Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5858-5

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

Genetic and metabolic analysis of the carbofuran catabolic pathway in Novosphingobium sp. KN65.2 Thi Phi Oanh Nguyen & Damian E. Helbling & Karolien Bers & Tekle Tafese Fida & Ruddy Wattiez & Hans-Peter E. Kohler & Dirk Springael & René De Mot

Received: 19 March 2014 / Revised: 27 May 2014 / Accepted: 27 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The widespread agricultural application of carbofuran and concomitant contamination of surface and ground waters has raised health concerns due to the reported toxic effects of this insecticide and its degradation products. Most bacteria that degrade carbofuran only perform partial degradation involving carbamate hydrolysis without Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5858-5) contains supplementary material, which is available to authorized users. T. P. O. Nguyen : K. Bers : T. T. Fida : D. Springael (*) Division of Soil and Water Management, Department of Earth and Environmental Sciences, KU Leuven, Kasteelpark Arenberg 20, Box 2459, 3001, Heverlee Leuven, Belgium e-mail: [email protected] T. P. O. Nguyen Department of Biology, College of Natural Sciences, Can Tho University, Can Tho, Vietnam D. E. Helbling : H.90 % amino acid identity) of the latter putative oxygenase from Novosphingobium sp. KN65.2 are encoded in the genomes of some phylogenetically close relatives (strains US6-1, PP1Y, LE124, LH128; Online resource 5), but the enzyme seems to be absent in most sphingomonads. Both group V mutants, showing no mineralization, no growth, and decreased degradation, combined with carbofuran phenol accumulation, were affected in cfdC (9G4) or in cftA (8A8). cfdC encodes a putative mono-oxygenase, and we suspect that it is involved in direct attack of carbofuran phenol. Indeed, although carbofuran phenol was found in all mutants analyzed, the highest level of carbofuran phenol accumulated upon incubation with mutants 9G4 and 8A8, and none of the seven other metabolites were detected, suggesting a true bottleneck for its further degradation in these mutants. Furthermore, several further metabolites were detected in the cfdD and cfdE mutants still producing CfdC, supporting a key role for this enzyme in carbofuran phenol degradation. cftA encodes a putative TonB-dependent receptor and makes part a four-gene module together with an RNA polymerase σ factor, a σ factor regulator, and a phosphatase (Fig. 5). The organization is reminiscent of the tripartite regulatory systems with an ECF-type σ factor, a σ factor regulator (membrane-bound anti-σ factor), and a TonB-dependent transporter mediating transenvelope signal transduction of iron-siderophore complexes (Koebnik 2005). The additional putative protein phosphatase gene of KN65.2 located downstream may have a regulatory role (Kaczmarczyk et al. 2011), possibly exerted in the periplasm since its gene product contains a predicted signal peptide for Sec-dependent transport. The strong effect of the cftA mutation on expression of all the genes of the cfdABCDEFGH operon and of cfdI points toward its importance. Indeed, this mutation strongly reduced the amount of the Cfd enzymes (CfdC through CfdF), as well as the CfdI monooxygenase, suggesting a pronounced adverse effect on expression of the respective operon and gene (Online resource 2). Concurrently, the amount of an ABC-type transporter protein (SPHv1_600010) increased about ~100-fold. A comparable strong increase of this cytoplasmic component of a putative efflux system (~140-fold) was noted in the mutant lacking CfdI. This might be rationalized by a regulatory function for this outer membrane protein as part of a module mediating transenvelope signal transduction responsive to some metabolite (Noinaj et al. 2010). The strong induction of the efflux

