Environment Health Techniques Cypermethrin biodegradation
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Research Paper Characterization of a cypermethrin-degrading Methylobacterium sp. strain A-1 and molecular cloning of its carboxylesterase gene Corinna Diegelmann, Joachim Weber, Regina Heinzel-Wieland and Michael Kemme Department of Chemical Engineering and Biotechnology, Hochschule Darmstadt, University of Applied Sciences, Darmstadt, Germany
A novel mesophilic bacterial strain, designated A-1, was isolated from microbially contaminated biopolymer microcapsules. The bacterium was able to withstand and grow in liquid cultures supplemented with the pyrethroid cypermethrin in concentrations up to 400 mg L1. Furthermore, strain A-1 could use cypermethrin as sole carbon source and could degrade >50% of it in 12 h. Based on phenotypic and chemotaxonomic characterization, and phylogenetic analysis of 16S rRNA gene sequence, the strain A-1 was identified as Methylobacterium sp., which is the first reported cypermethrin degrader of methylotrophic bacteria. A role for esterase activity in cypermethrin biodegradation was presumed. Therefore, the carboxylesterase gene mse1 was amplified from the Methylobacterium sp. strain A-1 genome and the resulting 1 kb amplicon cloned into E. coli. Sequence analysis of the mse1-DNA insert revealed an open reading frame of 633 bp encoding for a putative carboxylesterase of 210 amino acid residues with a predicted molecular mass of 22 kDa. The amino acid sequence of the deduced enzyme MsE1 with the catalytic triad Ser106, Asp156, and His187 was found to be similar to that of a/b-hydrolase fold proteins. The active site Ser106 residue is located in the consensus pentapeptide motif Gly-X-Ser-X-Gly that is typical of esterases. Abbreviations: MsE – Methylobacterium sp. esterase; MSM – mineral salts medium
PLGA – poly(lactic-co-glycolic acid) Keywords: Cypermethrin / Pyrethroid / Biodegradation / Methylobacterium / Carboxylesterase Received: March 23, 2015; accepted: June 14, 2015 DOI 10.1002/jobm.201500186
Introduction During the last three decades, pyrethroid insecticides have been utilized extensively as a replacement for the more toxic and environmentally persistent organochlorine, carbamate, and organophosphate compounds [1]. Among these agents, cypermethrin belongs to the fourth generation of synthetic pyrethroids and constitutes a lipophilic ester, with an a-cyano-3-phenoxybenzyl alcohol and a dichlorovinylcyclopropane-carboxylic acid moiety [2]. Cypermethrin
Correspondence: Michael Kemme, Department of Chemical Engineering and Biotechnology, Hochschule Darmstadt, University of Applied Sciences, Schnittspahnstrasse 12, D-64287 Darmstadt, Germany E-mail: [email protected] Phone: þ49-6151-16 8631 Fax: þ49-6151-16 8404 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
has become one of the most important insecticides globally and as an example of high consumption rate, total usage of cypermethrin in India is about 7300 tonnes of active ingredient per year [3]. This insecticide is registered world-wide for commercial agricultural application on food and feed crops but also to livestock as an ectoparasiticide and controversially discussed, it is authorized for the treatment of sea lice on salmon farms in the European Union [4]. In the USA, over 80% of cypermethrin usage occurs in a wide range of nonagricultural pest control for indoor and outdoor applications such as wood preservation in forestry and timber, disinfection of buildings and transportation means, household residual spraying, biocide impregnation of textiles, and in public health programms, respectively [5]. Cypermethrin is a long-lasting insecticide with contact and stomach action on chewing and
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sucking insects that interferes primarily with voltagegated sodium channels in their neuronal axons. Due to its a-cyano group, cypermethrin binding correlates with prolonged Naþ-tail currents along with depolarization of nerve membranes resulting in eventual failure of the neurons to transmit spastic muscular paralysis and death of insect pests [6]. Cypermethrin is highly toxic to some fish species and to aquatic invertebrates, but has low acute toxicity to birds and mammals. Subacute and chronic exposure of the insecticide might cause human health risks, including reproductive and developmental toxicity, neurobehavioral side effects, suppression of the immune system, and cypermethrin is considered as possible human carcinogen [7]. The progressive increase in cypermethrin consumption represents an environmental risk because a variable portion of the applied insecticide is released into terrestrial or aquatic sites through spray drift, accidential spillage, or surface runoff. As a hydrophobic substance, the water solubility of cypermethrin is very low (50% sequence identity), and includes proteins with demonstrated or predicted carboxylesterase/hydrolase activity. It was found that the MsE1 sequence had the highest degree of identity (98%) to a putative carboxylesterase from Methylobacterium oryzae (AIQ93377.1, Fig. 5). Also, it
Figure 4. Time course of growth of Methylobacterium sp. strain A-1 in MSM at different initial concentrations of cypermethrin as sole carbon source. Symbols: (open circle) 50 mg L1; (closed circle) 100 mg L1; (open triangle) 200 mg L1; (closed triangle) 400 mg L1; (open square) 1% methanol as control. Values showing total viable count are the means of three replications with standard deviation.
