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Sample records for broad range 16s

  1. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    DOE PAGESBeta

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more » 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

  2. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    SciTech Connect

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  3. Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis

    PubMed Central

    Lekang, Katrine; Langeland, Nina; Wiker, Harald G.

    2011-01-01

    The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing. PMID:21436365

  4. Absolute calibration for a broad range single shot electron spectrometer

    SciTech Connect

    Glinec, Y.; Faure, J.; Guemnie-Tafo, A.; Malka, V.; Monard, H.; Larbre, J. P.; De Waele, V.; Marignier, J. L.; Mostafavi, M.

    2006-10-15

    This article gives a detailed description of a single shot electron spectrometer which was used to characterize electron beams produced by laser-plasma interaction. Contrary to conventional electron sources, electron beams from laser-plasma accelerators can produce a broad range of energies. Therefore, diagnosing these electron spectra requires specific attention and experimental development. Here, we provide an absolute calibration of the Lanex Kodak Fine screen on a laser-triggered radio frequency picosecond electron accelerator. The efficiency of scintillating screens irradiated by electron beams has never been investigated so far. This absolute calibration is then compared to charge measurements from an integrating current transformer for quasimonoenergetic electron spectra from laser-plasma interaction.

  5. Method for detection and imaging over a broad spectral range

    DOEpatents

    Yefremenko, Volodymyr; Gordiyenko, Eduard; Pishko, legal representative, Olga; Novosad, Valentyn; Pishko, deceased; Vitalii

    2007-09-25

    A method of controlling the coordinate sensitivity in a superconducting microbolometer employs localized light, heating or magnetic field effects to form normal or mixed state regions on a superconducting film and to control the spatial location. Electron beam lithography and wet chemical etching were applied as pattern transfer processes in epitaxial Y--Ba--Cu--O films. Two different sensor designs were tested: (i) a 3 millimeter long and 40 micrometer wide stripe and (ii) a 1.25 millimeters long, and 50 micron wide meandering-like structure. Scanning the laser beam along the stripe leads to physical displacement of the sensitive area, and, therefore, may be used as a basis for imaging over a broad spectral range. Forming the superconducting film as a meandering structure provides the equivalent of a two-dimensional detector array. Advantages of this approach are simplicity of detector fabrication, and simplicity of the read-out process requiring only two electrical terminals.

  6. Absolute calibration for a broad range single shot electron spectrometer

    NASA Astrophysics Data System (ADS)

    Glinec, Y.; Faure, J.; Guemnie-Tafo, A.; Malka, V.; Monard, H.; Larbre, J. P.; De Waele, V.; Marignier, J. L.; Mostafavi, M.

    2006-10-01

    This article gives a detailed description of a single shot electron spectrometer which was used to characterize electron beams produced by laser-plasma interaction. Contrary to conventional electron sources, electron beams from laser-plasma accelerators can produce a broad range of energies. Therefore, diagnosing these electron spectra requires specific attention and experimental development. Here, we provide an absolute calibration of the Lanex Kodak Fine screen on a laser-triggered radio frequency picosecond electron accelerator. The efficiency of scintillating screens irradiated by electron beams has never been investigated so far. This absolute calibration is then compared to charge measurements from an integrating current transformer for quasimonoenergetic electron spectra from laser-plasma interaction.

  7. Approaches to a broad range of high performance PDT sensitizers

    NASA Astrophysics Data System (ADS)

    d'A. Rocha Gonsalves, António M.; Serra, Arménio C.; Pineiro, Marta; Botelho, M. Filomena

    2009-02-01

    Starting from expertise in the area of chemical synthesis, particularly in tetrapyrrolic macrocycles and an interest in modelling structures for particular objectives, we came to the point of aiming at modelling photochemical sensitizers designed for photodynamic therapy (PDT) purposes. Our endeavours were gratifying when it was proved that our synthetic methodologies allowed for the easy availability of properly halogenated porphyrins with high quantum yield singlet oxygen efficiency. Joining the presence of this heavy atom and other functionalities as substituents in selected positions of macrocyclic structures we were able to generate novel porphyrin structures whose photophysical and photochemical properties, singlet oxygen formation quantum yields, photobleaching and logP were measured. Cellular uptake measurements and cytotoxicity assays on WiDr adenocarcinoma and A375 tumor cell lines were carried out and some of our porphyrins demonstrated very high performance as PDT sensitizers comparatively to known compounds approved for clinical use and in the market. Further developments of our studies allowed for the generation of different and more efficient structures, easily made available by our own synthetic methodologies. Our studies in this area allowed us to reach a stage which we believe to correspond to a significant knowledge and capacity to synthesise a broad range of simple structures, whose selectivity and efficiency as PDT sensitizers can be modulated for different cellular and tissue specificities. Our most recent developments in this area will be presented in this communication.

  8. Characterization and potential of three temperature ranges for hydrogen fermentation of cellulose by means of activity test and 16s rRNA sequence analysis.

    PubMed

    Gadow, Samir I; Jiang, Hongyu; Li, Yu-You

    2016-06-01

    A series of standardized activity experiments were performed to characterize three different temperature ranges of hydrogen fermentation from different carbon sources. 16S rRNA sequences analysis showed that the bacteria were close to Enterobacter genus in the mesophilic mixed culture (MMC) and Thermoanaerobacterium genus in the thermophilic and hyper-thermophilic mixed cultures (TMC and HMC). The MMC was able to utilize the glucose and cellulose to produce methane gas within a temperature range between 25 and 45 °C and hydrogen gas from 35 to 60°C. While, the TMC and HMC produced only hydrogen gas at all temperature ranges and the highest activity of 521.4mlH2/gVSSd was obtained by TMC. The thermodynamic analysis showed that more energy is consumed by hydrogen production from cellulose than from glucose. The experimental results could help to improve the economic feasibility of cellulosic biomass energy using three-phase technology to produce hythane. PMID:26954308

  9. Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling.

    PubMed

    Barbier, Mariette; Damron, F Heath

    2016-01-01

    Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications. PMID:26937640

  10. Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling

    PubMed Central

    Barbier, Mariette; Damron, F. Heath

    2016-01-01

    Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications. PMID:26937640

  11. Laser-guide-stars used for cophasing broad capture ranges

    NASA Astrophysics Data System (ADS)

    Martinez, P.; Janin-Potiron, P.

    2016-08-01

    Context. Segmented primary mirrors are indispensable to master the steady increase in spatial resolution. Phasing optics systems must reduce segment misalignments to guarantee the high optical quality required for astronomical science programs. Aims: Modern telescopes routinely use adaptive optics systems to compensate for the atmosphere and use laser-guide-stars to create artificial stars as bright references in the field of observation. Because multiple laser-guide-star adaptive optics are being implemented in all major observatories, we propose to use man-made stars not only for adaptive optics, but for phasing optics. Methods: We propose a method called the doublet-wavelength coherence technique (DWCT), exploiting the D lines of sodium in the mesosphere using laser guide-stars. The signal coherence properties are then used. Results: The DWCT capture range exceeds current abilities by a factor of 100. It represents a change in paradigm by improving the phasing optics capture range from micrometric to millimetric. It thereby potentially eliminates the need of a man-made mechanical pre-phasing step. Conclusions: Extremely large telescopes require hundreds of segments, several of which need to be substituted on a daily basis to be recoated. The DWCT relaxes mechanical integration requirements and speeds up integration and re-integration process.

  12. BRAHMS (Broad Range Hadron Magnetic Spectrometer) Figures and Data Archive

    DOE Data Explorer

    The BRAHMS experiment was designed to measure charged hadrons over a wide range of rapidity and transverse momentum to study the reaction mechanisms of the relativistic heavy ion reactions at the Relativistic Heavy Ion Collider at Brookhaven National Laboratory and the properties of the highly excited nuclear matter formed in these reactions. The experiment took its first data during the RHIC 2000 year run and completed data taking in June 2006. The BRAHMS archive makes publications available and also makes data and figures from those publications available as separate items. See also the complete list of publications, multimedia presentations, and related papers at http://www4.rcf.bnl.gov/brahms/WWW/publications.html

  13. Bacterial diversity of soil in the vicinity of Pindari glacier, Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones.

    PubMed

    Shivaji, S; Pratibha, M S; Sailaja, B; Hara Kishore, K; Singh, Ashish K; Begum, Z; Anarasi, Uttam; Prabagaran, S R; Reddy, G S N; Srinivas, T N R

    2011-01-01

    Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected near Pindari glacier, Himalayas. The three libraries yielded a total of 703 clones. Actinobacteria, Firmicutes and Proteobacteria were common to the three libraries. In addition to the above P1L and P8L shared the phyla Acidobacteria, Bacteroidetes, Gemmatimonadetes and Planctomycetes. Phyla Chlamydiae, Chlorobi, Chloroflexi, Dictyoglomi, Fibrobacteres, Nitrospirae, Verrucomicrobia, candidate division SPAM and candidate TM7s TM7a phylum were present only in P1L. Rarefaction analysis indicated that the bacterial diversity in P4S and P8S soil samples was representative of the sample. Principal component analysis (PCA) revealed that P1S and P8S were different from P4S soil sample. PCA also indicated that arsenic content, pH, Cr and altitude influence the observed differences in the percentage of specific OTUs in the three 16S rRNA gene clone libraries. The observed bacterial diversity was similar to that observed for other Himalayan and non-polar cold habitats. A total of 40 strains of bacteria were isolated from the above three soil samples and based on the morphology 20 bacterial strains were selected for further characterization. The 20 bacteria belonged to 12 different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase and urease activities were detected in majority of the strains but lipase and protease activities were not detected. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria. PMID:21061031

  14. Complete genome sequence of the broad-host-range strain Sinorhizobium fredii USDA257

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we announce the complete genome sequence of the symbiotic and nitrogen fixing bacterium Sinorhizobium fredii USDA257. The genome shares a high degree of similarity with the closely related broad-host-range strains S. fredii NGR234 and HH103. Most striking, the USDA257 genome encodes for a wealt...

  15. Monolithic front-end preamplifiers for a broad range of calorimetry applications

    SciTech Connect

    Radeka, V.; Rescia, S.; Manfredi, P.F.; Speziali, V. |

    1993-12-31

    The present paper summarizes the salient results of a research and development activity in the area of low noise preamplifiers for different applications in calorimetry. Design target for all circuits considered here are low noise, ability to cope with broad energy ranges and radiation hardness.

  16. Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

    PubMed Central

    Brom, Susana; Martinez, Esperanza; Dávila, Guillermo; Palacios, Rafael

    1988-01-01

    Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix+ phenotype to the Agrobacterium transconjugants. A Fix− phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris. Images PMID:16347637

  17. Infective Endocarditis Caused by Panton-Valentine Leukocidin-producing Methicillin-susceptible Staphylococcus aureus Identified by the Broad-range PCR Method.

    PubMed

    Nakajima, Hiroshi; Dohi, Kaoru; Tanabe, Masaki; Nakamura, Akiko; Kanemitsu, Shinji; Wada, Hideo; Yamada, Norikazu; Nobori, Tsutomu; Shinpo, Hideto; Ito, Masaaki

    2016-01-01

    A 76-year-old man was admitted to a community hospital due to a persistent high fever. He became afebrile after the administration of broad-spectrum antibiotics, but developed heart failure due to progressive aortic and mitral valve insufficiency and was transferred to our hospital. Although sequential blood cultures were negative, a broad-range polymerase chain reaction targeting the bacterial 16S-rRNA gene followed by the direct sequencing of whole blood revealed spa(+), mecA(-) and Panton-Valentine leukocidin (PVL)(+). He was finally diagnosed with infective endocarditis (IE) caused by PVL-producing methicillin-susceptible Staphylococcus aureus (MSSA), and underwent cardiac surgery. This is the first reported case of IE due to MSSA producing PVL. PMID:27432095

  18. Broad Detection Range Rhenium Diselenide Photodetector Enhanced by (3-Aminopropyl)Triethoxysilane and Triphenylphosphine Treatment.

    PubMed

    Jo, Seo-Hyeon; Park, Hyung-Youl; Kang, Dong-Ho; Shim, Jaewoo; Jeon, Jaeho; Choi, Seunghyuk; Kim, Minwoo; Park, Yongkook; Lee, Jaehyeong; Song, Young Jae; Lee, Sungjoo; Park, Jin-Hong

    2016-08-01

    The effects of triphenylphosphine and (3-aminopropyl)triethoxysilane on a rhenium diselenide (ReSe2 ) photodetector are systematically studied by comparing with conventional MoS2 devices. This study demonstrates a very high performance ReSe2 photodetector with high photoresponsivity (1.18 × 10(6) A W(-1) ), fast photoswitching speed (rising/decaying time: 58/263 ms), and broad photodetection range (possible above 1064 nm). PMID:27167366

  19. Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range

    PubMed Central

    Tabakman, Scott M.; Lau, Lana; Robinson, Joshua T.; Price, Jordan; Sherlock, Sarah P.; Wang, Hailiang; Zhang, Bo; Chen, Zhuo; Tangsombatvisit, Stephanie; Jarrell, Justin A.; Utz, Paul J.; Dai, Hongjie

    2012-01-01

    Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications. PMID:21915108

  20. High-sensitive and broad-dynamic-range quantitative phase imaging with spectral domain phase microscopy.

    PubMed

    Yan, Yangzhi; Ding, Zhihua; Shen, Yi; Chen, Zhiyan; Zhao, Chen; Ni, Yang

    2013-11-01

    Spectral domain phase microscopy for high-sensitive and broad-dynamic-range quantitative phase imaging is presented. The phase retrieval is realized in the depth domain to maintain a high sensitivity, while the phase information obtained in the spectral domain is exploited to extend the dynamic range of optical path difference. Sensitivity advantage of phase retrieved in the depth domain over that in the spectral domain is thoroughly investigated. The performance of the proposed depth domain phase based approach is illustrated by phase imaging of a resolution target and an onion skin. PMID:24216799

  1. Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994.

    PubMed Central

    Gutell, R R

    1994-01-01

    A collection of diverse 16S and 16S-like rRNA secondary structure diagrams are available. This set of rRNAs contains representative structures from all of the major phylogenetic groupings--Archaea, (eu)Bacteria, and the nucleus, mitochondrion, and chloroplast of Eucarya. Within this broad phylogenetic sampling are examples of the major forms of structural diversity currently known for this class of rRNAs. These structure diagrams are available online through our computer-network WWW server and anonymous ftp, as well as from the author in hardcopy format. PMID:7524024

  2. Transcriptome dynamics of a broad host-range cyanophage and its hosts.

    PubMed

    Doron, Shany; Fedida, Ayalla; Hernández-Prieto, Miguel A; Sabehi, Gazalah; Karunker, Iris; Stazic, Damir; Feingersch, Roi; Steglich, Claudia; Futschik, Matthias; Lindell, Debbie; Sorek, Rotem

    2016-06-01

    Cyanobacteria are highly abundant in the oceans and are constantly exposed to lytic viruses. The T4-like cyanomyoviruses are abundant in the marine environment and have broad host-ranges relative to other cyanophages. It is currently unknown whether broad host-range phages specifically tailor their infection program for each host, or employ the same program irrespective of the host infected. Also unknown is how different hosts respond to infection by the same phage. Here we used microarray and RNA-seq analyses to investigate the interaction between the Syn9 T4-like cyanophage and three phylogenetically, ecologically and genomically distinct marine Synechococcus strains: WH7803, WH8102 and WH8109. Strikingly, Syn9 led a nearly identical infection and transcriptional program in all three hosts. Different to previous assumptions for T4-like cyanophages, three temporally regulated gene expression classes were observed. Furthermore, a novel regulatory element controlled early-gene transcription, and host-like promoters drove middle gene transcription, different to the regulatory paradigm for T4. Similar results were found for the P-TIM40 phage during infection of Prochlorococcus NATL2A. Moreover, genomic and metagenomic analyses indicate that these regulatory elements are abundant and conserved among T4-like cyanophages. In contrast to the near-identical transcriptional program employed by Syn9, host responses to infection involved host-specific genes primarily located in hypervariable genomic islands, substantiating islands as a major axis of phage-cyanobacteria interactions. Our findings suggest that the ability of broad host-range phages to infect multiple hosts is more likely dependent on the effectiveness of host defense strategies than on differential tailoring of the infection process by the phage. PMID:26623542

  3. Scattering of an ultrashort electromagnetic radiation pulse by an atom in a broad spectral range

    SciTech Connect

    Astapenko, V. A.

    2011-02-15

    The scattering of an ultrashort electromagnetic pulse by atomic particles is described using a consistent quantum-mechanical approach taking into account excitation of a target and nondipole electromagnetic interaction, which is valid in a broad spectral range. This approach is applied to the scattering of single- and few-cycle pulses by a multielectron atom and a hydrogen atom. Scattering spectra are obtained for ultrashort pulses of different durations. The relative contribution of 'elastic' scattering of a single-cycle pulse by a hydrogen atom is studied in the high-frequency limit as a function of the carrier frequency and scattering angle.

  4. Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria

    PubMed Central

    Taton, Arnaud; Unglaub, Federico; Wright, Nicole E.; Zeng, Wei Yue; Paz-Yepes, Javier; Brahamsha, Bianca; Palenik, Brian; Peterson, Todd C.; Haerizadeh, Farzad; Golden, Susan S.; Golden, James W.

    2014-01-01

    Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains. PMID:25074377

  5. Time-of-flight electron spectrometer for a broad range of kinetic energies.

    PubMed

    Kothe, Alexander; Metje, Jan; Wilke, Martin; Moguilevski, Alexandre; Engel, Nicholas; Al-Obaidi, Ruba; Richter, Clemens; Golnak, Ronny; Kiyan, Igor Yu; Aziz, Emad F

    2013-02-01

    A newly constructed time-of-flight electron spectrometer of the magnetic bottle type is characterized for electron detection in a broad range of kinetic energies. The instrument is designed to measure the energy spectra of electrons generated from liquids excited by strong laser fields and photons in the range of extreme ultra violet and soft X-rays. Argon inner shell electrons were recorded to calibrate the spectrometer and investigate its characteristics, such as energy resolution and collection efficiency. Its energy resolution ΔE/E of 1.6% allows resolving the Ar 2p spin orbit structure at kinetic energies higher than 100 eV. The collection efficiency is determined and compared to that of the spectrometer in its field-free configuration. PMID:23464194

  6. Time-of-flight electron spectrometer for a broad range of kinetic energies

    NASA Astrophysics Data System (ADS)

    Kothe, Alexander; Metje, Jan; Wilke, Martin; Moguilevski, Alexandre; Engel, Nicholas; Al-Obaidi, Ruba; Richter, Clemens; Golnak, Ronny; Kiyan, Igor Yu.; Aziz, Emad F.

    2013-02-01

    A newly constructed time-of-flight electron spectrometer of the magnetic bottle type is characterized for electron detection in a broad range of kinetic energies. The instrument is designed to measure the energy spectra of electrons generated from liquids excited by strong laser fields and photons in the range of extreme ultra violet and soft X-rays. Argon inner shell electrons were recorded to calibrate the spectrometer and investigate its characteristics, such as energy resolution and collection efficiency. Its energy resolution ΔE/E of 1.6% allows resolving the Ar 2p spin orbit structure at kinetic energies higher than 100 eV. The collection efficiency is determined and compared to that of the spectrometer in its field-free configuration.

  7. Time-of-flight electron spectrometer for a broad range of kinetic energies

    SciTech Connect

    Kothe, Alexander; Metje, Jan; Wilke, Martin; Moguilevski, Alexandre; Engel, Nicholas; Al-Obaidi, Ruba; Richter, Clemens; Golnak, Ronny; Kiyan, Igor Yu.; Aziz, Emad F.

    2013-02-15

    A newly constructed time-of-flight electron spectrometer of the magnetic bottle type is characterized for electron detection in a broad range of kinetic energies. The instrument is designed to measure the energy spectra of electrons generated from liquids excited by strong laser fields and photons in the range of extreme ultra violet and soft X-rays. Argon inner shell electrons were recorded to calibrate the spectrometer and investigate its characteristics, such as energy resolution and collection efficiency. Its energy resolution {Delta}E/E of 1.6% allows resolving the Ar 2p spin orbit structure at kinetic energies higher than 100 eV. The collection efficiency is determined and compared to that of the spectrometer in its field-free configuration.

  8. Development of a gene silencing DNA vector derived from a broad host range geminivirus

    PubMed Central

    Golenberg, Edward M; Sather, D Noah; Hancock, Leandria C; Buckley, Kenneth J; Villafranco, Natalie M; Bisaro, David M

    2009-01-01

    Background Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems. Results The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant. Conclusion The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility. PMID:19573239

  9. Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines from a Broad Range of Culturable Bacterial Species

    PubMed Central

    Moor, Kathrin; Wotzka, Sandra Y.; Toska, Albulena; Diard, Médéric; Hapfelmeier, Siegfried; Slack, Emma

    2016-01-01

    Our mucosal surfaces are the main sites of non-vector-borne pathogen entry, as well as the main interface with our commensal microbiota. We are still only beginning to understand how mucosal adaptive immunity interacts with commensal and pathogenic microbes to influence factors such as infectivity, phenotypic diversity, and within-host evolution. This is in part due to difficulties in generating specific mucosal adaptive immune responses without disrupting the mucosal microbial ecosystem itself. Here, we present a very simple tool to generate inactivated mucosal vaccines from a broad range of culturable bacteria. Oral gavage of 1010 peracetic acid-inactivated bacteria induces high-titer-specific intestinal IgA in the absence of any measurable inflammation or species invasion. As a proof of principle, we demonstrate that this technique is sufficient to provide fully protective immunity in the murine model of invasive non-typhoidal Salmonellosis, even in the face of severe innate immune deficiency. PMID:26904024

  10. Mechanical characteristics of alloy AMg6M in broad temperature and strain-rate ranges

    SciTech Connect

    Krashchenko, V.P.; Dvoeglazov, G.A.; Ermolaev, G.V.; Rudnitskii, N.P.

    1986-02-01

    The authors study the mechanical properties of an aluminum-magnesium alloy in broad ranges of temperature and strain rate, and the change in hardness and dynamic modulus of elasticity in relation to temperature. The material chosen for study was alloy AMg6M. The heat treating process is described. The experimental data obtained on Micro-6, UVT-2, and UP-7 units is shown graphically. It is apparent that an increase in temperature is accompanied by a decrease in hardness, elastic modulus, and strength in general. Models are obtained and additional tests of specimens at the strain rate 3.3 X 10/sup -2/ sec/sup -1/ and different temperatures are conducted. The dependences must be calculated using all possible models, and the lowest resulting value must be chosen.

  11. Monoglyceride-based organogelator for broad-range oil uptake with high capacity.

    PubMed

    Wang, Dong; Niu, Jian; Wang, Zhenggong; Jin, Jian

    2015-02-10

    Oil/water separation has been a worldwide subject because of increasing release of oil-containing wastewater as well as several marine oil spills. The phase-selective organogelators (PSOGs) are thought to offer a potential and effective implement for addressing this issue. An ideal PSOG for oil adsorption must fulfill some requirements involving effective gelation, easy synthesis, low cost, and recyclable for reuse. However, beyond those, the ability of gelation for a broad-range oil phase without selectivity is also important. However, most of the reported PSOGs have limitation in this respect thus far. In this paper, a new class of saturated 1-monoglyceride-derived organogelators can efficiently uptake almost all of the common fuel oils from water and gelate organic solvents with extremely low minimum gelation concentration (MGC). In addition, the oils in the gel and gelator molecules can be recovered quantitatively through simple vacuum distillation. PMID:25604733

  12. Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines from a Broad Range of Culturable Bacterial Species.

    PubMed

    Moor, Kathrin; Wotzka, Sandra Y; Toska, Albulena; Diard, Médéric; Hapfelmeier, Siegfried; Slack, Emma

    2016-01-01

    Our mucosal surfaces are the main sites of non-vector-borne pathogen entry, as well as the main interface with our commensal microbiota. We are still only beginning to understand how mucosal adaptive immunity interacts with commensal and pathogenic microbes to influence factors such as infectivity, phenotypic diversity, and within-host evolution. This is in part due to difficulties in generating specific mucosal adaptive immune responses without disrupting the mucosal microbial ecosystem itself. Here, we present a very simple tool to generate inactivated mucosal vaccines from a broad range of culturable bacteria. Oral gavage of 10(10) peracetic acid-inactivated bacteria induces high-titer-specific intestinal IgA in the absence of any measurable inflammation or species invasion. As a proof of principle, we demonstrate that this technique is sufficient to provide fully protective immunity in the murine model of invasive non-typhoidal Salmonellosis, even in the face of severe innate immune deficiency. PMID:26904024

  13. Nanoporous Anodic Alumina 3D FDTD Modelling for a Broad Range of Inter-pore Distances.

    PubMed

    Bertó-Roselló, Francesc; Xifré-Pérez, Elisabet; Ferré-Borrull, Josep; Pallarès, Josep; Marsal, Lluis F

    2016-12-01

    The capability of the finite difference time domain (FDTD) method for the numerical modelling of the optical properties of nanoporous anodic alumina (NAA) in a broad range of inter-pore distances is evaluated. FDTD permits taking into account in the same numerical framework all the structural features of NAA, such as the texturization of the interfaces or the incorporation of electrolyte anions in the aluminium oxide host. The evaluation is carried out by comparing reflectance measurements from two samples with two very different inter-pore distances with the simulation results. Results show that considering the texturization is crucial to obtain good agreement with the measurements. On the other hand, including the anionic layer in the model leads to a second-order contribution to the reflectance spectrum. PMID:27518230

  14. High-Sensitivity, Broad-Range Vacuum Gauge Using Nanotubes for Micromachined Cavities

    NASA Technical Reports Server (NTRS)

    Manohara, Harish; Kaul, Anupama B.

    2011-01-01

    A broad-range vacuum gauge has been created by suspending a single-walled carbon nanotube (SWNT) (metallic or semiconducting) in a Schottky diode format or in a bridge conductor format, between two electrically charged mesas. SWNTs are highly sensitive to molecular collisions because of their extremely small diameters in the range of 1 to 3 nanometers. The measurement parameter will be the change in conductivity of SWNT due to decreasing rate of molecular collisions as the pressure inside a chamber decreases. The rate of heat removal approaches a saturation limit as the mean free path (m.f.p.) lengths of molecules increase due to decreasing pressure. Only those sensing elements that have a long relaxation time can produce a measureable response when m.f.p. of molecules increases (or time between two consecutive collisions increases). A suspended SWNT offers such a capability because of its one-dimensional nature and ultrasmall diameter. In the initial approach, similar architecture was used as that of a SWNT-Schottky diode that has been developed at JPL, and has its changing conductivity measured as the test chamber is pumped down from atmospheric pressure to high vacuum (10(exp -7) Torr). Continuous response of decreasing conductivity has been measured as a function of decreasing pressure (SWNT is a negative thermal coefficient material) from atmosphere to less than 10(exp -6) Torr. A measureable current change in the hundreds of nA range has been recorded in the 10(exp -6) Torr regime.

  15. Noradrenaline transmission reducing drugs may protect against a broad range of diseases.

    PubMed

    Fitzgerald, P J

    2015-04-01

    1 A growing body of evidence suggests that the signalling molecule, noradrenaline (NA), plays a pathophysiological role in a broad range of psychiatric, neurological and peripheral disorders. Both preclinical and clinical data suggest that elevated NA signalling may be involved in the aetiology of major diseases such as depression, Alzheimer's disease and diabetes mellitus. 2 The molecular pathways by which NA may cause the manifestation of disease remain poorly understood, although they may include G protein-coupled receptor modulation of the Ras/MAP kinase, Stat3 and PI3K pathways, among others. In both individual animals and humans, NA tone may be elevated largely due to genetics, but also because of the exposure to marked psychological stress or trauma, or other environmental factors. 3 As NA is involved in the 'fight or flight' response by the sympathetic nervous system, this transmitter may be elevated in a large number of organisms due to evolutionary selection of enhancing responses to immediate environmental dangers. Likewise, acetylcholine signalling by the parasympathetic ('rest and digest') nervous system may be relatively diminished. This putative autonomic imbalance may result in diminished engagement in homeostatic processes, resulting in the emergence and progression of a number of diseases throughout the body. 4 In this scenario, a large number of individuals may benefit from chronic use of pharmacological agents - such as clonidine, guanfacine, propranolol or prazosin - that diminish NA signalling throughout the body. If so, NA transmission lowering drugs may protect against a wide range of diseases. PMID:25271382

  16. Radiometric calibration method for large aperture infrared system with broad dynamic range.

    PubMed

    Sun, Zhiyuan; Chang, Songtao; Zhu, Wei

    2015-05-20

    Infrared radiometric measurements can acquire important data for missile defense systems. When observation is carried out by ground-based infrared systems, a missile is characterized by long distance, small size, and large variation of radiance. Therefore, the infrared systems should be manufactured with a larger aperture to enhance detection ability and calibrated at a broader dynamic range to extend measurable radiance. Nevertheless, the frequently used calibration methods demand an extended-area blackbody with broad dynamic range or a huge collimator for filling the system's field stop, which would greatly increase manufacturing costs and difficulties. To overcome this restriction, a calibration method based on amendment of inner and outer calibration is proposed. First, the principles and procedures of this method are introduced. Then, a shifting strategy of infrared systems for measuring targets with large fluctuations of infrared radiance is put forward. Finally, several experiments are performed on a shortwave infrared system with Φ400  mm aperture. The results indicate that the proposed method cannot only ensure accuracy of calibration but have the advantage of low cost, low power, and high motility. Hence, it is an effective radiometric calibration method in the outfield. PMID:26192499

  17. Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans

    PubMed Central

    2013-01-01

    Background Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called “universal” primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms. Results Amplification tests conducted on a wide range of marine animal taxa, rendered possible the first–time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species. Conclusions These primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory. PMID:24020880

  18. A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

    PubMed Central

    Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca

    2015-01-01

    Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. PMID:25746986

  19. Personal glucose meters for detection and quantification of a broad range of analytes

    SciTech Connect

    Lu, Yi; Xiang, Yu

    2015-02-03

    A general methodology for the development of highly sensitive and selective sensors that can achieve portable, low-cost and quantitative detection of a broad range of targets using only a personal glucose meter (PGM) is disclosed. The method uses recognition molecules that are specific for a target agent, enzymes that can convert an enzyme substrate into glucose, and PGM. Also provided are sensors, which can include a solid support to which is attached a recognition molecule that permits detection of a target agent, wherein the recognition molecule specifically binds to the target agent in the presence of the target agent but not significantly to other agents as well as an enzyme that can catalyze the conversion of a substance into glucose, wherein the enzyme is attached directly or indirectly to the recognition molecule, and wherein in the presence of the target agent the enzyme can convert the substance into glucose. The disclosed sensors can be part of a lateral flow device. Methods of using such sensors for detecting target agents are also provided.

  20. Streptolysin S of Streptococcus anginosus exhibits broad-range hemolytic activity.

    PubMed

    Asam, Daniela; Mauerer, Stefanie; Spellerberg, Barbara

    2015-04-01

    Streptococcus anginosus is a commensal of mucous membranes and an emerging human pathogen. Some strains, including the type strain, display a prominent β-hemolytic phenotype. A gene cluster (sag), encoding a variant of streptolysin S (SLS) has recently been identified as the genetic background for β-hemolysin production in S. anginosus. In this study, we further characterized the hemolytic and cytolytic activity of the S. anginosus hemolysin in comparison with other streptococcal hemolysins. The results indicate that SLS of S. anginosus is a broad-range hemolysin able to lyse erythrocytes of different species, including horse, bovine, rabbit and even chicken. The hemolytic activity is temperature dependent, and a down-regulation of the hemolysin expression is induced in the presence of high glucose levels. Survival assays indicate that in contrast to other streptococcal species, S. anginosus does not require SLS for survival in the presence of human granulocytes. Cross-complementation studies using the sagB and sagD genes of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis demonstrated functional similarities to the S. anginosus SLS. Nevertheless, distinct differences to other streptolysin S variants were noted and provide further insights into the molecular mechanisms of SLS pathogen host interactions. PMID:25381594

  1. Oxidative Stress and Mitochondrial Dysfunction across Broad-Ranging Pathologies: Toward Mitochondria-Targeted Clinical Strategies

    PubMed Central

    d'Ischia, Marco; Gadaleta, Maria Nicola; Pallardó, Federico V.; Petrović, Sandra; Tiano, Luca; Zatterale, Adriana

    2014-01-01

    Beyond the disorders recognized as mitochondrial diseases, abnormalities in function and/or ultrastructure of mitochondria have been reported in several unrelated pathologies. These encompass ageing, malformations, and a number of genetic or acquired diseases, as diabetes and cardiologic, haematologic, organ-specific (e.g., eye or liver), neurologic and psychiatric, autoimmune, and dermatologic disorders. The mechanistic grounds for mitochondrial dysfunction (MDF) along with the occurrence of oxidative stress (OS) have been investigated within the pathogenesis of individual disorders or in groups of interrelated disorders. We attempt to review broad-ranging pathologies that involve mitochondrial-specific deficiencies or rely on cytosol-derived prooxidant states or on autoimmune-induced mitochondrial damage. The established knowledge in these subjects warrants studies aimed at elucidating several open questions that are highlighted in the present review. The relevance of OS and MDF in different pathologies may establish the grounds for chemoprevention trials aimed at compensating OS/MDF by means of antioxidants and mitochondrial nutrients. PMID:24876913

  2. A diffraction-limited scanning system providing broad spectral range for laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Yu, Jiun-Yann; Liao, Chien-Sheng; Zhuo, Zong-Yan; Huang, Chen-Han; Chui, Hsiang-Chen; Chu, Shi-Wei

    2009-11-01

    Diversified research interests in scanning laser microscopy nowadays require broadband capability of the optical system. Although an all-mirror-based optical design with a suitable metallic coating is appropriate for broad-spectrum applications from ultraviolet to terahertz, most researchers prefer lens-based scanning systems despite the drawbacks of a limited spectral range, ghost reflection, and chromatic aberration. One of the main concerns is that the geometrical aberration induced by off-axis incidence on spherical mirrors significantly deteriorates image resolution. Here, we demonstrate a novel geometrical design of a spherical-mirror-based scanning system in which off-axis aberrations, both astigmatism and coma, are compensated to reach diffraction-limited performance. We have numerically simulated and experimentally verified that this scanning system meets the Marechà l condition and provides high Strehl ratio within a 3°×3° scanning area. Moreover, we demonstrate second-harmonic-generation imaging from starch with our new design. A greatly improved resolution compared to the conventional mirror-based system is confirmed. This scanning system will be ideal for high-resolution linear/nonlinear laser scanning microscopy, ophthalmoscopic applications, and precision fabrications.

  3. A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

    PubMed

    Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca; Muñoz, Rosario

    2015-05-01

    Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. PMID:25746986

  4. Forest response to elevated CO2 is conserved across a broad range of productivity

    SciTech Connect

    Norby, Richard J; DeLucia, E. H.; Gielen, Birgit; Califapietra, Carlo; Giardina, Christian P; King, John S.; Childs, Joanne; McCarthy, Heather R; Moore, D J; Ceulemans, Reinhart; DeAngelis, Paolo; Finzi, Adrien C; Karnosky, David; Kubiske, Mark E; Lukac, Martin; Pregitzer, Kurt; Scarascia-Mugnozza, Giuseppe E; Oren, Ram; Schlesinger, William H

    2005-12-01

    Climate change predictions derived from coupled carbon-climate models are highly dependent on assumptions about feedbacks between the biosphere and atmosphere. One critical feedback occurs if C uptake by the biosphere increases in response to the fossil-fuel driven increase in atmospheric [CO2] ('CO2 fertilization'), thereby slowing the rate of increase in atmospheric [CO2]. Carbon exchanges between the terrestrial biosphere and atmosphere are often first represented in models as net primary productivity (NPP). However, the contribution of CO2 fertilization to the future global C cycle has been uncertain, especially in forest ecosystems that dominate global NPP, and models that include a feedback between terrestrial biosphere metabolism and atmospheric [CO2] are poorly constrained by experimental evidence. We analyzed the response of NPP to elevated CO2 ({approx}550 ppm) in four free-air CO2 enrichment experiments in forest stands. We show that the response of forest NPP to elevated [CO2] is highly conserved across a broad range of productivity, with a stimulation at the median of 23 {+-} 2%. At low leaf area indices, a large portion of the response was attributable to increased light absorption, but as leaf area indices increased, the response to elevated [CO2] was wholly caused by increased light-use efficiency. The surprising consistency of response across diverse sites provides a benchmark to evaluate predictions of ecosystem and global models and allows us now to focus on unresolved questions about carbon partitioning and retention, and spatial variation in NPP response caused by availability of other growth limiting resources.

  5. Theoretical Study of Radiation from a Broad Range of Impurity Ions for Magnetic Fusion Diagnostics

    SciTech Connect

    Safronova, Alla

    2014-03-14

    Spectroscopy of radiation emitted by impurities plays an important role in the study of magnetically confined fusion plasmas. The measurements of these impurities are crucial for the control of the general machine conditions, for the monitoring of the impurity levels, and for the detection of various possible fault conditions. Low-Z impurities, typically present in concentrations of 1%, are lithium, beryllium, boron, carbon, and oxygen. Some of the common medium-Z impurities are metals such as iron, nickel, and copper, and high-Z impurities, such as tungsten, are present in smaller concentrations of 0.1% or less. Despite the relatively small concentration numbers, the aforementioned impurities might make a substantial contribution to radiated power, and also influence both plasma conditions and instruments. A detailed theoretical study of line radiation from impurities that covers a very broad spectral range from less than 1 Å to more than 1000 Å has been accomplished and the results were applied to the LLNL Electron Beam Ion Trap (EBIT) and the Sustained Spheromak Physics Experiment (SSPX) and to the National Spherical Torus Experiment (NSTX) at Princeton. Though low- and medium-Z impurities were also studied, the main emphasis was made on the comprehensive theoretical study of radiation from tungsten using different state-of-the-art atomic structure codes such as Relativistic Many-Body Perturbation Theory (RMBPT). The important component of this research was a comparison of the results from the RMBPT code with other codes such as the Multiconfigurational Hartree–Fock developed by Cowan (COWAN code) and the Multiconfiguration Relativistic Hebrew University Lawrence Atomic Code (HULLAC code), and estimation of accuracy of calculations. We also have studied dielectronic recombination, an important recombination process for fusion plasma, for variety of highly and low charged tungsten ions using COWAN and HULLAC codes. Accurate DR rate coefficients are needed for

  6. Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data

    PubMed Central

    Aßhauer, Kathrin P.; Wemheuer, Bernd; Daniel, Rolf; Meinicke, Peter

    2015-01-01

    Motivation: The characterization of phylogenetic and functional diversity is a key element in the analysis of microbial communities. Amplicon-based sequencing of marker genes, such as 16S rRNA, is a powerful tool for assessing and comparing the structure of microbial communities at a high phylogenetic resolution. Because 16S rRNA sequencing is more cost-effective than whole metagenome shotgun sequencing, marker gene analysis is frequently used for broad studies that involve a large number of different samples. However, in comparison to shotgun sequencing approaches, insights into the functional capabilities of the community get lost when restricting the analysis to taxonomic assignment of 16S rRNA data. Results: Tax4Fun is a software package that predicts the functional capabilities of microbial communities based on 16S rRNA datasets. We evaluated Tax4Fun on a range of paired metagenome/16S rRNA datasets to assess its performance. Our results indicate that Tax4Fun provides a good approximation to functional profiles obtained from metagenomic shotgun sequencing approaches. Availability and implementation: Tax4Fun is an open-source R package and applicable to output as obtained from the SILVAngs web server or the application of QIIME with a SILVA database extension. Tax4Fun is freely available for download at http://tax4fun.gobics.de/. Contact: kasshau@gwdg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25957349

  7. Broad host range vectors derived from an RSF1010::Tn1 plasmid.

    PubMed

    Chistoserdov, A Y; Tsygankov, Y D

    1986-11-01

    Plasmid vector derivatives of the IncQ/P4 plasmid RSF1010 available for cloning DNA into a broad range of bacterial species were constructed. The plasmid pAYC31 constructed for the positive selection of inserted fragments contains part of transposon Tn1 inserted into the sequence of the gene sul. Gene aph transcription in pAYC31 can be initiated from the promoter for the transposase gene tnpA which is under the negative control of the gene tnpR product (Heffron, 1983). The insertion of a BamHI fragment, or of fragments generated by Sau3A, BclI, BglII, or XhoII digestion into the unique BamHI site within the gene tnpR sequence, leads to initiation of transcription from the promoter of the tnpA gene toward the aph gene. Expression of the aph gene upon insertion of a BamHI restriction fragment provides a positive selection for hybrid plasmids by plating the transformed bacteria on media with streptomycin. Versatile cloning vectors pAYC32 of 9.7 kb in length and pAYC39 of 11.3 kb in length were also constructed. Insertion into the BamHI site of vector pAYC32 of a 1.6-kb BglII fragment that contains the lambda cos site produced cosmid vectors pAYC51 and pAYC52. The two 11.3-kb cosmids differ only by the orientation of the 1.6-kb BglII fragment. By insertion into the BamHI site of pAYC32 of a BglII-BamHI fragment of plasmid pHC79 that contains the gene tet and lambda cos site cosmid vector pAYC53 was constructed. Vector pAYC31 was used to construct a gene bank from the chromosomal DNA of an obligate methylotrophic strain Methylomicrobium flagellatum KT. PMID:3027724

  8. Broad-Host Range Vector-Particle: Gene Transfer Particles From Thermal Vents

    NASA Astrophysics Data System (ADS)

    Chiura, H. X.; Nakamura, K.; Fukazawa, Y.; Nakata, D.; Tomaru, A.; Okita, N.; Hoaki, T.

