Isolation and characterization of a 2,4-dichlorophenoxyacetic acid-degrading soil bacterium

Summary A 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, Xanthobacter sp. CP, was isolated after enrichment in aerated soil columns. A limited number of chlorinated phenols and chlorinated phenoxyalkanoic acids with an even number of carbon atoms in the side chain served as substrates for growth, although whole cells exhibited oxygen uptake with a wide range of those compounds. The maximal growth rate with 2,4-D was 0.13·h^-1 at a growth yield of 0.1 g biomass/g 2,4-D. Chloride ions were released quantitatively from 2,4-D and related chlorinated aromatic compounds which served as growth substrates. No by-products of 2,4-D metabolism were detected in oxygen-sufficient cultures of Xanthobacter sp. CP and catechols were cleaved exclusively by catechol 1,2-dioxygenase.     

Isolation and characterization of a phorate degrading bacterium

Aims:  To study the degradation of phorate by a bacterium isolated from phorate-contaminated sites.
Methods and Results:  Ralstonia eutropha strain AAJ1 isolated from soil was found to degrade phorate (supplied as sole carbon source) upto 85% in 10 days in liquid medium. Half-life ( t _½) of phorate in the liquid medium in control (uninoculated) and in experimental (inoculated with R. eutropha , strain AAJ1) samples was recorded as 36·49 and 6·29 days, respectively. Kinetics revealed that phorate degradation depends on time and the reaction follows the first order kinetics. Diethyl dithiophosphate was one of the degradation products, which is markedly less toxic than the parent compound; other degradation products included phorate sulfoxide and phorate sulfone. Release of inorganic phosphates and sulfates indicated the potential of the isolate to further degrade the above-mentioned metabolites to simpler forms. The strain was also found to posses phosphomonoesterase and phosphodiesterase enzymatic activity, which are involved in biodegradation of organophosphorus compounds.
Conclusions:  Ralstonia eutropha AAJ1 could degrade and detoxify phorate upto 85% in 10 days in laboratory conditions.
Significance and Impact of the Study:  The isolate has the potential to be utilized for remediation of phorate-contaminated water and soil.     

一株菊酯类农药降解菌的分离鉴定及其降解酶基因的克隆

摘要：【目的】筛选分离高效降解菊酯类农药的光合细菌,研究其降解特性,并对该菌株中降解酶基因进行克隆与初步分析。【方法】根据分离菌株的细胞形态结构、活细胞光吸收特征、生理生化特征及其16S rDNA序列系统发育分析鉴定降解菌,气相色谱法测定该菌株降解菊酯类农药的能力,PCR方法克隆降解酶基因。【结果】菌株PSB07-21属红假单胞菌属（Rhodopseudomonas sp.）,其降解最佳条件为3000 lx、35℃、pH 7,在此条件下培养15 d对600 mg/L甲氰菊酯、氯氰菊酯、联苯菊酯降解率分别为     

Isolation and characterization of a furfural degrading sulfate-reducing bacterium from an anaerobic digester

A sulfate-reducing bacterium (SRB) was isolated from a continuous anaerobic digester, which converted the furfural-containing wastewater to methane and CO₂. This SRB isolate could use furfural, furfuryl alcohol, and 2-furoic acid as sole source of carbon and energy in a defined mineral sulfate medium. Acetic acid was the major end product of furfural degradation. This organism also used wide varieties of other carbon sources, including ethanol, pyruvate, lactate, succinate, propanol, formate, and malate. The SRB isolate contained the electron carrier desulfoviridin. It used SO₄, NO₃, and thiosulfate as electron acceptors. This isolate used ammonium chloride, nitrate and glutamate as nitrogen source. The characteristics of the SRB isolate were closely similar toDesulfovibrio sp.     

Isolation and characterization of a thermophilic bacterium, Geobacillus thermocatenulatus, degrading nylon 12 and nylon 66

A thermophilic bacterium, identified as a neighboring species to Geobacillus thermocatenulatus, having a growth optimum at 55 °C and, capable of degrading nylon 12, was isolated from soil by enrichment culture technique at 60 °C. At this temperature, the strain grew on 5 g nylon 12 l^–1 with a decrease in its molecular weight from 41000 to 11000 over 20 d. The degradation was assumed to be due to endogenous hydrolysis of amide bond in nylon 12. The strain degraded also nylon 66 with a decrease in its molecular weight from 43000 to 17000 in 20 d at 60 °C. Nylon 6 was not degraded.     

Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production.

An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.     

新型争论贪噬菌Variovorax paradoxus S110的细胞色素P450酶的异源表达及催化特性分析

细胞色素P450单加氧酶(Cytochrome P450 monooxygenases)是一种广谱催化剂,可以催化多种类型反应而参与生物体外源物质代谢与天然产物的合成。为丰富P450作为合成生物学的酶元件库,并探索新型催化反应,利用生物信息学手段从争论贪噬菌Variovorax paradoxus S110中挖掘出一种新型电子自供体细胞色素P45(VpMO)单加氧酶,属于CYP116B家族,它可以在大肠杆菌Escherichia coli异源可溶表达。酶学性质研究表明P450_(VpMO)最适pH和最适温度分别为8.0和45℃,并且在温度低于35℃时具有良好的稳定性,K_m值为0.458 mmol/L,k_(cat)为2.438 min~(-1);重要的是重组P450_(VpMO)可以催化一系列包含污染物的含甲氧基底物进行脱甲基反应,其中对4-甲氧基苯乙酮的脱甲基反应转化率高达91%。相比于其他CYP116B家族的P450酶,P450_(VpMO)表现出较强的酶活性,这为后期进一步研究P450_(VpMO)提供了基础。     

The ambiguous role of 2,4-dichlorophenoxyacetic acid in wheat tissue culture

The very basal, highly immature regions of dissected young leaves of Triticum aestivum L. cv. Kite formed adventitious roots on a nutrient medium supplemented with comparatively low concentrations (0.16 to 0.63 μ M ) of 2,4-dichlorophenoxyacetic acid (2,4-D). Higher concentrations (up to 640 μ M ) had to be applied to stimulate growth from more mature regions higher up the leaf. Yet, already at 2.5 μ M roots were less distinct and more callus-like, and eventually (at 10 to 640 μ M ) only a subculturable callus of apparently suppressed, slowly proliferating root primordia developed. Furthermore, at the most basal, highly immature regions growth was significantly retarded when the auxin concentration was raised. The leaf culture system appears to reflect the dual action of 2,4-D known from herbicide research, namely growth stimulation from differentiating (or differentiated) cells, but growth suppression at or in the vicinity of apical meristems. Correspondingly, when the callus of apparently suppressed, slowly proliferating root primordia was transferred to media without 2,4-D or with low concentrations (0.16–2.5 μ M ) rapid proliferation commenced, leading to profuse root outgrowth. The system demonstrates the ambiguous role which this auxin appears to have, at least in wheat tissue culture.     

Modeling available 2,4-dichlorophenoxyacetic acid in a tissue culture medium containing activated carbon

Clonal propagation of high-value forest trees by somatic embryogenesis can help meet industry needs for uniform and high quality raw materials. Low embryogenic tissue initiation frequencies for loblolly pine (Pinus taeda L.) pose a limitation in work towards commercialization of this technology. At the time our research began most work on somatic embryo culture initiation in loblolly pine reported success in the range of 1–5%. Activated carbon (AC) has been reported to improve many tissue culture systems including embryogenic tissue initiation in Douglas-fir. To improve initiation frequencies in loblolly pine, the development of an AC-containing system was explored. In order to better understand the availability of 2,4-dichlorophenoxyacetic acid (2,4-D) in initiation medium, we tracked media surface concentrations of free or available 2,4-D. Media containing 1/2 modified P6 salts, 1.5% maltose, 2% myo-inositol, case amino acids, glutamine, vitamins, and 0.4% Gelrite were modified to include 0.625 – 2.5 g l^–1 of activated carbon (Sigma C-9157, acid washed) and 110 –440 mg l^–1 2,4-D. Adsorption and availability of 2,4-D in AC-containing medium was tracked by C¹⁴ labeled 2,4-D present in surface moisture absorbed into filter paper. High correlations were found between–available 2,4-D and time when AC and initial 2,4-D concentrations were held constant,–available 2,4-D and AC concentration when initial added 2,4-D and time were held constant, and–available 2,4-D and initial 2,4-D when AC and time were held constant.All of these relationships were exponential, not linear. Multiple regression models inputting initial 2,4-D added to medium in mg l^–1, activated carbon added to medium in %, and time in days, were able to explain 85–88% of the variability in available 2,4-D. These models can be used to achieve target levels of available 2,4-D by adjustment of initial 2,4-D levels or AC content.     

