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Sebastian R Sorensen - One of the best experts on this subject based on the ideXlab platform.
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the novel bacterial n demethylase pdmab is responsible for the initial step of n n dimethyl substituted Phenylurea Herbicide degradation
Applied and Environmental Microbiology, 2013Co-Authors: Tao Gu, Peiwen Yu, Sebastian R Sorensen, Jian He, Ji Zhang, Chaoyang Zhou, Shunpeng LiAbstract:The environmental fate of Phenylurea Herbicides has received considerable attention in recent decades. The microbial metabolism of N,N-dimethyl-substituted Phenylurea Herbicides can generally be initiated by mono-N-demethylation. In this study, the molecular basis for this process was revealed. The pdmAB genes in Sphingobium sp. strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly used N,N-dimethyl-substituted Phenylurea Herbicides. PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase (RO) system. The genes pdmAB, encoding the α subunit PdmA and the β subunit PdmB, are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1. Furthermore, this transposable element is highly conserved among Phenylurea Herbicide-degrading sphingomonads originating from different areas of the world. However, there was no evidence of a gene for an electron carrier (a ferredoxin or a reductase) located in the immediate vicinity of pdmAB. Without its cognate electron transport components, expression of PdmAB in Escherichia coli, Pseudomonas putida, and other sphingomonads resulted in a functional enzyme. Moreover, coexpression of a putative [3Fe-4S]-type ferredoxin from Sphingomonas sp. strain RW1 greatly enhanced the catalytic activity of PdmAB in E. coli. These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the [3Fe-4S] type. PdmA exhibited low homology to the α subunits of previously characterized ROs (less than 37% identity) and did not cluster with the RO group involved in O- or N-demethylation reactions, indicating that PdmAB is a distinct bacterial RO N-demethylase.
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A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16
Applied and environmental microbiology, 2011Co-Authors: Karolien Bers, Sebastian R Sorensen, Jens Aamand, Ruddy Wattiez, R. De Mot, Baptiste Leroy, Philip Breugelmans, Pieter Albers, Rob Lavigne, Dirk SpringaelAbstract:ABSTRACT The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the Phenylurea Herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to Phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested Phenylurea Herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a Phenylurea Herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.
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c n and h isotope fractionation of the Herbicide isoproturon reflects different microbial transformation pathways
Environmental Science & Technology, 2010Co-Authors: Holger Penning, Sebastian R Sorensen, Jens Aamand, Armin H Meyer, Martin ElsnerAbstract:The fate of pesticides in the subsurface is of great interest to the public, industry, and regulatory authorities. Compound-specific isotope analysis (CSIA) is a promising tool complementary to existing methods for elucidating pesticide degradation reactions. Here, we address three different initial biotransformation reactions of the Phenylurea Herbicide isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) in pure culture experiments with bacterial and fungal strains. When analyzing isotopic changes in different parts of the isoproturon molecule, hydroxylation of the isopropyl group by fungi was found to be associated with C and H isotope fractionation. In contrast, hydrolysis by Arthrobacter globiformis D47 caused strong C and N isotope fractionation, albeit in a different manner than abiotic hydrolysis so that isotope measurements can distinguish between both modes of transformation. No significant isotope fractionation was observed during N-demethylation by Sphingomonas sp. SRS2. The observed isotope fractionation patterns were in agreement with the type of reactions and elements involved. Moreover, their substantially different nature suggests that isotope changes in natural samples may be uniquely attributed to either pathway, allowing even to distinguish the abiotic versus biotic nature of hydrolysis. Our investigations show how characteristic isotope patterns may significantly add to the present understanding of the environmental fate of pesticides.
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Characterization of an isoproturon mineralizing bacterial culture enriched from a French agricultural soil
Chemosphere, 2009Co-Authors: Sabir Hussain, Talaat El-sebai, Marion Devers-lamrani, Sebastian R Sorensen, Fabrice Martin-laurentAbstract:The Phenylurea Herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized by a bacterial culture isolated from an agricultural soil regularly exposed to IPU. Molecular analysis of the bacterial culture by DNA fingerprinting, cloning and sequencing of the 16S rRNA genes revealed that it consisted of six different members among whom the dominant was related to Sphingomonas sp. Six bacterial strains belonging to genera Ancylobacter, Pseudomonas, Stenotrophomonas, Methylobacterium, Variovorax and Agrobacterium were isolated from the IPU-degrading culture. None of these were able to degrade IPU in pure culture and only the intact culture sustained the ability to mineralize IPU. The composition of the culture appeared stable suggesting that yet unknown interactions are involved in the IPU mineralization. IPU degradation involved the transitory accumulation of three known IPU metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropylaniline and their further degradation. Thus, it indicates a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain. This culture did not degrade other structurally related Phenylurea Herbicides. The degrading activity of the bacterial culture was deeply influenced by the pH, being completely inhibited at pH 5.5 and optimal at pH 7.5.
