Biodegradation potential of the genus Rhodococcus

Environment International 35 (2009) 162–177

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Environment International j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e n v i n t

Review article

Biodegradation potential of the genus Rhodococcus Ludmila Martínková ⁎, Bronislava Uhnáková, Miroslav Pátek, Jan Nešvera, Vladimír Křen Centre of Biocatalysis and Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic

a r t i c l e

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Article history: Received 29 February 2008 Accepted 22 July 2008 Available online 11 September 2008 Keywords: Rhodococcus Aromatics Nitriles Biodegradation

a b s t r a c t A large number of aromatic compounds and organic nitriles, the two groups of compounds covered in this review, are intermediates, products, by-products or waste products of the chemical and pharmaceutical industries, agriculture and the processing of fossil fuels. The majority of these synthetic substances (xenobiotics) are toxic and their release and accumulation in the environment pose a serious threat to living organisms. Bioremediation using various bacterial strains of the genus Rhodococcus has proved to be a promising option for the clean-up of polluted sites. The large genomes of rhodococci, their redundant and versatile catabolic pathways, their ability to uptake and metabolize hydrophobic compounds, to form biofilms, to persist in adverse conditions and the availability of recently developed tools for genetic engineering in rhodococci make them suitable industrial microorganisms for biotransformations and the biodegradation of many organic compounds. The peripheral and central catabolic pathways in rhodococci are characterized for each type of aromatics (hydrocarbons, phenols, halogenated, nitroaromatic, and heterocyclic compounds) in this review. Pathways involved in the hydrolysis of nitrile pollutants (aliphatic nitriles, benzonitrile analogues) and the corresponding enzymes (nitrilase, nitrile hydratase) are described in detail. Examples of regulatory mechanisms for the expression of the catabolic genes are given. The strains that efficiently degrade the compounds in question are highlighted and examples of their use in biodegradation processes are presented. © 2008 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . The genus Rhodococcus . . . . . . . . . . . . . . . Degradation of aromatic compounds . . . . . . . . . 3.1. The central pathways. . . . . . . . . . . . . 3.2. Monoaromatic and polyaromatic hydrocarbons 3.3. Phenols and aromatic acids. . . . . . . . . . 3.4. Halogenated compounds . . . . . . . . . . . 3.5. Nitroaromatics. . . . . . . . . . . . . . . . 3.6. Other aromatics . . . . . . . . . . . . . . . 3.7. Desulfurization . . . . . . . . . . . . . . . 4. Biodegradation of nitriles . . . . . . . . . . . . . . 4.1. Nitrile-converting enzymes in rhodococci . . . 4.2. Acrylonitrile . . . . . . . . . . . . . . . . . 4.3. Saturated aliphatic nitriles . . . . . . . . . . 4.4. Aromatic nitriles . . . . . . . . . . . . . . . 5. Conclusions and prospects . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

⁎ Corresponding author. Tel.: +420 296 442 569; fax: +420 296 442 509. E-mail address: [email protected] (L. Martínková). 0160-4120/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2008.07.018

In the 1980s, rapidly increasing environmental contamination raised concerns about the health of ecosystems and humans and interest in biological methods of pollution cleanup (bioremediation). The genus Rhodococcus was soon regarded as one of the most

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promising groups of organisms suitable for the biodegradation of compounds that cannot be easily transformed by other organisms (Warhurst and Fewson, 1994). So far only pseudomonads and related bacteria have been reported to possess comparable biodegradation abilities. The xenobiotic compounds metabolized by rhodococci cover a wide range of structural groups, including aliphatic and aromatic hydrocarbons, oxygenates, halogenated compounds, including polychlorinated biphenyls, nitroaromatics, heterocyclic compounds, nitriles and various herbicides. Many of these substrates are complex synthetic molecules with remarkable stability and toxicity. The significance of rhodococci in environmental biotechnology was discussed in the review characterizing the genus Rhodococcus (Bell et al., 1998) and in the reviews concerned primarily with the use of enzymes from rhodococci as biocatalysts (Banerjee et al., 2002; Martínková and Křen, 2002; Singh et al., 2006). The biodegradations of recalcitrant compounds by rhodococci were briefly summarized by Larkin et al. (2005). Another review from the same group discussed the biochemical, physiological and genetic aspects of the catabolic versatility of the genus Rhodococcus (Larkin et al., 2006). Examples of the new tools for gene engineering in rhodococci were described by van der Geize and Dijkhuizen (2004). Research into the biotechnological use of rhodococci has intensified significantly within the last 25 years, as illustrated by citation analysis (Fig. 1). Both the numbers of publications and patents show the same trend, increasing dramatically after 1995. Among the chemicals degraded by rhodococci, the most interest was shown in hydrocarbons, followed by nitriles and phenolic compounds, while the impact of these bacteria in desulfurization was distinguished later. In this review, we focus on two large groups of compounds transformed by rhodococci, namely aromatics and nitriles. The two groups of different size with different technological applications may conveniently illustrate the current directions of research in rhodococci. Many of the aromatics and nitriles became widespread environmental pollutants, a number of them being on the Priority Pollutant List of United States Environmental Protection Agency (USEPA, 1996) and Priority List of Hazardous Substances of Agency for Toxic Substances and Disease Registry (ATSDR, 1997). These two groups of compounds belong to the most studied contaminants as well as substrates for practically important biotransformations. The usage, environmental impact and toxicity of these contaminants are described here and suitable bioremediation tools (strains, enzymes) for each compound group are highlighted. Special attention is paid to biotransformation products and their potential toxicity. Background knowledge of the biodegradation pathways at the protein and gene level is also presented.

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Fig. 1. Number of publications (A) and patents (B) concerned with nitriles and aromatic compounds transformed by rhodococci (according to www.scopus.com). Queries: Title/ Abstract/Keywords: Rhodococcus and (□) nitril⁎; ( ) benzene or toluene or xylene or naphthalene or ⁎biphenyl or hydrocarbon⁎; ( ) ⁎phenol; (■) ⁎thiophene or desulfurization.

2. The genus Rhodococcus Rhodococci are non-sporulating, aerobic bacteria classified into mycolate-containing nocardioform actinomycetes (Finnerty, 1992). The genus Rhodococcus is comprised of genetically and physiologically diverse bacteria, which have been isolated from various habitats, for example soils and sea water. Since they are equipped with a large number of enzymatic activities, unique cell wall structure and suitable biotechnological properties, Rhodococcus strains may be utilized as industrial organisms, primarily for biotransformations and the biodegradation of many organic compounds (Bell et al., 1998). Rhodococci can thus be applied in environmental remediation and in the pharmaceutical and chemical industries (van der Geize and Dijkhuizen, 2004; Larkin et al., 2005). The cell wall of rhodococci contains mycolic acids, like that of the genera Mycobacterium, Nocardia and Corynebacterium. The long aliphatic chains of mycolic acids in the cell envelope facilitate the uptake of hydrophobic substrates into the cells. The potential to metabolize hydrophobic substrates is further supported by the production of surfactants. These compounds (e.g. trehalose-containing glycolipids)

facilitate the adhesion of cells to hydrophobic phases in two-phase systems, decrease the interfacial tension between phases and disperse hydrophobic compounds (Finnerty, 1992; Bell et al., 1998; de Carvalho and da Fonseca, 2005). The presence of various substrates in the growth medium induces changes in the fatty acid composition of the membrane lipids and can thus alter the fluidity of the cell envelope. The ability to modulate the fatty acid composition in the cell envelope is probably also important to the resistance of Rhodococcus cells to many toxic compounds (de Carvalho and da Fonseca, 2005). The capacity of Rhodococcus strains to form biofilms on carriers suitable for biotechnological purposes further enhances the tolerance of their cells to such compounds (Čejková et al., 2005). Their cells can persist in starvation conditions and the degradation of pollutants is in many cases not repressed in the presence of more easily degradable carbon sources (Bell et al., 1998). The large genomes of Rhodococcus strains (e.g. Rhodococcus jostii RHA1; 9.7 Mb) provide a redundancy of catabolic pathways based on gene homologues (McLeod et al., 2006). Rhodococci typically harbour large linear and circular plasmids, which may accommodate a large number of catabolic genes. The high

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frequency of recombination described in some Rhodococcus strains may contribute to the flexibility of their genome and ability to easily acquire new genes and consequently, new enzymatic activities (Larkin et al., 2006). However, analyses of the first sequenced genome of R. jostii RHA1 revealed that this strain has acquired few genes by recent horizontal transfer and the genome contains few recent gene duplications (McLeod et al., 2006). Improved methods and tools for genetic engineering in rhodococci, which have appeared in recent years, provide new means for constructing strains for biotechnological applications (van der Geize and Dijkhuizen, 2004). Aromatics and nitriles, two large groups of compounds that are used in a vast array of practical applications and simultaneously contribute an alarming amount to environmental pollution are particularly metabolized by rhodococci. 3. Degradation of aromatic compounds Large amounts of natural aromatic compounds (molecules containing one or more benzene rings) in the biosphere originate from decaying plant material (e.g. from lignin, a complex phenolic polymer). In addition, xenobiotic aromatic compounds are released

due to leaks and spills from various industrial processes, and thus form hazardous environmental contaminations. Major sources of aromatic pollutants are the chemical and pharmaceutical industries, intensive agriculture, pulp and paper bleaching, mining and fossil fuels. There is great concern about the harmful effects of aromatic xenobiotics on natural ecosystems. Although immediate lethal toxicity is rare, the sublethal toxicity of these substances may cause a wide range of effects on living organisms, including humans, from subtle biochemical and physiological disturbances to severe toxic and mutagenic effects on development and reproduction (Diaz, 2004; Andreoni and Gianfreda, 2007). Moreover, many of these synthetic aromatic compounds persist in the environment due to their recalcitrant nature. The abilities of microorganisms to degrade natural aromatic compounds (e.g., aromatic amino acids and amines, hydroxylated benzoates and phenylcarboxylic acids) are widespread among microbial species. Xenobiotic aromatic compounds are degraded by a smaller group of bacteria, particularly by strains of the Gram-negative bacteria of Pseudomonas, Sphingomonas, Acinetobacter, Ralstonia and Burkholderia and the Gram-positive genus Rhodococcus. Bioremediation of the polluted soil and water by such bacteria has become a generally accepted procedure. Rhodococcus strains were found to be

Fig. 2. Peripheral pathways of biodegradation of aromatic compounds in rhodococci. R = alkyl.