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Figure 5. Multiple alignment of the predicted amino acid sequence of MsE1 with other related carboxylesterases or hydrolases from bacteria. Identical residues are shown as white letters on a black background. The amino acid residues predicted to be members of the catalytic triade are marked with an asterisk, those of the conserved motif Gly-X-Ser-X-Gly are boxed. Numbers to the right of sequences represent the true amino acid positions in the protein. The compared proteins were from Methylobacterium oryzae CBMB20 (AIQ93377.1), M. radiotolerans JCM 2831 (ACB26894.1), M. mesophilicum SR1.6/6 (EMS42449.1), Skermanella stibiiresistens SB22 (EWY37830.1), and Ochrobactrum anthropi YZ-1 (AEY11370.1).
scored significant similarity to the predicted carboxylesterases of M. radiotolerans (ACB26894.1, 89% identity) and M. mesophilicum (EMS42449.1, 85% identity), respectively, and to the putative hydrolase of Skermanella stibiiresistens (EWY37830.1, 65% identity), an antimonyresistant bacterium [29]. The experimentally characterized pyrethroid-hydrolyzing carboxylesterase PytZ (AEY11370.1) from Ochrobactrum anthropi YZ-1 [23] showed moderate identity of 41% with MsE1. Sequence alignments of MsE1 with the most homologous proteins revealed the presence of three blocks containing five or more conserved residues (Fig. 5). On the basis of sequence comparison with other known esterase or lipase proteins, it can be concluded that Ser106, Asp156, and His187 comprise a typical catalytic triade. The Ser106 residue is in the center of a conserved pentapeptide motif of Gly-X-Ser-X-Gly (residues from 104 to 108) which is a typical feature of esterase family VI proteins with an a/b-hydrolase fold [30]. The presence of a short hydrophobic region upstream of a conserved His26-Gly27 dipeptide indicated a putative oxyanion hole [31]. No potential signal sequence was found, which suggests that the esterase MsE1 is located intracellularly. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Discussion So far, most of the efforts regarding the isolation of pyrethroid-degrading bacteria were concentrated on contaminated soil or activated sludge samples [16, 19] but in the present study, the isolation was attempted from a biopolymer scaffold polluted by chlorinated hydrocarbon. Screening of microbial contaminants by enchrichment culture enabled us to isolate a pinkpigmented facultatively methylotrophic bacterial strain, designated A-1, capable of utilizing the chlorinated pyrethroid cypermethrin as sole carbon source. Combined analysis of morphological, biochemical, physiological, and phylogenetic characteristics allowed the assignment of strain A-1 to the genus Methylobacterium, but determination to the species level was not accomplished. 16S rRNA gene sequence analysis of the isolate showed highest identity (99.3%) to M. fujisawaense, however, the fatty acid profile of strain A-1 was significantly different from those of its phylogenetic relatives M. fujisawaense, M. mesophilicum, and M. radiotolerans, respectively [32]. Differentiation of strain A-1 from other members of the genus Methylobacterium was
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verified by antibiotics susceptibility testing. The majority of Methylobacterium isolates are susceptible to blactam drugs, cotrimoxazole, and aminoglycosides such as gentamycin and amikacin [33]. Discordant susceptibilities, with high sensitivity to amikacin and tetracyline, respectively, and resistance to all other antibiotics seem to be a distinctive feature of strain A-1. This indicates that strain A-1 is possibly a new species of the genus Methylobacterium. The genus Methylobacterium currently consists of 49 different species [33] which are widely distributed in the environment, colonizing the rhizosphere of soils, aquatic sediments, freshwater, or biofilms, and are isolated from air samples [34]. Methylotrophic bacteria are well known for symbiotic interactions with plants and for