Microbes Environ. Vol. 22, No. 2, 186–189, 2007
http://wwwsoc.nii.ac.jp/jsme2/
Short Communication
Ethylene Chemotaxis in Pseudomonas aeruginosa and Other Pseudomonas Species HYE-EUN KIM1, MAIKO SHITASHIRO1, AKIO KURODA1, NOBORU TAKIGUCHI1, and JUNICHI KATO1* 1
Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739–8530, Japan
(Received December 4, 2006—Accepted January 22, 2007) Pseudomonas aeruginosa strain PAO1 was attracted by ethylene. Assays of a set of mutants containing deletions in 26 potential methyl-accepting chemotaxis protein genes revealed that tlpQ (PA2654) encodes the chemoreceptor for positive chemotaxis to ethylene. Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas syringae strains also exhibited positive chemotaxis toward ethylene. Key words: chemotaxis, ethylene, Pseudomonas, chemotactic transducer
Chemotaxis is the movement of an organism toward chemical attractants and away from repellents. Motile bacteria are attracted by a wide variety of chemical stimuli, including sugars, amino acids, organic acids, inorganic phosphate, and aromatic compounds. Bacterial chemotaxis can be viewed as an important prelude to metabolism, preypredator relationships, symbiosis, and other ecological interactions in microbial communities3). Toluene-oxidizing bacteria Pseudomonas putida strain F1, Pseudomonas stutzeri strain OX1, and Burkholderia cepacia strain G4 are capable of cooxidizing chlorinated ethylenes such as trichloroethylene (TCE) and dichloroethylens4,20,27). Parales et al.21) and Varder et al.26) demonstrated that these chlorinated-ethylene-degrading bacteria are attracted by chlorinated ethylenes as well as toluene although they cannot utilize chlorinated ethylenes as growth substrates. Parales et al. discussed that bacterial chemotaxis towards compounds that are not growth substrates is probably a fortuitous consequence of a broad-substrate-specificity chemoreceptor that detects a variety of chlorinated ethylenes21). These findings and discussion led * Corresponding author. E-mail address:
[email protected]; Tel.: +81–82–424–7757; Fax: +81–82–424–7047.
us to speculate that the chemoreceptor for chloroethylenes also detects ethylene and that bacteria possessing the chemoreceptor are also attracted by ethylene. However, there are no reports on chemotaxis towards ethylene probably because gaseous compounds are difficult to handle in assays. Recently, we found that Pseudomonas aeruginosa strain PAO1 is repelled by TCE23). Genetic analysis revealed that the methyl-accepting chemotaxis proteins (MCPs) PstA, PctB, and PctC, which were identified as MCPs for amino acids15,25), serve as the major chemoreceptors for the negative chemotaxis to TCE24). Interestingly, the pctA pctB pctC triple mutant of P. aeruginosa strain PAO1 shows an attractive response to TCE13). The CttP protein was found to be a MCP responsible for the positive chemotaxis towards TCE in P. aeruginosa strain PAO1. In this study, we report that P. aeruginosa strain PAO1 showed attractive responses to ethylene and that unexpectedly, a MCP other than CttP was responsible for ethylene chemotaxis. We also demonstrate that several Pseudomonas strains also exhibited attractive responses to ethylene. P. aeruginosa strain PAO110) was provided by A.M. Chakrabarty. Chemotactic responses were assayed by the agarose plug method. Agarose plug assays were carried out
Ethylene Chemotaxis in Pseudomonas Species
as previously described29) with modifications. Ten milliliters of chemotaxis buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid], pH 7.0) and a magnetic stirring bar were put into a serum bottle (50 ml) and sealed with a butyl rubber stopper after introduction of gas chromatography-grade pure ethylene gas (GL Science, Tokyo, Japan) to the headspace. The chemotaxis buffer in the bottle was equilibrated with ethylene by magneticstirring overnight and the resulting buffer was used as a 4.8 mM ethylene solution. Molten 3% (wt/vol) agarose (Agarose S, Nippon Gene Inc., Toyama, Japan) in the chemotaxis buffer was kept at 60°C. The agarose was mixed with the same