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suitable for these biotechnological processes due to their resistance to a number of toxic xenobiotics and their ability to degrade many of these compounds, including the recalcitrant ones. Various strains of rhodococci degrade benzene and its derivatives (toluene, ethylbenzene, xylenes, biphenyl), polycyclic and heterocyclic aromatic compounds, phenolic compounds, aromatic acids, halogenated aromatics, including polychlorinated biphenyls (PCBs), amino- and nitro-derivatives of aromatic compounds (e.g., aniline and nitrophenol), ethers and pesticides and desulfurize coal and petroleum products (van der Geize and Dijkhuizen, 2004; Larkin et al., 2005). Members of the genus Rhodococcus represent an abundant part of indigenous bacterial communities occuring in localities contaminated with various aromatic pollutants (Fahy et al., 2006; Leigh et al., 2006). 3.1. The central pathways A wide range of natural and xenobiotic aromatic compounds are aerobically catabolized by bacteria via a large variety of upper or peripheral pathways resulting in a limited number of central intermediates. These common metabolites are further degraded through a few central pathways to finally provide intermediates of the citrate cycle (Harwood and Parales, 1996). Such arrangements for the catabolism of aromatic compounds were found in both the longerstudied pseudomonads (Jimenez et al., 2002) and in the recently characterized R. jostii RHA1 (McLeod et al., 2006). According to the R. jostii RHA1 genome sequence, 26 peripheral pathways and 8 central pathways are involved in the catabolism of aromatic compounds (McLeod et al., 2006). Within the peripheral pathway, the aromatic compound is modified in a number of steps, including the action of monooxygenase or dioxygenase resulting in the formation of a

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dihydroxylated benzene ring. The main resulting metabolites are catechol and protocatechuate with hydroxyl groups at positions 1,2 and gentisate with hydroxyl groups at positions 1,4 (Fig. 2). The ring cleavage of both catechol and protocatechuate catalyzed by dioxygenases occurs either between the hydroxyl groups (intradiol cleavage or ortho-cleavage) or adjacent to one of the hydroxyl groups (extradiol-cleavage or meta-cleavage) (Fig. 3). The ortho-cleavage pathway of catechol and protocatechuate converging at 3-oxoadipate is also called the 3-oxoadipate (β-ketoadipate) pathway (Harwood and Parales, 1996). In gentisate, cleavage occurs between the carboxyl group and adjacent hydroxyl group (Dagley, 1971). Whereas the upper pathways are only present in a limited number of bacterial strains, the central pathways of aromatic catabolism are common in bacteria (Harwood and Parales, 1996). The catechol, protocatechuate and gentisate dioxygenases play the central roles in degradation of aromatic compounds because they catalyze the critical and chemically difficult aromatic ring-cleavage reaction (Broderick, 1999). Chlorocatechols resulting from the degradation of various chlorinated aromatics like chlorobenzenes and chlorobenzoates are mainly catabolized by a modified ortho-cleavage pathway, whereas methylated catecholic intermediates generated from the breakdown of aromatic hydrocarbons like naphthalene and alkylated compounds like toluene and xylene, are mostly degraded via the meta-pathway (Maruyama et al., 2005). The enzymes, genes and regulatory mechanisms of the 3oxoadipate pathway were found to be similar in various bacteria, e.g., in Acinetobacter, Arthrobacter, Burkholderia, Pseudomonas and Rhodococcus. Catechol or protocatechuate are catabolized in four reactions into 3-oxoadipate (Fig. 3), which is in two further reactions converted to acetyl-coenzyme A and succinyl-coenzyme A. In many

Fig. 3. Central pathways of catabolism of aromatic compounds in rhodococci.

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bacteria the catA, catB and catC genes, coding for catechol 1,2dioxygenase (C1,2O), cis,cis-muconate cycloisomerase and muconolactone isomerase respectively, form operons and are clustered with the catR gene coding for a LysR-type transcriptional regulator (Chugani et al., 1997; Murakami et al., 2004). These LysR-type regulators are activators and expression of the operons is induced by cis,cis-muconate. In contrast, the CatR regulators in some Rhodococcus strains were found to be of the IclR-type (Eulberg and Schlömann, 1998; Veselý et al., 2007) and CatR was shown to function as a repressor in R. erythropolis CCM 2595 (Veselý et al., 2007). IclRtype regulators in most cases control the protocatechuate catabolic operons (Eulberg et al., 1998b; Gerischer et al., 1998). In anilinedegrading Rhodococcus sp. AN22, catABC operon expression was found to be constitutive because the insertion element IS204 disrupted the regulatory catR gene (Matsumura et al., 2006). The subfamily of C1,2O enzymes initiating the cleavage of the aromatic ring shows high enzymatic activity for chlorocatechols (Eulberg et al., 1997, 1998a; Suvorova et al., 2006). Another subfamily of C1,2O exhibits a high specificity for methylcatechols (Murakami et al., 1997; Kolomytseva et al., 2007). Enzymes initiating the meta-cleavage, catechol 2,3-dioxygenases, were described in 2-methylanilinedegrading R. rhodochrous (Candidus et al., 1994) and in strains degrading benzene (Na et al., 2005b), xylenes (Kim et al., 2005) and naphthalene (Kulakov et al., 2005). Gentisate is an intermediate of naphthalene, salicylate and 3hydroxybenzoate degradation. Gentisate is oxidized by 1,2-dioxygenase to maleylpyruvate as shown in R. erythropolis (Suemori et al., 1995). Two further reactions catalyzed by maleylpyruvate isomerase and fumarylpyruvate hydrolase finally convert the intermediates to pyruvate and fumarate (Fig. 3). 3.2. Monoaromatic and polyaromatic hydrocarbons Gasoline contains a high proportion of benzene and toluene as well as smaller amounts of mixed xylene isomers and ethylbenzene (collectively known as BTEX), which are regulated hazardous compounds. Thousands of tons of gasoline are lost each year due to leaking storage tanks. Due to their relatively high solubility, BTEX compounds are frequently reported as groundwater contaminants. Bioremediation by natural bacterial communities, in which rhodococci represent an abundant group, is increasingly applied in the elimination of BTEX from groundwater (Fahy et al., 2006). Initial oxidative attack on BTEX converting the compound into a catechol structure and the cleavage of the catechol structure are the key steps in aerobic BTEX degradation (Hendrickx et al., 2006b). Among the bacterial degraders of BTEX compounds (isolated at the oil-refinery site in the Czech Republic), a number of Rhodococcus strains were identified, in addition to the most frequent Pseudomonas strains (Hendrickx et al., 2005, 2006a,b). The set of primers for PCR were designed to detect the presence of (1) monooxygenase and dioxygenase genes involved in the initial attack of bacterial aerobic BTEX degradation and (2) catechol 2,3-dioxygenase genes responsible for the meta-cleavage of the aromatic ring (Hendrickx et al., 2006b). In another screening of benzene-utilizing bacteria, the strains R. opacus B-4 and B-9, highly tolerant to benzene, were isolated from the soil at chemical plants and from roadsides in Japan (Na et al., 2005a). R. opacus B-4 was found to efficiently assimilate benzene, toluene, styrene, ethylbenzene, xylenes, phthalates and naphthalene. The genes of the benzene dioxygenase operon from R. opacus B-4 chromosome were cloned and characterized and an upper pathway for benzene oxidation was proposed. In the first step, benzene is converted to catechol by benzene dioxygenase and the aromatic ring is then cleaved by the action of catechol 2,3-dioxygenase (meta-cleavage). The native plasmid pKNR01 isolated from R. opacus B-4 was used for the construction of a host-vector system for rhodococci, which may facilitate the development of a technological process for bioremediation on the basis of this powerful degrader

