Xiufen Zhou

Shanghai Jiao Tong University, Shanghai, Shanghai Shi, China

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Publications (56)235.23 Total impact

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    ABSTRACT: Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.
    Nature Communications 01/2014; 5:3951. · 10.02 Impact Factor
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    ABSTRACT: Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study, we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high resolution clear native/native PAGE, and native/SDS-PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high resolution clear native/SDS-PAGE is much higher than that of blue native/SDS-PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.
    Analytical Biochemistry 01/2014; · 2.58 Impact Factor
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    ABSTRACT: DNA phosphorothioate modification means substituting a non-bridging oxygen with a sulfur in DNA. The modification endows DNA with such chemical property that protects the hosting bacteria against peroxide. The modification is controlled by a dnd gene cluster. Salmonella entericaserovar Cerro 87 is one of the bacteria that harbor the DNA phosphorothioate modification. The modification is carried out by dptB, C, DandE. Ourstudy is designed to clone and express dptC, to optimize the expressing condition, and then to purify the DptC. dptC DNA fragment was amplified by KOD PCR with the special primers and S. entericaserovar Cerro 87 genomic DNA template. A fusion expression vector pJTU3622 was constructed by inserting the dptC DNA fragment into pGEX-6P-1 inSmaI and XhoI sites. The positive clone was verified by antibiotics resistance gene screening and sequenced, and then transferred into host strain E. coli BL21 (DE3) pLysS to producean engineering bacterium Anxh103. After optimizing the expression condition for dptC, we purified DptC from Anxh103 by Aikta FPLC with a GST-Trap column. A fusion expression vector pJTU3622 and an engineering bacterium Anxh103 were produced. The optimizing expressing condition for dptC is as follows: induced at 18 degrees C for 8 - 18 h; 0.6 mmol/L IPTG, LB with 50 micromol/L Fe2+. The anchor redeemed for high throughput expression of dptC. The TEV site in pJTU3622 made the process of purifying DptC easier and simpler. This helps lay the ground work for future study on the function of DptC. Also, the light brown color of DptC and the medium with 50 micromol/L Fe2+ showed us DptC has the same character with DndC which belongs to an iron-sufur protein with 4Fe - 4S.
    ACTA MICROBIOLOGICA SINICA 10/2013; 53(10):1111-6.
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    ABSTRACT: The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
    Applied Microbiology and Biotechnology 05/2013; · 3.69 Impact Factor
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    ABSTRACT: Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/ Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.
    Biochimie 04/2013; · 3.14 Impact Factor
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    ABSTRACT: DNA phosphorothioate modification (DNA sulfur modification, a non-bridging oxygen swapped with a sulfur) exists in diverse bacteria. Salmonella enterica serovar Cerro 87 is one of the bacteria that harbor the DNA sulfur modification. The modification is carried out by the products of a four-membered gene cluster, dptBCDE. Transformation of Escherichia coli DH10B with the dptBCDE gene cluster endows the strain with DNA sulfur modification capability. Deletion of dptC abolished the modification. Here, we studied the function of dptC in DNA sulfur modification. Six cysteine residues in dptC were mutated individually within the dptBCDE gene cluster. Mutants were then tested for DNA sulfur modification. Among the 6 cysteine mutations (C39, C146, C262, C273, C280, and C283), 5 abolished DNA modification except for C39, suggesting that C146, C262, C273, C280, and C283 are essential for DNA sulfur modification. Sequence alignment shows that these five cysteine residues are conserved among different strains. Mutation at anyone of C146, C262, C273, C280 and C283 of dptC abolished DNA modification. Our results shed light on further study of DNA sulfur modification biochemical pathway.
    ACTA MICROBIOLOGICA SINICA 02/2013; 53(2):204-9.
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    ABSTRACT: Streptomyces hygroscopicus 5008 has been used for the production of the antifungal validamycin/jinggangmycin for more than 40 years. A high yield of validamycin is achieved by culturing the strain at 37°C, rather than at 30°C for normal growth and sporulation. The mechanism(s) of its thermo-regulated biosynthesis was largely unknown. The 10,383,684-bp genome of strain 5008 was completely sequenced and composed of a linear chromosome, a 164.57-kb linear plasmid, and a 73.28-kb circular plasmid. Compared with other Streptomyces genomes, the chromosome of strain 5008 has a smaller core region and shorter terminal inverted repeats, encodes more α/β hydrolases, major facilitator superfamily transporters, and Mg2+/Mn2+-dependent regulatory phosphatases. Transcriptomic analysis revealed that the expression of 7.5% of coding sequences was increased at 37°C, including biosynthetic genes for validamycin and other three secondary metabolites. At 37°C, a glutamate dehydrogenase was transcriptionally up-regulated, and further proved its involvement in validamycin production by gene replacement. Moreover, efficient synthesis and utilization of intracellular glutamate were noticed in strain 5008 at 37°C, revealing glutamate as the nitrogen source for validamycin biosynthesis. Furthermore, a SARP-family regulatory gene with enhanced transcription at 37°C was identified and confirmed to be positively involved in the thermo-regulation of validamycin production by gene inactivation and transcriptional analysis. Strain 5008 seemed to have evolved with specific genomic components to facilitate the thermo-regulated validamycin biosynthesis. The data obtained here will facilitate future studies for validamycin yield improvement and industrial bioprocess optimization.
