukumura, T., Kuroda, A., Ikeda, T., Takiguchi, N., and Ohtake, H.: Evaluation of bacterial aerotaxis for its potential use in detecting the toxicity of chemicals to microorganisms. J. Biotechnol., 101, 11–18 (2003). 46. Parales, R. E., Ditty, J. L., and Harwood, C. S.: Toluenedegrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene. Appl. Environ. Microbiol., 66, 4098–4104 (2000). 47. Parales, R. E. and Harwood, C. S.: Bacterial chemotaxis to pollutants and plant-derived aromatic molecules. Curr. Opin. Microbiol., 5, 266–273 (2002). 48. Masduki, A., Nakamura, J., Ohga, T., Umezaki, R., Kato, J., and Ohtake, H.: Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa. J. Bacteriol., 177, 948–952 (1995). 49. Kato, J., Nakamura, T., Kuroda, A., and Ohtake, H.: Cloning and characterization of chemotaxis genes in Pseudomonas aeruginosa. Biosci. Biotechnol. Biochem., 63, 155– 161 (1999). 50. Ferrández, A., Hawkins, A. C., Summerfield, D. T., and Harwood, C. S.: Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response. J. Bacteriol., 184, 4374–4383 (2002). 51. Darzins, A.: The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY. J. Bacteriol., 175, 5934–5944 (1993). 52. Darzins, A.: Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus. Mol. Microbiol., 11, 137–153 (1994). 53. Whitchurch, C. B., Leech, A. J., Young, M. D., Kennedy, D., Sargent, J. L., Bertrand, J. J., Semmler, A. B., Mellick,
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A. S., and other 10 authors: Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa. Mol. Microbiol., 52, 873–893 (2004). Hickman, J. W., Tifrea, D. F., and Harwood, C. S.: A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc. Natl. Acad. Sci. USA, 102, 14422–14427 (2005). Morgan, R., Kohn, S., Hwang, S. H., Hassett, D. J., and Sauer, K.: BdlA, a chemotaxis regulator essential for biofilm dispersion in Pseudomonas aeruginosa. J. Bacteriol., 188, 7335–7343 (2006). Moulton, R. C. and Montie, T. C.: Chemotaxis by Pseudomonas aeruginosa. J. Bacteriol., 137, 274–280 (1979). Nikata, T., Sumida, K., Kato, J., and Ohtake, H.: Rapid method for analyzing bacterial behavioral responses to chemical stimuli. Appl. Environ. Microbiol., 58, 2250–2254 (1992). Nelson, J. W., Tredgett, M. W., Sheehan, J. K., Thornton, D. J., Notman, D., and Govan, J. R.: Mucinophilic and chemotactic properties of Pseudomonas aeruginosa in relation to pulmonary colonization in cystic fibrosis. Infect. Immun., 58, 1489–1495 (1990). Kennedy, M. J. and Lawless, J. G.: Role of chemotaxis in the ecology of denitrifiers. Appl. Environ. Microbiol., 49, 109–114 (1985). Kelly-Wintenberg, K. and Montie, T. C.: Chemotaxis to oligopeptides by P. aeruginosa. Appl. Environ. Microbiol., 60, 363–367 (1994). Caetano-Anollés, G., Crist-Estes, D. K., and Bauer, W. D.: Chemotaxis of Rhizobium meliloti to the plant flavone luteolin requires functional nodulation genes. J. Bacteriol., 170, 3164–3169 (1988). Kim, H. and Farrand, S.: Opine catabolic loci from Agrobacterium plasmid confer chemotaxis to their cognate substrates. Mol. Plant-Microbe Interact., 11, 131–143 (1998).
