Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

ABSTRACT

Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is a filamentous cyanobacterium, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen fixation. A widely used type strain [wild type (WT) in this article] of this species has not been reported to show any motile activity. However, we isolated a spontaneous mutant strain that shows active motility (gliding activity) to give rise to complicated colony patterns, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations in the genome of the mutant strain. We confirmed that inactivation of the candidate gene dgc2 ( LBDG_02920 ) in the WT background was sufficient to give rise to motility and morphologically complex colony patterns. This gene encodes a protein containing the GGDEF motif which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the dgc2 mutant significantly facilitated biofilm formation, suggesting a role for the dgc2 gene in suppressing both gliding motility and biofilm formation. Thus, Leptolyngbya is expected to be an excellent genetic model for studying dynamic colony pattern formation and to provide novel insights into the role of DGC family genes in biofilm formation.

IMPORTANCE

Self-propelled bacteria often exhibit complex collective behaviors, such as formation of dense-moving clusters, which are exemplified by wandering comet-like and rotating disk-like colonies; however, the molecular details of how these structures are formed are scant. We found that a strain of the filamentous cyanobacterium Leptolyngbya deficient in the GGDEF protein gene dgc2 elicits motility and complex and dynamic colony pattern formation, including comet-like and disk-like clusters. Although c-di-GMP has been reported to activate biofilm formation in some bacterial species, disruption of dgc2 unexpectedly enhanced it, suggesting a novel role for this GGDEF protein for inhibiting both colony pattern formation and biofilm formation.

DOI： 10.1128/spectrum.04837-22

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

A bacterial chromosome that was naturally fused with the secondary chromosome, or “chromid,” and presented as an unexpectedly large single replicon was discovered in the genome of
<named-content content-type="genus-species">Cupriavidus necator</named-content>
strain KK10, a biotechnologically useful member of the family
<italic>Burkholderiaceae</italic>
. Although
<italic>Burkholderiaceae</italic>
is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family.

DOI： 10.1128/spectrum.02225-21

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

The genus Thalassospira has often been studied as a potential major contributing member of aromatic hydrocarbon-exposed microbial communities. Here, the complete genome sequence of a new isolate of Thalassospira , strain GO-4, was obtained and was confirmed to possess functional genes that are responsible for its metabolism of phthalic acid.

DOI： 10.1128/mra.00532-22

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.mimet.2022.106468

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Comprehensive investigation of multiple genomic data sets from targeted microbial taxa deposited in databases may provide substantial information to predict metabolic capabilities and ecological roles in different environments. This study is the first report that details the functional profiling of Thalassospira spp. that have repeatedly been found in polycyclic aromatic hydrocarbon (PAH)-exposed marine bacterial communities by using genomic data from a new isolate, Thalassospira strain GO-4, and other strains in databases.

DOI： 10.1128/spectrum.03149-22

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ibiod.2022.105500

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

In many bacteria, cyclic diguanosine monophosphate (c-di-GMP), synthesized by diguanylate cyclase (DGC), serves as a second messenger involved in the regulation of biofilm formation. Although studies have suggested that c-di-GMP also regulates the formation of electrochemically active biofilms (EABFs) by <italic>Shewanella oneidensis</italic> MR-1, DGCs involved in this process remained to be identified. Here we report that the SO_1646 gene, hereafter named <italic>dgcS</italic>, is upregulated under medium-flow conditions in electrochemical flow cells (EFCs), and its product (DgcS) functions as a major DGC in MR-1. <italic>In vitro</italic> assays demonstrated that purified DgcS catalyzed the synthesis of c-di-GMP from GTP. Comparisons of intracellular c-di-GMP levels in the wild-type strain and a <italic>dgcS</italic>-deletion mutant (Δ<italic>dgcS</italic>) showed that production of c-di-GMP was markedly reduced in Δ<italic>dgcS</italic> when cells were grown in batch cultures and on electrodes in EFCs. Cultivation of Δ<italic>dgcS</italic> in EFCs also revealed that the loss of DgcS resulted in impaired biofilm formation and decreased current generation. These findings demonstrate that MR-1 uses DgcS to synthesize c-di-GMP under medium-flow conditions, thereby activating biofilm formation on electrodes.


<bold>IMPORTANCE</bold>


Bioelectrochemical systems (BESs) have attracted wide attention owing to their utility in sustainable biotechnology processes, such as microbial fuel cells and electro-fermentation systems. In BESs, electrochemically active bacteria (EAB) form biofilms on electrode surfaces, thereby serving as effective catalysts for the interconversion between chemical and electric energy. It is therefore important to understand mechanisms for the formation of biofilm by EAB grown on electrodes. Here we show that a model EAB, <italic>S. oneidensis</italic> MR-1, expresses DgcS as a major DGC, thereby activating the formation of biofilms on electrodes via c-di-GMP-dependent signal transduction cascades. The findings presented herein provide the molecular basis for improving electrochemical interactions between EAB and electrodes in BESs. The results also offer molecular insights into how <italic>Shewanella</italic> regulates biofilm formation on solid surfaces in the natural environment.

DOI： 10.1128/aem.00201-21

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.hazadv.2021.100018

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.petrol.2021.108870

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ibiod.2020.104993

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：English   Publishing type：Part of collection (book)   Publisher：CRC Press  

DOI： 10.1201/9781315372303

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

To detect metalloproteinase-7 (MMP7), zymography is conducted using a casein substrate and conventional CBB stain. It has disadvantages because it is time consuming and has low sensitivity. Previously, a sensitive method to detect MMP7 up to 30 pg was reported, however it required special substrates and complicated handlings. RAMA casein zymography described herein is rapid, sensitive, and reproducible. By applying high-sensitivity staining with low substrate conditions, the staining process is completed within 1 h and sensitivity was increased 100-fold. The method can detect 10 pg MMP7 by using commercially available casein without complicated handlings. Moreover, it increases detection sensitivity for trypsin.

DOI： 10.1002/elps.201600346

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Language：English   Publishing type：Part of collection (book)   Publisher：CRC Press  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The cyanobacterial circadian-related protein, Pex, accumulates in the dark period of the diurnal light-dark cycle. After the diurnal cycle, an approximately 3 h advance in the phase of the circadian bioluminescence rhythm is observed in pex-deficient mutants, as compared with the wild type. However, it is unclear what type of photosensing mechanism regulates the accumulation and the phase change. In monochromatic light irradiation experiments, Pex accumulation was strongly repressed under blue light conditions; however, only small reductions in Pex accumulation were observed under red or green light conditions. After the diurnal cycle of 12 h of white fluorescent light and 12 h of blue light, the phase advance was repressed more than that of the cycle of 12 h red (or green) light. The phase advance also occurred after 16 h light/8 h dark cycles (long-day cycles) but did not occur after 8 h light/16 h dark cycles (short-day cycles). While Pex is a unique winged helix transcription factor harboring secondary structures (alpha 0 and alpha 4 helices), the importance of the structures is not understood. In in vivo experiments with site-directed mutations in the alpha 0 helix, the obtained mutants, in which Pex was missing the hydrophobic side chain at the 28th or 32nd amino acid residue, exhibited no phase delay after the light/dark cycle. In in vitro DNA binding assays, the mutant proteins showed no binding to the promoter region of the clock gene kaiA. From these results, we propose a molecular model which describes the phase delay in cyanobacteria.

