A novel esterase DacApva from Comamonas sp. strain NyZ500 with deacetylation activity for acetylated polymer polyvinyl alcohol.

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.

Physiological Role of the Previously Unexplained Benzenetriol Dioxygenase Homolog in the Burkholderia sp. Strain SJ98 4-Nitrophenol Catabolism Pathway.

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.

Tahibacter caeni sp. nov., isolated from activated sludge.

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 °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-6(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-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-6(T) was 71.7 mol%. On the basis of the data from the polyphasic taxonomic study presented, strain BUT-6(T) 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-6(T) ( = CCTCC AB 2013266(T) = KACC 17139(T)).

Novel three-component Rieske non-heme iron oxygenase system catalyzing the N-dealkylation of chloroacetanilide herbicides in sphingomonads DC-6 and DC-2.

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-His6 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.

Rhodoligotrophos jinshengii sp. nov., isolated from activated sludge.

A Gram-stain-negative, non-spore-forming, non-motile, ovoid, aerobic bacterial strain, designated BUT-3(T), was isolated from activated sludge from the wastewater treatment facility of a herbicide-manufacturing plant in Kunshan city, Jiangsu province, PR China. Strain BUT-3(T) grew 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-3(T) was 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-3(T) clustered closely with Rhodoligotrophos appendicifer 120-1(T) (98.32 % similarity), with a bootstrap confidence level of 100 %. The major fatty acids (>5 % of total fatty acids) were C19 : 0 cyclo ω8c, C18 : 1ω7c, C16 : 0, anteiso-C15 : 0 and iso-C15 : 0. Strain BUT-3(T) contained 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-3(T) and R. appendicifer 120-1(T) was 44.1±0.6 %. Based on the polyphasic taxonomic data, strain BUT-3(T) should be classified as a representative of a novel species of the genus Rhodoligotrophos, for which the name Rhodoligotrophos jinshengii sp. nov. is proposed. The type strain is BUT-3(T) ( = CCTCC AB2013083(T) = KACC 17220(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)).

Discovery of the 1-naphthylamine biodegradation pathway reveals a broad-substrate-spectrum enzyme catalyzing 1-naphthylamine glutamylation.

1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in the isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a five gene-cluster encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. A glutamine synthetase-like (GS-like) enzyme (NpaA1) initiates 1NA glutamylation, and this enzyme exhibits a broad substrate selectivity toward a variety of anilines and naphthylamine derivatives. Structural analysis revealed that the aromatic residues in the 1NA entry tunnel and the V201 site in the large substrate-binding pocket significantly influence NpaA1's substrate preferences. The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.

A cytochrome P450 system initiates 4-nitroanisole degradation in Rhodococcus sp. strain JS3073.

Nitroanisoles are used widely as synthetic intermediates and explosives. Although bacteria have been reported to degrade 4-nitroanisole (4NA) under aerobic conditions, the key enzymes and the catalytic mechanism have remained elusive. Rhodococcus sp. strain JS3073 was isolated for its ability to grow on 4NA as the sole carbon and energy source. In this study, whole cell biotransformation experiments indicated that 4NA degradation is initiated by O-demethylation to form 4-nitrophenol (PNP), which undergoes subsequent degradation by a previously established pathway involving formation of 1,2,4-benzenetriol and release of nitrite. Based on comparative transcriptomics and heterologous expression, a novel three-component cytochrome P450 system encoded by pnaABC initiates the O-demethylation of 4NA to yield formaldehyde and PNP. The pnaABC genes encode a phthalate dioxygenase type reductase (PnaA), a cytochrome P450 monooxygenase (PnaB), and an EthD family protein (PnaC) with putative function similar to ferredoxins. This unusual P450 system also has a broad substrate specificity for nitroanisole derivatives. Sequence analysis of PnaAB revealed high identity with multiple self-sufficient P450s of the CYP116B subfamily. The findings revealed the molecular basis of the catabolic pathway for 4NA initiated by an unusual O-demethylase PnaABC and extends the understanding of the diversity among P450s and their electron transport chains.