transporter may be due to toxic effects of accumulating carbofuran phenol when its further degradation is blocked. The gene context of cftA in strains KN65.2, LE124, and BAL3 is very similar, composing the same four-gene module (Fig. 5). An increased level of certain TonB-dependent receptors, detected by differential proteomic analysis in dioxindegrading Sphingomonas wittichii RW1, suggests that this type of transporters plays a significant but as yet unresolved role in xenobiotic degradation (Hartmann and Armengaud 2014). Constitutive expression of the carbofuran degradation genes Different observations indicate that at least part of the genes are constitutively expressed. The cfd genes that appear crucial for metabolism are transcribed constitutively and not subject to induction by carbofuran. This is in line with the observed very similar carbofuran mineralization kinetics of carbofuran-induced and -noninduced resting cells. On the other hand, several genes were identified whose products can be linked to some kind of regulation such as the cfdA in the cfdABCDEFGH operon and the gene cluster containing cftA. Especially, the role of the latter in regulation of carbofuran metabolism is of interest since an insertion in cftA resulted into a complete abolishment of carbofuran phenol degradation and growth on carbofuran in mutant 8A8 and affected expression of all genes of the cfdABCDEFGH operon and cfdI. However, currently, it is unclear how this observation can be linked to the apparent constitutive expression of carbofuran mineralization and of the cfdABCDEFGH operon. Proposed pathway for carbofuran biodegradation in Novosphingobium sp. KN65.2 The tentative pathway for carbofuran degradation shown in Fig. 6 is based on the identified metabolites and integrates current information from the phenotypic characterization of the carbofuran degradation mutants. Overall, both the metabolite analysis and the genetic analysis point toward three major steps in carbofuran degradation by strain KN65.2, i.e., hydrolysis of the carbamate bond, processing of the aromatic moiety, and further degradation of the cleaved aromatic ring through CoA-activated metabolites. In line with several reports for other carbofurandegrading bacteria, the initial step is hydrolysis of the carbamate to the corresponding carbofuran phenol, methylamine, and carbon dioxide. However, the constitutively expressed hydrolase gene involved was not identified in this study. The Mcd enzyme might be a candidate for this initial step, but no homolog of the Achromobacter mcd gene is present in the Novosphingobium sp. KN65.2 draft genome. This enzyme also hydrolyzes carbaryl (1-naphthyl methylcarbamate) (Naqvi et al. 2009), even more efficiently than the carbaryl hydrolase CehA from Rhizobium sp. AC100 does (Hashimoto et al. 2002). The mobile element carrying cehA is also present in Novosphingobium sp. KN65.2, encoding a nearly identical

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copy of CehA (99.5 % amino acid identity), but the enzyme identified in strain AC100 lacks detectable activity on carbofuran (Hashimoto et al. 2002). The possible involvement of this CehA homolog in carbofuran degradation in strain KN65.2 is currently investigated. A homolog of another carbaryl hydrolase, CahA from Arthrobacter sp. RC100 (Hashimoto et al. 2006), is absent from the KN65.2 genome. Shin et al. (2012) isolated several sphingomonads degrading both carbofuran and carbaryl, but the enzymatic or genetic basis of these combined activities was not studied. As discussed above, the methylamine released from carbofuran can be likely metabolized by strain KN65.2 although we currently do not know whether this is indeed the case. Mutant analysis assigns a crucial role for the enzymes encoded by the cfdABCDEFGH operon. Based on the current data, the most likely candidate for opening of the furan ring of carbofuran phenol is the enzyme encoded by cfdC. CfdC is distantly related to the flavin-dependent mono-oxygenase HsaA catalyzing hydroxylation during cholesterol metabolism by M. tuberculosis (Dresen et al. 2010). Ipso-hydroxylation would produce quinone 2 via the putative intermediate 8. This reaction is analogous to ipso-substitution reactions with paraalkoxyphenols in Sphingobium xenophagum (Gabriel et al. 2007) and with sulfonamides in Microbacterium sp. strain BR1 (Ricken et al. 2013). A reductase for CfdC is not encoded within the cfd operon, but such function may be provided by an unlinked gene. A homolog of the HsaA-flavin reductase HsaB (Dresen et al. 2010) is actually encoded by the gene located in the genomic region with two plasposon insertions (group II mutants 6C1, 21D7), immediately upstream of the aldehyde dehydrogenase gene inactivated in mutant 21D7 (Online resource 8 and 9, gene 6.6). Alternatively, CfdC may represent a single-component flavin-dependent monooxygenase (van Berkel et al. 2006). Meta-cleavage of the previously described 3-substituted catechol 3, resulting from the reduction of quinone 2, would then yield intermediate 4, which can be hydrolyzed to products 5 and 9. Formation of the latter compound was not demonstrated, but 2-oxopent-4-enoate is a common intermediate in the metabolism of aromatics. A putative operon encoding the three enzymes for its conversion to pyruvate and acetyl-CoA (2-oxopent-4-enoatehydratase, 4-hydroxy-2oxovalerate aldolase, and acylating acetaldehyde dehydrogenase) is located ~17 kb upstream of cluster 2 (data not shown). CfdE, a putative dioxygenase with significant homology to HsaC that catalyzes meta-cleavage of a 3-substituted catechol during cholesterol degradation (Dresen et al. 2010), can be proposed for this step. Metabolite 4 was indeed not detected in the corresponding mutant (11A4), but a novel side-chain hydroxylation product of carbofuran (6) accumulated (Fig. 6). Two candidates for the hydrolysis of compound 4 to produce metabolite 5 are potentially encoded by the