    2002-12-01

    Viruses or virus-like particles (VLPs) are common in aquatic ecosystems, however, VLP-host interactions and its commitments to gene transfer in the environment is yet unclear. We have proposed that at least some of the widely distributed VLPs could be general gene transfer agents among a wide range of microbial host cells, and might function as a universal vector (1-4). To elucidate such a broad host range gene transfer mediated by "VLP", the sampling site was extended to the hyper hydrothermal vent, and boring cores. VLP (v) and cell (b) abundances per ml water samples from drilling holes of Suiyo seamount were: APSK04 (28°34.303'N, 140°38.618'E, 1385 m deep, 21°C, b = 8.26 *E^{6}, v = 6.03 x 10^{6}); APSK07 (28°34.299'N, 140°38.690'E, 1386 m deep, 250.5°C, b = 5.33 \\times 104, v = 2.52 \\times 104); a natural vent near APSK05 (28°34.322'N, 140°38.594'E, 1382 m deep, 304.7°C, b = 3.23 x 10^{4}, v = 1.85 x 10^{4}). A boring core sample was obtained from APSK06 (28°34.313'N, 140°38.617', 1386 m deep), from which a hyper thermophilic Archaean, Thermococcus kodakaraensis was successfully cultivated in sulphur supplemented medium between 70 and 90°C. VLP production was observed from T. kodakaraensis, whose VLP (v) and cell (b) abundances per ml at 480 h culture at 70°C were: b = 3.61 *E^{9}, v = 3.46 *E^{9}. Transduction experiment at multiplicity of infection of ca 0.2 using particles from APSK07 and T. kodakaraensis showed a plate efficiency on recipient Escherichia coli AB1157 by ca 72 % and ca 89 % regardless of UV treatment of the particle. Gene transfer frequency of APSK07 particle was (x 10^{-5} cfu/particle) between 2.4 and 0.92, and that of T. kodakaraensis particle was between x 10^{-4} and x 10^{-5}$ cfu/particle. These findings suggest the non-specific gene transfer by such particles may be a ubiquitous event in the natural environment. Such gene transfer particles may have mediated gene flux among phylogenetically diverse microbial

  9. Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing

    PubMed Central

    Siala, Mariam; Jaulhac, Benoit; Gdoura, Radhouane; Sibilia, Jean; Fourati, Hela; Younes, Mohamed; Baklouti, Sofien; Bargaoui, Naceur; Sellami, Slaheddine; Znazen, Abir; Barthel, Cathy; Collin, Elody; Hammami, Adnane; Sghir, Abdelghani

    2008-01-01

    Introduction Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing. Methods We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples. Conclusion This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic

  10. Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples.

    PubMed

    Budding, Andries E; Hoogewerf, Martine; Vandenbroucke-Grauls, Christina M J E; Savelkoul, Paul H M

    2016-04-01

    Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. PMID:26763956

  11. Development of Broad Range Scan Capabilities with Jet Cooled Cavity Ringdown Spectroscopy

    NASA Astrophysics Data System (ADS)

    Codd, Terrance J.; Chen, Ming-Wei; Miller, Terry A.

    2011-06-01

    We have developed a technique for obtaining broad scans, >100 Cm-1, for jet cooled cavity ringdown spectroscopy (CRDS) spectra. Previously the scans of the jet cooled, CRDS apparatus were limited to <10 Cm-1 due to the use of a narrow linewidth radiation source. However, by coupling our jet cooled, CRDS apparatus with a moderate resolution (≃q 0.05 Cm-1) dye laser we are able to greatly increase our rate of data acquisition thereby gaining the capability to perform broad spectral surveys of jet cooled molecules. As a test of the capabilities of the technique we have scanned the tilde{A}-tilde{X} transition of NO_3 previously reported by Deev et al. at room temperature. We believe that this will be a very useful technique to search for transitions of cold molecules whose frequencies are not well known and which later can be studied using high resolution methods. A. Deev, J. Sommar, and M. Okumura, J. Chem. Phys. 122, 224305 (2005).

  12. Study of transmission line attenuation in broad band millimeter wave frequency range

    NASA Astrophysics Data System (ADS)

    Pandya, Hitesh Kumar B.; Austin, M. E.; Ellis, R. F.

    2013-10-01

    Broad band millimeter wave transmission lines are used in fusion plasma diagnostics such as electron cyclotron emission (ECE), electron cyclotron absorption, reflectometry and interferometry systems. In particular, the ECE diagnostic for ITER will require efficient transmission over an ultra wide band, 100 to 1000 GHz. A circular corrugated waveguide transmission line is a prospective candidate to transmit such wide band with low attenuation. To evaluate this system, experiments of transmission line attenuation were performed and compared with theoretical loss calculations. A millimeter wave Michelson interferometer and a liquid nitrogen black body source are used to perform all the experiments. Atmospheric water vapor lines and continuum absorption within this band are reported. Ohmic attenuation in corrugated waveguide is very low; however, there is Bragg scattering and higher order mode conversion that can cause significant attenuation in this transmission line. The attenuation due to miter bends, gaps, joints, and curvature are estimated. The measured attenuation of 15 m length with seven miter bends and eighteen joints is 1 dB at low frequency (300 GHz) and 10 dB at high frequency (900 GHz), respectively.

  13. Broad range of adverse cutaneous eruptions in patients on TNF-alpha antagonists.

    PubMed

    Hawryluk, Elena B; Linskey, Katy R; Duncan, Lyn M; Nazarian, Rosalynn M

    2012-05-01

    Biologic therapies targeting tumor necrosis factor (TNF)-alpha have become a mainstay in the management of a number of autoimmune diseases. We report a series of adverse skin eruptions in six patients (four females, two males, age: 21-58 years, mean: 39) receiving 4 months to 10 years (mean 3.1 years) of anti-TNF-alpha therapies (infliximab, n = 4; adalimumab, n = 1 or etanercept, n = 1). The following drug-associated diagnoses were made in eight skin biopsies performed at Massachusetts General Hospital between 3/2007 and 10/2010: pustular folliculitis, psoriasis, interface dermatitis, neutrophilic eccrine hidradenitis, Sweet's syndrome, lupus, vasculitis and palmoplantar pustulosis. The descriptions of neutrophilic eccrine hidradenitis-like and Sweet's-like hypersensitivity eruptions induced by anti-TNF-alpha therapies are the first such cases described in the literature. Each cutaneous eruption improved or resolved with switching to a different TNF-alpha inhibitor, discontinuation of the anti-TNF-alpha agent, and/or topical or systemic steroids. There was a clear chronologic relationship with, and clinical remission upon withdrawal or steroid suppression of the anti-TNF-alpha agents. The mechanism for such diverse cutaneous eruptions among this class of medications remains poorly understood. The cutaneous adverse reaction profile of TNF-alpha inhibitors is broad and should be considered in the histopathologic differential in this clinical setting. PMID:22515220

  14. Determination of metal ions by fluorescence anisotropy exhibits a broad dynamic range

    NASA Astrophysics Data System (ADS)

    Thompson, Richard B.; Maliwal, Badri P.; Fierke, Carol A.

    1998-05-01

    Recently, we have shown that metal ions free in solution may be determined at low levels by fluorescence anisotropy (polarization) measurements. Anisotropy measurements enjoy the advantages of wavelength ratiometric techniques for determining metal ions such as calcium, because anisotropy measurements are ratiometric as well. Furthermore, fluorescence anisotropy may be imaged in the microscope. An advantage of anisotropy not demonstrated for wavelength ratiometric approaches using indicators such as Fura-2 and Indo-1 is that under favorable circumstances anisotropy-based determinations exhibit a much broader dynamic range in metal ion concentration. Determinations of free Zn(II) in the picomolar range are demonstrated.

  15. Investigation of pulsed X-ray radiation of a plasma focus in a broad energy range

    SciTech Connect

    Savelov, A. S. Salakhutdinov, G. Kh.; Koltunov, M. V.; Lemeshko, B. D.; Yurkov, D. I.; Sidorov, P. P.

    2011-12-15

    The results of the experimental investigations of the spectral composition of plasma focus X-ray radiation in the photon energy range of 1.5 keV-400 keV are presented. Three regions in the radiation spectrum where the latter is of a quasi-thermal nature with a corresponding effective temperature are distinguished.

  16. Vent and relief valve maintains low leakage rate over broad temperature range

    NASA Technical Reports Server (NTRS)

    Weitenbeck, R. G.

    1968-01-01

    Low leakage rate, large diameter vent and relief valve operates satisfactorily over a large temperature range by a design that accommodates waviness and distortions due to thermal gradients. It is based on a fixed sealing member having an inclined lapped surface to which a flexible flow gate conforms.

  17. Design, calibration and application of broad-range optical nanosensors for determining intracellular pH.

    PubMed

    Søndergaard, Rikke V; Henriksen, Jonas R; Andresen, Thomas L

    2014-12-01

    Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks. PMID:25411952

  18. Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

    PubMed Central

    Waheed, Syed Mohsin; Ghosh, Arnab; Chakravarti, Ritu; Biswas, Ashis; Haque, Mohammad Mahfuzul; Panda, Koustubh; Stuehr, Dennis J.

    2010-01-01

    Although heme insertion into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process is not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3 h time period, and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structure, heme ligation, and function (three NO synthases, two cytochrome P450’s, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme level, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process. PMID:20211245

  19. Discovery of parvovirus-related sequences in an unexpected broad range of animals

    PubMed Central

    François, S.; Filloux, D.; Roumagnac, P.; Bigot, D.; Gayral, P.; Martin, D. P.; Froissart, R.; Ogliastro, M.

    2016-01-01

    Our knowledge of the genetic diversity and host ranges of viruses is fragmentary. This is particularly true for the Parvoviridae family. Genetic diversity studies of single stranded DNA viruses within this family have been largely focused on arthropod- and vertebrate-infecting species that cause diseases of humans and our domesticated animals: a focus that has biased our perception of parvovirus diversity. While metagenomics approaches could help rectify this bias, so too could transcriptomics studies. Large amounts of transcriptomic data are available for a diverse array of animal species and whenever this data has inadvertently been gathered from virus-infected individuals, it could contain detectable viral transcripts. We therefore performed a systematic search for parvovirus-related sequences (PRSs) within publicly available transcript, genome and protein databases and eleven new transcriptome datasets. This revealed 463 PRSs in the transcript databases of 118 animals. At least 41 of these PRSs are likely integrated within animal genomes in that they were also found within genomic sequence databases. Besides illuminating the ubiquity of parvoviruses, the number of parvoviral sequences discovered within public databases revealed numerous previously unknown parvovirus-host combinations; particularly in invertebrates. Our findings suggest that the host-ranges of extant parvoviruses might span the entire animal kingdom. PMID:27600734

  20. Discovery of parvovirus-related sequences in an unexpected broad range of animals.

    PubMed

    François, S; Filloux, D; Roumagnac, P; Bigot, D; Gayral, P; Martin, D P; Froissart, R; Ogliastro, M

    2016-01-01

    Our knowledge of the genetic diversity and host ranges of viruses is fragmentary. This is particularly true for the Parvoviridae family. Genetic diversity studies of single stranded DNA viruses within this family have been largely focused on arthropod- and vertebrate-infecting species that cause diseases of humans and our domesticated animals: a focus that has biased our perception of parvovirus diversity. While metagenomics approaches could help rectify this bias, so too could transcriptomics studies. Large amounts of transcriptomic data are available for a diverse array of animal species and whenever this data has inadvertently been gathered from virus-infected individuals, it could contain detectable viral transcripts. We therefore performed a systematic search for parvovirus-related sequences (PRSs) within publicly available transcript, genome and protein databases and eleven new transcriptome datasets. This revealed 463 PRSs in the transcript databases of 118 animals. At least 41 of these PRSs are likely integrated within animal genomes in that they were also found within genomic sequence databases. Besides illuminating the ubiquity of parvoviruses, the number of parvoviral sequences discovered within public databases revealed numerous previously unknown parvovirus-host combinations; particularly in invertebrates. Our findings suggest that the host-ranges of extant parvoviruses might span the entire animal kingdom. PMID:27600734

  1. Near-linear response of mean monsoon strength to a broad range of radiative forcings.

    PubMed

    Boos, William R; Storelvmo, Trude

    2016-02-01

    Theoretical models have been used to argue that seasonal mean monsoons will shift abruptly and discontinuously from wet to dry stable states as their radiative forcings pass a critical threshold, sometimes referred to as a "tipping point." Further support for a strongly nonlinear response of monsoons to radiative forcings is found in the seasonal onset of the South Asian summer monsoon, which is abrupt compared with the annual cycle of insolation. Here it is shown that the seasonal mean strength of monsoons instead exhibits a nearly linear dependence on a wide range of radiative forcings. First, a previous theory that predicted a discontinuous, threshold response is shown to omit a dominant stabilizing term in the equations of motion; a corrected theory predicts a continuous and nearly linear response of seasonal mean monsoon strength to forcings. A comprehensive global climate model is then used to show that the seasonal mean South Asian monsoon exhibits a near-linear dependence on a wide range of isolated greenhouse gas, aerosol, and surface albedo forcings. This model reproduces the observed abrupt seasonal onset of the South Asian monsoon but produces a near-linear response of the mean monsoon by changing the duration of the summer circulation and the latitude of that circulation's ascent branch. Thus, neither a physically correct theoretical model nor a comprehensive climate model support the idea that seasonal mean monsoons will undergo abrupt, nonlinear shifts in response to changes in greenhouse gas concentrations, aerosol emissions, or land surface albedo. PMID:26811462

  2. Broad-range neutron spectra identification in ultraintense laser interactions with carbon-deuterated plasma

    SciTech Connect

    Youssef, A.; Kodama, R.; Habara, H.; Tanaka, K.A.; Sentoku, Y.; Tampo, M.; Toyama, Y.

    2005-11-15

    Detailed neutron energy spectra produced from a CD2 target irradiated by a 450 fs, 20 J, 1053 nm laser at an intensity of 3x10{sup 18} W/cm{sup 2} have been studied. Wide-ranging neutron spectra were observed from two different observation angles 20 deg. and 70 deg. relative to the rear-side target normal. The experiment and numerically calculated spectra, by a three-dimensional Monte Carlo code, indicate that the range of the measured spectra is larger than that produced by the D(d,n){sup 3}He reaction. An interpretation for the measured spectra is introduced by considering the {sup 12}C(d,n){sup 13}N and D({sup 12}c,n){sup 13}N reactions. In addition, the study revealed that the neutron spectra produced by the D-C and C-D reactions can overlap that produced by the D-D reaction, and due to their high cross sections, comparing to the D-D reaction, both of them effectively participate in the neutron yield.

  3. Virtual Acoustic Testing of Spacecraft Over a Broad Frequency Range Using FEM, BEM and Sea

    NASA Astrophysics Data System (ADS)

    Vansant, K.; Borello, G.; De Langhe, K.; Courjal, A.

    2012-07-01

    During launch, a spacecraft is exposed to high levels of structural and acoustical loading. Acceptance tests are carried out before actual launch mimicking these loading conditions to validate that vibration, force and stress levels, which could damage the payload and lead to mission failure, remain below the design envelopes. The acceptance tests themselves, carried out on the actual payload, imply a risk of overtesting. Simulation models make it possible to carry out these tests in the virtual world. They can be used to derive specifications for the desired shaker, loudspeaker or horn excitation signals and to upfront quantify the risk of overtesting. Simulation also allows to uncover and rectify flaws or sensitivities in the spacecraft design rather quickly and at a low cost. This paper will discuss several simulation models (FEM, BEM and SEA) which can be used to mimic an acoustic acceptance test for different frequency ranges of interest.

  4. Near-linear response of mean monsoon strength to a broad range of radiative forcings

    PubMed Central

    Boos, William R.; Storelvmo, Trude

    2016-01-01

    Theoretical models have been used to argue that seasonal mean monsoons will shift abruptly and discontinuously from wet to dry stable states as their radiative forcings pass a critical threshold, sometimes referred to as a “tipping point.” Further support for a strongly nonlinear response of monsoons to radiative forcings is found in the seasonal onset of the South Asian summer monsoon, which is abrupt compared with the annual cycle of insolation. Here it is shown that the seasonal mean strength of monsoons instead exhibits a nearly linear dependence on a wide range of radiative forcings. First, a previous theory that predicted a discontinuous, threshold response is shown to omit a dominant stabilizing term in the equations of motion; a corrected theory predicts a continuous and nearly linear response of seasonal mean monsoon strength to forcings. A comprehensive global climate model is then used to show that the seasonal mean South Asian monsoon exhibits a near-linear dependence on a wide range of isolated greenhouse gas, aerosol, and surface albedo forcings. This model reproduces the observed abrupt seasonal onset of the South Asian monsoon but produces a near-linear response of the mean monsoon by changing the duration of the summer circulation and the latitude of that circulation’s ascent branch. Thus, neither a physically correct theoretical model nor a comprehensive climate model support the idea that seasonal mean monsoons will undergo abrupt, nonlinear shifts in response to changes in greenhouse gas concentrations, aerosol emissions, or land surface albedo. PMID:26811462

  5. Effect of spectral range in surface inactivation of Listeria innocua using broad-spectrum pulsed light.

    PubMed

    Woodling, Sarah E; Moraru, Carmen I

    2007-04-01

    Pulsed light (PL) treatment is an alternative to traditional thermal treatment that has the potential to achieve several log-cycle reductions in the concentration of microorganisms. One issue that is still debated is related to what specifically causes cell death after PL treatments. The main objective of this work was to elucidate which portions of the PL range are responsible for bacterial inactivation. Stainless steel coupons with controlled surface properties were inoculated with a known concentration of Listeria innocua in the stationary growth phase and treated with 1 to 12 pulses of light at a pulse rate of 3 pulses per s and a pulse width of 360 micros. The effects of the full spectrum (lambda = 180 to 1,100 nm) were compared with the effects obtained when only certain regions of UV, visible, and near-infrared light were used. The effectiveness of the treatments was determined in parallel by the standard plate count and most-probable-number techniques. At a fluence of about 6 J/cm(2), the full-spectrum PL treatment resulted in a 4.08-log reduction of L. innocua on a Mill finish surface, the removal of lambda < 200 nm diminished the reduction to only 1.64 log, and total elimination of UV light resulted in no lethal effects on L. innocua. Overwhelmingly, the portions of the PL spectrum responsible for bacterial death are the UV-B and UV-C spectral ranges (X < 300 nm), with some death taking place during exposure to UV-A radiation (300 < lambda < 400 nm) and no observable death upon exposure to visible and near-infrared light (lambda > 400 nm). This work provides additional supporting evidence that cell death in PL treatment is due to exposure to UV light. Additionally, it was shown that even a minor modification of the light path or the UV light spectrum in PL treatments can have a significant negative impact on the treatment intensity and effectiveness. PMID:17477260

  6. Passively-Switched, Non-Contact Energy Harvester for Broad Operational Range and Enhanced Durability

    NASA Astrophysics Data System (ADS)

    Zhu, William Z.; Livermore, Carol

    2015-12-01

    Impact-based vibrational energy harvesters that switch passively among dynamical modes to best match the ambient conditions and perform frequency up-conversion to maximize power output can increase the operational range over which harvesters output useful power. The disadvantage of impact-based harvesting is that it can lead to premature system failure. This paper presents modelling and experimental validation of a harvester with passively- switched dynamics in which a magnetic non-contact interaction replaces the impact-based interaction of previous systems for greater robustness. A low-frequency driving beam couples to a higher-frequency generating beam via tip magnets. Simulations predict that the noncontact interaction drives a diversity of dynamical modes in which the tip of the driving beam variously is repelled from and passes the tip of generating beam. Experiments validate the predicted dynamical behaviour of the system. Output power levels are similar to predictions, with a power of 903 μW on the primary resonance at 1 g acceleration.

  7. Conjugative transfer of broad host range plasmids to an acidobacterial strain, Edaphobacter aggregans.

    PubMed

    Bouhajja, Emna; Efthymiopoulos, Theocharis; George, Isabelle F; Moreels, David; Van Houdt, Rob; Mergeay, Max; Agathos, Spiros N

    2016-03-10

    The Acidobacteria phylum is of high ecological interest. Its members are ubiquitous and particularly abundant in soils but many are recalcitrant to cultivation in the laboratory. Thus, the ability of Acidobacteria to capture and maintain plasmids remains largely unexplored. In this work we tested the transfer and the stability of (i) the PromA plasmid pMOL98 and (ii) the IncQ plasmid pKT230 to the acidobacterial strain Edaphobacter aggregans DSM 19364. To this end quantitative conjugation assays were performed and transconjugants were scored for plasmid-borne antibiotic selection markers. The tested plasmids were transferred and maintained in the new host. Plasmid pMOL98 was more stable than pKT230 in Ed. aggregans in the absence of positive selection. Thus, from an ecological point of view, we have extended the host range of PromA and IncQ plasmids for the first time to an acidobacterial strain. Furthermore, we have uncovered the potential of Acidobacteria to capture as-yet-unknown plasmids and to foster the development of new cloning and expression systems for the exploitation of biotechnologically valuable soil resources. PMID:26808872

  8. Generation of high-titer pseudotyped retroviral vectors with very broad host range.

    PubMed

    Yee, J K; Friedmann, T; Burns, J C

    1994-01-01

    Encapsidation of the VSV G protein into the virions of MoMLV-derived retroviral vectors in the absence of other VSV-encoded proteins is shown to be an efficient process, although the exact mechanism for this process is currently unclear. Unlike the conventional retroviral vectors bearing the amphotropic envelope protein, the pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation. This property of the pseudotyped virus enables the generation of high-titer retroviral vector stocks and has potential application for in vivo gene therapy studies. We have found as many as four copies of a pseudotyped vector to integrate into the genome of a single cell when a high multiplicity of infection was used to infect the cells. Multiple integration events were not observed with amphotropic retroviral vectors, probably because of their low virus titers. In addition, when retroviral vectors are pseudotyped with the VSV G protein, they acquire the host range of VSV and are able to infect nonmammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera. Since techniques for efficient gene transfer in some of these nonmammalian systems are not currently available, retrovirus-mediated gene transfer described here should be useful for transgenic and other genetic studies in lower vertebrate species. The inability to establish a stable cell line expressing the VSV G protein, however, limits large-scale production of the pseudotyped retroviral vectors. Generation of stable packaging cell lines for the pseudotyped retroviral vectors is a major challenge for the future. PMID:7823872

  9. Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida.

    PubMed

    Troeschel, Sonja Christina; Thies, Stephan; Link, Olga; Real, Catherine Isabell; Knops, Katja; Wilhelm, Susanne; Rosenau, Frank; Jaeger, Karl-Erich

    2012-10-15

    Novel shuttle vectors named pEBP were constructed to allow the gene expression in different bacterial hosts including Escherichia coli, Bacillus subtilis and Pseudomonas putida. These vectors share the inducible promoters P(T7) and P(Xyl) and a cos site to enable packaging of plasmid DNA into phage, and carry different multiple cloning sites and antibiotic resistance genes. Vector pEBP41 generally replicates episomally while pEBP18 replicates episomally in Gram-negative bacteria only, but integrates into the chromosome of B. subtilis. Plasmid copy numbers determined for E. coli and P. putida were in the range of 5-50 per cell. The functionality of pEBP18 and pEBP41 was confirmed by expression of two lipolytic enzymes, namely lipase A from B. subtilis and cutinase from the eukaryotic fungus Fusarium solani pisi in three different host strains. Additionally, we report here the construction of a T7 RNA polymerase-based expression strain of P. putida. PMID:22440389

  10. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

    PubMed Central

    Monson, Rita; Smith, Debra S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P. C.

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways. PMID:26733980

  11. Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

    PubMed Central

    Ahern, Stephen J.; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry

    2014-01-01

    The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ∼4 × 10−12 ml cell−1 min−1 for X. fastidiosa strain Temecula 1 and ∼5 × 10−10 to 7 × 10−10 ml cell−1 min−1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa. PMID:24214944

  12. Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

    PubMed

    Ahern, Stephen J; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry; Gonzalez, Carlos F

    2014-01-01

    The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa. PMID:24214944

  13. Annotated receipts capture household food purchases from a broad range of sources

    PubMed Central

    French, Simone A; Wall, Melanie; Mitchell, Nathan R; Shimotsu, Scott T; Welsh, Ericka

    2009-01-01

    Background Accurate measurement of household food purchase behavior (HFPB) is important for understanding its association with household characteristics, individual dietary intake and neighborhood food retail outlets. However, little research has been done to develop measures of HFPB. The main objective of this paper is to describe the development of a measure of HFPB using annotated food purchase receipts. Methods Households collected and annotated food purchase receipts for a four-week period as part of the baseline assessment of a household nutrition intervention. Receipts were collected from all food sources, including grocery stores and restaurants. Households (n = 90) were recruited from the community as part of an obesity prevention intervention conducted in 2007–2008 in Minneapolis, Minnesota, USA. Household primary shoppers were trained to follow a standardized receipt collection and annotation protocol. Annotated receipts were mailed weekly to research staff. Staff coded the receipt data and entered it into a database. Total food dollars, proportion of food dollars, and ounces of food purchased were examined for different food sources and food categories. Descriptive statistics and correlations are presented. Results A total of 2,483 receipts were returned by 90 households. Home sources comprised 45% of receipts and eating-out sources 55%. Eating-out entrees were proportionally the largest single food category based on counts (16.6%) and dollars ($106 per month). Two-week expenditures were highly correlated (r = 0.83) with four-week expenditures. Conclusion Receipt data provided important quantitative information about HFPB from a wide range of sources and food categories. Two weeks may be adequate to reliably characterize HFPB using annotated receipts. PMID:19570234

  14. Investigations of Saturn’s Main Rings over Broad Range of Wavelengths

    NASA Astrophysics Data System (ADS)

    Spilker, Linda J.; Deau, Estelle; Morishima, Ryuji; Filacchione, Gianrico; Hedman, Matt; Nicholson, Phil; Colwell, Josh; Bradley, Todd; Showalter, Mark; Pilorz, Stu; Brooks, Shawn; Ciarniello, Mauro

    2015-11-01

    An abundance of information about the characteristics of Saturn’s ring particles and their regolith can be obtained by comparing the changes in their brightness, color and temperature with changing viewing geometry over a wide range of wavelengths from ultraviolet through the thermal infrared. Data from Cassini’s Composite Infrared Spectrometer (CIRS), Visual and Infrared Mapping Spectrometer (VIMS), Imaging Science Subsystem (ISS) and Ultraviolet Imaging Spectrograph (UVIS) are jointly studied using data from the lit and unlit main rings at multiple geometries and solar elevations over 11 years of the Cassini mission. Using multi-wavelength data sets allows us to test different thermal models by combining the effects of particle albedo, regolith grain size and surface roughness with thermal emissivity and inertia, particle spin rate and spin axis orientation.CIRS temperatures, ISS colors and UVIS brightness appear to vary noticeably with phase angle, but are not a strong function of spacecraft elevation angle. Color, temperature and brightness dependence on solar elevation angle are also observed. VIMS observations show that the infrared ice absorption band depths change with the solar phase angle, in particular between 0-20° and at high phase. This trend indicates that single scattering approximation is correct only at low phases (<20°) while at high phase multiple scattering must be taken into account.These results imply that the individual properties of the ring particles may play a larger role than the collective properties of the rings, in particular at visible wavelengths. The temperature and color variation with phase angle may be a result of scattering within the regolith, as well as scattering between individual particles or clumps in a many-particle-thick layer. Initial results from our joint studies will be presented.This research was carried out in part at the Jet Propulsion Laboratory, California Institute of Technology, under contract with NASA

  15. Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics

    PubMed Central

    Menger, Marcus; Eckstein, Fritz; Porschke, Dietmar

    2000-01-01

    The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to ~200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A→G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (τ ≈200 ms). Addition of Mg2+ ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg2+ are very similar to those induced by Ca2+. The relaxation data do not provide any evidence for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, because a Mg2+-specific relaxation effect was not visible. However, a Mg2+-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere

  16. Miniature long-range light beam transmitter resorting to a high-power broad area laser diode

    NASA Astrophysics Data System (ADS)

    Yue, Wenjing; Lee, Sang-Shin

    2014-08-01

    A miniature long-range light beam transmitter, which taps into a high-power broad area laser diode (BALD), was realized to exhibit a uniform detectable width. An effective model was proposed to practically emulate the multimode characteristics of the beam generated by the BALD. The model, solely based on the emitting region and far-field divergence angle pertaining to the LD, is established through an incoherent superposition of multiple normalized Hermit-Gaussian modes. The feasibility of the proposed model was successfully verified in terms of the calculated and observed irradiance distributions of the light beams. A long-range light beam transmitter was then designed and constructed taking advantage of the BALD source in conjunction with a beam shaper. The manufactured transmitter was corroborated to provide an infrared beam with a constant detectable width of ~1 m, over a distance ranging up to 400 m, for a predefined threshold level.

  17. Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures.

    PubMed

    Theriot, Casey M; Du, Xuelian; Tove, Sherry R; Grunden, Amy M

    2010-08-01

    Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application for decontamination of organophosphorus compounds in certain pesticides and chemical warfare agents under harsh conditions. However, current applications that use an enzyme-based cocktail are limited by poor long-term enzyme stability and low reactivity over a broad range of temperatures. To obtain a better enzyme for OP nerve agent decontamination and to investigate structural factors that influence protein thermostability and thermoactivity, randomly mutated P. furiosus prolidases were prepared by using XL1-red-based mutagenesis and error-prone PCR. An Escherichia coli strain JD1 (lambdaDE3) (auxotrophic for proline [DeltaproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P. furiosus prolidase expression plasmids. JD1 (lambdaDE3) cells were transformed with mutated prolidase expression plasmids and plated on minimal media supplemented with 50 muM Leu-Pro as the only source of proline. By using this positive selection, Pyrococcus prolidase mutants with improved activity over a broader range of temperatures were isolated. The activities of the mutants over a broad temperature range were measured for both Xaa-Pro dipeptides and OP nerve agents, and the thermoactivity and thermostability of the mutants were determined. PMID:20422176

  18. Tandem photonic-crystal thin films surpassing Lambertian light-trapping limit over broad bandwidth and angular range

    SciTech Connect

    Oskooi, Ardavan Tanaka, Yoshinori; Noda, Susumu

    2014-03-03

    Random surface texturing of an optically thick film to increase the path length of scattered light rays, first proposed nearly thirty years ago, has thus far remained the most effective approach for photon absorption over the widest set of conditions. Here, using recent advances in computational electrodynamics, we describe a general strategy for the design of a silicon thin film applicable to photovoltaic cells based on a quasi-resonant approach to light trapping where two partially disordered photonic-crystal slabs, stacked vertically on top of each other, have large absorption that surpasses the Lambertian limit over a broad bandwidth and angular range.

  19. A hydrogel based nanosensor with an unprecedented broad sensitivity range for pH measurements in cellular compartments.

    PubMed

    Zhang, M; Søndergaard, R V; Kumar, E K P; Henriksen, J R; Cui, D; Hammershøj, P; Clausen, M H; Andresen, T L

    2015-11-01

    Optical pH nanosensors have been applied for monitoring intracellular pH in real-time for about two decades. However, the pH sensitivity range of most nanosensors is too narrow, and measurements that are on the borderline of this range may not be correct. Furthermore, ratiometric measurements of acidic intracellular pH (pH < 4) in living cells are still challenging due to the lack of suitable nanosensors. In this paper we successfully developed a multiple sensor, a fluorophore based nanosensor, with an unprecedented broad measurement range from pH 1.4 to 7.0. In this nanosensor, three pH-sensitive fluorophores (difluoro-Oregon Green, Oregon Green 488, and fluorescein) and one pH-insensitive fluorophore (Alexa 568) were covalently incorporated into a nanoparticle hydrogel matrix. With this broad range quadruple-labelled nanosensor all physiological relevant pH levels in living cells can be measured without being too close to the limits of its pH-range. The nanosensor exhibits no susceptibility to interference by other intracellular ions at physiological concentrations. Due to its positive surface charge it is spontaneously internalized by HeLa cells and localizes to the lysosomes where the mean pH was measured at 4.6. This quadruple-labelled nanosensor performs accurate measurements of fluctuations of lysosomal pH in both directions, which was shown by treatment with the V-ATPase inhibitor bafilomycin A1 or its substrate ATP in HeLa cells. These measurements indicate that this novel quadruple-labelled nanosensor is a promising new tool for measuring the pH of acidic compartments in living cells. PMID:26393332

  20. Antiviral Activity of Favipiravir (T-705) against a Broad Range of Paramyxoviruses In Vitro and against Human Metapneumovirus in Hamsters.

    PubMed

    Jochmans, D; van Nieuwkoop, S; Smits, S L; Neyts, J; Fouchier, R A M; van den Hoogen, B G

    2016-08-01

    The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses. PMID:27185803

  1. Emerging Trends in Molecular Interactions between Plants and the Broad Host Range Fungal Pathogens Botrytis cinerea and Sclerotinia sclerotiorum.

    PubMed

    Mbengue, Malick; Navaud, Olivier; Peyraud, Rémi; Barascud, Marielle; Badet, Thomas; Vincent, Rémy; Barbacci, Adelin; Raffaele, Sylvain

    2016-01-01

    Fungal plant pathogens are major threats to food security worldwide. Sclerotinia sclerotiorum and Botrytis cinerea are closely related Ascomycete plant pathogens causing mold diseases on hundreds of plant species. There is no genetic source of complete plant resistance to these broad host range pathogens known to date. Instead, natural plant populations show a continuum of resistance levels controlled by multiple genes, a phenotype designated as quantitative disease resistance. Little is known about the molecular mechanisms controlling the interaction between plants and S. sclerotiorum and B. cinerea but significant advances were made on this topic in the last years. This minireview highlights a selection of nine themes that emerged in recent research reports on the molecular bases of plant-S. sclerotiorum and plant-B. cinerea interactions. On the fungal side, this includes progress on understanding the role of oxalic acid, on the study of fungal small secreted proteins. Next, we discuss the exchanges of small RNA between organisms and the control of cell death in plant and fungi during pathogenic interactions. Finally on the plant side, we highlight defense priming by mechanical signals, the characterization of plant Receptor-like proteins and the hormone abscisic acid in the response to B. cinerea and S. sclerotiorum, the role of plant general transcription machinery and plant small bioactive peptides. These represent nine trends we selected as remarkable in our understanding of fungal molecules causing disease and plant mechanisms associated with disease resistance to two devastating broad host range fungi. PMID:27066056

  2. Construction of a broad host range shuttle vector for gene cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae.

    PubMed

    Frey, J

    1992-01-01

    We have constructed a pair of broad host range expression vectors, pJFF224-NX and pJFF224-XN, based on plasmid RSF1010, which enable cloning and efficient expression of genes in Actinobacillus pleuropneumoniae and Pasteurella haemolytica and in Escherichia coli. The vectors consist of the minimal autonomous replicon of the broad host range plasmid RSF1010 and a type II chloramphenicol acetyl transferase gene for chloramphenicol resistance selection. In addition, they contain a gene expression cassette based on the E. coli bacteriophage T4 gene 32 promoter region and a transcription stop signal, which are separated by a segment of multiple cloning sites in both orientations. Electroporation and subsequent selection for chloramphenicol resistance was used for the introduction of the vectors in A. pleuropneumoniae and P. haemolytica. A promoterless xy/E gene from the Pseudomonas putida TOL plasmid was cloned onto pJFF224-NX. This plasmid enabled efficient expression of active catechol2,3oxygenase in A. pleuropneumoniae and P. haemolytica. It was stably maintained in A. pleuropneumoniae without antibiotic selection, showing less than 0.1% loss after 100 generations, while native RSF1010 and other RSF1010-based vectors were unstable in this host. PMID:1448612

  3. DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies.

    PubMed

    Hugerth, Luisa W; Wefer, Hugo A; Lundin, Sverker; Jakobsson, Hedvig E; Lindberg, Mathilda; Rodin, Sandra; Engstrand, Lars; Andersson, Anders F

    2014-08-01

    The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples. PMID:24928874

  4. Emerging Trends in Molecular Interactions between Plants and the Broad Host Range Fungal Pathogens Botrytis cinerea and Sclerotinia sclerotiorum

    PubMed Central

    Mbengue, Malick; Navaud, Olivier; Peyraud, Rémi; Barascud, Marielle; Badet, Thomas; Vincent, Rémy; Barbacci, Adelin; Raffaele, Sylvain

    2016-01-01

    Fungal plant pathogens are major threats to food security worldwide. Sclerotinia sclerotiorum and Botrytis cinerea are closely related Ascomycete plant pathogens causing mold diseases on hundreds of plant species. There is no genetic source of complete plant resistance to these broad host range pathogens known to date. Instead, natural plant populations show a continuum of resistance levels controlled by multiple genes, a phenotype designated as quantitative disease resistance. Little is known about the molecular mechanisms controlling the interaction between plants and S. sclerotiorum and B. cinerea but significant advances were made on this topic in the last years. This minireview highlights a selection of nine themes that emerged in recent research reports on the molecular bases of plant-S. sclerotiorum and plant-B. cinerea interactions. On the fungal side, this includes progress on understanding the role of oxalic acid, on the study of fungal small secreted proteins. Next, we discuss the exchanges of small RNA between organisms and the control of cell death in plant and fungi during pathogenic interactions. Finally on the plant side, we highlight defense priming by mechanical signals, the characterization of plant Receptor-like proteins and the hormone abscisic acid in the response to B. cinerea and S. sclerotiorum, the role of plant general transcription machinery and plant small bioactive peptides. These represent nine trends we selected as remarkable in our understanding of fungal molecules causing disease and plant mechanisms associated with disease resistance to two devastating broad host range fungi. PMID:27066056

  5. Recombinant broad-range phospholipase C from Listeria monocytogenes exhibits optimal activity at acidic pH.

    PubMed

    Huang, Qiongying; Gershenson, Anne; Roberts, Mary F

    2016-06-01

    The broad-range phospholipase C (PLC) from Listeria monocytogenes has been expressed using an intein expression system and characterized. This zinc metalloenzyme, similar to the homologous enzyme from Bacillus cereus, targets a wide range of lipid substrates. With monomeric substrates, the length of the hydrophobic acyl chain has significant impact on enzyme efficiency by affecting substrate affinity (Km). Based on a homology model of the enzyme to the B. cereus protein, several active site residue mutations were generated. While this PLC shares many of the mechanistic characteristics of the B. cereus PLC, a major difference is that the L. monocytogenes enzyme displays an acidic pH optimum regardless of substrate status (monomer, micelle, or vesicle). This unusual behavior might be advantageous for its role in the pathogenicity of L. monocytogenes. PMID:26976751

  6. Single-ion polymer electrolyte membranes enable lithium-ion batteries with a broad operating temperature range.

    PubMed

    Cai, Weiwei; Zhang, Yunfeng; Li, Jing; Sun, Yubao; Cheng, Hansong

    2014-04-01

    Conductive processes involving lithium ions are analyzed in detail from a mechanistic perspective, and demonstrate that single ion polymeric electrolyte (SIPE) membranes can be used in lithium-ion batteries with a wide operating temperature range (25-80 °C) through systematic optimization of electrodes and electrode/electrolyte interfaces, in sharp contrast to other batteries equipped with SIPE membranes that display appreciable operability only at elevated temperatures (>60 °C). The performance is comparable to that of batteries using liquid electrolyte of inorganic salt, and the batteries exhibit excellent cycle life and rate performance. This significant widening of battery operation temperatures coupled with the inherent flexibility and robustness of the SIPE membranes makes it possible to develop thin and flexible Li-ion batteries for a broad range of applications. PMID:24623577

  7. Virulence of Broad- and Narrow-Host-Range Salmonella enterica Serovars in the Streptomycin-Pretreated Mouse Model

    PubMed Central

    Suar, Mrutyunjay; Jantsch, Jonathan; Hapfelmeier, Siegfried; Kremer, Marcus; Stallmach, Thomas; Barrow, Paul A.; Hardt, Wolf-Dietrich

    2006-01-01

    Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data. PMID:16369020

  8. High-resolution continuum source electrothermal atomic absorption spectrometry: Linearization of the calibration curves within a broad concentration range

    NASA Astrophysics Data System (ADS)

    Katskov, Dmitri; Hlongwane, Miranda; Heitmann, Uwe; Florek, Stefan

    2012-05-01

    The calculation algorithm suggested provides linearization of the calibration curves in high-resolution continuum source electrothermal atomic absorption spectrometry. The algorithm is based on the modification of the function wavelength-integrated absorbance vs. concentration of analyte vapor in the absorption volume. According to the suggested approach, the absorption line is represented by a triangle for low and trapezium for high analyte vapor concentration in the absorption volume. The respective semi-empirical formulas include two linearization parameters, which depend on properties of the absorption line and characteristics of the atomizer and spectrometer. The parameters can be approximately evaluated from the theory and determined in practice from the original broad-range calibration curve. The parameters were found and the proposed calculation algorithm verified in the experiments on direct determination of Ag, Cd, Cu, Fe, Mn and Pb in the solutions within a concentration ranges from 0.15 to 625 μg·L- 1 using tube, platform tube and filter furnace atomizers. The use of various atomizers, lines, elements and atomization temperatures made possible the simulation of various practical analytical conditions. It was found that the algorithm and optimal linearization parameters made it possible to obtain for each line and atomizer linear approximations of the calibration curves within 3-4 orders of magnitude with correlation coefficients close to 0.999. The algorithm makes possible to employ a single line for the direct element determination over a broad concentration range. The sources of errors and the possibility of a priori theoretical evaluation of the linearization parameters are discussed.

  9. Fluorescent pH Sensors for Broad-Range pH Measurement Based on a Single Fluorophore.

    PubMed

    Qi, Jing; Liu, Daying; Liu, Xiaoyan; Guan, Shiquan; Shi, Fengli; Chang, Hexi; He, Huarui; Yang, Guangming

    2015-06-16

    We constructed a series of novel optical sensors for determination of broad-range pH based on a single fluorophore and multi-ionophores with different pK(a) values. These optical sensors use photoinduced electron transfer (PET) as the signal transduction and follow the design concept of "fluorophore-spacer-receptor (ionophore)" which employs 4-amino-1,8-naphthalimide as the single fluorophore, ethyl moiety as the spacer, and a series of phenols and anilines as the receptors. Key to the successful development of this sensor system is that coupling the receptors with six different pK(a) values with a single fluorophore produces the correct optical properties. This rational design affords a series of optical pH sensors with unique fluorescence property and accurately tunable pH measurement ranging from 1 to 14 pH units. Because of covalent immobilization of the indicators, these sensors demonstrate excellent stability, adequate reversibility, and satisfactory dynamic range up to full pH ranges (pH 1-14). PMID:25893705

  10. Population structure over a broad spatial scale driven by nonanthropogenic factors in a wide-ranging migratory mammal, Alaskan caribou.

