Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

Differential degradation of bicyclics with aromatic and alicyclic rings by Rhodococcus sp. strain DK17

The metabolically versatile Rhodococcus sp. strain DK17 is able to grow on tetralin and indan but cannot use their respective desaturated counterparts, 1,2-dihydronaphthalene and indene, as sole carbon and energy sources. Metabolite analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry clearly show that (i) the meta-cleavage dioxygenase mutant strain DK180 accumulates 5,6,7,8-tetrahydro-1,2-naphthalene diol, 1,2-indene diol, and 3,4-dihydro-naphthalene-1,2-diol from tetralin, indene, and 1,2-dihydronaphthalene, respectively, and (ii) when expressed in Escherichia coli, the DK17 o-xylene dioxygenase transforms tetralin, indene, and 1,2-dihydronaphthalene into tetralin cis-dihydrodiol, indan-1,2-diol, and cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, respectively. Tetralin, which is activated by aromatic hydroxylation, is degraded successfully via the ring cleavage pathway to support growth of DK17. Indene and 1,2-dihydronaphthalene do not serve as growth substrates because DK17 hydroxylates them on the alicyclic ring and further metabolism results in a dead-end metabolite. This study reveals that aromatic hydroxylation is a prerequisite for proper degradation of bicyclics with aromatic and alicyclic rings by DK17 and confirms the unique ability of the DK17 o-xylene dioxygenase to perform distinct regioselective hydroxylations.     

Draft Genome Sequence and Comparative Analysis of the Superb Aromatic-Hydrocarbon Degrader Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 is capable of utilizing various derivatives of benzene and bicyclics containing both aromatic and alicyclic moieties as sole carbon and energy sources. Here, we present the 9,107,362-bp draft genome sequence of DK17 and its genomic analysis in comparison with other members of the genus Rhodococcus.     

Monocyclic Aromatic Hydrocarbon Degradation by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

Requirement of duplicated operons for maximal metabolism of phthalate by Rhodococcus sp. strain DK17

The operons encoding the transformation of phthalate to protocatechuate are duplicated and present on two different megaplasmids [pDK2 (330 kb) and pDK3 (750 kb)] in Rhodococcus sp. strain DK17. RT-PCR experiments using gene-specific primers showed that both the pDK2- and the pDK3-encoded dihydroxyphthalate decarboxylase genes are simultaneously expressed during growth on phthalate. The doubling time of the pDK2-cured mutant strain DK176 in minimal liquid medium with 5mM phthalate is 52.5% of that of the wild-type strain DK17. The data indicate that both copies of the phthalate operon are equally functional in DK17, and gene dosage is the main reason for slower growth of DK176 on phthalate.     

Regioselective oxidation of xylene isomers by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is able to utilize a variety of monocyclic aromatic hydrocarbons, including benzene, phenol, toluene, and o-xylene, as growth substrates. Although DK17 is unable to grow on m- and p-xylene, this strain could transform these two xylene isomers to some extent after induction by o-xylene. The major accumulating compounds formed during the degradation of m- and p-xylene by DK17 were isolated by high-pressure liquid chromatography and identified by gas chromatography-mass spectrometric and (1)H nuclear magnetic resonance spectral techniques. Both xylene isomers were transformed to dihydroxylated compounds by what must be two successive hydroxylation events: m-xylene was converted to 2,4-dimethylresorcinol and p-xylene was converted to 2,5-dimethylhydroquinone. The rigorous structural identification of 2,4-dimethylresorcinol and 2,5-dimethylhydroquinone demonstrates that DK17 can perform distinct regioselective hydroxylations depending on the position of the substituent groups on the aromatic ring.     

A new variant of the Es-4 locus in the rat

A new variant of kidney esterase in the DK/Nac rat strain is reported. The new esterase was tentatively named ES-4C determined by a third allele of the Es-4 locus of Linkage Group V (LGV). Strain distribution was surveyed using 17 inbred strains, but no strain except for the DK/Nac strain possessed the ES-4C type. Although we surveyed outbred stocks (Jcl: Wistar and Jcl: SD) we could not find rats carrying the ES-4C type. Genetic analysis of the ES-4C type was carried out using mating experiments between DK/Nac and BUF/Nac (ES-4B). The results indicated that the new variant was controlled by the Es-4 locus and it was named the Es-4c allele.     

