Introduction

The efficiency of in situ bioremediation can be increased if the bacteria and soil/groundwater contaminant are brought into close contact. Chemotaxis has an important role in enhancing the biodegradation of pollutants as it can overcome some of the limitations of in situ bioremediation such as poor bioavailability due to mass transfer limitations, low solubility, or sequestration of a chemical to a matrix surface (Parales and Harwood 2002; Samanta et al. 2002). For example, Pseudomonas putida G7 degrades naphthalene more rapidly than either a chemotaxis or a motility mutant (Marx and Aitken 2000).

Very little work has been done on bacterial chemotaxis toward chlorinated ethenes. P. putida F1 has been shown to exhibit positive chemotaxis to chlorinated ethenes, tetrachloroethylene (PCE), trichloroethylene (TCE), and cis-1,2-dichloroethylene (cis-DCE) (Parales et al. 2000). Negative chemotaxis of P. aeruginosa PAO1 has been observed with TCE, PCE, chloroform, and dichloromethane (Kato et al. 2001).

Chlorinated ethenes are a group of health-threatening pollutants. For example, PCE is one of 14 volatile organic compounds on the United States EPA’s Priority Pollutant List (Carter and Jewell 1993). Vinyl chloride (VC) is a known human carcinogen (McCarty 1997), and both VC and cis-DCE are US EPA priority pollutants (Bradley and Chapelle 1998).

To accelerate aerobic attack of these pollutants, the chemotactic responses toward chlorinated ethenes as well as toluene were studied for P. stutzeri OX1 [(expressing toluene-o-xylene monooxygenase (ToMO)] and Burkholderia cepacia G4. P. stutzeri OX1 was isolated from activated sludge of a wastewater treatment plant (Baggi et al. 1987) and is the only known strain that degrades aerobically PCE (Ryoo et al. 2000); it also oxidizes TCE, 1,1-dichloroethylene (1,1-DCE), cis-DCE, trans-1,2-dichloroethylene (trans-DCE), VC, and chloroform (Chauhan et al. 1998; Shim and Wood 2000). ToMO consists touARE (a three-component hydroxylase with a catalytic oxygen-bridged dinuclear center), TouC (ferredoxin), TouD (a mediating protein), and TouF (NADH-ferredoxin oxidoreductase) (Bertoni et al. 1998).

B. cepacia G4 was the first pure culture that could degrade TCE (Nelson et al. 1986), and toluene o-monooxygenase (TOM) of B. cepacia G4 oxidizes TCE, 1,1-DCE, cis-DCE, trans-DCE, and VC, and converts naphthalene to naphthol (Shields et al. 1994; Shields and Francesconi 1996; Shim and Wood 2000). Six genes encode TOM (tomA012345), including tomA1A3A4 (encodes a three-component hydroxylase with two catalytic oxygen-bridged binuclear iron centers), tomA5 (encodes NADH oxidoreductase), and tomA2 (encodes a protein involved in electron transfer between the hydroxylase and reductase) (Canada et al. 2002).

The chemotactic responses of B. cepacia G4, P. putida F1, and P. stutzeri OX1 were investigated here for TCE, PCE, cis-DCE, trans-DCE, 1,1-DCE, and VC as well as for toluene. This is the first report of chemotaxis toward VC, 1,1-DCE, and trans-DCE by any bacterial strain, chemotaxis with P. stutzeri OX1, and chemotaxis of B. cepacia G4 toward chlorinated ethenes. Furthermore, the involvement of tou genes and the touR (ToMO regulatory) gene in the role of positive chemotaxis toward PCE or o-xylene was investigated.