© 2008, The Society for Biotechnology, Japan
REVIEW Pseudomonas aeruginosa as a Model Microorganism for Investigation of Chemotactic Behaviors in Ecosystem Junichi Kato,1* Hye-Eun Kim,1 Noboru Takiguchi,1 Akio Kuroda,1 and Hisao Ohtake2 Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan1 and Department of Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan2 Received 18 February 2008/Accepted 14 April 2008
Motile bacteria sense changes in the concentration of chemicals in environments and respond in a behavioral manner. This behavioral response is called chemotaxis. Bacterial chemotaxis can be viewed as an important prelude to metabolism, prey–predator relationships, symbiosis, infections, and other ecological interactions in biological communities. Genome analysis reveals that a large number of environmental motile bacteria possess a number of genes involved in chemosensing and chemotatic signal transduction. Pseudomonas aeruginosa has a very complex chemosensory system with more than 20 chemotaxis (che) genes in five distinct clusters and 26 chemoreceptor (methyl-accepting chemotaxis protein [mcp]) genes. Among the 26 MCPs of P. aeruginosa, nine have been identified as MCPs for amino acids, inorganic phosphate, oxygen, ethylene, and volatile chlorinated aliphatic hydrocarbons, whereas 3 MCPs were demonstrated to be involved in biofilm formation and biosynthesis of type IV pilus. Six che genes are essential for chemotactic responses, while genes in Pil-Chp cluster and Wsp cluster are involved in type IV pilus synthesis and twitching motility and biofilm formation, respectively. P. aeruginosa, with its complex chemotaxis system, is a better model microorganism for investigating ecological aspects of chemotaxis in environmental bacteria than Escherichia coli and Salmonella enterica serovar Typhimurium, which possess a relatively simpler chemotaxis system. [Key words: chemotaxis, ecological interactions, Pseudomonas aeruginosa, chemosensing]
The molecular mechanisms that underlie bacterial chemotaxis have been studied extensively in enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium (10). Chemotactic ligands are detected by cell surface chemoreceptors called methyl-accepting chemotaxis proteins (MCPs) (Fig. 1). Upon binding a chemotactic ligand, MCPs generate chemotactic signals that are communicated to the flagellar motor via a series of chemotaxis (Che) proteins. E. coli possesses 6 Che proteins and 5 MCPs. Of these, CheA, a histidine protein kinase autophosphorylates at a specific histidine residue to form CheA-P. The phosphoryl group of CheA-P is transferred to a specific aspartate residue of CheY, which is a response regulator of a two-component regulatory system, to form activated CheY-P that interacts directly with the flagellar motor switch protein to control the direction of flagellar rotation. CheZ is involved in inactivation of CheY-P by dephosphorylation of CheY-P to CheY. MCPs, with help from CheW, modulate the autophosphorylation activity of CheA in response to temporal changes in stimuli intensity. MCPs undergo reversible methylation at several glutamate residues. CheR and CheB are methyltransferase and methyl-
Motile bacteria sense changes in the concentration of chemicals in environments and exhibit a behavioral response (1). This behavioral response is called chemotaxis. It is suggested that most microorganisms in heterogeneous environments are motile (2). The fact that a large number of genes are involved in motility and chemotaxis (3), and that environmental bacteria have retained motility and chemotaxis under natural selection for long periods of time indicate that motility and chemotaxis provide a selective advantage for motile bacteria in natural environments. Bacterial chemotaxis can be viewed as an important prelude to metabolism, prey–predator relationships, symbiosis, infections, and other ecological interactions in biological communities (4). It has been reported that chemotaxis has important roles in colonization of plant roots by plant growth-promoting Pseudomonas fluorescens (5, 6), infections of plants by Pseudomonas syringae and Ralstonia solanacearum (7, 8), and invasive infections of animals by Pseudomonas aeruginosa (9). * Corresponding author. e-mail: [email protected] phone: +81-(0)82-424-7757 fax: +81-(0)82-424-7047 1
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research on chemotaxis and che and mcp genes in P. aeruginosa. CHEMOTACTIC RESPONSES BY P. AERUGINOSA TO VARIOUS CHEMICAL COMPOUNDS AND THEIR MOLECULAR MECHANISMS
FIG. 1. Interactions of MCP, Che proteins, and the flagellar motor in E. coli (A) and the structure of typical MCP (B). HCD denotes highly conserved domain.