(Na et al., 2005b). In another benzene-utilizing strain, Rhodococcus sp. 33, the resulting catechol is degraded by catechol 1,2-dioxygenase (ortho-cleavage) and via the 3-oxoadipate pathway (Paje and Couperwhite, 1996). This strain was found to grow on an extraordinary high concentration of benzene (2% v/v) in a continuous culture, which was maintained for more than 30 days and resulted in 89% benzene degradation (Paje et al., 1997). Linear conjugative catabolic plasmid pBD2 was found to be responsible for isopropylbenzene, ethylbenzene and toluene degradation in R. erythropolis BD2 (Kesseler et al., 1996). The sequencing of this 210-kb plasmid revealed that many of the isopropylbenzene pathway genes are flanked by transposon-related sequences (Stecker et al., 2003). This finding, together with the significant sequence similarities of the ipb genes to the genes of the plasmid-encoded biphenyl pathway in other rhodococci, suggests that the ipb genes were acquired via transposition events and subsequently distributed among rhodococci via horizontal transfer. The o-, m- and p-xylenes contained in petroleum are used as solvents, thinners for paints and in the chemical and pharmaceutical industries and cause widespread contaminations of soil and water. Strains of Rhodococcus (e.g., R. opacus and R. koreensis) degrading o-xylene, one of the most recalcitrant pollutants, were isolated from contaminated soils (Di Gennaro et al., 2001; Kim et al., 2004; Taki et al., 2007). A megaplasmid pDK2, carrying the genes for the initial steps of alkylbenzene catabolism, was found in Rhodococcus sp. DK17 (Kim et al., 2004). The DK17 strain (with a 37-kb gene cluster involved in the degradation of aromatic compounds) was shown to degrade o-xylene and toluene via 3,4dimethylcatechol and 3- and 4-methylcatechol, respectively. The catechol 2,3-dioxygenase in this strain, which starts the meta-cleavage step, was very active, particularly for alkylated catechols, which is unusual among these enzymes (Kim et al., 2005). The strain R. opacus R7 (Di Gennaro et al., 2001), isolated from soil contaminated with polycyclic aromatic hydrocarbons, degrades naphthalene as well as o-xylene. The degradation of naphthalene occurs through the dioxygenation of the aromatic ring, via 1,2-dihydro-1,2-dihydroxynaphthalene, that is further oxidized to salicylate and gentisate. The final aromatic ring cleavage thus proceeds through the gentisate pathway in this case (Di Gennaro et al., 2001). Analysis of the nid gene cluster responsible for the degradation of o-xylene and naphthalene in R. opacus TKN14 showed that in addition to the nidAB genes coding for the subunits of oxygenase, the nidE (rubredoxin) and nidF (auxiliary protein) genes are essential for the oxidation of o-xylene (Maruyama et al., 2005). The nidABEF genes were found to be induced by o-xylene. The authors assume that the nidABEF genes for o-xylene monooxygenase-naphthalene dioxygenase are involved in the degradation pathways of a wide range of aromatic hydrocarbons in Rhodococcus species. A new type of cis-naphthalene dihydrodiol dehydrogenase coded by the narB gene was found in the naphthalene-assimilating strain Rhodococcus sp. NCIMB112038 (Kulakov et al., 2000). Whereas Rhodococcus sp. NCIMB12038 degrades naphthalene via salicylate and gentisate, Rhodococcus strains P200 and P400 were shown to degrade naphthalene via catechol and catechol-2,3-dioxygenase activity (Kulakov et al., 2005). Although the arrangement of the nar genes in several transcription units differs in the strains investigated, all are induced by naphthalene. The organization of the nar genes in modules and their presence on plasmids (in NCIMB12038 and P400) or the chromosome (in P200) document that genetic exchange and reshuffling of these genes occurred in Rhodococcus strains during the evolution of the naphthalene degradation pathways (Kulakov et al., 2005). Substituted benzenes, like ethylbenzene (ETB), isopropylbenzene (IPB) and biphenyl (BPH) are also degraded via ring hydroxylation and oxygenolytic cleavage of a catecholic metabolite. In R. jostii RHA1, these compounds are metabolized via a common pathway (Goncalves et al., 2006). BPH is converted to benzoate and 2-hydroxypenta-2,4-dienoate (HPD) in four steps catalyzed by (1) four-component biphenyl dioxygenase (ferredoxin reductase, ferredoxin and two-subunit

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terminal oxygenase coded by bphA1A2A3A4), (2) dihydrodiol dehydrogenase (bphB), (3) 2,3-dihydroxybiphenyl dioxygenase (bphC) and (4) 2hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD). ETB degradation through the same pathway results in HPD and propionate. HPD is further metabolized in three steps (encoded by bphGFE in R. jostii RHA1) to pyruvate and acetyl-coenzyme A (Sakai et al., 2003). Thanks to the knowledge of the complete R. jostii RHA1 genome sequence, a large number of genes potentially involved in the degradation of ETB, IPB and BPH have been identified (McLeod et al., 2006). RHA1 was isolated from soil contaminated with the insecticide lindane (hexachlorocyclohexane) and the strain proved capable of efficiently degrading polychlorinated biphenyls (PCBs), which are cometabolized concurrently with BPH (Seto et al., 1995a) and ETB (Seto et al., 1995b). Three gene clusters (bph, etb1 and etb2) encoding homologous ring-hydroxylating dioxygenase systems were found on the large linear plasmids pRHL1 (1,123 kb) and pRHL2 (443 kb). The genes involved in the further steps of the BPH and ETB pathways are scattered over the RHA1 genome (Goncalves et al., 2006). The genes of the lower or central pathways ben, cat and pca involved in benzoate, catechol and protocatechuate degradation, respectively, are located on the chromosome. DNA microarray analysis revealed that the transcription of bph, etb1 and etb2 gene clusters are induced by BPH, ETB and IPB, which indicates that all these substituted benzenes are catabolized by the same enzymes and the genes are controlled by a common regulatory system. The analysis showed that as many as 22 enzymes may be involved in the upper BPH and ETB pathway. The bphS and bphT genes, coding for a two-component signal transduction system involved in regulating BPH/PCB metabolism were discovered on R. jostii RHA1 chromosome. The bphS and bphT genes are required for transcriptional induction of the bphA1 promoter by biphenyl, benzene, alkylbenzenes and chlorinated benzenes (Takeda et al., 2004a). The transcriptome and proteome analysis of RHA1 showed that the styrene catabolic pathway in Rhodococcus differs from the pathway described in all other bacteria. In R. rhodochrous NCIMB 13259, styrene catabolism was found analogous to biphenyl catabolism, in which a vinylcatechol is produced by direct oxidation of the aromatic ring and subjected to meta-ring cleavage. Pseudomonads and corynebacteria contain specific Sty and Ben pathways and the styrene degradation is initiated via the side chain. In R. jostii RHA1, styrene is oxidized by the same enzymes as BPH and ETB and the resulting vinylcatechol is also further metabolized by meta-cleavage (Patrauchan et al., 2008). The multiplicity and varying activities of Bph and Etb enzyme homologues with various specificities may contribute to the exceptional ability of R. jostii RHA1 to degrade a large number of PCB congeners (see Section 3.4), which are cometabolized with BPH or ETB (Goncalves et al., 2006). Some Rhodococcus strains are capable of degrading polyaromatic hydrocarbons (PAHs) with 3 or 4 benzene rings. Rhodococcus sp. UW1 was shown to use phenanthrene, anthracene, pyrene, fluoranthene and chrysene as sole carbon sources, whereas further PAHs were cometabolized (Walter et al., 1991). The other Rhodococcus sp. degraded anthracene, phenanthrene, pyrene and fluoranthene (Dean-Ross et al., 2001). This strain was found to metabolize fluoranthene via ortho as well as meta cleavage (Dean-Ross et al., 2002). Three strains of rhodococci (R. rhodochrous 172, R. opacus 4a and R. rhodni 135) use fluorene as the sole carbon source (Finkelstein et al., 2003). The latter strain was also reported to oxidize phenanthren into 3-hydroxyphenanthren as the sole product (Baboshin et al., 2005). 3.3. Phenols and aromatic acids Phenols and phenolic compounds (e.g. cresols) are often contained in wastewaters from oil refineries, coal gasification plants, coking plants and the phenol resin industry. Phenolic compounds are among the most frequently found pollutants in rivers, industrial effluents, and

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landfill runoff waters. Their toxicity often reduces the efficiency of wastewater biotreatment. Aerobic biodegradation of phenolic compounds resulting in complete mineralization of these xenobiotics is generally preferred in wastewater clean-up. Cells of R. erythropolis immobilized on inorganic carriers (diatomaceous earth, zeolite) were found to be a suitable basis for an efficient phenol biodegradation process (Prieto et al., 2002; Čejková et al., 2005). Phenol is first converted by phenol hydroxylase to catechol, which is further degraded through ortho- or meta-cleavage. Phenol hydroxylase of R. erythropolis CCM 2595 consists of two subunits encoded by the pheA1pheA2 genes clustered with the pheR gene coding for an AraC-type transcriptional regulator (GenBank Accession Number AJ973228), which was found to function as an activator (Pátek et al., unpublished results). This FAD-dependent phenol hydroxylase was found to be induced by phenol and other aromatic compounds (Fialová et al., 2003; Čejková et al., 2005). The induction of the pheA1pheA2 genes on the transcription level was proved by the studies of the transcriptional fusions of the identified pheA1pheA2 and pheR promoters (Pátek et al., unpublished results). The broad substrate specificity enables this enzyme to converse also p-chlorophenol, pnitrophenol, resorcinol and p-cresol, although these compounds do not support growth of R. erythropolis CCM 2595 (Fialová et al., 2003). The pheA1pheA2–pheR gene cluster is duplicated on the chromosome of R. jostii RHA1 and another copy of the pheA1pheA2 genes is present on the plasmid pRHL1 carried also by this strain (McLeod et al., 2006). The only other Rhodococcus strain, in which the genes coding for phenol hydroxylase were sequenced, is R. erythropolis UPV-1 (GenBank Accession Numbers EU004078 and EU004079). Rhodococcus strains were shown to be efficient degraders of the phenolic compounds contained in creosote (PAHs, phenol and a mixture of o-, m- and p-cresols) used as a wood preservative. Rhodococcus strains growing on all cresol isomers contained as many as five different extradiol dioxygenases (Irvine et al., 2000). In R. opacus 1CP, p-cresol is degraded via 4-methylcatechol and through the orthopathway. This strain was thus shown to possess another catechol-1,2dioxygenase activity, in addition to dioxygenases with specificities for 4-chlorophenol and p-toluate (Kolomytseva et al., 2007). Decomposition of the mixtures of p-cresol, phenol and 2chlorophenol in activated sludge was found to be most efficient when mixed cultures of R. erythropolis and Pseudomonas fluorescens were used. In the presence of p-cresol in the mixture, none of the strains dominated the population and the degradation rate was higher than with pure cultures (Goswami et al., 2005). Rhodococcus strains were also found to degrade a variety of aromatic carboxylic acids like benzoate, phthalate, terephthalate, hydroxybenzoates, protocatechuate and gentisate (Fig. 2). Salicylate (o-hydroxybenzoate) and m-hydroxybenzoate were shown to be catabolized via the gentisate pathway, whereas p-hydroxybenzoate was catabolized via the protocatechuate ortho-pathway in R. erythropolis S1 (Suemori et al., 1995). The first step of p-hydroxybenzoate catabolism, resulting in formation of 3,4-dihydroxybenzoate, is catalyzed by p-hydroxybenzoate hydroxylase which was characterized in detail in R. rhodnii 135 and R. opacus 557 (Jadan et al., 2001, 2004). Benzoate and chlorobenzoates are intermediates of biphenyl and PCB catabolism and their degradation, which proceeds via the catechol or chlorocatechol branch of the ortho-pathway, is therefore essential for the complete breakdown of biphenyl and PCBs (Kitagawa et al., 2001). Phthalate, terephthalate and 4-hydroxybenzoate were found to be degraded via protocatechuate ortho-cleavage in R. jostii RHA1 (Patrauchan et al., 2005). Extensive transcriptomic analysis of R. jostii RHA1 genes showed that terephthalate can also be degraded via the catechol branch of the 3-oxoadipate pathway (Hara et al., 2006). Many strains of Rhodococcus and other related actinomycetes were shown to mostly utilize the 3-oxoadipate pathway for the degradation of benzoate and 4-hydroxybenzoate (Hammann and Kutzner, 1998). The enzymes of the meta-cleavage pathway probably operate mostly in

