Sphingomonads DC-6 and DC-2 degrade the chloroacetanilide herbicides alachlor, acetochlor, and butachlor via N-dealkylation. In this study, we report a three-component Rieske non-heme iron oxygenase (RHO) system catalyzing the N-dealkylation of these herbicides. The oxygenase component gene cndA is located in a transposable element that is highly conserved in the two strains. CndA shares 24 to 42% amino acid sequence identities with the oxygenase components of some RHOs that catalyze N-or O-demethylation. Two putative [2Fe-2S] ferredoxin genes and one glutathione reductase (GR)-type reductase gene were retrieved from the genome of each strain. These genes were not located in the immediate vicinity of cndA. The four ferredoxins share 64 to 72% amino acid sequence identities to the ferredoxin component of dicamba O-demethylase (DMO), and the two reductases share 62 to 65% amino acid sequence identities to the reductase component of DMO. cndA, the four ferredoxin genes, and the two reductases genes were expressed in Escherichia coli, and the recombinant proteins were purified using Ni-affinity chromatography. The individual components or the components in pairs displayed no activity; the enzyme mixture showed Ndealkylase activities toward alachlor, acetochlor, and butachlor only when CndA-His 6 was combined with one of the four ferredoxins and one of the two reductases, suggesting that the enzyme consists of three components, a homo-oligomer oxygenase, a [2Fe-2S] ferredoxin, and a GR-type reductase, and CndA has a low specificity for the electron transport component (ETC). The N-dealkylase utilizes NADH, but not NADPH, as the electron donor.

A Gram-reaction-negative, aerobic, rod-shaped, non-spore-forming, non-motile bacterial strain, designated BUT-6 T , was isolated from activated sludge of a wastewater-treatment facility. The strain grew at 15-35 6C (optimum 30 6C), pH 4.0-10.0 (optimum pH 7.0) and 0-3.0 % (w/v) NaCl (optimum 1.0 %). Phylogenetic analysis based on 16S rRNA sequences showed that strain T was most closely related to Tahibacter aquaticus PYM5-11 T (98.6 % similarity). However, the DNA-DNA relatedness between strain BUT-6 T and T. aquaticus PYM5-11 T was 47.1 %. The major fatty acids (.10 % of total fatty acids) of strain BUT-6 T were iso-C 15 : 0 , iso-C 17 : 1 v9c and iso-C 17 : 0 . The major respiratory quinone was ubiquinone Q-8. The profile of polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, an unidentified aminophospholipid, three unknown aminolipids and unidentified phospholipids. The DNA G+C content of strain BUT-6 T was 71.7 mol%. On the basis of the data from the polyphasic taxonomic study presented, strain BUT-6

A Gram-stain-negative, aerobic, short rod-shaped, non-endospore-forming, ivory-pigmented and non-motile bacterium, designated strain BUT-5 T , was isolated from activated sludge of an herbicides-manufacturing wastewater treatment facility in Jiangsu Province, China. The major fatty acids (.5 % of total fatty acids) were C 16 : 0 , C 18 : 1 2-OH and summed feature 8 (C 18 : 1 v7c and/or C 18 : 1 v6c). The predominant respiratory quinone was ubiquinone Q-10. The polar lipids profile of strain T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and two unknown aminolipids. The DNA G+C content was 67.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BUT-5 T showed the highest sequence similarities to Roseomonas soli 5N26 T (97.5 % 16S rRNA gene sequence similarity), followed by Roseomonas lacus TH-G33 T (97.3 %) and

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 (5CCTCC AB2013082 T 5KACC 17219 T ).

As a water-soluble polymer, the widely used polyvinyl alcohol (PVA) is produced from hydrolysis of polyvinyl acetate. Microbial PVA carbon backbone cleavage via a two-step reaction of dehydrogenation and hydrolysis has been well studied. Content of acetyl group is a pivotal factor affecting performance of PVA derivatives in industrial application, and deacetylation is a non-negligible part in PVA degradation. However, the genetic and biochemical studies of its deacetylation remain largely elusive. Here, Comamonas sp. strain NyZ500 was isolated for its capability of growing on acetylated PVA from activated sludge. A spontaneous PVA-utilization deficient mutant strain NyZ501 was obtained when strain NyZ500 was cultured in rich media. Comparative analysis between the genomes of these two strains revealed a fragment (containing a putative hydrolase gene dacApva) deletion in NyZ501 and dacApva-complemented strain NyZ501 restored the ability to grow on PVA. DacApva, which shares 21% identity with xylan esterase AxeA1 from Prevotella ruminicola 23, is a unique deacetylase catalyzing the conversion of acetylated PVA and its derivatives to deacetylated counterparts. This indicates that strain NyZ500 utilizes acetylated PVA via acetate as a carbon source to grow. DacApva also possessed the deacetylation ability for acetylated xylan and the antibiotic intermediate 7-aminocephalosporanic acid (7ACA) but the enzymes for the above two compounds had no activities against PVA derivatives. This study enhanced our understanding of the diversity of microbial degradation of PVA and DacApva characterized here is also a potential biocatalyst for the eco-friendly biotransformation of PVA derivatives and other acetylated compounds. IMPORTANCE: Water-soluble PVA, which possesses a very robust ability to accumulate in the environment, has a very grave environmental impact due to its widespread use in industrial and household applications. On the other hand, chemical transformation of PVA derivatives is currently being carried out at high energy consumption and high pollution conditions using hazardous chemicals (such as NaOH, methanol) under high temperatures. The DacApva reported here performs PVA deacetylation under mild conditions, then it has a great potential to be developed into an eco-friendly biocatalyst for biotransformation of PVA derivatives. DacApva also has deacetylation activity for compounds other than PVA derivatives, which facilitates its development into a broad-spectrum deacetylation biocatalyst for production of certain desired compounds.