esterase enzymes, respectively, responsible for methylation modification of MCPs. CheB is another response regulator, which is phosphorylated by CheA-P to form CheB-P. CheB-P exhibits higher methylesterase activity than CheB and is not affected by environmental stimuli. CheR continually adds methyl groups to MCPs. The methylation level of MCPs is controlled in response to environmental stimuli and affects their conformation. This reversible methylation of MCPs is required for temporal sensing of chemical gradients. The best characterized of the chemotactic systems in environmental bacteria is that of P. aeruginosa PAO1. P. aeruginosa is an obligately aerobic bacterium and is capable of locomotion by rotating a single polar flagellum. It inhabits a wide range of environmental niches, including soil, water, and human hosts. Sequence analysis of the P. aeruginosa PAO1 genome suggested that the P. aeruginosa chemotaxis system is very complex, with more than 20 che genes in five distinct clusters and 26 mcp-like genes scattered throughout the genome (Fig. 2) (11–13). Whole genome sequences of several environmental motile bacteria have been determined. Sequence analysis reveals that several environmental bacteria possess a number of che and MCP genes, like P. aeruginosa. Therefore, P. aeruginosa is thought to be a better model microorganism for investigation of ecological aspects of chemotaxis in environmental bacteria than E. coli, which has a relatively simple chemotactic system. In this paper, we provide an overview of molecular biological
MCPs are major chemosensory proteins in bacterial chemotaxis. They are membrane-spanning homo dimers and exhibit typical structural features (14) (Fig. 1) including a positively charged N terminus followed by a hydrophobic membrane-spanning region, a hydrophilic periplasmic domain, a second hydrophobic membrane-spanning region, and a hydrophilic cytoplasmic domain. Periplasmic domains contain variable sequences to which chemotactic ligands bind. In contrast, cytoplasmic domains are relatively conserved. Particularly, a 44-amino acid domain between two reversible methylation sites is highly conserved. MCPs from phylogenetically diverse bacteria have been shown to possess this highly conserved domain (HCD) (15), which is involved in the interaction between MCPs and CheW as well as CheA (16). Putative mcp genes are easily found within genomic sequences by similarity search using HCD as a query sequence. However, a limited number of MCPs have been characterized due to difficulty in identification of their cognate ligands. In P. aeruginosa, 12 of 26 MCPs have been characterized (17–22). Here, we describe chemotactic responses by P. aeruginosa PAO1 of which MCPs have been identified and characterized. Amino acids P. aeruginosa is attracted by twenty commonly occurring amino acids (17). Kuroda et al. isolated a P. aeruginosa mutant defective in chemotaxis toward L-serine but responsive to peptone from N-methyl-N′-nitro-N-nitrosoguanidine-mutagenized cells and cloned the pctA gene (AAG07697 [protein ID in the DDBJ/EMBL/NCBI databases]) encoding a MCP for L-serine by phenotypic complementation of the mutant. DNA sequence analysis of regions upstream and downstream of pctA showed two mcp genes (pctB [AAG07698] and pctC [AAG07695]) that were highly homologous to pctA (18). Chemotaxis assays of pctA pctB pctC triple mutant containing each one of these MCP genes
FIG. 2. Putative mcp genes and Che gene clusters on the genome of P. aeruginosa PAO1 (A) and genetic organization of Che gene clusters (B). (A) The putative mcp genes are shown outside of the circle. The mcp genes which have been characterized are underlined. (B) A, B, R. W, and Y in parentheses denote homologues of cheA, cheB, cheR, cheW, and cheY, respectively.