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the degradations of certain substituted phenols, like 3-methylcatechol and 3-methyl-4-(methylthio)phenol. With m-hydroxybenzoate, the degradation generally proceeds via the gentisate pathway (Hammann and Kutzner, 1998). The complete sequence of the R. jostii RHA1 genome as well as extensive transcriptomic and proteomic analyses have shown that the multiplicity of catabolic pathways for many aromatic compounds (e.g., carboxylic acids, biphenyl and alkylbenzenes) is most likely the basis for the high and versatile degradative potential of Rhodococcus species. 3.4. Halogenated compounds Large amounts of halogenated aromatic compounds have been released to the environment as solvents, pesticides and fire retardants. The substitution of hydrogen with fluorine, chlorine or bromine results in stable compounds that resist to chemical as well as microbial degradation. Mono-, di- and trifluorophenols are degraded by various strains of rhodococci (Bondar et al., 1998; Finkelstein et al., 2000). Ortho hydroxylation of fluorophenols, resulting in formation of the respective fluorocatechols, is the initial step of fluorophenol biodegradation pathway. In R. opacus 1CP, trihydroxyfluorobenzenes (fluoropyrogallols) were identified as further reaction intermediates in biotransformation of monofluorophenols (Finkelstein et al., 2000). R. corallinus 135 was found to be the most efficient strain for degrading various diand trifluorophenols (Bondar et al., 1998). Chlorophenols, chlorobenzenes and polychlorinated biphenyls are among the toxic chloroaromatic compounds efficiently degraded by various Rhodococcus strains. The members of the genus Rhodococcus were found to be the most abundant bacteria in communities applied to degradation of chlorobenzenes in contaminated ground water in situ (Vogt et al., 2004). The aerobic catabolism of chlorophenols, chlorobenzenes and chlorobenzoates is initiated by their hydroxylation and results finally in the respective chlorocatechols. The degradation of chlorocatechols via a modified ortho-cleavage pathway completes the catabolic pathway. R. opacus 1CP was found to efficiently degrade 4-chlorophenol and 2,4-dichlorophenol (Eulberg et al., 1998a) and, after prolonged adaptation, 2-chlorophenol as well. Their hydroxylation leads to 4-chlorocatechol, 3,5-dichlorocatechol and 3chlorocatechol, respectively. Analysis of the R. opacus 1CP genes responsible for the chlorocatechol catabolic pathways showed that the clcBRAD operon is involved in 4-chlorocatechol and 3,5-dichlorocatechol degradation, whereas the clcA2D2B2F operon is involved in 3chlorocatechol degradation. In contrast to the cat and pca operons for the catabolism of catechol and protocatechuate, which are located on the chromosome, the clc and clc2 operons for chlorocatechol degradation were identified on the large linear plasmid p1CP (740 kb) (König et al., 2004). The location of genes involved in the catabolism of halogenated compounds on large plasmids has also been observed in Pseudomonas putida (McFall et al., 1998), Ralstonia eutropha (Ogawa et al., 1999) and Burkholderia sp. (Liu et al., 2001). The new pathway for 3-chlorocatechol degradation (encoded by the clcA2D2B2F operon of R. opacus 1CP) differs from all other known chlorocatechol pathways, because the dechlorinating enzyme is related to muconolactone isomerase, whereas in other pathways it is chloromuconate cycloisomerase (Moiseeva et al., 2002). Apparently, two chlorocatechol catabolic pathways in R. opacus 1CP originated through independent but convergent evolution (Eulberg et al., 1998a). The isolation of R. percolatus from sludge and sediments contaminated by various chlorophenols showed that Rhodococcus strains are able to utilize various mono- di- and trichlorophenols as sole carbon sources (Briglia et al., 1996). The growth of R. opacus 1G on 2,3,5trichlorophenol showed that its phenol hydroxylase catalyzes the oxidative dechlorination of 2,3,5-trichlorophenol at the ortho position, which produces 3,5-dichlorocatechol (Bondar et al., 1999). Such

oxidative dechlorination at the ortho position makes this phenol hydroxylase exceptional, since all chlorophenol o-hydroxylases studied so far prefer monooxygenation of the non-halogenated ortho position. Degradation of pentachlorophenol by the strains Rhodococcus sp. CG-1 and Rhodococcus sp. CP-2 proceeds via para hydroxylation producing tetrachlorohydroquinone (Häggblom et al., 1988). Tetrachlorohydroquinone is then completely dechlorinated through another hydroxylation and three reductive dechlorinations producing trihydroxybenzene, which is a substrate for aromatic ring cleavage (Häggblom et al., 1989). An unusually broad range of haloaromatic compounds, including chlorobenzenes and dichlorobenzenes are utilized as carbon sources by R. opacus GM-14 (Zaitsev et al., 1995). Another efficient degrader of pollutants, Rhodococcus sp. MS11, grows on highly chlorinated benzenes (di-, tri- and tetrachlorobenzenes) (Rapp and GabrielJürgens, 2003). Chlorobenzene and dichlorobenzene are also utilized as the sole carbon sources by R. phenolicus, which is a novel species in the genus Rhodococcus (Rehfuss and Urban, 2005). Polychlorinated biphenyls are a large group of recalcitrant chlorinated pollutants. Commercial PCB products are mixtures of 50 or more PCB congeners, whereas as many as 209 different PCB congeners (containing 1 to 10 chlorines in the molecule) may theoretically arise. PCB mixtures have been used for a variety of applications, including common additives in paints, plastics and insulating fluids. Due to their remarkable stability, they persist in the environment decades after their production was halted. In natural bacterial communities degrading many PCB congeners, the majority of PCB degraders were identified as members of the genus Rhodococcus (Leigh et al., 2006) and several strong degraders of this genus were isolated. PCBs are cometabolized with BPH by R. globerulus P6 (Asturias et al., 1995), R. erythropolis TA421 (Maeda et al., 1995), R. jostii RHA1 (Masai et al., 1995), Rhodococcus sp. M5, (Lau et al., 1996), R. rhodochrous K37 (Taguchi et al., 2004) and Rhodococcus sp. R04 (Yang et al., 2007). The enzymes of the BPH degradation pathway are described in Section 3.2. Chlorobenzoates produced by the degradation of PCBs instead of benzoate in the BPH pathway may be further degraded to chlorocatechols and mineralized completely by a modified 3-oxoadipate pathway. The presence of multiple enzyme systems involved in biphenyl and PCB catabolism seems to be a typical feature of rhodococci (see Section 3.2). Seven bphC genes coding for 2,3-dihydroxybiphenyl dioxygenases were found in R. erythropolis TA421 (Maeda et al., 1995). As many as eight bphC genes were identified in R. rhodochrous K37 (Taguchi et al., 2007). The multiplicity of enzymes with differing induction mechanisms, substrates and activities probably enables rhodococci to metabolize a wide range of compounds. To evaluate the role of isoenzymes in biphenyl and PCB degradation by R. jostii RHA1, various recombinant gene clusters based on bphA and etbA/ebdA biphenyl dioxygenase operons were expressed in another Rhodococcus strain. The wide spectrum of PCB congeners attacked by EtbA/EbdA dioxygenase suggests that this enzyme is essential in the catabolism of highly chlorinated biphenyls (Iwasaki et al., 2007). Various degraders utilize different and in some cases complementary ranges of PCB substrates. To construct a more efficient degrader with wider spectrum of attacked PCB congeners, the bphA1A2A3A4 gene cluster encoding biphenyl dioxygenase from R. globerulus P6 (dioxygenating m- and p-monochlorinated rings) was expressed in Pseudomonas putida (attacking chlorinated ortho-carbons, thereby dechlorinating ortho-monochlorinated rings) (McKay et al., 1997). To determine the catabolic activity of isoenzymes BphC1, BphC2, and BphC3 coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases in R. globerulus P6, the respective genes were expressed in E. coli. BphC2 and BphC3 showed similar specificity for all monochlorinated as well as for more highly chlorinated 2,3-dihydroxybiphenyls. Since in R. globerulus P6, BphC2 is constitutively expressed, whereas BphC1 expression is induced by biphenyl, it seems that the differing substrate specificities of BphC1 and BphC2 contribute to the versatile PCB-degrading phenotype (McKay et al., 2003). In another attempt to extend the

