Ralstonia pickettii in environmental biotechnology: potential and applications

Xenobiotic pollutants such as toluene and trichloroethylene are released into the environment by various industrial processes. Ralstonia pickettii possess significant biotechnological potential in the field of bioremediation and has demonstrated the ability to breakdown many of these toxic substances. Here, we provide a description of the major compounds that various strains of R. pickettii are capable of degrading and a brief review of their breakdown pathways and an argument for its use in bioremediation.


Introduction
Ralstonia is a newly designated genus that includes former members of Burkholderia species (Burkholderia pickettii and Burkholderia solanacearum). These organisms have been renamed as Ralstonia pickettii and Ralstonia solanacearum, respectively (Yabuuchi et al. 1995). Ralstonia pickettii is an aerobic Gram-negative, oxidase-positive, nonfermentative rod and is a ubiquitous micro-organism found in water and soil (Gilligan et al. 2003). Ralstonia pickettii has been identified in biofilm formation in plastic water piping (Anderson et al. 1990). The bacterium has been identified in ultrapure water in industrial systems , in the Space Shuttle water system (Koenig and Pierson 1997) and in laboratory-based purified water systems (Adley et al. 2005). The organism has the ability to survive and thrive in low nutrient (oligotrophic) conditions ; it is theorized that in ultrapure water systems, the bacteria may be able to scavenge from the polymers in plastic piping. In addition, R. pickettii has been shown to have biodegradative abilities, demonstrating its large metabolic diversity (Table 1). Many different species of bacteria are being investigated for bioremediation capabilities; three of the best characterized are Burkholderia vietnamiensis, Pseudomonas putida and Pseudomonas fluorescens. However, these species have drawbacks that may limit their use. Burkholderia vietnamiensis strain G4 (O'Sullivan and Mahenthiralingam 2005) is part of the Burkholderia cepacia complex (Bcc) (Genomovar V), which in cystic fibrosis (CF) patients leads to a deterioration of prognosis and an increased risk of death (Isles et al. 1984;Tablan et al. 1985;Corey and Farewell 1996). In a study carried out by LiPuma et al. (2001), B. vietnamiensis accounted for 5AE1% of all Bcc cases found in 606 CF patients. Burkholderia vietnamiensis can also cause a phenomenon called plant tissue water soaking, which can cause disease and tissue damage in onions. As a result of the clinical relevance of Bcc species and their close interspecies relatedness, the biotechnological applications of all Bcc species have been severely restricted by the US EPA (Anon 2003). Pseudomonas fluorescens has been demonstrated to cause fin rot in fish (Sakai et al. 1989) and P. putida has also been shown to cause disease in fish (Nakatsugawa and Iida 1996;Wakabayashi et al. 1996). The use of these bacteria as bioremediators is, therefore, not advisable as environmental release could lead to environmental damage, such as disease in plants and depletion of fish stocks, and the potential cause of disease in humans. The use of these organisms could also cause public concern. Ralstonia pickettii demonstrates many advantages over these bacteria. a number of clinical situations in unusual circumstances (Ryan et al. 2006), it has never been detected as a phytopathogen or as an animal pathogen (Gilligan et al. 2003). This may allow more widespread environmental release. The organism's ability to survive in low nutrient environments like water and soil would also help it to survive on environmental release. These factors illustrate why R. pickettii is a good candidate for the study of its bioremediation capabilities and potential applications. A complete list of known compounds that can be degraded by various strains of R. pickettii are included in Table 1.