Isolation and characterization of comprehensive polychlorinated biphenyl degrading bacterium, Enterobacter sp. LY402

A Gram-negative bacterium, named LY402, was isolated from contaminated soil. 16S rDNA sequencing and measurement of the physiological and biochemical characteristics identified it as belonging to the genus Enterobacter. Degradation experiments showed that LY402 had the ability to aerobically transform 79 of the 91 major congeners of Aroclor 1242, 1254, and 1260. However, more interestingly, the strain readily degraded certain highly chlorinated and recalcitrant polychlorinated biphenyls (PCBs). Almost all the tri- and tetra-chlorobiphenyls (CBs), except for 3,4,3',4'-CB, were degraded in 3 days, whereas 73% of 3,4,3',4'-, 92% of the penta-, 76% of the hexa-, and 37% of the hepta-CBs were transformed after 6 days. In addition, among 12 octa-CBs, 2,2',3,3',5,5',6,6- CB was obviously degraded, and 2,2',3,3',4,5,6,6'- and 2,2',3,3',4,5,5',6'-CB were slightly transformed. In a metabolite analysis, mono- and di-chlorobenzoic acids (CBAs) were identified, and parts of them were also transformed by strain LY402. Analysis of PCB degradation indicated that strain LY402 could effectively degrade PCB congeners with chlorine substitutions in both ortho- and para-positions. Consequently, this is the first report of an Enterobacteria that can efficiently degrade both low and highly chlorinated PCBs under aerobic conditions.     

Isolation and characterization of 2,4-dichlorophenoxyacetic acid-catabolizing bacteria and their biodegradation efficiency in soil

Bacterial isolates (NJ 10 and NJ 15) capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were isolated from agricultural soil by enrichment culture technique. The isolates exhibited substantial growth in mineral salt medium supplemented with 0.1–0.5% of 2,4-D as a sole source of carbon and energy. Based on their morphological, cultural and biochemical characteristics, the isolates NJ 10 and NJ 15 have been identified as Pseudomonas species and Pseudomonas aeruginosa, respectively. Biodegradation studies in a soil microcosm enriched with pure cultures of the isolates demonstrated a time-dependent disappearance of 2,4-D from the 100 mg/kg herbicide-amended soil. The HPLC data analysis revealed 96.6 and 99.8% degradation in the soil inoculated with the pure cultures of isolates NJ 10 and NJ 15, respectively with in 20 days of incubation at 30 °C. Both the isolates showed significant solubilization of inorganic phosphate [Ca₃(PO₄)₂] on the specific Pikovskaya's medium.     

Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat.

To determine the effect of a secondary carbon source on biodegradation of a chloroaromatic compound, Pseudomonas cepacia DBO1(pRO101) was grown in continuous cultures on basal salts media containing various mixtures of 2,4-dichlorophenoxyacetic acid (2,4-D) and succinate. Both succinate and 2,4-D were metabolized over the entire range of dilution rates and compositions analyzed (0.05 to 0.6 h-1). 2,4-Dichlorophenol (DCP), the only intermediate detected, accumulated to significant amounts (10 to 21 mg/liter) in the chemostat only when the dilution rate was 0.4 h-1 or greater. At these concentrations, DCP reduced the apparent growth rate of P. cepacia DBO1(pRO101) in batch cultures by 15 to 35% over the apparent growth rate on succinate alone. Succinate fed to the chemostat increased the cell density as well as the percentage of 2,4-D that was consumed at each dilution rate. When the amount of succinate in the feed exceeded the amount of 2,4-D, the specific rates of 2,4-D degradation in the chemostat or by washed cells were significantly lower than the specific rates for cells grown on 2,4-D alone, suggesting repression by succinate. However, when the amount of 2,4-D in the feed exceeded the amount of succinate, the specific rates of 2,4-D degradation remained at values equivalent to or higher than the specific rate for cells grown on 2,4-D alone. DCP accumulated significantly in the washed-cell assay, suggesting that the level of DCP hydroxylase is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation,characterization, and metabolic activities of Bacillus brevis degrading isonicotinic acid