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Characterization of a bacterial consortium mineralizing isoproturon (IPU)
2009Co-Authors: Sabir Hussain, Marion Devers-lamrani, Sebastian R Sorensen, Fabrice MartinAbstract:A bacterial consortium, isolated from a French agricultural soil, was shown to rapidly mineralize the Phenylurea Herbicide isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea). Study of the mineralization kinetics by ultra performance liquid chromatography (UPLC)had shown the transitory accumulation and degradation of the three known isoproturon metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropylaniline, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain. This bacterial consortium did not degrade other Phenylurea Herbicides like diuron, linuron, monolinuron and chlorotoluron suggesting the existence of pathway specific for IPU. Six bacterial strains were isolated from the consortium by successive inoculations on mineral salt medium but none of them was able to degrade isoproturon in isolated form. Based on partial sequencing of the 16S rDNA sequence, the isolated bacterial strains were found to be belonging to genera Ancylobacter, Pseudomonas, Stenotrophomonas, Methylobacterium, variovorax and Agrobacterium. A BAC genomic library was established and is currently under functional screeing searching for BAC clone harbouring IPU-degrading capability. positive BAC clone will then be fully sequenced for identifying gene coding for the enzyme responsible for IPU transformation which up to now remains unknown.
Jens Aamand - One of the best experts on this subject based on the ideXlab platform.
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Strains of the soil fungus Mortierella show different degradation potentials for the Phenylurea Herbicide diuron
Biodegradation, 2013Co-Authors: Lea Ellegaard-jensen, Anders H Johnsen, Birthe B Kragelund, Jens Aamand, Soren RosendahlAbstract:Microbial pesticide degradation studies have until now mainly focused on bacteria, although fungi have also been shown to degrade pesticides. In this study we clarify the background for the ability of the common soil fungus Mortierella to degrade the Phenylurea Herbicide diuron. Diuron degradation potentials of five Mortierella strains were compared, and the role of carbon and nitrogen for the degradation process was investigated. Results showed that the ability to degrade diuron varied greatly among the Mortierella strains tested, and the strains able to degrade diuron were closely related. Degradation of diuron was fastest in carbon and nitrogen rich media while suboptimal nutrient levels restricted degradation, making it unlikely that Mortierella utilize diuron as carbon or nitrogen sources. Degradation kinetics showed that diuron degradation was followed by formation of the metabolites 1-(3,4-dichlorophenyl)-3-methylurea, 1-(3,4-dichlorophenyl)urea and an hitherto unknown metabolite suggested to be 1-(3,4-dichlorophenyl)-3-methylideneurea.
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A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16
Applied and environmental microbiology, 2011Co-Authors: Karolien Bers, Sebastian R Sorensen, Jens Aamand, Ruddy Wattiez, R. De Mot, Baptiste Leroy, Philip Breugelmans, Pieter Albers, Rob Lavigne, Dirk SpringaelAbstract:ABSTRACT The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the Phenylurea Herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to Phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested Phenylurea Herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a Phenylurea Herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.
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c n and h isotope fractionation of the Herbicide isoproturon reflects different microbial transformation pathways
Environmental Science & Technology, 2010Co-Authors: Holger Penning, Sebastian R Sorensen, Jens Aamand, Armin H Meyer, Martin ElsnerAbstract:The fate of pesticides in the subsurface is of great interest to the public, industry, and regulatory authorities. Compound-specific isotope analysis (CSIA) is a promising tool complementary to existing methods for elucidating pesticide degradation reactions. Here, we address three different initial biotransformation reactions of the Phenylurea Herbicide isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) in pure culture experiments with bacterial and fungal strains. When analyzing isotopic changes in different parts of the isoproturon molecule, hydroxylation of the isopropyl group by fungi was found to be associated with C and H isotope fractionation. In contrast, hydrolysis by Arthrobacter globiformis D47 caused strong C and N isotope fractionation, albeit in a different manner than abiotic hydrolysis so that isotope measurements can distinguish between both modes of transformation. No significant isotope fractionation was observed during N-demethylation by Sphingomonas sp. SRS2. The observed isotope fractionation patterns were in agreement with the type of reactions and elements involved. Moreover, their substantially different nature suggests that isotope changes in natural samples may be uniquely attributed to either pathway, allowing even to distinguish the abiotic versus biotic nature of hydrolysis. Our investigations show how characteristic isotope patterns may significantly add to the present understanding of the environmental fate of pesticides.