cfdABCDEFGH operon. CfdD belongs to the fumarylacetoacetase family whereas CfdF is a putative α/βhydrolase. A cfdD mutant was isolated, but the polar effect on the downstream genes prevents reliable conclusions to be drawn. CfdF belongs to the same α/β-hydrolase subfamily as HsaD that splits a metabolite, similar in structure to compound 4, during mycobacterial cholesterol catabolism (van der Geize et al. 2007). During degradation of carbofuran by strain KN65.2, a red color formed, as well as for most of the mutants except for those of group V (8A8, 9G4; data not shown). Formation of a carbofuran metabolite with a nominal mass of 343.4 Da similar to compound X identified in this study and generating a red color in solution was previously reported during degradation of carbofuran by Sphingomonas sp. SB5 (Kim et al. 2004). Another red compound produced by this strain was identified as 5-(2-hydroxy-2-methyl-propyl)-2,2-dimethyl2,3-dihydro-naphtho[2,3-6]furan-4,6,7,9-tetrone (C18H18O6) (Park et al. 2006). These authors suggested that this pigmented compound is a product of the condensation of several carbofuran degradation metabolites. The identity of this red compound in our study is currently further explored. A number of observations suggest that, in addition to the route shown in Fig. 6, a second catabolic pathway may be operating in strain KN65.2. The amount of carbofuran phenol accumulating in nonmineralizing mutants showing complete conversion of carbofuran is much less than expected from a stoichiometric conversion (Fig. 3f and Online resource 6f). The low amount of colored metabolite(s) accumulating in the medium rules out that a major fraction of carbofuran would end up in these pigmented products (data not shown). A parallel pathway may proceed through side-chainhydroxylated carbofuran (compound 6) that is not detected in the wild type and, hence, does not represent a dead-end metabolite. Moreover, mutant 9G4 does not degrade carbofuran phenol (data not shown). A good candidate for catalyzing this conversion is the mono-oxygenase CfdI, and the hydroxylation product may then be further metabolized by sequential activity of carbofuran hydrolase and CfdC as proposed in Online resource 10. This concurrent route could account for the formation of a side-chain-hydroxylated analog of metabolite 3, i.e., putative compound 7. In conclusion, a combined genomic-metabolomicproteomic approach revealed a number of steps and the corresponding catabolic genes in the biodegradation pathway of carbofuran by Novosphingobium sp. KN65.2, but the hydrolase gene required for initiating mineralization remains to be identified. Further analysis using nonpolar mutants affected in specific genes identified here and biochemical characterization of the respective encoded enzymes are required to further elucidate the tentative scheme currently envisaged. Of particular interest is how TonB-dependent uptake is involved in catabolism of this insecticide.

Appl Microbiol Biotechnol Acknowledgments This research was funded by the Flemish Interuniversity Council (VLIR-UOS) of Belgium (BBTP2007-0012-1087), the joint support of the International Foundation for Science and Organisation for the Prohibition of Chemical Weapons (IFS/OPCW) (C/4563-1), and the EU FP7 projects BIOTREAT (EU grant 266039) and AQUAREHAB (EU grant ENV 2008.3.1.1.1.). We thank Kenneth Simoens for technical support.