utilizing different one-carbon pollutants, including halogenated hydrocarbons such as dichloromethane [35]. Reports on biodegradative abilities of Methylobacteria concerning complex xenobiotics are rare. One phytosymbiotic Methylobacterium sp. BJ001 was isolated from poplar tissues and shown to degrade the toxic explosives trinitrotoluene, hexagen, and octogen in the presence of fructose [25]. Furthermore, a carbazoledegrading Methylobacterium sp. GPE1 has been identified in groundwater samples of a gasworks site [36]. Recently, Methylobacterium populi demonstrated multiple plant growth promotion activities by producing indole-3acetic acid and simultaneously, exhibited the ability to degrade polycyclic aromatic hydrocarbons (PAHs) in biofilms on plant seeds [35]. Thus, Methylobacteria are metabolically active species that possess the biochemical and physiological capacity to degrade complex molecules of environmental organic chemicals. To the best of our knowledge, no report on the isolation of a pyrethroidhydrolyzing Methylobacterium species is available yet. The bacterial strain A-1 isolated in this study is the first member of the genus Methylobacterium that was found highly effective in degrading the pyrethroid cypermethrin. The low water solubility of cypermethrin [8] limits its availability to microorganisms and could potentially lower the microbial capacity to degrade this hydrophobic compound in aqueous medium. During growth on cypermethrin, strain A-1 quickly emulsified the pyrethroid, possibly due to the production of biosurfactants, as other studies have described [15, 36]. The formation of stable emulsions can enhance the bioavailability of cypermethrin and consequently, can help direct absorption of the lipophilic molecule into the microbial cells. The amount of cypermethrin utilized by strain A-1 was approximately equal to 60% of the initial dose within 3 days of incubation. However, the strong ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
depletion trend of the degradation curve indicated that complete catabolism of the pyrethroid substrate could not be achieved, even over an extended duration. A possible explanation of the observed resistance for further degradation could be attributed to the fact that cypermethrin-metabolizing bacteria are isomer selective [37]. The technical cypermethrin used in this study consists of a mixture of eight stereoisomers [5] that differ greatly in their biological activity. Such selectivity in biodegradation might lead to relative accumulation of the most persistent diastereomers or enantiomers uncleaved in solution. Cypermethrin-degrading microorganisms usually need an adaptation period to produce the appropriate degradative enzymes followed by accelerated biodegradation. The addition of supplementary carbon sources and nutrients (e.g., glucose, beef extract, or yeast extract) was necessary to enhance their degradative abilities [12]. In contrast, strain A-1 could directly utilize and degrade cypermethrin without any apparent lag phase of degradation in the absence of other carbon sources. This is an important feature of an insecticide-degrading microorganism to be used in bioremediation of different contaminated environments. For survival in soil or aquatic substrates, resistance to a wide range of antibiotics is an important factor determining proliferation of an organism ahead of other members of microbial communities [38]. Antibiotics produced by the environmental microbiota or discharged by anthropogenic input can be treated as an ecological factor, offering a survival benefit in bacterial community structures [39]. Shared resistance to quinolone antibiotics, sulfonamide drugs, and some aminoglycosides generally confers Methylobacterium sp. strain A-1 an evolutionary advantage for use as seeds in bioaugmentation techniques. Therefore, Methylobacterium sp. strain A-1 might be most helpful for in situ biodegradation of pyrethroid contaminants under natural conditions and for the development of