Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides.

The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with K(m) and k(cat) values of 29.5 µM and 49.2 s(-1), respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.

SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113.

De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag(+), Cd(2+), Zn(2+), methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.

Catellibacterium nanjingense sp. nov., a propanil-degrading bacterium isolated from activated sludge, and emended description of the genus Catellibacterium.

A novel facultatively anaerobic, non-spore-forming, non-motile, catalase- and oxidase-positive, Gram-negative and rod-shaped bacterial strain, designated Y12(T), was isolated from activated sludge of a wastewater bio-treatment facility. The strain was able to degrade about 90% of added propanil (100 mg l(-1)) within 3 days of incubation. Growth occurred in the presence of 0-4.5% (w/v) NaCl (optimum 0.5%), at 10-40 °C (optimum 28 °C) and at pH 5.5-10.0 (optimum pH 7.0). Vesicular internal membrane structures and photoheterotrophic growth were not observed. The major respiratory quinone was ubiquinone-10 and the major cellular fatty acid was summed feature 8 (C(18:1)ω6c and/or C(18:1)ω7c). The genomic DNA G+C content of strain Y12(T) was 63.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Y12(T) was a member of the genus Catellibacterium, as it showed highest sequence similarities to Catellibacterium caeni DCA-1(T) (99.1%) and <96.0% similarities with other species of the genus Catellibacterium. Strain Y12(T) showed low DNA-DNA relatedness values with C. caeni DCA-1(T). Based on phenotypic, genotypic and phylogenetic properties, strain Y12(T) represents a novel species of the genus Catellibacterium, for which the name Catellibacterium nanjingense sp. nov. is proposed. The type strain is Y12(T) (=CCTCC AB 2010218(T) =KCTC 23298(T)). An emended description of the genus Catellibacterium is also presented.

Sphingobium jiangsuense sp. nov., a 3-phenoxybenzoic acid-degrading bacterium isolated from a wastewater treatment system.

A non-sporulating, non-motile, catalase- and oxidase-positive, Gram-negative, rod-shaped bacterial strain, designated BA-3T, was isolated from activated sludge of a wastewater treatment facility. The strain was able to degrade about 95 % of 100 mg 3-phenoxybenzoic acid l(-1) within 2 days of incubation. Growth occurred in the presence of 0-2 % (w/v) NaCl [optimum, 0.5 % (w/v) NaCl], at pH 5.5-9.0 (optimum, pH 7.0) and at 10-37 °C (optimum, 28 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain BA-3T was a member of the genus Sphingobium; it showed highest gene sequence similarity to Sphingobium qiguonii X23T (98.2 %), and similarities of <97.0 % with strains of other Sphingobium species. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of summed feature 8 (C18:1ω6c and/or C18:1ω7c) in the cellular fatty acid profile and the DNA G+C content also supported affiliation of the isolate to the genus Sphingobium. Strain BA-3T showed low DNA-DNA relatedness values (21.3±0.8 %) with Sphingobium qiguonii X23(T). Based on phenotypic, genotypic and phylogenetic data, strain BA-3T represents a novel species of the genus Sphingobium, for which the name Sphingobium jiangsuense sp. nov. is proposed; the type strain is BA-3T (=CCTCC AB 2010217T= KCTC 23196T=KACC 16433T).

Degradation of cyhalofop-butyl (CyB) by Pseudomonas azotoformans strain QDZ-1 and cloning of a novel gene encoding CyB-hydrolyzing esterase.

Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, whereas the substitutions in the aromatic ring had only a slight influence.

[Characteristics of a 3-phenoxybenzoic acid degrading-bacterium and the construction of a engineering bacterium].