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FIG. 3. Amino acids detected by PctA, PctB, and PctC of P. aeruginosa PAO1.
revealed that PctA, PctB, and PctC detected 18 amino acids, 6 amino acids, and 2 amino acids, respectively (Fig. 3). Blast search revealed that Pseudomonas species, Sinorhizobium meliloti, Agrobacterium tumefaciens, and Chromobacterium violaceum also possessed 1 to 3 PctA homologues. Many of these bacteria are known to interact with plants, and amino acids are one of the major constituents in plant exudates. Therefore, it would be interesting to investigate the involvement of PctA homologues in interaction between these bacteria and plants. Inorganic phosphate Phosphorus compounds are essential constituents in organisms. In nature, inorganic phosphate (Pi) is often found to be a growth-limiting factor for organisms. Therefore, it is not surprising that motile bacteria show chemotactic responses to Pi. Such responses were first reported in 1992 (23), even though the first reports on bacterial chemotaxis emerged at the end of the 19th century (24, 25). This is because researchers most commonly analyzed chemotactic responses by Adler’s capillary method (26) where Pi buffer is recommended as the cell suspension buffer, and E. coli does not have the ability to respond chemotactically to Pi (27). Pi-starved cells of P. aeruginosa are attracted to Pi, whereas no positive response to Pi is observed with P. aeruginosa cells grown under Pi-sufficient conditions (23). A series of mutants that have deletion-insertion mutations in individual mcp-like genes in the P. aeruginosa PAO1 genome were constructed to identify the MCP for Pi. Chemotaxis assays of the mcp mutant library identified CtpH (AAG05949) and CtpL (AAG08229) as MCPs for Pi (12). CtpH and CtpL are functional at different Pi concentrations. CtpH serves as the major chemoreceptor for Pi at high concentrations, while CtpL is required for exhibiting Pi chemotaxis at low Pi concentrations (Fig. 4). Pho regulon is known to be the major regulatory system responding to Pi limitation in many bacteria (28) including P. aeruginosa. It is a two-component regulatory system comprising His protein kinase PhoR and response regulator PhoB (29). It increases autophosphorylation activity of PhoR; the phosphoryl group of PhoR-P is transferred to PhoB. PhoB-P activates transcription of the Pho regulon genes upon binding to a specific sequence called Pho box in their promoter regions. Sufficient Pi stimulates dephosphorylation
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FIG. 4. Model for Pi taxis in P. aeruginosa PAO1. CtpH and CtpL are MCPs responsible for detection of high concentrations and low concentrations of Pi, respectively. Pi chemotaxis in P. aeruginosa is induced by Pi limitation. PhoB and PhoR, which are a response regulator and a sensor kinase of Pho regulon, respectively, positively control the transcription of ctpL, while Pi-specific transport (Pst) system and PhoU are involved in the negative regulation of ctpL transcription. Pst system and PhoU are essential for Pi detection by CtpL. Pst-PhoU complex negatively regulates CtpH expression at the posttranscriptional level.
of PhoB-P, likely by PhoR (28, 30, 31), forming unphosphorylated or inactive form of PhoB. Phosphate-specific transport (Pst) system is required for negative regulation of Pho regulon (32–34). Pst system is an ATP-binding cassette (ABC) membrane transporter that transports Pi into the cell. It is composed of four proteins (PstS, PstC, PstA, and PstB) encoded by the pstSCAB-phoU operon (34). The PhoU protein is predicted to be a cytoplasmic protein and is involved in negative control of Pho regulon (33). Although it is speculated that Pst system and PhoU negatively regulate Pho regulon through stimulating phosphatase activity of PhoR (28, 29), its molecular mechanism has not been elucidated yet. The ctpL gene belongs to Pho regulon and its inducible transcription is dependent on PhoB/PhoR, while transcription of ctpH is not dependent on PhoB/PhoR and ctpH is constitutively transcribed (12). The ctpL gene is constitutively transcribed in pst and phoU mutants of P. aeruginosa, but these mutant strains fail to show chemotactic responses to low concentrations of Pi, suggesting that the Pst complex and PhoU are required for Pi detection by CtpL. Mutations of pst and phoU do not affect transcription of ctpH. However, CtpH-dependent Pi chemotaxis is constitutive in pst and