Materials and methods

Bacteria and growth conditions

P. stutzeri OX1 ATCC BAA-172 (Baggi et al. 1987), P. putida F1 (Finette et al. 1984), and B. cepacia G4 (Shields et al. 1989) were grown initially at 30°C and 250 rpm in 250-ml Erlenmeyer flasks containing 20 ml of Luria-Bertani (LB) (Sambrook et al. 1989) medium. After growth in LB medium, P. stutzeri OX1 was grown in 25 ml minimal M9 medium (Sambrook et al. 1989) supplemented with o-xylene or o-cresol vapors as the only carbon source. P. putida F1 and B. cepacia G4 were grown in M9 with toluene vapors as the only carbon source (30°C, 250 rpm) to an optical density at 600 nm (OD) of 0.5–1.0 in sealed 250-ml Erlenmeyer flasks. Dead cells for negative controls were prepared by autoclaving at 121°C for 30 min (control for non-chemotactic aggregation). To verify the involvement of the tou genes in chemotaxis, P. stutzeri M1 (Lecce et al. 1997) and P. stutzeri OX1 were grown only in LB medium or M9 medium containing 20 mM malate. P. stutzeri M1 does not grow on o-xylene due to an insertion in touA of ToMO (Lecce et al. 1997). P. putida PaW340 (Franklin and Williams 1980) was grown at 30°C in LB medium supplemented with streptomycin (50 μg/ml). P. putida PaW340 [with pKGB4213 (Arenghi et al. 1999) containing the whole ToMO locus touABCDEF] was grown at 30°C in LB medium supplemented with kanamycin (30 μg/ml), and P. putida PaW340 [with pFP3028 (Arenghi et al. 1999), containing the touR (ToMO regulatory) gene, pFB1112 (Bertoni et al. 1996), containing both touABCDEF and touR, and pLAFR1 (Friedman et al. 1982), which is the control plasmid of both pFP3028 and pFB1112], were grown in LB medium supplemented with tetracycline (25 μg/ml) at 30°C to an OD of 0.5–1.0.

Chemicals

TCE, toluene, and o-cresol were obtained from Fisher Scientific (Pittsburgh, Pa.); 1,1-DCE, cis-DCE, trans-DCE, and o-xylene were obtained from Aldrich Chemical (Milwaukee, Wis.); VC was obtained from Supelco (Bellefonte, Pa.); and PCE was obtained from Sigma Chemical (St Louis, Mo.).

Chemotaxis assay

The agarose plug assay was used as described previously (Yu and Alam 1997). Plugs were prepared with 2% low-melting temperature NuSieve GTG agarose in chemotaxis buffer (Parales et al. 2000), and contained 10% (v/v) of volatile aliphatic organics (Parales et al. 2000) including toluene, TCE, PCE, trans-DCE, cis-DCE, 1,1-DCE, and VC. The temperature of the agarose was kept between 60°C and 65°C to avoid its solidification before addition of the volatile compounds. The preheated agarose mixture (10 μl) with aliphatic organics was injected between the space created by two cover glasses lying flat on the slide and separated by 1.5–2 mm on a microscope slide (kept at 60–65°C with a hot block). The agarose mixture was covered after 1 s to reduce the evaporation of volatile compounds with a third cover glass that resides on the top of the first two cover glasses and bridges the 1.5–2 mm gap. The slide was removed from the hot block, and, after 2–4 min, 100–120 μl of fresh cells harvested in the exponential growth phase in minimal medium or LB (OD between 0.5 and 1.0) was directly pipetted [without washing with chemotaxis buffer (Parales et al. 2000)] between the microscope slide and the third cover glass. For the negative controls, chemotaxis buffer was added to the agarose mixture instead of volatile compounds, and dead P. putida F1 and dead P. stutzeri OX1 were added instead of exponentially growing cells. The chemotaxis assay was performed with at least two independent cultures for each strain and for each culture, and there were at least two replicates (Tables 1, 2).

Table 1 Chemotaxis of Pseudomonas stutzeri OX1, Burkholderia cepacia G4, and Pseudomonas putida F1 toward chlorinated ethenes
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Table 2 Chemotaxis of Pseudomonas putida PaW340 expressing touABCDEF and touR toward o-xylene. Antibiotics used for growth were streptomycin at 50 μg/ml (Sm 50), tetracycline at 25 μg/ml (Tc 25), and kanamycin at 30 μg/ml (Kan 30)
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Microscopy and photography

After pipetting the cells, chemotactic rings toward organic pollutants were observed in 5–30 min. Slides were viewed with a microscope (Zeiss Axioskop20, Munchen-Hallbergmoos, Germany) equipped with a CCD camera (Hitachi KP-D50 Color Digital, Tokyo, Japan) and Axio-Vision software. Chemotactic responses were observed at 100× or 400× total magnification with an eyepiece magnification of 10× and objective of 10× or 40×. The size of the chemotactic ring was determined using Axio-Vision software.

Results

Agarose plug chemotaxis assay

To study chemotaxis, the agarose plug assay (Yu and Alam 1997) was used. The assay is an easy and useful technique to work with volatile compounds, since the evaporation losses of the volatile compounds are reduced (Parales et al. 2000). The positive chemotactic cells moved toward the agarose plug containing the chlorinated aliphatic and formed a ring structure (Fig. 1). When only chemotaxis buffer was present in the agarose plug, the cells were completely insensitive, and no ring was found (Fig. 1); also, dead bacteria failed to respond.

