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.     

Pyridine degradation characteristics of a newly isolated bacterial strain and its application with a novel reactor for the further treatment in pyridine wastewater

A pyridine-degrading strain Gemmobacter sp. ZP-12, isolated from an activated sludge, was able to use pyridine as the sole carbon and nitrogen source for the growth. The strain could effectively degrade pyridine and remove TOC over a wide range of initial pyridine concentrations. The pyridine degradation rate for 100, 500, 1000, 1500 and 2000 mg/L was 2.90 ± 0.17; 13.72 ± 0.21, 20.40 ± 0.24, 31.09 ± 0.26, 27.63 ± 0.17 mg/L/h, respectively. During the pyridine degraded, a large amount of NH₄⁺-N was released and accumulated. The accumulation of NH₄⁺-N increased with the increase of pyridine concentration. For further removing the NH₄⁺-N producing in pyridine degradation, an aerobic-moving bed biofilm reactor coupled with intermittent-aeration membrane biological reactor (a-MBBR-IMBR) was constructed, in which the strain and the aerobic / anoxic mixed sludge combined to remove the pollutants in the wastewater containing 500 mg/L pyridine. After 96 h of operation, the final TOC removal efficiency was 96.5 ± 1.05 %. The average residual concentration of NO₃⁻-N and NH₄⁺-N was respectively 9.09 ± 4.13 mg/L and 7.85 ± 3.88 mg/L. The study provides a viable option for treating pyridine wastewater.     

生防细菌T132的鉴定及其对采后柑橘炭疽病的抑制效果

【目的】柑橘(Citri)是世界上重要的果树。由胶孢炭疽菌[Colletotrichum gloeosporioides(Penz.)]引起的柑橘炭疽病是柑橘生产的主要病害之一。为探索对采后柑橘炭疽病有效的生防措施,分离鉴定柑橘根围土壤中一株细菌T132,并研究其特性及生防效果。【方法】根据菌株T132的形态特征、生理生化特性以及16S rDNA序列对其进行鉴定;通过连续8次在人工培养基上传代培养,测定该菌株的遗传稳定性;采用柑橘果实刺伤挑战接种和拮抗菌液直接浸泡健康果实两种方法研究该菌株对柑橘炭疽病的抑菌防病效果;利用洋葱伯克霍尔德氏菌致病因子的特异性引物检测菌株T132是否为潜在的人类致病菌。【结果】菌株T132鉴定为越南伯克霍尔德氏菌(Burkholderia vietnamiensis)。连续8次在人工培养基上传代培养,菌株T132抑制胶孢炭疽病菌生长的能力没有发生明显改变。菌株T132对胶孢炭疽菌C.gloeosporioides引起的柑橘炭疽病有明显的防治作用,刺伤接种的防效为88.2%,自然发病的防效为54.9%。未检测到该拮抗菌株有人体致病相关的洋葱伯克霍尔德氏菌致病因子(BCESM)毒力基因。【结论】首次报道对柑橘采后炭疽病具有生防效果、对人类相对安全的越南伯克霍尔德氏菌生防菌株。     

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.     

生防菌株1404的鉴定及其对采后柑橘炭疽病的防治效果

【目的】柑橘是世界上重要的果树。由胶孢炭疽菌[Colletotrichum gloeosporioides(Penz.)Sacc.]引起的柑橘炭疽病是柑橘生产的主要病害。为开发对采后柑橘炭疽病有效的生防措施,对从辣椒根际土壤中分离的一株生防细菌1404进行了鉴定,并对其特性及生防效果进行了研究。【方法】根据菌株1404的形态特征、生理生化特性以及16SrDNA序列对其进行鉴定;通过连续在人工培养基上转代培养,测定该菌株拮抗活性的稳定性;用果实刺伤接种法对采后柑橘炭疽病的防效进行测定。【结果】菌株1404与来自GenBank的短短芽孢杆菌[Brevibacillus brevis(Migula)Shidaetal.]以100%bootstrap水平类聚一群。该菌株的形态特征及生理生化特性与Brevibacillus brevis相符。连续4次在人工培养基上转代培养,菌株1404对柑橘炭疽病菌生长的抑制力没有发生明显改变。该生防菌对柑橘炭疽病的防治效果明显,处理后第20天防效达到64.9%。【结论】根据16SrDNA序列、形态特征、生理生化特性,将菌株1404鉴定为短短芽孢杆菌。本文首次报道对柑橘采后炭疽病具有较好防效的生防菌Brevibacillus brevis。     

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.     