References Aylward FO, McDonald BR, Adams SM, Valenzuela A, Schmidt RA, Goodwin LA, Woyke T, Currie CR, Suen G, Poulsen M (2013) Comparison of 26 sphingomonad genomes reveals diverse environmental adaptations and biodegradative capabilities. Appl Environ Microbiol 79(12):3724–3733. doi:10.1128/Aem.00518-13 Bermingham A, Derrick JP (2002) The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery. Bioessays 24:637–648. doi:10.1002/Bies.10114 Caldas SS, Demoliner A, Costa FP, D'Oca MGM, Primel EG (2010) Pesticide residue determination in groundwater using solid-phase extraction and high-performance liquid chromatography with diode array detector and liquid chromatography-tandem mass spectrometry. J Braz Chem Soc 21(4):642–650. doi:10.1590/S010350532010000400009 Castellanos J, Sánchez J, Uribe D, Moreno L, Melgarejo LM (2013) Characterization of carbofuran degrading bacteria obtained from potato cultivated soils with different pesticide application records. Rev Fac Nal Agr Medellín 66:6899–6908 Chapalamadugu S, Chaudhry GR (1992) Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates. Crit Rev Biotechnol 12(5–6):357–389. doi:10.3109/ 07388559209114232 Chaudhry GR, Ali HD (1988) Bacterial metabolism of carbofuran. Appl Environ Microbiol 54(1414):1419 Chaudhry GR, Mateen A, Kaskar B, Sardessai M, Bloda M, Bhatti AR, Walia SK (2002) Induction of carbofuran oxidation to 4hydroxycarbofuran by Pseudomonas sp. 50432. FEMS Microbiol Lett 214(2):171–176. doi:10.1016/s0378-1097(02)00851-0 Chen HP, Zhu SH, Casabon I, Hallam SJ, Crocker FH, Mohn WW, Indest KJ, Eltis LD (2012) Genomic and transcriptomic studies of an RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)degrading actinobacterium. Appl Environ Microbiol 78(21): 7798–7800. doi:10.1128/Aem.02120-12 Chiron S, Valverde A, FernandezAlba A, Barcelo D (1995) Automated sample preparation for monitoring groundwater pollution by carbamate insecticides and their transformation products. J AOAC Int 78(6):1346–1352 Chowdhury MAZ, Banik S, Uddin B, Moniruzzaman M, Karim N, Gan SH (2012) Organophosphorus and carbamate pesticide residues detected in water samples collected from paddy and vegetable fields of the Savar and Dhamrai Upazilas in Bangladesh. Int J Environ Res Public Health 9(9):3318–3329. doi:10.3390/ijerph9093318 Cuthbertson L, Nodwell JR (2013) The TetR family of regulators. Microbiol Mol Biol Rev 77(3):440–475. doi:10.1128/Mmbr. 00018-13 D'Argenio V, Petrillo M, Cantiello P, Naso B, Cozzuto L, Notomista E, Paolella G, Di Donato A, Salvatore F (2011) De novo sequencing and assembly of the whole genome of Novosphingobium sp. strain PP1Y. J Bacteriol 193(16):4296 Dasgupta S, Meisner C, Wheeler D, Xuyen K, Lam NT (2007) Pesticide poisoning of farm workers - implications of blood test results from Vietnam. Int J Hyg Environ Health 210(2):121–132. doi:10.1016/j. ijheh.2006.08.006

