Phenylurea herbicides (PHs) are frequently detected as major water contaminants in areas where there is extensive use. In this study, Diaphorobacter sp. strain LR2014-1, which initially hydrolyzes linuron to 3,4-dichloroanaline, and Achromobacter sp. strain ANB-1, which further mineralizes the produced aniline derivatives, were isolated from a linuronmineralizing consortium despite being present at low abundance in the community. The synergistic catabolism of linuron by the consortium containing these two strains resulted in more efficient catabolism of linuron and growth of both strains. Strain LR2014-1 harbors two evolutionary divergent hydrolases from the amidohydrolase superfamily Phh and the amidase superfamily TccA2, which functioned complementarily in the hydrolysis of various types of PHs, including linuron (N-methoxy-N-methyl-substituted), diuron, chlorotoluron, fluomethuron (N,N-dimethyl-substituted), and siduron. These findings show that a bacterial consortium can contain catabolically synergistic species for PH mineralization, and one strain could harbor functionally complementary hydrolases for a broadened substrate range.

The phenylurea herbicide linuron is globally used and has caused considerable concern because it leads to environmental pollution. In this study, a highly efficient linuron-transforming strain Sphingobium sp. SMB was isolated, and a gene (lahB) responsible for the hydrolysis of linuron to 3,4-dichloroaniline and N,O-dimethylhydroxylamine was cloned from the genome of strain SMB. The lahB gene encodes an amidohydrolase, which shares 20−53% identity with other biochemically characterized amidohydrolases, except for the newly reported linuron hydrolase Phh (75%). The optimal conditions for the hydrolysis of linuron by LahB were determined to be pH 7.0 and 30 °C, and the K m value of LahB for linuron was 37.3 ± 1.2 μM. Although LahB and Phh shared relatively high identity, LahB exhibited a narrow substrate spectrum (specific for linuron) compared to Phh (active for linuron, diuron, chlortoluron, etc.). Sequence analysis and site-directed mutagenesis revealed that Ala261 of Phh was the key amino acid residue affecting the substrate specificity. Our study provides a new amidohydrolase for the specific hydrolysis of linuron.

Background: Swep is an excellent carbamate herbicide that kills weeds by interfering with metabolic processes and inhibiting cell division at the growth point. Due to the large amount of use, swep residues in soil and water not only cause environmental pollution but also accumulate through the food chain, ultimately pose a threat to human health. This herbicide is degraded in soil mainly by microbial activity, but no studies on the biotransformation of swep have been reported. Results:In this study, a consortium consisting of two bacterial strains, Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34, was enriched from a contaminated soil sample and shown to be capable of mineralizing swep. Swep was first transformed by Comamonas sp. SWP-3 to the intermediate 3,4-dichloroaniline (3,4-DCA), after which 3,4-DCA was mineralized by Alicycliphilus sp. PH-34. An amidase gene, designated as ppa, responsible for the transformation of swep into 3,4-DCA was cloned from strain SWP-3. The expressed Ppa protein efficiently hydrolyzed swep and a number of other structural analogues, such as propanil, chlorpropham and propham. Ppa shared less than 50% identity with previously reported arylamidases and displayed maximal activity at 30 °C and pH 8.6. Gly449 and Val266 were confirmed by sequential error prone PCR to be the key catalytic sites for Ppa in the conversion of swep. Conclusions:These results provide additional microbial resources for the potential remediation of swep-contaminated sites and add new insights into the catalytic mechanism of amidase in the hydrolysis of swep.