Screening of a beta-cypermethrin-degrading bacterial strain <Emphasis Type="Italic">Brevibacillus parabrevis</Emphasis> BCP-09 and its biochemical degradation pathway

A novel beta-cypermethrin (Beta-CP)-degrading strain isolated from activated sludge was identified as Brevibacillus parabrevis BCP-09 based on its morphological and physio-biochemical characteristics, and 16S rRNA gene analysis. Strain BCP-09 could effectively degrade Beta-CP at pH 5.0–9.0, 20–40 °C, and 10–500 mg L^?1 Beta-CP. Under optimal conditions (pH 7.41, 38.9 °C, 30.9 mg L^?1 Beta-CP), 75.87% Beta-CP was degraded within 3 days. Beta-CP degradation (half-life, 33.45 h) and strain BCP-09 growth were respectively described using first-order-kinetic and logistic-kinetic models. Seven metabolites were detected by high-performance liquid chromatography and gas chromatography-mass spectrometry- methyl salicylate, catechol, phthalic acid, salicylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzaldehyde, and 3-phenoxybenzoic acid (3-PBA). The major Beta-CP metabolite, 3-PBA was further degraded into phenol, benzoic acid, and 4-methylhexanoic acid. BCP-09 also degraded aromatic compounds such as phenol, catechol, and protocatechuic acid. Beta-CP appears to be mainly degraded into 3-PBA, which is continuously degraded into smaller benzene or chain compounds. Thus, strain BCP-09 could form a complete degradation system for Beta-CP and might be considered a promising strain for application in the bioremediation of environments and agricultural products polluted by Beta-CP.     

一株萘降解菌的筛选及其降解途径

从胜利油田被污染土壤中筛选出一株能够以萘为唯一碳源的菌株W1,经形态和生理生化以及16S rD-NA测序分析,初步鉴定为沙雷氏菌属。其最适生长条件为35℃,pH 7.5。该菌对盐及萘有较好的耐受性。当培养基盐质量浓度为30 g/L,底物萘质量浓度为100 mg/L时,培养3 d后,其萘降解率仍可达到80.9%。当萘浓度为800 mg/L时,仍具有一定的降解作用,降解率为15.8%。通过对菌株降解原油前后组分的GC-MS分析,以及检测其降解多种底物后的吸光度,得出该菌能利用苯酚、甲苯、苯甲酸、1-萘酚、丙酮、辛烷生长,对原油中组分C20～C23、C33～C36的直链烃有较好的降解效果。经UV-Vis扫描其降解中间产物,初步判断其萘降解生物途径为邻苯二酚途径,萘首先被其降解生成水杨酸,而后转化为邻苯二酚,开环并生成一系列小分子物质,最后进入三羧酸循环。     

Structural and biochemical identification of a novel bacterial oxidoreductase

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.     

Characterization of a novel antarctic plant growth-promoting bacterial strain and its interaction with antarctic hair grass (Deschampsia antarctica Desv)

Deschampsia antarctica is the only hair grass that has been able to successfully colonize the Antarctic continent. However, there is little research on the role of microorganisms associated with the rhizosphere that may participate in its growth and development. The objective of this research was to characterize a psychrotolerant bacterial strain isolated from the rhizosphere of D. antarctica. Biochemical and molecular studies were performed to characterize this bacterium. It was determined that this strain secretes a neutral polysaccharide that presents different compositions at different temperatures (4 and 20 °C). Based on biochemical and phylogenetic analyses, the Antarctic rhizobacterium could be a new species of Pseudomonas. To determine their ability to solubilize different sources of inorganic phosphate, qualitative and quantitative analyses were conducted to determine P released at 4 °C. The Antarctic strain of Pseudomonas sp. was able to solubilize all sources of phosphates, and 34.2 mg P/L was released from rock phosphate. Growth physiological parameters were evaluated for seedlings of D. antarctica inoculated with the rhizobacteria. It was found that the bacterial inoculation promoted plant root development. SEM analysis of the roots showed that the bacterium is mainly located in the root hairs of D. antarctica.     

Characterization of strain HY99, a novel microorganism capable of aerobic and anaerobic degradation of aniline

We have characterized a novel microorganism, strain HY99, which is capable of aerobic and anaerobic degradation of aniline. Strain HY99 was found to aerobically metabolize aniline via catechol and 2-hydroxymuconic semialdehyde intermediates, and to transform aniline via p-aminobenzoate in anaerobic environments. Physiological and biochemical tests revealed that strain HY99 was most similar to Delftia acidovorans, but unlike D. acidovorans, strain HY99 was able to metabolize aniline under anaerobic conditions linked with nitrate reduction. Phylogenetic analysis based on 16S rDNA sequencing also revealed that strain HY99 was closely related to D. acidovorans, with 96% overall similarity.     

Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway

Nicotine, a major alkaloid in tobacco plants and the main toxic chemical in tobacco wastes, can be transformed by bacteria into hydroxylated-pyridine intermediates, which are important precursors for the chemical synthesis of valuable drugs and insecticides. Such biotransformation could be a useful approach to utilize tobacco and its wastes. In this study, we explored nicotine degradation by a recently isolated Agrobacterium tumefaciens S33 by identifying the intermediates during its growth on nicotine and during transformation of nicotine with its resting cells. Five hydroxylated-pyridine intermediates were detected through multiple approaches, including GC-HR-MS, HPLC, and ESI-Q-TOF MS analyses. Surprisingly, these identified intermediates suggest that strain S33 employs a novel pathway that is different from the two characterized pathways described in Arthrobacter and Pseudomonas. Based on these findings, we propose that strain S33 is able to transform nicotine to 6-hydroxy-pseudooxynicotine first via the pyridine pathway through 6-hydroxy-L: -nicotine and 6-hydroxy-N-methylmyosmine, and then, it turns to the pyrrolidine pathway with the formation of 6-hydroxy-3-succinoylpyridine and 2,5-dihydroxypyridine. The activities of the key enzymes, nicotine dehydrogenase, 6-hydroxy-L: -nicotine oxidase, and 6-hydroxy-3-succinoylpyridine hydroxylase, were demonstrated in the cell extract of strain S33 and by partially enriched enzymes. Moreover, the cell extract could transform 6-hydroxy-pseudooxynicotine into 6-hydroxy-3-succinoylpyridine by coupling with 6-hydroxy-L: -nicotine oxidation reaction by 6-hydroxy-L: -nicotine oxidase. These results indicated that strain S33 can transform nicotine into renewable hydroxylated-pyridine intermediates by the special pathway, in which at least three intermediates, 6-hydroxy-L: -nicotine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine, have potential to be further chemically modified into useful compounds.     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Molecular and biochemical characterization of the 5-nitroanthranilic acid degradation pathway in Bradyrhizobium sp. strain JS329

Biodegradation pathways of synthetic nitroaromatic compounds and anilines are well documented, but little is known about those of nitroanilines. We previously reported that the initial step in 5-nitroanthranilic acid (5NAA) degradation by Bradyrhizobium sp. strain JS329 is a hydrolytic deamination to form 5-nitrosalicylic acid (5NSA), followed by ring fission catalyzed by 5NSA dioxygenase. The mechanism of release of the nitro group was unknown. In this study, we subcloned, sequenced, and expressed the genes encoding 5NAA deaminase (5NAA aminohydrolase, NaaA), 5NSA dioxygenase (NaaB) and lactonase (NaaC), the key genes responsible for 5NAA degradation. Sequence analysis and enzyme characterization revealed that NaaA is a hydrolytic metalloenzyme with a narrow substrate range. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation. The elimination of the nitro group during lactone formation is a previously unreported mechanism for denitration of nitro aliphatic compounds.     

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.     

The metabolic pathway of metamifop degradation by consortium ME-1 and its bacterial community structure

Metamifop is universally used in agriculture as a post-emergence aryloxyphenoxy propionate herbicide (AOPP), however its microbial degradation mechanism remains unclear. Consortium ME-1 isolated from AOPP-contaminated soil can degrade metamifop completely after 6 days and utilize it as the carbon source for bacterial growth. Meanwhile, consortium ME-1 possessed the ability to degrade metamifop stably under a wide range of pH (6.0–10.0) or temperature (20–42 °C). HPLC–MS analysis shows that N-(2-fluorophenyl)-2-(4-hydroxyphenoxy)-N-methyl propionamide, 2-(4-hydroxyphenoxy)-propionic acid, 6-chloro-2-benzoxazolinone and N-methyl-2-fluoroaniline, were detected and identified as four intermediate metabolites. Based on the metabolites identified, a putative metabolic pathway of metamifop was proposed for the first time. In addition, the consortium ME-1 was also able to transform or degrade other AOPP such as fenoxaprop-p-ethyl, clodinafop-propargyl, quizalofop-p-ethyl and cyhalofop-butyl. Moreover, the community structure of ME-1 with lower microbial diversity compared with the initial soil sample was investigated by high throughput sequencing. β-Proteobacteria and Sphingobacteria were the largest class with sequence percentages of 46.6% and 27.55% at the class level. In addition, 50 genera were classified in consortium ME-1, of which Methylobacillus, Sphingobacterium, Bordetella and Flavobacterium were the dominant genera with sequence percentages of 25.79, 25.61, 14.68 and 9.55%, respectively.     