phoU mutants. Thus, the Pst complex and PhoU are likely to exert a negative control on CtpH expression at the posttranscriptional level. Blast search reveals that Pseudomonas species, including P. putida, P. entomophila, P. stutzeri, P. fluorescens, P. mendocina, and P. syringae, have homologues of ctpH and ctpL. Among them, P. putida was experimentally demonstrated to exhibit Pi taxis (35). It was reported that Enterobacter cloacae is also attracted by Pi and that the Pst system is essential for Pi taxis, although a MCP for Pi has not been identified (36). Thus, Pi chemotaxis seems to be a prevalent trait in environmental motile bacteria. Aerotaxis Aerotaxis is the movement of a cell toward
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or away from oxygen. P. aeruginosa is an aerobic bacterium. Therefore, it is not surprising that this bacterium shows positive aerotaxis. P. aeruginosa possesses two MCPs for aerotaxis, Aer (AAG04950) and Aer-2 (AAG03566) (19). P. aeruginosa Aer is 45% identical to E. coli Aer (37, 38), and has only one hydrophobic sequence, which may serve to anchor Aer to the cytoplasmic side of the inner membrane. We observed that the Aer-GFP fusion protein mainly localized to the cell pole in P. aeruginosa (Kato and Hong, unpublished data). The N-terminal domain of Aer 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, which is known to comprise a binding pocket for a prosthetic group. It was shown that E. coli Aer contains high levels of non-covalently associated flavin adenine dinucleotide (FAD) (37, 39). It is postulated that E. coli Aer uses FAD to monitor altered redox conditions in the cytoplasm. Aer-2 lacks transmembrane domains and it is thought to be a soluble MCP. However, Güvener et al. demonstrated that an Aer-2-YFP fusion protein localized to the cell pole (40). Aer-2 may associate with the inner membrane via other membrane bound proteins. Aer-2 also contains a PAS motif in the N-terminal region, suggesting this MCP may monitor changes of redox potential via the PAS motif. Expressions of aer and aer-2 are controlled by different regulators. The aer promoter contains two FNR/ANR boxes, to which the anaerobic regulator ANR binds; transcription of aer is induced under oxygen-limited conditions by ANR (41). The cheY2, cheA2, cheW2, and aer-2 genes are expressed as a single transcript (42, 43). This transcription is dependent on alternative sigma factor RpoS and is induced in the stationary phase. Chloroethylenes Chloroethylenes, including trichloroethylene (TCE), tetrachloroethylene (PCE), and dichloroethylene (DCE), are xenobiotics and the most frequently detected groundwater contaminants (44). P. aeruginosa is repelled by chloroethylenes (45). This reaction is thought to occur because of toxic compounds. Screening of the mcp mutant library revealed that MCPs for amino acids, PctA, PctB, and PctC, are chemosensory proteins of negative chemotaxis to chloroethylenes (20). Among the three MCPs, PctA is the major MCP for chloroethylenes. TCE-degrading bacterium P. putida F1 exhibits chemotactic responses to TCE. In contrast to P. aeruginosa, P. putida F1 is attracted by TCE (46). The response by P. putida F1 to TCE is induced by toluene in the presence of TodS and TodT, which are regulatory proteins of toluene dioxygenase operon. Chemotactic responses by P. aeruginosa to chloroethylenes are complex. Kim et al. investigated chemotactic responses by the pctA pctB pctC triple mutant of P. aeruginosa to TCE (21). As expected, the mutant showed weaker repelled responses to high concentrations of TCE than the wild-type strain. However, unexpectedly, the mutant was attracted by low concentrations of TCE (Fig. 5). This result suggests that P. aeruginosa has the ability to both negatively and positively respond to TCE and that the positive responses are detected only after disruption of the pctABC genes encoding the MCPs for negative chemotaxis to TCE. CttP
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FIG. 5. Chemotactic responses of P. aeruginosa and P. putida strains to TCE in agarose plug assays. Photographs were taken 10 min after addition of bacterial suspensions to solidified agarose plugs containing 1 mM and 3.8 mM TCE. (A) Responses of the wild-type P. aeruginosa PAO1 and the pctA pctB pctC triple mutant of PAO1. (B) Responses of the wild-type P. putida F1 and the F1 transformant harboring the P. aeruginosa cttP gene. P. putida strains were grown in the absence of toluene.