Aromatic hydrocarbons
Aromatic hydrocarbons are volatile organic compounds (VOC's), which include phenol (C 6 H 5 OH), cresol (C 7 H 8 O), benzene (C 6 H 6 ) and toluene (C 7 H 8 ). They are found in a variety of household and industrial products such as germicides, antiseptics and several different household cleaners. Many of the aromatic hydrocarbons are toxic, carcinogenic and otherwise hazardous compounds that are frequently found as contaminants of soil and ground water (Siegrist 1992;Anon 2002). Strains of R. pickettii are capable of degrading many of these aromatic hydrocarbons and using them as both a carbon and an energy source Olsen 1990, 1992). This is achieved by means of multi-enzyme pathways such as the Tbu pathway of R. pickettii PKO1, a soil isolate, which can convert aromatic hydrocarbons into catechols Olsen 1990, 1991;Olsen et al. 1994). The tbu pathway of R. pickettii PKO1 has been cloned as a 26AE5-kbp DNA fragment designated pRO1957 and expressed in Pseudomonas aeruginosa (Olsen et al. 1994). The genes encoding enzymes for this catabolic pathway have been shown to be organized into three operons. The first is the tbuA1UBVA2C and tbuT (Genbank accession numbers AY541701 and U04052) (Genbank accession number U72645) operon encoding the initial toluene-3-monooxygenase and the transcriptional activator TbuT . The second is the tbuD operon encoding phenol ⁄ cresol hydroxylase (Genbank accession number M98806) Olsen 1990 1992), and the third is the tbuWEFGKIHJ operon encoding enzymes of the meta cleavage pathway for conversion of catechol and methylcatechols to tricarboxylic acid cycle intermediates (Kukor and Olsen 1991). The first step in the degradation pathway is the hydroxylation of toluene or benzene into the intermediates cresol or phenol by a monooxygenase enzyme ( Fig. 1) Olsen 1990, 1992;Olsen et al. 1994). Toluene-3-monooxygenase (T3MO) was first reported to hydroxylate toluene at the meta position, producing primarily m-cresol (Olsen et al. 1994). It has subsequently been discovered that T3MO actually hydroxylates mono-substituted benzenes predominantly at the para position (Fishman et al. 2004). Therefore, the enzyme T3MO was renamed toluene para-monooxygenase (TpMO). TpMO is regulated by the tbuT locus and is induced by other compounds such as ethylbenzene (C 8 H 10 ), xylene (C 8 H 10 ) and trichloroethylene (C 2 HCl 2 ) . The phenol or p-cresol is then hydroxylated into catechol by a phenol ⁄ cresol hydroxylase (Fig. 1). The tbuD operon encodes phenol hydroxylase  Kiyohara et al. (1992), Takizawa et al. (1995) (EC Number 1AE14AE13AE7) and is regulated by the tbuR gene. This phenol hydroxylase is a flavoprotein that is capable of degrading a wide assortment of phenols. It exists in the form of a dimer and uses NADPH as a co-substrate (Kukor and Olsen 1992 (Kukor and Olsen 1991). The steps of this pathway can be seen in Fig. 1. The tbuWEFGKIHJ operon is controlled by tbuS, which represses the operon in the absence of phenol and activates it when the effector molecules are present by forming a transcription activator complex. Ralstonia pickettii also has the ability to metabolize toluene and other aromatic hydrocarbons under hypoxic conditions. Ralstonia pickettii PKO1 can metabolize toluene when oxygen levels are 25% of air-saturated water which distinguishes it from other toluene metabolizing bacteria. PKO1 can degrade toluene at oxygen levels as low as 2 mg of dissolved O 2 per litre (Kukor and Olsen 1996); this can be attributed to kinetic and binding differences within the enzyme catechol 2, 3-dioxygenase. The R. pickettii enzyme has a higher turnover rate and a twofold greater affinity for the substrate than non-hypoxic strains (Kukor et al. 1993;Kukor and Olsen 1996). PKO1 adapts to hypoxic environments by its ability to use nitrate as an alternative electron acceptor to oxygen for the catabolism of aromatic hydrocarbons (Kukor and Olsen 1996). The bioremediation of polluted groundwater and toxic waste sites requires that the bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis, where R. pickettii PKO1 is attracted to toluene, but the response is dependent on induction by growth with toluene (Parales et al. 2000).
Ralstonia pickettii PKO1 was shown to degrade 99AE9% of benzene, toluene, ethylbenzene, ortho-, meta-and paraxylene, and styrene, and 75AE9% of 1, 2, 4-trimethylbenzene at a concentration of 0AE2 mg l )1 after 48 h in a hydrocarbon degradation assay indicating that this organism has a wide range and high activity against many potential substrates (Leahy et al. 2003).