DOI： 10.1093/pcp/pcv177

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Toward the development of ecotoxicology methods to investigate microbial markers of impacts of hydrocarbon processing activities, DNA adductomic analyses were conducted on a sphingomonad soil bacterium. From growing cells that were exposed or unexposed to acrolein, a commonly used biocide in hydraulic fracturing processes, DNA was extracted, digested to 2'-deoxynucleosides and analyzed by liquid chromatography-positive ionization electrospray-tandem mass spectrometry in selected reaction monitoring mode transmitting the [M + H](+) &gt; [M + H - 116](+) transition over 100 transitions. Overall data shown as DNA adductome maps revealed numerous putative DNA adducts under both conditions with some occurring specifically for each condition. Adductomic analyses of triplicate samples indicated that elevated levels of some targeted putative adducts occurred in exposed cells. Two exposure-specific adducts were identified in exposed cells as 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxy- (and 8-hydroxy-)pyrimido[1,2-a]-purine-(3H)-one (6- and 8-hydroxy-PdG) following synthesis of authentic standards of these compounds and subsequent analyses. A time course experiment showed that 6- and 8-hydroxy-PdG were detected in bacterial DNA within 30 min of acrolein exposure but were not detected in unexposed cells. This work demonstrated the first application of DNA adductomics to examine DNA damage in a bacterium and sets a foundation for future work.

DOI： 10.1002/mbo3.283

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

A Gram-stain-negative, yellow, rod-shaped bacterium, designated strain KK22(T), was isolated from a microbial consortium that grew on diesel fuel originally recovered from cattle pasture soil. Strain KK22(T) has been studied for its ability to biotransform high molecular weight polycyclic aromatic hydrocarbons. On the basis of 16S rRNA gene sequence phylogeny, strain KK22(T) was affiliated with the genus Sphingobium in the phylum Proteobacteria and was most closely related to Sphingobium fuliginis TKPT (99.8 %) and less closely related to Sphingobium quisquiliarum P25(T) (97.5 %). Results of DNA DNA hybridization (DDH) revealed relatedness values between strain KK22(T) and strain TKPT and between strain KK22(T) and strain P25(T) of 21 +/- 4 % (reciprocal hybridization, 27 +/- 2 %) and 15 +/- 2 % (reciprocal hybridization, 17 +/- 1 %), respectively. Chemotaxonomic analyses of strain KK22(T) showed that the major respiratory quinone was ubiquinone Q-10, that the polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidyl-N-methylethylethanolamine and sphingoglycolipid, and that C-18 : 1 omega 7c and C-14 :0 2-OH were the main fatty acid and hydroxylated fatty acids, respectively. This strain was unable to reduce nitrate and the genomic DNA G+C content was 64.7 mol%. Based upon the results of the DDH analyses, the fact that strain KK22(T) was motile, and its biochemical and physiological characteristics, strain KK22(T) could be separated from recognized species of the genus Sphingobium. We conclude that strain KK22(T) represents a novel species of this genus for which the name Sphingobium barthaii sp. nov. is proposed; the type strain is KK22(T) (=DSM 29313(T)=JCM 30309(T)).

DOI： 10.1099/ijs.0.000356

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

3-Methylindole, also referred to as skatole, is a pollutant of environmental concern due to its persistence, mobility and potential health impacts. Petroleum refining, intensive livestock production and application of biosolids to agricultural lands result in releases of 3-methylindole to the environment. Even so, little is known about the aerobic biodegradation of 3-methylindole and comprehensive biotransformation pathways have not been established. Using glycerol as feedstock, the soil bacterium Cupriavidus sp. strain KK10 biodegraded 100 mg/L of 3-methylindole in 24 h. Cometabolic 3-methylindole biodegradation was confirmed by the identification of biotransformation products through liquid chromatography electrospray ionization tandem mass spectrometry analyses. In all, 14 3-methylindole biotransformation products were identified which revealed that biotransformation occurred through different pathways that included carbocyclic aromatic ring-fission of 3-methylindole to single-ring pyrrole carboxylic acids. This work provides first comprehensive evidence for the aerobic biotransformation mechanisms of 3-methylindole by a soil bacterium and expands our understanding of the biodegradative capabilities of members of the genus Cupriavidus towards heteroaromatic pollutants.

DOI： 10.1007/s10532-015-9739-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Comprehensive analyses of the biotransformation of the N-heteroaromatic environmental pollutant, indole, by a newly isolated soil bacterium designated strain KK10, were conducted by liquid chromatography negative electrospray ionization tandem mass spectrometry (LC/ESI(-)-MS/MS). Numerous indole bioproducts were revealed and provided evidence for multiple pathways of indole biotransformation by this strain including unreported pathways. Oxidation of the N-heterocyclic ring of indole and ring cleavage through N-formylanthranilic acid and gentisic acid was characterized in addition to a carbocyclic ring-cleavage pathway which was documented for the first time. Three carbocyclic aromatic ring cleavage products of indole were proposed and the structure of 2,3-pyrrole-dicarboxylic acid was confirmed following synthesis and analyses of an authentic standard of this compound. Eight downstream ring-fission products were identified overall and these results confirmed the lower pathways of indole biotransformation. Indigo, indirubin and other indigoid compounds were produced by strain KK10 and confirmed N-heterocyclic-ring oxidations in the upper pathways. Nearly complete sequencing of the 16S rRNA gene of strain KK10 and phylogenetic analysis revealed that it was a new member of the genus Cupriavidus. Cupriavidus strains that biotransform N-heteroaromatic pollutants have not been described. These results expand both our understanding of the versatile catabolic capabilities of members of the genus Cupriavidus and provide evidence for alternative biotransformation mechanisms for indole in the environment by bacteria. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ibiod.2014.11.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