bioremediation approaches to restore ecological pyrethroid damage. Pyrethroids are a large group of ester-containing compounds whose main degradation route involves cleavage of the central ester bond by carboxylesterases [3]. The carboxylesterase family (EC 3.1.1.1) comprises a group of enzymes hydrolyzing ester molecules with relatively broad substrate specificity. They are believed to be involved in the detoxification of different xenobiotics [21]. Previous studies have proven that carboxylesterases from a-Proteobacteria are the first protagonists in catabolic pathways of pyrethroids, and the hydrolysis products of these enzymes are likely further catabolized, as each of these microbial organisms mineralize pyrethroids [40]. To
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further substantiate that Methylobacterium sp. strain A-1 endows the enzymatic machinery necessary for pyrethroid degradation, a PCR-based approach was performed to identify a carboxylesterase gene within the A-1 genome. After sequencing and BLAST identification at NCBI, it turned out that the PCR-derived clones harbored the gene mse1 coding for an enzyme of the family VI esterases according to the classification of Arpigny and Jaeger [30]. The deduced amino acid sequence of MsE1 showed high identity with putative carboxylesterases of different Methylobacterium species (Fig. 5), but it shared only 41% amino acid sequence identity with the characterized carboxylesterase PytZ from Ochrobactrum anthropi YZ-1 [23]. MsE1 has a predicted molecular mass of 22 kDa, which is similar to PytZ with 24 kDa [23]. However, it is smaller than other reported cypermethrin-hydrolyzing enzymes, such as PytY (42 kDa) from O. anthropi YZ1 [22] and PytH (31 kDa) from Sphingobium sp. JZ-1 [24]. Taken together, the above findings suggest that MsE1 could be an new member of the pyrethroid-degrading carboxylesterases. Therefore, the gene mse1, which we isolated in this study, enriches the genetic resources for bioremediation of pyrethroid-contaminated ecosystems.
[3] Verma, J.P., Jaiswal, D.K., Sagar, R., 2014. Pesticide relevance and their microbial degradation: a-state-of-art. Rev. Environ. Sci. Biotechnol., 13, 429–466. [4] Willis, K.J., Gillibrand, P.A., Cromey, C.J., Black, K.D., 2005. Sea lice treatment on salmon farms have no adverse effects on zooplankton communities: a case study. Mar. Pollut. Bull., 50, 806–816. [5] U.S. Environmental Protection Agency, 2008. Revised reregistration eligibility decision for cypermethrin. EPA OPP-2005-0293, Washington, DC. [6] Gammon, D.W., Leggett, M.F., Clark, J.M., 2011. Pyrethroid mode(s) of action in the context of food quality protection act (FQPA) regulation. J. Agric. Food Chem., 59, 2773–2785. [7] Tsuji, R., Yamada, T., Kawamura, S., 2012. Mammal toxicology of synthetic pyrethroids. Top. Curr. Chem., 314, 83–112. [8] Feo, M.L., Ginebreda, A., Eljarrat, E., Barcelo, D., 2010. Presence of pyrethroid pesticides in water and sediments of Ebro River Delta. J. Hydrol., 393, 156–162. [9] Meyer, B.N., Lam, C., Moore, S., Jones, R.L., 2013. Laboratory degradation rates of 11 pyrethroids under aerobic and anaerobic conditions. J. Agric. Food Chem., 61, 4702–4708. [10] Chen, S., Geng, P., Xiao, Y., Hu, M., 2012. Bioremediation of b-cypermethrin and 3-phenoxybenzaldehyde contaminated soils using Streptomyces aureus HP-S-01. Appl. Microbiol. Biotechnol., 94, 505–515. [11] Ma, J., Chen, L., Qiu, J., 2013. Biodegradation of betacypermethrin by a novel Azoarcus indigens strainHZ5. J. Environ. Sci. Health B, 48, 851–859.
Acknowledgments The authors owe special thanks to Roland Klein (ErnstBerl Institute, Darmstadt University of Technology, Germany) for providing PLGA microcapsules. We also acknowledge the expert contribution of Susanne Verbarg (DSMZ, Braunschweig, Germany) for the analysis of cellular fatty acids. This work was financially supported €r by a grant-in-aid (403 76000) from zfe (Zentrum fu Forschung und Technologie) of Darmstadt University of Applied Sciences.