Trichloroethylene
Ralstonia pickettii strain PK01 is one of the most extensively studied degraders of trichloroethylene (TCE) (C 2 HCl 2 ), which is a suspected carcinogen (Anon 1976) and US EPA priority pollutant (Anon 1980 (Rajagopal 1986). The key enzyme in the breakdown of TCE in R. pickettii PKO1 is toluene para-monooxygenase. This then follows the same pathway as the aromatic hydrocarbons. The biodegradation of toluene and TCE by the organism is induced by TCE at high concentrations via the tbuT locus . Ralstonia pickettii itself demonstrates a tolerance towards TCE toxicity through two different systems (Park et al. 2002). The first system is responsible for the tolerance to solvent stress (e.g. disturbance of bacterial membrane by hydrophobic compounds), and the other system allows the bacteria to tolerate toxic intermediate stress (i.e. the ability of the organisms to survive toxic intermediates produced from the oxidation of TCE). PKO1 also has the ability to degrade TCE under hypoxic conditions using nitrate as an alternative electron acceptor to oxygen as with aromatic hydrocarbons (Leahy et al. 1996). In recent trials, R. pickettii PKO1 achieved 83AE9% removal of TCE at a concentration of 0AE2 mg l )1 after 48 h in a hydrocarbon degradation assay (Leahy et al. 2003).

1, 4-Dioxane
is likely a human carcinogen (DeRosa et al. 1996) and a significant emerging water contaminant. It is extensively used as a stabilizer for chlorinated solvents such as 1, 1, 1-trichloroethane (TCA). It is used in manufacturing several organic chemicals including polystyrene, wood stains, varnishes and SBR latex production (Zenker et al. 2003) and as a wetting agent in paper and textile processing industries. It has been detected as a contaminant in both surface waters and groundwaters (Johns et al. 1998;Abe 1999;Jackson and Dwarkanath 1999). Ralstonia pickettii PKO1 has been shown to degrade 50 mg l )l of 1, 4-Dioxane in the presence of a hydrocarbon inducer at a rate of 0AE31 ± 0AE007 mg h )1 mg )1 protein (Mahendra and Alvarez-Cohen 2006).

Chlorinated phenolic compounds
Chlorophenol compounds are used widely as pesticides and biocides, and some, especially monochlorophenols, can be formed during the chlorination of wastewaters and by the breakdown of chlorinated aromatic compounds (Pritchard et al. 1987). Chlorinated compounds have a high degree of environmental persistence as well as a high solubility in water (Smith and Novak 1987). Finding ways to remove chlorophenol compounds presents an important challenge. Ralstonia pickettii strain LD1 (purified from a bacterial biofilm) can utilize monochlorophenols as carbon sources and catabolize these compounds without the use of supplemental nutrients or cofactors (Fava et al. 1995). LD1 can utilize 2-chlorophenol (1AE51 mmol l )1 ), 3-chlorophenol (0AE57 mmol l )1 ) and 4-chlorophenol (0AE75 mmol l )1 ) (C 6 H 5 ClO). The chlorophenol compounds are metabolized into chlorocatechols. The mechanism of degradation, however, is still unclear (Fava et al. 1995). The LD1 strains' ability to mineralize monochlorophenols can be enhanced with the addition of vitamins to the culture medium. The addition to culture medium of a vitamin solution [containing biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, niacin, pantothenic acid, cyanocobalamin, p-aminobenzoic acid and thioctic acid (total final concentration £ 600 ppb)] resulted in a 7-16% increase in the amount of target compounds degraded over the incubation period required for the concentration of the compound in the cultures to drop to approximately zero (Kafkewitz et al. 1996). 2, 4, 6-Trichlorophenol (2, 4, 6-TCP) (C 6 HCl 2 O) is used widely as a biocide, a wood preservative, an antiseptic, a glue preservative, in anti-mildew treatment and in manufacturing other chemicals (Anon 1990). The US EPA has classified 2, 4, 6-TCP as a possible human carcinogen. These compounds are toxic and can accumulate in the environment as they are highly resistant to degradation (Anon 1999). The ability of R. pickettii and other organisms, e.g. Rhodococcus chlophenolicus and Streptomyces rochei, to degrade this CP compound has been examined (Apajalahti and Salkinoja-Salonen 1987;Zabolina et al. 1995). Three known strains of R. pickettii (DTP0309, DTP0405, DTP0602), all isolated from soil, are capable of using 2, 4, 6-TCP as a carbon source, with strain DTP0602 being the most rapid degrader (Kiyohara et al. 1992). These soil isolates were able to use 0AE5 mmol l )1 2, 4, 6-TCP as a growth substrate and resting cell suspensions degraded up to 2AE5 mmol l )1 2, 4, 6-TCD. These bacteria consume 2, 4, 6-TCP and leave residual chloride ions in the culture media. They can also degrade other isomers of TCP as well as pentachlorophenol (PCP) (Kiyohara et al. 1992). In R. pickettii, the had locus encodes the genes responsible for degradation of 2, 4, 6-TCP. Moreover, 2, 4, 6-trichlorophenol-4-dechlorinase (hydroxylase) (Genbank accession number: D86544) isolated from the strain DTP0602 is responsible for the first step in the degradation of 2, 4, 6-TCP (Takizawa et al. 1995). The pathway is shown in Fig. 2. A hydroxyquinol 1, 2-dioxygenase enzyme (Genbank accession number: D86544, EC Number 1AE13AE11AE37) then degrades the intermediate products created as shown in Fig. 2 (Hatta et al. 1999). Homology between the 2, 4, 6-trichlorophenol-4-dechlorinase in R. pickettii and the 2, 4, 6-trichlorophenol-4-monooxygenase in Azotobacter sp. GP1 has been found, particularly in the NH 2 -terminal amino region (Wieser et al. 1997). The genes responsible for the degradation of 2, 4, 6-Trichlorophenol from R. pickettii have been cloned in both Escherichia coli and P. putida using a transposon tagging strategy (Takizawa et al. 1995).