A pathway for the biotransformation of the environmental pollutant and high-molecular weight polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene by a soil bacterium was constructed through analyses of results from liquid chromatography negative electrospray ionization tandem mass spectrometry (LC/ESI(-)-MS/MS). Exposure of Sphingobium sp. strain KK22 to benzo[k]fluoranthene resulted in transformation to four-, three- and two-aromatic ring products. The structurally similar four- and three-ring non-alternant PAHs fluoranthene and acenaphthylene were also biotransformed by strain KK22, and LC/ESI(-)-MS/MS analyses of these products confirmed the lower biotransformation pathway proposed for benzo[k]fluoranthene. In all, seven products from benzo[k]fluoranthene and seven products from fluoranthene were revealed and included previously unreported products from both PAHs. Benzo[k]fluoranthene biotransformation proceeded through ortho-cleavage of 8,9-dihydroxy-benzo[k]fluoranthene to 8-carboxyfluoranthenyl-9-propenic acid and 9-hydroxy-fluoranthene-8-carboxylic acid, and was followed by meta-cleavage to produce 3-(2-formylacenaphthylen-1-yl)-2-hydroxy-prop-2-enoic acid. The fluoranthene pathway converged with the benzo[k]fluoranthene pathway through detection of the three-ring product, 2-formylacenaphthylene-1-carboxylic acid. Production of key downstream metabolites, 1,8-naphthalic anhydride and 1-naphthoic acid from benzo[k]fluoranthene, fluoranthene and acenaphthylene biotransformations provided evidence for a common pathway by strain KK22 for all three PAHs through acenaphthoquinone. Quantitative analysis of benzo[k]fluoranthene biotransformation by strain KK22 confirmed biodegradation. This is the first pathway proposed for the biotransformation of benzo[k]fluoranthene by a bacterium.

DOI： 10.1111/1751-7915.12102

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A pathway for the biotransformation of the environmental pollutant and high-molecular weight polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene by a soil bacterium was constructed through analyses of results from liquid chromatography negative electrospray ionization tandem mass spectrometry (LC/ESI(-)-MS/MS). Exposure of Sphingobium sp. strain KK22 to benzo[k]fluoranthene resulted in transformation to four-, three- and two-aromatic ring products. The structurally similar four- and three-ring non-alternant PAHs fluoranthene and acenaphthylene were also biotransformed by strain KK22, and LC/ESI(-)-MS/MS analyses of these products confirmed the lower biotransformation pathway proposed for benzo[k]fluoranthene. In all, seven products from benzo[k]fluoranthene and seven products from fluoranthene were revealed and included previously unreported products from both PAHs. Benzo[k]fluoranthene biotransformation proceeded through ortho-cleavage of 8,9-dihydroxy-benzo[k]fluoranthene to 8-carboxyfluoranthenyl-9-propenic acid and 9-hydroxy-fluoranthene-8-carboxylic acid, and was followed by meta-cleavage to produce 3-(2-formylacenaphthylen-1-yl)-2-hydroxy-prop-2-enoic acid. The fluoranthene pathway converged with the benzo[k]fluoranthene pathway through detection of the three-ring product, 2-formylacenaphthylene-1-carboxylic acid. Production of key downstream metabolites, 1,8-naphthalic anhydride and 1-naphthoic acid from benzo[k]fluoranthene, fluoranthene and acenaphthylene biotransformations provided evidence for a common pathway by strain KK22 for all three PAHs through acenaphthoquinone. Quantitative analysis of benzo[k]fluoranthene biotransformation by strain KK22 confirmed biodegradation. This is the first pathway proposed for the biotransformation of benzo[k]fluoranthene by a bacterium.

DOI： 10.1111/1751-7915.12102

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

Transformation of 9,10-phenanthrenedione, a cytotoxic derivative of phenanthrene, was shown to occur by a soil bacterium belonging to the genus Sphingobium. Phenanthrene-grown cells of this strain were exposed to 50mgL-1 9,10-phenanthrenedione in liquid cultures, extracted, and extracts were analyzed by liquid chromatography electrospray ionization mass spectrometry in negative ionization mode. Full scan analyses of exposed cells over the range from m/z 50 to m/z 500 were compared to abiotic and biotic controls. Product and precursor ion scan mode analyses indicated that at least three aromatic ring-cleavage transformation products of 9,10-phenanthrenedione were present and structures for these products, corresponding to [M-H]-=271, [M-H]-=241, and [M-H]-=339 were proposed to be 4-(1-hydroxy-3,4-dioxo-2-naphthyl)-2-oxo-but-3-enoic acid, 2,2'-diphenic acid and 2-[(6-carboxy-2,3-dihydroxy-phenyl)-hydroxy-methyl]-5-oxo-hex-3-enedioic acid. The identity of 2,2'-diphenic acid was confirmed by comparison to an authentic standard and when the strain was exposed to 50mgL-1 2,2'-diphenic acid in separate assays, a transformation product with a similar mass spectrum as 9,10-phenanthrenedione-derived [M-H]-=339 was revealed. Based upon these results, pathways for the transformation of 9,10-phenanthrenedione by strain KK22 were proposed. Strain KK22 appeared unable to use 9,10-phenanthrenedione as a growth substrate under these conditions. This is the first report of potential biotransformation pathways of 9,10-phenanthrenedione by a bacterium. © 2013 Elsevier Ltd.

DOI： 10.1016/j.chemosphere.2013.03.054

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Arsenic immobilization and release in the environment is significantly influenced by bacterial oxidation and reduction of arsenic and arsenic-bearing minerals. In this study, we tested three iron-reducing bacteria, Shewanella oneidensis MR-1, Shewanella sp. FIN-41, and Shewanella putrefaciens 200, which have diverse arsenate-reducing activities with regard to reduction of an As-bearing ferrihydrite slurry. In the cultures of S. oneidensis MR-1 and Shewanella sp. HN-41, which are not capable of respiratory reduction of As(V) to As(III), arsenic was maintained predominantly in its pentavalent form, existing in particulate poorly crystalline As-bearing ferrihydrite and formed small quantities of a stable ferrous arsenate [Fe-3(AsO4)(2)] precipitate. However, in the culture of the As(V) reducer, S. putrefaciens 200, As(V) was reduced to As(III) and a small fraction of As-bearing ferrihydrite was transformed into ribbon-shaped siderite that subsequently re-released arsenic into the liquid phase. Our results indicated that release of arsenic and formation of diverse secondary nanoscale Fe-As minerals are specifically closely related to the arsenic-reducing abilities of different bacteria. Therefore, bacterial arsenic reduction appears to significantly influence As mobilization in soils, minerals, and other Fe-rich environments.