Isolation and characterization of a rhodococcus species strain able to grow on ortho- and para-xylene

Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1.     

Complete Genome Sequences of a Novel Reassortant H4N2 Avian Influenza Virus Isolated from a Live Poultry Market in Eastern China

A/duck/Shanghai/28-1/2009(H4N2) (DK28) was isolated from a live poultry market in Shanghai, China. Using PCR and sequencing analysis, we obtained the complete genome sequences of the DK28 virus. The sequence analysis demonstrated that this H4N2 virus was a novel multiple-gene reassortant avian influenza virus (AIV) whose genes originated from H1N1, H1N3, H3N3, H4N2, and H4N6. Knowledge regarding the complete genome sequences of the DK28 virus will be useful for epidemiological surveillance.     

Lactobacillus plantarum DK119 as a Probiotic Confers Protection against Influenza Virus by Modulating Innate Immunity

Lactobacillus plantarum DK119 (DK119) isolated from the fermented Korean cabbage food was used as a probiotic to determine its antiviral effects on influenza virus. DK119 intranasal or oral administration conferred 100% protection against subsequent lethal infection with influenza A viruses, prevented significant weight loss, and lowered lung viral loads in a mouse model. The antiviral protective efficacy was observed in a dose and route dependent manner of DK119 administration. Mice that were treated with DK119 showed high levels of cytokines IL-12 and IFN-γ in bronchoalveolar lavage fluids, and a low degree of inflammation upon infection with influenza virus. Depletion of alveolar macrophage cells in lungs and bronchoalveolar lavages completely abrogated the DK119-mediated protection. Modulating host innate immunity of dendritic and macrophage cells, and cytokine production pattern appeared to be possible mechanisms by which DK119 exhibited antiviral effects on influenza virus infection. These results indicate that DK119 can be developed as a beneficial antiviral probiotic microorganism.     

犬2型腺病毒E1转化细胞系的特性研究

对筛选到的一株CAV—2 E1转化细胞(DK／E1)进行了初步的特性研究。以DK细胞为对照,观察到转化细胞的形态与DK细胞有明显区别,细胞变长,易聚集生长。通过测定DK及DK／E1细胞分别在2％、5％和10％牛血清培养基中的生长曲线,结果发现DK／E1细胞的生长速度比DK细胞快。用蚀斑和TCID50测定了病毒在DK和DK／E1细胞上的病毒滴度,结果表明DK／E1细胞产生的病毒蚀斑比DK细胞的大,TCID50测得的病毒滴度比DK细胞产生的病毒滴度高。CAV—2全基因组DNA对DK／E1细胞和DK细胞的转染试验结果表明,5μg和10μg的CAV—2 DNA转染的DK／E1细胞,分别在转染后第14天和第11天均出现了典型的腺病毒细胞病变,而相同量CAV—2 DNA转染的DK细胞未出现病变,说明DK／E1细胞有助于CAV—2 DNA的复制和病毒粒子的包装。在DK／E1细胞内的重组试验表明,DK／E1细胞也有利于重组病毒的产生。以上转化特性在DK／E1细胞传至80代时仍保持不变,说明本试验获得了一株CALV—2 E1基因的转化细胞系。     

Atypical Aeromonas salmonicida Isolated from Diseased Turbot (Scophtalmus maximus L.)