Lantadenes
The lantana plant has encroached on many forests, orchards and pastureland in many parts of the world (Sharma et al. 1988;Pass 1991). Lantadenes are pentacyclic triterpenoid compounds derived from the lantana plant. If consumed by grazing animals, they cause cholestasis, hepatotoxicity and photosentisation (Sharma et al. 1988). These cyclic triterpenoids are difficult compounds to degrade (Krasnobajew 1984). They have shown promise as pharmacological compounds as they inhibit the Epstein Barr virus (Inada et al. 1995) and demonstrate anti-HIV, anti-tumour, anti-bacterial and anti-inflammatory activities (Li et al. 1993;Fujioka et al. 1994;Pengsuparp et al. 1994). A strain of R. pickettii isolated from soil sampled proximal to lantana plants has demonstrated the ability to use lantadene A as a carbon source; however, this utilization was inhibited when other carbon-containing compounds were added to the growth medium (Sharma et al. 1997). This may lead to the exploitation of this bacterium in combating toxicity in grazing animals and in biotransforming lantadene into useful bioactive compounds.

3, 4-Dichloropropionanilide
3, 4-Dichloropropionanilide (C 9 H 9 Cl 2 NO) is the chemical name for a family of herbicides known as amides. It is used extensively in rice production for the control of grasses, especially barnyard grass and broadleaf weeds in rice (Barnes et al. 1987). It has been shown to have neurotoxic and immunotoxic effects in mice (Cuff et al. 1996). Approximately 70% of the US rice crop is treated with propanil, accounting for more than 95% of the 2AE7-3AE6 million kilogram of this active ingredient (AI) produced annually (Gianessi and Anderson 1995a,b;Aspelin and Grube 1999). A soil isolate of R. pickettii found demonstrated resistance to 3, 4-dichloropropionanilide because of the enzyme propanil hydrolase (Hirase and Matsunaka 1991).
Research is being carried out on the mechanism of resistance of R. pickettii to these herbicides with the aim of using the genes responsible to produce herbicide-resistant transgenic rice plants (Piruzian et al. 1988).

