A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1.

Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C-S bond, generating diethylcarbamothioic S-acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum, alkanesulfonate monooxygenase from Pseudomonas savastanoi, and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C-S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC, was located 7,129 bp downstream of tmoAB, and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD+ as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis.IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation in soil. In previous studies, thiobencarb was degraded initially via N-deethylation, sulfoxidation, hydroxylation, and dechlorination. However, enzymes and genes involved in the microbial degradation of thiobencarb have not been studied. This study revealed a new thiobencarb degradation pathway in Acidovorax sp. strain T1 and identified a novel two-component FMN-dependent monooxygenase system, TmoAB. Under TmoAB-mediated catalysis, thiobencarb was cleaved at the C-S bond, producing diethylcarbamothioic S-acid and 4CDA. Furthermore, the downstream degradation pathway of thiobencarb was proposed. Our study provides the physiological, biochemical, and genetic foundation of thiobencarb degradation in this microorganism.

Roseomonas chloroacetimidivorans sp. nov., a chloroacetamide herbicide-degrading bacterium isolated from activated sludge.

A Gram-negative, aerobic, short rod-shaped, pink-pigmented, non-motile bacterium, designated BUT-13(T), was isolated from activated sludge of an herbicide-manufacturing wastewater treatment facility in Jiangsu province, China. Growth was observed at 0-5.5 % NaCl, pH 6.0-9.0 and 12-37 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BUT-13(T) is a member of the genus Roseomonas, and shows high sequence similarities to R. pecuniae N75(T) (98.0 %) and R. rosea 173-96(T) (97.5 %), and lower (<97 %) sequence similarities to all other Roseomonas species. Chemotaxonomic analysis revealed that strain BUT-13(T) possesses Q-10 as the predominant ubiquinone; summed feature 8 (C18:1 w7c and/or C18:1 w6c; 38.8 %), C18:0 (16.6 %), C16:0 (15.2 %), summed feature 3 (C16:1 ω6c and/or C16:1 ω7; 7.9 %) and C18:1 w9c (4.7 %) as the major fatty acids. The polar lipids were found to consist of two aminolipids, a glycolipid, a phospholipid, a phosphoglycolipid, phosphatidylcholine, phosphatidylethanolamine and diphosphatidylglycerol. Strain BUT-13(T) showed low DNA-DNA relatedness with R. pecuniae N75(T) (45.2 %) and R. rosea 173-96(T) (51.2 %). The DNA G+C content was determined to be 67.6 mol%. Based on the phylogenetic analysis, DNA-DNA hybridization and chemotaxonomic analysis, as well as biochemical characteristics, strain BUT-13(T) can be clearly distinguished from all currently recognised Roseomonas species and should be classified as a novel species of the genus Roseomonas, for which the name Roseomonas chloroacetimidivorans sp. nov. is proposed. The type strain is BUT-13(T) (CCTCC AB 2015299(T) = JCM 31050(T)).

Lysobacter caeni sp. nov., isolated from the sludge of a pesticide manufacturing factory.

Strain BUT-8(T), a Gram-stain-negative, non-motile and rod-shaped aerobic bacterium, was isolated from the activated sludge of a herbicide-manufacturing wastewater treatment facility. Comparative 16S rRNA gene sequence analysis revealed that strain BUT-8(T) clustered with species of the genus Lysobacter and was closely related to Lysobacter ruishenii DSM 22393(T) (98.3 %) and Lysobacter daejeonensis KACC 11406(T) (98.7 %). The DNA G+C content of the genomic DNA was 70.6 mol%. The major respiratory quinone was ubiquinone-8, and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an aminolipid. The major cellular fatty acids were iso-C15 : 0, iso-C16 : 0, iso-C17 : 0, iso-C11 : 0, iso-C11 : 0 3OH and summed feature 9 (comprising iso-C17 : 1ω9c and/or C16 : 010-methyl). The DNA-DNA relatedness between strain BUT-8(T) and its closest phylogenetic neighbours was below 70 %. Phylogenetic, chemotaxonomic and phenotypic results clearly demonstrated that strain BUT-8(T) belongs to the genus Lysobacter and represents a novel species for which the name Lysobacter caeni sp. nov. is proposed. The type strain is BUT-8(T) ( = CCTCC AB 2013087(T) = KACC 17141(T)).

Chryseomicrobium aureum sp. nov., a bacterium isolated from activated sludge.

A Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, aerobic bacterial strain, designated BUT-2(T), was isolated from activated sludge of one herbicide-manufacturing wastewater-treatment facility in Kunshan, Jiangsu province, China, and subjected to polyphasic taxonomic studies. Analysis of the 16S rRNA gene sequence indicated that strain BUT-2(T) shared the highest similarity with Chryseomicrobium amylolyticum (98.98%), followed by Chryseomicrobium imtechense (98.88%), with less than 96% similarlity to members of the genera Paenisporosarcina, Planococcus, Sporosarcina and Planomicrobium. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain BUT-2(T) clustered with C. amylolyticum JC16(T) and C. imtechense MW10(T), occupying a distinct phylogenetic position. The major fatty acid (>10% of total fatty acids) type of strain BUT-2(T) was iso-C(15 : 0). The quinone system comprised menaquinone MK-7 (77.8%), MK-6 (11.9%) and MK-8 (10.3%). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and some unidentified phospholipids. The cell-wall peptidoglycan type of strain BUT-2(T) was L-Orn-D-Glu. The genomic DNA G+C content of strain BUT-2(T) was 48.5 mol%. Furthermore, the DNA-DNA relatedness in hybridization experiments against the reference strain was lower than 70%, confirming that strain BUT-2(T) did not belong to previously described species of the genus Chryseomicrobium. On the basis of its morphological, physiological and chemotaxonomic characteristics as well as phylogenetic analysis, strain BUT-2(T) is considered to represent a novel species of the genus Chryseomicrobium, for which the name Chryseomicrobium aureum sp. nov. is proposed. The type strain is BUT-2(T) ( = CCTCC AB2013082(T) = KACC 17219(T)).