DOI： 10.1021/es400534z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A soil bacterium, designated strain KK22, was isolated from a phenanthrene enrichment culture of a bacterial consortium that grew on diesel fuel, and it was found to biotransform the persistent environmental pollutant and high-molecular-weight polycyclic aromatic hydrocarbon (PAH) benz[a]anthracene. Nearly complete sequencing of the 16S rRNA gene of strain KK22 and phylogenetic analysis revealed that this organism is a new member of the genus Sphingobium. An 8-day time course study that consisted of whole-culture extractions followed by high-performance liquid chromatography (HPLC) analyses with fluorescence detection showed that 80 to 90% biodegradation of 2.5 mg liter(-1) benz[a]anthracene had occurred. Biodegradation assays where benz[a]anthracene was supplied in crystalline form (100 mg liter(-1)) confirmed biodegradation and showed that strain KK22 cells precultured on glucose were equally capable of benz[a]anthracene biotransformation when precultured on glucose plus phenanthrene. Analyses of organic extracts from benz[a]anthracene biodegradation by liquid chromatography negative electrospray ionization tandem mass spectrometry [LC/ESI(-)-MS/MS]revealed 10 products, including two o-hydroxypolyaromatic acids and two hydroxy-naphthoic acids. 1-Hydroxy-2- and 2-hydroxy-3-naphthoic acids were unambiguously identified, and this indicated that oxidation of the benz[a]anthracene molecule occurred via both the linear kata and angular kata ends of the molecule. Other two-and single-aromatic-ring metabolites were also documented, including 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid and salicylic acid, and the proposed pathways for benz[a]anthracene biotransformation by a bacterium were extended.

DOI： 10.1128/AEM.01129-13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Sphingobium sp. strain KK22 was isolated from a bacterial consortium that originated from cattle pasture soil from Texas. Strain KK22 grows on phenanthrene and has been shown to biotransform the high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) benz[a]anthracene. The genome of strain KK22 was sequenced to investigate the genes involved in aromatic pollutant biotransformation.

DOI： 10.1128/genomeA.00911-13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, reduced tellurite (Te( IV), TeO32-) to elemental tellurium under anaerobic conditions resulting in the intracellular accumulation of needle shaped crystalline Te(0) nanorods. Fatty acid analyses showed that toxic Te(IV) increased the unsaturated fatty acid composition of the lipid components of the cell membrane, implying a deconstruction of the integrity of the cellular membrane structure. The current results suggest that dissimilatory metal reducing bacteria such as S. oneidensis MR-1 may play an important role in recycling toxic tellurium elements, and may be applied as a novel selective biological filter via the accumulation of industry-applicable rare materials, Te(0) nanorods, in the cell. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biortech.2012.08.129

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1. Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed TAO and PAADH in E. coli, the engineered strain has potential to be applied in the fragrance industry.

DOI： 10.1021/jf303531u

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A novel lectin structure was found for a 17-kDa alpha-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of similar to 50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Gal alpha 1-4Gal beta 1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an alpha-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

DOI： 10.1074/jbc.M112.418012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A plasmid, pTA163, in Escherichia coli contained an approximately 34-kb gene fragment from Pseudomonas putida JYR-1 that included the genes responsible for the metabolism of trans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyze trans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter in E. coli catalyzed oxidative cleavage of a propenyl group of trans-anethole to an aldehyde group, resulting in the production of para-anisaldehyde, and this gene was designated tao (trans-anethole oxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein of Agrobacterium vitis S4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water into p-anisaldehyde using O-18-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase from Pseudomonas putida IE27 and Pseudomonas nitroreducens Jin1, TAO from P. putida JYR-1 catalyzed isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts of E. coli (pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

DOI： 10.1128/AEM.00781-12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

A divalent cation-independent lectin-HOL-18, with cytotoxic activity against leukemia cells, was purified from a demosponge, Halichondria okadai. HOL-18 is a 72 kDa tetrameric lectin that consists of four non-covalently bonded 18 kDa subunits. Hemagglutination activity of the lectin was strongly inhibited by chitotriose (GlcNAc beta 1-4GlcNAc beta 1-4GlcNAc), fetuin and mucins from porcine stomach and bovine submaxillary gland. Lectin activity was stable at pH 4-12 and temperatures lower than 60 degrees C. Frontal affinity chromatography with 16 types of pyridylaminated oligosaccharides indicated that the lectin had an affinity for N-linked complex-type and sphingolipid-type oligosaccharides with N-acetylated hexosamines and neuramic acid at the non-reducing termini. The lectin killed Jurkat leukemia T cells and K562 erythroleukemia cells in a dose- and carbohydrate-dependent manner.

DOI： 10.3390/toxins4050323

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A novel anticancer mechanism of catfish (Silurus asotus) egg lectin (SAL) was found to occur via the down-regulation of the membrane transopter protein, MRP1 (multidrug resistance associate protein-1) on Burkitt's lymphoma cells through Gb3(Gal alpha 1-4Gal beta 1-4Glc)-glycosphingolipid. Although SAL did not influence the viability of the cells directly, only 10 and 100 ng/mL of vincristine and etoposide, respectively induced anticancer effects when the lectin was applied in conjunction with these drugs. These phenomena were specifically inhibited by the co-presence of the alpha-galactoside, melibiose, which is a strong haptenic sugar of SAL that mimicks Gb3. The degree of expression regulation of the transporter proteins on the cells surface was investigated through the examination of the binding between SAL and Gb3-glycosphingolipid by immunological and molecular biological procedures. PCR data showed that MRP1 was more highly expressed when compared to another ATP-binding cassette family, multi-drug resistant protein and the expression levels of MRP1 on the cells were specifically dose- and time-dependently depleted by the addition of SAL. These results were also evaluated by immunological procedures using FACS and western-blotting. Small interfering RNA coding a part of MRP1 was transfected to Raji cells to knock down the protein, and cell death was increased by 10% when vincristine was administered at a concentration as low as 10 ng/mL compared to non-transfected cells. These results indicated that SAL possesses the potential to enhance the anticancer activites of low-concentrations of vincristine by the down-regulating the MRP1 gene expression to inhibit the multidrug resistance by binding to the target ligand Gb3-glycosphingolipid on Burkitt's lymphoma cells.

DOI： 10.1007/s10930-011-9369-2

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 A degrees C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Gal alpha 1-4Gal beta 1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k (ass) and k (diss) values are 2.4 x 10(3) M(-1) s(-1) and 3.8 x 10(-3) s(-1), respectively. AKL-2 appeared cytotoxicity against both Burkitt&apos;s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.