    PubMed

    Mager, Karen H; Colson, Kevin E; Groves, Pam; Hundertmark, Kris J

    2014-12-01

    Wide-ranging mammals face significant conservation threats, and knowledge of the spatial scale of population structure and its drivers is needed to understand processes that maintain diversity in these species. We analysed DNA from 655 Alaskan caribou (Rangifer tarandus granti) from 20 herds that vary in population size, used 19 microsatellite loci to document genetic diversity and differentiation in Alaskan caribou, and examined the extent to which genetic differentiation was associated with hypothesized drivers of population subdivision including landscape features, population size and ecotype. We found that Alaskan caribou are subdivided into two hierarchically structured clusters: one group on the Alaska Peninsula containing discrete herds and one large group on the Mainland lacking differentiation between many herds. Population size, geographic distance, migratory ecotype and the Kvichak River at the nexus of the Alaska Peninsula were associated with genetic differentiation. Contrary to previous hypotheses, small Mainland herds were often differentiated genetically from large interconnected herds nearby, and genetic drift coupled with reduced gene flow may explain this pattern. Our results raise the possibility that behaviour helps to maintain genetic differentiation between some herds of different ecotypes. Alaskan caribou show remarkably high diversity and low differentiation over a broad geographic scale. These results increase information for the conservation of caribou and other migratory mammals threatened by population reductions and landscape barriers and may be broadly applicable to understanding the spatial scale and ecological drivers of population structure in widespread species. PMID:25403098

  11. Synthesis and Characterization of ZnS:Eu3+ - CMC nanophosphors emitting white light over broad excitation range

    NASA Astrophysics Data System (ADS)

    de, Dilip; Ahemen, Ikorya; Bruno, Viena

    In this paper we report for the first time the synthesis and characterization of nanophosphors of ZnS:Eu3+ - embedded in sodium carboxymethyl cellulose matrix (CMC) that emits high quality white light over broad range of excitation. The nano-phosphors of cubic (zinc blende) structure were synthesized using precipitation technique with doping concentrations of Eu3+ ions 1 mol% and 5 mol%. The crystal sizes were 2.56 nm and 2.91 nm respectively. Annealing at 300 oC in a sulfur-rich atmosphere altered the crystal size to 4.35 nm and 3.65 nm respectively and the band gap from 4.2 eV to 3.76 eV and 3.81 eV respectively. The as-synthesized samples gave pure orange-red emission when excited at wavelengths of 394 nm and 465 nm. After thermal annealing of the samples, a broad emission band in the blue-green region assigned to defect related states emerged or were enhanced. Also enhanced were the emission lines of Eu3+ ions in the orange-red region. A combination of these two transitions gave white light of different shades (recorded on the CIE 1931 chromaticity diagram) from cool white through Day-light to warm white light, depending on Eu3+ concentration and the excitation wavelengths (UV-330 to blue 465 nm), thus showing great potential applications of these nano-phosphors.

  12. Predicted and measured EMI shielding effectiveness of a metallic mesh coating on a sapphire window over a broad frequency range

    NASA Astrophysics Data System (ADS)

    Jacoby, Keith T.; Pieratt, Matthew W.; Halman, Jennifer I.; Ramsey, Keith A.

    2009-05-01

    Metallic mesh thin film coatings have been used for many years to provide electromagnetic interference (EMI) shielding on infrared windows and domes. The level of EMI shielding effectiveness (SE) of metallic mesh coatings when used in a high frequency application is understood and characterized. Conversely, the level of SE of these metallic mesh coatings when used in a low frequency application has been called into question. In a recent study, we applied an appropriately designed metallic mesh coating to a sapphire window, mounted that window in a fixture, and tested the SE of the window assembly over a frequency range that envelopes the various military platforms covered in MIL-STD-461 (10 kHz to 18 GHz) for a radiated emissions test. The test plan was devised in such a way as to independently assess the individual contributions of the aperture, the mounting, and the metallic mesh coating to the total shielding. The results of our testing will be described in this paper. Additionally, the test results will be compared to the predicted SE for both the aperture and the metallic mesh coated window in order to validate the predictive model. Finally, an assessment of the appropriateness of the use of metallic mesh coatings for EMI shielding in a low and/or broad range frequency application will be made.

  13. Development and Use of a Selectable, Broad-Host-Range Reporter Transposon for Identifying Environmentally Regulated Promoters in Bacteria

    PubMed Central

    Spinler, Jennifer K.; Zajdowicz, Sheryl L. W.; Haller, Jon C.; Oram, Diana Marra; Gill, Ronald E.; Holmes, Randall K.

    2009-01-01

    This report describes the development and use of TnKnXSp, a selectable broad-host-range reporter transposon with a promoterless aphA gene. Insertion of TnKnXSp into the chromosome of a kanamycin-susceptible bacterium confers resistance to kanamycin only if aphA is transcribed from an active promoter adjacent to the insertion site. We designed TnKnXSp as a tool for identifying environmentally regulated promoters in bacteria and developed general methods for initial characterization of any TnKnXSp integrant. To identify putative iron-regulated promoters in Corynebacterium diphtheriae, we constructed TnKnXSp integrants and identified a subgroup that expressed kanamycin resistance under low-iron, but not high-iron, conditions. We characterized representative integrants with this phenotype, located the TnKnXSp insertion in each, and demonstrated that transcription of aphA was repressed under high-iron vs. low-iron growth conditions. We also demonstrated that TnKnXSp can be used in bacteria other than C. diphtheriae, including Escherichia coli and Bacillus subtilus. Our findings validate TnKnXSp as a useful tool for identifying environmentally regulated promoters in bacteria. PMID:19146571

  14. Plasmids captured in C. metallidurans CH34: defining the PromA family of broad-host-range plasmids.

    PubMed

    Van der Auwera, Géraldine A; Król, Jaroslaw E; Suzuki, Haruo; Foster, Brian; Van Houdt, Rob; Brown, Celeste J; Mergeay, Max; Top, Eva M

    2009-08-01

    The self-transmissible, broad-host-range (BHR) plasmid pMOL98 was previously isolated from polluted soil using a triparental plasmid capture approach and shown to possess a replicon similar to that of the BHR plasmids pSB102 and pIPO2. Here, complete sequence analysis and comparative genomics reveal that the 55.5 kb nucleotide sequence of pMOL98 shows extensive sequence similarity and synteny with the BHR plasmid family that now includes pIPO2, pSB102, pTER331, and pMRAD02. They share a plasmid backbone comprising replication, partitioning and conjugative transfer functions. Comparison of the variable accessory regions of these plasmids shows that the majority of natural transposons, as well as the mini-transposon used to mark the plasmids, are inserted in the parA locus. The transposon unique to pMOL98 appears to have inserted from the chromosome of the recipient strain used in the plasmid capture procedure. This demonstrates the necessity for careful screening of plasmids and host chromosomes to avoid mis-interpretation of plasmid genome content. The presence of very similar BHR plasmids with different accessory genes in geographically distinct locations suggests an important role in horizontal gene exchange and bacterial adaptation for this recently defined plasmid group, which we propose to name "PromA". PMID:19259779

  15. Broad-Host-Range Yersinia Phage PY100: Genome Sequence, Proteome Analysis of Virions, and DNA Packaging Strategy▿

    PubMed Central

    Schwudke, Dominik; Ergin, Asgar; Michael, Kathrin; Volkmar, Sven; Appel, Bernd; Knabner, Dorothea; Konietzny, Antje; Strauch, Eckhard

    2008-01-01

    PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2. PMID:17965162

  16. Construction of the recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidation ability.

    PubMed

    Drewniak, Lukasz; Ciezkowska, Martyna; Radlinska, Monika; Sklodowska, Aleksandra

    2015-02-20

    The plasmid pSinA of Sinorhizobium sp. M14 was used as a source of functional phenotypic modules, encoding proteins involved in arsenite oxidation and arsenic resistance, to obtain recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidative ability. An arsenite oxidation module was cloned into pBBR1MCS-2 vector yielding plasmid vector pAIO1, while an arsenic resistance module was cloned into pCM62 vector yielding plasmid pARS1. Both plasmid constructs were introduced (separately and together) into the cells of phylogenetically distant (representing Alpha-, Beta-, and Gammaproteobacteria) and physiologically diversified (unable to oxidize arsenite and susceptible/resistant to arsenite and arsenate) bacteria. Functional analysis of the modified strains showed that: (i) the plasmid pARS1 can be used for the construction of strains with an increased resistance to arsenite [up to 20mM of As(III), (ii) the presence of the plasmid pAIO1 in bacteria previously unable to oxidize As(III) to As(V), contributes to the acquisition of arsenite oxidation abilities by these cells, (iii) the highest arsenite utilization rate are observed in the culture of strains harbouring both the plasmids pAIO1 and pARS1, (iv) the strains harbouring the plasmid pAIO1 were able to grow on arsenic-contaminated mine waters (∼ 3.0 mg As L(-1)) without any supplementation. PMID:25617684

  17. Broad-range PCR coupled with mass-spectrometry for the detection of Mycobacterium tuberculosis drug resistance

    PubMed Central

    Florea, Dragoş; Oţelea, Dan; Olaru, Ioana D.; Hristea, Adriana

    2016-01-01

    Background The need to limit the spread of drug-resistant Mycobacterium tuberculosis requires rapid detection of resistant strains. The present study aimed to evaluate a commercial assay using broad-range PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) for the rapid detection of isoniazid (INH) and rifampin (RIF) resistance in M. tuberculosis strains isolated from Romanian patients with pulmonary tuberculosis. Methods PCR/ESI-MS was used to detect genotypic resistance to RIF and INH in a panel of 63 M. tuberculosis isolates phenotypically characterized using the absolute concentration method on Löwenstein-Jensen medium. Results Thirty-eight (60%) strains were susceptible to both drugs, 22 (35%) were RIF and INH resistant, one was INH mono-resistant and two were RIF mono-resistant. The sensitivity for INH and RIF resistance mutations detection were 100% and 92% respectively, with a specificity of more than 95% for each drug. Conclusion PCR/ESI-MS is a good method for the detection of RIF and INH resistance and might represent an alternative to other rapid diagnostic tests for the detection of genetic markers of resistance in M. tuberculosis isolates. PMID:27019827

  18. Comparative Analysis of Chlamydia psittaci Genomes Reveals the Recent Emergence of a Pathogenic Lineage with a Broad Host Range

    PubMed Central

    Read, Timothy D.; Joseph, Sandeep J.; Didelot, Xavier; Liang, Brooke; Patel, Lisa; Dean, Deborah

    2013-01-01

    ABSTRACT Chlamydia psittaci is an obligate intracellular bacterium. Interest in Chlamydia stems from its high degree of virulence as an intestinal and pulmonary pathogen across a broad range of animals, including humans. C. psittaci human pulmonary infections, referred to as psittacosis, can be life-threatening, which is why the organism was developed as a bioweapon in the 20th century and is listed as a CDC biothreat agent. One remarkable recent result from comparative genomics is the finding of frequent homologous recombination across the genome of the sexually transmitted and trachoma pathogen Chlamydia trachomatis. We sought to determine if similar evolutionary dynamics occurred in C. psittaci. We analyzed 20 C. psittaci genomes from diverse strains representing the nine known serotypes of the organism as well as infections in a range of birds and mammals, including humans. Genome annotation revealed a core genome in all strains of 911 genes. Our analyses showed that C. psittaci has a history of frequently switching hosts and undergoing recombination more often than C. trachomatis. Evolutionary history reconstructions showed genome-wide homologous recombination and evidence of whole-plasmid exchange. Tracking the origins of recombinant segments revealed that some strains have imported DNA from as-yet-unsampled or -unsequenced C. psittaci lineages or other Chlamydiaceae species. Three ancestral populations of C. psittaci were predicted, explaining the current population structure. Molecular clock analysis found that certain strains are part of a clonal epidemic expansion likely introduced into North America by South American bird traders, suggesting that psittacosis is a recently emerged disease originating in New World parrots. PMID:23532978

  19. Comparative analysis of Chlamydia psittaci genomes reveals the recent emergence of a pathogenic lineage with a broad host range.

    PubMed

    Read, Timothy D; Joseph, Sandeep J; Didelot, Xavier; Liang, Brooke; Patel, Lisa; Dean, Deborah

    2013-01-01

    Chlamydia psittaci is an obligate intracellular bacterium. Interest in Chlamydia stems from its high degree of virulence as an intestinal and pulmonary pathogen across a broad range of animals, including humans. C. psittaci human pulmonary infections, referred to as psittacosis, can be life-threatening, which is why the organism was developed as a bioweapon in the 20th century and is listed as a CDC biothreat agent. One remarkable recent result from comparative genomics is the finding of frequent homologous recombination across the genome of the sexually transmitted and trachoma pathogen Chlamydia trachomatis. We sought to determine if similar evolutionary dynamics occurred in C. psittaci. We analyzed 20 C. psittaci genomes from diverse strains representing the nine known serotypes of the organism as well as infections in a range of birds and mammals, including humans. Genome annotation revealed a core genome in all strains of 911 genes. Our analyses showed that C. psittaci has a history of frequently switching hosts and undergoing recombination more often than C. trachomatis. Evolutionary history reconstructions showed genome-wide homologous recombination and evidence of whole-plasmid exchange. Tracking the origins of recombinant segments revealed that some strains have imported DNA from as-yet-unsampled or -unsequenced C. psittaci lineages or other Chlamydiaceae species. Three ancestral populations of C. psittaci were predicted, explaining the current population structure. Molecular clock analysis found that certain strains are part of a clonal epidemic expansion likely introduced into North America by South American bird traders, suggesting that psittacosis is a recently emerged disease originating in New World parrots. PMID:23532978

  20. Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

    PubMed Central

    2013-01-01

    Background Comparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail. Results Here we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1. Conclusions Bc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G. PMID:23388049

  1. Forest response to elevated CO2 is conserved across a broad range of productivity

    SciTech Connect

    Norby, Richard J; DeLucia, E. H.; Gielen, Birgit; Califapietra, Carlo; Giardina, Christian P; King, John S.; Childs, Joanne; McCarthy, Heather R; Moore, D J; Ceulemans, Reinhart; DeAngelis, Paolo; Finzi, Adrien C; Karnosky, David; Kubiske, Mark E; Lukac, Martin; Pregitzer, Kurt; Scarascia-Mugnozza, Giuseppe E; Schlesinger, William H; Oren, Ram

    2005-11-01

    Climate change predictions derived from coupled carbon-climate models are highly dependent on assumptions about feedbacks between the biosphere and atmosphere. One critical feedback occurs if C uptake by the biosphere increases in response to the fossil-fuel driven increase in atmospheric [CO{sub 2}] ('CO{sub 2} fertilization'), thereby slowing the rate of increase in atmospheric [CO{sub 2}]. Carbon exchanges between the terrestrial biosphere and atmosphere are often first represented in models as net primary productivity (NPP). However, the contribution of CO{sub 2} fertilization to the future global C cycle has been uncertain, especially in forest ecosystems that dominate global NPP, and models that include a feedback between terrestrial biosphere metabolism and atmospheric [CO{sub 2}] are poorly constrained by experimental evidence. We analyzed the response of NPP to elevated CO{sub 2} ({approx}550 ppm) in four free-air CO{sub 2} enrichment experiments in forest stands. We show that the response of forest NPP to elevated [CO{sub 2}] is highly conserved across a broad range of productivity, with a stimulation at the median of 23 {+-} 2%. At low leaf area indices, a large portion of the response was attributable to increased light absorption, but as leaf area indices increased, the response to elevated [CO{sub 2}] was wholly caused by increased light-use efficiency. The surprising consistency of response across diverse sites provides a benchmark to evaluate predictions of ecosystem and global models and allows us now to focus on unresolved questions about carbon partitioning and retention, and spatial variation in NPP response caused by availability of other growth limiting resources.

  2. Type IV Pilus Assembly in Pseudomonas aeruginosa over a Broad Range of Cyclic di-GMP Concentrations

    PubMed Central

    Jain, Ruchi; Behrens, Anna-Janina; Kaever, Volkhard

    2012-01-01

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that utilizes polar type IV pili (T4P) for twitching motility and adhesion in the environment and during infection. Pilus assembly requires FimX, a GGDEF/EAL domain protein that binds and hydrolyzes cyclic di-GMP (c-di-GMP). Bacteria lacking FimX are deficient in twitching motility and microcolony formation. We carried out an extragenic suppressor screen in PA103ΔfimX bacteria to identify additional regulators of pilus assembly. Multiple suppressor mutations were mapped to PA0171, PA1121 (yfiR), and PA3703 (wspF), three genes previously associated with small-colony-variant phenotypes. Multiple independent techniques confirmed that suppressors assembled functional surface pili, though at both polar and nonpolar sites. Whole-cell c-di-GMP levels were elevated in suppressor strains, in agreement with previous studies that had shown that the disrupted genes encoded negative regulators of diguanylate cyclases. Overexpression of the regulated diguanylate cyclases was sufficient to suppress the ΔfimX pilus assembly defect, as was overexpression of an unrelated diguanylate cyclase from Caulobacter crescentus. Furthermore, under natural conditions of high c-di-GMP, PA103ΔfimX formed robust biofilms that showed T4P staining and were structurally distinct from those formed by nonpiliated bacteria. These results are the first demonstration that P. aeruginosa assembles a surface organelle, type IV pili, over a broad range of c-di-GMP concentrations. Assembly of pili at low c-di-GMP concentrations requires a polarly localized c-di-GMP binding protein and phosphodiesterase, FimX; this requirement for FimX is bypassed at high c-di-GMP concentrations. Thus, P. aeruginosa can assemble the same surface organelle in distinct ways for motility or adhesion under very different environmental conditions. PMID:22685276

  3. Why to measure a broad range of city sizes? Analysis of globally pooled data of urban GHG measurements for sustainability

    NASA Astrophysics Data System (ADS)

    Rybski, Diego; Sterzel, Till; Reusser, Dominik E.; Fichter, Christina; Kropp, Jürgen P.

    2013-04-01

    We have assembled a database of urban GHG emissions from various published sources, including about 200 cities globally. Analyzing this CO2 emission inventory from multiple countries we find power-law relations between the emissions and city size, measured in population. The results suggest that in developing countries large cities emit more CO2 per capita compared to small cities, i.e. they tend to comprise super-linear correlations. For developed countries the results suggest the opposite, i.e. linear or sub-linear correlations, implying better efficiency of large cities. We derive how the total emissions of an entire country relate with the power-law correlations and find that the size of the most populated city is dominating in the case of linear and super-linear correlations, while a transition occurs to sub-linear correlations, where the size of the largest city has no influence. It is important to further substantiate an overview of city emission inventories across a broad range of city sizes and types to further clarify the complex relationships between cities and GHG emissions. On the one hand, we propose a minimum set of meta-information to be reported together with the emission inventories, e.g. for determining comparability among inventories. On the other hand, we propose to fill evident gaps with respect to regions (e.g. sub-Saharan African and South American cities) and types of cities (e.g. small medium and low-income country cities) to allow for a better global overview of city sizes, income, and emissions. We conclude that from the climate change mitigation point of view, urbanization is desirable in developed countries and should be avoided in developing countries, if effinciency increasing mechanisms can not be established. More data acquisition is needed to support our empirical findings.

  4. [Isolation and characterization of petroleum catabolic broad-host-range plasmids from Shen-Fu wastewater irrigation zone].

    PubMed

    Wang, Ya-Fei; Wang, Ya-Fei; Li, Hui; Li, Xiao-Bin

    2013-11-01

    Based on triparental mating, we isolated a total of eight broad host range (BHR) petroleum hydrocarbon catabolic plasmids from the soils, sediments, and wastewater samples in the Shen-Fu irrigation zone. The antibiotic resistance of the plasmids was tested, and then, the plasmids were transferred to Escherichia coli EC100. The plasmids carrying no antibiotic resistance were tagged by miniTn5 transposon consisting of antibiotic resistant genes. The PCR-based incompatibility test revealed that the pS3-2C and pS4-6G belonged to Inc P group, the pS3-2G, pW22-3G, and pA15-7G belonged to Inc N group, the pS7-2G was identified as Inc W plasmid, and the pA23-1G and pA10-1C were placed into Inc Q group. By adopting the reported PCR amplification methods of petroleum hydrocarbon-degrading catabolic genes, the petroleum-degrading capability of these BHR plasmids were preliminarily analyzed. The plasmids pS3-2G, pS7-2G, pA23-1G, pW22-3G, and pA10-1C carried aromatic ring- hydroxylating dioxygenase gene phdA and toluene monooxygenase gene touA; the plasmid pA15-7G carried touA and toluene dioxygenase gene tod; the plasmid pS3-2C carried ben, phdA, and tod; whereas the pS4-6G only carried ben. The host range test showed that all the isolated plasmids except pS3-2C could be transferred and maintained stably in the representative strains Agrobacterium tumefaciens C58, Cupriavidus necator JMP228, and E. coli EC100 of the alpha-, beta-, and gamma-Proteobacteria, respectively. PMID:24564162

  5. Prospective Study of Use of PCR Amplification and Sequencing of 16S Ribosomal DNA from Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis in a Clinical Setting

    PubMed Central

    Schuurman, Tim; de Boer, Richard F.; Kooistra-Smid, Anna M. D.; van Zwet, Anton A.

    2004-01-01

    We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 × 102 to 2 × 102 CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains. PMID:14766845

  6. Complete Genome Sequences of Broad-Host-Range Pseudomonas aeruginosa Bacteriophages ΦR18 and ΦS12-1

    PubMed Central

    Furusawa, Takaaki; Higuchi, Hidetoshi; Usui, Masaru; Maruyama, Fumito; Nakagawa, Ichiro; Yokota, Hiroshi; Tamura, Yutaka

    2016-01-01

    Pseudomonas aeruginosa is an important cause of racehorse keratitis. Bacteriophage therapy has the potential to aid in the prevention and treatment of diseases caused by P. aeruginosa. We present here the complete genome sequences of two phages, ΦR18 and ΦS12-1, which exhibit infectivity for a broad range of P. aeruginosa isolates. PMID:27151780

  7. Bio-inspired anti-oil-fouling chitosan-coated mesh for oil/water separation suitable for broad pH range and hyper-saline environments.

    PubMed

    Zhang, Shiyan; Lu, Fei; Tao, Lei; Liu, Na; Gao, Changrui; Feng, Lin; Wei, Yen

    2013-11-27

    Here, we report a bio-inspired chitosan (CS)-based mesh with high separation efficiency, oil-fouling repellency, and stability in a complex liquid environment. The surface of the CS coating maintains underwater superoleophobicity and low oil adhesion (<1 μN) in pure water and hyper-saline solutions, and it can keep stable special wettability in broad pH range environments after the CS mesh is fully cross-linked with glutaraldehyde and then reduced by sodium borohydride to form a stable carbon-nitrogen single bond. The separation process is solely gravity-driven, and the mesh can separate a range of different oil/water mixtures with >99% separation efficiency in hyper-saline and broad pH range conditions. We envision that such a separation method will be useful in oil spill cleanup and industrial oily wastewater treatment in extreme environments. PMID:24180691

  8. Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types

    NASA Astrophysics Data System (ADS)

    Clark, Imram; Hanania, Elie G.; Stevens, Janine; Gallina, Marijo; Fieck, Annabeth; Brandes, Rolf; Palsson, Bernhard O.; Koller, Manfred R.

    2006-01-01

    Efficient delivery of compounds and macromolecules into living cells is essential in many fields including basic research, applied drug discovery, and clinical gene therapy. Unfortunately, current delivery methods, such as cationic lipids and electroporation, are limited by the types of macromolecules and cells that can be employed, poor efficiency, and/or cell toxicity. To address these issues, novel methods were developed based on laser-mediated delivery of macromolecules into cells through optoinjection. An automated high-throughput instrument, the laser-enabled analysis and processing (LEAPTM) system, was utilized to elucidate and optimize several parameters that influence optoinjection efficiency and toxicity. Techniques employing direct cell irradiation (i.e., targeted to specific cell coordinates) and grid-based irradiation (i.e., without locating individual cells) were both successfully developed. With both techniques, it was determined that multiple, sequential low radiant exposures produced more favorable results than a single high radiant exposure. Various substances were efficiently optoinjected-including ions, small molecules, dextrans, siRNAs (small interfering RNAs), plasmids, proteins, and semiconductor nanocrystals-into numerous cell types. Notably, cells refractory to traditional delivery methods were efficiently optoinjected with lower toxicity. We establish the broad utility of optoinjection, and furthermore, are the first to demonstrate its implementation in an automated, high-throughput manner.

  9. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background and Aims: Vulnerability of the leaf hydraulic pathway to water-stress-induced dysfunction is a key component of drought tolerance in plants and may be important in defining species’ climatic range. However, the generality of the association between leaf hydraulic vulnerability and climate...

  10. LTP in Hippocampal Area CA1 Is Induced by Burst Stimulation over a Broad Frequency Range Centered around Delta

    ERIC Educational Resources Information Center

    Grover, Lawrence M.; Kim, Eunyoung; Cooke, Jennifer D.; Holmes, William R.

    2009-01-01

    Long-term potentiation (LTP) is typically studied using either continuous high-frequency stimulation or theta burst stimulation. Previous studies emphasized the physiological relevance of theta frequency; however, synchronized hippocampal activity occurs over a broader frequency range. We therefore tested burst stimulation at intervals from 100…

  11. Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens.

    PubMed

    Bombaci, Mauro; Grifantini, Renata; Mora, Marirosa; Reguzzi, Valerio; Petracca, Roberto; Meoni, Eva; Balloni, Sergio; Zingaretti, Chiara; Falugi, Fabiana; Manetti, Andrea G O; Margarit, Immaculada; Musser, James M; Cardona, Francesco; Orefici, Graziella; Grandi, Guido; Bensi, Giuliano

    2009-01-01

    The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen

  12. Controlling the red boundary of the tunneling photoeffect in nanodimensional carbon structures in a broad (UV-IR) wavelength range

    NASA Astrophysics Data System (ADS)

    Akchurin, G. G.; Yakunin, A. N.; Aban'shin, N. P.; Gorfinkel', B. I.; Akchurin, G. G.

    2013-06-01

    The tunneling photoeffect (PE) has been studied in a microdiode with an electrostatic field localized at an emitter based on a nanodimensional carbon structure. It is established that, when the carbon nanoemitter is exposed to laser and LED radiation photons of low energy (below work function) in the spectral range from near-UV (380 nm) to near-IR (1150 nm) at micro- and milliwatt optical power, a tunneling photocurrent can be initiated by controlling the electric field strength in the emitter-anode gap. The observed phenomenon can be adequately interpreted using a modified Fowler-Nordheim equation for non-equilibrium photoelectrons. Specific features of the construction and operation of photodetectors based on the tunneling PE with a controlled long-wavelength threshold (red boundary) of photoelectron emission are considered. The bandwidth of photoelectron emitters is evaluated, and the possibility of their operation in the wavelength range from UV up to far-IR is predicted.

  13. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates

    PubMed Central

    Blackman, Chris J.; Gleason, Sean M.; Chang, Yvonne; Cook, Alicia M.; Laws, Claire; Westoby, Mark

    2014-01-01

    Background and Aims Vulnerability of the leaf hydraulic pathway to water-stress-induced dysfunction is a key component of drought tolerance in plants and may be important in defining species' climatic range. However, the generality of the association between leaf hydraulic vulnerability and climate across species and sites remains to be tested. Methods Leaf hydraulic vulnerability to drought (P50leaf, the water potential inducing 50 % loss in hydraulic function) was measured in a diverse group of 92 woody, mostly evergreen angiosperms from sites across a wide range of habitats. These new data together with some previously published were tested against key climate indices related to water availability. Differences in within-site variability in P50leaf between sites were also examined. Key Results Values of hydraulic vulnerability to drought in leaves decreased strongly (i.e. became more negative) with decreasing annual rainfall and increasing aridity across sites. The standard deviation in P50leaf values recorded within each site was positively correlated with increasing aridity. P50leaf was also a good indicator of the climatic envelope across each species' distributional range as well as their dry-end distributional limits within Australia, although this relationship was not consistently detectable within sites. Conclusions The findings indicate that species sorting processes have influenced distributional patterns of P50leaf across the rainfall spectrum, but alternative strategies for dealing with water deficit exist within sites. The strong link to aridity suggests leaf hydraulic vulnerability may influence plant distributions under future climates. PMID:25006181

  14. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

    SciTech Connect

    Igarashi, Aki; Yamagata, Kousuke; Sugai, Tomokazu; Takahashi, Yukari; Sugawara, Emiko; Tamura, Akihiro; Yaegashi, Hajime; Yamagishi, Noriko; Takahashi, Tsubasa; Isogai, Masamichi; Takahashi, Hideki; Yoshikawa, Nobuyuki

    2009-04-10

    Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

  15. Can job redesign interventions influence a broad range of employee outcomes by changing multiple job characteristics? A quasi-experimental study.

    PubMed

    Holman, David; Axtell, Carolyn

    2016-07-01

    Many job redesign interventions are based on a multiple mediator-multiple outcome model in which the job redesign intervention indirectly influences a broad range of employee outcomes by changing multiple job characteristics. As this model remains untested, the aim of this study is to test a multiple mediator-multiple outcome model of job redesign. Multilevel analysis of data from a quasi-experimental job redesign intervention in a call center confirmed the hypothesized model and showed that the job redesign intervention affected a broad range of employee outcomes (i.e., employee well-being, psychological contract fulfillment, and supervisor-rated job performance) through changes in 2 job characteristics (i.e., job control and feedback). The results provide further evidence for the efficacy and mechanisms of job redesign interventions. (PsycINFO Database Record PMID:26641482

  16. Promoters of the Broad Host Range Plasmid Rk2: Analysis of Transcription (Initiation) in Five Species of Gram-Negative Bacteria

    PubMed Central

    Greener, A.; Lehman, S. M.; Helinski, D. R.

    1992-01-01

    A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined. PMID:1732166

  17. Construction of a self-luminescent cyanobacterial bioreporter that detects a broad range of bioavailable heavy metals in aquatic environments

    PubMed Central

    Martín-Betancor, Keila; Rodea-Palomares, Ismael; Muñoz-Martín, M. A.; Leganés, Francisco; Fernández-Piñas, Francisca

    2015-01-01

    A self-luminescent bioreporter strain of the unicellular cyanobacterium Synechococcus sp. PCC 7942 was constructed by fusing the promoter region of the smt locus (encoding the transcriptional repressor SmtB and the metallothionein SmtA) to luxCDABE from Photorhabdus luminescens; the sensor smtB gene controlling the expression of smtA was cloned in the same vector. The bioreporter performance was tested with a range of heavy metals and was shown to respond linearly to divalent Zn, Cd, Cu, Co, Hg, and monovalent Ag. Chemical modeling was used to link bioreporter response with metal speciation and bioavailability. Limits of Detection (LODs), Maximum Permissive Concentrations (MPCs) and dynamic ranges for each metal were calculated in terms of free ion concentrations. The ranges of detection varied from 11 to 72 pM for Hg2+ (the ion to which the bioreporter was most sensitive) to 1.54–5.35 μM for Cd2+ with an order of decreasing sensitivity as follows: Hg2+ >> Cu2+ >> Ag+ > Co2+ ≥ Zn2+ > Cd2+. However, the maximum induction factor reached 75-fold in the case of Zn2+ and 56-fold in the case of Cd2+, implying that Zn2+ is the preferred metal in vivo for the SmtB sensor, followed by Cd2+, Ag+ and Cu2+ (around 45–50-fold induction), Hg2+ (30-fold) and finally Co2+ (20-fold). The bioreporter performance was tested in real environmental samples with different water matrix complexity artificially contaminated with increasing concentrations of Zn, Cd, Ag, and Cu, confirming its validity as a sensor of free heavy metal cations bioavailability in aquatic environments. PMID:25806029

  18. Construction of a self-luminescent cyanobacterial bioreporter that detects a broad range of bioavailable heavy metals in aquatic environments.

    PubMed

    Martín-Betancor, Keila; Rodea-Palomares, Ismael; Muñoz-Martín, M A; Leganés, Francisco; Fernández-Piñas, Francisca

    2015-01-01

    A self-luminescent bioreporter strain of the unicellular cyanobacterium Synechococcus sp. PCC 7942 was constructed by fusing the promoter region of the smt locus (encoding the transcriptional repressor SmtB and the metallothionein SmtA) to luxCDABE from Photorhabdus luminescens; the sensor smtB gene controlling the expression of smtA was cloned in the same vector. The bioreporter performance was tested with a range of heavy metals and was shown to respond linearly to divalent Zn, Cd, Cu, Co, Hg, and monovalent Ag. Chemical modeling was used to link bioreporter response with metal speciation and bioavailability. Limits of Detection (LODs), Maximum Permissive Concentrations (MPCs) and dynamic ranges for each metal were calculated in terms of free ion concentrations. The ranges of detection varied from 11 to 72 pM for Hg(2+) (the ion to which the bioreporter was most sensitive) to 1.54-5.35 μM for Cd(2+) with an order of decreasing sensitivity as follows: Hg(2+) > Cu(2+) > Ag(+) > Co(2+) ≥ Zn(2+) > Cd(2+). However, the maximum induction factor reached 75-fold in the case of Zn(2+) and 56-fold in the case of Cd(2+), implying that Zn(2+) is the preferred metal in vivo for the SmtB sensor, followed by Cd(2+), Ag(+) and Cu(2+) (around 45-50-fold induction), Hg(2+) (30-fold) and finally Co(2+) (20-fold). The bioreporter performance was tested in real environmental samples with different water matrix complexity artificially contaminated with increasing concentrations of Zn, Cd, Ag, and Cu, confirming its validity as a sensor of free heavy metal cations bioavailability in aquatic environments. PMID:25806029

  19. Compton scattering for spectroscopic detection of ultra-fast, high flux, broad energy range X-rays

    SciTech Connect

    Cipiccia, S.; Wiggins, S. M.; Brunetti, E.; Vieux, G.; Yang, X.; Welsh, G. H.; Anania, M.; Islam, M. R.; Ersfeld, B.; Jaroszynski, D. A.; Maneuski, D.; Montgomery, R.; Smith, G.; Hoek, M.; Hamilton, D. J.; Shea, V. O.; Issac, R. C.; Lemos, N. R. C.; Dias, J. M.; and others

    2013-11-15

    Compton side-scattering has been used to simultaneously downshift the energy of keV to MeV energy range photons while attenuating their flux to enable single-shot, spectrally resolved, measurements of high flux X-ray sources to be undertaken. To demonstrate the technique a 1 mm thick pixelated cadmium telluride detector has been used to measure spectra of Compton side-scattered radiation from a Cobalt-60 laboratory source and a high flux, high peak brilliance X-ray source of betatron radiation from a laser-plasma wakefield accelerator.

  20. Optimal foraging strategies: Lévy walks balance searching and patch exploitation under a very broad range of conditions.

    PubMed

    Humphries, Nicolas E; Sims, David W

    2014-10-01

    While evidence for optimal random search patterns, known as Lévy walks, in empirical movement data is mounting for a growing list of taxa spanning motile cells to humans, there is still much debate concerning the theoretical generality of Lévy walk optimisation. Here, using a new and robust simulation environment, we investigate in the most detailed study to date (24×10(6) simulations) the foraging and search efficiencies of 2-D Lévy walks with a range of exponents, target resource distributions and several competing models. We find strong and comprehensive support for the predictions of the Lévy flight foraging hypothesis and in particular for the optimality of inverse square distributions of move step-lengths across a much broader range of resource densities and distributions than previously realised. Further support for the evolutionary advantage of Lévy walk movement patterns is provided by an investigation into the 'feast and famine' effect, with Lévy foragers in heterogeneous environments experiencing fewer long 'famines' than other types of searchers. Therefore overall, optimal Lévy foraging results in more predictable resources in unpredictable environments. PMID:24882791

  1. Qualification of a Plant Disease Simulation Model: Performance of the LATEBLIGHT Model Across a Broad Range of Environments.

    PubMed

    Andrade-Piedra, Jorge L; Forbes, Gregory A; Shtienberg, Dani; Grünwald, Niklaus J; Chacón, María G; Taipe, Marco V; Hijmans, Robert J; Fry, William E

    2005-12-01

    ABSTRACT The concept of model qualification, i.e., discovering the domain over which a validated model may be properly used, was illustrated with LATEBLIGHT, a mathematical model that simulates the effect of weather, host growth and resistance, and fungicide use on asexual development and growth of Phytophthora infestans on potato foliage. Late blight epidemics from Ecuador, Mexico, Israel, and the United States involving 13 potato cultivars (32 epidemics in total) were compared with model predictions using graphical and statistical tests. Fungicides were not applied in any of the epidemics. For the simulations, a host resistance level was assigned to each cultivar based on general categories reported by local investigators. For eight cultivars, the model predictions fit the observed data. For four cultivars, the model predictions overestimated disease, likely due to inaccurate estimates of host resistance. Model predictions were inconsistent for one cultivar and for one location. It was concluded that the domain of applicability of LATEBLIGHT can be extended from the range of conditions in Peru for which it has been previously validated to those observed in this study. A sensitivity analysis showed that, within the range of values observed empirically, LATEBLIGHT is more sensitive to changes in variables related to initial inoculum and to weather than to changes in variables relating to host resistance. PMID:18943552

  2. Effect of Gd polarization on the large magnetocaloric effect of GdCrO4 in a broad temperature range

    NASA Astrophysics Data System (ADS)

    Palacios, E.; Tomasi, C.; Sáez-Puche, R.; Dos santos-García, A. J.; Fernández-Martínez, F.; Burriel, R.

    2016-02-01

    The ferromagnetic zircon-type phase of GdCrO4 presents high values for the magnetocaloric (MC) parameters. This compound has large isothermal entropy changes Δ ST under the magnetic field action in a wide temperature range, from 5 to 35 K, reaching a maximum |Δ ST|=29.0 ±0.1 J /kg K at 22 K, for a field increment Δ B =9 T. It orders ferromagnetically at TC=21.3 K via the Cr-Cr exchange interaction and shows a second transition at 4.8 K due to the ordering of the Gd sublattice. The large MC effect is enhanced by the polarization of the Gd3 + ions by the Cr5 + ones via a weaker Gd-Cr interaction. This effect is an interesting feature to be considered in the search for new compounds with a high MC effect in the range of liquid hydrogen or natural gas, regarding the liquefaction of gases by magnetization-demagnetization cycles. This paper contains experimental measurements of magnetization, heat capacity, and direct determinations of the MC effect. The magnetic contribution to the heat capacity Cm has been obtained after subtracting the lattice component. Approximate values for the exchange constants J1 (Cr-Cr) and J3 (Gd-Cr) have been deduced from Cm.

  3. Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles.

    PubMed

    Ceuppens, Siele; Boon, Nico; Uyttendaele, Mieke

    2013-06-01

    Bacillus cereus comprises a highly versatile group of bacteria, which are of particular interest because of their capacity to cause disease. Emetic food poisoning is caused by the toxin cereulide produced during the growth of emetic B. cereus in food, while diarrhoeal food poisoning is the result of enterotoxin production by viable vegetative B. cereus cells in the small intestine, probably in the mucus layer and/or attached to the host's intestinal epithelium. The numbers of B. cereus causing disease are highly variable, depending on diverse factors linked to the host (age, diet, physiology and immunology), bacteria (cellular form, toxin genes and expression) and food (nutritional composition and meal characteristics). Bacillus cereus group strains show impressive ecological diversity, ranging from their saprophytic life cycle in soil to symbiotic (commensal and mutualistic) lifestyles near plant roots and in guts of insects and mammals to various pathogenic ones in diverse insect and mammalian hosts. During all these different ecological lifestyles, their toxins play important roles ranging from providing competitive advantages within microbial communities to inhibition of specific pathogenic organisms for their host and accomplishment of infections by damaging their host's tissues. PMID:23488744

  4. Characterization of extended range Bonner Sphere Spectrometers in the CERF high-energy broad neutron field at CERN

    NASA Astrophysics Data System (ADS)

    Agosteo, S.; Bedogni, R.; Caresana, M.; Charitonidis, N.; Chiti, M.; Esposito, A.; Ferrarini, M.; Severino, C.; Silari, M.

    2012-12-01

    The accurate determination of the ambient dose equivalent in the mixed neutron-photon fields encountered around high-energy particle accelerators still represents a challenging task. The main complexity arises from the extreme variability of the neutron energy, which spans over 10 orders of magnitude or more. Operational survey instruments, which response function attempts to mimic the fluence-to-ambient dose equivalent conversion coefficient up to GeV neutrons, are available on the market, but their response is not fully reliable over the entire energy range. Extended range rem counters (ERRC) do not require the exact knowledge of the energy distribution of the neutron field and the calibration can be done with a source spectrum. If the actual neutron field has an energy distribution different from the calibration spectrum, the measurement is affected by an added uncertainty related to the partial overlap of the fluence-to-ambient dose equivalent conversion curve and the response function. For this reason their operational use should always be preceded by an "in-field" calibration, i.e. a calibration made against a reference instrument exposed in the same field where the survey-meter will be employed. In practice the extended-range Bonner Sphere Spectrometer (ERBSS) is the only device which can serve as reference instrument in these fields, because of its wide energy range and the possibility to assess the neutron fluence and the ambient dose equivalent (H*(10)) values with the appropriate accuracy. Nevertheless, the experience gained by a number of experimental groups suggests that mandatory conditions for obtaining accurate results in workplaces are: (1) the use of a well-established response matrix, thus implying validation campaigns in reference monochromatic neutrons fields, (2) the expert and critical use of suitable unfolding codes, and (3) the performance test of the whole system (experimental set-up, elaboration and unfolding procedures) in a well

  5. A Multivalent Adsorption Apparatus Explains the Broad Host Range of Phage phi92: a Comprehensive Genomic and Structural Analysis

    PubMed Central

    Buettner, Falk F. R.; Browning, Christopher; Nazarov, Sergey; Rabsch, Wolfgang; Bethe, Andrea; Oberbeck, Astrid; Bowman, Valorie D.; Stummeyer, Katharina; Mühlenhoff, Martina; Gerardy-Schahn, Rita

    2012-01-01

    Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent. PMID:22787233

  6. Hemoglobin niobate composite based biosensor for efficient determination of hydrogen peroxide in a broad pH range.

    PubMed

    Gao, Lu; Gao, Qiuming

    2007-02-15

    Inorganic layered niobates (HCa2Nb3O10) were used as immobilization matrices of hemoglobin (Hb) because of their tunable interlayer spaces, large surface areas and good biocompatibilities. A pair of well-defined, quasi-reversible cycle voltammertric peaks were obtained at the Hb-HCa2Nb3O10 modified pyrolytic graphite electrode, suggesting that the layered niobates facilitate the electron transfer between the proteins and the electrode. Hb-HCa2Nb3O10 modified electrode exhibited electrocatalytic response for monitoring H2O2 with a large linear detection range from 25 microM to 3.0 mM and a relatively high sensitivity of 172 microA mM-1 cm-2. Based on the stabilizing effect of the layered niobates, Hb-HCa2Nb3O10 modified electrode can detect H2O2 in strongly acidic and basic solutions with pH of 1-12, which greatly expands the application fields of biosensors. PMID:16887346

  7. Broad host range of human T-cell leukemia virus type 1 demonstrated with an improved pseudotyping system.

    PubMed Central

    Sutton, R E; Littman, D R

    1996-01-01

    Studies of human T-cell leukemia virus type 1 (HTLV-1) have been hampered by the difficulty of achieving high cell-free and cell-associated infectious titers. Current retroviral pseudotyping systems using the HTLV-1 envelope generate titers of less than 200 infectious particles per ml. We describe here an improved system for pseudotyping using a defective human immunodeficiency virus (HIV) type 1 genome in combination with HTLV-1 env in 293T producer cells. Introduction of additional copies of rev and treatment of cells with sodium butyrate resulted in a cell-associated titer of 10(5)/ml and cell-free titers of greater than 10(4)/ml . By using this system, we found that the host range of HTLV-1 is even greater than previously suspected. Earlier studies which assigned a chromosomal location for the HTLV-1 receptor may therefore reflect cell-to-cell variation in receptor number rather than the absolute presence or absence of a receptor. The generation of higher-titer HIV(HTLV-1) may facilitate identification of the cellular receptor and investigations of the pathophysiology of HTLV-1 infection. PMID:8794391

  8. Femtosecond laser nanostructuring of titanium metal towards fabrication of low-reflective surfaces over broad wavelength range

    NASA Astrophysics Data System (ADS)

    Dar, Mudasir H.; Kuladeep, R.; Saikiran, V.; Narayana Rao, D.