[12] Chen, S., Hu, M., Liu, J., Zhong, G., et al., 2011. Biodegradation of beta-cypermethrin and 3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-02. J. Hazard. Mater., 187, 433–440. [13] Wang, B.Z., Ma, Y., Zhou, W.J., Zheng, J.W., et al., 2011. Biodegradation of synthetic pyrethroids by Ochrobactrum tritici strain pyd-1. World J. Microbiol. Biotechnol., 27, 2315–2324. [14] Grant, R.J., Daniell, T.J., Betts, W.B., 2002. Isolation and identification of synthetic pyrethroid-degrading bacteria. J. Appl. Microbiol., 92, 534–540. [15] Zhang, C., Wang, S., Yan, Y., 2011. Isomerization and biodegradation of beta-cypermethrin by Pseudomonas aeruginosa CH7 with biosurfactant production. Bioresour. Technol., 102, 7139–7146.
Conflicts of interest All authors have no financial or commercial conflicts of interest concerning the current work or its publication.
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Research Paper Characterization of a cypermethrin-degrading Methylobacterium sp. strain A-1 and molecular cloning of its carboxylesterase gene Corinna Diegelmann, Joachim Weber, Regina Heinzel-Wieland and Michael Kemme Department of Chemical Engineering and Biotechnology, Hochschule Darmstadt, University of Applied Sciences, Darmstadt, Germany
A novel mesophilic bacterial strain, designated A-1, was isolated from microbially contaminated biopolymer microcapsules. The bacterium was able to withstand and grow in liquid cultures supplemented with the pyrethroid cypermethrin in concentrations up to 400 mg L1. Furthermore, strain A-1 could use cypermethrin as sole carbon source and could degrade >50% of it in 12 h. Based on phenotypic and chemotaxonomic characterization, and phylogenetic analysis of 16S rRNA gene sequence, the strain A-1 was identified as Methylobacterium sp., which is the first reported cypermethrin degrader of methylotrophic bacteria. A role for esterase activity in cypermethrin biodegradation was presumed. Therefore, the carboxylesterase gene mse1 was amplified from the Methylobacterium sp. strain A-1 genome and the resulting 1 kb amplicon cloned into E. coli. Sequence analysis of the mse1-DNA insert revealed an open reading frame of 633 bp encoding for a putative carboxylesterase of 210 amino acid residues with a predicted molecular mass of 22 kDa. The amino acid sequence of the deduced enzyme MsE1 with the catalytic triad Ser106, Asp156, and His187 was found to be similar to that of a/b-hydrolase fold proteins. The active site Ser106 residue is located in the consensus pentapeptide motif Gly-X-Ser-X-Gly that is typical of esterases. Abbreviations: MsE – Methylobacterium sp. esterase; MSM – mineral salts medium
PLGA – poly(lactic-co-glycolic acid) Keywords: Cypermethrin / Pyrethroid / Biodegradation / Methylobacterium / Carboxylesterase Received: March 23, 2015; accepted: June 14, 2015 DOI 10.1002/jobm.201500186
Introduction During the last three decades, pyrethroid insecticides have been utilized extensively as a replacement for the more toxic and environmentally persistent organochlorine, carbamate, and organophosphate compounds [1]. Among these agents, cypermethrin belongs to the fourth generation of synthetic pyrethroids and constitutes a lipophilic ester, with an a-cyano-3-phenoxybenzyl alcohol and a dichlorovinylcyclopropane-carboxylic acid moiety [2]. Cypermethrin
Correspondence: Michael Kemme, Department of Chemical Engineering and Biotechnology, Hochschule Darmstadt, University of Applied Sciences, Schnittspahnstrasse 12, D-64287 Darmstadt, Germany E-mail: [email protected] Phone: þ49-6151-16 8631 Fax: þ49-6151-16 8404 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
has become one of the most important insecticides globally and as an example of high consumption rate, total usage of cypermethrin in India is about 7300 tonnes of active ingredient per year [3]. This insecticide is registered world-wide for commercial agricultural application on food and feed crops but also to livestock as an ectoparasiticide and controversially discussed, it is authorized for the treatment of sea lice on salmon farms in the European Union [4]. In the USA, over 80% of cypermethrin usage occurs in a wide range of nonagricultural pest control for indoor and outdoor applications such as wood preservation in forestry and timber, disinfection of buildings and transportation means, household residual spraying, biocide impregnation of textiles, and in public health programms, respectively [5]. Cypermethrin is a long-lasting insecticide with contact and stomach action on chewing and