DOI： 10.1007/s10930-011-9356-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2&apos; and C3&apos; on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol &gt; (3R,4S)-trans-isoflavan-4-ol &gt; (3S,4R)-trans-isoflavan-4-ol &gt; (3R, 4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

DOI： 10.1007/s00253-010-2989-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12 ng within 45 min, and because it is non-alcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2010.06.035

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The spread of antibiotics resistance among bacteria is a threat to human health. Since South Korea uses approximately 1.5 times more antibiotics than do other OECD countries, this is likely to impact the numbers and types of antibiotic-resistant bacteria found in the environment. In this study we examined feces from domesticated animals and humans for the diversity and abundance of antibiotic-resistant Escherichia coli. Abundant antibiotic-resistant E. coli were isolated from all the tested animals and humans and were examined by horizontal, fluorophore-enhanced, rep-PCR (HFERP) DNA fingerprint analysis. A total of 793 unique, non-clonal, E. coli isolates were obtained from the 513 human and animal hosts examined. Antibiotic resistance analysis, done using 14 antibiotics, indicated that 72.3% of the isolates (573 of 793) were found resistant to more than one antibiotic. The E. coli isolated from swine were resistant to the greatest number of antibiotics. Tetracycline resistant E. coli were routinely isolated from all animal hosts (36 to 77% per host), except for dairy cattle (9.3%). Twenty nine E. coli isolates from all hosts, except for duck, were resistant to more than 10 antibiotics. Gene transfer and southern hybridization studies revealed that resistance to 13 of the antibiotics was self-transmissible, and likely mediated by plasmids and integrons. Since genetically diverse and numerically abundant antibiotic-resistant E. coli were consistently recovered from chicken, swine and other domesticated animals in South Korea, our results suggest that the use of sub-therapeutic levels of antibiotics for disease prophylaxis and growth promotion should be curtailed. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scitotenv.2010.04.046

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Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 incorporated dioxygen at the C7 and C8 positions on the A-rings of flavone and isoflavone with different stereoselectivity, resulting in the formation of (7S,8S)-dihydroxy-2-phenyl-7,8-dihydro-4H-chromen-4-one (flavone-cis-(7S,8S)-dihydrodiol) and (7R,8R)-dihydroxy-3-phenyl-7,8-dihydro-4H-chromen-4-one (isoflavone-cis-(7R,8R)-dihydrodiol), respectively. In addition, NDO was shown to incorporate dioxygen at the C5 and C6 positions on the A-ring and the C2&apos; and C3&apos; positions on the B-ring of isoflavone, resulting in the production of (5S,6R)-dihydroxy-3-phenyl-5,6-dihydro-4H-chromen-4-one (isoflavone-cis-(5S,6R)-dihydrodiol) and 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one (isoflavone-cis-(2&apos;R,3&apos;S)-dihydrodiol), respectively. The metabolites were identified by LC/MS, (1)H, and (13)C NMR analyses and TD-SCF calculations combined with CD spectroscopy. In the case of flavone biotransformation, formation of flavone-(7S,8S)-dihydrodiol is likely to be the result of hydrogen bond interactions between the substrate and the active site of the dioxygenase. On the contrary, regioselective dioxygenation of isoflavone was found not to occur, and this may be due to the fact that the same hydrogen bonds that occur in the case of the flavone reaction cannot be established due to steric hindrance caused by the position of the B-ring. It is therefore proposed that the regioselectivity and stereoselectivity of NDO from strain NCIB 9816-4 are controlled by the position of the phenyl ring on flavone molecules.

DOI： 10.1007/s00253-009-2389-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Interest in understanding prokaryotic biotransformation of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) has continued to grow and the scientific literature shows that studies in this field are originating from research groups from many different locations throughout the world. In the last 10 years, research in regard to HMW PAH biodegradation by bacteria has been further advanced through the documentation of new isolates that represent diverse bacterial types that have been isolated from different environments and that possess different metabolic capabilities. This has occurred in addition to the continuation of in-depth comprehensive characterizations of previously isolated organisms, such as Mycobacterium vanbaalenii PYR-1. New metabolites derived from prokaryotic biodegradation of four-and five-ring PAHs have been characterized, our knowledge of the enzymes involved in these transformations has been advanced and HMW PAH biodegradation pathways have been further developed, expanded upon and refined. At the same time, investigation of prokaryotic consortia has furthered our understanding of the capabilities of microorganisms functioning as communities during HMW PAH biodegradation.

DOI： 10.1111/j.1751-7915.2009.00130.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Escherichia coli cells containing the biphenyl dioxygenase genes bphA1A2A3A4 from Pseudomonas pseudoalcaligenes KF707 were found to biotransform isoflavone and produced a metabolite that was not found in a control experiment. Liquid chromatography/mass spectrometry (LC/MS) and (1)H and (13)C nuclear magnetic resonance (NMR) analyses indicated that biphenyl dioxygenase induced 2&apos;,3&apos;-cis-dihydroxylation of the B-ring of isoflavone. In a previous report, the same enzyme showed dioxygenase activity toward flavone, producing flavone 2&apos;,3&apos;-cis-dihydrodiol. Due to growing interest in flavone chemistry and the absolute configuration of natural products, time-dependent density functional theory (TD-DFT) calculations were combined with circular dichroism (CD) spectroscopy to determine the absolute configuration of the isoflavone dihydrodiol. By computational methods, the structure of the isoflavone metabolite was determined to be 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. This structure was confirmed further by the modified Mosher&apos;s method. The same protocol was applied to the flavone metabolite, and the absolute Configuration was determined to be 2-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. After determination of the absolute configurations of the biotransformation products, we suggest the binding mode of these substrate analogs to the enzyme active site. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2009.10.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Amorphous selenium nanospheres, originally produced by Shewanella sp. strain HN-41 under anaerobic conditions, can be rapidly transformed into extensive, long and thin, polycrystalline Se nanowires and nanoribbons (&gt;100 mu m x 57 nm) in 80% DMSO with bacterial pellets at physiological temperature. Scanning and transmission electron microscopic analyses indicated that the Se nanowires and nanospheres were crystalline structures indexed into the hexagonal plane of Se. The structures possessed an unusually high crystalline peak ( 100), suggesting a preferential [ 001] growth direction. Electron micrographic analyses and incubation studies suggested that the cell membrane of the Shewanella sp. strain HN-41 likely plays an important role in the formation of amorphous Se nanospheres from soluble Se(IV) and the formation of long and thin h-Se nanowires and nanoribbons. The formation of zero- and one-dimensional h-Se nanostructures by this bacterium may provide a facile strategy to recover soluble Se(IV) from the environment and generate new materials that will be useful for advanced nanotechnologies.