Atypical Aeromonas salmonicida were isolated from 3 outbreaks of disease among farmed turbot (Scophthalmus maximus L.) in 3 different farms, 1 from Norway (Nl) and 2 from Denmark (DK1 and DK2). In all 3 cases, the incidence of disease and mortality was high and the main characteristic pathological finding was skin ulcers and septicaemia. The isolated bacteria were subjected to a thorough phenotypic and genotypic examination and comparison in the laboratory. All 3 isolates belonged to A. salmonicida but dis-played some very different biochemical properties. However, the 2 Danish strains, DK1 and DK2 had identical ribotypes but different from that of Nl, whereas the plasmid pro-files of DK1 and Nl were identical but different from that of DK2. These observations emphasize the need for an improvement of our understanding of the taxonomy and epi-demiology of atypical A. salmonicida.     

Drought-induced site-specific DNA methylation and its association with drought tolerance in rice (Oryza sativa L.)

An indica pyramiding line, DK151, and its recurrent parent, IR64, were evaluated under drought stress and non-stress conditions for three consecutive seasons. DK151 showed significantly improved tolerance to drought. The DNA methylation changes in DK151 and IR64 under drought stress and subsequent recovery were assessed using methylation-sensitive amplified polymorphism analysis. Our results indicate that drought-induced genome-wide DNA methylation changes accounted for ～12.1% of the total site-specific methylation differences in the rice genome. This drought-induced DNA methylation pattern showed three interesting properties. The most important one was its genotypic specificity reflected by large differences in the detected DNA methylation/demethylation sites between DK151 and IR64, which result from introgressed genomic fragments in DK151. Second, most drought-induced methylation/demethylation sites were of two major types distinguished by their reversibility, including 70% of the sites at which drought-induced epigenetic changes were reversed to their original status after recovery, and 29% of sites at which the drought-induced DNA demethylation/methylation changes remain even after recovery. Third, the drought-induced DNA methylation alteration showed a significant level of developmental and tissue specificity. Together, these properties are expected to have contributed greatly to rice response and adaptation to drought stress. Thus, induced epigenetic changes in rice genome can be considered as a very important regulatory mechanism for rice plants to adapt to drought and possibly other environmental stresses.     

Preferential Inhibition of I h in Rat Trigeminal Ganglion Neurons by an Organic Blocker

The potency and specificity of a novel organic I _h current blocker DK-AH 268 (DK, Boehringer) was studied in cultured rat trigeminal ganglion neurons using whole-cell patch-clamp recording techniques. In neurons current-clamped at the resting potential, the application of 10 μm DK caused a slight hyperpolarization of the membrane potential and a small increase in the threshold for action potential discharge without any major change in the shape of the action potential. In voltage-clamped neurons, DK caused a reduction of a hyperpolarization-activated current. Current subtraction protocols revealed that the time-dependent, hyperpolarization-activated currents blocked by 10 μm DK or external Cs⁺ (3 mm) had virtually identical activation properties, suggesting that DK and Cs⁺ caused blockade of the same current, namely I _h . The block of I _h by DK was dose-dependent. At the intermediate and higher concentrations of DK (10 and 100 μm) a decrease in specificity was observed so that time-independent, inwardly rectifying and noninactivating, voltage-gated outward potassium currents were also reduced by DK but to a much lesser extent than the time-dependent, hyperpolarization-activated currents. Blockade of the time-dependent, hyperpolarization-activated currents by DK appeared to be use-dependent since it required hyperpolarization for the effect to take place. Relief of DK block was also aided by membrane hyperpolarization. Since both the time-dependent current blocked by DK and the Cs⁺-sensitive time-dependent current behaved as I _h , we conclude that 10 μm DK can preferentially reduce I _h without a major effect on other potassium currents. Thus, DK may be a useful agent in the investigation of the function of I _h in neurons. Received: 3 March 1995/Revised: 8 July 1997     

Hymenobacter qilianensis sp. nov., isolated from a subsurface sandstone sediment in the permafrost region of Qilian Mountains,China and emended description of the genus Hymenobacter