volume of chemotaxis buffer containing a known concentration of ethylene. Immediately after mixing, 12 µl of the mixture was placed on a microscope slide, and a coverslip supported by two staples (0.5 mm in diameter) was placed on top to form a chamber. Bacteria were cultivated in T2 minimal medium9) containing glucose as the solo source of carbon at 37°C for P. aeruginosa strains or at 28°C for other Pseudomonas strains. Bacterial cells harvested in the early stationary phase of growth were resuspended in chemotaxis buffer to turbidity at 600 nm of approximately 1, and 120 µl of the cell suspension was pipetted between the microscope slide and the coverslip. The chemotaxis chambers were incubated for 10 min at 37°C for P. aeruginosa strains or at 28°C for other Pseudomonas strains and photographs were then taken. P. aeruginosa strain PAO1 cells were attracted by ethylene and formed dense zones around agarose plugs containing 1 mM ethylene but not the control agarose plug (Fig. 1). The cheR gene encoding the chemotaxis-specific methyltransferase is required for MCP-dependent chemotaxis17). Strain PC4 (the cheR mutant of strain PAO112)) failed to respond to ethylene, indicating that a MCP(s) is involved in the chemotaxis in P. aeruginosa strain PAO1. The PAO1 strain possesses 26 potential mcp genes in its genome6,28). We previously constructed a series of mutants that have deletion-insertion mutations in individual mcp-like genes in the PAO1 genome11,28). We first examined the cttP mutant PAO-dF for a chemotactic response to ethylene, since CttP was demonstrated to detect tetrachloroethylene and dichloroethylene isomers as well as TCE13). The deletion of cttP did not affect the response to ethylene (Fig. 1), suggesting that CttP is not involved in the chemotaxis. We then tested 25 mcp mutants to identify the MCP mediating positive chemotaxis. The agarose plug assays revealed that among the mcp mutants, the tlpQ (gene ID number PA2654 in the P. aeruginosa genome sequencing project [http:// www.pseudomonas.com/]) mutant PAO-dQ showed mark-
187
Fig. 1. Chemotactic responses of P. aeruginosa strains to ethylene in agarose plug assays. Photographs were taken 10 min after the strains were added. (A) Responses of the wild-type P. aeruginosa strain PAO1 to an agarose plug without ethylene (control). (B)– (E) Responses of strain PAO1 (B), the cheR mutant PC4 (C), the cttP mutant PAO-dF (D), the tlpQ mutant PAO-dQ (E), and strain PAO-dQ harboring pHEK03 (carrying tlpQ) (F) to 1 mM ethylene.
edly decreased responses to ethylene. We cloned the tlpQ gene into the broad-host-range vector pUCP1822) to construct pHEK03 and introduced it into strain PAO-dQ. pHEK03 restored the ability of strain PAO-dQ to respond to ethylene (Fig. 1), indicating that tlpQ complements the mutation in strain PAO-dQ and that the mutant phenotype was not due to polar effects of the kanamycin-resistant gene cassette’s insertion. The potential product of tlpQ is a 714-amino-acid TlpQ (predicted size 77.1 kDa). It exhibits typical structural features of MCPs7): a positively charged N terminus followed by a hydrophobic membrane-spanning region, a hydrophilic periplasmic domain, a second hydrophobic membranespanning region, and a hydrophilic cytoplasmic domain. TlpQ residues 547 to 590 are 73% identical to the 44amino-acid highly conserved domain of the Escherichia coli MCP Tsr1). Other P. aeruginosa strains (PA7, PA14, and 2192) possess TlpQ homologs (ZP 01293049, ZP 00135963, and ZP 00975006) and their sequences are 96– 100% identical to that of the P. aeruginosa strain PAO1 TlpQ. A protein-protein BLAST search of public databases revealed that the potential periplasmic domain of TlpQ (residues 24 to 363) is 29–57% identical to the N-terminal region of several putative MCPs from pseudomonads other than P. aeruginosa. These MCPs include those of P. fluorescens strain Pf-5 (AAY93274), P. fluorescens strain PfO-1 (ABA75508), P. putida strain KT2440 (AAN69158), P. putida strain F1 (EAP52327), and P. syringae pv. syringae strain B728a (AAY37721). These Pseudomonas species
KIM et al.
