Short Note: Biodegradation of chlorobenzoates by Actinomycetes

Of the nine actinomycete strains screened for their ability to grow on isomeric chlorobenzoates (Cba), Corynebacterium liquefaciens, a sewage isolate, was able to maximally metabolize 3.2mM 2- and 3-Cba in presence of 0.25mM glucose as co-substrate. The degradation of 2-Cba and 3-Cba was 70.3% and 79.37% (w/v), respectively, under optimized conditions.     

Biodegradation of cis-1,4-polyisoprene rubbers by distinct actinomycetes: microbial strategies and detailed surface analysis

Several actinomycetes isolated from nature were able to use both natural rubber (NR) and synthetic cis-1,4-polyisoprene rubber (IR) as a sole source of carbon. According to their degradation behavior, they were divided into two groups. Representatives of the first group grew only in direct contact to the rubber substrate and led to considerable disintegration of the material during cultivation. The second group consisted of weaker rubber decomposers that did not grow adhesively, as indicated by the formation of clear zones (translucent halos) around bacterial colonies after cultivation on NR dispersed in mineral agar. Taxonomic analysis of four selected strains based on 16S rRNA similarity examinations revealed two Gordonia sp. strains, VH2 and Kb2, and one Mycobacterium fortuitum strain, NF4, belonging to the first group as well as one Micromonospora aurantiaca strain, W2b, belonging to the second group. Schiff's reagent staining tests performed for each of the strains indicated colonization of the rubber surface, formation of a bacterial biofilm, and occurrence of compounds containing aldehyde groups during cultivation with NR latex gloves. Detailed analysis by means of scanning electron microscopy yielded further evidence for the two different microbial strategies and clarified the colonization efficiency. Thereby, strains VH2, Kb2, and NF4 directly adhered to and merged into the rubber material, while strain W2b produced mycelial corridors, especially on the surface of IR. Fourier transform infrared spectroscopy comprising the attenuated total reflectance technique was applied on NR latex gloves overgrown by cells of the Gordonia strains, which were the strongest rubber decomposers. Spectra demonstrated the decrease in number of cis-1,4 double bonds, the formation of carbonyl groups, and the change of the overall chemical environment, indicating that an oxidative attack at the double bond is the first metabolic step of the biodegradation process.     

内生放线菌对鬼臼毒素的微生物转化

调查桃儿七根茎内生放线菌对鬼臼毒素的微生物转化,以期获得一些鬼臼毒素的结构类似物或衍生物。利用表面消毒法分离内生放线菌;采用薄层层析和高效液相色谱(HPLC)方法筛选转化鬼臼毒素的内生放线菌;利用硅胶柱层析和制备HPLC分离纯化生物转化产物;应用波谱技术解析转化产物的化学结构;通过形态学、生理生化特征和16S rRNA基因序列分析对内生放线菌进行初步鉴定。从桃儿七根茎中分离出20株内生放线菌,经筛选发现其中1株放线菌能转化鬼臼毒素,其产物为4’-去甲基表鬼臼毒素。初步鉴定该内生放线菌为Streptomyces sp.。内生放线菌Streptomyces sp.能对鬼臼毒素进行去甲基和异构化修饰,推测其可能具有O-去甲基化酶和异构化酶。     

Physiological and Chemical Investigations into Microbial Degradation of Synthetic Poly(cis-1,4-isoprene)

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6,10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of β-oxidation, (iii) oxidation of the conjugated double bond resulting in a β-keto acid, and (iv) decarboxylation.     

Enhanced Biodegradation of Methyl tert-butyl-ether by a Microbial Consortium

The widespread use of Methyl tert-butyl-ether (MTBE) as a gasoline additive has resulted in a higher detection rate of MTBE in groundwater systems. Therefore, the researchers show more concern about the bioremediation of MTBE-impacted aquifers. In this paper, a MTBE-direct-degrading bacterial consortium was enriched (named RS1) and further studied. In order to identify the microbial community of the consortium, 17 and 12 different single strains were isolated from nutrient medium and MSM media (with MTBE as the sole carbon source), respectively. 16S rDNA-based phylogenetic analysis revealed that these diverse bacteria belonged to 14 genera, in which Pseudomonas was dominant. Several strains which can grow with MTBE as the sole carbon and energy source were also identified, such as M1, related to MTBE-degrading Arthrobacter sp. ATCC27778. Furthermore, the appropriate addition of certain single strain in consortium RS1 (M1:RS1 = 1:2) facilitates MTBE degradation by increasing the quantity of efficient MTBE-degrading bacteria. This work will provide microbial source and theoretical fundament for further bioremediation of MTBE-contaminated aquifers, which has applied potential and environmental importance.     