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sucking insects that interferes primarily with voltagegated sodium channels in their neuronal axons. Due to its a-cyano group, cypermethrin binding correlates with prolonged Naþ-tail currents along with depolarization of nerve membranes resulting in eventual failure of the neurons to transmit spastic muscular paralysis and death of insect pests [6]. Cypermethrin is highly toxic to some fish species and to aquatic invertebrates, but has low acute toxicity to birds and mammals. Subacute and chronic exposure of the insecticide might cause human health risks, including reproductive and developmental toxicity, neurobehavioral side effects, suppression of the immune system, and cypermethrin is considered as possible human carcinogen [7]. The progressive increase in cypermethrin consumption represents an environmental risk because a variable portion of the applied insecticide is released into terrestrial or aquatic sites through spray drift, accidential spillage, or surface runoff. As a hydrophobic substance, the water solubility of cypermethrin is very low (50% sequence identity), and includes proteins with demonstrated or predicted carboxylesterase/hydrolase activity. It was found that the MsE1 sequence had the highest degree of identity (98%) to a putative carboxylesterase from Methylobacterium oryzae (AIQ93377.1, Fig. 5). Also, it
Figure 4. Time course of growth of Methylobacterium sp. strain A-1 in MSM at different initial concentrations of cypermethrin as sole carbon source. Symbols: (open circle) 50 mg L1; (closed circle) 100 mg L1; (open triangle) 200 mg L1; (closed triangle) 400 mg L1; (open square) 1% methanol as control. Values showing total viable count are the means of three replications with standard deviation.
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Figure 5. Multiple alignment of the predicted amino acid sequence of MsE1 with other related carboxylesterases or hydrolases from bacteria. Identical residues are shown as white letters on a black background. The amino acid residues predicted to be members of the catalytic triade are marked with an asterisk, those of the conserved motif Gly-X-Ser-X-Gly are boxed. Numbers to the right of sequences represent the true amino acid positions in the protein. The compared proteins were from Methylobacterium oryzae CBMB20 (AIQ93377.1), M. radiotolerans JCM 2831 (ACB26894.1), M. mesophilicum SR1.6/6 (EMS42449.1), Skermanella stibiiresistens SB22 (EWY37830.1), and Ochrobactrum anthropi YZ-1 (AEY11370.1).
scored significant similarity to the predicted carboxylesterases of M. radiotolerans (ACB26894.1, 89% identity) and M. mesophilicum (EMS42449.1, 85% identity), respectively, and to the putative hydrolase of Skermanella stibiiresistens (EWY37830.1, 65% identity), an antimonyresistant bacterium [29]. The experimentally characterized pyrethroid-hydrolyzing carboxylesterase PytZ (AEY11370.1) from Ochrobactrum anthropi YZ-1 [23] showed moderate identity of 41% with MsE1. Sequence alignments of MsE1 with the most homologous proteins revealed the presence of three blocks containing five or more conserved residues (Fig. 5). On the basis of sequence comparison with other known esterase or lipase proteins, it can be concluded that Ser106, Asp156, and His187 comprise a typical catalytic triade. The Ser106 residue is in the center of a conserved pentapeptide motif of Gly-X-Ser-X-Gly (residues from 104 to 108) which is a typical feature of esterase family VI proteins with an a/b-hydrolase fold [30]. The presence of a short hydrophobic region upstream of a conserved His26-Gly27 dipeptide indicated a putative oxyanion hole [31]. No potential signal sequence was found, which suggests that the esterase MsE1 is located intracellularly. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Discussion So far, most of the efforts regarding the isolation of pyrethroid-degrading bacteria were concentrated on contaminated soil or activated sludge samples [16, 19] but in the present study, the isolation was attempted from a biopolymer scaffold polluted by chlorinated hydrocarbon. Screening of microbial contaminants by enchrichment culture enabled us to isolate a pinkpigmented facultatively methylotrophic bacterial strain, designated A-1, capable of utilizing the chlorinated pyrethroid cypermethrin as sole carbon source. Combined analysis of morphological, biochemical, physiological, and phylogenetic characteristics allowed the assignment of strain A-1 to the genus Methylobacterium, but determination to the species level was not accomplished. 16S rRNA gene sequence analysis of the isolate showed highest identity (99.3%) to M. fujisawaense, however, the fatty acid profile of strain A-1 was significantly different from those of its phylogenetic relatives M. fujisawaense, M. mesophilicum, and M. radiotolerans, respectively [32]. Differentiation of strain A-1 from other members of the genus Methylobacterium was