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Rapid Mineralization of the Phenylurea Herbicide Diuron by Variovorax sp. Strain SRS16 in Pure Culture and within a Two-Member Consortium
Applied and environmental microbiology, 2008Co-Authors: Sebastian R Sorensen, Christian Nyrop Albers, Jens AamandAbstract:The Phenylurea Herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of Herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-14C]diuron to 14CO2, depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted Phenylurea Herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-14C]diuron was mineralized to 14CO2 when 15.5 to 38.9 μg liter−1 diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.
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growth in coculture stimulates metabolism of the Phenylurea Herbicide isoproturon by sphingomonas sp strain srs2
Applied and Environmental Microbiology, 2002Co-Authors: Sebastian R Sorensen, Zeev Ronen, Jens AamandAbstract:Metabolism of the Phenylurea Herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the Herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of 14C-labeled isoproturon to 14CO2 and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.
Dirk Springael - One of the best experts on this subject based on the ideXlab platform.
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comparative genomics suggests mechanisms of genetic adaptation toward the catabolism of the Phenylurea Herbicide linuron in variovorax
Genome Biology and Evolution, 2020Co-Authors: Basak Ozturk, Johannes Werner, Jan P Meierkolthoff, Boyke Bunk, Cathrin Sproer, Dirk SpringaelAbstract:Biodegradation of the Phenylurea Herbicide linuron appears a specialization within a specific clade of the Variovorax genus. The linuron catabolic ability is likely acquired by horizontal gene transfer but the mechanisms involved are not known. The full-genome sequences of six linuron-degrading Variovorax strains isolated from geographically distant locations were analyzed to acquire insight into the mechanisms of genetic adaptation toward linuron metabolism. Whole-genome sequence analysis confirmed the phylogenetic position of the linuron degraders in a separate clade within Variovorax and indicated that they unlikely originate from a common ancestral linuron degrader. The linuron degraders differentiated from Variovorax strains that do not degrade linuron by the presence of multiple plasmids of 20-839 kb, including plasmids of unknown plasmid groups. The linuron catabolic gene clusters showed 1) high conservation and synteny and 2) strain-dependent distribution among the different plasmids. Most of them were bordered by IS1071 elements forming composite transposon structures, often in a multimeric array configuration, appointing IS1071 as a key element in the recruitment of linuron catabolic genes in Variovorax. Most of the strains carried at least one (catabolic) broad host range plasmid that might have been a second instrument for catabolic gene acquisition. We conclude that clade 1 Variovorax strains, despite their different geographical origin, made use of a limited genetic repertoire regarding both catabolic functions and vehicles to acquire linuron biodegradation.
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comparative genomics unravels mechanisms of genetic adaptation for the catabolism of the Phenylurea Herbicide linuron in variovorax
bioRxiv, 2019Co-Authors: Basak Ozturk, Johannes Werner, Jan P Meierkolthoff, Boyke Bunk, Cathrin Sproer, Dirk SpringaelAbstract:Abstract Biodegradation of the Phenylurea Herbicide linuron appears a specialization within a specific clade of the Variovorax genus. The linuron catabolic ability is likely acquired by horizontal gene transfer but the mechanisms involved are not known. The full genome sequences of six linuron degrading Variovorax strains isolated from geographically distant locations were analyzed to acquire insight in the mechanisms of genetic adaptation towards linuron metabolism in Variovorax. Whole genome sequence analysis confirmed the phylogenetic position of the linuron degraders in a separate clade within Variovorax and indicated their unlikely origin from a common ancestral linuron degrader. The linuron degraders differentiated from non-degraders by the presence of multiple plasmids of 20 to 839 kb, including plasmids of unknown plasmid groups. The linuron catabolic gene clusters showed (i) high conservation and synteny and (ii) strain-dependent distribution among the different plasmids. All were bordered by IS1071 elements forming composite transposon structures appointing IS1071 as key for catabolic gene recruitment. Most of the strain carried at least one broad host range plasmid that might have been a second instrument for catabolic gene acquisition. We conclude that clade 1Variovorax strains, despite their different geographical origin, made use of a limited genetic repertoire to acquire linuron biodegradation. Importance The genus Variovorax and especially a clade of strains that phylogenetically separates from the majority of Variovorax species, appears to be a specialist in the biodegradation of the phenyl urea Herbicide linuron. Horizontal gene transfer (HGT) likely played an essential role in the genetic adaptation of those strain to acquire the linuron catabolic genotype. However, we do not know the genetic repertoire involved in this adaptation both regarding catabolic gene functions as well as gene functions that promote HGT neither do we know how this varies between the different strains. These questions are addressed in this paper by analyzing the full genome sequences of six linuron degrading Variovorax strains. This knowledge is important for understanding the mechanisms that steer world-wide genetic adaptation in a particular species and this for a particular phenotypic trait as linuron biodegradation.