    BMC Genomics 07/2012; 13:337. · 4.40 Impact Factor
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    ABSTRACT: Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioation). We found that in vitro oxidation of phosphorothioate (PT) DNA by hydrogen peroxide (H(2)O(2)) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physiological relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H(2)O(2). DNA phosphorothioation was found to correlate with increasing resistance to the growth inhibition by H(2)O(2). Resistance to H(2)O(2) was abolished when each of the three dnd genes, required for phosphorothioation, was inactivated. In vivo, PT DNA is more resistant to the double-strand break damage caused by H(2)O(2) than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was converted to PT-free state when the bacteria were incubated with H(2)O(2). These findings are consistent with a hypothesis that phosphorothioation modification endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is maintained by diverse bacteria.
    Nucleic Acids Research 07/2012; 40(18):9115-24. · 8.81 Impact Factor
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    ABSTRACT: Many bacterial species modify their DNA with the addition of sulfur to phosphate groups, a modification known as DNA phosphorothioation. DndA is known to act as a cysteine desulfurase, catalyzing a key biochemical step in phosphorothioation. However, bioinformatic analysis revealed that 19 out of the 31 known dnd gene clusters, contain only four genes (dndB-E), lacking a key cysteine desulfurase corresponding gene. There are multiple cysteine desulfurase genes in Escherichia coli, but which one of them participates into DNA phosphorothioation is unknown. Here, by employing heterologous expression of the Salmonella enterica dnd gene cluster named dptBCDE in three E. coli mutants, each of which lacked a different cysteine desulfurase gene, we show that IscS is the only cysteine desulfurase that collaborates with dptB-E, resulting in DNA phosphorothioation. Using a bacterial two-hybrid system, protein interactions between IscS and DptC, and IscS and DptE were identified. Our findings revealed IscS as a key participant in DNA phosphorothioation and lay the basis for in-depth analysis of the DNA phosphorothioation biochemical pathway.
    PLoS ONE 01/2012; 7(12):e51265. · 3.73 Impact Factor
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    ABSTRACT: During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.
    Metabolic Engineering 11/2011; 13(6):768-76. · 6.86 Impact Factor
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    ABSTRACT: Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8 g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.
    Applied Microbiology and Biotechnology 06/2011; 92(2):337-45. · 3.69 Impact Factor
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    ABSTRACT: Muraymycin, a potent translocase I inhibitor with clinical potential, is produced by Streptomyces sp. NRRL 30471. The structure of muraymycin is highly unusual and contains the hexahydro-2-imino-4-pyrimidylglycyl moiety (epicapreomycidine) and an ureido bond. Here we report the identification of the muraymycin gene cluster from Streptomyces sp. NRRL 30471. Sequencing analysis of a 43.4-kb contiguous region revealed 33 ORFs, 26 of which were proposed to be involved in muraymycin biosynthesis. Independent targeted inactivation of mur16 and mur17 directly abolished muraymycin production, demonstrating the role of the genes essential for muraymycin biosynthesis. These data provide insights into the molecular mechanisms for muraymycin biosynthesis, and lay a foundation for the generation of muraymycin derivatives with enhanced bioactivity via the strategies of combinatorial biosynthesis.
    Molecular BioSystems 03/2011; 7(3):920-7. · 3.35 Impact Factor
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    ABSTRACT: The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.
    Antimicrobial Agents and Chemotherapy 12/2010; 55(3):974-82. · 4.57 Impact Factor
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    ABSTRACT: A novel, site-specific, DNA backbone S-modification (phosphorothioation) has been discovered, but its in vivo function(s) have remained obscure. Here, we report that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolated from Escherichia coli, while protecting its own DNA by site-specific phosphorothioation. A cloned 15-kb gene cluster from S. enterica conferred both host-specific restriction and DNA S-modification on E. coli. Mutational analysis of the gene cluster proved unambiguously that the S-modification prevented host-specific restriction specified by the same gene cluster. Restriction activity required three genes in addition to at least four contiguous genes necessary for DNA S-modification. This functional overlap ensures that restriction of heterologous DNA occurs only when the host DNA is protected by phosphorothioation. Meanwhile, this novel type of host-specific restriction and modification system was identified in many diverse bacteria. As in the case of methylation-specific restriction systems, targeted inactivation of this gene cluster should facilitate genetic manipulation of these bacteria, as we demonstrate in Salmonella.