Biodegradation of Metal-EDTA Complexes by an Enriched Microbial Population

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera Methylobacterium, Variovorax, Enterobacter, Aureobacterium, and Bacillus. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe>Cu>Co>Ni>Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources.     

Biodegradation Processes in a Laboratory-Scale Groundwater Contaminant Plume Assessed by Fluorescence Imaging and Microbial Analysis

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O₂(aq)] concentration field quantified the depletion of O₂(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O₂(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.     

Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.     

五氯苯酚厌氧生物降解及降解体系中细菌种群结构分析

研究了不同外加碳源作为共代谢基质及氢气作为电子供体条件下PCP的厌氧生物降解特性,并借助末端限制性片段长度多态性技术(T-RFLP)分析了PCP降解菌群的微生物群落结构.结果表明,添加外加碳源及以氢气作为电子供体均对PCP降解有显著促进作用.添加葡萄糖、丙酮酸、酵母膏和氢气时的降解率分别为71%、56%、51%和74%.微生物群落结构分析表明,不同处理条件下PCP降解菌群微生物群落结构不同.PCP降解菌群中可能存在Clostridium.Frankia和Desulfitobacterium等属的微生物.     

Anoxic Biodegradation of Isosaccharinic Acids at Alkaline pH by Natural Microbial Communities

One design concept for the long-term management of the UK’s intermediate level radioactive wastes (ILW) is disposal to a cementitious geological disposal facility (GDF). Under the alkaline (10.0<pH>13.0) anoxic conditions expected within a GDF, cellulosic wastes will undergo chemical hydrolysis. The resulting cellulose degradation products (CDP) are dominated by α- and β-isosaccharinic acids (ISA), which present an organic carbon source that may enable subsequent microbial colonisation of a GDF. Microcosms established from neutral, near-surface sediments demonstrated complete ISA degradation under methanogenic conditions up to pH 10.0. Degradation decreased as pH increased, with β-ISA fermentation more heavily influenced than α-ISA. This reduction in degradation rate was accompanied by a shift in microbial population away from organisms related to Clostridium sporosphaeroides to a more diverse Clostridial community. The increase in pH to 10.0 saw an increase in detection of Alcaligenes aquatilis and a dominance of hydrogenotrophic methanogens within the Archaeal population. Methane was generated up to pH 10.0 with acetate accumulation at higher pH values reflecting a reduced detection of acetoclastic methanogens. An increase in pH to 11.0 resulted in the accumulation of ISA, the absence of methanogenesis and the loss of biomass from the system. This study is the first to demonstrate methanogenesis from ISA by near surface microbial communities not previously exposed to these compounds up to and including pH 10.0.     

Biodegradation kinetics of 1,4-benzoquinone in batch and continuous systems

Combining chemical and biological treatments is a potentially economic approach to remove high concentration of recalcitrant compounds from wastewaters. In the present study, the biodegradation of 1,4-benzoquinone, an intermediate compound formed during phenol oxidation by chlorine dioxide, was investigated using Pseudomonas putida (ATCC 17484) in batch and continuous bioreactors. Batch experiments were conducted to determine the effects of 1,4-benzoquinone concentration and temperature on the microbial activity and biodegradation kinetics. Using the generated data, the maximum specific growth rate and biodegradation rate were determined as 0.94 h⁻¹ and 6.71 mg of 1,4-benzoquinone l⁻¹ h⁻¹. Biodegradation in a continuous bioreactor indicated a linear relationship between substrate loading and biodegradation rates prior to wash out of the cells, with a maximum biodegradation rate of 246 mg l⁻¹ h⁻¹ observed at a loading rate of 275 mg l⁻¹ h⁻¹ (residence time: 1.82 h). Biokinetic parameters were also determined using the steady state substrate and biomass concentrations at various dilution rates and compared to those obtained in batch cultures.     

Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.     

Effect of Concentration of Organic Chemicals on Their Biodegradation by Natural Microbial Communities

The effect of concentration on the biodegradation of synthetic organic chemicals by natural microbial communities was investigated by adding individual ¹⁴C-labeled organic compounds to stream water at various initial concentrations and measuring the formation of ¹⁴CO₂. The rate of degradation of p-chlorobenzoate and chloroacetate at initial concentrations of 47 pg/ml to 47 μg/ml fell markedly with lower initial concentrations, although half or more of the compound was converted to CO₂ in 8 days or less. On the other hand, little mineralization of 2,4-dichlorophenoxyacetate and 1-naphthyl-N-methylcarbamate, or the naphthol formed from the latter, occurred when these compounds were present at initial concentrations of 2 to 3 ng/ml or less, although 60% or more of the chemical initially present at higher concentrations was converted to CO₂ in 6 days. It is concluded that laboratory tests of biodegradation involving chemical concentrations greater than those in nature may not correctly assess the rate of biodegradation in natural ecosystems and that low substrate concentration may be important in limiting biodegradation in natural waters.     