    2016-05-01

    We investigated experimentally the formation of laser induced periodic surface structures (LIPSS) on titanium (Ti) metal upon irradiation with linearly polarized Ti:Sapphire femtosecond (fs) laser pulses of ∼110 fs pulse width and 800 nm wavelength in air and water environments. It is observed that initially formed random and sparsely distributed nano-roughness (nanoholes, nanoparticles and nanoprotrusions) gets periodically structured with increase in number of laser pulses. In air at lower fluence, we observed the formation of high spatial frequency-LIPSS (HSFL) oriented parallel to the laser polarization direction, whereas at higher fluence formation of low spatial frequency-LIPSS (LSFL) were observed that are oriented perpendicular to the incident laser polarization. In water two types of subwavelength structures were observed, one with spatial periodicity of ∼λ/15 and oriented parallel to laser polarization, while the other oriented perpendicular to laser polarization with feature size of λ/4. The optimal conditions for fabricating periodic sub-wavelength structures are determined by controlling the fluence and pulse number. The fs laser induced surface modifications were found to suppress the specular reflection of the Ti surface over a wide wavelength range of 250-2000 nm to a great extent.

  9. Two Measurement Methods of Leaf Dry Matter Content Produce Similar Results in a Broad Range of Species

    PubMed Central

    Vaieretti, María Victoria; Díaz, Sandra; Vile, Denis; Garnier, Eric

    2007-01-01

    Background and Aims Leaf dry matter content (LDMC) is widely used as an indicator of plant resource use in plant functional trait databases. Two main methods have been proposed to measure LDMC, which basically differ in the rehydration procedure to which leaves are subjected after harvesting. These are the ‘complete rehydration’ protocol of Garnier et al. (2001, Functional Ecology 15: 688–695) and the ‘partial rehydration’ protocol of Vendramini et al. (2002, New Phytologist 154: 147–157). Methods To test differences in LDMC due to the use of different methods, LDMC was measured on 51 native and cultivated species representing a wide range of plant families and growth forms from central-western Argentina, following the complete rehydration and partial rehydration protocols. Key Results and Conclusions The LDMC values obtained by both methods were strongly and positively correlated, clearly showing that LDMC is highly conserved between the two procedures. These trends were not altered by the exclusion of plants with non-laminar leaves. Although the complete rehydration method is the safest to measure LDMC, the partial rehydration procedure produces similar results and is faster. It therefore appears as an acceptable option for those situations in which the complete rehydration method cannot be applied. Two notes of caution are given for cases in which different datasets are compared or combined: (1) the discrepancy between the two rehydration protocols is greatest in the case of high-LDMC (succulent or tender) leaves; (2) the results suggest that, when comparing many studies across unrelated datasets, differences in the measurement protocol may be less important than differences among seasons, years and the quality of local habitats. PMID:17353207

  10. [Characterization of mid-subtropical evergreen broad-leaved forest gap based on light detection and ranging (LiDAR)].

    PubMed

    Liu, Feng; Tan, Chang; Wang, Hong; Zhang, Jiang; Wan, Ying; Long, Jiang-ping; Liu, Rui-xi

    2015-12-01

    Light Detection and Ranging (LiDAR) is an active remote sensing technology for acqui- ring three-dimensional structure parameters of vegetation canopy with high accuracy over multiple spatial scales, which is greatly important to the promotion of forest disturbance ecology and the ap- plication on gaps. This paper focused on mid-subtropical evergreen broadleaved forest in Hunan Province, and small footprint LiDAR point data were adopted to identify canopy gaps. and measure geomagnetic characteristics of gaps. The optimal grid model resolution and interpolation methods were chosen to generate canopy height model, and the computer graphics processing was adopted to estimate characteristics of gaps which involved gap size, canopy height and gap shape index, then field investigation was utilized to validate the estimation results. The results showed that the gap rec- ognition rate was 94.8%, and the major influencing factors were gap size and gap maker type. Line- ar correlation was observed between LiDAR estimation and field investigation, and the R² values of gap size and canopy height case were 0.962 and 0.878, respectively. Compared with field investiga- tion, the size of mean estimated gap was 19.9% larger and the mean estimated canopy height was 9.9% less. Gap density was 12.8 gaps · hm⁻² and the area of gaps occupied 13.3% of the forest area. The average gap size, canopy height and gap shape index were 85.06 m², 15.33 m and 1.71, respectively. The study site usually contained small gaps in which the edge effect was not obvious. PMID:27111996

  11. Laboratory study on the kinetics of CO2 hydrates in a broad p-T range relevant to Mars

    NASA Astrophysics Data System (ADS)

    Falenty, A.; Kuhs, W. F.

    2007-08-01

    , temporary gas outbursts are conceivable. Between 190K and 240K neither annealing of defective ice Ih nor the crystal regrowth is fast enough to effectively slow down outward diffusing gas molecules. In such a scenario slow decomposition is to be expected and therefore the impact on the surface will be very limited. Surprisingly we also have found "self preservation" in a narrow pressure range. The sealing effect is, however, less pronounced as the preservation mechanism differs from the higher temperature one. Therefore only large agglomerations of CO2 hydrates may be effectively saved from further decomposition. [1] J.S.Kargel Mars: A Warmer Wetter Planet, Springer Berlin, 2004. [2] R. Greve, R.A. Mahajan (2005), Icarus 176, 475-485 [3] D.K. Staykova et al. (2003) J. Phys. Chem. B 107,10299-10311 [4] G. Genov et al. (2004), Am. Miner. 89, 1228-1239 [5] W.F. Kuhs et al. (2006) J.Phys.Chem. B 110 (26), 13283-13295 [6] G. Genov PhD thesis, Georg-August Universität, Göttingen, 2004 [7] W. F. Kuhs et al. (2004), Phys. Chem. Chem. Phys. 6, 4917-4920 [8] A. Falenty et al. (2007) In: Physics and Chemistry of Ice (ed. W. F. Kuhs), RSC Publishing, Cambridge, 2007, pp. 171-179

  12. Hierarchical MoS2@MoP core-shell heterojunction electrocatalysts for efficient hydrogen evolution reaction over a broad pH range

    NASA Astrophysics Data System (ADS)

    Wu, Aiping; Tian, Chungui; Yan, Haijing; Jiao, Yanqing; Yan, Qing; Yang, Guoyu; Fu, Honggang

    2016-05-01

    A low-cost catalyst for the hydrogen evolution reaction (HER) over a broad pH range is highly desired to meet the practical needs in different areas. In this study, hierarchical flower-like MoS2@MoP core-shell heterojunctions (HF-MoSP) are designed as a promising catalyst for HER over a broad pH range. The materials are obtained by the controllable phosphidation of the hierarchical MoS2 flower (HF-MoS2) composed of thin silk belt-like sheets. The phosphidation degree, P/S ratio and work function (WF) of HF-MoSP can be tuned easily over broad range by changing the phosphidation temperature. Under optimized condition, HF-MoSP exhibits excellent electrocatalytic activity for HER with a low onset overpotential of 29 mV and η of 108 mV at 10 mA cm-2 in 0.5 M H2SO4 and retains its good activity for 30 h. In addition, the catalyst shows excellent activity in 1 M KOH with an onset overpotential of 42 mV and η of 119 mV at 10 mA cm-2. The catalysts also exhibit obvious activity in neutral, weak acid and weak alkaline conditions. The good performance is relative to the synergy of the MoP shell and MoS2 core and the high WF of HF-MoSP close to Pt, and the large SBET of HF-MoSP benefited from the hierarchical structure. This study represents the construction of the core-shell heterojunction and provides a new way to provide the low-cost and high-performance catalyst for HER.A low-cost catalyst for the hydrogen evolution reaction (HER) over a broad pH range is highly desired to meet the practical needs in different areas. In this study, hierarchical flower-like MoS2@MoP core-shell heterojunctions (HF-MoSP) are designed as a promising catalyst for HER over a broad pH range. The materials are obtained by the controllable phosphidation of the hierarchical MoS2 flower (HF-MoS2) composed of thin silk belt-like sheets. The phosphidation degree, P/S ratio and work function (WF) of HF-MoSP can be tuned easily over broad range by changing the phosphidation temperature. Under optimized

  13. Hierarchical MoS2@MoP core-shell heterojunction electrocatalysts for efficient hydrogen evolution reaction over a broad pH range.

    PubMed

    Wu, Aiping; Tian, Chungui; Yan, Haijing; Jiao, Yanqing; Yan, Qing; Yang, Guoyu; Fu, Honggang

    2016-06-01

    A low-cost catalyst for the hydrogen evolution reaction (HER) over a broad pH range is highly desired to meet the practical needs in different areas. In this study, hierarchical flower-like MoS2@MoP core-shell heterojunctions (HF-MoSP) are designed as a promising catalyst for HER over a broad pH range. The materials are obtained by the controllable phosphidation of the hierarchical MoS2 flower (HF-MoS2) composed of thin silk belt-like sheets. The phosphidation degree, P/S ratio and work function (WF) of HF-MoSP can be tuned easily over broad range by changing the phosphidation temperature. Under optimized condition, HF-MoSP exhibits excellent electrocatalytic activity for HER with a low onset overpotential of 29 mV and η of 108 mV at 10 mA cm(-2) in 0.5 M H2SO4 and retains its good activity for 30 h. In addition, the catalyst shows excellent activity in 1 M KOH with an onset overpotential of 42 mV and η of 119 mV at 10 mA cm(-2). The catalysts also exhibit obvious activity in neutral, weak acid and weak alkaline conditions. The good performance is relative to the synergy of the MoP shell and MoS2 core and the high WF of HF-MoSP close to Pt, and the large SBET of HF-MoSP benefited from the hierarchical structure. This study represents the construction of the core-shell heterojunction and provides a new way to provide the low-cost and high-performance catalyst for HER. PMID:27172989

  14. Nitrogen-rich functional groups carbon nanoparticles based fluorescent pH sensor with broad-range responding for environmental and live cells applications.

    PubMed

    Shi, Bingfang; Su, Yubin; Zhang, Liangliang; Liu, Rongjun; Huang, Mengjiao; Zhao, Shulin

    2016-08-15

    A nitrogen-rich functional groups carbon nanoparticles (N-CNs) based fluorescent pH sensor with a broad-range responding was prepared by one-pot hydrothermal treatment of melamine and triethanolamine. The as-prepared N-CNs exhibited excellent photoluminesence properties with an absolute quantum yield (QY) of 11.0%. Furthermore, the N-CNs possessed a broad-range pH response. The linear pH response range was 3.0 to 12.0, which is much wider than that of previously reported fluorescent pH sensors. The possible mechanism for the pH-sensitive response of the N-CNs was ascribed to photoinduced electron transfer (PET). Cell toxicity experiment showed that the as-prepared N-CNs exhibited low cytotoxicity and excellent biocompatibility with the cell viabilities of more than 87%. The proposed N-CNs-based pH sensor was used for pH monitoring of environmental water samples, and pH fluorescence imaging of live T24 cells. The N-CNs is promising as a convenient and general fluorescent pH sensor for environmental monitoring and bioimaging applications. PMID:27085956

  15. Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing

    PubMed Central

    Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

    2015-01-01

    The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0–9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community. PMID:25798135

  16. Monitoring the Dissemination of the Broad-Host-Range Plasmid pB10 in Sediment Microcosms by Quantitative PCR▿ †

    PubMed Central

    Bonot, Sébastien; Merlin, Christophe

    2010-01-01

    Studying the transfer of specific mobile genetic elements in complex environmental matrices remains difficult because suitable molecular tools are not yet available to back up classical culture-dependent approaches. In this report, we show that quantitative PCR could be used to monitor the dissemination of the broad-host-range plasmid pB10 in sediment microcosms. This approach lies in the differential measurement of the host and plasmid DNAs used to inoculate the microcosms, using a particular design of quantitative PCR primers/probes where we took advantage of the mosaic aspect of the bacterial genomes to achieve a highly specific quantitative PCR detection system. PMID:19897757

  17. Photodetectors: Broad Detection Range Rhenium Diselenide Photodetector Enhanced by (3-Aminopropyl)Triethoxysilane and Triphenylphosphine Treatment (Adv. Mater. 31/2016).

    PubMed

    Jo, Seo-Hyeon; Park, Hyung-Youl; Kang, Dong-Ho; Shim, Jaewoo; Jeon, Jaeho; Choi, Seunghyuk; Kim, Minwoo; Park, Yongkook; Lee, Jaehyeong; Song, Young Jae; Lee, Sungjoo; Park, Jin-Hong

    2016-08-01

    The effects of triphenylphosphine (PPh3 ) and (3-amino-propyl)triethoxysilane (APTES) on a rhenium diselenide (ReSe2 ) photodetector are systematically studied by J.-H. Park and co-workers on page 6711 in comparison with a conventional MoS2 device. A very high performance ReSe2 photodetector is demonstrated, which has a broad photodetection range, high photoresponsivity (1.18 × 10(6) A W(-1) ), and fast photoswitching speed (rising/decaying time: 58/263 ms). PMID:27511529

  18. Thermodynamical features of Verlinde's approach for a non-commutative Schwarzschild-anti-deSitter black hole in a broad range of scales

    NASA Astrophysics Data System (ADS)

    Mehdipour, S. Hamid

    2014-09-01

    We try to study the thermodynamical features of a non-commutative inspired Schwarzschild-anti-de Sitter black hole in the context of the entropic gravity model, particularly for the model that is employed in a broad range of scales, from the short distances to the large distances. At small length scales, the Newtonian force fails because one finds a linear relation between the entropic force and the distance. In addition, there are some deviations from the standard Newtonian gravity at large length scales.

  19. Broad-range TRP channel inhibitors (2-APB, flufenamic acid, SKF-96365) affect differently contraction of resistance and conduit femoral arteries of rat.

    PubMed

    Bencze, Michal; Behuliak, Michal; Vavřínová, Anna; Zicha, Josef

    2015-10-15

    Transient receptor potential (TRP) channels are proposed to contribute to membrane depolarization and Ca2+ influx into vascular smooth muscle (VSM) cells. Our aim was to study the effects of widely used broad-range TRP channel inhibitors--2-aminoethoxydiphenyl borate (2-APB), flufenamic acid (FFA) and SKF-96365--on the contraction of freshly isolated small and large arteries. Endothelium-denuded resistance (≈250 µm) and conduit (≈1000 µm) femoral arteries were isolated from adult Wistar rats and mounted in wire myograph. The effects of the above mentioned TRP channel inhibitors and voltage-dependent calcium channel inhibitor nifedipine were studied on arterial contractions induced by phenylephrine, U-46619 or K+. Phenylephrine-induced contractions were also studied in the absence of extracellular Na+. mRNA expression of particular canonical and melastatin TRP channel subunits in femoral vascular bed was determined. TRP channel inhibitors attenuated K+-induced contraction less than nifedipine. Phenylephrine-induced contraction was more influenced by 2-APB in resistance arteries, while FFA completely prevented U-46619-induced contraction in both sizes of arteries. The absence of extracellular Na+ prevented the inhibitory effects of 2-APB, but not those of FFA. The observed effects of broad-range TRP channel inhibitors, which were dependent on the size of the artery, confirmed the involvement of TRP channels in agonist-induced contractions. The inhibitory effects of 2-APB (but not those of FFA or SKF-96365) were dependent on the presence of extracellular Na+. PMID:26384458

  20. Microwave-assisted synthesis of novel non-peripherally substituted metallophthalocyanines and their sensing behaviour for a broad range of Lewis bases.

    PubMed

    Duruk, E Gülruh; Yenilmez, H Yasemin; Altındal, Ahmet; Altuntaş Bayır, Zehra

    2015-06-01

    The synthesis of novel, symmetrical, tetrasubstituted metallophthalocyanines (cobalt, zinc, and manganese) bearing four 2-(4-methyl-1,3-thiazol-5-yl)ethoxy units is reported. The new compounds have been characterized using UV-Vis, IR, (1)H NMR, (13)C NMR, and mass spectroscopy data. Photophysical properties of zinc(ii) phthalocyanines were found, including electronic absorption and fluorescence quantum yields. The fluorescence of the complexes was investigated in DMF and it was found that benzoquinone (BQ) was an effective quencher. The response and recovery behaviours of the spin coated films to different analytes, which span a broad range of Lewis bases, have been investigated by means of conductivity measurements. It was observed that the operating temperature had a considerable effect on the gas sensing performance of the sensors investigated. The sensing behaviour of the films for a broad range of Lewis bases and the correlation between the sensor sensitivity and Lewis base enthalpies were investigated. Results show that the sensitivity of the films may be correlated exponentially with the binding enthalpy. PMID:25947943

  1. High-density universal 16S rRNA microarray analysis revealsbroader diversity than typical clone library when sampling theenvironment

    SciTech Connect

    DeSantis, Todd Z.; Brodie, Eoin L.; Moberg, Jordan P.; Zubieta,Ingrid X.; Piceno, Yvette M.; Andersen, Gary L.

    2006-06-15

    Molecular approaches aimed at detection of a broad-range ofprokaryotes in the environment routinely rely upon classifyingheterogeneous 16S rRNA genes amplified by PCR using primers with broadspecificity. The general method of sampling and categorizing DNA has beento clone then sequence the PCR products. However, the number of clonesrequired to adequately catalogue the majority of taxa in a sample isunwieldy. Alternatively, hybridizing target sequences to a universal 16SrRNA gene microarray may provide a more rapid and comprehensive view ofprokaryotic community composition. This study investigated the breadthand accuracy of a microarray in detecting diverse 16S rRNA gene sequencetypes compared to clone-and-sequencing using three environmental samples:urban aerosol, subsurface soil and subsurface water. PCR productsgenerated from universal 16S rRNA gene-targeted primers were classifiedusing either the clone-and-sequence method or by hybridization to a novelhigh-density microarray of 297,851 probes complementary to 842prokaryotic sub-families. The three clone libraries comprised 1,391high-quality sequences. Approximately 8 percent of the clones could notbe placed into a known sub-family and were considered novel. Themicroarray results confirmed the majority of clone-detected sub-familiesand additionally demonstrated greater amplicon diversity extending intophyla not observed by the cloning method. Sequences matching OTUs withinthe phyla Nitrospira, Planctomycetes, and TM7, which were uniquelydetected by the array, were verified with specific primers and subsequentamplicon sequencing. Sub-family richness detected by the arraycorresponded well with non-parametric richness predictions extrapolatedfrom clone libraries except in the water community where clone-basedrichness predictions were greatly exceeded. It was concluded thatalthough the microarray is unreliable inidentifying novel prokaryotictaxa, it reveals greater diversity in environmental samples thansequencing a

  2. Detection of novel organisms associated with salpingitis, by use of 16S rDNA polymerase chain reaction.

    PubMed

    Hebb, Jennifer K; Cohen, Craig R; Astete, Sabina G; Bukusi, Elizabeth A; Totten, Patricia A

    2004-12-15

    Although Chlamydia trachomatis and Neisseria gonorrhoeae are established causes of salpingitis, the majority of cases have no known etiology. We used broad-range 16S rDNA polymerase chain reaction to identify novel, possibly uncultivable, bacteria associated with salpingitis and identified bacterial 16S sequences in Fallopian-tube specimens from 11 (24%) of 45 consecutive women with laparoscopically confirmed acute salpingitis (the case patients) and from 0 of 44 women seeking tubal ligations (the control subjects) at Kenyatta National Hospital, Nairobi, Kenya. Bacterial phylotypes most closely related to Leptotrichia spp. were detected as the sole phylotypes in 1, and mixed with other bacterial phylotypes in 2, specimens. Novel bacterial phylotypes and those associated with bacterial vaginosis, including Atopobium vaginae, were identified in 3 specimens. N. gonorrhoeae and Streptococcus pyogenes were identified in 2 and 1 specimens, respectively. The finding of novel phylotypes associated with salpingitis has important implications for the etiology, pathogenesis, and treatment of this important reproductive-tract disease syndrome. PMID:15551209

  3. Broad energy range neutron spectroscopy using a liquid scintillator and a proportional counter: Application to a neutron spectrum similar to that from an improvised nuclear device

    NASA Astrophysics Data System (ADS)

    Xu, Yanping; Randers-Pehrson, Gerhard; Marino, Stephen A.; Garty, Guy; Harken, Andrew; Brenner, David J.

    2015-09-01

    A novel neutron irradiation facility at the Radiological Research Accelerator Facility (RARAF) has been developed to mimic the neutron radiation from an Improvised Nuclear Device (IND) at relevant distances (e.g. 1.5 km) from the epicenter. The neutron spectrum of this IND-like neutron irradiator was designed according to estimations of the Hiroshima neutron spectrum at 1.5 km. It is significantly different from a standard reactor fission spectrum, because the spectrum changes as the neutrons are transported through air, and it is dominated by neutron energies from 100 keV up to 9 MeV. To verify such wide energy range neutron spectrum, detailed here is the development of a combined spectroscopy system. Both a liquid scintillator detector and a gas proportional counter were used for the recoil spectra measurements, with the individual response functions estimated from a series of Monte Carlo simulations. These normalized individual response functions were formed into a single response matrix for the unfolding process. Several accelerator-based quasi-monoenergetic neutron source spectra were measured and unfolded to test this spectroscopy system. These reference neutrons were produced from two reactions: T(p,n)3He and D(d,n)3He, generating neutron energies in the range between 0.2 and 8 MeV. The unfolded quasi-monoenergetic neutron spectra indicated that the detection system can provide good neutron spectroscopy results in this energy range. A broad-energy neutron spectrum from the 9Be(d,n) reaction using a 5 MeV deuteron beam, measured at 60 degrees to the incident beam was measured and unfolded with the evaluated response matrix. The unfolded broad neutron spectrum is comparable with published time-of-flight results. Finally, the pair of detectors were used to measure the neutron spectrum generated at the RARAF IND-like neutron facility and a comparison is made to the neutron spectrum of Hiroshima.

  4. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    SciTech Connect

    Walters , William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada , Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, Greg; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob

    2015-12-22

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of datasets amplified with varied primers requires attention. Here we examine the performance of modified 16S rRNA gene and ITS primers for archaea/bacteria and fungi, respectively, with non-aquatic samples. We moved primer barcodes to the 5’-end, allowing for a range of different 3’ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4-5 of the 16S rRNA gene. We additionally demonstrate that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

  5. Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p-Coumaric Acid Production in Pseudomonas putida KT2440.

    PubMed

    Calero, Patricia; Jensen, Sheila I; Nielsen, Alex T

    2016-07-15

    Pseudomonas putida KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set of ProUSER-reporter vectors was further created to characterize different inducible promoters. The PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine and p-coumaric acid. Pm and PrhaB were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale p-coumaric acid production of 1.2 mM, the highest achieved in Pseudomonads under comparable conditions. With broad-host-range compatibility, the ProUSER vectors will serve as useful tools for optimizing gene expression in a variety of bacteria. PMID:27092814

  6. Fluorescence signaling of hydrogen sulfide in broad pH range using a copper complex based on BINOL-benzimidazole ligands.

    PubMed

    Sun, Mingtai; Yu, Huan; Li, Huihui; Xu, Hongda; Huang, Dejian; Wang, Suhua

    2015-04-20

    A weakly fluorescent complex derived from a binaphthol-benzimidazole ligand was designed and synthesized for hydrogen sulfide at different pH conditions. It was demonstrated that the probe showed the same reactivity to various hydrogen sulfide species in a broad range of pH values to generate highly fluorescent product through a displacement reaction mechanism, whereas the product's fluorescence spectrum exhibited a hypsochromic shift of ∼73 nm (2393 cm(-1)) as pH increased from neutral to basic, which can be used for distinguishing the various species of hydrogen sulfide. This turn-on fluorescence probe was highly selective and sensitive to hydrogen sulfide with a detection limit of 0.11 μM. It was then applied for evaluating the total content of sulfide (including hydrogen sulfide, hydrosulfide, and sulfide) as well as for the visual detection of gaseous H2S in air using a simple test paper strip. PMID:25839192

  7. Temperature-dependent dynamic correlations in suspensions of magnetic nanoparticles in a broad range of concentrations: a combined experimental and theoretical study.

    PubMed

    Ivanov, Alexey O; Kantorovich, Sofia S; Zverev, Vladimir S; Elfimova, Ekaterina A; Lebedev, Alexander V; Pshenichnikov, Alexander F

    2016-07-21

    The interweave of competing individual relaxations influenced by the presence of temperature and concentration dependent correlations is an intrinsic feature of superparamagnetic nanoparticle suspensions. This unique combination gives rise to multiple applications of such suspensions in medicine, nanotechnology and microfluidics. Here, using theory and experiment, we investigate dynamic magnetic susceptibility in a broad range of temperatures and frequencies. Our approach allows, for the first time to our knowledge, to separate clearly the effects of superparamagnetic particle polydispersity and interparticle magnetic interactions on the dynamic spectra of these systems. In this way, we not only provide a theoretical model that can predict well the dynamic response of magnetic nanoparticles systems, but also deepen the understanding of the dynamic nanoparticle self-assembly, opening new perspectives in tuning and controlling the magnetic behaviour of such systems in AC fields. PMID:27334549

  8. Dry desulfurization of simulated flue gas in a fluidized-bed reactor for a broad range of SO{sub 2} concentration and temperature: A comparison of models

    SciTech Connect

    Suyadal, Y.; Oguz, H.

    1999-08-01

    In this work, dry desulfurization of simulated flue gas was investigated in a batchwise operated laboratory-scale stainless steel fluidized-bed reactor (46 x 500 mm{sup 2}) by using calcium-containing local Turkish limestone (Karaagach/MUS) which was calcined at 900 C with 5% H{sub 2}O vapor. The sulfation reaction was carried out in a broad range of temperature (200 {le} T({degree}C) {le} 900) and SO{sub 2} feedstock concentration (1000 {le} C(ppm SO{sub 2}) {le} 6000). The experimental sulfation conversion-time data were tested according to unreacted shrinking core model (SCM), changing the grain size model (GM) and random pore model (RPM). It was found that the random pore model with control of product layer (CaSO{sub 3}/CaSO{sub 4}) diffusion described the experimental data best.

  9. Characterization of 6H-SiC JFET Integrated Circuits Over A Broad Temperature Range from -150 C to +500 C

    NASA Technical Reports Server (NTRS)

    Neudeck, Philip G.; Krasowski, Michael J.; Chen, Liang-Yu; Prokop, Norman F.

    2009-01-01

    The NASA Glenn Research Center has previously reported prolonged stable operation of simple prototype 6H-SiC JFET integrated circuits (logic gates and amplifier stages) for thousands of hours at +500 C. This paper experimentally investigates the ability of these 6H-SiC JFET devices and integrated circuits to also function at cold temperatures expected to arise in some envisioned applications. Prototype logic gate ICs experimentally demonstrated good functionality down to -125 C without changing circuit input voltages. Cascaded operation of gates at cold temperatures was verified by externally wiring gates together to form a 3-stage ring oscillator. While logic gate output voltages exhibited little change across the broad temperature range from -125 C to +500 C, the change in operating frequency and power consumption of these non-optimized logic gates as a function of temperature was much larger and tracked JFET channel conduction properties.

  10. Complete Genome sequence of Burkholderia phymatum STM815T, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

    PubMed Central

    Moulin, Lionel; Klonowska, Agnieszka; Caroline, Bournaud; Booth, Kristina; Vriezen, Jan A.C.; Melkonian, Rémy; James, Euan K.; Young, J. Peter W.; Bena, Gilles; Hauser, Loren; Land, Miriam; Kyrpides, Nikos; Bruce, David; Chain, Patrick; Copeland, Alex; Pitluck, Sam; Woyke, Tanja; Lizotte-Waniewski, Michelle; Bristow, Jim; Riley, Margaret

    2014-01-01

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp). PMID:25197461

  11. Complete Genome sequence of Burkholderia phymatum STM815, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

    SciTech Connect

    Moulin, Lionel; Klonowska, Agnieszka; Caroline, Bournaud; Booth, Kristina; Vriezen, Jan A.C.; Melkonian, Remy; James, Euan; Young, Peter W.; Bena, Gilles; Hauser, Loren John; Land, Miriam L; Kyrpides, Nikos C; Bruce, David; Chain, Patrick S. G.; Copeland, A; Pitluck, Sam; Woyke, Tanja; Lizotte-Waniewski, Michelle; Bristow, James; Riley, Monica

    2014-01-01

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).

  12. The 16S ribosomal RNA mutation database (16SMDB).

    PubMed Central

    Triman, K L

    1996-01-01

    The 16S ribosomal RNA mutation database (16SMDB) provides a list of mutated positions in 16S ribosomal RNA from Escherichia coli and the identity of each alteration. Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation; (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods; (iii) relevant literature citations. The database is available via ftp and on the World Wide Web. PMID:8594570

  13. Bacillus polymachus sp. nov., with a broad range of antibacterial activity, isolated from forest topsoil samples by using a modified culture method.

    PubMed

    Nguyen, Tuan Manh; Kim, Jaisoo

    2015-02-01

    A new, modified culture method that utilizes a transwell plate with a 0.4 µm pore-size microporous membrane was developed. This system allows only trace nutrients from the soil into the liquid culture through the microporous membrane. The method is a more powerful tool for the discovery of novel species from soils than are traditional methods. Such newly identified species could potentially produce useful metabolites. A bacterial strain, T515(T), was isolated using this modified culture method. Growth of strain T515(T) occurred at pH 4-9 in a temperature range between 20 °C and 40 °C and in the presence of 0-2 % (w/v) NaCl on R2A agar. Colonies on the agar plates were tiny, white, and convex after 5 days incubation at 28 °C. Comparative analysis of the nearly full-length 16S rRNA gene sequence of strain T515(T) revealed close pairwise similarity with species of the genus Bacillus, and strain T515(T) was most closely related to Bacillus panaciterrae Gsoil 1517(T) (96.7 %) and Bacillus funiculus NAF001(T) (96.0 %). The major quinone of strain T515(T) was menaquinone-7 (MK-7) and the major fatty acids were iso-C15 : 0 (45.5 %), anteiso-C15 : 0 (23.2 %) and C16 : 0 (10.9 %). The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Strain T515(T) was sensitive to streptomycin and tetracycline, but resistant to rifampicin (0.125 µg ml(-1)), ampicillin (0.5 µg ml(-1)) and chloramphenicol (1 µg ml(-1)). The strain showed antimicrobial activities against the six strains tested: Bacillus subtilis KEMB 51201-001, Staphylococcus aureus KEMB 4659, Pseudomonas aeruginosa KACC 10185, Staphylococcus epidermidis KACC 13234, Paenibacillus larvae KACC 14031 and Escherichia coli KEMB 212-234. Based on these results, strain T515(T) represents a novel species of the genus Bacillus with the proposed name, Bacillus polymachus sp. nov. The type strain is T515(T) ( = KEMB 9005-168(T) = KACC 18242(T) = NBRC 110614(T)). PMID

  14. Creation of a Broad-Range and Highly Stereoselective d-Amino Acid Dehydrogenase for the One-Step Synthesis of d-Amino Acids

    PubMed Central

    Vedha-Peters, Kavitha; Gunawardana, Manjula; Rozzell, J. David; Novick, Scott J.

    2008-01-01

    Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective d-amino acid dehydrogenase. This new enzyme is capable of producing d-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate d-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight- and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity towards the d-enantiomer (95 to > 99% e.e) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for d-amino acid synthesis. PMID:16910688

  15. Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system.

    PubMed Central

    Winstanley, C; Morgan, J A; Pickup, R W; Jones, J G; Saunders, J R

    1989-01-01

    Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species. Images PMID:2729979

  16. Carbon dots with strong excitation-dependent fluorescence changes towards pH. Application as nanosensors for a broad range of pH.

    PubMed

    Barati, Ali; Shamsipur, Mojtaba; Abdollahi, Hamid

    2016-08-10

    In this study, preparation of novel pH-sensitive N-doped carbon dots (NCDs) using glucose and urea is reported. The prepared NCDs present strong excitation-dependent fluorescence changes towards the pH that is a new behavior from these nanomaterials. By taking advantage of this unique behavior, two separated ratiometric pH sensors using emission spectra of the NCDs for both acidic (pH 2.0 to 8.0) and basic (pH 7.0 to 14.0) ranges of pH are constructed. Additionally, by considering the entire Excitation-Emission Matrix (EEM) of NCDs as analytical signal and using a suitable multivariate calibration method, a broad range of pH from 2.0 to 14.0 was well calibrated. The multivariate calibration method was independent from the concentration of NCDs and resulted in a very low average prediction error of 0.067 pH units. No changes in the predicted pH under UV irradiation (for 3 h) and at high ionic strength (up to 2 M NaCl) indicated the high stability of this pH nanosensor. The practicality of this pH nanosensor for pH determination in real water samples was validated with good accuracy and repeatability. PMID:27282748

  17. Wavelength-resolved optical extinction measurements of aerosols using broad-band cavity-enhanced absorption spectroscopy over the spectral range of 445-480 nm.

    PubMed

    Zhao, Weixiong; Dong, Meili; Chen, Weidong; Gu, Xuejun; Hu, Changjin; Gao, Xiaoming; Huang, Wei; Zhang, Weijun

    2013-02-19

    Despite the significant progress in the measurements of aerosol extinction and absorption using spectroscopy approaches such as cavity ring-down spectroscopy (CRDS) and photoacoustic spectroscopy (PAS), the widely used single-wavelength instruments may suffer from the interferences of gases absorption present in the real environment. A second instrument for simultaneous measurement of absorbing gases is required to characterize the effect of light extinction resulted from gases absorption. We present in this paper the development of a blue light-emitting diode (LED)-based incoherent broad-band cavity-enhanced spectroscopy (IBBCEAS) approach for broad-band measurements of wavelength-resolved aerosol extinction over the spectral range of 445-480 nm. This method also allows for simultaneous measurement of trace gases absorption present in the air sample using the same instrument. On the basis of the measured wavelength-dependent aerosol extinction cross section, the real part of the refractive index (RI) can be directly retrieved in a case where the RI does not vary strongly with the wavelength over the relevant spectral region. Laboratory-generated monodispersed aerosols, polystyrene latex spheres (PSL) and ammonium sulfate (AS), were employed for validation of the RI determination by IBBCEAS measurements. On the basis of a Mie scattering model, the real parts of the aerosol RI were retrieved from the measured wavelength-resolved extinction cross sections for both aerosol samples, which are in good agreement with the reported values. The developed IBBCEAS instrument was deployed for simultaneous measurements of aerosol extinction coefficient and NO(2) concentration in ambient air in a suburban site during two representative days. PMID:23320530

  18. Broad Habitat Range of the Phylogenetically Narrow R-BT065 Cluster, Representing a Core Group of the Betaproteobacterial Genus Limnohabitans▿ †

    PubMed Central

    Šimek, Karel; Kasalický, Vojtěch; Jezbera, Jan; Jezberová, Jitka; Hejzlar, Josef; Hahn, Martin W.

    2010-01-01

    The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats. PMID:19948856

  19. Elevated Norepinephrine may be a Unifying Etiological Factor in the Abuse of a Broad Range of Substances: Alcohol, Nicotine, Marijuana, Heroin, Cocaine, and Caffeine.

    PubMed

    Fitzgerald, Paul J

    2013-01-01

    A wide range of commonly abused drugs have effects on the noradrenergic neurotransmitter system, including alterations during acute intoxication and chronic use of these drugs. It is not established, however, that individual differences in noradrenergic signaling, which may be present prior to use of drugs, predispose certain persons to substance abuse. This paper puts forth the novel hypothesis that elevated noradrenergic signaling, which may be raised largely due to genetics but also due to environmental factors, is an etiological factor in the abuse of a wide range of substances, including alcohol, nicotine, marijuana, heroin, cocaine, and caffeine. Data are reviewed for each of these drugs comprising their interaction with norepinephrine during acute intoxication, long-term use, subsequent withdrawal, and stress-induced relapse. In general, the data suggest that these drugs acutely boost noradrenergic signaling, whereas long-term use also affects this neurotransmitter system, possibly suppressing it. During acute withdrawal after chronic drug use, noradrenergic signaling tends to be elevated, consistent with the observation that norepinephrine lowering drugs such as clonidine reduce withdrawal symptoms. Since psychological stress can promote relapse of drug seeking in susceptible individuals and stress produces elevated norepinephrine release, this suggests that these drugs may be suppressing noradrenergic signaling during chronic use or instead elevating it only in reward circuits of the brain. If elevated noradrenergic signaling is an etiological factor in the abuse of a broad range of substances, then chronic use of pharmacological agents that reduce noradrenergic signaling, such as clonidine, guanfacine, lofexidine, propranolol, or prazosin, may help prevent or treat drug abuse in general. PMID:24151426

  20. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase.

    PubMed

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-01

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1-2.5 µg) conferring optimal levels of long-term expression (>10(11) photons/second/cm(2)). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>10(10) photons/second/cm(2)) was achieved at a transposon dose of 5-125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver. PMID:26784638

  1. Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

    PubMed Central

    Van Puyvelde, Sandra; De Block, Tessa; Maltha, Jessica; Palpouguini, Lompo; Tahita, Marc; Tinto, Halidou; Jacobs, Jan; Deborggraeve, Stijn

    2016-01-01

    Background Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. Methodology/Principal Findings The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Conclusions/Significance Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination. PMID:26927306

  2. Highly Active and Robust Metalloporphyrin Catalysts for the Synthesis of Cyclic Carbonates from a Broad Range of Epoxides and Carbon Dioxide.

    PubMed

    Maeda, Chihiro; Shimonishi, Junta; Miyazaki, Ray; Hasegawa, Jun-Ya; Ema, Tadashi

    2016-05-01

    Bifunctional metalloporphyrins with quaternary ammonium bromides (nucleophiles) at the meta, para, or ortho positions of meso-phenyl groups were synthesized as catalysts for the formation of cyclic carbonates from epoxides and carbon dioxide under solvent-free conditions. The meta-substituted catalysts exhibited high catalytic performance, whereas the para- and ortho-substituted catalysts showed moderate and low activity, respectively. DFT calculations revealed the origin of the advantage of the meta-substituted catalyst, which could use the flexible quaternary ammonium cation at the meta position to stabilize various anionic species generated during catalysis. A zinc(II) porphyrin with eight nucleophiles at the meta positions showed very high catalytic activity (turnover number (TON)=240 000 at 120 °C, turnover frequency (TOF)=31 500 h(-1) at 170 °C) at an initial CO2 pressure of 1.7 MPa; catalyzed the reaction even at atmospheric CO2 pressure (balloon) at ambient temperature (20 °C); and was applicable to a broad range of substrates, including terminal and internal epoxides. PMID:26990557

  3. Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection.

    PubMed

    Baert, Leen; Uyttendaele, Mieke; Debevere, Johan

    2008-03-31

    Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform. PMID:18258325

  4. Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

    PubMed Central

    Taliaferro, Lanyn P.; Galvin, Teresa A.; Ma, Hailun; Shaheduzzaman, Syed; Williams, Dhanya K.; Glasner, Dustin R.; Khan, Arifa S.

    2014-01-01

    Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences. PMID:24777034

  5. Electrospray synthesis of monodisperse polymer particles in a broad (60 nm-2 μm) diameter range: guiding principles and formulation recipes.

    PubMed

    Almería, Begoña; Gomez, Alessandro

    2014-03-01

    This study reports on a methodology to control the size of polymer particles generated by the electrospray (ES) drying route, with emphasis on the generation of biodegradable polymer nanoparticles that are well suited for biomedical applications. The ability to produce spherical poly(lactic-co-glycolic) acid (PLGA) particles with and without encapsulated active agent, with relative standard deviation not exceeding 15%, was demonstrated over a remarkably broad (60 nm-2 μm) diameter range. By judiciously choosing ES parameters and solution properties, we can control the monodispersity and the size of the obtained particles, tailoring it to a specific application. The main parameters affecting particle size include solution electrical conductivity, flow rate and initial polymer volume fraction. Quasi-monodispersity at both the micro- and the more challenging nano-scale was achieved by avoiding Coulomb fission in the spray droplets, via entanglement of the polymer chains within the droplets rather than by charge neutralization. Guiding principles in the formulation of the solutions to satisfy a multiplicity of constraints are provided along with an extensive database of "recipes". PMID:24407667

  6. Detection and validation of a small broad-host-range plasmid pBBR1MCS-2 for use in genetic manipulation of the extremely acidophilic Acidithiobacillus sp.

    PubMed

    Hao, Likai; Liu, Xiangmei; Wang, Huiyan; Lin, Jianqun; Pang, Xin; Lin, Jianqiang

    2012-09-01

    An efficient genetic system for introducing genes into biomining microorganisms is essential not only to experimentally determine the functions of genes predicted based on bioinformatic analysis, but also for their genetic breeding. In this study, a small broad-host-range vector named pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups, was studied for the feasibility of its use in conjugative gene transfer into extremely acidophilic strains of Acidithiobacillus. To do this, a recombinant plasmid pBBR-tac-Sm, a derivative of pBBR1MCS-2, was constructed and the streptomycin resistant gene (Sm(r)) was used as the reporter gene. Using conjugation, pBBR-tac-Sm was successfully transferred into three tested strains of Acidithiobacillus. Then we measured its transfer frequency, its stability in Acidithiobacillus cells, and the level of resistance to streptomycin of the transconjugants and compared this with the IncQ plasmid pJRD215 control. Our results indicate that pBBR1MCS-2 provides a new and useful tool in the genetic manipulation of Acidithiobacillus strains. PMID:22705922

  7. Application of a Broad-Range Resequencing Array for Detection of Pathogens in Desert Dust Samples from Kuwait and Iraq ▿

    PubMed Central

    Leski, Tomasz A.; Malanoski, Anthony P.; Gregory, Michael J.; Lin, Baochuan; Stenger, David A.