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HylA, an alternative hydrolase for initiation of catabolism of the Phenylurea Herbicide linuron in Variovorax sp. strains.
Applied and environmental microbiology, 2013Co-Authors: Karolien Bers, Isabelle Batisson, Paul Proost, Ruddy Wattiez, R. De Mot, Dirk SpringaelAbstract:Variovorax sp. strain WDL1, which mineralizes the Phenylurea Herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from Phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in Km, temperature optimum, and Phenylurea Herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.
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A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16
Applied and environmental microbiology, 2011Co-Authors: Karolien Bers, Sebastian R Sorensen, Jens Aamand, Ruddy Wattiez, R. De Mot, Baptiste Leroy, Philip Breugelmans, Pieter Albers, Rob Lavigne, Dirk SpringaelAbstract:ABSTRACT The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the Phenylurea Herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to Phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested Phenylurea Herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a Phenylurea Herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.
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a molecular toolbox to estimate the number and diversity of variovorax in the environment application in soils treated with the Phenylurea Herbicide linuron
FEMS Microbiology Ecology, 2011Co-Authors: Karolien Bers, R. De Mot, Philip Breugelmans, Pieter Albers, Kristel Sniegowski, Larissa Hendrickx, Dirk SpringaelAbstract:Real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE) approaches that specifically target the Variovorax 16S rRNA gene were developed to estimate the number and diversity of Variovorax in environmental ecosystems. PCR primers suitable for both methods were selected as such that the enclosed sequence showed maximum polymorphism. PCR specificity was maximized by combining PCR with a targeted endonuclease treatment of template DNA to eliminate 16S rRNA genes of the closely related Acidovorax. DGGE allowed the grouping of PCR amplicons according to the phylogenetic grouping within the genus Variovorax. The toolbox was used to assess the Variovorax community dynamics in agricultural soil microcosms (SMs) exposed to the Phenylurea Herbicide linuron. Exposure to linuron resulted in an increased abundance within the Variovorax community of a subgroup previously linked to linuron degradation through cultivation-dependent isolation. SMs that were treated only once with linuron reverted to the initial community composition 70 days after linuron exposure. In contrast, SMs irrigated with linuron on a long-term base showed a significant increase in Variovorax number after 70 days. Our data support the hypothesis that the genus Variovorax is involved in linuron degradation in linuron-treated agricultural soils.
Springael Dirk - One of the best experts on this subject based on the ideXlab platform.
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Culture-independent analysis of linuron-mineralizing microbiota and functions in on-farm biopurification systems via DNA-stable isotope probing : comparison with enrichment culture
'American Chemical Society (ACS)', 2020Co-Authors: Lerner Harry, De Mot René, Öztürk Başak, Dohrmann, Anja B., Thomas Joice, Marchal Kathleen, Dehaen Wim, Tebbe, Christoph C., Springael DirkAbstract:Our understanding of the microorganisms involved in in situ biodegradation of xenobiotics, like pesticides, in natural and engineered environments is poor. On-farm biopurification systems (BPSs) treat farm-produced pesticide-contaminated wastewater to reduce surface water pollution. BPSs are a labor and cost-efficient technology but are still mainly operated as black box systems. We used DNA-stable isotope probing (DNA-SIP) and classical enrichment to be informed about the organisms responsible for in situ degradation of the Phenylurea Herbicide linuron in a BPS matrix. DNA-SIP identified Ramlibacter, Variovorax, and an unknown Comamonadaceae genus as the dominant linuron assimilators. While linuron-degrading Variovorax strains have been isolated repeatedly, Ramlibacter has never been associated before with linuron degradation. Genes and mobile genetic elements (MGEs) previously linked to linuron catabolism were enriched in the heavy DNA-SIP fractions, suggesting their involvement in in situ linuron assimilation. BPS material free cultivation of linuron degraders from the same BPS matrix resulted in a community dominated by Variovorax, while Ramlibacter was not observed. Our study provides evidence for the role of Variovorax in in situ linuron biodegradation in a BPS, alongside other organisms like Ramlibacter, and further shows that cultivation results in a biased representation of the in situ linuron-assimilating bacterial populations
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Molecular processes underlying synergistic linuron mineralization in a triple-species bacterial consortium biofilm revealed by differential transcriptomics