(AAG03570) was identified as an MCP for positive chemotaxis to TCE through screening of the mcp mutant library. CttP detects PCE and three isomers of DCE as well as TCE. P. aeruginosa cannot utilize chloroethylenes as carbon and energy sources. Although P. putida F1 is capable of degrading TCE in the presence of toluene, it cannot use TCE as a growth substrate. These findings lead one to a question as to why these bacteria possess MCPs for positive chemotaxis to chloroethylenes. Parales et al. speculated that chemotaxis towards chloroethylenes, which are not growth substrates, is a fortuitous consequence of a broad-substrate-specificity chemoreceptor (46). P. putida F1 harboring the P. aeruginosa cttP gene showed positive chemotaxis to TCE even when the cells were grown in the absence of toluene (Fig. 5). The migration of environmental pollutant-degrading bacteria toward environmental pollutants might speed up the biodegradation process because it would bring the cells into contact with environmental pollutants (47). The cttP gene may be used for improvement of the ability of TCE-degrading bacteria to migrate to TCE. Blast search did not find cttP homologues in the bacterial genomes other than those of P. aeruginosa strains. Ethylene Based on the chemotactic responses by P. aeruginosa and P. putida to chlorinated ethylenes, questions were raised about the ability of environmental motile bacteria to respond to ethylene. Ethylene is one of plant hormones and is essentially produced by all plant tissues. Therefore, chemotaxis to ethylene may provide an advantage for initiation of microbe-plant interactions. Kim et al. found that P. aeruginosa was attracted by ethylene and that CttP was not involved in ethylene chemotaxis (22). They identi-
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fied TlpQ (AAG06402) as a MCP for ethylene. Plant-growth promoting bacteria P. fluorescens and P. putida and plant pathogen P. syringae contain tlpQ homologues in their genomes. Chemotaxis assays revealed that these Pseudomonas strains are also attracted by ethylene. Further studies may elucidate a role of ethylene chemotaxis in microbe-plant interactions. FUNCTIONAL ANALYSIS OF che GENE CLUSTERS IN P. AERUGINOSA P. aeruginosa possesses more than 20 che genes in 5 gene clusters (Che, Che2, Pil-Chp, Wsp, and cheVR clusters) in its genome (Fig. 2). Genetic analysis revealed that each of 5 che genes (cheY [AAG04845], cheZ [AAG04846], cheA [AAG04847], cheB [AAG04848], and cheW [AAG04853]) in the Che cluster and cheR (AAG06736) are essential for chemotaxis in P. aeruginosa (48, 49). Deletion of other che genes does not affect chemotactic responses (19). Among Che proteins in P. aeruginosa, gene products of cheY2 (AAG03569), cheA2 (AAG03568), cheW2 (AAG03567), cheB2 (AAG03565), and cheW2 (AAG03563) in the Che2 cluster show the highest similarities to E. coli counterparts. Overexpression of cheB2 complemented cheB mutation in P. aeruginosa (50), suggesting the possibility that this gene product is involved in chemotaxis under certain conditions. Overexpression of cheA2 and cheW2 inhibited chemotaxis in E. coli, suggesting that these gene products compete with endogenous E. coli chemotaxis proteins. Pil-Chp and Wsp clusters contain cheA, cheB, cheR, cheW, and cheY homologues and one mcp gene (Fig. 2). Recent researches reported implications of Pil-Chp and Wsp clusters in biological functions other than chemotaxis. Pil-Chp cluster is involved in biosynthesis of type IV pilus (51, 52) and control of twitching motility (53), while Wsp cluster is involved in biofilm formation (54). WspR (AAG07089) and WspF (AAG07090) are a cyclic diguanylate (c-diGMP)synthesizing enzyme and a negative regulator of WspR, respectively, which control intracellular levels of c-diGMP (54). Deletion of wspF resulted in increased levels of intracellular c-diGMP, which led to enhancement of biofilm formation. BdlA (AGG04812), one of MCPs in P. aeruginosa, also