    2011-01-01

    A significant percentage of the human population is exposed to high levels of naturally occurring airborne dusts. Although the link between airborne particulate inhalation and a variety of respiratory diseases has long been established, little is known about the pathogenic role of the microbial component of the dust. In this study, we applied highly multiplexed PCR and a high-density resequencing microarray (RPM-TEI version 1.0) to screen samples of fine topsoil particles and airborne dust collected in 19 locations in Iraq and Kuwait for the presence of a broad range of human pathogens. The results indicated the presence of potential human pathogens, including Mycobacterium, Brucella, Coxiella burnetii, Clostridium perfringens, and Bacillus. The presence of Coxiella burnetii, a highly infectious potential biowarfare agent, was confirmed and detected in additional samples by use of a more sensitive technique (real-time PCR), indicating a high prevalence of this organism in the analyzed samples. The detection of potentially viable pathogens in breathable dusts from arid regions of Iraq and Kuwait underscores the importance of further study of these environments. PMID:21571877

  8. Radiometric calibration of optical microscopy and microspectroscopy apparata over a broad spectral range using a special thin-film luminescence standard

    SciTech Connect

    Valenta, J. Greben, M.

    2015-04-15

    Application capabilities of optical microscopes and microspectroscopes can be considerably enhanced by a proper calibration of their spectral sensitivity. We propose and demonstrate a method of relative and absolute calibration of a microspectroscope over an extraordinary broad spectral range covered by two (parallel) detection branches in visible and near-infrared spectral regions. The key point of the absolute calibration of a relative spectral sensitivity is application of the standard sample formed by a thin layer of Si nanocrystals with stable and efficient photoluminescence. The spectral PL quantum yield and the PL spatial distribution of the standard sample must be characterized by separate experiments. The absolutely calibrated microspectroscope enables to characterize spectral photon emittance of a studied object or even its luminescence quantum yield (QY) if additional knowledge about spatial distribution of emission and about excitance is available. Capabilities of the calibrated microspectroscope are demonstrated by measuring external QY of electroluminescence from a standard poly-Si solar-cell and of photoluminescence of Er-doped Si nanocrystals.

  9. The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone.

    PubMed

    Konieczny, I; Helinski, D R

    1997-12-23

    Initiation and control of replication of the broad-host-range plasmid RK2 requires two plasmid-encoded elements, the replication origin (oriV) and the initiation protein TrfA. Purified TrfA is largely in the form of a dimer; however, only the monomeric form of the protein can bind specifically to the direct repeats (iterons) at the RK2 origin. The largely dimeric form of wild-type TrfA is inactive in the initiation of replication of RK2 in an in vitro replication system reconstituted from purified components. However, preincubation of the TrfA protein with the ClpX molecular chaperone isolated from Escherichia coli activates the initiator protein for replication in the purified system. We further observed that ClpX, in an ATP-dependent reaction, greatly increases the proportion of TrfA monomers and, therefore, the ability of this protein to bind to iterons localized within RK2 origin. Finally, a copy-up mutant of the TrfA protein which is largely in the monomer form is active in the reconstituted in vitro replication system, and its activity is not affected by ClpX. PMID:9405620

  10. Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors.

    PubMed Central

    Simon, R; Hötte, B; Klauke, B; Kosier, B

    1991-01-01

    On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different. Images PMID:1847366

  11. Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastoris▿

    PubMed Central

    Viader-Salvadó, José M.; Gallegos-López, Juan A.; Carreón-Treviño, J. Gerardo; Castillo-Galván, Miguel; Rojo-Domínguez, Arturo; Guerrero-Olazarán, Martha

    2010-01-01

    Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets. PMID:20693453

  12. Efficient Detection of Pathogenic Leptospires Using 16S Ribosomal RNA

    PubMed Central

    Lindow, Janet; Wunder, Elsio A.; Reis, Mitermayer G.; Usmani-Brown, Sahar; Ledizet, Michel; Ko, Albert; Pal, Utpal

    2015-01-01

    Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted. PMID:26091292

  13. Macroscopic force experienced by extended objects in granular flows over a very broad Froude-number range : Macroscopic granular force on extended object.

    PubMed

    Faug, Thierry

    2015-05-01

    This paper revisits a great number of data from previous studies about the macroscopic force experienced by either objects moving at constant speed and depth inside static granular materials or motionless objects subject to steady granular flows. It focuses on extended objects whose immersed height is equal or close to the thickness of the surrounding granular medium. A simple scaling argument allows demarcating quasi-static from speed-squared force contributions for all the data from different geometries over a very broad range of Froude number. However, a wide scatter of the data is observed in the quasi-static regime. In the first step, a mean-field model is proposed to describe the average force. Mass and momentum balances are applied to a control volume, namely the expected volume of grains disturbed by the object, which is assumed to extend across the whole width and the entire height of the granular system. This allows defining an equivalent length scale which is computed by fitting the force predicted by the model to the available force data. In the second step, a circular shape is assumed for the effective mobilized domain and the associated diameter can be directly extracted from the computed equivalent length scale. This effective diameter is found to vary linearly with both the object width and the thickness of the granular layer moving around the extended object or the immersed depth of the object. The scaling highlights the key role played by the geometry which may enhance the force in the quasi-static regime. PMID:25957179

  14. Modulation of Enzymatic Activity and Biological Function of Listeria monocytogenes Broad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with the Bacillus cereus Ortholog

    PubMed Central

    Zückert, Wolfram R.; Marquis, Hélène; Goldfine, Howard

    1998-01-01

    The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens α-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells. PMID:9746585

  15. The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

    PubMed Central

    Li, Xiaobin; Top, Eva M.; Wang, Yafei; Brown, Celeste J.; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

    2015-01-01

    A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent “essential” plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world. PMID:25628616

  16. The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC.

    PubMed Central

    Kornacki, J A; Burlage, R S; Figurski, D H

    1990-01-01

    Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC. Images PMID:2160936

  17. Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

    PubMed Central

    Verstrepen, Kevin J.; Van Laere, Stijn D. M.; Vanderhaegen, Bart M. P.; Derdelinckx, Guy; Dufour, Jean-Pierre; Pretorius, Isak S.; Winderickx, Joris; Thevelein, Johan M.; Delvaux, Freddy R.

    2003-01-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Δ atf2Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes. PMID:12957907

  18. Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales.

    PubMed

    Merriman, Dawn K; Xue, Yi; Yang, Shan; Kimsey, Isaac J; Shakya, Anisha; Clay, Mary; Al-Hashimi, Hashim M

    2016-08-16

    Helix-junction-helix (HJH) motifs are flexible building blocks of RNA architecture that help define the orientation and dynamics of helical domains. They are also frequently involved in adaptive recognition of proteins and small molecules and in the formation of tertiary contacts. Here, we use a battery of nuclear magnetic resonance techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) trinucleotide bulge (U23-C24-U25) affects dynamics over a broad range of time scales. Shortening the bulge has an effect on picosecond-to-nanosecond interhelical and local bulge dynamics similar to that casued by increasing the Mg(2+) and Na(+) concentration, whereby a preexisting two-state equilibrium in TAR is shifted away from a bent flexible conformation toward a coaxial conformation, in which all three bulge residues are flipped out and flexible. Surprisingly, the point deletion minimally affects microsecond-to-millisecond conformational exchange directed toward two low-populated and short-lived excited conformational states that form through reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond time scale, in which U23 is intrahelical, mimicking the expected conformation of residue C24 in the excited conformational state of wild-type TAR. Thus, minor changes in HJH topology preserve motional modes in RNA occurring over the picosecond-to-millisecond time scales but alter the relative populations of the sampled states or cause subtle changes in their conformational features. PMID:27232530

  19. Spectral Modulation Effect in Teleseismic P-waves from North Korean Nuclear Tests Recorded in Broad Azimuthal Range and Possible Source Depth Estimation

    NASA Astrophysics Data System (ADS)

    Gitterman, Y.; Kim, S. G.; Hofstetter, R.

    2016-04-01

    Three underground nuclear explosions, conducted by North Korea in 2006, 2009 and 2013, are analyzed. The last two tests were recorded by the Israel Seismic Network. Pronounced coherent minima (spectral nulls) at 1.2-1.3 Hz were revealed in the spectra of teleseismic P -waves. For a ground-truth explosion with a shallow source depth, this phenomenon can be interpreted in terms of the interference between the down-going P-wave and the pP phase reflected from the Earth's surface. This effect was also observed at ISN stations for a Pakistan nuclear explosion at a different frequency 1.7 Hz and the PNE Rubin-2 in West Siberia at 1 Hz, indicating a source-effect and not a site-effect. Similar spectral minima having essentially the same frequency, as at ISN, were observed in teleseismic P-waves for all the three North Korean explosions recorded at networks and arrays in Kazakhstan (KURK), Norway (NNSN), Australia (ASAR, WRA) and Canada (YKA), covering a broad azimuthal range. Data of 2009 and 2013 tests at WRA and KURK arrays showed harmonic spectral modulation with three multiple minima frequencies, evidencing the clear interference effect. These observations support the above-mentioned interpretation. Based on the null frequency dependency on the near-surface acoustic velocity and the source depth, the depth of the North Korean tests was estimated about 2.0-2.1 km. It was shown that the observed null frequencies and the obtained source depth estimates correspond to P- pP interference phenomena in both cases of a vertical shaft or a horizontal drift in a mountain. This unusual depth estimation needs additional validation based on more stations and verification by other methods.

  20. Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA.

    PubMed

    Duineveld, B M; Kowalchuk, G A; Keijzer, A; van Elsas, J D; van Veen, J A

    2001-01-01

    The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of

  1. Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

    PubMed

    Small, J; Call, D R; Brockman, F J; Straub, T M; Chandler, D P

    2001-10-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  2. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides.

    PubMed

    Doi, Yohei; Arakawa, Yoshichika

    2007-07-01

    Methylation of 16S ribosomal RNA (rRNA) has recently emerged as a new mechanism of resistance against aminoglycosides among gram-negative pathogens belonging to the family Enterobacteriaceae and glucose-nonfermentative microbes, including Pseudomonas aeruginosa and Acinetobacter species. This event is mediated by a newly recognized group of 16S rRNA methylases, which share modest similarity to those produced by aminoglycoside-producing actinomycetes. Their presence confers a high level of resistance to all parenterally administered aminoglycosides that are currently in clinical use. The responsible genes are mostly located on transposons within transferable plasmids, which provides them with the potential to spread horizontally and may in part explain the already worldwide distribution of this novel resistance mechanism. Some of these organisms have been found to coproduce extended-spectrum beta-lactamases or metallo-beta-lactamases, contributing to their multidrug-resistant phenotypes. A 2-tiered approach, consisting of disk diffusion tests followed by confirmation with polymerase chain reaction, is recommended for detection of 16S rRNA methylase-mediated resistance. PMID:17554708

  3. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  4. Detection of a broad range of class I and II Newcastle disease viruses using a multiplex real time RT-PCR assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prompt detection of virulent strains of Newcastle disease virus (vNDV) using real time RT-PCR (rRT-PCR) is challenging due to the broad genetic variability across two clades comprising 18 recognized genotypes. A large proportion of class I low virulence Newcastle disease viruses (loNDV) recently id...

  5. Enhanced radiative Auger emission from lithiumlike 16S13+

    NASA Astrophysics Data System (ADS)

    Bernstein, E. M.; Clark, M. W.; Oglesby, C. S.; Tanis, J. A.; Graham, W. G.; McFarland, R. H.; Morgan, T. J.; Johnson, B. M.; Jones, K. W.

    1990-03-01

    The radiative Auger emission (RAE) from 0.94-6.25-MeV/u 16S13+ (lithiumlike) projectiles excited in collisions with He target atoms has been measured. For these highly stripped ions the intensity of RAE photons relative to Kα x-ray emission is enhanced by about a factor of five compared with theoretical calculations and an earlier experimental measurement for S ions with few electron vacancies. The enhancement of RAE for S13+ is qualitatively similar to results reported previously for lithiumlike 23V20+; however, some differences between S and V are evident.

  6. Broad-Range Direct Detection and Identification of Fungi by Use of the PLEX-ID PCR-Electrospray Ionization Mass Spectrometry (ESI-MS) System

    PubMed Central

    Simner, Patricia J.; Uhl, James R.; Hall, Leslie; Weber, Michelle M.; Walchak, Robert C.; Buckwalter, Seanne

    2013-01-01

    The PLEX-ID system is a novel technology that couples PCR amplification and electrospray ionization-mass spectrometry to identify pathogens directly in clinical specimens. The analytical performance of the PLEX-ID Broad Fungal assay was compared with that of traditional culture identification by using 91 characterized fungal culture isolates (64 manufacturer-claimed and 27 nonclaimed organisms) and directly by using 395 respiratory specimens. Discordant results were resolved by D2 large-subunit ribosomal DNA fungal sequencing. Environmental studies were performed to monitor for potential contamination. The PLEX-ID Broad Fungal assay correctly identified 95.6% (87/91) and 81.3% (74/91) of the culture isolates to the genus and species levels, respectively. Of the manufacturer-claimed organisms, 100% (64/64) and 92.2% (59/64) were correctly identified to the genus and species levels, respectively. Direct analysis of respiratory specimens resulted in 67.6% (267/395) and 66.6% (263/395) agreement with culture results to the genus and species levels, respectively, with 16.2% (64/395) of the results discordant with culture and 16.2% (64/395) not detected by the system. The majority (>95%) of the isolates not detected directly by the PLEX-ID system ultimately grew in low quantities in culture (≤20 colonies). In 20.3% (35/172) of the respiratory specimens where no growth was observed in culture, the PLEX-ID system identified a fungus, suggesting a potential increase in sensitivity over culture in some instances. The PLEX-ID system provides a rapid method for the detection of a broad array of fungi directly in respiratory specimens and has the potential of impacting turnaround times and patient care by reducing the need to wait for the growth of an organism in culture. PMID:23515540

  7. Universal bacterial identification by mass spectrometry of 16S ribosomal RNA cleavage products

    NASA Astrophysics Data System (ADS)

    Jackson, George W.; McNichols, Roger J.; Fox, George E.; Willson, Richard C.

    2007-03-01

    The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using nucleic acid hybridization and other molecular approaches. Species-specific PCR, microarrays, and in situ hybridization are based on the presence of unique subsequences in the target sequence and therefore require prior knowledge of what organisms are likely to be present in a sample. Mass spectrometry is not limited by a pre-synthesized inventory of probe/primer sequences. It has already been demonstrated that organism identification can be recovered from mass spectra using various methods including base-specific cleavage of nucleic acids. The feasibility of broad bacterial identification by comparing such mass spectral patterns to predictive databases derived from virtually all previously sequenced strains has yet to be demonstrated, however. Herein, we present universal bacterial identification by base-specific cleavage, mass spectrometry, and an efficient coincidence function for rapid spectral scoring against a large database of predicted "mass catalogs". Using this approach in conjunction with universal PCR of the 16S rDNA gene, four bacterial isolates and an uncultured clone were successfully identified against a database of predicted cleavage products derived 6rom over 47,000 16S rRNA sequences representing all major bacterial taxaE At present, the conventional DNA isolation and PCR steps require approximately 2 h, while subsequent transcription, enzymatic cleavage, mass spectrometric analysis, and database comparison require less than 45 min. All steps are amenable to high-throughput implementation.

  8. Using an intervening sequence of Faecalibacterium 16S rDNA to identify poultry feces.

    PubMed

    Shen, Zhenyu; Duan, Chuanren; Zhang, Chao; Carson, Andrew; Xu, Dong; Zheng, Guolu

    2013-10-15

    This study was designed to identify poultry feces-specific marker(s) within sequences of Faecalibacterium 16S rDNA for detecting poultry fecal pollution in water. Bioinformatics tools were used in the comparative analysis of 7,458 sequences of Faecalibacterium 16S rDNA, reportedly associated with various poultry (chicken and turkey) and animal species. One intervening sequence (IVS) within between the hypervariable region 1 and the conserved region 2, designated as IVS-p, was found to be unique to poultry feces. Based on this sequence, a PCR assay (PCR-p) was developed. The PCR-p produced an amplicon of 132 bp only in the test when fecal or wastewater samples from poultry were used, but not when using fecal or wastewater samples from other sources. The non-poultry sources included feces of beef or dairy cattle, dog, horse, human, domestic or wild geese, seagull, sheep, swine, and wild turkey. These data indicate that IVS-p may prove to be a useful genetic marker for the specific identification of poultry fecal pollution in environmental waterways. Furthermore, results of data mining and PCR assay indicate that the IVS-p may have a broad geographic distribution. This report represents initial evidence of the potential utility of ribosomal intervening sequences as genetic markers for tracking host sources of fecal pollution in waterways. PMID:24011842

  9. Broad-Scale Latitudinal Variation in Female Reproductive Success Contributes to the Maintenance of a Geographic Range Boundary in Bagworms (Lepidoptera: Psychidae)

    PubMed Central

    Rhainds, Marc; Fagan, William F.

    2010-01-01

    Background Geographic range limits and the factors structuring them are of great interest to biologists, in part because of concerns about how global change may shift range boundaries. However, scientists lack strong mechanistic understanding of the factors that set geographic range limits in empirical systems, especially in animals. Methodology/Principal Findings Across dozens of populations spread over six degrees of latitude in the American Midwest, female mating success of the evergreen bagworm Thyridopteryx ephemeraeformis (Lepidoptera: Psychidae) declines from ∼100% to ∼0% near the edge of the species range. When coupled with additional latitudinal declines in fecundity and in egg and pupal survivorship, a spatial gradient of bagworm reproductive success emerges. This gradient is associated with a progressive decline in local abundance and an increased risk of local population extinction, up to a latitudinal threshold where extremely low female fitness meshes spatially with the species' geographic range boundary. Conclusions/Significance The reduction in fitness of female bagworms near the geographic range limit, which concords with the abundant centre hypothesis from biogeography, provides a concrete, empirical example of how an Allee effect (increased pre-reproductive mortality of females in sparsely populated areas) may interact with other demographic factors to induce a geographic range limit. PMID:21152445

  10. Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics

    PubMed Central

    Poretsky, Rachel; Rodriguez-R, Luis M.; Luo, Chengwei; Tsementzi, Despina; Konstantinidis, Konstantinos T.

    2014-01-01

    This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ. PMID:24714158

  11. Technologically important extremophile 16S rRNA sequence Shannon entropy and fractal property comparison with long term dormant microbes

    NASA Astrophysics Data System (ADS)

    Holden, Todd; Gadura, N.; Dehipawala, S.; Cheung, E.; Tuffour, M.; Schneider, P.; Tremberger, G., Jr.; Lieberman, D.; Cheung, T.

    2011-10-01

    Technologically important extremophiles including oil eating microbes, uranium and rocket fuel perchlorate reduction microbes, electron producing microbes and electrode electrons feeding microbes were compared in terms of their 16S rRNA sequences, a standard targeted sequence in comparative phylogeny studies. Microbes that were reported to have survived a prolonged dormant duration were also studied. Examples included the recently discovered microbe that survives after 34,000 years in a salty environment while feeding off organic compounds from other trapped dead microbes. Shannon entropy of the 16S rRNA nucleotide composition and fractal dimension of the nucleotide sequence in terms of its atomic number fluctuation analyses suggest a selected range for these extremophiles as compared to other microbes; consistent with the experience of relatively mild evolutionary pressure. However, most of the microbes that have been reported to survive in prolonged dormant duration carry sequences with fractal dimension between 1.995 and 2.005 (N = 10 out of 13). Similar results are observed for halophiles, red-shifted chlorophyll and radiation resistant microbes. The results suggest that prolonged dormant duration, in analogous to high salty or radiation environment, would select high fractal 16S rRNA sequences. Path analysis in structural equation modeling supports a causal relation between entropy and fractal dimension for the studied 16S rRNA sequences (N = 7). Candidate choices for high fractal 16S rRNA microbes could offer protection for prolonged spaceflights. BioBrick gene network manipulation could include extremophile 16S rRNA sequences in synthetic biology and shed more light on exobiology and future colonization in shielded spaceflights. Whether the high fractal 16S rRNA sequences contain an asteroidlike extra-terrestrial source could be speculative but interesting.

  12. White LEDs as broad spectrum light sources for spectrophotometry: demonstration in the visible spectrum range in a diode-array spectrophotometric detector.

    PubMed

    Piasecki, Tomasz; Breadmore, Michael C; Macka, Mirek

    2010-11-01

    Although traditional lamps, such as deuterium lamps, are suitable for bench-top instrumentation, their compatibility with the requirements of modern miniaturized instrumentation is limited. This study investigates the option of utilizing solid-state light source technology, namely white LEDs, as a broad band spectrum source for spectrophotometry. Several white light LEDs of both RGB and white phosphorus have been characterized in terms of their emission spectra and energy output and a white phosphorus Luxeon LED was then chosen for demonstration as a light source for visible-spectrum spectrophotometry conducted in CE. The Luxeon LED was fixed onto the base of a dismounted deuterium (D(2) ) lamp so that the light-emitting spot was geometrically positioned exactly where the light-emitting spot of the original D(2) lamp is placed. In this manner, the detector of a commercial CE instrument equipped with a DAD was not modified in any way. As the detector hardware and electronics remained the same, the change of the deuterium lamp for the Luxeon white LED allowed a direct comparison of their performances. Several anionic dyes as model analytes with absorption maxima between 450 and 600 nm were separated by CE in an electrolyte of 0.01 mol/L sodium tetraborate. The absorbance baseline noise as the key parameter was 5 × lower for the white LED lamp, showing clearly superior performance to the deuterium lamp in the available, i.e. visible part of the spectrum. PMID:21077241

  13. Influenza B-cells Protective Epitope Characterization: A Passkey for the Rational Design of New Broad-Range Anti-Influenza Vaccines

    PubMed Central

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    The emergence of new influenza strains causing pandemics represents a serious threat to human health. From 1918, four influenza pandemics occurred, caused by H1N1, H2N2 and H3N2 subtypes. Moreover, in 1997 a novel influenza avian strain belonging to the H5N1 subtype infected humans. Nowadays, even if its transmission is still circumscribed to avian species, the capability of the virus to infect humans directly from avian reservoirs can result in fatalities. Moreover, the risk that this or novel avian strains could adapt to inter-human transmission, the development of resistance to anti-viral drugs and the lack of an effective prevention are all incumbent problems for the world population. In this scenario, the identification of broadly neutralizing monoclonal antibodies (mAbs) directed against conserved regions shared among influenza isolates has raised hopes for the development of monoclonal antibody-based immunotherapy and “universal” anti-influenza vaccines. PMID:23202517

  14. Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

    PubMed Central

    Aasa-Chapman, Marlèn; Gorlani, Andrea; Forsman Quigley, Anna; Hulsik, David Lutje; Chen, Lei; Weiss, Robin; de Haard, Hans; Verrips, Theo

    2012-01-01

    Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides. PMID:22438910

  15. Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.

    PubMed

    Singer, John T; Phennicie, Ryan T; Sullivan, Matthew J; Porter, Laura A; Shaffer, Valerie J; Kim, Carol H

    2010-06-01

    To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen. PMID:20363780

  16. Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System▿ †

    PubMed Central

    Singer, John T.; Phennicie, Ryan T.; Sullivan, Matthew J.; Porter, Laura A.; Shaffer, Valerie J.; Kim, Carol H.

    2010-01-01

    To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacIq carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen. PMID:20363780

  17. Genetic mapping of the Batten disease locus (CLN3) to the interval D16S288-D16S383 by analysis of haplotypes and allelic association

    SciTech Connect

    Mitchison, H.M.; O`Rawe, A.M.; Gardiner, R.M.

    1994-07-15

    CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten disease, has been localized by genetic linkage analysis to chromosome 16p between loci D16S297 and D16S57. The authors have now further refined the localization of CLN3 by haplotype analysis using two new microsatellite markers from loci D16S383 and SPN in the D16S297-D16S57 interval on a larger collaborative family resource consisting of 142 JNCL pedigrees. Crossover events in 3 maternal meioses define new flanking markers for CLN3 and localize the gene to the interval at 16p12.1-11.2 between D16S288 and D16S383, which corresponds to a genetic distance of 2.1 cM. Within this interval 4 microsatellite loci are in strong linkage disequilibrium with CLN3, and extended haplotype analysis of the associated alleles indicates that CLN3 is in closest proximity to loci D16S299 and D16S298. 6 refs., 1 fig., 2 tabs.

  18. Electronic band structure of highly mismatched GaN{sub 1−x}Sb{sub x} alloys in a broad composition range

    SciTech Connect

    Segercrantz, N.; Yu, K. M.; Ting, M.; Sarney, W. L.; Svensson, S. P.; Novikov, S. V.; Foxon, C. T.; Walukiewicz, W.

    2015-10-05

    In this letter, we study the optical properties of GaN{sub 1−x}Sb{sub x} thin films. Films with an Sb fraction up to 42% were synthesized by alternating GaN-GaSb layers at a constant temperature of 325 °C. The measured optical absorption data of the films are interpreted using a modified band anticrossing model that is applicable to highly mismatched alloys such as GaN{sub 1−x}Sb{sub x} in the entire composition range. The presented model allows us to more accurately determine the band gap as well as the band edges over the entire composition range thereby providing means for determining the composition for, e.g., efficient spontaneous photoelectrochemical cell applications.

  19. Evaluation of the Broad-Range PCR/ESI-MS Technology in Blood Specimens for the Molecular Diagnosis of Bloodstream Infections

    PubMed Central

    Jordana-Lluch, Elena; Giménez, Montserrat; Quesada, Mª Dolores; Rivaya, Belén; Marcó, Clara; Domínguez, Mª Jesús; Arméstar, Fernando; Martró, Elisa; Ausina, Vicente

    2015-01-01

    Background Rapid identification of the etiological agent in bloodstream infections is of vital importance for the early administration of the most appropriate antibiotic therapy. Molecular methods may offer an advantage to current culture-based microbiological diagnosis. The goal of this study was to evaluate the performance of IRIDICA, a platform based on universal genetic amplification followed by mass spectrometry (PCR/ESI-MS) for the molecular diagnosis of sepsis-related pathogens directly from the patient’s blood. Methods A total of 410 whole blood specimens from patients admitted to Emergency Room (ER) and Intensive Care Unit (ICU) with clinical suspicion of sepsis were tested with the IRIDICA BAC BSI Assay (broad identification of bacteria and Candida spp.). Microorganisms grown in culture and detected by IRIDICA were compared considering blood culture as gold standard. When discrepancies were found, clinical records and results from other cultures were taken into consideration (clinical infection criterion). Results The overall positive and negative agreement of IRIDICA with blood culture in the analysis by specimen was 74.8% and 78.6%, respectively, rising to 76.9% and 87.2% respectively, when compared with the clinical infection criterion. Interestingly, IRIDICA detected 41 clinically significant microorganisms missed by culture, most of them from patients under antimicrobial treatment. Of special interest were the detections of one Mycoplasma hominis and two Mycobacterium simiae in immunocompromised patients. When ICU patients were analyzed separately, sensitivity, specificity, positive and negative predictive values compared with blood culture were 83.3%, 78.6%, 33.9% and 97.3% respectively, and 90.5%, 87.2%, 64.4% and 97.3% respectively, in comparison with the clinical infection criterion. Conclusions IRIDICA is a promising technology that offers an early and reliable identification of a wide variety of pathogens directly from the patient’s blood

  20. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  1. Incorporating graphene oxide and gold nanoclusters: a synergistic catalyst with surprisingly high peroxidase-like activity over a broad pH range and its application for cancer cell detection.

    PubMed

    Tao, Yu; Lin, Youhui; Huang, Zhenzhen; Ren, Jinsong; Qu, Xiaogang

    2013-05-14

    A synergistic graphene oxide-gold nanocluster (GO-AuNC) hybrid has been constructed as an enzyme mimic that is able to show high catalytic activity over a broad pH range, especially at neutral pH. Importantly, the target-functionalized hybrid has been applied as a robust nanoprobe for selective, quantitative, and fast colorimetric detection of cancer cells. PMID:23418013

  2. Influence of CO molecular impurity on the structural and thermodynamic properties of fullerite C60, in a broad range of sorption temperatures

    NASA Astrophysics Data System (ADS)

    Meleshko, V. V.; Legchenkova, I. V.; Stetsenko, Y. E.; Prokhvatilov, A. I.

    2016-02-01

    An x-ray diffraction study of how sorption of CO gas at a pressure of 30 atm in the temperature range of 150-600 °C influences the structural characteristics of polycrystalline and single crystal fullerite C60. The sorption kinetics are studied by constructing a dependence of the lattice parameter on the time it takes for fullerite to be saturated by CO molecules. At temperatures Tsorb > 300 °C there is an observed dissociation of carbon monoxide, accompanied by the precipitation of carbon powder and the chemical interaction of atomic oxygen with C60 and CO molecules, and possibly with the carbon condensate. These processes have a strong influence on the structural characteristics of fullerite, thus creating, in part, a nonmonotonic dependence of the parameter and lattice matrix volume on the impurity saturation temperature. The concentrations of solid solutions C60(CO)x poly- and single crystal samples are determined in the physisorption range for two modes (150 and 250 °C). It is found that the CO impurity has a linear effect on the lattice parameter and the temperature of the orientational transition of fullerite C60.

  3. Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

    PubMed Central

    Theriot, Casey M.; Semcer, Rebecca L.; Shah, Saumil S.; Grunden, Amy M.

    2011-01-01

    Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases. PMID:22162664

  4. Improving the catalytic activity of hyperthermophilic Pyrococcus horikoshii prolidase for detoxification of organophosphorus nerve agents over a broad range of temperatures.

    PubMed

    Theriot, Casey M; Semcer, Rebecca L; Shah, Saumil S; Grunden, Amy M

    2011-01-01

    Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases. PMID:22162664

  5. Liquid-phase epitaxy grown PbSnTe distributed feedback laser diodes with broad continuous single-mode tuning range

    NASA Technical Reports Server (NTRS)

    Hsieh, H.-H.; Fonstad, C. G.

    1980-01-01

    Distributed feedback (DFB) pulsed laser operation has been demonstrated in stripe geometry Pb(1-x)Sn(x)Te double-heterostructures grown by liquid-phase epitaxy. The grating structure of 0.79 micron periodicity operates in first order near 12.8 microns and was fabricated prior to the liquid-phase epitaxial growth using holographic exposure techniques. These DFB lasers had moderate thresholds, 3.6 kA/sq cm, and the output power versus current curves exhibited a sharp turn-on free of kinks. Clean, single-mode emission spectra, continuously tunable over a range in excess of 20 per cm, centered about 780 per cm (12.8 microns), and at an average rate of 1.2 per cm-K from 9 to 26 K, were observed. While weaker modes could at times be seen in the spectrum, substantially single-mode operation was obtained over the entire operating range and to over 10 times threshold.

  6. Responses Of Alpine Vegetation To Global Warming: Insights From Comparing Alpine-Restricted And Broad-Ranging Herbs Along Snowmelt Gradients

    NASA Astrophysics Data System (ADS)

    Butz, R. J.; Reinhardt, K. S.; Germino, M. J.; Kueppers, L. M.

    2009-12-01

    Many alpine plant species face habitat fragmentation and loss, and even extinction because their narrow elevation, precipitation, and temperature tolerances limit their geographic distribution. In order to assess the impacts of climate change on sensitive native alpine communities we used a variety of methods to look at the seasonal timing of life stages (phenology) and the stress responses (physiology) of alpine species along a natural environmental gradient at Niwot Ridge in the Colorado Rocky Mountains to address the following question: Will alpine plants be impaired in their existing range as a result of climate change? We collected data on date of snowmelt and vegetative and flowering phenology of all alpine species present from snowmelt to senescence in 80 1m2 plots above treeline. In addition, we measured soil temperature and moisture, plant water potential and leaf-level gas exchange early, mid, and late-season on three alpine-restricted and three broader-ranging alpine species: Geum rossii, Artemisia scopulorum, Carex rupestris, Lewisia pygmaea, Tetraneuris grandiflora, and Sibbaldia procumbens. In 2009, the natural variation in snowmelt timing was 40 days (approximately 5.5 weeks) over the 80 plots. Our results suggest that with earlier snowmelt, the number of vascular species per plot increases. However, this increase is almost exclusively attributable to wider ranging species not restricted to the alpine. Plots with intermediate natural snowmelt dates had a higher diversity of alpine-restricted species, photosynthesis, and water-use efficiency, thereby potentially increasing long-term survival rates amongst alpine species. Water stress increased in all species as the season progressed, especially in plots where snow melted earliest. Photosynthetic productivity and diversity of alpine-restricted species was greatest in plots having intermediate melt dates. These findings suggest that shifts in snowmelt date under a warming climate will likely impact the

  7. A measurement of the energy spectra and relative abundance of the cosmic-ray H and He isotopes over a broad energy range

    NASA Technical Reports Server (NTRS)

    Webber, W. R.; Yushak, S. M.

    1983-01-01

    The measurements reported of these isotopes were made using two sets of detectors during the same minimum modulation period in 1977. One measurement was made with a balloon-borne telescope, the other with telescopes on the Voyager spacecraft. It is noted that together they provide the widest energy range yet available for studying these isotopes: 14-150 MeV per nucleon for H2 and 10-290 MeV per nucleon for He-3. The simultaneous helium isotope observations are used to give a mutually consistent picture of galactic propagation and solar modulation. The data define the form of the interstellar H-1 and He-4 spectra, an interstellar matter path length for both H-1 and He-4, and a total residual modulation for He-4. The H-2 observations suggest a picture that is very similar for the galactic propagation of H-1 and He-4.

  8. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

  9. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.

    PubMed Central

    Greisen, K; Loeffelholz, M; Purohit, A; Leong, D

    1994-01-01

    A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF. Images PMID:7512093

  10. The AusD Study: a population-based study of the determinants of serum 25-hydroxyvitamin D concentration across a broad latitude range.

    PubMed

    Brodie, A M; Lucas, R M; Harrison, S L; van der Mei, I A F; Armstrong, B; Kricker, A; Mason, R S; McMichael, A J; Nowak, M; Whiteman, D C; Kimlin, M G

    2013-05-01

    Observational studies suggest that people with a high serum 25-hydroxyvitamin D (25(OH)D) concentration may have reduced risk of chronic diseases such as osteoporosis, multiple sclerosis, type 1 diabetes, cardiovascular disease, and some cancers. The AusD Study (A Quantitative Assessment of Solar UV Exposure for Vitamin D Synthesis in Australian Adults) was conducted to clarify the relationships between ultraviolet (UV) radiation exposure, dietary intake of vitamin D, and serum 25(OH)D concentration among Australian adults residing in Townsville (19.3°S), Brisbane (27.5°S), Canberra (35.3°S), and Hobart (42.8°S). Participants aged 18-75 years were recruited from the Australian Electoral Roll between 2009 and 2010. Measurements were made of height, weight, waist:hip ratio, skin, hair, and eye color, blood pressure, and grip strength. Participants completed a questionnaire on sun exposure and vitamin D intake, together with 10 days of personal UV dosimetry and an associated sun-exposure and physical-activity diary that was temporally linked to a blood test for measurement of 25(OH)D concentration. Ambient solar UV radiation was also monitored at all study sites. We collected comprehensive, high-quality data from 1,002 participants (459 males, 543 females) assessed simultaneously across a range of latitudes and through all seasons. Here we describe the scientific and methodological issues considered in designing the AusD Study. PMID:23524036

  11. An rRT-PCR assay to detect the matrix gene of a broad range of avian paramyxovirus serotype-1 strains.

    PubMed

    Hines, Nichole L; Killian, Mary Lea; Pedersen, Janice C; Reising, Monica M; Mosos, Nestor A; Mathieu-Benson, Christian; Miller, Cathy L

    2012-06-01

    The current U.S. Department of Agriculture (USDA)-validated real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay designed to detect the matrix gene of avian paramyxovirus serotype-1 (APMV-1) is the primary screening assay used in the United States. It has previously been shown to be unable to consistently detect all members of class I APMV-1. Diagnostic testing relies on rRT-PCR to quickly detect APMV-1 in wild birds, backyard flocks, live bird markets, commercial poultry, and for export testing. Limitations of the current USDA assay have raised concerns about the potential for some strains of APMV-1 to remain undetected by the primary screening assay. Mismatches in the probe were shown to cause a loss in template binding efficiency, resulting in lack of detection by the assay. Here, we describe the development and analytical validation of a new rRT-PCR assay designed to target a highly conserved region of the matrix gene across a wide range of APMV-1 strains. Limit of detection testing revealed a 3 log10 decrease in sensitivity for one low-virulence strain when compared to the USDA validated assay. Conversely, the assay showed increased sensitivity for a class I isolate and two virulent strains of APMV-1 that were not detected by the USDA-validated assay. The new assay also demonstrated a high degree of specificity by the lack of detection of 43 non-APMV-1 viruses. PMID:22856199

  12. Using a Pareto-optimal solution set to characterize trade-offs between a broad range of values and preferences in climate risk management

    NASA Astrophysics Data System (ADS)

    Garner, Gregory; Reed, Patrick; Keller, Klaus

    2015-04-01

    Integrated assessment models (IAMs) are often used to inform the design of climate risk management strategies. Previous IAM studies have broken important new ground on analyzing the effects of parametric uncertainties, but they are often silent on the implications of uncertainties regarding the problem formulation. Here we use the Dynamic Integrated model of Climate and the Economy (DICE) to analyze the effects of uncertainty surrounding the definition of the objective(s). The standard DICE model adopts a single objective to maximize a weighted sum of utilities of per-capita consumption. Decision makers, however, are often concerned with a broader range of values and preferences that may be poorly captured by this a priori definition of utility. We reformulate the problem by introducing three additional objectives that represent values such as (i) reliably limiting global average warming to two degrees Celsius and minimizing (ii) the costs of abatement and (iii) the climate change damages. We use advanced multi-objective optimization methods to derive a set of Pareto-optimal solutions over which decision makers can trade-off and assess performance criteria a posteriori. We illustrate the potential for myopia in the traditional problem formulation and discuss the capability of this multiobjective formulation to provide decision support.

  13. 16S classifier: a tool for fast and accurate taxonomic classification of 16S rRNA hypervariable regions in metagenomic datasets.

    PubMed

    Chaudhary, Nikhil; Sharma, Ashok K; Agarwal, Piyush; Gupta, Ankit; Sharma, Vineet K

    2015-01-01

    The diversity of microbial species in a metagenomic study is commonly assessed using 16S rRNA gene sequencing. With the rapid developments in genome sequencing technologies, the focus has shifted towards the sequencing of hypervariable regions of 16S rRNA gene instead of full length gene sequencing. Therefore, 16S Classifier is developed using a machine learning method, Random Forest, for faster and accurate taxonomic classification of short hypervariable regions of 16S rRNA sequence. It displayed precision values of up to 0.91 on training datasets and the precision values of up to 0.98 on the test dataset. On real metagenomic datasets, it showed up to 99.7% accuracy at the phylum level and up to 99.0% accuracy at the genus level. 16S Classifier is available freely at http://metagenomics.iiserb.ac.in/16Sclassifier and http://metabiosys.iiserb.ac.in/16Sclassifier. PMID:25646627

  14. Segmentation of brain magnetic resonance images based on multi-atlas likelihood fusion: testing using data with a broad range of anatomical and photometric profiles

    PubMed Central

    Tang, Xiaoying; Crocetti, Deana; Kutten, Kwame; Ceritoglu, Can; Albert, Marilyn S.; Mori, Susumu; Mostofsky, Stewart H.; Miller, Michael I.

    2015-01-01

    We propose a hierarchical pipeline for skull-stripping and segmentation of anatomical structures of interest from T1-weighted images of the human brain. The pipeline is constructed based on a two-level Bayesian parameter estimation algorithm called multi-atlas likelihood fusion (MALF). In MALF, estimation of the parameter of interest is performed via maximum a posteriori estimation using the expectation-maximization (EM) algorithm. The likelihoods of multiple atlases are fused in the E-step while the optimal estimator, a single maximizer of the fused likelihoods, is then obtained in the M-step. There are two stages in the proposed pipeline; first the input T1-weighted image is automatically skull-stripped via a fast MALF, then internal brain structures of interest are automatically extracted using a regular MALF. We assess the performance of each of the two modules in the pipeline based on two sets of images with markedly different anatomical and photometric contrasts; 3T MPRAGE scans of pediatric subjects with developmental disorders vs. 1.5T SPGR scans of elderly subjects with dementia. Evaluation is performed quantitatively using the Dice overlap as well as qualitatively via visual inspections. As a result, we demonstrate subject-level differences in the performance of the proposed pipeline, which may be accounted for by age, diagnosis, or the imaging parameters (particularly the field strength). For the subcortical and ventricular structures of the two datasets, the hierarchical pipeline is capable of producing automated segmentations with Dice overlaps ranging from 0.8 to 0.964 when compared with the gold standard. Comparisons with other representative segmentation algorithms are presented, relative to which the proposed hierarchical pipeline demonstrates comparative or superior accuracy. PMID:25784852

  15. Prevalent presence of periodic actin–spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species

    PubMed Central

    He, Jiang; Zhou, Ruobo; Wu, Zhuhao; Carrasco, Monica A.; Kurshan, Peri T.; Farley, Jonathan E.; Simon, David J.; Wang, Guiping; Han, Boran; Hao, Junjie; Heller, Evan; Freeman, Marc R.; Shen, Kang; Maniatis, Tom; Tessier-Lavigne, Marc

    2016-01-01

    Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens. PMID:27162329

  16. Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures

    PubMed Central

    Vakulskas, Christopher A.; Pannuri, Archana; Cortés-Selva, Diana; Zere, Tesfalem R.; Ahmer, Brian M.; Babitzke, Paul; Romeo, Tony

    2014-01-01

    Summary In Escherichia coli, activity of the global regulatory RNA binding protein CsrA is antagonized by two noncoding sRNAs, CsrB and CsrC, which sequester it away from its lower affinity mRNA targets. Transcription of csrB/C requires the BarA-UvrY two component signal transduction system, which responds to short chain carboxylates. We show that two DEAD-box RNA helicases, DeaD and SrmB, activate csrB/C expression by different pathways. DeaD facilitates uvrY translation by counteracting the inhibitory effect of long distance basepairing between the uvrY mRNA leader and coding region, while SrmB does not affect UvrY or UvrY-phosphate levels. Contrary to the prevailing notion that these helicases act primarily at low temperatures, DeaD and SrmB activated csrB expression over a wide temperature range. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) revealed in vivo interactions of DeaD with 39 mRNAs, including those of uvrY and 9 other regulatory genes. Studies on the expression of several of the identified genes revealed regulatory effects of DeaD in all cases and diverse temperature response patterns. Our findings uncover an expanded regulatory role for DeaD, which is mediated through novel mRNA targets, important global regulators and under physiological conditions that were considered to be incompatible with its function. PMID:24708042

  17. Purification, biochemical characterization and antifungal activity of a novel Aspergillus tubingensis glucose oxidase steady on broad range of pH and temperatures.