De Llasera MPG, Bernal-Gonzalez M (2001) Presence of carbamate pesticides in environmental waters from the northwest of Mexico: determination by liquid chromatography. Water Res 35(8):1933–1940 Desaint S, Hartmann A, Parekh NR, Fournier JC (2000) Genetic diversity of carbofuran-degrading soil bacteria. FEMS Microbiol Ecol 34(2): 173–180. doi:10.1111/j.1574-6941.2000.tb00767.x Desaint S, Arrault S, Siblot S, Fournier JC (2003) Genetic transfer of the mcd gene in soil. J Appl Microbiol 95(1):102–108. doi:10.1046/j. 1365-2672.2003.01965.x Dresen C, Lin LY, D'Angelo I, Tocheva EI, Strynadka N, Eltis LD (2010) A flavin-dependent monooxygenase from Mycobacterium tuberculosis involved in cholesterol catabolism. J Biol Chem 285(29):22264–22275. doi:10.1074/jbc.M109.099028 Fahmy MAH, Fukuto TR, Myers RO, March RB (1970) Selective toxicity of new N-phosphorothioylcarbamate esters. J Agric Food Chem 18(5):793–796. doi:10.1021/jf60171a014 Feng XH, Ou LT, Ogram A (1997) Plasmid-mediated mineralization of carbofuran by Sphingomonas sp. strain CF06. Appl Environ Microbiol 63(4):1332–1337 Fida TT, Breugelmans P, Lavigne R, van der Meer JR, De Mot R, Vaysse PJ, Springael D (2014) Identification of opsA, a gene involved in solute stress mitigation and survival in soil, in the polycyclic aromatic hydrocarbon-degrading Novosphingobium sp. strain LH128. Appl Environ Microbiol 80(11):3350–3361. doi:10.1128/AEM.00306-14 Gabriel FL, Cyris M, Jonkers N, Giger W, Guenther K, Kohler HP (2007) Elucidation of the ipso-substitution mechanism for side-chain cleavage of alpha-quaternary 4-nonylphenols and 4-t-butoxyphenol in Sphingobium xenophagum Bayram. Appl Environ Microbiol 73(10):3320–3326. doi:10.1128/AEM.02994-06 Gao JF, Ellis LBM, Wackett LP (2011) The university of Minnesota pathway prediction system: multi-level prediction and visualization. Nucleic Acids Res 39:W406–W411. doi:10.1093/Nar/Gkr200 Goad RT, Goad JT, Atieh BH, Gupta RC (2004) Carbofuran-induced endocrine disruption in adult male rats. Toxicol Mech Methods 14(4):233–239. doi:10.1080/15376520490434476 Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52(5): 696–704. doi:10.1080/10635150390235520 Gupta RC (1994) Carbofuran toxicity. J Toxicol Environ Health 43(4): 383–418 Hamed RB, Batchelar ET, Clifton IJ, Schofield CJ (2008) Mechanisms and structures of crotonase superfamily enzymes—how nature controls enolate and oxyanion reactivity. Cell Mol Life Sci 65:2507– 2527. doi:10.1007/s00018-008-8082-6 Hartmann EM, Armengaud J (2014) Shotgun proteomics suggests involvement of additional enzymes in dioxin degradation by Sphingomonas wittichii RW1. Environ Microbiol 16:162–176. doi: 10.1111/1462-2920.12264 Hashimoto M, Fukui M, Hayano K, Hayatsu M (2002) Nucleotide sequence and genetic structure of a novel carbaryl hydrolase gene (cehA) from Rhizobium sp. strain AC100. Appl Environ Microbiol 68(3):1220–1227. doi:10.1128/Aem.68.3.1220-1227.2002 Hashimoto M, Mizutani A, Tago K, Ohnishi-Kameyama M, Shimojo T, Hayatsu M (2006) Cloning and nucleotide sequence of carbaryl hydrolase gene (cahA) from Arthrobacter sp. RC100. J Biosci Bioeng 101:410–414. doi:10.1263/Jbb.101.410 Head IM, Cain RB, Suett DL (1992) Characterization of a carbofurandegrading bacterium and investigation of the role of plasmids in catabolism of the insecticide carbofuran. Arch Microbiol 158(4): 302–308. doi:10.1007/Bf00245249 Heider J (2001) A new family of CoA-transferases. FEBS Lett 509(3): 345–349. doi:10.1016/s0014-5793(01)03178-7 Helbling DE, Hollender J, Kohler HPE, Singer H, Fenner K (2010) Highthroughput identification of microbial transformation products of organic micropollutants. Environ Sci Technol 44:6621–6627. doi: 10.1021/es100970m