Fig. 1
figure 1

Representative chemotactic responses toward the volatile organic compounds (a), and the role of tou genes and touR on positive chemotaxis response toward tetrachloroethylene (PCE) or o-xylene (b). Chemotaxis of toluene-grown Burkholderia cepacia G4 toward chemotaxis buffer (a1), vinyl chloride [(VC), a2], and trichloroethylene [(TCE), a3]. Chemotaxis of toluene-grown Pseudomonas putida F1 toward toluene (a4) and 1,1-dichloroethylene [(1,1-DCE), a5]. Chemotaxis of o-cresol-grown Pseudomonas stutzeri OX1 toward chemotaxis buffer (a6) and VC (a7), and o-xylene-grown P. stutzeri OX1 toward TCE (a8), 1,1-DCE (a9), and toluene (a10). Chemotaxis of malate-grown P. stutzeri M1 (b1) and malate-grown P. stutzeri OX1 (b2) toward PCE. Chemotaxis of P. putida PaW340/pKGB4213 (touABCDEF; b3, b4) and P. putida PaW340/pFB1112 (touABCDEF + touR; b5, b6) toward chemotaxis buffer and o-xylene. Scale bar 100 μm

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In the plug assays, a concentration gradient of the dissolved volatile compound existed. After diffusion of the volatile compounds into the cells, positive chemotactic bacteria swam toward the plug, and accumulated near the plug without touching it. This indicated that cells responded to an optimal concentration of the volatile organic compound (Parales et al. 2000). Accumulation of cells around only half of the agarose, which was observed toward trans-DCE, indicated that the volatile compound trans-DCE might not have diffused homogeneously (Table 1). After 1–3 h, the chemotactic rings disappeared.

The strength of the bacterial chemotactic response was quantified by measuring the size of the ring (4–40 μm) in order to judge its significance for bioremediation (Table 1). (Chemotactic rings more than 20 μm thick indicate a strong response; rings less than 20 μm indicate a weak response.) Bacteria were originally observed in the microscope, and the size of the ring was determined using the Axio-Vision program. A strong response indicated that more cells accumulated near the plug containing the attractant and formed a thicker ring.

Chemotactic responses of P. putida F1 and B. cepacia G4

The chemotactic response of toluene-grown P. putida F1 to the chlorinated aliphatics and toluene are shown in Fig. 1 and Table 1. Previously it was observed that P. putida F1 grown with toluene and pyruvate showed positive chemotactic responses to PCE, TCE, and cis-DCE (Parales et al. 2000). Here, P. putida F1 was chemotactic toward the chlorinated ethenes PCE, TCE, and cis-DCE as well as toward trans-DCE, 1,1-DCE, VC, and toluene, and formed a ring structure around the agarose plug. Strong chemotaxis was observed toward toluene, TCE, cis-DCE, and VC, and weak chemotaxis was observed toward PCE, 1,1-DCE, and trans-DCE. Formation of only a half ring was observed with trans-DCE, which might be due to uneven diffusion of trans-DCE.

Previously it was shown that B. cepacia G4 grown with toluene and succinate was chemotactic toward toluene (Parales et al. 2000), and the same result was found here. In addition, B. cepacia G4 grown on toluene vapors without succinate was found by us to be chemotactic toward the chlorinated ethenes PCE, TCE, 1,1-DCE, cis-DCE, and VC (Fig. 1; Table 1). The chemotaxis was strongest toward PCE, TCE, and 1,1-DCE, and weakest toward toluene, cis-DCE, and VC. No chemotaxis was found for trans-DCE. The addition of succinate or pyruvate during growth was not required for positive chemotaxis attraction.

Chemotactic responses of P. stutzeri OX1

P. stutzeri OX1 grown on o-xylene or o-cresol vapors was chemotactic and formed a ring structure around agarose plugs with TCE, PCE, toluene, all DCEs, and VC. Chemotaxis toward TCE and cis-DCE of P. stutzeri OX1 cells grown on o-xylene vapors was better than that of cells grown on o-cresol vapors (no ring formed for o-cresol grown cells). On the other hand, chemotaxis toward VC was stronger with o-cresol-grown cells. Similar chemotactic responses were observed with o-xylene-grown and o-cresol-grown P. stutzeri OX1 cells toward PCE, trans-DCE, and 1,1-DCE.

ToMO plays a role in chemotaxis

To investigate the role of tou genes in chemotaxis, the differences in the chemotactic responses toward PCE were checked for mutant P. stutzeri M1, which has an inactive touA (alpha fragment of ToMO) but active touR (Bolognese et al. 1999), and wild-type P. stutzeri OX1 (Fig. 1). Malate-grown M1 cells did not form a chemotactic ring around the PCE-containing agarose plug but malate-grown OX1 cells did. The same results were obtained when the strains were grown on LB; hence, touA is important for chemotaxis attraction, and the carbon source used during cell growth does not seem to matter for chemotaxis toward PCE.