A bacterium capable of utilizing 3-phenoxybenzoic acid (3-PBA) as sole carbon source was isolated from petroleum-contaminated soil. This bacterium, designated as BA3, was identified as Sphingobium sp. according to its physiological & biochemical characteristic and the similarity analysis of its 16S rDNA sequence. Strain BA3 was able to degrade 99% of 100 mg x L(-1) 3-phenoxybenzoic acid within 60 h. The optimal pH and temperature for the degradation were 7.0 and 30 degrees C, respectively. The degradation efficiency was related positively to initial inoculum size. The pyrethroid hydrolase gene (pytH) gene was amplified from the genomic DNA of Sphingobium sp. JZ-2 by PCR. Recombinant plasmids pPYTH was constructed by ligating pytH gene into the broad host vector pBBRMCS- 5. Under the help of plasmid RK600, pPYTH was transferred into Sphingobium sp. BA3 to construct engineering strain BA3-pytH; Fenpropathrin degradation experiments showed that strain JZ-2 was able to degrade only 60% of 50 mg x L(-1) fenpropathrin in 48 h while engineering strain BA3-pytH was able to degrade over 95% of 50 mg x L(-1) fenpropathrin under the same conditions. Even more, BA3-pytH could rapidly degrade 3-PBA, metabolic products of pyrethroid insecticides, eliminating the inhibition of 3-PBA to pyrethroid hydrolase. Therefore, in contrast to strain JZ-2, engineering strain BA3-pytH had more advantages in bioremediation of pyrethroid insecticides contaminated environment.

Rhodanobacter xiangquanii sp. nov., a novel anilofos-degrading bacterium isolated from a wastewater treating system.

A Gram-staining-negative, oxidase-positive, catalase-positive, non-motile, non-spore-forming, and rod-shaped bacterium, designated BJQ-6(T), was isolated from activated sludge of a waste-water treatment plant in Jiangsu Province, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BJQ-6(T) belonged to the genus Rhodanobacter, and shared 98.7% similarity with Rhodanobacter lindaniclasticus LMG 18385(T) and <97% similarities with other Rhodanobacter species. The major fatty acids were iso-C15:0 (17.6%), iso-C16:0 (19.3%), and Summed feature 9 (isoC17:1 ω9c and/or C16:0 10-methyl) (25.8%). The DNA G+C content of strain BJQ-6(T) was 64.8 mol%. Based on the phenotypic and phylogenetic considerations, strain BJQ-6(T) represents a novel species of the genus Rhodanobacter, for which the name Rhodanobacter xiangquanii sp. nov. is proposed. The type strain is BJQ-6(T) (=CCTCC AB 2010106(T) =KCTC 23100(T)).

Sphingobium wenxiniae sp. nov., a synthetic pyrethroid (SP)-degrading bacterium isolated from activated sludge in an SP-manufacturing wastewater treatment facility.

A synthetic pyrethroid (SP)-degrading bacterial strain, designated JZ-1(T), was isolated from activated sludge of a SP-manufacturing wastewater treatment facility and studied using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-1(T) belonged to the genus Sphingobium, showing highest sequence similarities to Sphingobium faniae DSM 21829(T) (98.6 %), Sphingobium cloacae JCM 10874(T) (98.5 %), Sphingobium vermicomposti DSM 21299(T) (97.4 %) and Sphingobium ummariense CCM 7431(T) (96.9 %). The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C(18 : 1)ω7c, C(19 : 0) cyclo ω8c, 11 methyl C(18 : 1)ω7c, C(16 : 0) and C(14 : 0) 2-OH, and the G+C content of the genomic DNA also supported the affiliation of the strain with the genus Sphingobium. Strain JZ-1(T) showed low DNA-DNA relatedness values with S. faniae DSM 21829(T) (30.2 %), S. cloacae JCM 10874(T) (23.3 %), S. vermicomposti DSM 21299(T) (10.9 %) and S. ummariense CCM 7431(T) (7.9 %). Based on its phylogenetic position and its phenotypic and genotypic properties, strain JZ-1(T) represents a novel species of the genus Sphingobium, for which the name Sphingobium wenxiniae sp. nov. is proposed. The type strain is JZ-1(T) ( = CGMCC 1.7748(T)  = DSM 21828(T)).

Methylopila jiangsuensis sp. nov., an aerobic, facultatively methylotrophic bacterium.

