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4-Nitrophenol, a priority pollutant, is degraded by Gram-positive and Gram-negative bacteria via 1,2,4-benzenetriol (BT) and hydroquinone (HQ), respectively. All enzymes involved in the two pathways have been functionally identified. So far, all Gram-negative 4-nitrophenol utilizers are from the genera Pseudomonas and Burkholderia. But it remains a mystery why pnpG, an apparently superfluous BT 1,2-dioxygenase-encoding gene, always coexists in the catabolic cluster (pnpABCDEF) encoding 4-nitrophenol degradation via HQ. Here, the physiological role of pnpG in Burkholderia sp. strain SJ98 was investigated. Deletion and complementation experiments established that pnpG is essential for strain SJ98 growing on 4-nitrocatechol rather than 4-nitrophenol. During 4-nitrophenol degradation by strain SJ98 and its two variants (pnpG deletion and complementation strains), 1,4-benzoquinone and HQ were detected, but neither 4-nitrocatechol nor BT was observed. When the above-mentioned three strains (the wild type and complementation strains with 2,2'-dipyridyl) were incubated with 4-nitrocatechol, BT was the only intermediate detected. The results established the physiological role of pnpG that encodes BT degradation in vivo. Biotransformation analyses showed that the pnpA-deleted strain was unable to degrade both 4-nitrophenol and 4-nitrocatechol. Thus, the previously characterized 4-nitrophenol monooxygenase PnpASJ98 is also essential for the conversion of 4-nitrocatechol to BT. Among 775 available complete genomes for Pseudomonas and Burkholderia, as many as 89 genomes were found to contain the putative pnpBCDEFG genes. The paucity of pnpA (3 in 775 genomes) implies that the extension of BT and HQ pathways enabling the degradation of 4-nitrophenol and 4-nitrocatechol is rarer, more recent, and likely due to the release of xenobiotic nitroaromatic compounds. IMPORTANCE An apparently superfluous gene (pnpG) encoding BT 1,2-dioxygenase is always found in the catabolic clusters involved in 4-nitrophenol degradation via HQ by Gram-negative bacteria. Our experiments reveal that pnpG is not essential for 4-nitrophenol degradation in Burkholderia sp. strain SJ98 but instead enables its degradation of 4-nitrocatechol via BT. The presence of pnpG genes broadens the range of growth substrates to include 4-nitrocatechol or BT, intermediates from the microbial degradation of many aromatic compounds in natural ecosystems. In addition, the existence of pnpCDEFG in 11.6% of the above-mentioned two genera suggests that the ability to degrade BT and HQ simultaneously is ancient. The extension of BT and HQ pathways including 4-nitrophenol degradation seems to be an adaptive evolution for responding to synthetic nitroaromatic compounds entering the environment since the industrial revolution.

4-Nitrophenol, a priority pollutant, is degraded by Gram-positive and Gram-negative bacteria via 1,2,4-benzenetriol (BT) and hydroquinone (HQ), respectively. All enzymes involved in the two pathways have been functionally identified. So far, all Gram-negative 4-nitrophenol utilizers are from the genera Pseudomonas and Burkholderia. But it remains a mystery why pnpG, an apparently superfluous BT 1,2-dioxygenase-encoding gene, always coexists in the catabolic cluster (pnpABCDEF) encoding 4-nitrophenol degradation via HQ. Here, the physiological role of pnpG in Burkholderia sp. strain SJ98 was investigated. Deletion and complementation experiments established that pnpG is essential for strain SJ98 growing on 4-nitrocatechol rather than 4-nitrophenol. During 4-nitrophenol degradation by strain SJ98 and its two variants (pnpG deletion and complementation strains), 1,4-benzoquinone and HQ were detected, but neither 4-nitrocatechol nor BT was observed. When the above-mentioned three strains (the wild type and complementation strains with 2,2′-dipyridyl) were incubated with 4-nitrocatechol, BT was the only intermediate detected. The results established the physiological role of pnpG that encodes BT degradation in vivo. Biotransformation analyses showed that the pnpA-deleted strain was unable to degrade both 4-nitrophenol and 4-nitrocatechol. Thus, the previously characterized 4-nitrophenol monooxygenase PnpA(SJ98) is also essential for the conversion of 4-nitrocatechol to BT. Among 775 available complete genomes for Pseudomonas and Burkholderia, as many as 89 genomes were found to contain the putative pnpBCDEFG genes. The paucity of pnpA (3 in 775 genomes) implies that the extension of BT and HQ pathways enabling the degradation of 4-nitrophenol and 4-nitrocatechol is rarer, more recent, and likely due to the release of xenobiotic nitroaromatic compounds. IMPORTANCE An apparently superfluous gene (pnpG) encoding BT 1,2-dioxygenase is always found in the catabolic clusters involved in 4-nitrophenol degradation via HQ by Gram-negative bacteria. Our experiments reveal that pnpG is not essential for 4-nitrophenol degradation in Burkholderia sp. strain SJ98 but instead enables its degradation of 4-nitrocatechol via BT. The presence of pnpG genes broadens the range of growth substrates to include 4-nitrocatechol or BT, intermediates from the microbial degradation of many aromatic compounds in natural ecosystems. In addition, the existence of pnpCDEFG in 11.6% of the above-mentioned two genera suggests that the ability to degrade BT and HQ simultaneously is ancient. The extension of BT and HQ pathways including 4-nitrophenol degradation seems to be an adaptive evolution for responding to synthetic nitroaromatic compounds entering the environment since the industrial revolution.

