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A novel hydrolase identified by genomic-proteomic analysis of phenylurea herbicide mineralization by Variovorax sp. strain SRS16.

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 K(m) and a V(max) for linuron of 5.8 ?M 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.

SUBMITTER: Bers K

PROVIDER: S-EPMC3233098 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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A novel hydrolase identified by genomic-proteomic analysis of phenylurea herbicide mineralization by Variovorax sp. strain SRS16.

Bers Karolien K Leroy Baptiste B Breugelmans Philip P Albers Pieter P Lavigne Rob R Sørensen Sebastian R SR Aamand Jens J De Mot René R Wattiez Ruddy R Springael Dirk D

Applied and environmental microbiology 20111014 24

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 ...[more]

PMID: 22003008

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Project description:Diuron (DUR) is a phenylurea herbicide widely used for the effective control of most annual and perennial weeds in farming areas. The extensive use of DUR has led to its widespread presence in soil, sediment, and aquatic environments, which poses a threat to non-target crops, animals, humans, and ecosystems. Therefore, the removal of DUR from contaminated environments has been a hot topic for researchers in recent decades. Bioremediation seldom leaves harmful intermediate metabolites and is emerging as the most effective and eco-friendly strategy for removing DUR from the environment. Microorganisms, such as bacteria, fungi, and actinomycetes, can use DUR as their sole source of carbon. Some of them have been isolated, including organisms from the bacterial genera Arthrobacter, Bacillus, Vagococcus, Burkholderia, Micrococcus, Stenotrophomonas, and Pseudomonas and fungal genera Aspergillus, Pycnoporus, Pluteus, Trametes, Neurospora, Cunninghamella, and Mortierella. A number of studies have investigated the toxicity and fate of DUR, its degradation pathways and metabolites, and DUR-degrading hydrolases and related genes. However, few reviews have focused on the microbial degradation and biochemical mechanisms of DUR. The common microbial degradation pathway for DUR is via transformation to 3,4-dichloroaniline, which is then metabolized through two different metabolic pathways: dehalogenation and hydroxylation, the products of which are further degraded via cooperative metabolism. Microbial degradation hydrolases, including PuhA, PuhB, LibA, HylA, Phh, Mhh, and LahB, provide new knowledge about the underlying pathways governing DUR metabolism. The present review summarizes the state-of-the-art knowledge regarding (1) the environmental occurrence and toxicity of DUR, (2) newly isolated and identified DUR-degrading microbes and their enzymes/genes, and (3) the bioremediation of DUR in soil and water environments. This review further updates the recent knowledge on bioremediation strategies with a focus on the metabolic pathways and molecular mechanisms involved in the bioremediation of DUR.

Project description:Isoprene monooxygenase (IsoMO, encoded by isoABCDEF) initiates the oxidation of the climate-active gas isoprene, with the genes isoGHIJ and aldH nearly always found adjacent to isoABCDEF in extant and metagenome-derived isoprene degraders. The roles of isoGHIJ and aldH are uncertain, although each is essential to isoprene degradation. We report here the characterization of these proteins from two model isoprene degraders, Rhodococcus sp. strain AD45 and Variovorax sp. strain WS11. The genes isoHIJ and aldH from Variovorax and aldH from Rhodococcus were expressed individually in Escherichia coli as maltose binding protein fusions to overcome issues of insolubility. The activity of two glutathione S-transferases from Variovorax, IsoI and IsoJ was assessed with model substrates, and the conversion of epoxyisoprene to the intermediate 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB) was demonstrated. The next step of the isoprene metabolic pathway of Variovorax is catalyzed by the dehydrogenase IsoH, resulting in the conversion of HGMB to 2-glutathionyl-2-methyl-3-butenoic acid (GMBA). The aldehyde dehydrogenases (AldH) from Variovorax and Rhodococcus were examined with a variety of aldehydes, with both exhibiting maximum activity with butanal. AldH significantly increased the rate of production of NADH when added to the IsoH-catalyzed conversion of HGMB to GMBA (via GMB), suggesting a synergistic role for AldH in the isoprene metabolic pathway. An in silico analysis of IsoG revealed that this protein, which is essential for isoprene metabolism in Variovorax, is an enzyme of the formyl CoA-transferase family and is predicted to catalyze the formation of a GMBA-CoA thioester as an intermediate in the isoprene oxidation pathway. IMPORTANCE Isoprene is a climate-active gas, largely produced by trees, which is released from the biosphere in amounts equivalent to those of methane and all other volatile organic compounds combined. Bacteria found in many environments, including soils and on the surface of leaves of isoprene-producing trees, can grow on isoprene and thus may represent a significant biological sink for this globally significant volatile compound and remove isoprene before it escapes to the atmosphere, thus reducing its potency as a climate-active gas. The initial oxidation of isoprene by bacteria is mediated by isoprene monooxygenase encoded by the genes isoABCDEF. In isoprene-degrading bacteria, a second gene cluster, isoGHIJ, is also present, although the exact role in isoprene degradation by the proteins encoded by these genes is uncertain. This investigation sheds new light on the roles of these proteins in the isoprene oxidation pathway in two model isoprene-degrading bacteria of the genera Rhodococcus and Variovorax.

Dataset Information

A novel hydrolase identified by genomic-proteomic analysis of phenylurea herbicide mineralization by Variovorax sp. strain SRS16.

Publications

A novel hydrolase identified by genomic-proteomic analysis of phenylurea herbicide mineralization by Variovorax sp. strain SRS16.

Similar Datasets

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