The taxonomic status was determined of an aerobic, facultatively methylotrophic strain, JZL-4(T), isolated from activated sludge. The cells were gram-negative, asporogenous, colourless, motile, short rods. The strain utilized methanol, methylamine, formate and a variety of polycarbon compounds, but not methane, dichloromethane or CO(2)/H(2), as carbon and energy sources. C(1) compounds were assimilated via the isocitrate lyase-negative serine pathway. Optimal growth occurred at 30 °C, pH 6.5-7.5 and 0.5 % (w/v) NaCl. The major cellular fatty acids were C(18 : 1)ω7c and C(18 : 0). The major phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine (PME); PME, the main phospholipid of strain JZL-4(T), was absent or present in only minor amounts in Methylopila capsulata IM1(T), Methylopila helvetica DM9(T) and Albibacter methylovorans DM10(T). The major ubiquinone was Q-10. The DNA G+C content of strain JZL-4(T) was 70.4 mol% (T(m)). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain showed high sequence similarities to M. capsulata IM1(T) (97.2 %), A. methylovorans DM10(T) (94.9 %) and M. helvetica DM9(T) (94.1 %), and showed less than 94 % similarity to strains of other species with validly published names. Strain JZL-4(T) had a low level of DNA-DNA relatedness (34 %) with M. capsulata IM1(T). On the basis of phenotypic, genetic and phylogenetic data, strain JZL-4(T) is proposed to represent a novel species of the genus Methylopila, with the name Methylopila jiangsuensis sp. nov. The type strain is strain JZL-4(T) ( = ACCC 05406(T)  = DSM 22718(T)  = VKM B-2555(T)).

Flavobacterium haoranii sp. nov., a cypermethrin-degrading bacterium isolated from a wastewater treatment system.

A Gram-negative, non-spore-forming, yellow-pigmented bacterium, strain LQY-7(T), was isolated from activated sludge treating synthetic pyrethroid-manufacturing wastewater. The taxonomic status of the strain was determined using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LQY-7(T) was a member of the genus Flavobacterium but had low similarities with other species of this genus (95.0 % similarity with Flavobacterium indicum GPTSA100-9(T) and <94 % similarities with other Flavobacterium species). On the basis of phenotypic, genetic and phylogenetic data, strain LQY-7(T) should be classified as a representative of a novel species of the genus Flavobacterium, for which the name Flavobacterium haoranii sp. nov. is proposed; the type strain is LQY-7(T) (=ACCC 05409(T) =KCTC 23008(T)).

Sphingobium faniae sp. nov., a pyrethroid-degrading bacterium isolated from activated sludge treating wastewater from pyrethroid manufacture.

A bacterial strain capable of degrading pyrethroid, designated JZ-2(T), was isolated from activated sludge treating pyrethroid-contaminated wastewater. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-2(T) belongs to the genus Sphingobium. It showed the highest levels of 16S rRNA gene sequence similarity to Sphingobium cloacae JCM 10874(T) (98.3 %) and Sphingobium ummariense CCM 7431(T) (97.1 %), and 94.8-96.9 % similarity to the type strains of other members of the genus Sphingobium. Strain JZ-2(T) contained C(18 : 1)omega7c as the predominant fatty acid, C(14 : 0) 2-OH as the major 2-hydroxy fatty acid, ubiquinone Q-10 as the main respiratory quinone, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and two sphingoglycolipids as the predominant polar lipids and spermidine as the major polyamine. DNA-DNA hybridization results showed that strain JZ-2(T) had low genomic relatedness with S. cloacae JCM 10874(T) (34 %) and S. ummariense CCM 7431(T) (23 %). Based on the phenotypic, genotypic and phylogenetic data presented, strain JZ-2(T) is considered to represent a novel species of the genus Sphingobium, for which the name Sphingobium faniae sp. nov. is proposed. The type strain is JZ-2(T) (=CGMCC 1.7749(T) =DSM 21829(T)).

Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. strain JZ-1 and characterization of the gene product.

A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many alpha/beta-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.