John Wiley & Sons Ltd., 2018Co-Authors: Albers Pieter, De Mot René, Marchal Kathleen, Weytjens Bram, Springael DirkAbstract:The proteobacteria Variovorax sp. WDL1, Comamonas testosteroni WDL7, and Hyphomicrobium sulfonivorans WDL6 compose a triple-species consortium that synergistically degrades and grows on the Phenylurea Herbicide linuron. To acquire a better insight into the interactions between the consortium members and the underlying molecular mechanisms, we compared the transcriptomes of the key biodegrading strains WDL7 and WDL1 grown as biofilms in either isolation or consortium conditions by differential RNAseq analysis. Differentially expressed pathways and cellular systems were inferred using the network-based algorithm PheNetic. Coculturing affected mainly metabolism in WDL1. Significantly enhanced expression of hylA encoding linuron hydrolase was observed. Moreover, differential expression of several pathways involved in carbohydrate, amino acid, nitrogen, and sulfur metabolism was observed indicating that WDL1 gains carbon and energy from linuron indirectly by consuming excretion products from WDL7 and/or WDL6. Moreover, in consortium conditions, WDL1 showed a pronounced stress response and overexpression of cell to cell interaction systems such as quorum sensing, contact-dependent inhibition, and Type VI secretion. Since the latter two systems can mediate interference competition, it prompts the question if synergistic linuron degradation is the result of true adaptive cooperation or rather a facultative interaction between bacteria that coincidentally occupy complementary metabolic niches.status: publishe
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Molecular processes underlying synergistic linuron mineralization in a triple-species bacterial consortium biofilm revealed by differential transcriptomics
'Wiley', 2018Co-Authors: Albers Pieter, De Mot René, Marchal Kathleen, Weytjens Bram, Springael DirkAbstract:The proteobacteria Variovorax sp. WDL1, Comamonas testosteroni WDL7, and Hyphomicrobium sulfonivorans WDL6 compose a triple-species consortium that synergistically degrades and grows on the Phenylurea Herbicide linuron. To acquire a better insight into the interactions between the consortium members and the underlying molecular mechanisms, we compared the transcriptomes of the key biodegrading strains WDL7 and WDL1 grown as biofilms in either isolation or consortium conditions by differential RNAseq analysis. Differentially expressed pathways and cellular systems were inferred using the network-based algorithm PheNetic. Coculturing affected mainly metabolism in WDL1. Significantly enhanced expression of hylA encoding linuron hydrolase was observed. Moreover, differential expression of several pathways involved in carbohydrate, amino acid, nitrogen, and sulfur metabolism was observed indicating that WDL1 gains carbon and energy from linuron indirectly by consuming excretion products from WDL7 and/or WDL6. Moreover, in consortium conditions, WDL1 showed a pronounced stress response and overexpression of cell to cell interaction systems such as quorum sensing, contact-dependent inhibition, and Type VI secretion. Since the latter two systems can mediate interference competition, it prompts the question if synergistic linuron degradation is the result of true adaptive cooperation or rather a facultative interaction between bacteria that coincidentally occupy complementary metabolic niches
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Abiotic and biotic processes governing the fate of Phenylurea Herbicides in soils : a review
'Informa UK Limited', 2015Co-Authors: Hussain Sabir, Springael Dirk, Sørensen, Sebastian R, Arshad Muhammad, Bending, Gary D., Devers Lamrani Marion, Maqbool Zahid, Martin Laurent FabriceAbstract:EA ECOLDUR CT3International audiencePhenylurea Herbicides are world widely used for controlling the development of various broad leaf weeds in cereal crops. Despite their agronomical interest, these Herbicides are of environmental concern because they are often detected at concentrations above the threshold concentration imposed by legislation in soil and water worldwide. Understanding the fate of these pollutants is as such of major importance and has been the subject of many scientific studies in the last decades. This review article presents an overview of the abiotic and biotic processes governing the environmental fate of Phenylurea Herbicides after their application to agricultural fields. More precisely, it highlights the pivotal role of biodegradation in natural attenuation of pesticides in agricultural soils.. The recent advances in the microbial aspects of their degradation in the field as well as in pure cultures are presented including metabolic pathways, processes and factors that control the biodegradation of Phenylurea Herbicide
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HylA, an alternative hydrolase for initiating catabolism of the Phenylurea Herbicide linuron in Variovorax
'American Society for Microbiology', 2013Co-Authors: Bers Karolien, Proost Paul, Wattiez R, De Mot René, Springael DirkAbstract:Variovorax sp. WDL1 that mineralizes the Phenylurea Herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from Phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in Km, temperature optimum and Phenylurea Herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families while LibA is a member of the unrelated Amidase Signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation which are very similar to those present in Variovorax sp. SRS16 that contains libA. Our results show that the expansion of a DCA catabolic pathway towards linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.status: publishe
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A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16
Applied and environmental microbiology, 2011Co-Authors: Karolien Bers, Sebastian R Sorensen, Jens Aamand, Ruddy Wattiez, R. De Mot, Baptiste Leroy, Philip Breugelmans, Pieter Albers, Rob Lavigne, Dirk SpringaelAbstract:ABSTRACT The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the Phenylurea Herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to Phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested Phenylurea Herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a Phenylurea Herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.