DOI： 10.1039/b923252d

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The hypolipidemic agent gemfibrozil (GEM), which has been studied for its metabolism in humans and animals, was investigated to elucidate its primary metabolism by Cunninghamella elegans. The fungus produced ten metabolites (FM1-FM9 and FM6&apos;) from the biotransformation of GEM. Based on LC/MS/MS and NMR analyses, a major metabolite, FM7, was identified as 2&apos;-hydroxymethyl GEM. FM6 was considered to be 5&apos;-hydroxymethyl GEM, after comparison of results LC/MS, LC/MS/MS, and UV absorption spectra to FM7. The combined concentration of FM6 and FM7 was found to increase up to 0.83 mM by day 2, and then decreased gradually with incubation time, followed by a noticeable increase in the biotransformation product, FM1, up to 0.86 mM by day 15. NMR analyses confirmed that FM1 was 2&apos;,5&apos;-dihydroxymethyl GEM. Further minor oxidations of the aromatic ring and carboxylic acid intermediates were also detected. Based upon these findings, the major fungal metabolic pathway for GEM is likely to occur via production of 2&apos;,5&apos;-dihydroxymethyl GEM from 2&apos;-hydroxymethyl GEM. These relatively rapid and diverse biotransformations of GEM by C. elegans suggest that depending upon conditions, it may also follow a similar biodegradation fate when released into the natural environment.

DOI： 10.1007/s00203-009-0480-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Methods for determining the differential susceptibility of human organs to DNA damage have not yet been explored to any large extent due to technical constraints. The development of comprehensive analytical approaches by which to detect intertissue variations in DNA damage susceptibility may advance our understanding of the roles of DNA adducts in cancer etiology and as exposure biomarkers at least. A strategy designed for the detection and comparison of multiple DNA adducts from different tissue samples was applied to assess esophageal and peripherally- and centrally-located lung tissue DNA obtained from the same person. This adductome approach utilized LC/ESI-MS/MS analysis methods designed to detect the neutral loss of 2'-deoxyribose from positively ionized 2'-deoxynucleoside adducts transmitting the [M+H](+) &gt; [M+H- 116](+) transition over 374 transitions. In the final analyses, adductome maps were produced which facilitated the visualization of putative DNA adducts and their relative levels of occurrence and allowed for comprehensive comparisons between samples, including a calf thymus DNA negative control. The largest putative adducts were distributed similarly across the samples, however, differences in the relative amounts of putative adducts in lung and esophagus tissue were also revealed. The largest-occurring lung tissue DNA putative adducts were 90% similar (n = 50), while putative adducts in esophagus tissue DNA were shown to be 80 and 84% similar to central and peripheral lung tissue DNA respectively. Seven DNA adducts, N-2-ethyl-2'-deoxyguanosine (N-2-ethyl-dG), 1,N-6-etheno-2'-deoxyadenosine (epsilon dA), alpha-S- and alpha-R-methyl-gamma-hydroxy-1,N-2-propano-2'-deoxyguanosine (1,N-2-PdG(1), 1,N-2-PdG(2)), 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxy-pyrimido[1,2-a]purine-(3H)-one (8-OH-PdG) and the two stereoisomers of 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (6-OH-PdG) were unambiguously detected in all tissue DNA samples by comparison to authentic adduct standards and stable isotope dilution and their identities were matched to putative adducts detected in the adductome maps. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mrfmmm.2007.05.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

N-2-ethylidene-2'-deoxyguanosine (N-2-ethylidene-dG) is a major DNA adduct induced by acetaldehyde. Although it is unstable in the nucleoside form, it is relatively stable when present in DNA. In this study, we analyzed three acetaldehyde-derived DNA adducts, N-2-ethylidene-dG, N-2-ethyl-2'-deoxyguanosine (N-2-Et-dG) and alpha-methyl-gamma-hydroxy-1,N-2-propano-2'-deoxyguanosine (alpha-Me-gamma-OH-PdG) in the liver DNA of aldehyde dehydrogenase (Aldh)-2-knockout mice to determine the influence of alcohol consumption and the Aldh2 genotype on the levels of DNA damage. In control Aldh2+/+ mice, the level of N-2-ethylidene-dG adduct in liver DNA was 1.9 +/- 0.7 adducts per 10(7) bases and was not significantly different than that of Aldh2+/- and -/- mice. In alcohol-fed mice (20% ethanol for 5 weeks), the adduct levels of Aldh2+/+, +/- and -/- mice were 7.9 +/- 1.8, 23.3 +/- 4.0 and 79.9 +/- 14.2 adducts per 10(7) bases, respectively, and indicated that adduct level was alcohol and Aldh2 genotype dependent. In contrast, an alcohol- or Aldh2 genotype-dependent increase was not observed for alpha-Me-gamma-OH-PdG, and N-2-Et-dG was not detected in any of the analyzed samples. In conclusion, the risk of formation of N-2-ethylidene-dG in model animal liver in vivo is significantly higher in the Aldh2-deficient population and these results may contribute to our understanding of in vivo adduct formation in humans.

DOI： 10.1093/carcin/bgm057

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Newly isolated soil bacterium strain Jin1 was able to grow on both eugenol and isoeugenol each as sole source of carbon and energy. Based on bacterial 16S rDNA analysis, Jin1 belongs to Pseudomonas nitroreducens with a similarity of 98.92% (14/1297). P. nitroreducens Jin1 was found to biotransform eugenol and isoeugenol to vanillin by different pathways. Eugenol was biotransformed to vanillin through coniferyl alcohol and ferulic acid similarly to the pathway shown previously by Pseudomonassp. HR1 99 and vanillin produced from eugenol was rapidly metabolized to vanillic acid. Contrastively, Pseudomonas nitroreducens Jin1 did not appear to produce metabolic intermediates during the biotransformation of isoeugenol to vanillin which was finally biotransformed to vanillic acid with much slower rate. These results indicate that there seems to be different metabolic regulation systems for the biotransformation of eugenol and isoeugenol by this bacterium. Herein, we report on Pseudomonas nitroreducens Jin1, a novel bacterium that produces vanillin from eugenol and isoeugenol by two different metabolic pathways.

DOI： 10.1021/jf0715929

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Language：English   Publishing type：Research paper (scientific journal)  

The complex and diverse structural configurations of polycyclic aromatic hydrocarbons (PAHs), combined with their low bioavailability, hydrophobic nature, strong sorption phenomena, and high persistence in soil makes the design of effective bioremediation methodologies a challenge. The multi-phasic nature of the bioremediation process, restricted mass transfer and non-availability of degrading soil microflora further compound the problem. In this direction, this communication presents a focused review of bioremediation technologies used recently for the treatment of PAH-contaminated soils. The specific roles of important factors affecting bioremediation process efficiency are discussed. Finally some of the recently used strategies to enhance bioremediation process efficiency, including bioaugmentation, biostimulation, rhizoremediation, the use of chemotaxins, the biomimetic catalytic system approach, and integrated techniques, are reviewed. © Springer Science+Business Media B.V. 2006.