affects biofilm dispersion via modulation of intracellular levels of c-diGMP (55). However, there is no clear evidence of any communication between BdlA and Wsp proteins. FUTURE PERSPECTIVES MCPs for amino acids, Pi, oxygen, ethylene, and volatile chlorinated aliphatic hydrocarbons have been identified, and 12 of 26 MCPs have been characterized in P. aeruginosa. It is shown that P. aeruginosa exhibits chemotactic responses to dicarboxylic acids such as succinate, citrate, and malate (56, 57), sugars (56, 58), nitrate and nitrite (59), oligopeptides (60), and aromatic compounds such as benzoate, p-hydroxybenzoate, vanillate, vanillin, benzene, toluene, phenol, and catechol (Kim and Kato, unpublished data). Some of these compounds are major constituents of plant exudates and secretions of animal tissues. Chemotaxis to these com-
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pounds may contribute to initiation of interactions of environmental bacteria with their hosts. It is expected that MCPs for these compounds will be identified in P. aeruginosa in the near future using the mcp mutant library. The symbiotic bacterium Sinorhizobium meliloti is attracted by the plant flavone luteolin which acts as an inducer of its nodulation genes (61), and the plant pathogenic bacterium Agrobacterium tumefaciens shows positive chemotaxis to opines which are produced by crown galls of dicotyledonous plants (62). Therefore, it is interesting to see whether P. aerguinosa responds to these compounds. Previous researches indicated that chemotaxis contributes to colonization of plant roots, infection of plant tissues, and invasive infections of animal tissues by motile bacteria (6– 10). Some compounds have been suggested as chemotactic ligands involved in these interactions. However, direct evidence has not been found. This is primarily because of a lack of information about MCPs in these bacteria and a difficulty in obtaining mutants deficient in chemotaxis to a specific compound. We believe that molecular biological information regarding chemotaxis in P. aeruginosa is of benefit for the investigation of ecological aspects of chemotaxis in environmental bacteria. REFERENCES 1. Adler, J.: Chemotaxis in bacteria. Science, 153, 708–716 (1966). 2. Fenchel, T.: Microbial behavior in a heterogeneous world. Science, 296, 1068–1071 (2002). 3. Macnab, R. M.: Flagella and motility, p. 123–145. In Cutiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Rezinikoff, W. S., Riley, M., Schaechter M., and Umbarger, H. E. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C. (1996). 4. Chet, I. and Mitchell, R.: Ecological aspects of microbial chemotactic behavior. Annu. Rev. Microbiol., 30, 221–239 (1979). 5. De Weger, L. A., van der Vlugt, C. I., Wijfjes, A. H., Bakker, P. A., Schippers, B., and Lugtenberg, B.: Flagella of a plant-growth-stimulating Pseudomonas fluorescens strain are required for colonization of potato roots. J. Bacteriol., 169, 2769–2773 (1987). 6. de Weert, S., Vermeiren, H., Mulders, I. H., Kuiper, I., Hendrickx, N., Bloemberg, G. V., Vanderleyden, J., De Mot, R., and Lugtenberg, B. J.: Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens. Mol. Plant Microbe Interact., 15, 1173–1180 (2002). 7. Panopoulos, N. J. and Schroth, M. N.: Role of flagellar motility in the invasion of bean leaves by Pseudomonas phaseolicola. Phytopathology, 64, 1389–1397 (1974). 8. Yao, J. and Allen, C.: Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum. J. Bacteriol., 188, 3697–3708 (2006). 9. Drake, D. and Montie, T. C.: Flagella, motility and invasive virulence of Pseudomonas aeruginosa. J. Gen. Microbiol., 134, 43–52 (1988). 10. Stock, J. B. and Surette, M. G.: Chemotaxis, p. 1103–1129. In Cutiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Rezinikoff, W. S., Riley, M., Schaechter, M., and Umbarger, H. E. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for
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