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- The phenylurea herbicide linuron is a nonselective preemergent herbicide that acts as a photosystem II inhibitor. The herbicide is globally used to control a wide variety of annual and perennial broadleaf and grassy weeds in agricultural land. Microbial degradation is considered an important mechanism in the dissipation of linuron and other phenylurea herbicides in the environment. Several bacterial strains (39, 46), as well as consortia (5, 10), able to degrade and even use the compound as a sole source of carbon and nitrogen have been reported. Although derived from different geographical locations, most of the linuron-catabolizing isolates, either individual strains or key members of linurondegrading consortia, belong to the genus Variovorax. This suggests that this genus plays an important role in linuron degradation in soil. The proposed pathway of linuron catabolism starts with amide hydrolysis to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine (N,O-DMHA) (Fig. 1). DCA is harmful and recalcitrant, while N,O-DMHA is not and degraded easily. Several linuron-degrading Variovorax strains, in addition to mediating linuron hydrolysis, are able to use DCA as the sole carbon source and mineralize it. To date, little is known about the genes and enzymes responsible for linuron and DCA degradation. Engelhardt et al. (13) described an arylacyl amidase responsible for conversion of linuron to DCA in Bacillus sphaericus ATCC 12123. In addition, phenylurea hydrolase-encoding genes puhA and puhB were identified in the linuron-degrading actinomycetes Arthrobacter globiformis D47 (52) and Mycobacterium brisbanense JK1 (23), respectively. PuhA and PuhB form a novel branch within the metal-dependent amidohydrolase superfamily (23). Regarding the degradation of DCA, Dejonghe (9) and Breugelmans et al. (6) found indications for the involvement of a multicomponent aniline dioxygenase enzyme in DCA degradation in Variovorax sp. strain WDL1. However, the genes responsible for DCA degradation in linuron-mineralizing bacteria have not yet been identified.We report here on the identification of the linuron and DCA degradation genes in the linuron-mineralizing strain Variovorax sp. strain SRS16 (46). The enzyme responsible for hydrolysis of linuron was purified and characterized. The expression of the catabolic genes under different conditions

Microbial community structure and function of Nitrobenzene reduction biocathode in response to carbon source switchover

“…Moreover, GeoChip analysis detected three carbon fixation genes (Gene ID 220718833 and 221082971 for propionylCoA carboxylase and 239801525 for ribulose-bisphosphate carboxylase) from V. paradoxus S110 and Paracoccus denitrificans PD1222 (Gene ID 69936694 and 119386345 for propionyl-CoA carboxylase and 69153892 for ribulosebisphosphate carboxylase) respectively, which further supported the function of Variovorax and Paracoccus in the autotrophic bBC. Based on the GeoChip analysis, a total of 592 carbon fixation genes involved in the reductive pentose phosphate cycle, reductive citric acid cycle, reductive acetylCoA (Wood-Ljungdahl, WL) pathway, 3-hydroxypropionate bicycle and 3-hydroxypropionate-4-hydroxybutyrate cycle (Berg et al, 2010;Fuchs, 2011) were detected in this study (Fig. S1).…”

“…S1), suggesting that the bBC species may uptake electrons from the electrode and use these electrons to generate ATP and then produce biomass under autotrophic condition. Additionally, Variovorax was capable of mineralizing of aromatic amine 3,4-dichloroaniline to Krebs cycle intermediates (Bers et al, 2011), suggesting that Variovorax of the bBC likely involved in the mineralization of aniline to support cathodophilic bacteria growth to a certain extent as the autotrophic bBC generated significantly lower aniline than that of the heterotrophic gBC ( Fig. 2A).…”

“…Fifty-five cytochrome c genes undetected in the initial inoculum were enriched in the biocathode. Cytochromes c belonging to the class III family are distinguished by their multi-heme nature and negative redox potential (À160 to À500 mV) (Bianco and Haladjian, 1994). Twelve class III cytochrome c genes were detected, and another 26 having multiheme were also found.…”

Role of IncP-1β Plasmids pWDL7:: rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses

“…In P. putida UCC22 and Delftia tsuruhatensis AD9, where these systems were first described, these components are encoded by, respectively, tdnQ-tadQ, tdnT-tadT, tdnA1-tadA1, tdnA2-tadA2, and tdnB-tadB (18,30). The genes dcaQ, -T, -A1, -A2, -B, and -R on pWDL7::rfp and pNB8c show high sequence similarity to genes from all previously described systems, which together are thought to code for conversion of (chloro)aniline to (chloro)catechols (5,17,18,35,59). No homologs of aromatic ring cleavage pathway genes were identified on either pWDL7::rfp or pNB8c.…”

The Novel BacterialN-Demethylase PdmAB Is Responsible for the Initial Step ofN,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

“…The second process for initiating degradation of phenylurea herbicides, demonstrated by bacteria such as Arthrobacter globiformis D47 (1), Mycobacterium brisbanense JK1 (35), and Variovorax sp. strain SRS16 (36), is direct hydrolysis of phenylurea herbicides to their aniline derivatives. The phenylurea hydrolase-encoding genes puhA, puhB, libA, and hylA were identified in strains D47 (1), JK1 (35), SRS16 (36), and WDL1 (37), respectively.…”