188
Fig. 2. Chemotactic responses of P. putida strain KT244019) (A), P. putida strain F18) (B), P. fluorescens IFO 14053 (C), P. fluorescens IFO 14086 (D), P. syringae IAM 12022 (E), and P. syringae IAM 12405 (F) to agarose plugs with and without ethylene (2.4 mM). IFO 14053 and IFO 14086 were obtained from the National Institute of Technology and Evaluation (Tokyo, Japan), and IAM 12022 and IAM 12405, from the Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
may have the ability to respond to ethylene. To investigate this possibility, we examined 6 strains of P. fluorescens, P. putida, and P. syringae for ethylene chemotaxis. Five strains exhibited chemotactic responses to ethylene (Fig. 2). Only P. fluorescens IFO 14053 failed to respond to ethylene under test conditions. It is worth mentioning that P. putida strain F1 cells grown in the absence of toluene showed positive chemotaxis in response to ethylene. Movement toward chloroethylenes is induced in P. putida strain F1 when it is grown in the presence of toluene, but cells grown in the absence of toluene do not respond to chloroethylenes21). Therefore, it is likely that a chemoreceptor for ethylene is different from that for chloroethylenes in P. putida strain F1 as is the case in P. aeruginosa strain PAO1. Since most bacterial chemoattractants are growth substrates and nutrients, ethylene may serve as a growth substrate for Pseudomonas strains that respond to ethylene. To assess this possibility, we cultivated these Pseudomonas strains in a mineral salt basal medium (T2 medium) containing ethylene as the sole source of energy and carbon, however, no strains grew on ethylene under test conditions (data not shown). We tested only one set of conditions for growth of Pseudomonas strains on ethylene in this study and it is possible that the strains do grow on ethylene in different conditions. Some Pseudomonas strains have been reported to utilize ethylene as the sole source of energy and carbon14). Therefore, the possibility that ethylene chemotaxis plays a role in bringing bacterial cells to a nutrient cannot be ruled out. However, we did not obtain any experimental evidence to support this possibility. Ethylene is a plant hormone and essentially all plant tissues produce it. P. fluorescens, P. putida, and P. syringae inhabit plant-associated environments. Bacterial chemotaxis is thought to be an early and essential event in most plantmicrobe interactions2). Therefore, ethylene chemotaxis may play an important role in plant-microbe interactions. P.
putida and P. fluorescens colonize the rhizosphere of a large number of plants16,18). Root colonization by P. fluorescens is being intensively investigated to understand the mechanism of its biocontrol of plant diseases caused by fungi16). P. fluorescens controls plant diseases via the synthesis of antibiotics and induction of systemic resistance in plants5). P. syringae is a plant pathogen causing chlorosis and necrotic lesions on leaves. If ethylene chemotaxis has a role in plantmicrobe interactions, its enhancement may contribute to help rhizosphere competence and as a consequence, the efficacy of the biocontrol agent. On the other hand, understanding ethylene chemotaxis in P. syringae may lead to the development of novel chemicals to control P. syringae infections. We are now planning to construct P. fluorescens, P. syringae and P. putida mutant strains containing a deletion in the tlpQ homolog to further investigate ethylene chemotaxis in these bacteria.
Acknowledgements We are grateful to J.L. Ramos for kindly providing P. putida strain KT2440.
References 1) Boyd, A.W., K. Kendall, and M.I. Simon. 1983. Structure of the serine chemoreceptor in Escherichia coli. Nature 301:623–626. 2) Brencic, A., and S.C. Winans. 2005. Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiol. Mol. Biol. Rev. 69:155–194. 3) Chet, I., and R. Mitchell. 1979. Ecological aspects of microbial chemotactic behavior. Annu. Rev. Microbiol. 30:221–239. 4) Chuahan, S., P. Barbieri, and T.K. Wood. 1998. Oxidation of trichloroethylene, 1,1-dichloroethylene, and chloroform by toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1. Appl. Environ. Microbiol. 64:3023–3024. 5) Compant, S., B. Duffy, J. Nowak, C. Clément, and E.A. Barka. 2005. Use of plant growth-promoting bacteria for biocontrol of