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a molecular toolbox to estimate the number and diversity of variovorax in the environment application in soils treated with the Phenylurea Herbicide linuron
FEMS Microbiology Ecology, 2011Co-Authors: Karolien Bers, R. De Mot, Philip Breugelmans, Pieter Albers, Kristel Sniegowski, Larissa Hendrickx, Dirk SpringaelAbstract:Real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE) approaches that specifically target the Variovorax 16S rRNA gene were developed to estimate the number and diversity of Variovorax in environmental ecosystems. PCR primers suitable for both methods were selected as such that the enclosed sequence showed maximum polymorphism. PCR specificity was maximized by combining PCR with a targeted endonuclease treatment of template DNA to eliminate 16S rRNA genes of the closely related Acidovorax. DGGE allowed the grouping of PCR amplicons according to the phylogenetic grouping within the genus Variovorax. The toolbox was used to assess the Variovorax community dynamics in agricultural soil microcosms (SMs) exposed to the Phenylurea Herbicide linuron. Exposure to linuron resulted in an increased abundance within the Variovorax community of a subgroup previously linked to linuron degradation through cultivation-dependent isolation. SMs that were treated only once with linuron reverted to the initial community composition 70 days after linuron exposure. In contrast, SMs irrigated with linuron on a long-term base showed a significant increase in Variovorax number after 70 days. Our data support the hypothesis that the genus Variovorax is involved in linuron degradation in linuron-treated agricultural soils.
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architecture and spatial organization in a triple species bacterial biofilm synergistically degrading the Phenylurea Herbicide linuron
FEMS Microbiology Ecology, 2008Co-Authors: Philip Breugelmans, Kim Bundvig Barken, Tim Tolkernielsen, Johan Hofkens, Winnie Dejonghe, Dirk SpringaelAbstract:Members of a triple-species 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea (linuron)-mineralizing consortium, i.e. the linuron- and 3,4-dichloroaniline-degrading Variovorax sp. WDL1, the 3,4-dichloroaniline-degrading Comamonas testosteroni WDL7 and the N,O-dimethylhydroxylamine-degrading Hyphomicrobium sulfonivorans WDL6, were cultivated as mono- or multi-species biofilms in flow cells irrigated with selective or nonselective media, and examined with confocal laser scanning microscopy. In contrast to mono-species biofilms of Variovorax sp. WDL1, the triple-species consortium biofilm degraded linuron completely through apparent synergistic interactions. The triple-species linuron-fed consortium biofilm displayed a heterogeneous structure with an irregular surface topography that most resembled the topography of linuron-fed mono-species WDL1 biofilms, indicating that WDL1 had a dominating influence on the triple-species biofilm architecture. This architecture was dependent on the carbon source supplied, as the biofilm architecture of WDL1 growing on alternative carbon sources was different from that observed under linuron-fed conditions. Linuron-fed triple-species consortium biofilms consisted of mounds composed of closely associated WDL1, WDL7 and WDL6 cells, while this association was lost when the consortium was grown on a nonselective carbon source. In addition, under linuron-fed conditions, microcolonies displaying associated growth developed rapidly after inoculation. These observations indicate that the spatial organization in the linuron-fed consortium biofilm reflected the metabolic interactions within the consortium.