An organism isolated from soil by enrichment on isonicotinic acid (INA) was characterized as Bacillus brevis. It used sugars more readily than amino acids as growth substrates. The organism also used isoniazid, 2-hydorxypyridine, and benzoic and p-hydroxybenzoic acids. This bacterium did not metabolize 2-hydroxy-INA, citrazinic acid, or other mono- and dihydroxypyridine compounds as well as intermediates of the maleamate pathway. Accumulation of hydroxylated pyridine compounds was not detected during fermentation, or incubation of INA with resting cells in the presence or absence of inhibitors. Succinic semialdehyde was isolated and characterized as a key intermediate and was rapidly oxidized by INA-adapted cells. Formate was detected as a product of INA metabolism, and formate but not formamide was oxidized by INA-adapted cells; γ-aminobutyrate or γ-aminocrotonate were oxidized. A pathway for INA degradation involving oxygenative cleavage of a partially reduced pyridine ring is proposed.     

Identification and characterization of a novel d-amidase gene from Variovorax paradoxus and its expression in Escherichia coli

The gene for the newly described d-amidase from Variovorax paradoxus (Krieg et al. 2002) was cloned and functionally expressed in Escherichia coli. Since native enzyme was available in minute amounts only, we determined the N-terminal sequence of the enzyme and utilized the Universal GenomeWalker Approach to make use of the common internal sequence of the amidase signature family. The high GC content of the gene made it necessary to employ an appropriate DNA polymerase in the amplification reactions. Thus, the sequence of the complete gene and the flanking regions was established. In independent experiments, the gene was then amplified from genomic DNA of V. paradoxus, expressed in E. coli, and characterized. The recombinant enzyme has a specific activity of 1.7 units/mg with racemic tert-leucine amide as substrate and is a homodimer of 49.6-kDa monomers.     

Gene cloning of alpha-methylserine aldolase from Variovorax paradoxus and purification and characterization of the recombinant enzyme

The alpha-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from alpha-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The Vmax and Km of the enzyme for the formaldehyde release reaction from alpha-methyl-L-serine were 1.89 micromol min(-1) mg(-1) and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of alpha-methyl-L-serine and alpha-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5'-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.     

一株氯霉素降解细菌的分离鉴定与代谢特性研究

微生物是介导环境中氯霉素降解转化的主要驱动者,但高效降解矿化菌株资源匮乏,氧化反应介导的代谢途径不清。为研究微生物介导下氯霉素的环境归趋过程,为氯霉素污染环境强化修复提供菌株资源,文中以受氯霉素污染的活性污泥为接种源,首先富集获得一个由红球菌Rhodococcus主导 (相对丰度>70%) 的氯霉素高效降解菌群,并从中分离获得一株能够高效降解氯霉素的菌株CAP-2,通过16S rRNA基因分析鉴定为红球菌Rhodococcus sp.。菌株CAP-2能在不同营养条件下高效降解氯霉素。基于菌株CAP-2对检测到的代谢产物对硝基苯甲酸和已报道的代谢产物对硝基苯甲醛和原儿茶酸的生物转化特征,提出其降解途径是由氯霉素侧链氧化断裂生成对硝基苯甲醛,进一步氧化为对硝基苯甲酸的新型氧化降解途径。该菌株对于氯霉素分解代谢的分子机制研究以及受氯霉素污染环境的原位生物修复应用具有巨大潜力。     

Isolation and characterization of an anaerobic ruminal bacterium capable of degrading hydrolyzable tannins.

An anaerobic diplococcoid bacterium able to degrade hydrolyzable tannins was isolated from the ruminal fluid of a goat fed desmodium (Desmodium ovalifolium), a tropical legume which contains levels as high as 17% condensed tannins. This strain grew under anaerobic conditions in the presence of up to 30 g of tannic acid per liter and tolerated a range of phenolic monomers, including gallic, ferulic, and p-coumaric acids. The predominant fermentation product from tannic acid breakdown was pyrogallol, as detected by high-performance liquid chromatography and mass spectrometry. Tannic acid degradation was dependent on the presence of a sugar such as glucose, fructose, arabinose, sucrose, galactose, cellobiose, or soluble starch as an added carbon and energy source. The strain also demonstrated resistance to condensed tannins up to a level of 4 g/liter.     

Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2–3 μg ml⁻¹ for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 μg ml⁻¹. This revised version was published online in July 2006 with corrections to the Cover Date.