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.

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. The content of the acetyl group is a pivotal factor affecting performance of PVA derivatives in industrial applications, and deacetylation is a nonnegligible part of PVA degradation. However, the genetics and biochemistry of its deacetylation remain largely elusive. Here, Comamonas sp. strain NyZ500 was isolated for its capability of growing on acetylated PVA from activated sludge. The 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, dacA(pva)) deletion in strain NyZ501, and in the dacA(pva)-complemented strain NyZ501 the ability to grow on PVA was restored. DacA(pva,) 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. DacA(pva) also possesses the ability to deacetylate acetylated xylan and the antibiotic intermediate 7-aminocephalosporanic acid (7ACA), but the enzymes responsible for the conversion of those two compounds have no activity against PVA derivatives. This study enhanced our understanding of the diversity of microbial degradation of PVA, and DacA(pva) 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 under high-energy-consumption and high-pollution conditions using hazardous chemicals (such as NaOH and methanol) under high temperatures. The DacA(pva) reported here performs PVA deacetylation under mild conditions, so it has great potential to be developed into an eco-friendly biocatalyst for biotransformation of PVA derivatives. DacA(pva) 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.

4-chloro-2-nitrophenol (4C2NP) is a typical chlorinated nitroaromatic pollutant and its environmental fate is of great concern due to its mutagenic and carcinogenic properties. So far, neither genes nor enzymes responsible for 4C2NP biodegradation have been elucidated. Here, a new 4C2NP degrader NyZ417 was isolated from the activated sludge collected from a wastewater treatment plant of China and identified as a member within Cupriavidus genus according to its 16S rRNA gene sequence analysis. Cupriavidus sp. strain NyZ417 utilized 4C2NP as the sole of nitrogen source for its growth in the presence of additional carbon source. The degradation of 4C2NP by strain NyZ417 was confirmed via an oxidative pathway and induced by substrate itself. For the elucidation of molecular mechanism of 4C2NP degradation in this newly isolated degrader, a monooxygenase encoding gene onpANyZ417 retrieved from its complete genome was confirmed to be involved in 4C2NP biotransformation. Resting cells expressing OnpANyZ417 depleted 4C2NP completely with stoichiometric formation of 4-chlorocatechol. This study not only elucidated a functional gene for 4C2NP degradation but also would lay a solid foundation for designing approaches for clean-up of 4C2NP residues in the environment.

A Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, aerobic bacterial strain, designated BUT-2T, 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-2Tshared the highest similarity withChryseomicrobium amylolyticum(98.98 %), followed byChryseomicrobium imtechense(98.88 %), with less than 96 % similarlity to members of the generaPaenisporosarcina,Planococcus,SporosarcinaandPlanomicrobium. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain BUT-2Tclustered withC. amylolyticumJC16TandC. imtechenseMW10T, occupying a distinct phylogenetic position. The major fatty acid (>10 % of total fatty acids) type of strain BUT-2Twas iso-C15 : 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-2Twasl-Orn-d-Glu. The genomic DNA G+C content of strain BUT-2Twas 48.5 mol%. Furthermore, the DNA–DNA relatedness in hybridization experiments against the reference strain was lower than 70 %, confirming that strain BUT-2Tdid not belong to previously described species of the genusChryseomicrobium. On the basis of its morphological, physiological and chemotaxonomic characteristics as well as phylogenetic analysis, strain BUT-2Tis considered to represent a novel species of the genusChryseomicrobium, for which the nameChryseomicrobiumaureumsp. nov. is proposed. The type strain is BUT-2T( = CCTCC AB2013082T = KACC 17219T).