Appl Microbiol Biotechnol Held JM, Schilling B, D'Souza AK, Srinivasan T, Behring JB, Sorensen DJ, Benz CC, Gibson BW (2013) Label-free quantitation and mapping of the ErbB2 tumor receptor by multiple protease digestion with data-dependent (MS1) and dataindependent (MS2) acquisitions. Int J Proteomics 2013: 791985. doi:10.1155/2013/791985 Kaczmarczyk A, Campagne S, Danza F, Metzger LC, Vorholt JA, Francez-Charlot A (2011) Role of Sphingomonas sp. strain Fr1 PhyR-NepR-sigmaEcfG cascade in general stress response and identification of a negative regulator of PhyR. J Bacteriol 193(23):6629– 6638. doi:10.1128/JB.06006-11 Karns JS, Tomasek PH (1991) Carbofuran hydrolase—purification and properties. J Agric Food Chem 39:1004–1008. doi:10.1021/ Jf00005a041 Karns JS, Mulbry WW, Nelson JO, Kearney PC (1986) Metabolism of carbofuran by a pure bacterial culture. Pestic Biochem Physiol 25(2):211–217. doi:10.1016/0048-3575(86)90048-9 Kaur B, Khera A, Sandhir R (2012) Attenuation of cellular antioxidant defense mechanisms in kidney of rats intoxicated with carbofuran. J Biochem Mol Toxicol 26(10):393–398. doi:10.1002/jbt.21433 Kim IS, Ryu JY, Hur HG, Gu MB, Kim SD, Shim JH (2004) Sphingomonas sp. strain SB5 degrades carbofuran to a new metabolite by hydrolysis at the furanyl ring. J Agric Food Chem 52:2309– 2314. doi:10.1021/jf035502l Koebnik R (2005) TonB-dependent trans-envelope signalling: the exception or the rule? Trends Microbiol 13:343–347. doi:10.1016/j.tim. 2005.06.005 Larsen RA, Wilson MM, Guss AM, Metcalf WW (2002) Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178(3):193– 201. doi:10.1007/s00203-002-0442-2 Luo YR, Kang SG, Kim SJ, Kim MR, Li N, Lee JH, Kwon KK (2012) Genome sequence of benzo(a)pyrene-degrading bacterium Novosphingobium pentaromativorans US6-1. J Bacteriol 194(4): 907. doi:10.1128/JB.06476-11 Masai E, Sasaki M, Minakawa Y, Abe T, Sonoki T, Miyauchi K, Katayama Y, Fukuda M (2004) A novel tetrahydrofolatedependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate. J Bacteriol 186(9):2757–2765 Milatovic D, Gupta RC, Dekundy A, Montine TJ, Dettbarn WD (2005) Carbofuran-induced oxidative stress in slow and fast skeletal muscles: prevention by memantine and atropine. Toxicol 208:13–24. doi:10.1016/j.tox.2004.11.004 Naqvi T, Cheesman MJ, Williams MR, Campbell PM, Ahmed S, Russell RJ, Scott C, Oakeshott JG (2009) Heterologous expression of the methyl carbamate-degrading hydrolase Mcd. J Biotechnol 144(2): 89–95. doi:10.1016/j.jbiotec.2009.09.009 Noinaj N, Guillier M, Barnard TJ, Buchanan SK (2010) TonBdependent transporters: regulation, structure, and function. Annu Rev Microbiol 64:43–60. doi:10.1146/annurev.micro. 112408.134247 Ogram AV, Duan YP, Trabue SL, Feng X, Castro H, Ou LT (2000) Carbofuran degradation mediated by three related plasmid systems. FEMS Microbiol Ecol 32(3):197–203. doi:10.1111/j.1574-6941. 2000.tb00712.x Parekh NR, Hartmann A, Charnay MP, Fournier JC (1995) Diversity of carbofuran-degrading soil bacteria and detection of plasmid-encoded sequences homologous to the mcd gene. FEMS Microbiol Ecol 17(3): 149–160. doi:10.1111/j.1574-6941.1995.tb00138.x Park MR, Lee S, Han TH, Oh BT, Shim JH, Kim IS (2006) A new intermediate in the degradation of carbofuran by Sphingomonas sp. strain SB5. J Microbiol Biotechnol 16(8):1306–1310 Plangklang P, Reungsang A (2012) Isolation and characterisation of a carbofuran degrading Burkholderia sp. PCL3 from carbofuran-