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capacities of PCB degraders, the technique of DNA shuffling was used to combine the activities of biphenyl dioxygenases from the strains of Burkhoderia sp., Comamonas testosteroni and R. globerulus P6. The resulting variants of biphenyl dioxygenase were more active towards 2,2′, 3,3′, 4,4′and 2,6′-dichlorobiphenyls (Barriault et al., 2002). The potential of metabolic engineering to produce strains with complete PCB degradation pathways has been documented by the construction of a recombinant strain of R. jostii RHA1 carrying the 4-chlorobenzoate degradation operon (fcb) from Arthrobacter globiformis (Rodrigues et al., 2001). The constructed recombinant strain R. jostii RHA1 was applied together with an engineered strain of Burkholderia xenovorans carrying the ortho dehalogenation ohb operon in the bioremediation of sediments contaminated with a commercial mixture of PCBs, Aroclor 1242 (Rodrigues et al., 2006). Broadening the spectrum of attacked PCB congeners and completing the pathways to avoid accumulating the toxic cleavage products are examples of metabolicengineering techniques that may be utilized for practical remediation biotechnology. With regard to the high biodegradation potential of rhodococci, it is of interest to examine whether these bacteria may also metabolize brominated aromatic compounds. These chemicals are widely used in organic chemistry, for instance in the production of polymers and pesticides. In particular, brominated compounds (e.g., tetrabromobisphenol A (TBBPA), hexabromocyclododecane, brominated diphenylethers) are used as reactive components or additives in plastics, electronic circuitry or textiles to prevent fires (de Wit, 2002). Notably, TBBPA is the most widely used flame retardant (as a reactive component in epoxy and polycarbonate resins) with an annual worldwide demand estimated at over 120 thousand tons (Zalko et al., 2006). The appearance of these persistent compounds in the environment is a cause for concern, as they are known for multiple adverse effects on living organisms (possible estrogenic effects, toxicity for aquatic life). We have tested several strains classified as R. erythropolis, R. opacus and Rhodococcus sp. for their ability to degrade mono-, di- and tribrominated phenols and tetrabromobisphenol A. In particular R. erythropolis CCM 2595, known for its activities towards chlorinated and other substituted aromatics (Čejková et al., 2005), exhibited a significant ability to deplete the cultivation medium of brominated compounds (2-bromophenol, 4bromophenol, 2,4-dibromophenol, 2,4,6-tribromophenol and TBBPA; Uhnáková, unpublished data). This finding is promising, as hardly any microbial biodegraders of these persistent contaminants have been found to date. Until now, the utilization of some bromophenols as sole carbon sources has only been found in R. opacus GM-14 (Zaitsev et al., 1995) and Achromobacter piechaudii TBPZ (Ronen et al., 2005). In addition, the strain Rhodococcus sp. MS11 grew on dibromobenzenes as sole carbon sources (Rapp and Gabriel-Jürgens, 2003). 3.5. Nitroaromatics Various nitroaromatic compounds (e.g., mono-, di- and trinitrophenols) have been used for the synthesis of dyes, explosives and pesticides. These compounds and their degradation metabolites may become serious contaminants of water and soil, due to their toxic and carcinogenic effects. Nitroaromatic compounds are quite resistant to biodegradation due to the presence of the nitro group(s), and are therefore considered recalcitrant contaminants. Several bacterial strains were found to degrade nitroaromatics and use them as nitrogen or carbon sources (Nishino and Spain, 2004). Rhodococci form a group of potent nitroaromatic degraders. The biodegradation of nitrophenols is initiated by oxidative or reductive reactions. The nitro group is removed from mononitrophenols (2-nitrophenol (2-NP) and 4-nitrophenol (4-NP)) by the action of monooxygenases in most cases. In the versatile R. opacus SAO101 strain, which degrades benzene, biphenyl, naphthalene, phenol, and 4-NP, the catabolic gene cluster for 4-NP was characterized (Kitagawa et al., 2004). The respective enzymes, converting 4-NP successively to

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4-nitrocatechol, hydroxyquinol and finally to maleylacetate, were described. The hydroxyquinol pathway was thus proved in R. opacus SAO101, whereas the hydroquinone pathway is usual in Gramnegative bacteria like Burkholderia (Prakash et al., 1996). The strain R. wratislaviensis J3 was shown to degrade 4-nitrocatechol, 3nitrophenol and 4-nitroguaiacol (Navrátilová et al., 2005). In contrast, a reductive mechanism was described for the initial reaction of 2,4-dinitrophenol (DNP) and 2.4,6-trinitrophenol (TNP, picric acid) biodegradation. Initial reduction (hydrogenation) of the aromatic ring of 2,4-DNP and 2,4,6-TNP resulting in the respective hydride and dihydride (Meisenheimer complexes) was described in R. opacus HL PM1. This strain utilizes DNP and TNP as the sole nitrogen source. The npd genes involved in the respective degradation pathway were characterized. The capacity of R. opacus HL PM1 to degrade DNP and TNP was shown to be induced by DNP (Heiss et al., 2003). The npdR gene coding for a transcriptional regulator of the IclR family was identified in the npd gene cluster. Binding the NpdR protein to the promoter regions of the orfb and npdI genes within the cluster and constitutive expression of npd genes in npdR knock-out mutants demonstrated that NpdR is a repressor involved in regulation of TNP degradation (Nga et al., 2004). Describing all the enzymes and metabolites of the pathway and understanding the mechanisms that control the expression of npd genes can facilitate establishing suitable DNP and TNP biodegradation processes for the efficient treatment of polluted water and soils. 3.6. Other aromatics The capacities of Rhodococcus strains to decompose other pollutants have frequently been investigated. Among the toxic and recalcitrant aromatic pollutants degraded by rhodococci are compounds with ether bonds and thiazols. Various ethers are used as solvents, pesticides and pharmaceuticals. An ether bond contributes to the chemical stability and high toxicity of tetrachlorodibenzodioxin. During attempts to isolate bacteria able to cleave ether bonds in aromatic compounds, R. rhodochrous 116 was found to catabolize 2-ethoxyphenol and 4-methoxybenzoate by two distinct cytochromes P450. The resulting catechol and protocatechuate, respectively, are further degraded by the corresponding branches of the ortho-cleavage pathway (Karlson et al., 1993). The strain Rhodococcus sp. DEE5151 degraded various ethers, including dibenzyl ether (giving rise to benzoate) and the alkyl phenyl ethers anisole and phenetole (producing phenol) (Kim and Engesser, 2004). Cleavage of the ether bond of the chlorinated phenoxybutyrate herbicides by a constitutively synthesized cytochrome P450 in R. erythropolis K2–3 leads to dichlorophenol as a reaction product (Sträuber et al., 2003). R. opacus SAO101 can utilize dibenzodioxin as the sole carbon source. High cooxidative potential for chlorinated dibenzodioxins was found in R. opacus SAO101 (Kimura and Urushigawa, 2001). Genes coding for the subunits of dioxygenase responsible for oxidizing dibenzodioxin and dibenzofuran were cloned and identified. This enzyme with a relaxed substrate specificity can also oxidize other aromatic compounds, like naphthalene and phenanthrene (Kimura et al., 2006). The genes for extradiol dioxygenase subunits involved in dibenzofuran degradation were cloned from Rhodococcus sp. YK2 (Iida et al., 2002). Benzothiazoles used e.g., as fungicides, herbicides and chemotherapeutics form another group of xenobiotics degraded by Rhodococcus strains. Various benzothiazoles, including toxic and recalcitrant 2-mercaptobenzothiazole used e.g. as a rubber additive were mineralized by R. rhodochrous OBT18 via catechol derivatives (Haroune et al., 2004). 3.7. Desulfurization The combustion of sulfur-containing fossil fuels (oil and coal) generates sulfur dioxide, which contributes to acid rain. Removing

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organic sulfur from these fuels by chemical methods is becoming inadequate, particularly for aromatic heterocyclic sulfur compounds, whereas specific biodesulfurization was shown to be more efficient in these cases. The microorganisms remove sulfur without degrading the carbon skeleton (aromatic rings) thus maintaining the energetic value of the fuel. Several strains of rhodococci were shown to efficiently desulfurize very stable benzothiophene (BT) and dibenzothiophene (DBT) and their derivatives found in fossil fuels. The dszABC operon (coding for two monooxygenases and a desulfinase) was shown to be responsible for the desulfurization of DBT to 2-hydroxybiphenyl in Rhodococcus sp. IGTS8, and these genes were cloned and characterized (Piddington et al., 1995). The substrate specificities of the enzymes involved in desulfurization in various Rhodococcus strains were found to be different. Rhodococcus sp. K462 can metabolize sulfur from both BT and its alkylated derivatives (Kobayashi et al., 2000), while R. erythropolis KA2-5-1 does not utilize BT but can desulfurize alkylated BT and DBT (Tanaka et al., 2002). Yet another strain, Rhodococcus sp. WUK2R, metabolizes sulfur from BT and from naphthol[2,1-b]thiophene (NTH) (Kirimura et al., 2002). The abilities of Rhodococcus strains to use these recalcitrant compounds as sole sources of sulfur are exceptional in bacteria, however, the activity of wild type strains is still too low to develop a commercially usable process. To improve the desulfurization activity of the natural Rhodococcus strains, recombinant biocatalysts were constructed by genetic engineering. The dszABC operon and the related reductase gene, dszD, from R. erythropolis KA2-5-1 were cloned on the plasmid vector pRHK1 and expressed in Rhodococcus cells. The DBT desulfurization ability of the resulting recombinant strain was about 4-fold higher than that of the parent strain (Hirasawa et al., 2001). Using another approach, the dsz genes were efficiently expressed from a strong sulfate non-repressible promoter, kap1 in R. erythropolis KA2-5-1 (Noda et al., 2003). Optimum function of the cloned dsz genes from Rhodococcus sp. DS7 was achieved by expressing the dszABC operon on a plasmid under the control of the strong Rhodococcus promoter P600, and expression of the dszD gene inserted in a single copy into the chromosome under the control of the P57 promoter (Franchi et al., 2003). Li et al. (2007) achieved a 12-fold increase in desulfurization activity by the rearrangement of the dszABC operon into dszBCA, removing the gene overlap within the operon and reconstructing the ribosome-binding site of dszB. The engineered operon was expressed from the plasmid vector pRHK1 in R. erythropolis 4.1491 (Li et al., 2007). The desulfurization efficiency of the strains constructed by various genetic engineering approaches is apparently increasing rapidly in recent years and their applications in an industrial scale process will probably soon reach a sufficient level of economic performance. 4. Biodegradation of nitriles Nitriles are compounds that have the general formula R-CN. Organic nitriles may be synthesized by a variety of reactions (Crosby et al., 1994) and can be in turn converted into acids, amides, amines and other compounds, having been applied in many organic syntheses. Acrylonitrile is a bulk commodity chemical mainly used in polymer production. Many other nitriles are also widely used in the chemical industry (aliphatic nitriles as solvents and intermediates for organic synthesis) and agriculture (aromatic nitrile herbicides). In addition, nitriles (cyanogenic glycosides) are found in many foods and industrial plants (cassava, Sorghum plants, lima beans, sweet potatoes, flax, bamboo, plants of the rose family, etc.). Therefore, the consumption of some of the food plants with a higher nitrile content represents something of a health hazard and may necessitate their pre-treatment or selective breeding for varieties with a lower nitrile content (Zöllner and Giebelmann, 2007). The processing of such plants may also produce nitrile-containing wastes.