    PubMed

    Kriaa, Mouna; Hammami, Inès; Sahnoun, Mouna; Azebou, Manel Cheffi; Triki, Mohamed Ali; Kammoun, Radhouane

    2015-11-01

    This study was carried out to evaluate the in vitro and in vivo antifungal efficiency of Aspergillus tubingensis CTM 507 glucose oxidase (GOD) against plant pathogenic fungi. GOD displayed a wide inhibitory spectrum toward different fungi at a concentration of 20 AU. The GOD had a strong inhibitor effect on mycelia growth and spore germination of Pythium ultimum. Interestingly, the GOD exhibited a potent in vivo antifungal effect against P. ultimum responsible for potato plants disease. The antifungal GOD was purified 13-fold with 27 % yield and a specific activity of 3435 U/mg. The relative molecular mass of the GOD was 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GOD activity was optimum at pH 4.5 and 60 °C. It was found to be stable over a large pH range (3-9). It also displayed a marked thermostability with a 50-min half-life at 65 °C. The 10 residues of the N-terminal sequence of the purified GOD (S-K-G-S-A-V-T-T-P-D) showed no homology to the other reported GOD, identifying a novel GOD. FTIR spectroscopic analysis revealed the presence of C-O and C=O groups corresponding to a D-glucono-lactone. The findings indicated that GOD is the first A. tubingensis-produced fungicide ever reported to exhibit such promising biological properties. It could become a natural alternative to synthetic fungicides to control certain important plant microbial diseases. PMID:26280215

  18. Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice

    PubMed Central

    Fouts, Derrick E.; Tyler, Heather L.; DeBoy, Robert T.; Daugherty, Sean; Ren, Qinghu; Badger, Jonathan H.; Durkin, Anthony S.; Huot, Heather; Shrivastava, Susmita; Kothari, Sagar; Dodson, Robert J.; Mohamoud, Yasmin; Khouri, Hoda; Roesch, Luiz F. W.; Krogfelt, Karen A.; Struve, Carsten; Triplett, Eric W.; Methé, Barbara A.

    2008-01-01

    We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category

  19. Prevalent presence of periodic actin-spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species.

    PubMed

    He, Jiang; Zhou, Ruobo; Wu, Zhuhao; Carrasco, Monica A; Kurshan, Peri T; Farley, Jonathan E; Simon, David J; Wang, Guiping; Han, Boran; Hao, Junjie; Heller, Evan; Freeman, Marc R; Shen, Kang; Maniatis, Tom; Tessier-Lavigne, Marc; Zhuang, Xiaowei

    2016-05-24

    Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens. PMID:27162329

  20. Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice.

    PubMed

    Fouts, Derrick E; Tyler, Heather L; DeBoy, Robert T; Daugherty, Sean; Ren, Qinghu; Badger, Jonathan H; Durkin, Anthony S; Huot, Heather; Shrivastava, Susmita; Kothari, Sagar; Dodson, Robert J; Mohamoud, Yasmin; Khouri, Hoda; Roesch, Luiz F W; Krogfelt, Karen A; Struve, Carsten; Triplett, Eric W; Methé, Barbara A

    2008-01-01

    We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category

  1. Broad range of inhibiting action of novel camphor-based compound with anti-hemagglutinin activity against influenza viruses in vitro and in vivo.

    PubMed

    Zarubaev, V V; Garshinina, A V; Tretiak, T S; Fedorova, V A; Shtro, A A; Sokolova, A S; Yarovaya, O I; Salakhutdinov, N F

    2015-08-01

    Influenza virus continues to remain one of the leading human respiratory pathogens causing significant morbidity and mortality around the globe. Due to short-term life cycle and high rate of mutations influenza virus is able to rapidly develop resistance to clinically available antivirals. This makes necessary the search and development of new drugs with different targets and mechanisms of activity. Here we report anti-influenza activity of camphor derivative 1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene-aminoethanol (camphecene). In in vitro experiments it inhibited influenza viruses A(H1, H1pdm09, H3 and H5 subtypes) and B with EC50's lying in micromolar range. Due to low cytotoxicity it resulted in high selectivity indices (74-661 depending on the virus). This effect did not depend on susceptibility or resistance of the viruses to adamantane derivatives amantadine and rimantadine. The compound appeared the most effective when added at the early stages of viral life cycle (0-2h p.i.). In direct hemagglutinin inhibition tests camphecene was shown to decrease the activity of HA's of influenza viruses A and B. The activity of camphecene was further confirmed in experiments with influenza virus-infected mice, in which, being used orally by therapeutic schedule (once a day, days 1-5 p.i.) it decreased specific mortality of animals infected with both influenza A and B viruses (highest indices of protection 66.7% and 88.9%, respectively). Taken together, these results are encouraging for further development of camphecene-based drug(s) and for exploration of camphor derivatives as highly prospective group of potential antivirals. PMID:26072310

  2. Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and pyrosequencing for the characterization of the dentine caries-associated microbiome

    PubMed Central

    Schulze-Schweifing, Kathrin; Banerjee, Avijit; Wade, William G.

    2014-01-01

    Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas, and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture. PMID:25429361

  3. Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and pyrosequencing for the characterization of the dentine caries-associated microbiome.

    PubMed

    Schulze-Schweifing, Kathrin; Banerjee, Avijit; Wade, William G

    2014-01-01

    Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas, and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture. PMID:25429361

  4. Aminoglycoside Resistance: The Emergence of Acquired 16S Ribosomal RNA Methyltransferases.

    PubMed

    Doi, Yohei; Wachino, Jun-Ichi; Arakawa, Yoshichika

    2016-06-01

    Aminoglycoside-producing Actinobacteria are known to protect themselves from their own aminoglycoside metabolites by producing 16S ribosomal RNA methyltransferase (16S-RMTase), which prevents them from binding to the 16S rRNA targets. Ten acquired 16S-RMTases have been reported from gram-negative pathogens. Most of them posttranscriptionally methylate residue G1405 of 16S rRNA resulting in high-level resistance to gentamicin, tobramycin, amikacin, and plazomicin. Strains that produce 16S-RMTase are frequently multidrug-resistant or even extensively drug-resistant. Although the direct clinical impact of high-level aminoglycoside resistance resulting from production of 16S-RMTase is yet to be determined, ongoing spread of this mechanism will further limit treatment options for multidrug-resistant and extensively drug-resistant gram-negative infections. PMID:27208771

  5. Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays.

    PubMed

    Shirafuji, Hiroaki; Yazaki, Ryu; Shuto, Yozo; Yanase, Tohru; Kato, Tomoko; Ishikura, Youji; Sakaguchi, Zenjiro; Suzuki, Moemi; Yamakawa, Makoto

    2015-12-01

    TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine β-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1. PMID:26341063

  6. Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at √{sNN} = 2.76 TeV

    NASA Astrophysics Data System (ADS)

    Adam, J.; Adamová, D.; Aggarwal, M. M.; Aglieri Rinella, G.; Agnello, M.; Agrawal, N.; Ahammed, Z.; Ahn, S. U.; Aiola, S.; Akindinov, A.; Alam, S. N.; Aleksandrov, D.; Alessandro, B.; Alexandre, D.; Alfaro Molina, R.; Alici, A.; Alkin, A.; Almaraz, J. R. M.; Alme, J.; Alt, T.; Altinpinar, S.; Altsybeev, I.; Alves Garcia Prado, C.; Andrei, C.; Andronic, A.; Anguelov, V.; Anielski, J.; Antičić, T.; Antinori, F.; Antonioli, P.; Aphecetche, L.; Appelshäuser, H.; Arcelli, S.; Arnaldi, R.; Arnold, O. W.; Arsene, I. C.; Arslandok, M.; Audurier, B.; Augustinus, A.; Averbeck, R.; Azmi, M. D.; Badalà, A.; Baek, Y. W.; Bagnasco, S.; Bailhache, R.; Bala, R.; Baldisseri, A.; Baral, R. C.; Barbano, A. M.; Barbera, R.; Barile, F.; Barnaföldi, G. G.; Barnby, L. S.; Barret, V.; Bartalini, P.; Barth, K.; Bartke, J.; Bartsch, E.; Basile, M.; Bastid, N.; Basu, S.; Bathen, B.; Batigne, G.; Batista Camejo, A.; Batyunya, B.; Batzing, P. C.; Bearden, I. G.; Beck, H.; Bedda, C.; Behera, N. K.; Belikov, I.; Bellini, F.; Bello Martinez, H.; Bellwied, R.; Belmont, R.; Belmont-Moreno, E.; Belyaev, V.; Bencedi, G.; Beole, S.; Berceanu, I.; Bercuci, A.; Berdnikov, Y.; Berenyi, D.; Bertens, R. A.; Berzano, D.; Betev, L.; Bhasin, A.; Bhat, I. R.; Bhati, A. K.; Bhattacharjee, B.; Bhom, J.; Bianchi, L.; Bianchi, N.; Bianchin, C.; Bielčík, J.; Bielčíková, J.; Bilandzic, A.; Biswas, R.; Biswas, S.; Bjelogrlic, S.; Blair, J. T.; Blau, D.; Blume, C.; Bock, F.; Bogdanov, A.; Bøggild, H.; Boldizsár, L.; Bombara, M.; Book, J.; Borel, H.; Borissov, A.; Borri, M.; Bossú, F.; Botta, E.; Böttger, S.; Bourjau, C.; Braun-Munzinger, P.; Bregant, M.; Breitner, T.; Broker, T. A.; Browning, T. A.; Broz, M.; Brucken, E. J.; Bruna, E.; Bruno, G. E.; Budnikov, D.; Buesching, H.; Bufalino, S.; Buncic, P.; Busch, O.; Buthelezi, Z.; Butt, J. B.; Buxton, J. T.; Caffarri, D.; Cai, X.; Caines, H.; Calero Diaz, L.; Caliva, A.; Calvo Villar, E.; Camerini, P.; Carena, F.; Carena, W.; Carnesecchi, F.; Castillo Castellanos, J.; Castro, A. J.; Casula, E. A. R.; Ceballos Sanchez, C.; Cepila, J.; Cerello, P.; Cerkala, J.; Chang, B.; Chapeland, S.; Chartier, M.; Charvet, J. L.; Chattopadhyay, S.; Chattopadhyay, S.; Chelnokov, V.; Cherney, M.; Cheshkov, C.; Cheynis, B.; Chibante Barroso, V.; Chinellato, D. D.; Cho, S.; Chochula, P.; Choi, K.; Chojnacki, M.; Choudhury, S.; Christakoglou, P.; Christensen, C. H.; Christiansen, P.; Chujo, T.; Chung, S. U.; Cicalo, C.; Cifarelli, L.; Cindolo, F.; Cleymans, J.; Colamaria, F.; Colella, D.; Collu, A.; Colocci, M.; Conesa Balbastre, G.; Conesa del Valle, Z.; Connors, M. E.; Contreras, J. G.; Cormier, T. M.; Corrales Morales, Y.; Cortés Maldonado, I.; Cortese, P.; Cosentino, M. R.; Costa, F.; Crochet, P.; Cruz Albino, R.; Cuautle, E.; Cunqueiro, L.; Dahms, T.; Dainese, A.; Danu, A.; Das, D.; Das, I.; Das, S.; Dash, A.; Dash, S.; De, S.; De Caro, A.; de Cataldo, G.; de Conti, C.; de Cuveland, J.; De Falco, A.; De Gruttola, D.; De Marco, N.; De Pasquale, S.; Deisting, A.; Deloff, A.; Dénes, E.; Deplano, C.; Dhankher, P.; Di Bari, D.; Di Mauro, A.; Di Nezza, P.; Diaz Corchero, M. A.; Dietel, T.; Dillenseger, P.; Divià, R.; Djuvsland, Ø.; Dobrin, A.; Domenicis Gimenez, D.; Dönigus, B.; Dordic, O.; Drozhzhova, T.; Dubey, A. K.; Dubla, A.; Ducroux, L.; Dupieux, P.; Ehlers, R. J.; Elia, D.; Engel, H.; Epple, E.; Erazmus, B.; Erdemir, I.; Erhardt, F.; Espagnon, B.; Estienne, M.; Esumi, S.; Eum, J.; Evans, D.; Evdokimov, S.; Eyyubova, G.; Fabbietti, L.; Fabris, D.; Faivre, J.; Fantoni, A.; Fasel, M.; Feldkamp, L.; Feliciello, A.; Feofilov, G.; Ferencei, J.; Fernández Téllez, A.; Ferreiro, E. G.; Ferretti, A.; Festanti, A.; Feuillard, V. J. G.; Figiel, J.; Figueredo, M. A. S.; Filchagin, S.; Finogeev, D.; Fionda, F. M.; Fiore, E. M.; Fleck, M. G.; Floris, M.; Foertsch, S.; Foka, P.; Fokin, S.; Fragiacomo, E.; Francescon, A.; Frankenfeld, U.; Fuchs, U.; Furget, C.; Furs, A.; Fusco Girard, M.; Gaardhøje, J. J.; Gagliardi, M.; Gago, A. M.; Gallio, M.; Gangadharan, D. R.; Ganoti, P.; Gao, C.; Garabatos, C.; Garcia-Solis, E.; Gargiulo, C.; Gasik, P.; Gauger, E. F.; Germain, M.; Gheata, A.; Gheata, M.; Ghosh, P.; Ghosh, S. K.; Gianotti, P.; Giubellino, P.; Giubilato, P.; Gladysz-Dziadus, E.; Glässel, P.; Goméz Coral, D. M.; Gomez Ramirez, A.; Gonzalez, V.; González-Zamora, P.; Gorbunov, S.; Görlich, L.; Gotovac, S.; Grabski, V.; Grachov, O. A.; Graczykowski, L. K.; Graham, K. L.; Grelli, A.; Grigoras, A.; Grigoras, C.; Grigoriev, V.; Grigoryan, A.; Grigoryan, S.; Grinyov, B.; Grion, N.; Gronefeld, J. M.; Grosse-Oetringhaus, J. F.; Grossiord, J.-Y.; Grosso, R.; Guber, F.; Guernane, R.; Guerzoni, B.; Gulbrandsen, K.; Gunji, T.; Gupta, A.; Gupta, R.; Haake, R.; Haaland, Ø.; Hadjidakis, C.

    2016-03-01

    The centrality dependence of the charged-particle pseudorapidity density measured with ALICE in Pb-Pb collisions at √{sNN} = 2.76 TeV over a broad pseudorapidity range is presented. This Letter extends the previous results reported by ALICE to more peripheral collisions. No strong change of the overall shape of charged-particle pseudorapidity density distributions with centrality is observed, and when normalised to the number of participating nucleons in the collisions, the evolution over pseudorapidity with centrality is likewise small. The broad pseudorapidity range (- 3.5 < η < 5) allows precise estimates of the total number of produced charged particles which we find to range from 162 ± 22(syst.) to 17170 ± 770(syst.) in 80-90% and 0-5% central collisions, respectively. The total charged-particle multiplicity is seen to approximately scale with the number of participating nucleons in the collision. This suggests that hard contributions to the charged-particle multiplicity are limited. The results are compared to models which describe dNch / dη at mid-rapidity in the most central Pb-Pb collisions and it is found that these models do not capture all features of the distributions.

  7. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences.

    PubMed

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-07-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences. PMID:26943621

  8. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences

    PubMed Central

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-01-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences. PMID:26943621

  9. HTCC: Broad Range Inhibitor of Coronavirus Entry

    PubMed Central

    Milewska, Aleksandra; Kaminski, Kamil; Ciejka, Justyna; Kosowicz, Katarzyna; Zeglen, Slawomir; Wojarski, Jacek; Nowakowska, Maria; Szczubiałka, Krzysztof; Pyrc, Krzysztof

    2016-01-01

    To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1) circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and its hydrophobically-modified derivative (HM-HTCC) as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses. PMID:27249425

  10. Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons

    PubMed Central

    2011-01-01

    Background Urine within the urinary tract is commonly regarded as "sterile" in cultivation terms. Here, we present a comprehensive in-depth study of bacterial 16S rDNA sequences associated with urine from healthy females by means of culture-independent high-throughput sequencing techniques. Results Sequencing of the V1V2 and V6 regions of the 16S ribosomal RNA gene using the 454 GS FLX system was performed to characterize the possible bacterial composition in 8 culture-negative (<100,000 CFU/ml) healthy female urine specimens. Sequences were compared to 16S rRNA databases and showed significant diversity, with the predominant genera detected being Lactobacillus, Prevotella and Gardnerella. The bacterial profiles in the female urine samples studied were complex; considerable variation between individuals was observed and a common microbial signature was not evident. Notably, a significant amount of sequences belonging to bacteria with a known pathogenic potential was observed. The number of operational taxonomic units (OTUs) for individual samples varied substantially and was in the range of 20 - 500. Conclusions Normal female urine displays a noticeable and variable bacterial 16S rDNA sequence richness, which includes fastidious and anaerobic bacteria previously shown to be associated with female urogenital pathology. PMID:22047020

  11. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    PubMed Central

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  12. Investigation of the spectral properties of LED-based MR16s for general illumination

    NASA Astrophysics Data System (ADS)

    Brown, David F.; Nicol, David B.; Payne, Adam; Ferguson, Ian T.

    2004-10-01

    The spectral properties of commercially available LED-based and halogen MR16s were investigated. The measurements taken include TLF (Total Luminous Flux), CCT (Correlated Color Temperature), CRI (Color Rendering Index), angular variation of CCT, and luminous efficacy. The halogen MR16s were used as a baseline for comparison with LED-based MR16s. It is shown at this time that LED-based MR16s are not suitable as a direct replacement for existing alternatives due to high initial cost, low power efficiency, poor CRIs, and undesirable CCTs.

  13. The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

    PubMed

    Jeng, R S; Svircev, A M; Myers, A L; Beliaeva, L; Hunter, D M; Hubbes, M

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. PMID:11166101

  14. Tetrathiobacter kashmirensis Strain CA-1 16S rRNA gene complete sequence.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used 1326 base pair 16S rRNA gene sequence methods to confirm the identification of a bacterium as Tetrathiobacter kashmirensis. Morphological, biochemical characteristics, and fatty acid profiles are consistent with the 16S rRNA gene sequence identification of the bacterium. The isolate...

  15. A New Protocol to Detect Multiple Foodborne Pathogens with PCR Dipstick DNA Chromatography after a Six-Hour Enrichment Culture in a Broad-Range Food Pathogen Enrichment Broth

    PubMed Central

    Hayashi, Masahiro; Natori, Tatsuya; Kubota-Hayashi, Sayoko; Miyata, Machiko; Ohkusu, Kiyofumi; Kurazono, Hisao; Makino, Souichi; Ezaki, Takayuki

    2013-01-01

    A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5–10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments. PMID:24364031

  16. Monitoring Precursor 16S rRNAs of Acinetobacter spp. in Activated Sludge Wastewater Treatment Systems

    PubMed Central

    Oerther, Daniel B.; Pernthaler, Jakob; Schramm, Andreas; Amann, Rudolf; Raskin, Lutgarde

    2000-01-01

    Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457–4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3′ region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems. PMID:10788395

  17. CATCh, an ensemble classifier for chimera detection in 16S rRNA sequencing studies.

    PubMed

    Mysara, Mohamed; Saeys, Yvan; Leys, Natalie; Raes, Jeroen; Monsieurs, Pieter

    2015-03-01

    In ecological studies, microbial diversity is nowadays mostly assessed via the detection of phylogenetic marker genes, such as 16S rRNA. However, PCR amplification of these marker genes produces a significant amount of artificial sequences, often referred to as chimeras. Different algorithms have been developed to remove these chimeras, but efforts to combine different methodologies are limited. Therefore, two machine learning classifiers (reference-based and de novo CATCh) were developed by integrating the output of existing chimera detection tools into a new, more powerful method. When comparing our classifiers with existing tools in either the reference-based or de novo mode, a higher performance of our ensemble method was observed on a wide range of sequencing data, including simulated, 454 pyrosequencing, and Illumina MiSeq data sets. Since our algorithm combines the advantages of different individual chimera detection tools, our approach produces more robust results when challenged with chimeric sequences having a low parent divergence, short length of the chimeric range, and various numbers of parents. Additionally, it could be shown that integrating CATCh in the preprocessing pipeline has a beneficial effect on the quality of the clustering in operational taxonomic units. PMID:25527546

  18. Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.)

    PubMed Central

    2012-01-01

    Background Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. Results Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. Conclusions Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean

  19. Mixed quantum/classical calculations of total and differential elastic and rotationally inelastic scattering cross sections for light and heavy reduced masses in a broad range of collision energies

    SciTech Connect

    Semenov, Alexander; Babikov, Dmitri

    2014-01-28

    The mixed quantum/classical theory (MQCT) for rotationally inelastic scattering developed recently [A. Semenov and D. Babikov, J. Chem. Phys. 139, 174108 (2013)] is benchmarked against the full quantum calculations for two molecular systems: He + H{sub 2} and Na + N{sub 2}. This allows testing new method in the cases of light and reasonably heavy reduced masses, for small and large rotational quanta, in a broad range of collision energies and rotational excitations. The resultant collision cross sections vary through ten-orders of magnitude range of values. Both inelastic and elastic channels are considered, as well as differential (over scattering angle) cross sections. In many cases results of the mixed quantum/classical method are hard to distinguish from the full quantum results. In less favorable cases (light masses, larger quanta, and small collision energies) some deviations are observed but, even in the worst cases, they are within 25% or so. The method is computationally cheap and particularly accurate at higher energies, heavier masses, and larger densities of states. At these conditions MQCT represents a useful alternative to the standard full-quantum scattering theory.

  20. Mixed quantum/classical calculations of total and differential elastic and rotationally inelastic scattering cross sections for light and heavy reduced masses in a broad range of collision energies

    NASA Astrophysics Data System (ADS)

    Semenov, Alexander; Babikov, Dmitri

    2014-01-01

    The mixed quantum/classical theory (MQCT) for rotationally inelastic scattering developed recently [A. Semenov and D. Babikov, J. Chem. Phys. 139, 174108 (2013)] is benchmarked against the full quantum calculations for two molecular systems: He + H2 and Na + N2. This allows testing new method in the cases of light and reasonably heavy reduced masses, for small and large rotational quanta, in a broad range of collision energies and rotational excitations. The resultant collision cross sections vary through ten-orders of magnitude range of values. Both inelastic and elastic channels are considered, as well as differential (over scattering angle) cross sections. In many cases results of the mixed quantum/classical method are hard to distinguish from the full quantum results. In less favorable cases (light masses, larger quanta, and small collision energies) some deviations are observed but, even in the worst cases, they are within 25% or so. The method is computationally cheap and particularly accurate at higher energies, heavier masses, and larger densities of states. At these conditions MQCT represents a useful alternative to the standard full-quantum scattering theory.

  1. Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

    PubMed Central

    Gaibani, Paolo; Mariconti, Mara; Bua, Gloria; Bonora, Sonia; Sassera, Davide; Landini, Maria Paola; Mulatto, Patrizia; Novati, Stefano; Bandi, Claudio; Sambri, Vittorio

    2013-01-01

    Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood. PMID:23586027

  2. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.

    PubMed Central

    Hartmann, R K; Erdmann, V A

    1989-01-01

    A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA. Images PMID:2722737

  3. Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets.

    PubMed

    Górska, Anna; Markowska-Zagrajek, Agnieszka; Równicki, Marcin; Trylska, Joanna

    2016-08-25

    We have designed a protocol and server to aid in the search for putative binding sites in 16S rRNA that could be targeted by peptide nucleic acid oligomers. Various features of 16S rRNA were considered to score its regions as potential targets for sequence-specific binding that could result in inhibition of ribosome function. Specifically, apart from the functional importance of a particular rRNA region, we calculated its accessibility, flexibility, energetics of strand invasion by an oligomer, as well as similarity to human rRNA. To determine 16S rRNA flexibility in the ribosome context, we performed all-atom molecular dynamics simulations of the 30S subunit in explicit solvent. We proposed a few 16S RNA target sites, and one of them was tested experimentally to verify inhibition of bacterial growth by a peptide nucleic acid oligomer. PMID:27105576

  4. Evaluation of 16S rDNA-Based Community Profiling for Human Microbiome Research

    PubMed Central

    2012-01-01

    The Human Microbiome Project will establish a reference data set for analysis of the microbiome of healthy adults by surveying multiple body sites from 300 people and generating data from over 12,000 samples. To characterize these samples, the participating sequencing centers evaluated and adopted 16S rDNA community profiling protocols for ABI 3730 and 454 FLX Titanium sequencing. In the course of establishing protocols, we examined the performance and error characteristics of each technology, and the relationship of sequence error to the utility of 16S rDNA regions for classification- and OTU-based analysis of community structure. The data production protocols used for this work are those used by the participating centers to produce 16S rDNA sequence for the Human Microbiome Project. Thus, these results can be informative for interpreting the large body of clinical 16S rDNA data produced for this project. PMID:22720093

  5. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

    PubMed Central

    Razzauti, Maria; Galan, Maxime; Bernard, Maria; Maman, Sarah; Klopp, Christophe; Charbonnel, Nathalie; Vayssier-Taussat, Muriel; Eloit, Marc; Cosson, Jean-François

    2015-01-01

    Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. Methodology/Principal Findings We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each

  6. Molecular identification of adulteration in mutton based on mitochondrial 16S rRNA gene.

    PubMed

    Xu, Jia; Zhao, Wei; Zhu, Mengru; Wen, Yuanju; Xie, Tao; He, Xiaoqian; Zhang, Yongfeng; Cao, Suizhong; Niu, Lili; Zhang, Hongping; Zhong, Tao

    2016-01-01

    The aim of this study is to set up a protocol for identification of the adulteration in mutton based on mitochondrial 16S rRNA gene. The multiplex polymerase chain reaction (multi-PCR) assay was carried out to trace the impure DNA in mutton. A universal primer pair yielded an approximate 610 bp fragment in mutton, pork, duck, chicken, horse and cat meats. The amplicons of multi-PCR assay represented the species-specific products, which could be discriminated by the size ranging from 106 bp to 532 bp. Subsequently, the authentication of each fragment was also confirmed by sequencing. Random analyses of adulterants with various meats yielded the identical results to their components, showing the suitability of the multi-PCR assay for tracing of adulterant meats with high-accuracy and precision. This assay was sensitive to detect the species-specific DNA in different proportional mixtures of mutton and duck/pork (9.1%-90.9%). In conclusion, this multi-PCR assay successfully discriminated the double-, triple-, quadruple-, and quintuple-mixtures containing variant counterparts. This method will be particularly useful in the detection of mutton adulteration in processed foods further. PMID:24739005

  7. Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

    PubMed Central

    Liefting, L W; Andersen, M T; Beever, R E; Gardner, R C; Forster, R L

    1996-01-01

    Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data. PMID:8795200

  8. Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization

    PubMed Central

    Witek, Marta A.; Conn, Graeme L.

    2016-01-01

    Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m1A1408) methyltransferase (‘CacKam’). The apo CacKam structure closely resembles other m1A1408 methyltransferases within its conserved SAM-binding fold but the region linking core β strands 6 and 7 (the ‘β6/7 linker’) has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the β6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover. PMID:26609134

  9. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  10. Eight-band k·p modeling of InAs/InGaAsSb type-II W-design quantum well structures for interband cascade lasers emitting in a broad range of mid infrared

    SciTech Connect

    Ryczko, K.; Sęk, G.; Misiewicz, J.

    2013-12-14

    Band structure properties of the type-II W-design AlSb/InAs/GaIn(As)Sb/InAs/AlSb quantum wells have been investigated theoretically in a systematic manner and with respect to their use in the active region of interband cascade laser for a broad range of emission in mid infrared between below 3 to beyond 10 μm. Eight-band k·p approach has been utilized to calculate the electronic subbands. The fundamental optical transition energy and the corresponding oscillator strength have been determined in function of the thickness of InAs and GaIn(As)Sb layers and the composition of the latter. There have been considered active structures on two types of relevant substrates, GaSb and InAs, introducing slightly modified strain conditions. Additionally, the effect of external electric field has been taken into account to simulate the conditions occurring in the operational devices. The results show that introducing arsenic as fourth element into the valence band well of the type-II W-design system, and then altering its composition, can efficiently enhance the transition oscillator strength and allow additionally increasing the emission wavelength, which makes this solution prospective for improved performance and long wavelength interband cascade lasers.

  11. Design of Vibrio 16S rRNA gene specific primers and their application in the analysis of seawater Vibrio community

    NASA Astrophysics Data System (ADS)

    Yong, Liu; Guanpin, Yang; Hualei, Wang; Jixiang, Chen; Xianming, Shi; Guiwei, Zou; Qiwei, Wei; Xiuqin, Sun

    2006-04-01

    The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene (rDNA) fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying, Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

  12. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

    PubMed Central

    Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  13. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    PubMed

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  14. Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

    PubMed Central

    Kitahara, Kei; Yasutake, Yoshiaki; Miyazaki, Kentaro

    2012-01-01

    The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome’s structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, we have recently shown that an active hybrid ribosome whose 16S rRNA has been specifically substituted with that from non–E. coli bacteria can be reconstituted in vivo. To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality, we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E. coli Δ7. Various functional genes were obtained from the Gammaproteobacteria and Betaproteobacteria lineages. Despite the large sequence diversity (80.9–99.0% identity with E. coli 16S rRNA) of the functional 16S rRNA molecules, the doubling times (DTs) of each mutant increased only modestly with decreasing sequence identity (average increase in DT, 4.6 s per mutation). The three-dimensional structure of the 30S ribosome showed that at least 40.7% (628/1,542) of the nucleotides were variable, even at ribosomal protein-binding sites, provided that the secondary structures were properly conserved. Our results clearly demonstrate that 16S rRNA functionality largely depends on the secondary structure but not on the sequence itself. PMID:23112186

  15. Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family).

    PubMed

    Kulinska, Anna; Godziszewska, Jolanta; Wojciechowska, Anna; Ludwiczak, Marta; Jagura-Burdzy, Grazyna

    2016-04-01

    The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions in Escherichia coli, since its overexpression in transnegatively interferes with mini-RA3 replication and stable maintenance of RA3. PMID:26850301

  16. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers

    PubMed Central

    Liu, Zongzhi; DeSantis, Todd Z.; Andersen, Gary L.; Knight, Rob

    2008-01-01

    The recent introduction of massively parallel pyrosequencers allows rapid, inexpensive analysis of microbial community composition using 16S ribosomal RNA (rRNA) sequences. However, a major challenge is to design a workflow so that taxonomic information can be accurately and rapidly assigned to each read, so that the composition of each community can be linked back to likely ecological roles played by members of each species, genus, family or phylum. Here, we use three large 16S rRNA datasets to test whether taxonomic information based on the full-length sequences can be recaptured by short reads that simulate the pyrosequencer outputs. We find that different taxonomic assignment methods vary radically in their ability to recapture the taxonomic information in full-length 16S rRNA sequences: most methods are sensitive to the region of the 16S rRNA gene that is targeted for sequencing, but many combinations of methods and rRNA regions produce consistent and accurate results. To process large datasets of partial 16S rRNA sequences obtained from surveys of various microbial communities, including those from human body habitats, we recommend the use of Greengenes or RDP classifier with fragments of at least 250 bases, starting from one of the primers R357, R534, R798, F343 or F517. PMID:18723574

  17. Mitochondrial 16S ribosomal RNA gene for forensic identification of crocodile species.

    PubMed

    Naga Jogayya, K; Meganathan, P R; Dubey, Bhawna; Haque, I

    2013-05-01

    All crocodilians are under various threats due to over exploitation and these species have been listed in Appendix I or II of CITES. Lack of molecular techniques for the forensic identification of confiscated samples makes it difficult to enforce the law. Therefore, we herein present a molecular method developed on the basis on 16S rRNA gene of mitochondrial DNA for identification of crocodile species. We have developed a set of 16S rRNA primers for PCR based identification of crocodilian species. These novel primers amplify partial 16S rRNA sequences of six crocodile species which can be later combined to obtain a larger region (1290 bp) of 16S rRNA gene. This 16S rRNA gene could be used as an effective tool for forensic authentication of crocodiles. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these species. PMID:23622485

  18. Broad spectrum bioactive sunscreens.

    PubMed

    Velasco, Maria Valéria Robles; Sarruf, Fernanda Daud; Salgado-Santos, Idalina Maria Nunes; Haroutiounian-Filho, Carlos Alberto; Kaneko, Telma Mary; Baby, André Rolim

    2008-11-01

    The development of sunscreens containing reduced concentration of chemical UV filters, even though, possessing broad spectrum effectiveness with the use of natural raw materials that improve and infer UV absorption is of great interest. Due to the structural similarities between polyphenolic compounds and organic UV filters, they might exert photoprotection activity. The objective of the present research work was to develop bioactive sunscreen delivery systems containing rutin, Passiflora incarnata L. and Plantago lanceolata extracts associated or not with organic and inorganic UV filters. UV transmission of the sunscreen delivery system films was performed by using diffuse transmittance measurements coupling to an integrating sphere. In vitro photoprotection efficacy was evaluated according to the following parameters: estimated sun protection factor (SPF); Boot's Star Rating category; UVA/UVB ratio; and critical wavelength (lambda(c)). Sunscreen delivery systems obtained SPF values ranging from 0.972+/-0.004 to 28.064+/-2.429 and bioactive compounds interacted with the UV filters positive and negatively. This behavior may be attributed to: the composition of the delivery system; the presence of inorganic UV filter and quantitative composition of the organic UV filters; and the phytochemical composition of the P. incarnata L. and P. lanceolata extracts. Among all associations of bioactive compounds and UV filters, we found that the broad spectrum sunscreen was accomplished when 1.68% (w/w) P. incarnata L. dry extract was in the presence of 7.0% (w/w) ethylhexyl methoxycinnamate, 2.0% (w/w) benzophenone-3 and 2.0% (w/w) TiO(2). It was demonstrated that this association generated estimated SPF of 20.072+/-0.906 and it has improved the protective defense against UVA radiation accompanying augmentation of the UVA/UVB ratio from 0.49 to 0.52 and lambda(c) from 364 to 368.6nm. PMID:18662760

  19. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research.

    PubMed

    Jo, Jay-Hyun; Kennedy, Elizabeth A; Kong, Heidi H

    2016-03-01

    Skin serves as a protective barrier and also harbors numerous microorganisms collectively comprising the skin microbiome. As a result of recent advances in sequencing (next-generation sequencing), our understanding of microbial communities on skin has advanced substantially. In particular, the 16S ribosomal RNA gene sequencing technique has played an important role in efforts to identify the global communities of bacteria in healthy individuals and patients with various disorders in multiple topographical regions over the skin surface. Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach. This article will also discuss some applications and challenges of 16S ribosomal RNA sequencing as well as directions for future development. PMID:26902128

  20. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  1. Depletion of pre-16S rRNA in starved Escherichia coli cells.

    PubMed

    Cangelosi, G A; Brabant, W H

    1997-07-01

    Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth. PMID:9226253

  2. Assessment of Three Mitochondrial Genes (16S, Cytb, CO1) for Identifying Species in the Praomyini Tribe (Rodentia: Muridae)

    PubMed Central

    Nicolas, Violaine; Schaeffer, Brigitte; Missoup, Alain Didier; Kennis, Jan; Colyn, Marc; Denys, Christiane; Tatard, Caroline; Cruaud, Corinne; Laredo, Catherine

    2012-01-01

    The Praomyini tribe is one of the most diverse and abundant groups of Old World rodents. Several species are known to be involved in crop damage and in the epidemiology of several human and cattle diseases. Due to the existence of sibling species their identification is often problematic. Thus an easy, fast and accurate species identification tool is needed for non-systematicians to correctly identify Praomyini species. In this study we compare the usefulness of three genes (16S, Cytb, CO1) for identifying species of this tribe. A total of 426 specimens representing 40 species (sampled across their geographical range) were sequenced for the three genes. Nearly all of the species included in our study are monophyletic in the neighbour joining trees. The degree of intra-specific variability tends to be lower than the divergence between species, but no barcoding gap is detected. The success rate of the statistical methods of species identification is excellent (up to 99% or 100% for statistical supervised classification methods as the k-Nearest Neighbour or Random Forest). The 16S gene is 2.5 less variable than the Cytb and CO1 genes. As a result its discriminatory power is smaller. To sum up, our results suggest that using DNA markers for identifying species in the Praomyini tribe is a largely valid approach, and that the CO1 and Cytb genes are better DNA markers than the 16S gene. Our results confirm the usefulness of statistical methods such as the Random Forest and the 1-NN methods to assign a sequence to a species, even when the number of species is relatively large. Based on our NJ trees and the distribution of all intraspecific and interspecific pairwise nucleotide distances, we highlight the presence of several potentially new species within the Praomyini tribe that should be subject to corroboration assessments. PMID:22574186

  3. 16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

    PubMed Central

    Auchtung, Thomas A.; Takacs-Vesbach, Cristina D.; Cavanaugh, Colleen M.

    2006-01-01

    The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity. PMID:16820509

  4. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

    NASA Technical Reports Server (NTRS)

    Rossler, D.; Ludwig, W.; Schleifer, K. H.; Lin, C.; McGill, T. J.; Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  5. Molecular analysis of 16S-23S spacer regions of Acetobacter species.

    PubMed

    Kretová, M; Grones, J

    2005-01-01

    16S-23S rDNA internal transcribed spacer regions (ITS) similarities were determined in 8 Acetobacter and 1 Gluconacetobacter strains. ITS-PCR amplification of the 16S-23S spacers showed 2 products of similar size in 7 strains; only 1 product of similar size was found in the 2 remaining strains. Analysis of the PCR products using restriction endonucleases HaeIII, HpaII and AluI revealed 3 different restriction groups of A. pasteurianus for AluI and HaeIII, and 4 restriction groups for HpaII. ITS nucleotide sequences of all studied strains exhibited a 52-98% similarity. PMID:16408846

  6. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m)with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  7. Effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s = 4, 5, 6) surfactants on the interaction of ninhydrin with chromium-glycylphenylalanine

    NASA Astrophysics Data System (ADS)

    Kumar, Dileep; Rub, Malik Abdul; Akram, Mohd.; Kabir-ud-Din

    2014-11-01

    The effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s = 4, 5, 6) surfactants on the interaction of ninhydrin with chromium(III) complex of glycylphenylalanine ([Cr(III)-Gly-Phe]2+) has been investigated using UV-visible spectrophotometer at different temperatures. The order of reaction with respect to [Cr(III)-Gly-Phe]2+ is unity while it is fractional with respect to ninhydrin. Whereas, the values of rate constant (kψ) increase and leveling-off regions, like conventional single chain cetyltrimethylammonium bromide (CTAB) surfactant, were observed with geminis, later produces a third region of increasing kψ at higher gemini surfactant concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The results obtained in micellar media were treated in terms of pseudo-phase model. The values of thermodynamic parameters (Ea, ΔH# and ΔS#) and binding constants (KA and KNin) have been evaluated.

  8. Effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium-glycylphenylalanine.

    PubMed

    Kumar, Dileep; Rub, Malik Abdul; Akram, Mohd; Kabir-ud-Din

    2014-11-11

    The effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium(III) complex of glycylphenylalanine ([Cr(III)-Gly-Phe]2+) has been investigated using UV-visible spectrophotometer at different temperatures. The order of reaction with respect to [Cr(III)-Gly-Phe]2+ is unity while it is fractional with respect to ninhydrin. Whereas, the values of rate constant (kψ) increase and leveling-off regions, like conventional single chain cetyltrimethylammonium bromide (CTAB) surfactant, were observed with geminis, later produces a third region of increasing kψ at higher gemini surfactant concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The results obtained in micellar media were treated in terms of pseudo-phase model. The values of thermodynamic parameters (Ea, ΔH# and ΔS#) and binding constants (KA and KNin) have been evaluated. PMID:24878435

  9. The structure of Aquifex aeolicus ribosomal protein S8 reveals a unique subdomain that contributes to an extremely tight association with 16S rRNA.

    PubMed

    Menichelli, Elena; Edgcomb, Stephen P; Recht, Michael I; Williamson, James R

    2012-01-20

    The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA. PMID:22079365

  10. Rapid Identification of Bacteria from Positive Blood Cultures by Fluorescence-Based PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene

    PubMed Central

    Turenne, Christine Y.; Witwicki, Evelyn; Hoban, Daryl J.; Karlowsky, James A.; Kabani, Amin M.

    2000-01-01

    Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR–single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections. PMID:10655337

  11. Molecular Diagnosis of Actinomadura madurae Infection by 16S rRNA Deep Sequencing

    PubMed Central

    SenGupta, Dhruba J.; Hoogestraat, Daniel R.; Cummings, Lisa A.; Bryant, Bronwyn H.; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W.; Chau, Mimosa; Barbee, Lindley A.; Rosenthal, Christopher; Cookson, Brad T.; Hoffman, Noah G.

    2013-01-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. PMID:24108607

  12. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    PubMed

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. PMID:25343859

  13. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Ovine foot rot is an infectious, contagious disease of sheep that causes severe lameness and economic loss from decreased flock produc...

  14. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects.

    PubMed

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic; Leese, Florian

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  15. The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline

    PubMed Central

    Païssé, Sandrine; Valle, Carine; Valière, Sophie; Kuchly, Claire; Vilchez, Gaëlle; Donnadieu, Cécile; Courtney, Michael; Burcelin, Rémy; Amar, Jacques; Bouchez, Olivier; Lelouvier, Benjamin

    2015-01-01

    Background Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples. Results We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart). Conclusion The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease. PMID:26544955

  16. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    PubMed Central

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  17. Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

  18. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra

    PubMed Central

    Ota-Tsuzuki, C.; Brunheira, A.T.P.; Mayer, M.P.A.