To investigate the importance of the ToMO regulatory gene touR for chemotaxis and confirm the necessity of the tou locus, the chemotactic response of P. putida PaW340 to o-xylene was investigated in the presence of both touABCDEF and touR. P. putida PaW340/pKGB4213 (Arenghi et al. 1999), which contains touABCDEF, was chemotactic toward o-xylene (Fig. 1; Table 2). P. putida PaW340 with pFP3028 (Arenghi et al. 1999), which contains the touR but not touABCDEF, was not chemotactic toward o-xylene. P. putida PaW340 with pFB1112 (Bertoni et al. 1996), which has touABCDEF and touR, showed a stronger positive chemotactic response and formed a thicker ring around the o-xylene-containing agarose plug (Fig. 1; Table 2). Negative chemotaxis responses were observed with P. putida PaW340 (Franklin and Williams 1980) cells and as well as P. putida PaW340 cells with pLAFR1 (control plasmid of pFP3028 and pFB1112, Friedman et al. 1982). As expected, no chemotactic ring was observed with P. putida PaW340 with pFB1112 for the negative control of a buffer-containing agarose plug. These results corroborate the importance of the tou operon for chemotaxis toward o-xylene, and indicate the response might be enhanced in the presence of touR.

Discussion

Our results of chemotaxis by P. putida F1 toward PCE, TCE, cis-DCE, and toluene and B. cepacia G4 toward toluene compare well to those reported previously (Parales et al. 2000). Here, energy sources such as succinate or pyruvate during cell growth in minimal media were not required for positive chemotaxis attraction. In addition to these compounds, it was also found here that P. putida F1 and B. cepacia G4 are chemotactic toward 1,1-DCE and VC, and that P. putida F1 is chemotactic toward trans-DCE; this is the first report of these compounds acting as attractants for these two strains. Since in P. stutzeri OX1, the carbon source used during cell growth did not affect chemotaxis toward PCE, 1,1-DCE, and trans-DCE but affected chemotaxis with cis-DCE and TCE, the growth medium might be important for some substrates.

It was reported previously that the genes required for positive chemotactic response to toluene are the regulatory proteins TodS and TodT, which regulate the todRXFC1C2BADEGIH operon for toluene degradation in P. putida F1 (Parales et al. 2000). Similarly, pcaK, which has a role in the degradation of aromatic acids, has been shown to be required for chemotaxis toward 4-hydroxybenzoate by P. putida PRS2000 (Ditty and Harwood 2001; Parales and Harwood 2002). nahY, which is part of an operon that contains genes for salicylate degradation, has been shown to be required for chemotaxis toward naphthalene by P. putida G7 (Grimm and Harwood 1999; Parales and Harwood 2002). tfdK, which is located on plasmid pJP4 within the cluster of tfd genes that are required for 2,4-dichlorophenoxyacetate (2,4-D) degradation, has been shown to be required for chemotaxis toward 2,4-D by Ralstonia eutropha JMP134 (pJP4) (Hawkins and Harwood 2002; Parales and Harwood 2002). Hence, the chemotactic response appears to be coordinated with catabolic-gene expression as shown from previous studies.

The importance of the whole ToMO locus (touABCDEF) and the regulatory gene (touR) for chemotaxis was investigated in this work. These results showed the importance of the tou operon for chemotaxis toward o-xylene or PCE and indicated the response might be enhanced with touR. Since positive chemotaxis was also observed with P. putida F1 expressing toluene 2,3-dioxygenase and with B. cepacia G4 expressing TOM, oxygenases might be playing an important role in chemotaxis, but still more data are required to understand the role of catabolic structural and regulatory genes in the chemotactic response.

Previously, it was shown that ToMO from P. stutzeri OX1 degrades the chlorinated ethenes PCE, TCE, 1,1-DCE, cis-DCE, trans-DCE, and VC, individually and as mixtures (Chauhan et al. 1998; Ryoo et al. 2000; Shim and Wood 2000). It was also shown that PCE and TCE induce their own degradation in this strain (Ryoo et al. 2000, 2001). Hence, the positive chemotaxis attraction toward PCE and other less chlorinated compounds that was discovered here makes P. stutzeri OX1 expressing ToMO very attractive for bioremediation of chlorinated ethenes, since these pollutants induce chemotaxis, induce expression of ToMO, and are degraded by ToMO with a stoichiometric release of chloride ions.