HylA, an Alternative Hydrolase for Initiation of Catabolism of the Phenylurea Herbicide Linuron in Variovorax sp. Strains

Abstract

f 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 K m , 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.is a phenylurea herbicide widely used in agriculture to control germinating and newly emerging grasses and broad-leafed weeds. Biodegradation contributes largely to the dissipation of linuron in the environment. Several single bacterial strains (1, 2) and consortia (3, 4) that degrade (3, 5) or even mineralize and use linuron as the sole source of carbon, nitrogen, and energy have been reported (1-4). Bacterial degradation of linuron is initiated by amide hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine (N,O-DMHA). In the case of linuron mineralization, DCA is further converted to water and carbon dioxide (Fig. 1). Bacteria belonging to the genus Variovorax appear to play a crucial role in linuron biodegradation. In linuron-degrading consortia, they are almost always responsible for at least the initial hydrolysis step in linuron degradation, and most linuron-mineralizing single-strain isolates are of the genus Variovorax (1, 2). The genetic basis of linuron degradation in the linuron-mineralizing Variovorax sp. strain SRS16 was recently elucidated (6) and involves three major catabolic gene modules. In strain SRS16, conversion of linuron to DCA is catalyzed by the hydrolase LibA, encoded by the libA gene. Further mineralization of DCA involves a multicomponent dioxygenase complex encoded by dcaQTA 1 A 2 BR, which degrades DCA to a chlorocatechol intermediate. The latter is further degraded by a modified ortho-cleava...

Targeted metagenomics demonstrates the ecological role of IS1071in bacterial community adaptation to pesticide degradation

“…Downstream pathway enzymes included aromatic ring cleavage enzymes and enzymes that are involved in the further conversion of the (substituted) aromatic cleavage products to Krebs cycle intermediates. Similar to the upstream catabolic functions, several of these proteins could be directly linked to pesticide biodegradation like proteins encoding chloroanaline dioxygenase (contig 250 in BPS Kortrijk and contig 23 in L SM A) involved in the degradation of several pesticide groups including phenylurea herbicides such as linuron (Bers et al ., ) and enzymes of the modified ortho ‐cleavage pathway for chlorocatechol degradation (contigs 70, 117, 293 and 309 in BPS Kortrijk and contigs 21 and 33 in L SM A) for instance involved in degradation of chlorinated aromatic pesticides (Kumar et al ., ). Other downstream pathway enzymes were enzymes that could be linked to lipid (like cholesterol) and fatty acid metabolism (e.g.…”

Functional Redundancy of Linuron Degradation in Microbial Communities in Agricultural Soil and Biopurification Systems

“…The linuron hydrolysis genes in both WDL1 and SRS16 are combined with highly similar catabolic gene modules encoding the downstream pathway for 3,4-DCA degradation. Apparently, the expansion of a 3,4-DCA catabolic pathway toward linuron degradation in the two strains involved divergent evolution and the independent acquisition of nonrelated isofunctional linuron hydrolytic gene functions by horizontal gene transfer (10). Moreover, in contrast to SRS16, WDL1 is a member of a commensal bacterial consortium in which most of the 3,4-DCA produced from HylA-dependent linuron hydrolysis is channeled to 3,4-DCA catabolic strains (4).…”

“…strain WDL1 (10). Interestingly, HylA is evolutionarily unrelated to LibA and shows different enzymatic kinetic properties (10). The linuron hydrolysis genes in both WDL1 and SRS16 are combined with highly similar catabolic gene modules encoding the downstream pathway for 3,4-DCA degradation.…”

“…Recently, we identified HylA as a second type of linuron hydrolase in Variovorax sp. strain WDL1 (10). Interestingly, HylA is evolutionarily unrelated to LibA and shows different enzymatic kinetic properties (10).…”

Molecular processes underlying synergistic linuron mineralization in a triple‐species bacterial consortium biofilm revealed by differential transcriptomics

“…Variovorax sp. WDL1 hydrolyzes linuron into 3,4‐dichloroaniline (3,4‐DCA) and N , O ‐dimethylhydroxylamine ( N , O‐ DMHA) using the phenylurea hydrolase HylA (Bers et al., ; Dejonghe et al., ). Although WDL1 contains dca and ccd clusters encoding for the further degradation of 3,4‐DCA to 3‐oxoadipate via a chlorocatechol intermediate, 3,4‐DCA is degraded inefficiently by WDL1.…”