Reaction of 2'-deoxyribonucleosides with cis- and trans-1,4-dioxo-2-butene

cis-1,4-Dioxo-2-butene is a toxic metabolite of furan, while the trans-isomer is a product of deoxyribose oxidation in DNA. It has recently been reported that both cis- and trans-1,4-dioxo-2-butene react with the 2'-deoxynucleosides dC, dG, and dA to form novel diastereomeric oxadiazabicyclo(3.3.0)octaimine adducts. We have now extended these studies with kinetic and spectroscopic analyses of the reactions of cis- and trans-1,4-dioxo-2-butene, as well as the identification of novel adducts of dA. The reaction of dC with trans-1,4-dioxo-2-butene was observed to be nearly quantitative and produced two interchanging diastereomers with a second-order rate constant of 3.66 x 10(-2)M(-1)s(-1), which is nearly 10-fold faster than the reaction with the cis-isomer (3.74 x 10(-3)M(-1)s(-1)). HPLC analyses of reactions of 1,4-dioxo-2-butene with both dA and 9-methyladenine (pH 7.4, 37 degrees C) revealed multiple products including a novel pair of closely eluting fluorescent species of identical mass ([M+H] m/z 420), each of which contains two molecules of 1,4-dioxo-2-butene, and a more abundant but unstable and non-fluorescent species ([M+H] m/z 414). Partial structural characterization of the fluorescent adducts of dA revealed the presence of the oxadiazabicyclo(3.3.0)octaimine ring system common to the dC adducts. These results support the genotoxic potential of furan metabolites and products of deoxyribose oxidation.     

Distinct Microbial Behavior of Re Compared to Tc: Evidence Against Microbial Re Fixation in Aquatic Sediments

Rhenium is enriched in suboxic and anoxic sediments relative to oxic sediments, a characteristic that is being exploited in its use as a paleoredox indicator. Rhenium is fixed at sediment depths where iron reduction and sulfate reduction are the dominant microbial terminal electron-accepting processes. In order to explore mechanisms of its fixation, we investigated perrhenate behavior in pure, batch cultures of two dissimilatory sulfate-reducing strains (Desulfovibrio desulfuricans subsp. desulfuricans and Desulfovibrio desulfuricans ND132) and two iron-reducing strains (Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1). Perrhenate concentrations tested ranged from 0.04 to 12 μM, roughly 4 to 7 orders of magnitude larger than seawater Re concentrations. Within this broad concentration range, none of the organisms tested actively removed Re from solution during one week's growth to stationary phase. Despite these results, the sulfate-reducing cultures appeared to have reached supersaturation relative to ReS_2(s), and the iron-reducing cultures may have reached supersaturation relative to ReO_2(s). We conclude that neither direct nor short-term indirect microbial processes involving these bacteria are likely to explain Re fixation in sediments. Our results cannot exclude the possibility that microbial metabolites, such as Fe(II) or sulfide, do drive abiotic Re fixation over longer periods of time. The lack of perrhenate reduction by dissimilatory sulfate-reducing bacteria and iron-reducing bacteria contrasts with published reports of pertechnetate behavior. Despite many qualitative similarities between Re and Tc, it is clear that these two elements are quantitatively dissimilar, with Re fixation requiring more intensely reducing conditions.     

Identification of Poly(cis-1,4-Isoprene) Degradation Intermediates during Growth of Moderately Thermophilic Actinomycetes on Rubber and Cloning of a Functional lcp Homologue from Nocardia farcinica Strain E1

The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50°C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.     

Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase

The unusual enzyme, Gracilariopsis alpha-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of alpha-1,4-glucans via a beta-elimination reaction involving a covalent glycosyl-enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124, 4948-4949). The classical bell-shaped pH dependence of k(cat)/K(m) indicates two ionizable groups in the active site with apparent pK(a) values of 3.05 and 6.66. Br?nsted relationships of log k(cat) versus pK(a) and log(k(cat)/K(m)) versus pK(a) for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK(a) with low beta(lg) values of 0.32 and 0.33, respectively. The combination of these low beta(lg) values with large secondary deuterium kinetic isotope effects (k(H)/k(D) = 1.16 - 1.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (K(i) = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-alpha-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large alpha-secondary deuterium kinetic isotope effect (k(H)/k(D) = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k(H)/k(D) = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining alpha-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.