2, 4-Dichlorophenoxyacetic acid
2, 4-Dichlorophenoxyacetic acid (C 8 H 6 Cl 2 O 3 ) is a systemic chlorophenoxy herbicide used widely in Canada (more than 4 million kilograms annually) to control broadleaf weeds in cereal cropland and in industrial property, lawns, turf, pastures, non-cropland and aquatic weeds (Anon 1986a). Commercial 2, 4-Dichlorophenoxyacetic acid products are marketed as alkali salts, amine salts and ester formulations. Many micro-organisms, including the members of the Pseudomonas family, can degrade 2, 4-Dichlorophenoxyacetic acid (Ka and Tiedje 1994;Ka et al. 1994aKa et al. , 1994b. The structural gene tfdA from plasmid pJP4 encoding the first of the functional enzymes necessary for the transformation of chlorocatechols into 3-oxoadipate (Don and Pemberton 1981) has been used as a probe to detect bacterial populations with 2, 4-Dichlorophenoxyacetic acid degrading capability. In R. pickettii, homologous tfd genes have been shown to be plasmid borne (Ka et al. 1994a). One R. pickettii isolate (designated 712) contains a 40AE9-kb plasmid that Ralstonia pickettii and environmental biotechnology M.P. Ryan et al. hybridizes to a 2, 4-Dichlorophenoxyacetic acid tfdA gene probe and shares the features in common with pKA2 from Variovorax paradoxus (formally Alcaligenes paradoxus) (Willems et al. 1991). pKA4 is self-transmissible, strongly hybridizes to pKA2 and has a similar restriction pattern. It is thought that these micro-organisms may have exchanged plasmids by intergeneric transfer (Ka and Tiedje 1994). The 2, 4-Dichlorophenoxyacetic acid degrading genes from the pKA2 plasmid may undergo recombination between the chromosome and the plasmid. This is well documented for aromatic hydrocarbon-degradative determinates on the Pseudomonas TOL plasmid that integrate into the host chromosome (Jeenes and Williams 1982;Sinclair et al. 1986;Ka and Tiedje 1994) and has been demonstrated with pKA2, which was transferred to Burkholderia cepacia and found to integrate into the chromosome of that organism.

Nitroaromatics
While nitrobenzene (C 6 H 5 NO 2 ) is primarily used in the production of aniline and aniline derivatives, such as methyl diphenyl diisocyanate (MDI), it also finds use in manufacturing rubber chemicals, pesticides, dyes and pharmaceuticals (Anon 1991). Nitrobenzene is also used in shoe and floor polishes, leather dressings, paint solvents and other materials in order to mask unpleasant odours. Substitution reactions with nitrobenzene are used to form m-derivatives (Anon 1991;Sittig 1991). Redistilled, as oil of mirbane, nitrobenzene has been used as an inexpensive perfume for soaps. A significant market for nitrobenzene is its use in the production of analgesic acetaminophen (Anon 1991). In 1992, releases of nitrobenzene to the environment reported to the Toxic Chemical Release Inventory by certain types of US industries totaled to about 917 000 pounds (Anon 1992). Because of its toxicity, nitrobenzene has been listed as a priority pollutant by the US EPA as far back as 1979 (Keith and Telliard 1979) and has been added to the list of compounds regulated under the Resource Conservation and Recovery Act (Hanson 1990). Two different pathways of degradation of nitroaromatics have been found in two different strains of R. pickettii. In R. pickettii strain YH105 (sludge isolate), degradation of nitroaromatics occurs via a two-step enzymatic process that uses two chromosomally encoded genes: p-nitrobenzoate reductase and p-hydroxylaminobenzoate lyase as outlined in Fig. 3 (Genbank accession number AF187879). YH105 is able to degrade up to 15 mmol l )1 p-nitrobenzoate to protocatechuate via p-hydroxylaminobenzoate; protocatechuate then enters the TCA cycle (Yabannavar and Zylstra 1995). The genes responsible have been cloned and expressed in Escherichia coli. Similar nitroreductases have been found in Comamonas acidovorans NBA-10 (  and Pseudomonas pseudoalcaligenes JS45 (Somerville et al. 1995); the enzyme hydroxylaminolyase; however; has only been purified from C. acidovorans NBA-10 . Ralstonia pickettii PKO1 degrades nitrobenzene, using TpMO, to 3-and 4-nitrocatechol via 3-and 4-nitrophenol, and these nitrocatechols are then slowly degraded to unidentified metabolites. Haigler and Spain (1991) had not identified the enzymes responsible for addition of the second hydroxyl group to the nitrophenols to form nitrocatechols; however, it has subsequently been shown that this reaction proceeds down the tbu pathway (Parales et al. 1997;Fishman et al. 2004).
Quinoline Quinoline (C 9 H 7 N) and its derivatives occur widely in coal tar, bone oil, oil shale and plant alkaloids, and serve as intermediates and solvents in the chemical industry. Quinoline and some of its derivatives are reported to be toxic, carcinogenic and mutagenic (Minako et al. 1977;Sideropoulos and Secht 1984). The widespread use of quinoline and its derivatives entails that these compounds, together with many other environmental chemicals, are distributed in the environment, thus polluting soil and water (Miethling et al. 1993;Sutton et al. 1996). Degradation of quinoline by microbial processes has attracted much interest in recent years. In 2002, a pure strain identified as B. pickettii was isolated from the activated sludge of a coke-oven wastewater treatment plant through enrichment using quinoline as sole source of carbon and nitrogen. The isolate was then used to test quinoline biodegradation by free cells, and this was shown to remove 500 mg l )1 of quinoline after 9 h (Jianlong et al. 2002).