A red–pink, Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain DK6-37 was isolated from the permafrost region of Qilian Mountains in northwest of China. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that this isolate represents a novel member of the genus Hymenobacter, with low sequence similarities (<97 %) to recognized Hymenobacter species. Optimum growth was observed at 28 °C, pH 7.0 and 0 % NaCl. The strain was found to contain MK-7 as the predominant menaquinone. The polar lipids were identified as phosphatidylethanolanmine, two unknown aminophospholipids, one unknown aminolipid and three unknown polar lipids. The major fatty acids were identified as summed feature 3 (C_16:1 ω7c/C_16:1 ω6c as defined by MIDI), summed feature 4 (anteiso-C_17:1 B/iso-C_17:1 I), C_16:1 ω5c, iso-C_17:0 3-OH, iso-C_15:0 and C_18:0. The DNA G + C content was determined to be 67.4 mol %. On the basis of the polyphasic evidence presented, it is proposed that strain DK6-37 represents a novel species of the genus Hymenobacter, for which the name Hymenobacter qilianensis sp. nov. is proposed. The type strain is DK6-37^T (= CGMCC 1.12720^T = JCM 19763^T).     

黄色黏细菌转录因子FruA对自身基因的表达不具自调节作用

 粘细菌是研究多细胞结构形态发生机制的良好模型.FruA是粘细菌发育所必需的一种 关键性转录因子, 调节一系列发育相关基因的表达,本文研究FruA对自身基因是否存在反馈调节从而导致发育后期fruA表达水平的下调.以野生型粘细菌模式菌株DK1622为基础构建fruA基因敲除突变株DK1622ΔfruA,再将fruA-lacZ转录融合载体pMF1A整合入fruA突变株染色体attB, 获得重组菌株DK1622ΔfruA/pMF1A,通过检测β-半乳糖苷酶活性来确认FruA对自身基因的表达水平是否有影响. 结果表明fruA调控序列完整的fruA-lacZ转录融合体β-半乳糖苷酶活性在DK1622/pMF1A和DK1622ΔfruA/pMF1A之间无明显差异, 即fruA表达产物作为一种转录因子对自身基因的转录没有调节作用,黏细菌发育后期fruA表达水平的下降存在其它调节机制.     

Functional characterization and molecular modeling of methylcatechol 2,3-dioxygenase from o-xylene-degrading Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is known to metabolize o-xylene and toluene through the intermediates 3,4-dimethylcatechol and 3- and 4-methylcatechol, respectively, which are further cleaved by a common catechol 2,3-dioxygenase. A putative gene encoding this enzyme (akbC) was amplified by PCR, cloned, and expressed in Escherichia coli. Assessment of the enzyme activity expressed in E. coli combined with sequence analysis of a mutant gene demonstrated that the akbC gene encodes the bona fide catechol 2,3-dioxygenase (AkbC) for metabolism of o-xylene and alkylbenzenes such as toluene and ethylbenzene. Analysis of the deduced amino acid sequence indicates that AkbC consists of a new catechol 2,3-dioxygenase class specific for methyl-substituted catechols. A computer-aided molecular modeling studies suggest that amino acid residues (particularly Phe177) in the beta10-beta11 loop play an essential role in characterizing the substrate specificity of AkbC.     

Effect of dikegulac on growth and alkaloid production inCatharanthus roseus (L.) G. Don. (pink flowered)

Application of sodium-dikegulac reduced plant height with associated increase in branch and leaf number and root biomass inC. roseus (L.) G. DON. Chlorophyll content reduced significantly after first month of 100 and 250 μg/ml DK application. However, such reduction was replaced by significant rise after forth month in 250 μg/ml DK application and fifth month in 100 μg/ml DK application followed by appreciable decline only in 250 μg/ml DK treatment but 100 μg/ml DK maintained higher level till harvest. Total sugar content was significantly high during forth and fifth month stage of growth after DK application. Amino acid content was higher during third to fifth month in 100 μg/ml DK treatment and during third to forth month in 250 μg/ml DK treatment. Tryptophan, on the other hand showed higher content at the fifth month stage of growth after application of DK in both the concentrations. Leaf and root dry weight as well as total alkaloid content were highest in 100 μg/ml DK application. DK, therefore, appears to be a potential chemical for increasing biomass and alkaloid content inC. roseus.