    2008-01-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes. PMID:24031274

  19. The Broad Way

    ERIC Educational Resources Information Center

    Butler, Kevin

    2008-01-01

    In the world of corporate philanthropy, there are those who give to educational causes, and this article describes one such philanthropist, Eli Broad, who shares his take on schools in America. Broad is in a category unto himself not only because of the amount of money he has given--more than $280 million since 1999--but also for his unique…

  20. The 2010 Broad Prize

    ERIC Educational Resources Information Center

    Education Digest: Essential Readings Condensed for Quick Review, 2011

    2011-01-01

    A new data analysis, based on data collected as part of The Broad Prize process, provides insights into which large urban school districts in the United States are doing the best job of educating traditionally disadvantaged groups: African-American, Hispanics, and low-income students. Since 2002, The Eli and Edythe Broad Foundation has awarded The…

  1. Analysis of the cystic fibrosis lung microbiota via serial Illumina sequencing of bacterial 16S rRNA hypervariable regions.

    PubMed

    Maughan, Heather; Wang, Pauline W; Diaz Caballero, Julio; Fung, Pauline; Gong, Yunchen; Donaldson, Sylva L; Yuan, Lijie; Keshavjee, Shaf; Zhang, Yu; Yau, Yvonne C W; Waters, Valerie J; Tullis, D Elizabeth; Hwang, David M; Guttman, David S

    2012-01-01

    The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients. PMID:23056217

  2. Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

    PubMed Central

    Vinuesa, Pablo; Rademaker, Jan L. W.; de Bruijn, Frans J.; Werner, Dietrich

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata. PMID:9603820

  3. Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes

    PubMed Central

    Baker, Brett J.; Hugenholtz, Philip; Dawson, Scott C.; Banfield, Jillian F.

    2003-01-01

    During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name “Candidatus Captivus acidiprotistae.” To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat. PMID:12957940

  4. Comparative sequence analyses of the genes coding for 16S rRNA of Lactobacillus casei-related taxa.

    PubMed

    Mori, K; Yamazaki, K; Ishiyama, T; Katsumata, M; Kobayashi, K; Kawai, Y; Inoue, N; Shinano, H

    1997-01-01

    The primary structures of the 16S rRNA genes of the type strains of Lactobacillus casei and related taxa were determined by PCR DNA-sequencing methods. The sequences of Lactobacillus casei, Lactobacillus zeae, Lactobacillus paracasei, and Lactobacillus rhamnosus were different. The Knuc values ranged from 0.0040 to 0.0126. On the basis of the Knuc values and the levels of DNA-DNA relatedness among the strains of these species, the L. casei-related taxa should be classified in the following three species: L. zeae, which includes the type strains of L. zeae and L. casei; a species that includes the strains of L. paracasei and L. casei ATCC 334; and L. rhamnosus. PMID:8995801

  5. Point mutations in the 3' minor domain of 16S rRNA of E.coli.

    PubMed Central

    Jemiolo, D K; Zwieb, C; Dahlberg, A E

    1985-01-01

    Point mutations were produced near the 3' end of E. coli 16S rRNA by bisulfite mutagenesis in a 121 base loop-out (1385 to 1505) in a heteroduplex of wild type (pKK3535) and deletion mutant plasmids. Two highly conserved, single stranded regions flank an irregular helix (1409-1491) in the area studied. Only a single mutation was isolated in the flanking regions, a transition at C1402, (normally methylated on the base and ribose in rRNA). Mutations occurred throughout the irregular helix. All mutant rRNAs were processed and assembled into 30S subunits capable of interacting with 50S subunits. Growth rates ranged from faster to significantly slower than cells with the wild type transcript. In particular, mutations at C1467 or C1469 cause slow growth. These two transitions (in a bulge region within the helix) reduced the bulge by additional base pairing. PMID:3909106

  6. A renaissance for the pioneering 16S rRNA gene

    SciTech Connect

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

    Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

  7. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  8. Rapid identification of Renibacterium salmoninarum using an oligonucleotide probe complementary to 16S rRNA.

    PubMed

    Mattsson, J G; Gersdorf, H; Jansson, E; Hongslo, T; Göbel, U B; Johansson, K E

    1993-02-01

    Bacterial kidney disease in salmonid fish is caused by the slow-growing Gram-positive rod, Renibacterium salmoninarum. The partial sequence of 16S rRNA from R. salmoninarum was determined and compared with published bacterial 16S rRNA sequences. From this sequence information, a 30-bases-long oligonucleotide was designed and used as a specific probe for identification of R. salmoninarum in filter hybridization experiments. Strong specific hybridization signals were observed for all strains of R. salmoninarum tested. Furthermore, no cross-hybridization could be seen against 22 other bacterial species, among them other salmonid fish pathogens. The detection limit for the probe in direct filter hybridization by the dot-blot technique was 2.5 x 10(4) bacteria. It was also possible to detect R. salmoninarum in clinical samples by direct filter hybridization. PMID:8455640

  9. Structural Analysis of Base Substitutions in Thermus thermophilus 16S rRNA Conferring Streptomycin Resistance

    PubMed Central

    Demirci, Hasan; Murphy, Frank V.; Murphy, Eileen L.; Connetti, Jacqueline L.; Dahlberg, Albert E.; Jogl, Gerwald

    2014-01-01

    Streptomycin is a bactericidal antibiotic that induces translational errors. It binds to the 30S ribosomal subunit, interacting with ribosomal protein S12 and with 16S rRNA through contacts with the phosphodiester backbone. To explore the structural basis for streptomycin resistance, we determined the X-ray crystal structures of 30S ribosomal subunits from six streptomycin-resistant mutants of Thermus thermophilus both in the apo form and in complex with streptomycin. Base substitutions at highly conserved residues in the central pseudoknot of 16S rRNA produce novel hydrogen-bonding and base-stacking interactions. These rearrangements in secondary structure produce only minor adjustments in the three-dimensional fold of the pseudoknot. These results illustrate how antibiotic resistance can occur as a result of small changes in binding site conformation. PMID:24820088

  10. The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans.

    PubMed

    Tomioka, N; Sugiura, M

    1983-01-01

    The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans, has been determined. Its coding region is estimated to be 1,487 base pairs long, which is nearly identical to those reported for chloroplast 16S rRNA genes and is about 4% shorter than that of the Escherichia coli gene. The 16S rRNA sequence of A. nidulans has 83% homology with that of tobacco chloroplast and 74% homology with that of E. coli. Possible stem and loop structures of A. nidulans 16S rRNA sequences resemble more closely those of chloroplast 16S rRNAs than those of E. coli 16S rRNA. These observations support the endosymbiotic theory of chloroplast origin. PMID:6412038

  11. Distinct genetic lineages of Bactrocera caudata (Insecta: Tephritidae) revealed by COI and 16S DNA sequences.

    PubMed

    Lim, Phaik-Eem; Tan, Ji; Suana, I Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies. PMID:22615962

  12. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    NASA Technical Reports Server (NTRS)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  13. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    SciTech Connect

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  14. Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences.

    PubMed

    Sakata, Shinji; Ryu, Chun Sun; Kitahara, Maki; Sakamoto, Mitsuo; Hayashi, Hidenori; Fukuyama, Masafumi; Benno, Yoshimi

    2006-01-01

    In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization. PMID:16428867

  15. Distinct Genetic Lineages of Bactrocera caudata (Insecta: Tephritidae) Revealed by COI and 16S DNA Sequences

    PubMed Central

    Lim, Phaik-Eem; Tan, Ji; Suana, I. Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies. PMID:22615962

  16. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring.

    PubMed

    Vierheilig, J; Savio, D; Ley, R E; Mach, R L; Farnleitner, A H; Reischer, G H

    2015-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multi-compartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

  17. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    PubMed Central

    Ziesemer, Kirsten A.; Mann, Allison E.; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T.; Brandt, Bernd W.; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C.; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A.; MacDonald, Sandy J.; Thomas, Gavin H.; Collins, Matthew J.; Lewis, Cecil M.; Hofman, Corinne; Warinner, Christina

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

  18. Interpreting 16S metagenomic data without clustering to achieve sub-OTU resolution.

    PubMed

    Tikhonov, Mikhail; Leach, Robert W; Wingreen, Ned S

    2015-01-01

    The standard approach to analyzing 16S tag sequence data, which relies on clustering reads by sequence similarity into Operational Taxonomic Units (OTUs), underexploits the accuracy of modern sequencing technology. We present a clustering-free approach to multi-sample Illumina data sets that can identify independent bacterial subpopulations regardless of the similarity of their 16S tag sequences. Using published data from a longitudinal time-series study of human tongue microbiota, we are able to resolve within standard 97% similarity OTUs up to 20 distinct subpopulations, all ecologically distinct but with 16S tags differing by as little as one nucleotide (99.2% similarity). A comparative analysis of oral communities of two cohabiting individuals reveals that most such subpopulations are shared between the two communities at 100% sequence identity, and that dynamical similarity between subpopulations in one host is strongly predictive of dynamical similarity between the same subpopulations in the other host. Our method can also be applied to samples collected in cross-sectional studies and can be used with the 454 sequencing platform. We discuss how the sub-OTU resolution of our approach can provide new insight into factors shaping community assembly. PMID:25012900

  19. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    PubMed

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  20. Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences.

    PubMed Central

    Wesley, I V; Wesley, R D; Cardella, M; Dewhirst, F E; Paster, B J

    1991-01-01

    Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA. Images PMID:1723076

  1. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    PubMed

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

  2. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene

    PubMed Central

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-01-01

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese’s complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity <98.0%), and further analysis revealed HGT events and potential donors of the heterogeneous copies (such as HGT from Chlamydia suis to Chlamydia trachomatis) and mutation events of some heterogeneous copies (such as Streptococcus suis JS14). Interestingly, HGT of the 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes. PMID:26220935

  3. Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays

    PubMed Central

    Warsen, Adelaide E.; Krug, Melissa J.; LaFrentz, Stacey; Stanek, Danielle R.; Loge, Frank J.; Call, Douglas R.

    2004-01-01

    We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 × 106 genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens. PMID:15240304

  4. PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats

    PubMed Central

    Camacho, H.; Tuero, A. D.; Bacardí, D.; Palenzuela, D. O.; Aguilera, A.; Silva, J. A.; Estrada, R.; Gell, O.; Suárez, J.; Ancizar, J.; Brown, E.; Colarte, A. B.; Castro, J.; Novoa, L. I.

    2016-01-01

    The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies. PMID:27382362

  5. PhylOPDb: a 16S rRNA oligonucleotide probe database for prokaryotic identification

    PubMed Central

    Jaziri, Faouzi; Parisot, Nicolas; Abid, Anis; Denonfoux, Jérémie; Ribière, Céline; Gasc, Cyrielle; Boucher, Delphine; Brugère, Jean-François; Mahul, Antoine; Hill, David R.C.; Peyretaillade, Eric; Peyret, Pierre

    2014-01-01

    In recent years, high-throughput molecular tools have led to an exponential growth of available 16S rRNA gene sequences. Incorporating such data, molecular tools based on target-probe hybridization were developed to monitor microbial communities within complex environments. Unfortunately, only a few 16S rRNA gene-targeted probe collections were described. Here, we present PhylOPDb, an online resource for a comprehensive phylogenetic oligonucleotide probe database. PhylOPDb provides a convivial and easy-to-use web interface to browse both regular and explorative 16S rRNA-targeted probes. Such probes set or subset could be used to globally monitor known and unknown prokaryotic communities through various techniques including DNA microarrays, polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), targeted gene capture or in silico rapid sequence identification. PhylOPDb contains 74 003 25-mer probes targeting 2178 genera including Bacteria and Archaea. Database URL: http://g2im.u-clermont1.fr/phylopdb/ PMID:24771669

  6. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

    PubMed

    Anahtar, Melis N; Bowman, Brittany A; Kwon, Douglas S

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  7. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

    PubMed Central

    Anahtar, Melis N.; Bowman, Brittany A.; Kwon, Douglas S.

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  8. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    PubMed

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  9. Refined localization of the Batten disease gene (CL3) by haplotype and linkage disequilibrium mapping to D16S288-D16S383 and exclusion from this region of a variant form of Batten disease with granular osmiophilic deposits

    SciTech Connect

    Mitchison, H.M.; O`Rawe, A.M.; Gormally, E.

    1995-06-05

    Haplotype analysis in a collaborative collection of 143 families with juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten (Spielmeyer-Vogt-Sjoegren) disease has permitted refined localization of the disease gene, CLN3, which was assigned to chromosome 16 in 1989. Recombination events in four maternal meioses delimit new flanking genetic markers for CLN3 which localize the gene to the chromosome interval 16p12.1-11.2 between microsatellite markers D16S288 and D16S383. This narrows the position of CLN3 to a region of 2.1 cM, a significant reduction from the previous best interval. Using haplotypes, analysis of the strong linkage disequilibrium that exists between genetic markers within the D16S288-D16S383 interval and CLN3 shows that CLN3 is in closest proximity to loci D16S299 and D16S298. Analysis of markers across the D16S288-D16S383 region in four families with a variant form of JNCL characterized histologically by cytosomal granular osmiophilic deposits (GROD) has excluded linkage of the gene locus to the CLN3 region of chromosome 16, suggesting that JNCL with GROD is not an allelic form of JNCL. 8 refs., 2 figs., 2 tabs.

  10. Bacterial Communities Associated with Host-Adapted Populations of Pea Aphids Revealed by Deep Sequencing of 16S Ribosomal DNA

    PubMed Central

    Gauthier, Jean-Pierre; Outreman, Yannick; Mieuzet, Lucie; Simon, Jean-Christophe

    2015-01-01

    Associations between microbes and animals are ubiquitous and hosts may benefit from harbouring microbial communities through improved resource exploitation or resistance to environmental stress. The pea aphid, Acyrthosiphon pisum, is the host of heritable bacterial symbionts, including the obligate endosymbiont Buchnera aphidicola and several facultative symbionts. While obligate symbionts supply aphids with key nutrients, facultative symbionts influence their hosts in many ways such as protection against natural enemies, heat tolerance, color change and reproduction alteration. The pea aphid also encompasses multiple plant-specialized biotypes, each adapted to one or a few legume species. Facultative symbiont communities differ strongly between biotypes, although bacterial involvement in plant specialization is uncertain. Here, we analyse the diversity of bacterial communities associated with nine biotypes of the pea aphid complex using amplicon pyrosequencing of 16S rRNA genes. Combined clustering and phylogenetic analyses of 16S sequences allowed identifying 21 bacterial OTUs (Operational Taxonomic Unit). More than 98% of the sequencing reads were assigned to known pea aphid symbionts. The presence of Wolbachia was confirmed in A. pisum while Erwinia and Pantoea, two gut associates, were detected in multiple samples. The diversity of bacterial communities harboured by pea aphid biotypes was very low, ranging from 3 to 11 OTUs across samples. Bacterial communities differed more between than within biotypes but this difference did not correlate with the genetic divergence between biotypes. Altogether, these results confirm that the aphid microbiota is dominated by a few heritable symbionts and that plant specialization is an important structuring factor of bacterial communities associated with the pea aphid complex. However, since we examined the microbiota of aphid samples kept a few generations in controlled conditions, it may be that bacterial diversity was

  11. Bacterial communities associated with host-adapted populations of pea aphids revealed by deep sequencing of 16S ribosomal DNA.

    PubMed

    Gauthier, Jean-Pierre; Outreman, Yannick; Mieuzet, Lucie; Simon, Jean-Christophe

    2015-01-01

    Associations between microbes and animals are ubiquitous and hosts may benefit from harbouring microbial communities through improved resource exploitation or resistance to environmental stress. The pea aphid, Acyrthosiphon pisum, is the host of heritable bacterial symbionts, including the obligate endosymbiont Buchnera aphidicola and several facultative symbionts. While obligate symbionts supply aphids with key nutrients, facultative symbionts influence their hosts in many ways such as protection against natural enemies, heat tolerance, color change and reproduction alteration. The pea aphid also encompasses multiple plant-specialized biotypes, each adapted to one or a few legume species. Facultative symbiont communities differ strongly between biotypes, although bacterial involvement in plant specialization is uncertain. Here, we analyse the diversity of bacterial communities associated with nine biotypes of the pea aphid complex using amplicon pyrosequencing of 16S rRNA genes. Combined clustering and phylogenetic analyses of 16S sequences allowed identifying 21 bacterial OTUs (Operational Taxonomic Unit). More than 98% of the sequencing reads were assigned to known pea aphid symbionts. The presence of Wolbachia was confirmed in A. pisum while Erwinia and Pantoea, two gut associates, were detected in multiple samples. The diversity of bacterial communities harboured by pea aphid biotypes was very low, ranging from 3 to 11 OTUs across samples. Bacterial communities differed more between than within biotypes but this difference did not correlate with the genetic divergence between biotypes. Altogether, these results confirm that the aphid microbiota is dominated by a few heritable symbionts and that plant specialization is an important structuring factor of bacterial communities associated with the pea aphid complex. However, since we examined the microbiota of aphid samples kept a few generations in controlled conditions, it may be that bacterial diversity was

  12. Comparison of Antifungal Activities and 16S Ribosomal DNA Sequences of Clinical and Environmental Isolates of Stenotrophomonas maltophilia

    PubMed Central

    Minkwitz, Arite; Berg, Gabriele

    2001-01-01

    In recent years, the gram-negative bacterium Stenotrophomonas maltophilia has become increasingly important in biotechnology and as a nosocomial pathogen, giving rise to a need for new information about its taxonomy and epidemiology. To determine intraspecies diversity and whether strains can be distinguished based on the sources of their isolation, 50 S. maltophilia isolates from clinical and environmental sources, including strains of biotechnological interest, were investigated. The isolates were characterized by in vitro antagonism against pathogenic fungi and the production of antifungal metabolites and enzymes. Phenotypically the strains showed variability that did not correlate significantly with their sources of isolation. Clinical strains displayed remarkable activity against the human pathogenic fungus Candida albicans. Antifungal activity against plant pathogens was more common and generally more severe from the environmental isolates, although not exclusive to them. All isolates, clinical and environmental, produced a range of antifungal metabolites including antibiotics, siderophores, and the enzymes proteases and chitinases. From 16S ribosomal DNA sequencing analysis, the isolates could be separated into three clusters, two of which consisted of isolates originating from the environment, especially rhizosphere isolates, and one of which consisted of clinical and aquatic strains. In contrast to the results of other recent investigations, these strains could be grouped based on their sources of isolation, with the exception of three rhizosphere isolates. Because there was evidence of nucleotide signature positions within the sequences that are suitable for distinguishing among the clusters, the clusters could be defined as different genomovars of S. maltophilia. Key sequences on the 16S ribosomal DNA could be used to develop a diagnostic method that differentiates these genomovars. PMID:11136762

  13. The Broad Foundations, 2006

    ERIC Educational Resources Information Center

    Broad Foundation, 2006

    2006-01-01

    The mission of the Broad Foundations is to transform K-12 urban public education through better governance, management, labor relations and competition; make significant contributions to advance major scientific and medical research; foster public appreciation of contemporary art by increasing access for audiences worldwide; and lead and…

  14. Broad Bandwidth Telecommunications Systems.

    ERIC Educational Resources Information Center

    Sodolski, John

    Broad bandwidth transmission systems have been around for years. They include microwave, assorted cable systems, and recently, satellites. With the exception of some privately owned systems, broadband services have been furnished by the common carriers. Recently, a new element has been added--Cable Antenna Television (CATV) distribution systems.…

  15. Rhizobium sp. strain BN4 (a selenium oxyanion-reducing bacterium) 16S rRNA gene complete sequence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used 1482 base pair 16S rRNA gene sequence methods in conjunction with other biochemical and morphological studies to confirm the identification of a bacterium (refer to as the BN4 strain) as a Rhizobium sp. The 16S rRNA gene sequence places it with the Rhizobium clade that includes R. d...

  16. PHYLOGENETIC TREE OF 16S RIBOSOMAL RNA SEQUENCES FROM SULFATE-REDUCING BACTERIA IN A SANDY MARINE ENVIRONMENT

    EPA Science Inventory

    Phylogenetic divergence among sulfate-reducing bacteria in an estuarine sediment sample was investigated by PCR amplification and comparison of partial 16S rDNA sequences. wenty unique 16S RDNA sequences were found, 12 from delta subclass bacteria based on overall sequence simila...

  17. Analysis, Optimization and Verification of Illumina-Generated 16S rRNA Gene Amplicon Surveys

    PubMed Central

    Nelson, Michael C.; Morrison, Hilary G.; Benjamino, Jacquelynn; Grim, Sharon L.; Graf, Joerg

    2014-01-01

    The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity. PMID:24722003

  18. Greengenes, a Chimera-checked 16S rRNA gene database and workbenchcompatible with ARB

    SciTech Connect

    DeSantis, Todd Z.; Hugenholtz, Philip; Larsen, Neils; Rojas,Mark; Brodie, Eoin L.; Keller, Keith; Huber, Thomas; Dalevi, Daniel; Hu,Ping; Andersen, Gary L.

    2006-04-10

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that in congruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3 percent of environmental sequences and 0.2 percent of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  19. Improved performance of the PacBio SMRT technology for 16S rDNA sequencing.

    PubMed

    Mosher, Jennifer J; Bowman, Brett; Bernberg, Erin L; Shevchenko, Olga; Kan, Jinjun; Korlach, Jonas; Kaplan, Louis A

    2014-09-01

    Improved sequencing accuracy was obtained with 16S amplicons from environmental samples and a known pure culture when upgraded Pacific Biosciences (PacBio) hardware and enzymes were used for the single molecule, real-time (SMRT) sequencing platform. The new PacBio RS II system with P4/C2 chemistry, when used with previously constructed libraries (Mosher et al., 2013) surpassed the accuracy of Roche/454 pyrosequencing platform. With accurate read lengths of >1400 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in environmental samples. PMID:24978594

  20. Circular code motifs in transfer and 16S ribosomal RNAs: a possible translation code in genes.

    PubMed

    Michel, Christian J

    2012-04-01

    In 1996, a common trinucleotide circular code, called X, is identified in genes of eukaryotes and prokaryotes (Arquès and Michel, 1996). This circular code X is a set of 20 trinucleotides allowing the reading frames in genes to be retrieved locally, i.e. anywhere in genes and in particular without start codons. This reading frame retrieval needs a window length l of 12 nucleotides (l ≥ 12). With a window length strictly less than 12 nucleotides (l < 12), some words of X, called ambiguous words, are found in the shifted frames (the reading frame shifted by one or two nucleotides) preventing the reading frame in genes to be retrieved. Since 1996, these ambiguous words of X were never studied. In the first part of this paper, we identify all the ambiguous words of the common trinucleotide circular code X. With a length l varying from 1 to 11 nucleotides, the type and the occurrence number (multiplicity) of ambiguous words of X are given in each shifted frame. Maximal ambiguous words of X, words which are not factors of another ambiguous words, are also determined. Two probability definitions based on these results show that the common trinucleotide circular code X retrieves the reading frame in genes with a probability of about 90% with a window length of 6 nucleotides, and a probability of 99.9% with a window length of 9 nucleotides (100% with a window length of 12 nucleotides, by definition of a circular code). In the second part of this paper, we identify X circular code motifs (shortly X motifs) in transfer RNA and 16S ribosomal RNA: a tRNA X motif of 26 nucleotides including the anticodon stem-loop and seven 16S rRNA X motifs of length greater or equal to 15 nucleotides. Window lengths of reading frame retrieval with each trinucleotide of these X motifs are also determined. Thanks to the crystal structure 3I8G (Jenner et al., 2010), a 3D visualization of X motifs in the ribosome shows several spatial configurations involving mRNA X motifs, A-tRNA and E-tRNA X

  1. Mutation analysis of 16S rRNA in patients with Rett syndrome.

    PubMed

    Armstrong, J; Pineda, M; Monrós, E

    2000-07-01

    Rett syndrome (RTT) is a progressive neurodevelopmental disorder that affects one in 10,000-15,000 females. RTT is mainly sporadic; familial cases have an estimated frequency of less than 1%. Before the recent identification of de novo dominant mutations in the X-linked MECP2 gene, many other hypotheses had been proposed to explain the particular pattern of inheritance and the phenotypic expression of the disease. The involvement of mitochondrial DNA had been investigated because of the structural and functional mitochondrial abnormalities evident in the patients. In 1997 the finding of mutations at 16S rRNA in several affected RTT females and their mothers was reported, suggesting that mitochondrial DNA might play a key role in the pathogenesis of RTT. To investigate the relevance of such mutations, we used the same methodologic approach to analyze RTT mitochondrial DNA in our series. No 16S rRNA alterations were evident in 27 Spanish patients with classic RTT. PMID:10963979

  2. Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics.

    PubMed

    Jovel, Juan; Patterson, Jordan; Wang, Weiwei; Hotte, Naomi; O'Keefe, Sandra; Mitchel, Troy; Perry, Troy; Kao, Dina; Mason, Andrew L; Madsen, Karen L; Wong, Gane K-S

    2016-01-01

    The advent of next generation sequencing (NGS) has enabled investigations of the gut microbiome with unprecedented resolution and throughput. This has stimulated the development of sophisticated bioinformatics tools to analyze the massive amounts of data generated. Researchers therefore need a clear understanding of the key concepts required for the design, execution and interpretation of NGS experiments on microbiomes. We conducted a literature review and used our own data to determine which approaches work best. The two main approaches for analyzing the microbiome, 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics, are illustrated with analyses of libraries designed to highlight their strengths and weaknesses. Several methods for taxonomic classification of bacterial sequences are discussed. We present simulations to assess the number of sequences that are required to perform reliable appraisals of bacterial community structure. To the extent that fluctuations in the diversity of gut bacterial populations correlate with health and disease, we emphasize various techniques for the analysis of bacterial communities within samples (α-diversity) and between samples (β-diversity). Finally, we demonstrate techniques to infer the metabolic capabilities of a bacteria community from these 16S and shotgun data. PMID:27148170

  3. An unusual case of Streptococcus anginosus group pyomyositis diagnosed using direct 16S ribosomal DNA sequencing

    PubMed Central

    Walkty, Andrew; Embil, John M; Nichol, Kim; Karlowsky, James

    2014-01-01

    Bacteria belonging to the Streptococcus anginosus group (Streptococcus intermedius, Streptococcus constellatus and Streptococcus anginosus) are capable of causing serious pyogenic infections, with a tendency for abscess formation. The present article reports a case of S anginosus group pyomyositis in a 47-year-old man. The pathogen was recovered from one of two blood cultures obtained from the patient, but speciation was initially not performed because the organism was considered to be a contaminant (viridans streptococci group). The diagnosis was ultimately confirmed using 16S ribosomal DNA sequencing of purulent fluid obtained from a muscle abscess aspirate. The present case serves to emphasize that finding even a single positive blood culture of an organism belonging to the S anginosus group should prompt careful evaluation of the patient for a pyogenic focus of infection. It also highlights the potential utility of 16S ribosomal DNA amplification and sequencing in direct pathogen detection from aspirated fluid in cases of pyomyositis in which antimicrobial therapy was initiated before specimen collection. PMID:24634686

  4. Molecular phylogeny of western Atlantic Farfantepenaeus and Litopenaeus shrimp based on mitochondrial 16S partial sequences.

    PubMed

    Maggioni, R; Rogers, A D; Maclean, N; D'Incao, F

    2001-01-01

    Partial sequences for the 16S rRNA mitochondrial gene were obtained from 10 penaeid shrimp species: Farfantepenaeus paulensis, F. brasiliensis, F. subtilis, F. duorarum, F. aztecus, Litopenaeus schmitti, L. setiferus, and Xiphopenaeus kroyeri from the western Atlantic and L. vannamei and L. stylirostris from the eastern Pacific. Sequences were also obtained from an undescribed morphotype of pink shrimp (morphotype II) usually identified as F. subtilis. The phylogeny resulting from the 16S partial sequences showed that these species form two well-supported monophyletic clades consistent with the two genera proposed in a recent systematic review of the suborder Dendrobranchiata. This contrasted with conclusions drawn from recent molecular phylogenetic work on penaeid shrimps based on partial sequences of the mitochondrial COI region that failed to support recent revisions of the Dendrobranchiata based on morphological analysis. Consistent differences observed in the sequences for morphotype II, coupled with previous allozyme data, support the conclusion that this is a previously undescribed species of Farfantepenaeus. PMID:11161743

  5. Greengenes: 16S rRNA Database and Workbench Compatible with ARB

    DOE Data Explorer

    DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E. L.; Keller, K.; Huber, T.; Dalevi, D. Hu, P. Andersen, G. L.

    Greengenes was developed, as the abstract of an AEM reprint states, to "addresse limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria....Greengenes is also a functional workbench to assist in analysis of user-generated 16S rRNA gene sequences. Batches of sequencing reads can be uploaded for quality-based trimming and creation of multiple-sequence alignments (9). Three types of non-MSA similarity searches are also available, seed extension by BLAST (1), similarity based on shared 7-mers by a tool called Simrank, and a direct degenerative pattern match for probe/primer evaluation. Results are displayed using user-preferred taxonomic nomenclature and can be saved between sessions. [Taken from DeSantis, T. Z., P. Hugenholtz, N. Larsen, M. Rojas, E. L. Brodie, K. Keller, T. Huber, D. Dalevi, P. Hu, and G. L. Andersen. 2006. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl Environ Microbiol 72:5069-72, pages 1 and 3] (Specialized Interface)

  6. The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation▿ †

    PubMed Central

    Lauro, Federico M.; Chastain, Roger A.; Blankenship, Lesley E.; Yayanos, A. Aristides; Bartlett, Douglas H.

    2007-01-01

    In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few γ-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions. PMID:17158629

  7. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences

    PubMed Central

    Langille, Morgan G. I.; Zaneveld, Jesse; Caporaso, J. Gregory; McDonald, Daniel; Knights, Dan; Reyes, Joshua A.; Clemente, Jose C.; Burkepile, Deron E.; Vega Thurber, Rebecca L.; Knight, Rob; Beiko, Robert G.; Huttenhower, Curtis

    2013-01-01

    Profiling phylogenetic marker genes, such as the 16S rRNA gene, is a key tool for studies of microbial communities but does not provide direct evidence of a community’s functional capabilities. Here we describe PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Our results demonstrate that phylogeny and function are sufficiently linked that this ‘predictive metagenomic’ approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available. PMID:23975157

  8. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

    PubMed Central

    Calvo-Bado, Leo A; Oakley, Brian B; Dowd, Scot E; Green, Laura E; Medley, Graham F; Ul-Hassan, Atiya; Bateman, Vicky; Gaze, William; Witcomb, Luci; Grogono-Thomas, Rose; Kaler, Jasmeet; Russell, Claire L; Wellington, Elizabeth MH

    2011-01-01

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (104–109 cells per g tissue) than those with H or VFR feet. PMID:21430786

  9. IM-TORNADO: A Tool for Comparison of 16S Reads from Paired-End Libraries

    PubMed Central

    Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

    2014-01-01

    Motivation 16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. Results We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. Availability and Implementation IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq. PMID:25506826

  10. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing.

    PubMed

    Naveed, Muhammad; Mubeen, Samavia; Khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization. PMID:25477935

  11. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

    PubMed Central

    Naveed, Muhammad; Mubeen, Samavia; khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization. PMID:25477935

  12. Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics

    PubMed Central

    Jovel, Juan; Patterson, Jordan; Wang, Weiwei; Hotte, Naomi; O'Keefe, Sandra; Mitchel, Troy; Perry, Troy; Kao, Dina; Mason, Andrew L.; Madsen, Karen L.; Wong, Gane K.-S.

    2016-01-01

    The advent of next generation sequencing (NGS) has enabled investigations of the gut microbiome with unprecedented resolution and throughput. This has stimulated the development of sophisticated bioinformatics tools to analyze the massive amounts of data generated. Researchers therefore need a clear understanding of the key concepts required for the design, execution and interpretation of NGS experiments on microbiomes. We conducted a literature review and used our own data to determine which approaches work best. The two main approaches for analyzing the microbiome, 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics, are illustrated with analyses of libraries designed to highlight their strengths and weaknesses. Several methods for taxonomic classification of bacterial sequences are discussed. We present simulations to assess the number of sequences that are required to perform reliable appraisals of bacterial community structure. To the extent that fluctuations in the diversity of gut bacterial populations correlate with health and disease, we emphasize various techniques for the analysis of bacterial communities within samples (α-diversity) and between samples (β-diversity). Finally, we demonstrate techniques to infer the metabolic capabilities of a bacteria community from these 16S and shotgun data. PMID:27148170

  13. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome.

    PubMed

    Calvo-Bado, Leo A; Oakley, Brian B; Dowd, Scot E; Green, Laura E; Medley, Graham F; Ul-Hassan, Atiya; Bateman, Vicky; Gaze, William; Witcomb, Luci; Grogono-Thomas, Rose; Kaler, Jasmeet; Russell, Claire L; Wellington, Elizabeth M H

    2011-09-01

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet. PMID:21430786

  14. Phylogenetic relationships between Bacillus species and related genera inferred from 16s rDNA sequences

    PubMed Central

    Wei Wang, Mi Sun

    2009-01-01

    Neighbor-joining, maximum-parsimony, minimum-evolution, maximum-likelihood and Bayesian trees constructed based on 16S rDNA sequences of 181 type strains of Bacillus species and related taxa manifested nine phylogenetic groups. The phylogenetic analysis showed that Bacillus was not a monophyletic group. B. subtilis was in Group 1. Group 4, 6 and 8 respectively consisted of thermophiles, halophilic or halotolerant bacilli and alkaliphilic bacilli. Group 2, 4 and 8 consisting of Bacillus species and related genera demonstrated that the current taxonomic system did not agree well with the 16S rDNA evolutionary trees. The position of Caryophanaceae and Planococcaceae in Group 2 suggested that they might be transferred into Bacillaceae, and the heterogeneity of Group 2 implied that some Bacillus species in it might belong to several new genera. Group 9 was mainly comprised of the genera (excluding Bacillus) of Bacillaceae, so some Bacillus species in Group 9: B. salarius, B. qingdaonensis and B. thermcloacae might not belong to Bacillus. Four Bacillus species, B. schlegelii, B. tusciae, B. edaphicus and B. mucilaginosus were clearly placed outside the nine groups. PMID:24031394

  15. Obtaining long 16S rDNA sequences using multiple primers and its application on dioxin-containing samples

    PubMed Central

    2015-01-01

    Background Next-generation sequencing (NGS) technology has transformed metagenomics because the high-throughput data allow an in-depth exploration of a complex microbial community. However, accurate species identification with NGS data is challenging because NGS sequences are relatively short. Assembling 16S rDNA segments into longer sequences has been proposed for improving species identification. Current approaches, however, either suffer from amplification bias due to one single primer or insufficient 16S rDNA reads in whole genome sequencing data. Results Multiple primers were used to amplify different 16S rDNA segments for 454 sequencing, followed by 454 read classification and assembly. This permitted targeted sequencing while reducing primer bias. For test samples containing four known bacteria, accurate and near full-length 16S rDNAs of three known bacteria were obtained. For real soil and sediment samples containing dioxins in various concentrations, 16S rDNA sequences were lengthened by 50% for about half of the non-rare microbes, and 16S rDNAs of several microbes reached more than 1000 bp. In addition, reduced primer bias using multiple primers was illustrated. Conclusions A new experimental and computational pipeline for obtaining long 16S rDNA sequences was proposed. The capability of the pipeline was validated on test samples and illustrated on real samples. For dioxin-containing samples, the pipeline revealed several microbes suitable for future studies of dioxin chemistry. PMID:26681335

  16. Broad band waveguide spectrometer

    DOEpatents

    Goldman, Don S.

    1995-01-01

    A spectrometer for analyzing a sample of material utilizing a broad band source of electromagnetic radiation and a detector. The spectrometer employs a waveguide possessing an entry and an exit for the electromagnetic radiation emanating from the source. The waveguide further includes a surface between the entry and exit portions which permits interaction between the electromagnetic radiation passing through the wave guide and a sample material. A tapered portion forms a part of the entry of the wave guide and couples the electromagnetic radiation emanating from the source to the waveguide. The electromagnetic radiation passing from the exit of the waveguide is captured and directed to a detector for analysis.

  17. PCR primers to amplify 16S rRNA genes from cyanobacteria.

    PubMed Central

    Nübel, U; Garcia-Pichel, F; Muyzer, G

    1997-01-01

    We developed and tested a set of oligonucleotide primers for the specific amplification of 16S rRNA gene segments from cyanobacteria and plastids by PCR. PCR products were recovered from all cultures of cyanobacteria and diatoms that were checked but not from other bacteria and archaea. Gene segments selectively retrieved from cyanobacteria and diatoms in unialgal but nonaxenic cultures and from cyanobionts in lichens could be directly sequenced. In the context of growing sequence databases, this procedure allows rapid and phylogenetically meaningful identification without pure cultures or molecular cloning. We demonstrate the use of this specific PCR in combination with denaturing gradient gel electrophoresis to probe the diversity of oxygenic phototrophic microorganisms in cultures, lichens, and complex microbial communities. PMID:9251225

  18. Use of 16S Ribosomal DNA for Delineation of Marine Bacterioplankton Species

    PubMed Central

    Hagström, Åke; Pommier, Thomas; Rohwer, Forest; Simu, Karin; Stolte, Willem; Svensson, Dominika; Zweifel, Ulla Li

    2002-01-01

    All of the marine bacterioplankton-derived 16S ribosomal DNA sequences previously deposited in GenBank were reanalyzed to determine the number of bacterial species in the oceanic surface waters. These sequences have been entered into the database since 1990. The rate of new additions reached a peak in 1999 and subsequently leveled off, suggesting that much of the marine microbial species richness has been sampled. When the GenBank sequences were dereplicated by using 97% similarity as a cutoff, 1,117 unique ribotypes were found. Of the unique sequences, 609 came from uncultured environmental clones and 508 came from cultured bacteria. We conclude that the apparent bacterioplankton species richness is relatively low. PMID:12089052

  19. Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences.

    PubMed Central

    Hiorns, W D; Methé, B A; Nierzwicki-Bauer, S A; Zehr, J P

    1997-01-01

    Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA. Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%). Additionally, a number of the sequences were similar to those of the order Verrucomicrobiales. However, few of the sequence types are closely related to those of characterized species. The relative contributions of the groups of sequences differed among the lakes, suggesting that bacterial population structure varies and that it may be possible to relate aquatic bacterial community structure to water chemistry. PMID:9212443

  20. Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

    NASA Astrophysics Data System (ADS)

    You, Feng; Liu, Jing; Zhang, Peijun; Xiang, Jianhai

    2005-09-01

    A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

  1. 16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae.

    PubMed

    Davis, Daniel J; Bryda, Elizabeth C; Gillespie, Catherine H; Ericsson, Aaron C

    2016-09-01

    Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these "conventionalized" larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled "Microbial modulation of behavior and stress responses in zebrafish larvae" (Davis et al., 2016) [1]. PMID:27508247

  2. [Analysis of DNA homology and 16S rDNA sequence of rhizobia, a new phenotypic subgroup, isolated from Xizang Autonomous Region of China].

    PubMed

    Wang, Su-ying; Yang, Xiao-li; Li, Hai-feng; Liu, Jie

    2006-02-01

    Based on the studies of numerical taxonomy, the seven rhizobial strains isolated from the root nodules of leguminous plants Trigonella spp. and Astragalus spp. growing in the Xizang Autonomous Region of China constituted a new phenotypic subgroup, where wide phenotypic and genotypic diversity among legume crops had been reported due to complex terrain and various climate. The new phenotypic subgroup were further identified to clarify its taxonomic position by DNA homology analysis and 16S rDNA gene sequencing. The mol% G + C ratio of the DNA among members of the new subgroup ranged from 59.5 to 63.3 mol% as determined by T (m) assay. The levels of DNA relatedness, determined by using the DNA liquid hybridization method, among the members of the new subgroup were between 74.3% and 92.3%, while level of DNA relatedness between the central strains XZ2-3 of the new subgroup and the type strains of known species of Rhizobium was less than 47.4%. These results indicated that the new phenotypic subgroup is a DNA homological group different from described species of Rhizobium. Therefore, this new phenotypic subgroup was supposed to be a new species in the genus of Rhizobium since the strains in the same species generally exhibit levels of DNA homology ranging from 70 to 100%. A systematic identification method-16S rDNA gene sequence comparison was carried out to determine the phylogenetic relationships of the new subgroup with the described species of Rhizobium. The GenBank accession number for the 16S rDNA sequence of the central strain XZ2-3 of the new subgroup is DQ099745. The full-length 16S rDNA gene sequence were sequenced by chain terminator techniques and analyzed with PHYLIP. The phylogenetic trees were constructed by using the programs DRAWTREE. The phylogenetic analysis indicated that new subgroup occupy a independent sub-branch in phylogenetic tree. The sequence similarities between the center strain XZ2-3 and the closest relatives, strain R. leguminosarum USDA

  3. Multi-site-specific 16S rRNA Methyltransferase RsmF from Thermus thermophilus

    SciTech Connect

    Demirci, H.; Larsen, L; Hansen, T; Rasmussen, A; Cadambi, A; Gregory, S; Kirpekar, F; Jogl, G

    2010-01-01

    Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m{sup 5}C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m{sup 5}C967. In contrast to E. coli RsmF, which introduces a single m{sup 5}C1407 modification, T. thermophilus RsmF modifies three positions, generating m{sup 5}C1400 and m{sup 5}C1404 in addition to m{sup 5}C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 {angstrom} resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.

  4. Identification of the Microbiota in Carious Dentin Lesions Using 16S rRNA Gene Sequencing

    PubMed Central

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4–76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating. PMID:25083880

  5. Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence.

    PubMed

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100% among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2-24.0%, 24.0-24.9%, and 22.9-23.2%, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai. PMID:23996126

  6. Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria.

    PubMed

    He, Zhanfei; Wang, Jiaqi; Hu, Jiajie; Zhang, Hao; Cai, Chaoyang; Shen, Jiaxian; Xu, Xinhua; Zheng, Ping; Hu, Baolan

    2016-06-01

    Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3) copies g(-1) dry sed. for the environmental samples compared to approximately 10(5) copies g(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems. PMID:27020287

  7. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing.