Pseudomonas sp. strain WBC-3 mineralizes para-nitrophenol (PNP) as a growth substrate. The genes responsible for PNP degradation constitute 3 operons: pnpA, pnpB, and pnpCDEFG. As a chlorinated PNP, 2-chloro-4-nitrophenol (2C4NP) serves as a substrate for the PNP 4-monooxygenase PnpA; however, strain WBC-3 is unable to utilize 2C4NP. The LysR-type transcriptional regulator (LTTR) PnpR activates the expression of the 3 catabolic operons in response to PNP. Furthermore, an additional LTTR PnpM responds to 2C4NP other than PNP to initiate the expression of pnpCDEFG operon. Using a long-term directed evolution selection, spontaneous mutants of strain WBC-3 that grew on 2C4NP were obtained. The 2C4NP+ strains had single mutation in codon 6 of the gene encoding PnpR, with substitution of cysteine for serine. The variant PnpRS6C drove the PNP-independent expression of pnpA and pnpB operons in an almost constitutive manner but allowed higher levels of induction upon addition of inducers. However, expression of pnpCDEFG operon was still PNP-dependent together with active PnpRS6C. When the pnpRS6C allele was introduced into a pnpR-deleted WBC-3 mutant, the corresponding strain acquired the ability to grow on 2C4NP but was still able to utilize PNP for growth.

A Gram-stain-negative, aerobic, short rod-shaped, non-endospore-forming, ivory-pigmented and non-motile bacterium, designated strain BUT-5T, 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 C16 : 0, C18 : 1 2-OH and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The predominant respiratory quinone was ubiquinone Q-10. The polar lipids profile of strain BUT-5T 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-5T showed the highest sequence similarities to Roseomonas soli 5N26T (97.5 % 16S rRNA gene sequence similarity), followed by Roseomonas lacus TH-G33T (97.3 %) and Roseomonas terrae DS-48T (97.1 %). Strain BUT-5T showed low DNA-DNA relatedness with Roseomonas soli KACC 16376T (41 %), Roseomonas lacus KACC 11678T (46 %) and Roseomonas terrae KACC 12677T (42 %), respectively. On the basis of phenotypic and genotypic properties, as well as chemotaxonomic data, strain BUT-5T represents a novel species of the genus Roseomonas, for which the name Roseomonas eburnea sp. nov. is proposed. The type strain is BUT-5T ( = CCTCC AB2013276T = KACC 17166T).

A Gram-reaction-negative, aerobic, rod-shaped, non-spore-forming, non-motile bacterial strain, designated BUT-6T, was isolated from activated sludge of a wastewater-treatment facility. The strain grew at 15–35 °C (optimum 30 °C), 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 BUT-6T was most closely related to Tahibacter aquaticus PYM5-11T (98.6 % similarity). However, the DNA–DNA relatedness between strain BUT-6T and T. aquaticus PYM5-11T was 47.1 %. The major fatty acids (>10 % of total fatty acids) of strain BUT-6T were iso-C15 : 0, iso-C17 : 1ω9c and iso-C17 : 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-6T was 71.7 mol%. On the basis of the data from the polyphasic taxonomic study presented, strain BUT-6T is considered to represent a novel species of the genus Tahibacter , for which the name Tahibacter caeni sp. nov. is proposed. The type strain is BUT-6T ( = CCTCC AB 2013266T = KACC 17139T).

A Gram-stain-negative, non-spore-forming, non-motile, ovoid, aerobic bacterial strain, designated BUT-3T, was isolated from activated sludge from the wastewater treatment facility of a herbicide-manufacturing plant in Kunshan city, Jiangsu province, PR China. Strain BUT-3Tgrew between 15 and 40 °C, with optimum growth at 30 °C. The pH range for growth was between 5.0 and 10.0 (optimum pH 7.0). The range of NaCl concentrations for growth of strain BUT-3Twas 0–7.0 % (w/v), with an optimum of 1.5–3.0 % (w/v). A phylogenetic tree based on 16S rRNA gene sequence analysis showed that strain BUT-3Tclustered closely withRhodoligotrophos appendicifer120-1T(98.32 % similarity), with a bootstrap confidence level of 100 %. The major fatty acids (>5 % of total fatty acids) were C19 : 0cyclo ω8c, C18 : 1ω7c, C16 : 0, anteiso-C15 : 0and iso-C15 : 0. Strain BUT-3Tcontained ubiquinone Q-10 as the predominant respiratory quinone. The polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, three unidentified aminolipids (AL1–3), two unknown phospholipids (PL1, 5), four unidentified glycolipids (GL1–4) and two unknown lipids (L1, 2). The G+C content of the genomic DNA was 67.7 mol%. The DNA–DNA relatedness between BUT-3TandR. appendicifer120-1Twas 44.1±0.6 %. Based on the polyphasic taxonomic data, strain BUT-3Tshould be classified as a representative of a novel species of the genusRhodoligotrophos, for which the nameRhodoligotrophosjinshengiisp. nov. is proposed. The type strain is BUT-3T( = CCTCC AB2013083T = KACC 17220T).

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 N -dealkylase 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.