Nitriles, in general, exhibit harmful effects on humans and the environment. Some of them (for example cyanohydrins, aliphatic saturated and unsaturated nitriles) are classified as toxic or even highly toxic compounds. Moreover, several nitriles are suspected of having carcinogenic effects, which is especially notable in the case of acrylonitrile. Since the discovery of nitrilase (Hook and Robinson, 1964; Thimann et al., 1964) and nitrile hydratase (Asano et al., 1981), the significant potential of several groups of microorganisms and plants to degrade nitriles has been demonstrated. Many of the nitrileconverting microbes and enzymes showed high specific activities and broad substrate specificities, which made them promising for biotechnological applications. Although the natural substrates of nitrilase or nitrile hydratase have not been unambiguously determined, it is reasonable to assume that they are the naturally occurring nitriles that are found in microorganisms, plants and insects, for example 3-indolylacetonitrile, 4-amino-4-cyanobutyric acid, mandelonitrile or 2-phenylacetonitrile, or those that are components of cyanogenic glycosides and cyanolipids (mainly cyanohydrins) in plants (Legras et al., 1990). The recent discovery of aldoxime dehydratases (Kato et al., 1998) suggests that the substrates of nitrile-converting enzymes could be formed in the same organisms from aldoximes, which, in turn, originate from amino acid precursors (Kato et al., 2004). Among nitrile degraders, strains of the genus Rhodococcus represent a prominent group. The selection of nitriles as sole nitrogen or carbon sources often resulted in the isolation of Rhodococcus strains (Layh et al., 1997; Brady et al., 2004; Prasad et al., 2007). Similarly, more than one fourth of nitrile-converting strains available from public collections (Martínková and Křen, 2002) were rhodococci, whereas other bacterial strains with significant nitrile-degrading capabilities belonged to the genera Arthrobacter, Alcaligenes, Bacillus, Comamonas and especially Pseudomonas. In addition to a remarkable diversity of genes coding for nitrilases and nitrile hydratases in rhodococci, the permeability of the rhodococcal cell wall for nitriles and its corresponding biotransformation products contributes to their efficient nitrile degradation. An important feature of most nitrile-converting enzymes is their affinity for a broad range of man-made nitriles. Therefore, they can be used as biocatalysts for a large number of organic synthetic reactions, which lead to valuable products and intermediates (first of all carboxylic acids or their amides). These reactions have been extensively studied and summarized in a number of previous reviews (Beard and Page, 1998; Bunch, 1998; Banerjee et al., 2002; Martínková and Křen, 2002; Wang and Lin, 2002; Wang, 2005; Singh et al., 2006). In some of them, the applications of microorganisms in biodegradation and bioremediation have also been mentioned. Another work summarized the fate of inorganic cyanide and some organic nitrile compounds in water and soil and briefly reviewed the distribution of cyanide-degrading pathways throughout bacteria and fungi (Baxter and Cummings, 2006a). Although the studies devoted to biodegradation and bioremediation formed only a small part of the extensive literature on nitrile-converting microorganisms and enzymes, it is obvious that these tools can serve to efficiently detoxify nitriles. The products of microbial nitrile degradation, carboxylic acids or their amides, are in most cases less toxic that the corresponding nitriles, and, furthermore, many of these intermediates can be further metabolized and mineralized by the same or other microbial strains. The following sections constitute an overview of rhodococcal enzymes catalyzing the initial step of nitrile metabolism (nitrilases, nitrile hydratases) and their role in the hydrolysis of the most prominent nitrile contaminants. Organisms and processes that have been examined as tools for the biodegradation of these nitriles (acrylonitrile, aliphatic nitriles and aromatic nitrile herbicides) are summarized. Additionally, strains and enzymes are mentioned that have not been used for bioremediation experiments but show promise for such applications due to their high ability to transform the nitriles of interest.

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Table 1 Catalytic properties of purified nitrilases from genus Rhodococcus Enzyme type Strain

Specific activity

Substrate specificity

Optimum

(U mg− 1 protein) (%)

Stability

Reference

pH

T (°C) pH

T (°C) b 50

Aromatic

R. rhodochrous J1

14.6 (BN)

BN 100, ACN 0/58.8b

7.5

45

Aliphatic

R. rhodochrous PA-34 Rhodococcus sp. ATCC 39484 Rhodococcus sp. NCIMB 11216a benzonitrile-induced Rhodococcus sp. NCIMB 11216a propionitrile-induced R. rhodochrous K22

24.5 (BN) 84 (BN) 2.0 (BN) 10.6 (BN) 2.6 (ACN)

BN 100, PN 5, BuN 5, ACN 22 BN 100, ACNb 1 BN 100, PN 5c BN 100, PN 5c ACN 100, AN 8, PN 4, BuN 5, BN 8

7.5 7.5 8.0 8.0 5.5

35 30 30 30 50

nd

Kobayashi et al., 1989; Nagasawa et al., 2000 6.0–9.0 30–50 Bhalla et al., 1992 5.5–9.5 25–40 Stevenson et al., 1992 n.a. n.a. Hoyle et al., 1998 n.a. n.a. Hoyle et al., 1998 6.0–8.0 b 50 Kobayashi et al., 1990

PN = propionitrile, AN = acetonitrile, BuN = butyronitrile, ACN = acrylonitrile, BN = benzonitrile. n.a. = not assayed. Note: Optima and stabilities of the enzymes were taken from a previous review (Banerjee et al., 2002). a Reclassified (previously Nocardia sp.). b Activity for acrylonitrile was recovered by subunit aggregation in presence of 10% saturated ammonium sulfate. c Assayed only with intact cells (Hoyle et al., 1998).

4.1. Nitrile-converting enzymes in rhodococci It has been well known for several decades that the hydrolysis of nitriles can proceed by two distinct routes, catalyzed by either nitrilase (RCN + 2 H2O → RCOOH + NH3) or nitrile hydratase (RCN + H2O → RCONH2) followed by amidase (RCONH2 + H2O → RCOOH + NH3). Both nitrile hydratases and nitrilases can be found in the genus Rhodococcus, sometimes even in a single strain. The members of both groups differ widely in their catalytic properties (Tables 1 and 2). Nitrilases are usually classified into three groups (aromatic, aliphatic and arylaliphatic) according to their substrate specificity (Kobayashi and Shimizu, 1994; Table 1). The amino acid sequence similarity between these groups is rather low (for example 48% between an aromatic nitrilase from R. rhodochrous J1 and an aliphatic nitrilase from R. rhodochrous K22 (O'Reilly and Turner, 2003). In rhodococci, nitrilases are hyperinduced by some nitriles, especially isovaleronitrile (Kobayashi et al., 1991). In R. rhodochrous J1, a regulatory gene (nitR) located downstream of the nitrilase gene (nitA) encodes a protein acting as a positive regulator of nitA gene transcription (Komeda et al., 1996b). Three types of nitrile hydratases differing in their cofactors exist in rhodococci. In addition to the well characterized nitrile hydratases containing an Fe3+ or Co3+ cofactor (for a review see Banerjee et al., 2002), a new nitrile hydratase containing three types of metal ions (Co, Cu, Zn) was recently described in R. jostii RHA1 (Okamoto and Eltis, 2007). All known nitrile hydratases from rhodococci consist of two subunits (α and β). Although most sequenced Fe3+-containing nitrile hydratases show a high level of similarity (N90% in most cases) in rhodococci, remarkable differences in substrate specificities have been reported for different enzymes (Table 2). However, it is not clear to what extent

the different results (especially concerning the different activities for benzonitrile) were due to the use of distinct enzymatic assays. Brandao et al. (2003) suggested that different amino acid residues located near the active site may affect enzyme activities and substrate specificities. Some of these enzymes like that from Rhodococcus sp. AJ270 (Song et al., 2007) and R. erythropolis A4 (Kubáč et al., 2008) exhibit wide-ranging substrate specificities for aromatic, (aryl) aliphatic, alicyclic and branched nitriles. The nitrile hydratases carrying the Co3+-cofactor are comprised of two subtypes, low- and high-molecular-mass nitrile hydratase (Wieser et al., 1998), which also differ in their substrate specificities. Therefore, rhodococcal nitrileconverting enzymes are suitable tools for the biotransformation of a wide variety of structurally different nitriles. The high specific activity for medium-chain-length saturated aliphatic nitriles and acrylonitrile is a typical property of rhodococcal nitrile hydratases (Table 2). Recently, a new nitrile hydratase (acetonitrile hydratase) acting on aliphatic nitriles has been purified from R. jostii RHA1 (Okamoto and Eltis, 2007), whose genome sequence is available (McLeod et al., 2006). There is an unusual homology between the α- and β-subunit of this enzyme (32%), and the size of these subunits (63 and 56 kDa) is higher than that of other nitrile hydratases (26–35 kDa). Moreover, there is no homology between this protein and other nitrile hydratases that have been described so far. This acetonitrile hydratase has a pronounced specificity for acetonitrile and acrylonitrile followed by propionitrile and butyronitrile (Table 2). The relative lability of nitrilases and, in particular, nitrile hydratases, may hamper biocatalytic and bioremediation applications. The optimum temperatures of the enzymes are generally moderate, while rhodococcal nitrile hydratases require lower temperatures (≤ 40 °C) than nitrilases from the same genus (≤50 °C; compare Tables 1 and 2).