联苯菊酯降解菌株BF-3的分离筛选、鉴定和降解特性

【背景】联苯菊酯是人工合成的类似天然除虫菊素的一种仿生杀虫剂,近年来被广泛应用于农业病虫害的防治。联苯菊酯化学性质较稳定,在环境中的残留期长,是我国出口果蔬、茶叶中残留较严重的农药之一。微生物降解具有降解速度快、无二次污染等优点,被认为是有效去除农药残留的绿色生产技术。【方法】通过富集驯化,从农药厂排污口的污泥中筛选分离能够降解联苯菊酯的菌株,经形态学观察、生理生化特性测定及16SrRNA序列分析对其进行鉴定,通过单因素试验对菌株的降解特性进行研究。【结果】分离到1株联苯菊酯的降解菌株BF-3。菌株BF-3为革兰氏阳性菌,被鉴定为蜡状芽孢杆菌;该菌株在联苯菊酯质量浓度为100mg·L^-1的无机盐液体培养基（MSM）中呈现s型生长,7d后对联苯菊酯的降解率达到87．9％。单因素试验表明,菌株最适降解条件为pH7．0、30oC,初始菌株D600am=1．0。【结论与意义】蜡状芽孢杆菌BF．3能够有效降解联苯菊酯,在环境中联苯菊酯残留的生物修复方面具有应用潜力。     

Escherichia hermanii--a new bacterial strain for chlorobenzene degradation

A new bacterial strain capable of chlorobenzene degradation has been isolated from sludge of an industrial wastewater treatment plant. The micro-organism is short, rod-shaped, Gram-negative, yellow-pigmented and has been identified as Escherichia hermanii. It was observed that high chlorobenzene concentrations (up to 394 mg l-1) had low toxic effects towards this strain, which was able to degrade chlorobenzene without any previous adaptation.     

Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate

Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.     

Identification of the biochemical degradation pathway of triazophos and its intermediate in Diaphorobacter sp. TPD-1

Triazophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A bacterial strain, Diaphorobacter sp. TPD-1, capable of using triazophos and its intermediate, 1-phenyl-3-hydroxy-1,2,4-triazole (PHT), as its sole carbon sources for growth was isolated from a triazophos-contaminated soil in China. This strain could completely degrade 50 mg l⁻¹ triazophos and PHT to non-detectable level in 24 and 56 h, respectively. During PHT degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) analysis. Using this information, a biochemical degradation pathway of triazophos by Diaphorobacter sp. TPD-1 was proposed. The first step involved in the degradation of triazophos is the hydrolysis of the P–O ester bond of triazophos to form PHT and o,o-diethyl phosphorothioic acid, then the triazol ring of PHT is subsequently cleaved to form (E)-1-formyl-2-phenyldiazene. Subsequently, (E)-1-formyl-2-phenyldiazene is transformed to 2-phenylhydrazinecarboxylic acid by adding one molecular of H₂O. Finally, the carboxyl group of 2-phenylhydrazinecarboxylic acid is decarboxylated to form phenylhydrazine.     

Characterization of a novel Dunaliella salina (Chlorophyta) strain and the assessment of its cultivation parameters

Physiological characteristics of a novel strain of the halophilic alga Dunaliella salina IPPAS D-232 isolated from the hypersaline lake Razval (Sol-Iletsk, Orenburg region) were studied. It was shown that the extract from the cells of this strain manifested antioxidant properties and also antibacterial activity relative to opportunistic bacteria. The influence of medium stirring and salinity on the level of D. salina antioxidant activity was assessed: it was established that medium salinization (93 g/L) and continuous stirring favored the highest accumulation of D. salina IPPAS D-232 biomass. It was found that the extremely halophilic archaea Halorubrum tebenquichense and moderately halophilic bacterium Marinococcus halophilus stimulated D. salina development and increased its antioxidant activity. The results obtained expand our knowledge of specific ecological features of the studied microalgal species; they could form the basis for the development of new approaches to the mass cultivation of D. salina.     

Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp.

N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The K_m and V_max values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min⁻¹ · mg protein⁻¹, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).     

The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway.

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide. The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.