N-Nitrosodimethylamine
N-nitrosodimethylamine (NDMA) (C 2 H 6 N 2 O) is considered a probable human carcinogen (Anon 1986b). This compound is regulated in USA waters with an US EPA cleanup level of 0AE7 ng l )1 (Anon 2001). Its presence in the environment has been linked to aerospace facilities through the decomposition of hydrazine-based rocket fuels (MacDonald 2002) and more generally to the discharge of water and wastewater disinfected with chlorine (Njam and Trussell 2001;Mitch et al. 2003). In the latter case, it appears that secondary amines react with chloramine to form a hydrazine intermediate that is in turn oxidized to NDMA (Mitch and Sedlak 2002). Its persistence in groundwater aquifers has been responsible for the closure of municipal drinking water wells and its listing as a priority pollutant (Mitch et al. 2003). Ralstonia pickettii PKO1 has been found to degrade this compound in the presence of toluene at a rate of 1 ng mg )1 min )1 (Sharp et al. 2005). It was not stated in the paper whether NDMA was completely degraded or just transformed.

Conclusions
Ralstonia pickettii has the ability to survive and prosper in oligotrophic environment and use a variety of compounds as energy and carbon sources. The organism already has demonstrated its capacity to degrade a number of toxic substances (Table 1), making it an excellent candidate for bioremediation. It has several advantages over other candidate strains being studied such as B. vietnamiensis G4 [which is currently undergoing intense study (O'Sullivan and Mahenthiralingam 2005)] or P. putida, in that it is only weakly pathogenic with no phytopathogenic or animal pathogenic incidents being reported. Several areas of application have the potential to use R. pickettii, which include treatment of contaminated groundwater and municipal and industrial waste and sewage. Examples include the removal of toluene from groundwater of which a successful test was carried out in Hanahan, South Carolina. The use of bioremediators prevented the contamination of residential areas from a massive fuel leak from a nearby military installation (Vroblesky et al. 1997). Natural microbial communities in the area were stimulated with nutrients to increase the biodegradation of toluene. Through the use of nutrient addition (Kafkewitz et al. 1996), bioremediation of compounds such as chlorophenols and pesticides found in sewage effluent and groundwater could be increased. The degradation of toxic compounds by micro-organisms that are part of the microflora of wastewater treatment plants could be augmented if plasmids and genes responsible for these properties in R. pickettii were transferred from R. pickettii to the indigenous micro-organisms. When genome sequence data of R. pickettii is available, the analysis of degradative processes will potentially allow optimization of the physiological state of R. pickettii strains during bioremediation applications and could potentially lead to the construction of novel or more proficient pathways for degradation. Ralstonia pickettii strain PKO1 could have the potential to be a super biodegrader with the introduction of plasmids bearing other degradative enzymes, e.g. pKA4, and integrating other genes from different bacteria into the chromosome to assist in the breakdown of toxic compounds. An example of the potential of genome analysis can be seen in the alteration of TpMO so that the enzyme hydroxylates all three positions of toluene as well as both positions of naphthalene. The mutation in the enzyme produced a toluene paramonooxygenase variant that formed 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene. This was the first time a true meta-hydroxylating toluene monooxygenase was created (Fishman et al. 2005). The demand for a safe organism makes R. pickettii a natural choice for bioremediation applications.