Table 2 Catalytic properties of purified nitrile hydratases from genus Rhodococcus Enzyme type

Strain

Specific activity (U mg

Fe-type

Co-type

Acetonitrile hydratase

Rhodococcus sp. N-771 Rhodococcus sp. R-312a R. erythropolis A4b R. erythropolis AJ270 R. rhodochrous J1 low-molecular mass R. rhodochrous J1 high-molecular mass Rhodococcus sp. YH3-3 R. jostii RHA1

−1

protein)

691 (PN) 1890 (PN) 400 (PN) 829 (ACN) 1200 (PN) 1600 (PN) 1280 (PN)c 5.9 (AN)

Substrate specificity

Optimum

(%)

pH

T (°C)

pH

T (°C)

PN 100, BuN 113, ACN 170, AN 4 PN 100, BuN 140, ACN 78 PN 100, BN 14

7.8 7.8 7.5 7.2–7.6 8.8 6.5

30 25 32–35 25 40 35–40 n.a. n.a.

6.0–8.0 6.5–8.5 n.a. n.a. 6.5–8.0 6.0–8.5 2.5–11.0 n.a.

b35 n.a. n.a. n.a. 30 50 40–60 n.a.

PN 100, BuN 242, ACN 69, BN 75 PN 100, BuN 81, ACN 110, BN 16 PN 100, BuN 162, BN 81 PN 100, AN 1577, ACN 1077, BuN 38

PN = propionitrile, AN = acetonitrile, BuN = butyronitrile, ACN = acrylonitrile, BN = benzonitrile. n.a. = not assayed. Note: Optima and stabilities of the enzymes were mostly taken from a previous review (Banerjee et al., 2002). a Reclassified (previously Brevibacterium sp.). b Reclassified (previously R. equi). c Vmax.

5.5–10.0

Stability

Reference

Nagamune et al., 1990 Nagasawa et al., 1986 Přepechalová et al., 2001 Song et al., 2007 Wieser et al., 1998 Wieser et al., 1998 Kato et al., 1999 Okamoto and Eltis, 2007

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The highly conserved genes coding for the α- and β-subunits of Fetype nitrile hydratases form a cluster with aldoxime dehydratase and amidase genes, as well as with the respective regulatory genes, in a number of rhodococci (Fig. 4). The nhr3 gene of this cluster codes for an activator whose interaction with nitrile hydratase is critical to the biogenesis of functional Fe-type nitrile hydratases (Lu et al., 2003). Genes coding for homologues of the Nhr1 regulator are also clustered with genes coding for the α- and β-subunits of both subtypes of Cotype nitrile hydratases (Komeda et al., 1996a) and with genes coding for the α- and β-subunits of acetonitrile hydratase, located on the large plasmid pRHL2 in R. jostii RHA1 (Okamoto and Eltis, 2007). A gene encoding an amidase active towards aliphatic amides (acetamide, propionamide, butyramide and phenylacetamide) is also located just upstream of the nitrile hydratase subunit genes on the plasmid pRHL2. 4.2. Acrylonitrile Acrylonitrile is a commodity chemical with a yearly worldwide production of almost 4 million tons. Its industrial applications (plastics, acrylic fibres, synthetic rubber manufacture) and occurrence in the environment (from waste water and accidental spillage) have recently been summarized (Baxter et al., 2006). It is classified as highly toxic due to its cyanide effect (LD50 in rats (p.o.) 93 mg/kg) and there are also indications of carcinogenic (O'Neil et al., 2006) and teratogenic activities (Saillenfait and Sabaté, 2000). The toxicity of acrylonitrile and other aliphatic nitriles is thought to be at least partly due to cyanide release in the body, but biotransformations of unsaturated nitriles by microsomal enzymes may also lead to toxic metabolites other than cyanide. For example, the carcinogenic effects of acrylonitrile may be ascribed to its biotransformation product, 2-cyanoethylene oxide (Saillenfait and Sabaté, 2000). The biotransformation of acrylonitrile has become attractive because of its industrial applicability in acrylamide production. Three generations of biocatalysts (Rhodococcus sp. N-774, Pseudomonas sp. B23, Rhodococcus rhodochrous J-1) with nitrile hydratase activity have been employed for this process, which is used by the Mitshubishi Rayon Co. at an annual output of about 30 000 tons per year (Kobayashi et al., 1992). The third-generation acrylonitrile-transforming biocatalyst R. rhodochrous J-1 harbours two types of enzymes bearing a Co3+cofactor, namely a high- and a low-molecular-mass nitrile hydratase, the former being one of the best nitrile hydratases in terms of thermostability (see Table 2). This property is probably due to the unique multimeric character of the enzyme (Nagasawa et al., 2000). The rhodococcal nitrile hydratases generally exhibit high specific activities towards acrylonitrile, irrespective of the enzyme type (Fe- or Co-type; see Table 2). The strain Rhodococcus sp. AJ270, which was widely used as a biocatalyst in organic chemistry (see Wang, 2005 for

review), was also applied to acrylonitrile bioremediation in situ (Baxter et al., 2006). It was found that the application of a pure Rhodococcus sp. AJ270 culture resulted in the same level of acrylonitrile degradation in the soil as that achieved by the whole of the indigenous bacterial flora. Moreover, this Rhodococcus strain became stably established within the soil bacterial community, becoming its most abundant member. However, no further increase in the level of acrylonitrile degradation was observed in this bio-augmented community (Baxter et al., 2006). The isolation of acrylonitrile-utilizing strains by enrichment culture techniques is, however, not straightforward, as acrylonitrile generally inhibits bacterial growth. In those cases structurally similar substrates (acetonitrile, isobutyronitrile) have successfully been used. Alternatively, acclimation cultures resulting from long-term adaptation of the microbes can be applied to the selection of strains utilizing toxic compounds (Asano, 2002). The selection and screening techniques used to isolate nitrile-converting enzymes have recently been reviewed (Martínková et al., 2008). In general, nitrile-hydrating whole-cell biocatalysts exhibit low or no amidase activity for acrylamide. This is beneficial in acrylamide production but becomes a disadvantage in bioremediation, since acrylamide, whose toxicity is well-known, can persist as a dead-end product in the environment. In this respect, rhodococci with nitrilase activity (see Table 1), which are able to hydrolyze acrylonitrile into ammonium acrylate, are convenient, as this compound is much less toxic than acrylamide and, moreover, is commercially valuable for polymer production (Hughes et al., 1998; Webster et al., 2001; Roach et al., 2004). The highest efficiency so far for this process was achieved with immobilized R. ruber cells (Roach et al., 2004). Whole cells of this strain immobilized by entrapment into porous dimethyl silicone rings were used for bioscrubbing acrylonitrile vapours in a conventional trickle-bed air biofilter. The assembly contained a water-jacketed column filled with the immobilized biocatalyst. The contaminated air source was simulated by sparging air through an acrylonitrilecontaining test-tube placed in a water bath. The scrubbing liquid (water) was continuously recycled through the biocatalyst bed. A high elimination capacity was achieved with more than 90% acrylonitrile removal from the vapour stream (Roach et al., 2004). The same enzyme and immobilization principle was used for the construction of a biosensor for the detection and quantitation of acrylonitrile in waste streams (Roach et al., 2003). 4.3. Saturated aliphatic nitriles Acetonitrile is widely used in organic synthesis, fatty acid extraction, the removal of colouring matter and aromatic alcohols from petroleum hydrocarbons, steroid recrystallization etc. (O'Neil et al.,

Fig. 4. Organization of the genes involved in nitrile biosynthesis and biodegradation in rhodococci and the respective pathway. (A) Gene cluster. Gene products: nhr4, nhr2 – transcriptional regulators, nhr1 – nitrile hydratase regulator, ami – amidase, nha1 – α-subunit of nitrile hydratase, nha2 – β-subunit of nitrile hydratase, nhr3 – nitrile hydratase activator. This gene organization was found in R. globerulus A-4 (Xie et al., 2003), R. erythropolis A4 (GenBank Accession Number AM946017) and Rhodococus sp. N-771 (Kato et al., 2004). The gene cluster lacking the nhr1 and nhr2 genes is present on the R. jostii RHA1 chromosome (GenBank Accesion Number CP000431). (B) Nitrile metabolism in rhodococci.