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verified by antibiotics susceptibility testing. The majority of Methylobacterium isolates are susceptible to blactam drugs, cotrimoxazole, and aminoglycosides such as gentamycin and amikacin [33]. Discordant susceptibilities, with high sensitivity to amikacin and tetracyline, respectively, and resistance to all other antibiotics seem to be a distinctive feature of strain A-1. This indicates that strain A-1 is possibly a new species of the genus Methylobacterium. The genus Methylobacterium currently consists of 49 different species [33] which are widely distributed in the environment, colonizing the rhizosphere of soils, aquatic sediments, freshwater, or biofilms, and are isolated from air samples [34]. Methylotrophic bacteria are well known for symbiotic interactions with plants and for utilizing different one-carbon pollutants, including halogenated hydrocarbons such as dichloromethane [35]. Reports on biodegradative abilities of Methylobacteria concerning complex xenobiotics are rare. One phytosymbiotic Methylobacterium sp. BJ001 was isolated from poplar tissues and shown to degrade the toxic explosives trinitrotoluene, hexagen, and octogen in the presence of fructose [25]. Furthermore, a carbazoledegrading Methylobacterium sp. GPE1 has been identified in groundwater samples of a gasworks site [36]. Recently, Methylobacterium populi demonstrated multiple plant growth promotion activities by producing indole-3acetic acid and simultaneously, exhibited the ability to degrade polycyclic aromatic hydrocarbons (PAHs) in biofilms on plant seeds [35]. Thus, Methylobacteria are metabolically active species that possess the biochemical and physiological capacity to degrade complex molecules of environmental organic chemicals. To the best of our knowledge, no report on the isolation of a pyrethroidhydrolyzing Methylobacterium species is available yet. The bacterial strain A-1 isolated in this study is the first member of the genus Methylobacterium that was found highly effective in degrading the pyrethroid cypermethrin. The low water solubility of cypermethrin [8] limits its availability to microorganisms and could potentially lower the microbial capacity to degrade this hydrophobic compound in aqueous medium. During growth on cypermethrin, strain A-1 quickly emulsified the pyrethroid, possibly due to the production of biosurfactants, as other studies have described [15, 36]. The formation of stable emulsions can enhance the bioavailability of cypermethrin and consequently, can help direct absorption of the lipophilic molecule into the microbial cells. The amount of cypermethrin utilized by strain A-1 was approximately equal to 60% of the initial dose within 3 days of incubation. However, the strong ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
depletion trend of the degradation curve indicated that complete catabolism of the pyrethroid substrate could not be achieved, even over an extended duration. A possible explanation of the observed resistance for further degradation could be attributed to the fact that cypermethrin-metabolizing bacteria are isomer selective [37]. The technical cypermethrin used in this study consists of a mixture of eight stereoisomers [5] that differ greatly in their biological activity. Such selectivity in biodegradation might lead to relative accumulation of the most persistent diastereomers or enantiomers uncleaved in solution. Cypermethrin-degrading microorganisms usually need an adaptation period to produce the appropriate degradative enzymes followed by accelerated biodegradation. The addition of supplementary carbon sources and nutrients (e.g., glucose, beef extract, or yeast extract) was necessary to enhance their degradative abilities [12]. In contrast, strain A-1 could directly utilize and degrade cypermethrin without any