Iprodione [3-(3,5-dichlorophenyl) -isopropyl-2,4-dioxoimidazolidine-1-carboxamide] is a highly effective broad-spectrum dicarboxamide fungicide. Several bacteria with iprodione-degrading capabilities have been reported; however, the enzymes and genes involved in this process have not been characterized. In this study, an iprodione-degrading strain, sp. strain YJN-5, was isolated and characterized. Strain YJN-5 degraded iprodione through the typical pathway, with hydrolysis of its N-1 amide bond to -(3,5-dichlorophenyl)-2,4-dioxoimidazolidine as the initial step. The gene, encoding a novel amidase responsible for this step, was cloned from strain YJN-5 by the shotgun method. IpaH shares the highest similarity (40%) with an indoleacetamide hydrolase (IAHH) from USDA 110. IpaH displayed maximal enzymatic activity at 35°C and pH 7.5, and it was not a metalloamidase. The and of IpaH against iprodione were 22.42 s and 7.33 μM, respectively, and the catalytic efficiency value ( ) was 3.09 μM s IpaH has a Ser-Ser-Lys motif, which is conserved among members of the amidase signature family. The replacement of Lys82, Ser157, and Ser181 with alanine in IpaH led to the complete loss of enzymatic activity. Furthermore, strain YJN-5M lost the ability to degrade iprodione, suggesting that is the only gene responsible for the initial iprodione degradation step. The gene could also be amplified from another previously reported iprodione-degrading strain, sp. strain YJN-G. The sequence similarity between the two IpaHs at the amino acid level was 98%, indicating that conservation of IpaH exists in different strains. Iprodione is a widely used dicarboxamide fungicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency has classified iprodione as moderately toxic to small animals and a probable carcinogen to humans. Bacterial degradation of iprodione has been widely investigated. Previous studies demonstrate that hydrolysis of its N-1 amide bond is the initial step in the typical bacterial degradation pathway of iprodione; however, enzymes or genes involved in iprodione degradation have yet to be reported. In this study, a novel gene encoding an amidase responsible for the initial degradation step of iprodione in sp. strain YJN-5 was cloned. In addition, the characteristics and key amino acid sites of IpaH were investigated. These findings enhance our understanding of the microbial degradation mechanism of iprodione.

Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, and , were predicted from the 84 open reading frames (ORFs) that share ≥95% nucleic acid similarities between strain CDS-1 and another sphingomonad sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that and are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehA hydrolyzes carbofuran to carbofuran phenol. CfdC, a reduced flavin mononucleotide (FMNH)- or reduced flavin adenine dinucleotide (FADH)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehA, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity. Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in sp. strain CDS-1. We speculate that the close homologues and are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

This study presents a bacterial combination of Aminobacter sp. MDW-2 and Afipia sp. MDW-3, which could degrade methomyl completely by biochemical cooperation. This study also proposes the biodegradation pathway of methomyl for the first time and highlights the application potential of a bacterial combination in the remediation of methomyl-contaminated environments.

A Gram-staining-negative, aerobic, non-motile and rod-shaped bacterium that produced yellow viscous colonies, designated FL-8, was isolated from farmland soil in Chuzhou, Anhui province, PR China. 16S rRNA gene sequence similarities between strain FL-8 and the type strains of species of the genus Terrimonas with validly published names ranged from 94.6 to 96.1 %. Strain FL-8 contained iso-C15 : 1 G, iso-C15 : 0 and iso-C17 : 0 3-OH as the predominant fatty acids. The predominant polar lipid of strain FL-8 was phosphatidylethanolamine. The sole respiratory quinone of strain FL-8 was MK-7 and the DNA G+C content was 44.8 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain FL-8 is considered to represent a novel species of the genus Terrimonas, for which the name Terrimonassoli sp. nov. is proposed. The type strain is FL-8 (=CCTCC AB 2017059=JCM 32095).

A novel Gram-stain-positive bacterium, designated djl-8, was isolated from farmland soil in Nanjing, Jiangsu province, PR China. Cells of strain djl-8 were aerobic, non-motile, non-spore-forming and rod-shaped. The organism grew at 25-37 °C, pH 5.5-8.0 and 0.5-4.0 % NaCl (w/v). The DNA G+C content was 69.3 mol%. The diagnostic diamino acid in the cell-wall peptidoglycan was LL-2, 6-diaminopimelic acid. The major fatty acids (>5 %) were iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0, 10-Me C17 : 0 and C17 : 1ω8c. The respiratory quinone was MK-8 (H4) and the major polar lipids were phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and unknown phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain djl-8 is a member of the genus Nocardioides and shared the highest similarity with Nocardioides ginkgobilobae SYP-A7303 (97.1 %), followed by Nocardioides soli mbc-2 (96.9 %), Nocardioide spyridinolyticus OS4 (96.6 %) and Nocardioides maradonensis RP-B30 (96.6 %). Strain djl-8 exhibited low DNA-DNA relatedness with Nocardioides ginkgobilobae SYP-A7303 (26.9±2.1 %). On the basis of the morphological, physiological, biochemical and chemotaxonomic characteristics presented in this study, strain djl-8 represents a novel species of the genus Nocardioides, for which the name Nocardioides agrisoli sp. nov. is proposed. The type strain is djl-8 (=KCTC 39844=CCTCC AB 2017058).