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2006) and high performance liquid chromatographic analysis. Propionitrile and butyronitrile are also used as solvents and chemical intermediates. All these aliphatic nitriles are poisonous substances, especially propionitrile and butyronitrile (LD50 in rats (p.o.) 39 and 140 mg/kg, respectively (O'Neil et al., 2006). Acetonitrile and propionitrile are also suspect teratogens (Saillenfait and Sabaté, 2000). Nitrile hydratases from various strains of rhodococci show similar relative activities for individual aliphatic nitriles. In most cases, propionitrile and butyronitrile are the best substrates for these enzymes, whereas acetonitrile is hydrolyzed at a much lower rate. The acetonitrile hydratase recently found in R. jostii RHA1, which differs from the other nitrile hydratases in many features (see Section 4.1), is an exception to this rule, as it strongly prefers acetonitrile over longerchain aliphatic nitriles (Okamoto and Eltis, 2007). There is a broad selection of Rhodococcus strains producing nitrile hydratases whose substrate specificity makes them suitable for the biodegradation of saturated aliphatic nitriles and, at the same time, of acrylonitrile. Those Fe-type nitrile hydratases which have so far been sequenced were all highly similar (Brandao et al., 2003, Kubáč et al., 2008). However, their detoxification ability can also be affected by other properties of the strains besides the structure and function of the key enzyme itself (e.g., properties of the cell envelope see Section 2.). Whole-cell biodegradation studies were performed with R. erythropolis BL1 (Langdahl et al., 1996) or R. pyridinivorans S85-2 (Kohyama et al., 2006). R. erythropolis BL1, which was isolated from marine sediment by an enrichment culture with acetonitrile as the sole C and N source, showed a high tolerance to this compound. Both growing and resting cells had a strong ability to degrade high concentrations of acetonitrile. The biodegradation by cell suspensions proceeded in the presence of all examined acetonitrile concentrations of up to 2000 mM and achieved a maximum rate at 500 mM of acetonitrile (Langdahl et al., 1996). Markedly higher still enzyme activities were recorded for propionitrile and butyronitrile. R. pyridinivorans S85-2 was even more resistant to high concentrations of acetonitrile, which was totally consumed at concentrations of 1 to 6 M, the rates of degradation being similar over this range. In both strains, the initial step of acetonitrile metabolism was catalyzed by nitrile hydratase. The respective gene of R. pyridinivorans was sequenced and the corresponding enzyme showed 99.8% similarity to that of the high-molecular-mass Co-type nitrile hydratase from R. rhodochrous J1. Accordingly, the production of the nitrile hydratase in R. pyridinivorans was markedly increased by the addition of CoCl2 (Kohyama et al., 2006). The products of acetonitrile conversion in R. erythropolis BL1 and R. pyridinivorans S85-2 were different. During the growth of R. erythropolis BL1 on 30 mM or 1 M acetonitrile, acetic acid appeared transiently in the culture medium and was later used by the organism as a source of carbon and energy. No significant amounts of acetamide were excreted by either growing or resting cells (Langdahl et al., 1996). In contrast, the product of 1 to 6 M acetonitrile conversion by R. pyridinivorans showed acetamide: acetic acid ratios of approx. 8:1 to 28:1. It is possible that the high concentrations of acetonitrile deactivated the amidase in R. pyridinivorans, but the differing metabolite profiles of the strains when converting 1 M acetonitrile suggest differing properties of their amidases. Therefore, R. erythropolis BL1 was more suitable for the detoxification of acetonitrile. Nevertheless, the disadvantage of R. pyridinivorans, which produced the carcinogenic compound acetamide, was overcome by coupling the acetonitrile hydration with a second step consisting of the enzymatic hydrolysis of acetamide. A strain of Brevundimonas diminuta with a high amidase activity for acetamide was selected as the best biocatalyst for the second step of this tandem process, which enabled the N90% hydrolysis of 6 M acetonitrile within 10 h (Kohyama et al., 2006). The biodegradation of toxic amides was also feasible using whole cells of Rhodococcus rhodochrous NMB-2 immobilized in agar. The catalyst was used to remove acetamide, propionamide or acrylamide from simulated wastewater in a plug flow reactor (Chand et al., 2004).

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Several deep-sea and terrestrial isolates belonging mostly to R. erythropolis were also tested for growth on acetonitrile (20 mM) as the sole C and N source and their metabolite profiles were measured (Brandão and Bull, 2003). These strains behaved in a similar way to R. erythropolis BL1, being able to metabolize acetonitrile into acetic acid and ammonia, but the transitory appearance of significant concentrations of acetamide was detected for some of the strains examined. The genes for nitrile hydratases and amidases of these and other strains of the genus Rhodococcus, which were isolated from different habitats by enrichment on acetonitrile or benzonitrile, were amplified and analyzed by PCR-restriction fragment length polymorphism–singlestrand conformation polymorphism (PCR-SSCP). This PCR-SSCP analysis revealed profiles, which correlated with the geographical origin of the isolates. A sequence analysis of the genes for Fe-type nitrile hydratases showed a high similarity of their products (N 92% aa identity) with the sequenced nitrile hydratases from other Rhodococcus strains (Brandao et al., 2003). 4.4. Aromatic nitriles The substituted benzonitrile derivatives 2,6-dichlorobenzonitrile (dichlobenil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-dichloro-4-hydroxybenzonitril (chloroxynil) and 3,5-diiodo-4hydroxybenzonitril (ioxynil) are the active compounds in a number of herbicides. Dichlobenil is used to manage a broad spectrum of weeds in plant nurseries and fruit orchards and on non-agricultural areas (Sørensen et al., 2006). Bromoxynil is widely used for the postemergence control of broadleaved weeds in cereals and other crops (Baxter and Cummings, 2006b; Nielsen et al., 2007). In soil, dichlobenil is largely degraded into 2,6-dichlorobenzamide, which is persistent with a half-life ranging from 106 to 2079 days. This metabolite is also highly mobile, so that it can easily contaminate groundwaters. The acute toxicity of 2,6-dichlorobenzamide is low to medium (with an LD50 ranging from 1144 to 2330 mg/kg in mice and rats), but its carcinogenicity and reproduction toxicity have not been sufficiently investigated. Dichlobenil is still in use in many countries, moreover, the contamination of groundwaters with its metabolite 2,6-dichlorobenzamide persists even after the use of dichlobenil was prohibited as in, e.g., 1997 in Denmark (Holtze et al., 2006; Sørensen et al., 2006). The biodegradation pathway for dichlobenil was examined using collection cultures of Rhodococcus, Pseudomonas and Rhizobium. Similarly to other bacteria, R. erythropolis DSM 9674 and R. erythropolis DSM 9675 transformed dichlobenil into 2,6-dichlorobenzamide as the dead-end product at low degradation rates, while the non-halogenated analogue benzonitrile was hydrolyzed into benzoic acid (Holtze et al., 2006). Therefore, the amidase of these bacteria seems to be unable to accept 2,6dichlorobenzamide due to steric hindrance. On the other hand, new isolates of Aminobacter spp. ASI1 and MSH1 are able to mineralize 2,6dichlorobenzamide, as well as related compounds - dichlobenil, 2chlorobenzonitrile, 2-chlorobenzoic acid, benzonitrile (former strain), 2chlorobenzamide, 2,6-dichlorobenzoic acid, benzamide and benzoic acid (both strains) (Simonsen et al., 2006; Sørensen et al., 2006). In contrast to dichlobenil, the hydration of bromoxynil or ioxynil by nitrile hydratases from the Rhodococcus genus has not been reported until now, as opposed to nitrile hydratases from other bacteria, i.e. Agrobacterium spp. (Věková et al., 1995) or Variovorax sp. (Nielsen et al., 2007). 5. Conclusions and prospects The known range of pollutants catabolized by rhodococci and the number of strains and even new Rhodococcus species found to be useful as degraders have been expanding rapidly over the last few years. Their suitable physiological and biotechnological properties (Larkin et al., 2005), large genomes with megaplasmids carrying sets of genes encoding arrays of catabolic pathways (McLeod et al., 2006), high levels of recombination that increase the adaptability and flexibility of their

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genome (Larkin et al., 1998), ability to acquire new genes by horizontal gene transfer (Larkin et al., 2006) and the recent development of tools for genetic engineering in rhodococci (van der Geize and Dijkhuizen, 2004) seemingly predestine these bacteria for practical use in the degradation of pollutants. However, the application of rhodococci in real bioremediation processes is not so straightforward. Although the general redundancy and diversity of catabolic pathways is considered a decisive factor in their successful use in the removal of composite mixtures of related compounds (e.g. various congeners of PCBs), it is difficult to predict the real range and redundancy of catabolic activities merely from their gene sequences, because homologous genes may code enzymes with different activities (McLeod et al., 2006). Moreover, expression of these genes may be regulated in a different way and the respective enzymes may function under different conditions. Large bacterial genomes possess a higher percentage of regulatory genes and the regulatory networks of specific and global control mechanisms may reach a high level of complexity. Expression of catabolic genes for the utilization of unfavourable substrates is determined by a hierarchy of substrate utilization and the preferred substrates (e.g. carboxylic acids) may repress the genes for the degradation of pollutants. The presence of toxic contaminants and the environmental conditions of polluted sites (e.g., pH, temperature and ionic strength) exert stress on the cells of the degraders and the respective promoters may be recognized by alternative sigma factors of RNA polymerase. Limited information is available on the transport and regulatory genes in Rhodococcus and almost no data on the function of 34 sigma factors in R. jostii RHA1. During their evolution, Rhodococcus strains were shown to acquire abilities to degrade various xenobiotics, but the rate and efficiency of their removal are mostly low, because the microorganisms evolve on the basis of environmental fitness rather than degradation efficiency (Diaz, 2004). All these cons suggest that much more information on physiology, enzymology and genetics of rhodococci will be required to better understand the regulatory mechanisms of their gene expression and the function of the respective enzymes. Such knowledge can be best obtained using transcriptomic and proteomic approaches (Goncalves et al., 2006; Hara et al., 2006; Patrauchan et al., 2008), analyses of regulator-operator interactions (Eulberg and Schlömann, 1998; Nga et al., 2004) and studies of transcription using reporters (Nga et al., 2004; Takeda et al., 2004b; Knoppová et al., 2007; Veselý et al., 2007). The multicopy plasmid vectors and genetic transfers that have been developed (Veselý et al., 2003; Lessard et al., 2004; Nakashima and Tamura, 2004; Na et al., 2005b) for gene manipulation in rhodococci may be utilized for the overexpression of the genes for the key catabolic pathways and enzymes, as well as the inactivation of undesirable pathways or enzymes (van der Geize and Dijkhuizen, 2004). However, this brute-force approach frequently fails due to the instability of the constructs and low ability of the resulting strains to survive in competition with the autochthonous microflora in the natural conditions of remediation in situ (Cases and de Lorenzo, 2005). Moreover, regulations on the release of genetically modified organisms (GMOs) into an open environment prevent use of plasmids with heterologous genes, which could be easily disseminated in the environment. Efforts to construct efficient degraders should therefore be focused on interventions that include the modulation of metabolic pathways and rearrangement of enzymatic, transport, and regulatory functions using the approaches of genetic engineering within the chromosomes of the manipulated strains (van der Geize et al., 2001; Čejková et al., 2005). Chromosomal integrations of all purposeful alterations would result in the modulation of metabolic pathways in genetically modified strains with high stability and low-level risk for the environment. A single breeded degrader would probably not be able to clean-up the polluted site with a mixture of various contaminants and pure cultures therefore fail (Bouchez et al., 2000). Suitable consortia of manipulated organisms should therefore be constructed with the members specialized in certain degradation steps or pathways (Diaz, 2004).

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