DOI： 10.1007/s11157-006-0004-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Drinking alcohol is a risk factor for cancers of the oral cavity, pharynx, larynx, and esophagus. Although many studies suggest that acetaldehyde, a major metabolite of orally ingested alcohol, plays a crucial role in cancer initiation, the link between the aldehyde dehydrogenase-2 (ALDH2) genotype and acetaldehyde-derived DNA damage has not yet been explored. We have developed a sensitive and quantitative method for detecting the acetaldehyde- derived DNA adducts, N-2-ethyl-2'-deoxyguanosine (N-2-Et-dG), alpha-S-and alpha-R-methyl-gamma-hydroxy-1, N-2-propano-2'-deoxyguanosine (alpha-S-Me-gamma-OH-PdG and alpha-R-Me-gamma-OH-PdG), and N-2-(2,6-dimethyl-1,3-dioxan-4-yl)-deoxyguanosine (N-2-Dio-dG), by using liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) and stable-isotope internal standards. We determined the DNA adducts in 44 blood DNA samples from Japanese alcoholic patients. The levels of three acetaldehyde-derived DNA adducts, N-2-Et-dG, alpha-S-Me-gamma-OH-PdG, and alpha-R-Me-gamma-OH-PdG, were significantly higher in alcoholics with the ALDH2* 1/2* 2 genotype compared to those with the ALDH2* 1/2* 1 genotype. N-2-Dio-dG was not detected in any of the DNA samples analyzed. These results provide molecular evidence that the ALDH2 genotype affects the genotoxic damage caused by acetaldehyde.

DOI： 10.1021/tx060113h

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

The development of new strategies designed to detect DNA damage caused by oxidative stress and other means may advance our understanding of the roles of such types of damage in the etiology of cancers, in aging processes, and as biomarkers; of exposure. A DNA adduct detection method that uses liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) to detect multiple DNA adducts in human lung tissue is reported herein. This adductome analysis strategy is designed to detect the neutral loss of 2'-deoxyribose from positively ionized 2'-deoxynucleoside adducts in multiple reaction ion monitoring mode (MRM) transmitting the [M + H](+) &gt; [M + H - 116](+) transition over a total of 374 transitions in the mass range from m/z 228.8 to m/z 602.8. Data analysis is optimized and coupled with a comprehensive manual screening process designed to minimize the number of artifactual adducts appearing in the final analysis. In the final analysis, putative adducts were organized into an adductome map and unambiguous confirmation of selected oxidative adducts were made by stable isotope dilution and comparison to authentic standards. The future applications of this method are discussed.

DOI： 10.1089/ars.2006.8.993

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Generally, the white-rot fungus Phanerochaete chrysosporium performs its biodegradative activities in liquid culture while growing on easily utilized carbon sources such as malt- or potato-extract. However, less is known about the potential of this organism to grow directly on environmental pollutants without regard to special conditions. Growth of P. chrysosporium on a middle fraction (MF) of diesel fuel at neutral pH in mineral medium under non-ligninolytic conditions was explored. After 14 d, the GC-analyzable n-alkanes of 1000 mg l(-1)MF were reduced to background, with most biodegradation occurring by day 7 when quantified relative to the biodegradation of the internal fuel biodegradation marker, pristane. Investigations with n-hexadecane and unmodified diesel fuel further confirmed these biodegradation results. Biomass production was monitored and indicated that fungal biomass was more than 10 times less than positive controls (potato dextrose broth, PDB) but that biomass increased relative to negative controls. When P. chrysosporium was incubated with diesel fuel and PDB, fuel biodegradation was delayed for at least 4 d and inhibited overall through 14 d. Experiments with P. chrysosporium growing on n-hexadecane in the presence of 1 mM 1-aminobenzotriazole (ABT), an inhibitor of the cytochrome P-450 enzyme system, resulted in inhibition of biomass production relative to positive controls implicating the utilization of this enzyme system in n-alkane metabolism. Finally, when P. chrysosporium was incubated in a non-aqueous phase liquid (NAPL) mixture of polycyclic aromatic hydrocarbons (PAHs) and MF, n-alkanes and phenanthrene were degraded in 2 weeks while anthracene, chrysene and benzo[a]pyrene were not. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2005.08.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：I W A PUBLISHING  

The high molecular weight polycyclic aromatic hydrocarbon (HMW PAH) benzo[a]pyrene is generally persistent in the environment and its persistence may be due to bioavailability limitations. However, the presence of degradation-capable microorganisms and a suitable cosubstrate are also necessary. This is especially the case for benzo[a]pyrene because it may only be degraded by fortuitous metabolism. Nonaqueous phase liquid (NAPL)-enhanced benzo[a]pyrene biodegradation and indicators of bioavailability were measured in soil and liquid culture. In soil, (CO2)-C-14 from 7-[C-14]benzo[a]pyrene mineralisation and overall CO2 production were monitored for 83 d after treatment with different types of NAPLs in bionneter flasks. Monitoring was followed by soil extraction and measurement of C-14 residues and of the remaining NAPL by gravimetry. In liquid culture, 7- [C-14]benzo[a]pyrene mineralisation was monitored after treatment with different NAPLs and followed by a radiocarbon-mass balance of C-14 residues. Results indicated that although benzo[a]pyrene may have been bioavailable in both media types, benzo[a]pyrene mineralisation only occurred when a suitable NAPL cosubstrate was present to facilitate biodegradation. In soil, rapid increases in the rate and onset of benzo[a]pyrene mineralisation were shown to occur in benzo[a]pyrenecontaminated soils that were treated with mineral oil, which was a relatively non-biodegradable NAPL cosolvent, plus a hexane fraction-NAPL which was biodegradable and contained suitable cosubstrate(s).

DOI： 10.2166/wst.2006.333

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Biotransformation of the environmental pollutant 3-methyl-4-nitrophenol (MNP), a newly characterized estrogenic chemical, and the primary breakdown product of the heavily used insecticide fenitrothlon was investigated using a common soil fungus. In 96 h, daily culture sacrifice, extraction, and analysis showed that the filamentous fungus, Aspergillus niger VKM F-1119, removed more than 85% of the MNP present in solution (original concentration = 25 mg/L), mostly through biodegradation. Additionally, in 16-day time-course studies, A. niger was capable of biotransformation of MNP at concentrations as high as 70 mg/L. Gas chromatography mass spectroscopy (MS) analyses of culture fluid extracts indicated the formation of four metabolites: 2-methyl-1,4-benzenediol, 4-amino-3-methylphenol, and two singly: hydroxylated derivatives of MNP. Culture scale up and metabolite analysis by liquid chromatography MS resulted in the confirmation of the original metabolites plus the detection of an azo derivative metabolite that has not been previously reported before during MNP biodegradation by any micro-organisms.