    Nucleic Acids Research 11/2010; 38(20):7133-41. · 8.81 Impact Factor
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    ABSTRACT: The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species that show diverse and important antibacterial, antitumor, and anti-human immunodeficiency virus activity. Oxazolomycin A is a peptide-polyketide hybrid compound containing a unique spiro-linked beta-lactone/gamma-lactam, a 5-substituted oxazole ring. The oxazolomycin biosynthetic gene cluster (ozm) was identified from Streptomyces albus JA3453 and localized to 79.5-kb DNA, consisting of 20 open reading frames that encode non-ribosomal peptide synthases, polyketide synthases (PKSs), hybrid non-ribosomal peptide synthase-PKS, trans-acyltransferases (trans-ATs), enzymes for methoxymalonyl-acyl carrier protein (ACP) synthesis, putative resistance genes, and hypothetical regulation genes. In contrast to classical type I polyketide or fatty acid biosynthases, all 10 PKS modules in the gene cluster lack cognate ATs. Instead, discrete ATs OzmM (with tandem domains OzmM-AT1 and OzmM-AT2) and OzmC were equipped to carry out all of the loading functions of both malonyl-CoA and methoxymalonyl-ACP extender units. Strikingly, only OzmM-AT2 is required for OzmM activity for OZM biosynthesis, whereas OzmM-AT1 seemed to be a cryptic AT domain. The above findings, together with previous results using isotope-labeled precursor feeding assays, are assembled for the OZM biosynthesis model to be proposed. The incorporation of both malonyl-CoA (by OzmM-AT2) and methoxymalonyl-ACP (by OzmC) extender units seemed to be unprecedented for this class of trans-AT type I PKSs, which might be fruitfully manipulated to create structurally diverse novel compounds.
    Journal of Biological Chemistry 06/2010; 285(26):20097-108. · 4.65 Impact Factor
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    ABSTRACT: Deletion of sti on Streptomyces plasmid pIJ101, made its derivative pHZ1358 an efficient vector for gene disruption and replacement. Here, we further optimized pHZ1358 by the construction of a derivative plasmid pJTU1278, in which a cassette carrying multiple cloning sites and a lacZ selection marker was introduced for convenient plasmid construction in E. coli. Furthermore, we also deleted oriT region of the pJTU1278, generated a vector (pJTU1289) that can be used specifically for PCR-targeting. The efficient usage of these vectors was demonstrated by the deletion of a gene involved in avermectin biosynthetic in S. avermitilis.
    Journal of Microbiology and Biotechnology 04/2010; 20(4):678-82. · 1.40 Impact Factor
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    ABSTRACT: Five meilingmycins, A to E, with A as the major component, were isolated from Streptomyces nanchangensis NS3226. Through nuclear magnetic resonance (NMR) characterization, meilingmycins A to E proved to be identical to reported milbemycins alpha11, alpha13, alpha14, beta1, and beta9, respectively. Sequencing of a previously cloned 103-kb region identified three modular type I polyketide synthase genes putatively encoding the last 11 elongation steps, three modification proteins, and one transcriptional regulatory protein for meilingmycin biosynthesis. However, the expected loading module and the first two elongation modules were missing. In meilingmycin, the presence of a methyl group at C-24 and a hydroxyl group at C-25 suggests that the elongation module 1 contains a methylmalonyl-coenzyme A (CoA)-specific acyltransferase (ATp) domain and a ketoreductase (KR) domain. Based on the conserved motifs of the ATp and KR domains, a pair of primers was designed for PCR amplification, and a 1.40-kb expected fragment was amplified, whose sequence shows significant homology with the elongation module 1 of the aveA1-encoded enzyme AVES1. A polyketide synthase (PKS) gene encoding one loading and two elongation modules, with a downstream C-5-O-methyltransferase gene, meiD, was subsequently localized 55 kb apart from the previously sequenced region, and its deletion abolishes meilingmycin production. A series of deletions within the 55-kb intercluster region rules out its involvement in meilingmycin biosynthesis. Furthermore, gene deletion of meiD eliminates meilingmycins D and E, with methyls at C-5. Our work provides a more specific strategy for the cloning of modular type I PKS gene clusters. The cloning of the meilingmycin gene clusters paves the way for its pathway engineering.
    Applied and Environmental Microbiology 03/2010; 76(10):3283-92. · 3.95 Impact Factor
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    ABSTRACT: Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.
    PLoS Genetics 01/2010; 6(12):e1001253. · 8.52 Impact Factor
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    ABSTRACT: Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps.
    Electrophoresis 08/2009; 30(14):2454-9. · 3.26 Impact Factor

Publication Stats

843 Citations
235.23 Total Impact Points

Institutions

  • 2002–2014
    • Shanghai Jiao Tong University
      • • State Key Laboratory of Microbial Metabolism
      • • School of Life Science and Biotechnology
      Shanghai, Shanghai Shi, China
    • Shanghai University
      Shanghai, Shanghai Shi, China
  • 2007
    • John Innes Centre
      Norwich, England, United Kingdom
  • 2002–2007
    • Huazhong Agricultural University
      Wu-han-shih, Hubei, China
  • 2006
    • University of California, Davis
      • Department of Chemistry
      Davis, California, United States
  • 2005
    • Oregon State University
      • College of Pharmacy
      Corvallis, Oregon, United States