Ethylene Chemotaxis in Pseudomonas Species
6)
7)
8)
9)
10) 11)
12)
13)
14) 15)
16)
17)
plant diseases: principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71:4951–4959. Croft, L., S.A. Beaston, C.B. Whiychurch, B. Huang, R.L. Blakeley, and J. Mattick. 2000. An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures. Microbiology 146:2351–2364. Dahl, M.K., W. Boos, and M.D. Manson. 1989. Evolution of chemotactic-signal transducers in enteric bacteria. J. Bacteriol. 171:2361–2371. Finnette, B.A., V. Subramanian, and D.T. Gibson. 1984. Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system. J. Bacteriol. 160:1003–1009. Harold, F.M., and S. Sylvan. 1963. Accumulation of inorganic polyphosphate in Aerobacter aerogenes. II. Environmental control and the role of sulfur compounds. J. Bacteriol. 86:222–231. Holloway, B.W., V. Krishnapillai, and A.F. Morgan. 1979. Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43:73–102. Hong, C.S., M. Shitashiro, A. Kuroda, T. Ikeda, N. Takiguchi, H. Ohtake, and J. Kato. 2004. Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 231:247–252. Kato, J., T. Nakamura, A. Kuroda, and H. Ohtake. 1999. Cloning and characterization of chemotaxis genes in Pseudomonas aeruginosa. Biosci. Biotechnol. Biochem. 63:151–161. Kim, H.-E., M. Shitashiro, A. Kuroda, N. Takiguchi, H. Ohtake, and J. Kato. 2006. Identification and characterization of chemotactic transducer for attractive response to trichloroethylene in Pseudomonas aeruginosa PAO1. J. Bacteriol. 188:61100–61102. Kim, J. 2006. Assessment of ethylene removal with Pseudomonas strains. J. Hazard. Mater. 131:131–136. Kuroda, A., T. Kumano, K. Taguchi, T. Nikata, J. Kato, and H. Ohtake. 1995. Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa. J. Bacteriol. 177:7019–7025. Lugtenberg, B.J.J., L. Dekkers, and G.V. Bloemberg. 2001. Molecular determinants of rhizosphere colonization by Pseudomonas. 2001. Annu. Rev. Phytopathol. 39:461–490. Macnab, R.M. 1987. Chapter 49. Motility and chemotaxis, pp. 732–759. In F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter, and H.E. Umbarger (eds.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular
189 Biology, American Society for Microbiology, Washington, DC. 18) Morina, L., C. Ramos, E. Duque, M.C. Ronchel, J.M. García, L. Wyke, and J.L. Ramos. 2000. Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions. Soil Biol. Biochem. 32:315–321. 19) Nakazawa, T. 2002. Travels of a Pseudomonas, from Japan around the world. Environ. Microbiol. 4:782–786. 20) Nelson, M.J.K., S.O. Montgomery, E.J. O’Niel, and P.H. Pritchard. 1986. Aerobic metabolism of trichloroethylene by a bacterial isolate. Appl. Environ. Microbiol. 52:383–384. 21) Parales, R.E., J.L. Ditty, and C.S. Harwood. 2000. Toluenedegrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene. Appl. Environ. Microbiol. 66:4098–4104. 22) Schweizer, H.P. 1991. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene 97:109–112. 23) Shitashiro, M., T. Fukumura, J. Kato, A. Kuroda, T. Ikeda, N. Takiguchi, and H. Ohtake. 2003. Evaluation of bacterial aerotaxis for its potential use in detecting toxicity of chemicals to microorganisms. J. Biotechnol. 101:11–18. 24) Shitashiro, M., H. Tanaka, C.S. Hong, A. Kuroda, N. Takiguchi, H. Ohtake, and J. Kato. 2005. Identification of chemosensory proteins for trichloroethylene in Pseudomonas aeruginosa. J. Biosci. Bioeng. 99:396–402. 25) Taguchi, K., H. Fukutomi, A. Kuroda, J. Kato, and H. Ohtake. 1997. Genetic identification of chemotactic transducers for amino acids in Pseudomonas aeruginosa. Microbiology 143:3223– 3229. 26) Varder, G., P. Barbieri, and T.K. Wood. 2005. Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes. Appl. Microbiol. Biotechnol. 66:696–701. 27) Wackett, L.P., and D.T. Gibson. 1988. Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1. Appl Environ Microbiol. 54:1703– 1708. 28) Wu, H., J. Kato, A. Kuroda, T. Ikeda, N. Takiguchi, and H. Ohtake. 2000. Identification of two chemotactic transducers for inorganic phosphate in Pseudomonas aeruginosa. J. Bacteriol. 182:3400–3404. 29) Yu, H.S., and M. Alam. 1997. An agarose-in-plug bridge method to study chemotaxis in the Archaeon Halobacterium salinarum. FEMS Microbiol. Lett. 156:265–269.