phytoremediated rhizosphere soil. Chem Ecol 28:253–266. doi:10. 1080/02757540.2011.645032 Ramanand K, Sharmila M, Singh N, Sethunathan N (1991) Metabolism of carbamate insecticides by resting cells and cell-free preparations of a soil bacterium, Arthrobacter sp. Bull Environ Contam Toxicol 46(3):380–386 Ran T, Gao Y, Marsh M, Zhu W, Wang M, Mao X, Xu L, Xu D, Wang W (2013) Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family. Biochem J 449(1):51–60. doi:10.1042/BJ20120913 Ricken B, Corvini PFX, Cichocka D, Parisi M, Lenz M, Wyss D, Martinez-Lavanchy PM, Muller JA, Shahgaldian P, Tulli LG, Kohler HPE, Kolvenbach BA (2013) ipso-Hydroxylation and subsequent fragmentation: a novel microbial strategy to eliminate sulfonamide antibiotics. Appl Environ Microbiol 79(18):5550–5558. doi:10.1128/Aem.00911-13 Salama AK (1998) Metabolism of carbofuran by Aspergillus niger and Fusarium graminearum. J Environ Sci Health B 33:253–266. doi: 10.1080/03601239809373142 Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Saxena A, Anand S, Dua A, Sangwan N, Khan F, Lal R (2013) Novosphingobium lindaniclasticum sp. nov., a hexachlorocyclohexane (HCH)-degrading bacterium isolated from an HCH dumpsite. Int J Syst Evol Microbiol 63:2160–2167. doi:10.1099/ijs.0.045443-0 Seo J, Jeon J, Kim S-D, Kang S, Han J, Hur H-G (2007) Fungal biodegradation of carbofuran and carbofuran phenol by the fungus Mucor ramannianus: identification of metabolites. Water Sci Technol 55(1–2):163–167. doi:10.2166/wst.2007.051 Shin DH, Kim DU, Seong CN, Song HG, Ka JO (2012) Genetic and phenotypic diversity of carbofuran-degrading bacteria isolated from agricultural soils. J Microbiol Biotechnol 22(4):448–456. doi:10. 4014/jmb.1108.08087 Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in-vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Nat Biotechnol 1:784–791. doi:10.1038/ nbt1183-784 Tariq MI, Afzal S, Hussain I (2006) Degradation and persistence of cotton pesticides in sandy loam soils from Punjab, Pakistan. Environ Res 100:184–196. doi:10.1016/j.envres.2005.05.002 Tomasek PH, Karns JS (1989) Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in Gramnegative bacteria. J Bacteriol 171(7):4038–4044 Topp E, Hanson RS, Ringelberg DB, White DC, Wheatcroft R (1993) Isolation and characterization of an N-methylcarbamate insecticidedegrading methylotrophic bacterium. Appl Environ Microbiol 59(10):3339–3349 Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3 - new capabilities and interfaces. Nucleic Acids Res 40(15):e115. doi:10.1093/nar/gks596 Uyttebroek M, Breugelmans P, Janssen M, Wattiau P, Joffe B, Karlson U, Ortega-Calvo JJ, Bastiaens L, Ryngaert A, Hausner M, Springael D (2006) Distribution of the Mycobacterium community and polycyclic aromatic hydrocarbons (PAHs) among different size fractions of a long-term PAH-contaminated soil. Environ Microbiol 8:836–847. doi:10.1111/j.1462-2920.2005.00970.x van Berkel WJH, Kamerbeek NM, Fraaije MW (2006) Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. J Biotechnol 124(4):670–689. doi:10.1016/j.jbiotec.2006.03.044 van der Geize R, Yam K, Heuser T, Wilbrink MH, Hara H, Anderton MC, Sim E, Dijkhuizen L, Davies JE, Mohn WW, Eltis LD (2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci U S A 104:1947–1952. doi:10.1073/ pnas.0605728104

Appl Microbiol Biotechnol Vryzas Z, Vassiliou G, Alexoudis C, Papadopoulou-Mourkidou E (2009) Spatial and temporal distribution of pesticide residues in surface waters in northeastern Greece. Water Res 43(1):1–10. doi:10.1016/j. watres.2008.09.021

Yan QX, Hong Q, Han P, Dong XJ, Shen YJ, Li SP (2007) Isolation and characterization of a carbofuran-degrading strain Novosphingobium sp. FND-3. FEMS Microbiol Lett 271(2):207–213. doi:10.1111/j. 1574-6968.2007.00718.x