DOI： 10.1021/jf050679w

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SETAC  

The presence of multicomponent nonaqueous-phase liquids (NAPLs) on contaminated sites critically alters the biodegradation susceptibility of many target pollutants, including polycyclic aromatic hydrocarbons. This study investigated the effects of petroleum-derived multicomponent NAPLs on biodegradation of benzo[a]pyrene by a bacterial consortium in liquid culture. When high-boiling point diesel fuel distillate (HBD)-NAPL was added to liquid culture, the consortium initiated benzo[a]pyrene mineralization after a lag period of several days. This lag period was not observed in the mineralization of phenanthrene, anthracene, and chrysene by the same consortium with HBD. Nonaqueous-phase liquids added to cultures pregrown before experimentation largely affected the extent of benzo[a]pyrene mineralization and the duration of lag period in subsequent experiments, suggesting that NAPL presence was important for maintaining the efficiency of the mineralizing consortium. Experiments using further fractionated oil components suggested that stimulation of benzo[a]pyrene mineralization by NAPLs was fraction dependent; an alkylated aromatic fraction was more effective than aromatic and aliphatic fractions. The effect of NAPL on benzo[a]pyrene biodegradation was determined to be multi mechanistic: that is, NAPL acted as a cosolvent for polycyclic aromatic hydrocarbon dissolution, as a substrate to induce cometabolic degradative pathways, and as an agent to formulate the effective microbial consortium. Data suggest that the third mechanism was of particular importance for rapid benzo[a]pyrene mineralization.

DOI： 10.1897/03-191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol. 66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them. was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter, whose type strain, Rhodanobacter lindaniclasticus RP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo [a] pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo [a] pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.

DOI： 10.1128/AEM.68.12.5826-5833.2002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SETAC  

Bacterial mineralization of [7-C-14]benzo[a]pyrene (BaP) to (CO2)-C-14 was enhanced by the presence of nonaqueous phase liquids (NAPLs). Mineralization of BaP was affected differently by different NAPLs, and the mode of enhancement of mineralization by a NAPL most likely occurred by a combination of cometabolic and physical effects. Mineralization was enhanced to the greatest extent when BaP was dissolved in a high-boiling distillation product of diesel fuel.

DOI： 10.1897/1551-5028(2001)020<0498:EMOBAP>2.0.CO;2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A microbial consortium which rapidly mineralized the environmentally persistent pollutant benzo[a]pyrene was recovered from soil. The consortium cometabolically converted [7-C-14]benzo[a]pyrene to (CO2)-C-14 when it was grown on diesel fuel, and the extent of benzo[a]pyrene mineralization was dependent on both diesel fuel and benzo[a]pyrene concentrations, Addition of diesel fuel at concentrations ranging from 0.007 to 0.2% (wt/vol) stimulated the mineralization of 10 mg of benzo[a]pyrene per liter 33 to 65% during a 2-week incubation period. When the benzo[a]pyrene concentration was 10 to 100 mg liter(-1) and the diesel fuel concentration was 0.1% (wt/vol), an inoculum containing 1 mg of cell protein per liter (small inoculum) resulted in mineralization of up to 17.2 mg of benzo[a]pyrene per liter in 16 days. This corresponded to 35% of the added radiolabel when the concentration of benzo[a]pyrene was 50 mg liter(-1). A radiocarbon mass balance analysis recovered 25% of the added benzo[a]pyrene solubilized in the culture suspension prior to mineralization. Populations growing on diesel fuel most likely promoted emulsification of benzo[a]pyrene through the production of surface-active compounds, The consortium was also analyzed by PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments, and 12 dominant bands, representing different sequence types, were detected during a 19-day incubation period. The onset of benzo[a]pyrene mineralization was compared to changes in the consortium community structure and was found to correlate with the emergence of at least four sequence types. DNA from 10 sequence types were successfully purified and sequenced, and that data revealed that eight of the consortium members were related to the class Proteobacteria but that the consortium also included members which were related to the genera Mycobacterium and Sphingobacterium.

DOI： 10.1128/AEM.66.10.4205-4211.2000

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

DOI： 10.1128/JB.182.8.2059-2067.2000

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SETAC PRESS  

The mineralization of [7-C-14]benzo[a]pyrene (BaP) in soil was investigated in response to additions of individual hydrocarbons, defined hydrocarbon mixtures, crude oil, and crude oil fractions. Neither substantial BaP mineralization nor enrichment of BaP degraders occurred in BaP-spiked soil in the absence of a suitable hydrocarbon supplement. Crude oil, the saturated and aromatic class components of crude oil, the distillates heating oil, jet fuel, and diesel fuel supported up to 60% mineralization of 80 mu g [7-C-14]BaP per gram of soil in 40 d. Neither single hydrocarbons nor defined hydrocarbon mixtures containing normal and branched alkanes, alicyclics, and aromatics supported comparable BaP mineralization. Evolution of (CO2)-C-14 occurred after lag periods characteristic to specific petroleum products and their concentrations. Time required for microbial proliferation, hydrocarbon toxicity, and competitive inhibition might have contributed to these lag periods, but the complete inhibition of BaP mineralization by diesel-fuel vapors pointed to a dominant role of competitive inhibition. A lack of radiocarbon incorporation into soil biomass from [7-C-14]BaP indicated that at least the initial steps of BaP biodegradation in soil were cometabolic in nature. Suitable hydrocarbon mixtures not only supported BaP mineralization by serving as primary substrates, but also enhanced BaP bioavailability by dissolving this hydrophobic solid.

DOI： 10.1897/1551-5028(1999)018<2186:CMOBAP>2.3.CO;2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

To investigate the possible cometabolic biodegradation of benzo[a]pyrene (BaP), crude oil spiked with [7-C-14] BaP and unlabeled BaP was added to soil with no known pollution history, to give 34 g of oil and 67 mg of BaP/kg of dry soil. The oil-soil mixture was amended with mineral nutrients and incubated in an airtight container with continuous forced aeration, Total CO2 and (CO2)-C-14, in the off-gas were trapped and quantified. Soil samples were Soxhlet extracted with dichloromethane at seven time points during the 150-day incubation period, and the extracted soil was subjected to further fractionation in order to recover reversibly and irreversibly bound radiocarbon, Radiocarbon recovery was 100% +/- 3% for each time point. During the first 50 days of incubation, no (CO2)-C-14 was evolved, but over the next 100 days, 50% of the BaP radiocarbon was evolved as (CO2)-C-14,. At 150 days, only 5% of the intact BaP and 23% of the crude oil remained. Of the remaining radiolabel, 20% was found in solvent extractable metabolites and 25% was incorporated into soil organic matter. Only 1/10 of this could be solubilized by chemical hydrolysis. An abiotic control experiment exhibited binding of only 2% of the BaP, indicating the microbial nature of the BaP transformations. We report that in soil containing suitable cosubstrates, BaP can be completely degraded.

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