apparent lag phase of degradation in the absence of other carbon sources. This is an important feature of an insecticide-degrading microorganism to be used in bioremediation of different contaminated environments. For survival in soil or aquatic substrates, resistance to a wide range of antibiotics is an important factor determining proliferation of an organism ahead of other members of microbial communities [38]. Antibiotics produced by the environmental microbiota or discharged by anthropogenic input can be treated as an ecological factor, offering a survival benefit in bacterial community structures [39]. Shared resistance to quinolone antibiotics, sulfonamide drugs, and some aminoglycosides generally confers Methylobacterium sp. strain A-1 an evolutionary advantage for use as seeds in bioaugmentation techniques. Therefore, Methylobacterium sp. strain A-1 might be most helpful for in situ biodegradation of pyrethroid contaminants under natural conditions and for the development of bioremediation approaches to restore ecological pyrethroid damage. Pyrethroids are a large group of ester-containing compounds whose main degradation route involves cleavage of the central ester bond by carboxylesterases [3]. The carboxylesterase family (EC 3.1.1.1) comprises a group of enzymes hydrolyzing ester molecules with relatively broad substrate specificity. They are believed to be involved in the detoxification of different xenobiotics [21]. Previous studies have proven that carboxylesterases from a-Proteobacteria are the first protagonists in catabolic pathways of pyrethroids, and the hydrolysis products of these enzymes are likely further catabolized, as each of these microbial organisms mineralize pyrethroids [40]. To
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further substantiate that Methylobacterium sp. strain A-1 endows the enzymatic machinery necessary for pyrethroid degradation, a PCR-based approach was performed to identify a carboxylesterase gene within the A-1 genome. After sequencing and BLAST identification at NCBI, it turned out that the PCR-derived clones harbored the gene mse1 coding for an enzyme of the family VI esterases according to the classification of Arpigny and Jaeger [30]. The deduced amino acid sequence of MsE1 showed high identity with putative carboxylesterases of different Methylobacterium species (Fig. 5), but it shared only 41% amino acid sequence identity with the characterized carboxylesterase PytZ from Ochrobactrum anthropi YZ-1 [23]. MsE1 has a predicted molecular mass of 22 kDa, which is similar to PytZ with 24 kDa [23]. However, it is smaller than other reported cypermethrin-hydrolyzing enzymes, such as PytY (42 kDa) from O. anthropi YZ1 [22] and PytH (31 kDa) from Sphingobium sp. JZ-1 [24]. Taken together, the above findings suggest that MsE1 could be an new member of the pyrethroid-degrading carboxylesterases. Therefore, the gene mse1, which we isolated in this study, enriches the genetic resources for bioremediation of pyrethroid-contaminated ecosystems.
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Acknowledgments The authors owe special thanks to Roland Klein (ErnstBerl Institute, Darmstadt University of Technology, Germany) for providing PLGA microcapsules. We also acknowledge the expert contribution of Susanne Verbarg (DSMZ, Braunschweig, Germany) for the analysis of cellular fatty acids. This work was financially supported €r by a grant-in-aid (403 76000) from zfe (Zentrum fu Forschung und Technologie) of Darmstadt University of Applied Sciences.
[12] Chen, S., Hu, M., Liu, J., Zhong, G., et al., 2011. Biodegradation of beta-cypermethrin and 3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-02. J. Hazard. Mater., 187, 433–440. [13] Wang, B.Z., Ma, Y., Zhou, W.J., Zheng, J.W., et al., 2011. Biodegradation of synthetic pyrethroids by Ochrobactrum tritici strain pyd-1. World J. Microbiol. Biotechnol., 27, 2315–2324. [14] Grant, R.J., Daniell, T.J., Betts, W.B., 2002. Isolation and identification of synthetic pyrethroid-degrading bacteria. J. Appl. Microbiol., 92, 534–540. [15] Zhang, C., Wang, S., Yan, Y., 2011. Isomerization and biodegradation of beta-cypermethrin by Pseudomonas aeruginosa CH7 with biosurfactant production. Bioresour. Technol., 102, 7139–7146.
Conflicts of interest All authors have no financial or commercial conflicts of interest concerning the current work or its publication.
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