Evolution of a new bacterial pathway for 4-nitrotoluene degradation

Bacteria that assimilate synthetic nitroarene compounds represent unique evolutionary models, as their metabolic pathways are in the process of adaptation and optimization for the consumption of these toxic chemicals. We used Acidovorax sp. strain JS42, which is capable of growth on nitrobenzene and 2-nitrotoluene, in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates. Although the same enzyme that initiates the degradation of nitrobenzene and 2-nitrotoluene also oxidizes 4-nitrotoluene to 4-methylcatechol, which is a growth substrate for JS42, the strain is incapable of growth on 4-nitrotoluene. Using long-term laboratory evolution experiments, we obtained JS42 mutants that gained the ability to grow on 4-nitrotoluene via a new degradation pathway. The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the dioxygenase that catalyses the initial oxidation of nitroarene substrates, but at positions distal to the active site and previously unknown to affect activity in this or related enzymes. We constructed additional mutant dioxygenases to identify the order of mutations that led to the improved enzymes. Biochemical analyses revealed a defined, step-wise pathway for the evolution of the improved dioxygenases.     

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.     

孔雀石绿脱色菌的分离鉴定及染料脱色后毒性研究

【目的】从印染污水处理厂污泥中筛选孔雀石绿脱色菌,并分析比较染料脱色前后毒性。【方法】采用平板稀释涂布法筛选分离纯化菌株,通过扫描电镜、16S r RNA基因序列分析对菌株进行初步鉴定,采用蚕豆根尖微核毒性试验和小球藻毒性试验对染料脱色前后毒性进行分析。【结果】从印染污水处理厂污泥中筛选出一株高效孔雀石绿脱色菌。经过16S r RNA基因序列分析,鉴定该菌株为Klebsiella sp.JD。菌株的菌落边缘规则,圆形,灰白色粘稠,以接种环挑之,易拉成丝,表面光滑。通过对比小球藻在脱色前后的生长率和抑制率以及蚕豆微核率和污染指数分析出脱色后的染料毒性大大降低。【结论】筛选得到一株新的孔雀石绿脱色菌,该菌降解效率高,可降低染料毒性,对修复染料污染的水体有一定的现实意义。     

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.     

一株蒽降解细菌的分离及降解特性研究

【目的】从盐碱土壤中筛选蒽降解菌株并分析其降解特性。【方法】采用极度稀释结果流式细胞检测法筛选分离纯化菌株,通过16S rRNA基因序列分析对菌株进行初步鉴定,采用气质联用仪(GC-MS)分析蒽的降解特性。【结果】从盐碱土壤中筛选出一株高效蒽降解菌株。经过16S rRNA基因序列分析,鉴定该菌株为Demequina salsinemorus BJ1。菌株可以利用蒽作为唯一碳源生长,降解率可达92%。在一定浓度范围内,随着蒽浓度的降低,细菌生长速率变快,降解率升高。添加外加碳源后,细菌生长速率明显变快,而对蒽降解率变低。对萃取中间代谢产物的质谱分析表明,降解蒽的中间代谢产物主要有9,10-anthracenedione (9,10-蒽醌)和Phthalic acid (邻苯二甲酸)等,说明它可能通过邻苯二甲酸途径降解蒽。【结论】筛选得到一株新的耐盐碱蒽降解菌,该菌降解效率高,对修复石油污染的土壤有一定的现实意义。     

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.