    PubMed

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating. PMID:25083880

  8. Bacterial Community Composition of South China Sea Sediments through Pyrosequencing-Based Analysis of 16S rRNA Genes

    PubMed Central

    Zhu, Daochen; Tanabe, Shoko-Hosoi; Yang, Chong; Zhang, Weimin; Sun, Jianzhong

    2013-01-01

    Background Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. Methodology/Principal Findings Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample) at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. Conclusions This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m. PMID:24205246

  9. High-resolution microbial community reconstruction by integrating short reads from multiple 16S rRNA regions.

    PubMed

    Amir, Amnon; Zeisel, Amit; Zuk, Or; Elgart, Michael; Stern, Shay; Shamir, Ohad; Turnbaugh, Peter J; Soen, Yoav; Shental, Noam

    2013-12-01

    The emergence of massively parallel sequencing technology has revolutionized microbial profiling, allowing the unprecedented comparison of microbial diversity across time and space in a wide range of host-associated and environmental ecosystems. Although the high-throughput nature of such methods enables the detection of low-frequency bacteria, these advances come at the cost of sequencing read length, limiting the phylogenetic resolution possible by current methods. Here, we present a generic approach for integrating short reads from large genomic regions, thus enabling phylogenetic resolution far exceeding current methods. The approach is based on a mapping to a statistical model that is later solved as a constrained optimization problem. We demonstrate the utility of this method by analyzing human saliva and Drosophila samples, using Illumina single-end sequencing of a 750 bp amplicon of the 16S rRNA gene. Phylogenetic resolution is significantly extended while reducing the number of falsely detected bacteria, as compared with standard single-region Roche 454 Pyrosequencing. Our approach can be seamlessly applied to simultaneous sequencing of multiple genes providing a higher resolution view of the composition and activity of complex microbial communities. PMID:24214960

  10. High-resolution microbial community reconstruction by integrating short reads from multiple 16S rRNA regions

    PubMed Central

    Amir, Amnon; Zeisel, Amit; Zuk, Or; Elgart, Michael; Stern, Shay; Shamir, Ohad; Turnbaugh, Peter J.; Soen, Yoav; Shental, Noam

    2013-01-01

    The emergence of massively parallel sequencing technology has revolutionized microbial profiling, allowing the unprecedented comparison of microbial diversity across time and space in a wide range of host-associated and environmental ecosystems. Although the high-throughput nature of such methods enables the detection of low-frequency bacteria, these advances come at the cost of sequencing read length, limiting the phylogenetic resolution possible by current methods. Here, we present a generic approach for integrating short reads from large genomic regions, thus enabling phylogenetic resolution far exceeding current methods. The approach is based on a mapping to a statistical model that is later solved as a constrained optimization problem. We demonstrate the utility of this method by analyzing human saliva and Drosophila samples, using Illumina single-end sequencing of a 750 bp amplicon of the 16S rRNA gene. Phylogenetic resolution is significantly extended while reducing the number of falsely detected bacteria, as compared with standard single-region Roche 454 Pyrosequencing. Our approach can be seamlessly applied to simultaneous sequencing of multiple genes providing a higher resolution view of the composition and activity of complex microbial communities. PMID:24214960

  11. Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

    PubMed

    Pandya, P R; Singh, K M; Parnerkar, S; Tripathi, A K; Mehta, H H; Rank, D N; Kothari, R K; Joshi, C G

    2010-01-01

    Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85-89% similar to 16S rDNA database sequences. For the remaining 3.14%; the similarity was lower than 85% Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of the Cytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good's coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen. PMID:20720314

  12. Updated 16S rRNA-RFLP method for the identification of all currently characterised Arcobacter spp

    PubMed Central

    2012-01-01

    Background Arcobacter spp. (family Campylobacteraceae) are ubiquitous zoonotic bacteria that are being increasingly recognised as a threat to human health. A previously published 16S rRNA-RFLP Arcobacter spp. identification method produced specific RFLP patterns for the six species described at that time, using a single endonuclease (MseI). The number of characterised Arcobacter species has since risen to 17. The aim of the present study was to update the 16S rRNA-RFLP identification method to include all currently characterised species of Arcobacter. Results Digestion of the 16S rRNA gene with the endonuclease MseI produced clear, distinctive patterns for 10 of the 17 species, while the remaining species shared a common or very similar RFLP pattern. Subsequent digestion of the 16S rRNA gene from these species with the endonucleases MnlI and/or BfaI generated species-specific RFLP patterns. Conclusions 16S rRNA-RFLP analysis identified 17 Arcobacter spp. using either polyacrylamide or agarose gel electrophoresis. Microheterogeneities within the 16S rRNA gene, which interfered with the RFLP identification, were also documented for the first time in this genus, particularly in strains of Arcobacter cryaerophilus isolated from animal faeces and aborted foetuses. PMID:23244705

  13. Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

    PubMed

    Soni, Dharmendra Kumar; Dubey, Suresh Kumar

    2014-12-01

    The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity. PMID:25205124

  14. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    PubMed

    Olson, Nathan D; Lund, Steven P; Zook, Justin M; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B

    2015-03-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

  15. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

  16. Phylogenetic relationship of phosphate solubilizing bacteria according to 16S rRNA genes.

    PubMed

    Javadi Nobandegani, Mohammad Bagher; Saud, Halimi Mohd; Yun, Wong Mui

    2015-01-01

    Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field. PMID:25632387

  17. Primer and platform effects on 16S rRNA tag sequencing

    SciTech Connect

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; Kirton, Edward S.; He, Shaomei; Woyke, Tanja; Lee, Janey; Chen, Feng; Dangl, Jeffery L.; Tringe, Susannah G.

    2015-08-04

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.

  18. Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing

    NASA Technical Reports Server (NTRS)

    Shi, T.; Reeves, R. H.; Gilichinsky, D. A.; Friedmann, E. I.

    1997-01-01

    Viable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is -10 degrees C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30 degrees C. The majority of the bacterial isolates were rod shaped and grew well at 30 degrees C; but two of them did not grow at or above 28 degrees C, and had optimum growth temperatures around 20 degrees C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, beta-proteobacteria, gamma-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and beta-proteobacteria, and all gamma-proteobacteria, came from samples with an estimated age of 1.8-3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000-8,000 years (Alas suite).

  19. Primer and platform effects on 16S rRNA tag sequencing

    PubMed Central

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; Kirton, Edward S.; He, Shaomei; Woyke, Tanja; Lee, Janey; Chen, Feng; Dangl, Jeffery L.; Tringe, Susannah G.

    2015-01-01

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. Beta diversity metrics are surprisingly robust to both primer and sequencing platform biases. PMID:26300854

  20. 16S rRNA gene mutations associated with decreased susceptibility to tetracycline in Mycoplasma bovis.

    PubMed

    Amram, E; Mikula, I; Schnee, C; Ayling, R D; Nicholas, R A J; Rosales, R S; Harrus, S; Lysnyansky, I

    2015-02-01

    Mycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 μg/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, ≥2 μg/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the two rrs alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis isolates. The data presented correlate (P<0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline. PMID:25403668

  1. Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes

    PubMed Central

    Javadi Nobandegani, Mohammad Bagher; Saud, Halimi Mohd; Yun, Wong Mui

    2015-01-01

    Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field. PMID:25632387

  2. Concurrent Nucleation of 16S Folding and Induced Fit in 30S Ribosome Assembly

    SciTech Connect

    Adilakshmi, T.; Bellur, D; Woodson, S

    2008-01-01

    Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting3 was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.

  3. Functional Specialization of Domains Tandemly Duplicated Witin 16S rRNA Methyltransferase RsmC

    SciTech Connect

    Sunita,S.; Purta, E.; Durawa, M.; Tkaczuk, K.; Swaathi, J.; Bujnicki, J.; Sivaraman, J.

    2007-01-01

    RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 Angstroms resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.

  4. Algae-bacteria association inferred by 16S rDNA similarity in established microalgae cultures.

    PubMed

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-06-01

    Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae-associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga-Flavobacterium-Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities. PMID:24799387

  5. 16S community profiling identifies proton pump inhibitor related differences in gastric, lung and oropharyngeal microflora

    PubMed Central

    Rosen, Rachel; Hu, Lan; Amirault, Janine; Khatwa, Umakanth; Ward, Doyle V.; Onderdonk, Andrew

    2015-01-01

    Objectives To test the hypothesis that PPI use results in changes in gastric microflora which, through full column reflux, results in lung and oropharyngeal microflora changes. Study design We performed a prospective, cross sectional cohort study of 116 children (57 off and 59 on PPIs) undergoing simultaneous bronchoscopy and upper endoscopy for the evaluation of chronic cough. We performed 16S sequencing on gastric, bronchoalveolar lavage and oropharyngeal fluid. Fifty patients also underwent multichannel intraluminal impedance (pH-MII) testing. Results Streptococcus was more abundant in the gastric fluid of patients taking proton pump inhibitors (PPIs) and there was a significant correlation with PPI dose (mg/kg/day) and abundance of gastric Streptococcus (p=0.01). There was also a significant difference in the abundance of oropharyngeal Streptococcus in PPI treated patients. Eight unique bacterial genera were found in the gastric and lung fluid but not in the oropharyngeal suggesting exchange between the two sites and two of the seven (Lactococcus, Acinetobacter) were more abundant in patients with more full column reflux, suggesting direct aspiration. Principal component analysis revealed greater overlap between gastric and lung than oropharyngeal microflora. Conclusions PPI use was associated with differences in gastric, lung and oropharyngeal microflora. Although microflora exchange can occur between all three sites, gastric and lung microflora are more closely related and the mechanism of exchange between sites may be aspiration of full column reflux. PMID:25661411

  6. [Determination of 16S rRNA gene sequence for a new ANAMMOX bacterial species].

    PubMed

    Zu, Bo; Zhang, Dai-jun; Yan, Qing

    2008-02-01

    The anaerobic ammonium oxidation (ANAMMOX) activity of the sludge was about 9.84 x 10(-4) mg x (mg x h)(-1) by measuring the simultaneous consumption of ammonium and nitrite under anoxic conditions in the batch tests. The consumption of NO2(-) -N and NH4+ -N was 1.311 for ANAMMOX bacteria. The partial 16S rDNA sequence was obtained by using molecule biology methods. Crude DNA of the total bacteria in granular sludge from EGSB reactor was extracted and purified. Then, PCR amplification by using specific primer, clone and sequence determination was performed. ANAMMOX bacterial species(anaerobic ammonium-oxidizing Planctomycete cquenviron-1) which was enrichment cultivated from EGSB reactor were the same genera with Candidatus "Anammoxoglobus propionicus" and Candidatus "Jettenia asiatica" by analyzing phylogenetic tree. The maximum identities of anaerobic ammonium-oxidizing Planctomycete cquenviron-1 with other ANAMMOX bacterial species was about 93%. The results showed that a new ANAMMOX bacterial species which was enrichment cultivated from EGSB reactor was found and anaerobic ammonium-oxidizing Planctomycete cquenviron-1 was denominated. PMID:18613522

  7. 16S rRNA Gene Mutations Associated with Decreased Susceptibility to Tetracycline in Mycoplasma bovis

    PubMed Central

    Amram, E.; Mikula, I.; Schnee, C.; Ayling, R. D.; Nicholas, R. A. J.; Rosales, R. S.; Harrus, S.

    2014-01-01

    Mycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 μg/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, ≥2 μg/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the two rrs alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis isolates. The data presented correlate (P < 0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline. PMID:25403668

  8. Algae–bacteria association inferred by 16S rDNA similarity in established microalgae cultures

    PubMed Central

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-01-01

    Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae-associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga–Flavobacterium–Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities. PMID:24799387

  9. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences

    PubMed Central

    Yadav, Shailendra; Kundu, Sharbadeb; Ghosh, Sankar K.; Maitra, S. S.

    2015-01-01

    Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process. PMID:26568700

  10. Primer and platform effects on 16S rRNA tag sequencing

    DOE PAGESBeta

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; Kirton, Edward S.; He, Shaomei; Woyke, Tanja; Lee, Janey; Chen, Feng; Dangl, Jeffery L.; Tringe, Susannah G.

    2015-08-04

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as wellmore » as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.« less

  11. 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates

    PubMed Central

    Drancourt, Michel; Bollet, Claude; Carlioz, Antoine; Martelin, Rolland; Gayral, Jean-Pierre; Raoult, Didier

    2000-01-01

    Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides

  12. Optical fiber voltage sensors for broad temperature ranges

    NASA Technical Reports Server (NTRS)

    Rose, A. H.; Day, G. W.

    1992-01-01

    We describe the development of an optical fiber ac voltage sensor for aircraft and spacecraft applications. Among the most difficult specifications to meet for this application is a temperature stability of +/- 1 percent from -65 C to +125 C. This stability requires a careful selection of materials, components, and optical configuration with further compensation using an optical-fiber temperature sensor located near the sensing element. The sensor is a polarimetric design, based on the linear electro-optic effect in bulk Bi4Ge3O12. The temperature sensor is also polarimetric, based on the temperature dependence of the birefringence of bulk SiO2. The temperature sensor output is used to automatically adjust the calibration of the instrument.

  13. Space Weather Enterprise Forum Includes Broad Range of Discussion

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2012-08-01

    With the 2013 solar maximum nearing, researchers and government agencies are focusing on how the increased solar activity could affect our increasingly technological society and what measures can be taken to help prevent or mitigate any threats to the electricity grid, GPS, and other potentially vulnerable technological soft spots.

  14. Optical fiber voltage sensors for broad temperature ranges

    NASA Astrophysics Data System (ADS)

    Rose, A. H.; Day, G. W.

    1992-12-01

    We describe the development of an optical fiber ac voltage sensor for aircraft and spacecraft applications. Among the most difficult specifications to meet for this application is a temperature stability of +/- 1 percent from -65 C to +125 C. This stability requires a careful selection of materials, components, and optical configuration with further compensation using an optical-fiber temperature sensor located near the sensing element. The sensor is a polarimetric design, based on the linear electro-optic effect in bulk Bi4Ge3O12. The temperature sensor is also polarimetric, based on the temperature dependence of the birefringence of bulk SiO2. The temperature sensor output is used to automatically adjust the calibration of the instrument.

  15. Genomic Analysis of Broad-Host-Range Enterobacteriophage Av-05

    PubMed Central

    Amarillas, Luis; López-Cuevas, Osvaldo; León-Félix, Josefina; Castro-del Campo, Nohelia; Gerba, Charles P.

    2015-01-01

    Lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phage relies on appropriate genomic characterization. In this study, we report the genome of bacteriophage Av-05 and its sequence analysis, which has strong lytic activity against Escherichia coli O157:H7 strains and several Salmonella serotypes. The analysis revealed that the phage Av-05 genome consists of 120,938 bp, containing 209 putative open reading frames (ORFs) and 9 tRNAs. PMID:26067947

  16. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

    PubMed Central

    Haas, Brian J.; Gevers, Dirk; Earl, Ashlee M.; Feldgarden, Mike; Ward, Doyle V.; Giannoukos, Georgia; Ciulla, Dawn; Tabbaa, Diana; Highlander, Sarah K.; Sodergren, Erica; Methé, Barbara; DeSantis, Todd Z.; Petrosino, Joseph F.; Knight, Rob; Birren, Bruce W.

    2011-01-01

    Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys. PMID:21212162

  17. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures.

    PubMed

    Boers, Stefan A; Hays, John P; Jansen, Ruud

    2015-01-01

    16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys). PMID:26373611

  18. [Strategy of selecting 16S rRNA hypervariable regions for metagenome-phylogenetic marker genes based analysis].

    PubMed

    Zhang, Jun-yi; Zhu, Bing-chuan; Xu, Chao; Ding, Xiao; Li, Jun-feng; Zhang, Xue-gong; Lu, Zu-hong

    2015-11-01

    The advent of next generation sequencing technology enables parallel analysis of the whole microbial community from multiple samples. Particularly, sequencing 16S rRNA hypervariable tags has become the most efficient and cost-effective method for assessing microbial diversity. Due to its short read length of the 2nd-generation sequencing methods that cannot cover the full 16S rRNA genomic region, specific hypervariable regions or V-regions must be selected to act as the proxy. Over the past decade, selection of V-regions has not been consistent in assessing microbial diversity. Here we evaluated the current strategies of selecting 16S rRNA hypervariable regions for surveying microbial diversity. The environmental condition was considered as one of the important factors for selection of 16S rRNA hypervariable regions. We suggested that a pilot study to test different V-regions is required in bacterial diversity studies based on 16S rRNA genes. PMID:26915214

  19. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes.

    PubMed

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-06-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  20. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice.

    PubMed

    Allen, Julie M; Burleigh, J Gordon; Light, Jessica E; Reed, David L

    2016-01-01

    Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

  1. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

    PubMed Central

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-01-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  2. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

    PubMed Central

    Burleigh, J. Gordon; Light, Jessica E.; Reed, David L.

    2016-01-01

    Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

  3. Specific multiplex analysis of pathogens using a direct 16S rRNA hybridization in microarray system.

    PubMed

    Hwang, Byeong Hee; Shin, Hwa Hui; Seo, Jeong Hyun; Cha, Hyung Joon

    2012-06-01

    For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA-DNA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21-41% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine-cytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10-10(3) cells mL(-1) and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples. PMID:22551354

  4. Broad spectrum solar cell

    DOEpatents

    Walukiewicz, Wladyslaw; Yu, Kin Man; Wu, Junqiao; Schaff, William J.

    2007-05-15

    An alloy having a large band gap range is used in a multijunction solar cell to enhance utilization of the solar energy spectrum. In one embodiment, the alloy is In.sub.1-xGa.sub.xN having an energy bandgap range of approximately 0.7 eV to 3.4 eV, providing a good match to the solar energy spectrum. Multiple junctions having different bandgaps are stacked to form a solar cell. Each junction may have different bandgaps (realized by varying the alloy composition), and therefore be responsive to different parts of the spectrum. The junctions are stacked in such a manner that some bands of light pass through upper junctions to lower junctions that are responsive to such bands.

  5. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

    PubMed Central

    Schmidt, T M; DeLong, E F; Pace, N R

    1991-01-01

    The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms. Images PMID:2066334

  6. Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

    PubMed Central

    2011-01-01

    Background Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods. Results Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations. Conclusions This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality

  7. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    PubMed

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods. PMID:24950754

  8. Vikodak - A Modular Framework for Inferring Functional Potential of Microbial Communities from 16S Metagenomic Datasets

    PubMed Central

    Nagpal, Sunil; Haque, Mohammed Monzoorul; Mande, Sharmila S.

    2016-01-01

    Background The overall metabolic/functional potential of any given environmental niche is a function of the sum total of genes/proteins/enzymes that are encoded and expressed by various interacting microbes residing in that niche. Consequently, prior (collated) information pertaining to genes, enzymes encoded by the resident microbes can aid in indirectly (re)constructing/ inferring the metabolic/ functional potential of a given microbial community (given its taxonomic abundance profile). In this study, we present Vikodak—a multi-modular package that is based on the above assumption and automates inferring and/ or comparing the functional characteristics of an environment using taxonomic abundance generated from one or more environmental sample datasets. With the underlying assumptions of co-metabolism and independent contributions of different microbes in a community, a concerted effort has been made to accommodate microbial co-existence patterns in various modules incorporated in Vikodak. Results Validation experiments on over 1400 metagenomic samples have confirmed the utility of Vikodak in (a) deciphering enzyme abundance profiles of any KEGG metabolic pathway, (b) functional resolution of distinct metagenomic environments, (c) inferring patterns of functional interaction between resident microbes, and (d) automating statistical comparison of functional features of studied microbiomes. Novel features incorporated in Vikodak also facilitate automatic removal of false positives and spurious functional predictions. Conclusions With novel provisions for comprehensive functional analysis, inclusion of microbial co-existence pattern based algorithms, automated inter-environment comparisons; in-depth analysis of individual metabolic pathways and greater flexibilities at the user end, Vikodak is expected to be an important value addition to the family of existing tools for 16S based function prediction. Availability and Implementation A web implementation of Vikodak

  9. Molecular Identification of Nontuberculous Mycobacteria in Humans in Zimbabwe Using 16S Ribosequencing

    PubMed Central

    Chin’ombe, Nyasha; Muzividzi, Boniface; Munemo, Ellen; Nziramasanga, Pasipanodya

    2016-01-01

    Background: Several nontuberculous mycobacteria (NTM) were previously isolated from diverse environments such as water, soil, sewage, food and animals. Some of these NTM are now known to be opportunistic pathogens of humans. Objective: The main purpose of the study was to identify NTM isolates stored at the National Microbiology Reference Laboratory (NMRL) and were previously isolated from humans during a national tuberculosis (TB) survey. Methods: Pure NTM cultures already isolated from human sputum samples during the national TB survey were retrieved from the NMRL and used for this study. DNA was extracted from the samples and 16S ribosomal RNA gene amplified by polymerase chain reaction. The amplicons were sequenced and bioinformatics tools were used to identify the NTM species. Results: Out of total of 963 NTM isolates stored at the NMRL, 81 were retrieved for speciation. Forty isolates (49.4%) were found to belong to Mycobacterium avium-intracellulare complex (MAC) species. The other 41 isolates (50.6%) were identified as M. lentiflavum (6.2%), M. terrae complex (4.9%), M. paraense (4.9%), M. kansasii (3.7%), M. moriokaense (3.7%), M. asiaticum (2.5%), M. novocastrense (2.5%), M. brasiliensis (2.5%), M. elephantis (2.5%), M. paraffinicum (1.2%), M. bohemicum (1.2%), M. manitobense (1.2%), M. intermedium (1.2%), M. tuberculosis complex (1.2%), M. parakoreense (1.2%), M. florentinum (1.2%), M. litorale (1.2%), M. fluoranthenivorans (1.2%), M. sherrisii (1.2%), M. fortuitum (1.2%) and M septicum (1.2%). Two isolates (2.5%) could not be identified, but were closely related to M. montefiorense and M. phlei respectively. Interestingly, the MAC species were the commonest NTM during the survey. Conclusion: The study emphasizes the importance of identifying species of NTM in Zimbabwe. Future studies need to ascertain their true diversity and clinical relevance. PMID:27335623

  10. The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

    PubMed Central

    Wong, Adam C-N; Chaston, John M; Douglas, Angela E

    2013-01-01

    The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans. PMID:23719154

  11. The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis.

    PubMed

    Wong, Adam C-N; Chaston, John M; Douglas, Angela E

    2013-10-01

    The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans. PMID:23719154

  12. 16S rRNA Gene Survey of Microbial Communities in Winogradsky Columns

    PubMed Central

    Rundell, Ethan A.; Banta, Lois M.; Ward, Doyle V.; Watts, Corey D.; Birren, Bruce; Esteban, David J.

    2014-01-01

    A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities. PMID:25101630

  13. [Cloning and sequencing of 16S rRNA gene of Phytoplasma CWB1 strain associated with cactus witches' broom].

    PubMed

    Cai, H; Li, F; Kong, B; Chen, H

    2001-12-01

    A 1.5 kb DNA fragment was amplified in DNA samples extracted from Opuntia salmiana porm showed witches'-broom symptom. The result indicates the existence of phytoplasma associated with this disease and this phytoplasma was designated as CWB1. The amplified fragment was ligated to pGEM-T easy vector and then transformed into JM109 strain of E. coli. Cloned DNA fragments were verified by PCR, restriction endonuclease (EcoRI) digestion and sequence analysis. The result revealed that the 16S rRNA gene of CWB1 consists of 1489 bp and shared 99.7% homology with Faba bean phyllody which belongs to phytoplasma 16S rII-C subgroup. So we can classify this strain into phytoplasma 16S rII-C subgroup. PMID:12552825

  14. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity

    NASA Technical Reports Server (NTRS)

    Fox, G. E.; Wisotzkey, J. D.; Jurtshuk, P. Jr

    1992-01-01

    16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.

  15. Bacterial Community Shift in Treated Periodontitis Patients Revealed by Ion Torrent 16S rRNA Gene Amplicon Sequencing

    PubMed Central

    Jünemann, Sebastian; Prior, Karola; Szczepanowski, Rafael; Harks, Inga; Ehmke, Benjamin; Goesmann, Alexander; Stoye, Jens; Harmsen, Dag

    2012-01-01

    Periodontitis, one of the most common diseases in the world, is caused by a mixture of pathogenic bacteria and inflammatory host responses and often treated by antimicrobials as an adjunct to scaling and root planing (SRP). Our study aims to elucidate explorative and descriptive temporal shifts in bacterial communities between patients treated by SRP alone versus SRP plus antibiotics. This is the first metagenomic study using an Ion Torrent Personal Genome Machine (PGM). Eight subgingival plaque samples from four patients with chronic periodontitis, taken before and two months after intervention were analyzed. Amplicons from the V6 hypervariable region of the 16S rRNA gene were generated and sequenced each on a 314 chip. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance), described by community metrics, and taxonomically classified. Reads ranging from 599,933 to 650,416 per sample were clustered into 1,648 to 2,659 non-singleton OTUs, respectively. Increased diversity (Shannon and Simpson) in all samples after therapy was observed regardless of the treatment type whereas richness (ACE) showed no correlation. Taxonomic analysis revealed different microbial shifts between both therapy approaches at all taxonomic levels. Most remarkably, the genera Porphyromonas, Tannerella, Treponema, and Filifactor all harboring periodontal pathogenic species were removed almost only in the group treated with SPR and antibiotics. For the species T. forsythia and P. gingivalis results were corroborated by real-time PCR analysis. In the future, hypothesis free metagenomic analysis could be the key in understanding polymicrobial diseases and be used for therapy monitoring. Therefore, as read length continues to increase and cost to decrease, rapid benchtop sequencers like the PGM might finally be used in routine diagnostic. PMID:22870235

  16. [Rapid detection of Pseudomonas aeruginosa by the fluorescence quantitative PCR assay targeting 16S rDNA].

    PubMed

    Xue, Li-Jun; Wang, Yong-Zhi; Ren, Hao; Tong, Yi-Min; Zhao, Ping; Zhu, Shi-Ying; Qi, Zhong-Tian

    2006-09-01

    The 16S rDNA specific primers were designed for rapid detection of Pseudomonas aeruginosa (PA) by the fluorescence quantitative PCR (FQ-PCR) assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20microL of reaction system with SYBR Green I. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/microL of bacterial DNA or (2.1 x 10(3) +/- 3.1 x 10(2)) copies/microL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future. PMID:17037203

  17. Molecular Evolution of Mycoplasma capricolum subsp. capripneumoniae Strains, Based on Polymorphisms in the 16S rRNA Genes

    PubMed Central

    Pettersson, Bertil; Bölske, Göran; Thiaucourt, François; Uhlén, Mathias; Johansson, Karl-Erik

    1998-01-01

    Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP. PMID:9573185

  18. 16S-gyrB-rpoB multilocus sequence analysis for species identification in the genus Microbispora.

    PubMed

    Savi, D C; Aluizio, R; Galli-Terasawa, L; Kava, V; Glienke, C

    2016-06-01

    The genus Microbispora has been considered difficult to define taxonomically. While 16S rRNA gene analysis is required to determine phylogenetic relationships among species in this genus, most 16S rRNA gene-based phylogenetic tree topologies are not reliable. The genus Microbispora currently contains eight species along with six reclassified species (Microbispora chromogenes, Microbispora diastatica, Microbispora parva, Microbispora indica, Microbispora karnatakensis, Microbispora rosea) and Microbispora rosea subsp. aerata, a taxon composed of three further reclassified species (Microbispora aerata, Microbispora thermodiastatica, and Microbispora thermorosea). 16S rRNA, 23S rRNA, gyrB, and rpoB gene sequences were obtained for the type strains of Microbispora species, and eleven endophytic isolates from a Brazilian medicinal plant, Vochysia divergens. Using the concatenated sequence, most Microbispora type strains could be distinguished with high probability support. Based on these analyses, we propose that five of the species reclassified within the subspecies of M. rosea (M. chromogenes, M. karnatakensis, M. parva, M. aerata and M. thermorosea) are distinct from M. rosea and so should be retained as distinct species. The concatenated 16S-gyrB-rpoB gene phylogenic tree had significant probability support and topology. We propose the use of concatenated 16S-gyrB-rpoB gene sequences to determine phylogenetic relationships within the genus Microbispora. We also suggest that strains sharing >98.1 % 16S-gyrB-rpoB gene sequences similarity be defined as a single species, based on results from this analysis. Seven of the strains isolated from V. divergens were not related to any previously described Microbispora species. PMID:26984252

  19. Persistent spread of the rmtB 16S rRNA methyltransferase gene among Escherichia coli isolates from diseased food-producing animals in China.

    PubMed

    Xia, Jing; Sun, Jian; Cheng, Ke; Li, Liang; Fang, Liang-Xing; Zou, Meng-Ting; Liao, Xiao-Ping; Liu, Ya-Hong

    2016-05-30

    A total of 963 non-duplicate Escherichia coli strains isolated from food-producing animals between 2002 and 2012 were screened for the presence of the 16S rRNA methyltransferase genes. Among the positive isolates, resistance determinants to extended spectrum β-lactamases, plasmid-mediated quinolone resistance genes as well as floR and fosA/A3/C2 were detected using PCR analysis. These isolates were further subjected to antimicrobial susceptibility testing, molecular typing, PCR-based plasmid replicon typing and plasmid analysis. Of the 963 E. coli isolates, 173 (18.0%), 3 (0.3%) and 2 (0.2%) were rmtB-, armA- and rmtE-positive strains, respectively. All the 16S rRNA methyltransferase gene-positive isolates were multidrug resistant and over 90% of them carried one or more type of resistance gene. IncF (especially IncFII) and non-typeable plasmids played the main role in the dissemination of rmtB, followed by the IncN plasmids. Plasmids that harbored rmtB ranged in size from 20kb to 340kb EcoRI-RFLP testing of the 109 rmtB-positive plasmids from different years and different origins suggested that horizontal (among diverse animals) and vertical transfer of IncF, non-typeable and IncN-type plasmids were responsible for the spread of rmtB gene. In summary, our findings highlight that rmtB was the most prevalent 16S rRNA methyltransferase gene, which present persistent spread in food-producing animals in China and a diverse group of plasmids was responsible for rmtB dissemination. PMID:27139028

  20. The genetic diversity of genus Bacillus and the related genera revealed by 16s rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey

    PubMed Central

    Cihan, Arzu Coleri; Tekin, Nilgun; Ozcan, Birgul; Cokmus, Cumhur

    2012-01-01

    Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4–100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies. PMID:24031834

  1. Molecular systematics of the genus Troglophilus (Rhaphidophoridae, Orthoptera) in Turkey: mitochondrial 16S rDNA evidences

    PubMed Central

    Taylan, Mehmet Sait; Russo, Claudio Di; Rampini, Mauro; Ketmaier, Valerio

    2013-01-01

    Abstract This study focuses on the evolutionary relationships among Turkish species of the cave cricket genus Troglophilus.Fifteen populations were studied for sequence variation in a fragment (543 base pairs) of the mitochondrial DNA (mtDNA) 16S rDNA gene (16S) to reconstruct their phylogenetic relationships and biogeographic history. Genetic data retrieved three main clades and at least three divergent lineages that could not be attributed to any of the taxa known for the area. Molecular time estimates suggest that the diversification of the group took place between the Messinian and the Plio-Pleistocene. PMID:23653493

  2. Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam

    PubMed Central

    2013-01-01

    Background 16S rRNA methylase-producing Gram-negative bacteria are highly resistant to all clinically important aminoglycosides. We analyzed clinical strains of 16S rRNA methylase-producing Acinetobactor baumannii and Pseudomonas aeruginosa obtained from clinical isolates in medical settings in Vietnam. Methods From 2008 to 2011, 101 clinical strains of A. baumannii and 15 of P. aeruginosa were isolated from patients in intensive care units (ICUs) in two medical settings in Vietnam. Antimicrobial susceptibilities were determined using the microdilution method and epidemiological analysis was performed by pulsed-field gel electrophoresis and MLST. Genes encoding the 16S rRNA methylases, OXAs and CTX-Ms were analyzed by PCR and sequence analysis. Results 16S rRNA methylase-producing Gram-negative pathogens were detected in two hospitals in Vietnam. Of the 101 clinical isolates of A. baumannii and the 15 of P. aeruginosa isolated from two ICUs in these hospitals, 72 (71.3%) were highly resistant to amikacin, arbekacin and gentamicin, with MICs greater than 1,024 mg/L. The 16S rRNA methylases ArmA and RmtB were produced by 61 and 9 isolates of A. baumannii, respectively, and RmtB was produced by 2 isolates of P. aeruginosa. Moreover, 52 of the A. baumannii isolates producing 16S rRNA methylases harbored both blaOXA-23-like and blaOXA-51-like genes. Most A. baumannii isolates producing 16S rRNA methylase obtained in hospital A in Hanoi were ST91 and ST231, whereas most from hospital B in Ho Chi Minh City were ST136, ST195, and ST254. The two P. aeruginosa isolates harboring rmtB showed different patterns on PFGE, one each corresponding to ST217 and ST313. Conclusions Gram-negative bacteria producing the 16S rRNA methylases ArmA and RmtB are emerging in medical settings in Vietnam. A. baumannii isolates in northern and southern regions of Vietnam may be of different lineages. PMID:23721359

  3. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

    PubMed Central

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  4. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    NASA Technical Reports Server (NTRS)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  5. The GREGOR Broad-Band Imager

    NASA Astrophysics Data System (ADS)

    von der Lühe, O.; Volkmer, R.; Kentischer, T. J.; Geißler, R.

    2012-11-01

    The design and characteristics of the Broad-Band Imager (BBI) of GREGOR are described. BBI covers the visible spectral range with two cameras simultaneously for a large field and with critical sampling at 390 nm, and it includes a mode for observing the pupil in a Foucault configuration. Samples of first-light observations are shown.

  6. Broad-band UHF dipole array

    NASA Technical Reports Server (NTRS)

    Bailey, M. C.

    1985-01-01

    A 6X6 array of fan-dipoles was designed to operate in the 510 to 660 MHz frequency range for aircraft flight test and evaluation of a UHF radiometer system. A broad-band dipole design operating near the first resonance is detailed. Measured VSWR and radiation patterns for the dipole array demonstrate achievable bandwidths in the 35 percent to 40 percent range.

  7. Broadly tunable terahertz source

    NASA Astrophysics Data System (ADS)

    Powers, Peter E.; Kramb, Kevan; Haus, Joseph W.

    2010-02-01

    We present the results of a terahertz (THz) source based on difference frequency generation (DFG) that tunes seamlessly from 1.4 to 13.3 THz. The outputs from two seeded periodically poled lithium niobate (PPLN) optical parametric generators (OPGs) are mixed in a DAST crystal to generate the THz frequencies. The OPG's have ~1 nsec pulse duration and an output energy of approximately 200 μJ. The corresponding high peak intensities in the DAST crystal leads to appreciable conversion efficiency such that a room temperature pyro-electric detector is used to measure the THz signal. In one of the OPGs a continuously varying periodicity PPLN crystal is used to tune the output wavelength by translating the crystal. The crystal position and seed laser are computer-controlled and synchronized such that any wavelength within the seed laser's tuning range is randomly accessible, and hence any THz difference frequency between the two seed lasers is also randomly accessible. Phase matching in DAST requires the DFG inputs to have the same polarization. We demonstrate a scheme where the output of one of the OPGs is sent through the second OPG such that the two beams are collinear with the same polarization without using a beam splitter.

  8. Molecular Methods for Identification of Acinetobacter Species by Partial Sequencing of the rpoB and 16S rRNA Genes

    PubMed Central

    Khosravi, Azar Dokht; Shahraki, Abdolrazagh Hashemi; Heidarieh, Parvin; Sheikhi, Nasrin

    2015-01-01

    Background Acinetobacter spp. is a diverse group of Gram-negative bacteria which are ubiquitous in soil and water, and an important cause of nosocomial infections. The purpose of this study was to identify a collection of Acinetobacter spp. clinical isolates accurately and to investigate their antibiotic susceptibility patterns. Materials and Methods A total of 197 non-duplicate clinical isolates of Acinetobacter spp. isolates identified using conventional biochemical tests. The molecular technique of PCR-RFLP and sequence analysis of rpoB and 16S rRNA genes was applied for species identification. Antimicrobial susceptibility test was performed with a disk diffusion assay. Results Based on 16S rRNA and rpoB genes analysis separately, most of clinical isolates can be identified with high bootstrap values. However, the identity of the isolate 555T was uncertain due to high similarity of A. grimontii and A. junii. Identification by concatenation of 16S rRNA and rpoB confirmed the identity of clinical isolates of Acenitobacer to species level confidently. Accordingly, the isolate 555T assigned as A. grimontii due to 100% similarity to A. grimontii. Moreover, this isolate showed 98.64% to A. junii. Besides, the identity of the isolates 218T and 364T was confirmed as Genomic species 3 and A. calcoaceticus respectively. So, the majority of Acinetobacter spp. isolates, were identified as: A. baumannii (131 isolates, 66%), A. calcoaceticus (9 isolates, 4.5%), and A. genomosp 16 (8 isolates, 4%). The rest of identified species showed the lower frequencies. In susceptibility test, 105 isolates (53%), presented high antibiotic resistance of 90% to ceftriaxone, piperacillin, piperacillin tazobactam, amikacin, and 81% to ciprofloxacin. Conclusion Sequence analysis of the 16S rRNA and rpoB spacer simultaneously was able to do identification of Acinetobacter spp. to species level. A.baumannii was identified as the most prevalent species with high antibiotic resistance. Other

  9. [Antimicrobial susceptibilities of clinical Nocardia isolates identified by 16S rRNA gene sequence analysis].

    PubMed

    Uner, Mahmut Celalettin; Hasçelik, Gülşen; Müştak, Hamit Kaan

    2016-01-01

    Nocardia species are ubiquitous in the environment and responsible for various human infections such as pulmonary, cutaneous, central nervous system and disseminated nocardiosis. Since the clinical pictures and antimicrobial susceptibilities of Nocardia species exhibit variability, susceptibility testing is recommended for every Nocardia isolates. The aims of this study was to determine the antimicrobial susceptibilities of Nocardia clinical isolates and to compare the results of broth microdilution and disc diffusion susceptibility tests. A total of 45 clinical Nocardia isolates (isolated from 17 respiratory tract, 8 brain abscess, 7 pus, 3 skin, 3 conjunctiva, 2 blood, 2 tissue, 2 pleural fluid and 1 cerebrospinal fluid samples) were identified by using conventional methods and 16S rRNA gene sequence analysis. Susceptibility testing was performed for amikacin, ciprofloxacin, ceftriaxone, linezolid and trimethoprim-sulfamethoxazole (TMP-SMX) by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) criteria recommended in 2011 approved standard (M24-A2) and disk diffusion method used as an alternative comparative susceptibility testing method. Among the 45 Nocardia strains, N.cyriacigeorgica (n: 26, 57.8%) was the most common species, followed by N.farcinica (n: 12, 26.7%), N.otitiscaviarum (n: 4, 8.9%), N.asteroides (n: 1, 2.2%), N.neocaledoniensis (n: 1, 2.2%) and N.abscessus (n: 1, 2.2%). Amikacin and linezolid were the only two antimicrobials to which all isolates were susceptible for both broth microdilution and disk diffusion tests. In broth microdilution test, resistance rates to TMP-SMX, ceftriaxone and ciprofloxacin were found as 15.6%, 37.8% and 84.4% respectively, whereas in the disk diffusion test, the highest resistance rate was observed against ciprofloxacin (n: 33, 73.3%), followed by TMP-SMX (n: 22, 48.9%) and ceftriaxone (n: 15, 33.3%). In both of these tests, N.cyriacigeorgica was the species with the

  10. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    PubMed

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria. PMID:27412167

  11. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  12. Distinct Ectomycorrhizospheres Share Similar Bacterial Communities as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes

    PubMed Central

    Oger, P.; Morin, E.; Frey-Klett, P.

    2012-01-01

    Analysis of the 16S rRNA gene sequences generated from Xerocomus pruinatus and Scleroderma citrinum ectomycorrhizospheres revealed that similar bacterial communities inhabited the two ectomycorrhizospheres in terms of phyla and genera, with an enrichment of the Burkholderia genus. Compared to the bulk soil habitat, ectomycorrhizospheres hosted significantly more Alpha-, Beta-, and Gammaproteobacteria. PMID:22307291

  13. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    EPA Science Inventory

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  14. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing

    PubMed Central

    Kvich, Lasse; Eickhardt, Steffen; Omland, Lars H.; Bjarnsholt, Thomas; Moser, Claus

    2015-01-01

    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii-specific PCR and immunohistochemistry. The patient was later diagnosed with HIV/AIDS. PMID:25854484

  15. Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

    PubMed Central

    Lay, Christophe; Ho, Eliza Xin Pei; Low, Louie; Hibberd, Martin Lloyd; Nagarajan, Niranjan

    2013-01-01

    The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization. PMID:23579286

  16. Phylogenetic relationships linking Duttaphrynus (Amphibia: Anura: Bufonidae) species based on 12S and 16S rDNA sequences.

    PubMed

    Pratihar, Suman; Bhattacharya, Manojit; Deuti, Kaushik

    2016-07-01

    Genus Duttaphrynus (Amphibia: Anura: Bufonidae) is endemic to southwestern and southern China and throughout southern Asia. Duttaphrynus phylogeny was also under debate for many years. 12S and 16S rDNAs help us to elucidate Duttaphrynus phylogeny. PMID:26155970

  17. IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

    EPA Science Inventory

    Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

  18. Bacterial diversity in Philippine fermented mustard (burong mustasa) as revealed by 16S rRNA gene analysis.

    PubMed

    Larcia, L L H; Estacio, R C; Dalmacio, L M M

    2011-12-01

    Previous studies on the bacterial profile of burong mustasa, a traditional Philippine fermented food, had been conducted using culture-dependent techniques. Since these methods may underestimate the total microbiota of a sample, a culture-independent study was done to determine the bacterial diversity in burong mustasa through molecular biology techniques. Bacterial DNA was isolated from fermented mustard samples at different stages of fermentation. The isolated genomic DNA was amplified by PCR using specific primers for the 16S ribosomal RNA gene (16S rDNA). The 1.5 kb amplicons obtained were subjected to nested PCR using primers for the internal variable region of the 16S rDNA. The 585 bp nested PCR amplicons were then subjected to denaturing gradient gel electrophoresis (DGGE) to separate the different bacteria present in each sample. Distinct and unique bands in the DGGE profile were excised, reamplified, purified and sequenced for bacterial identification. Molecular cloning of the 1.5 kb 16S rDNA was also performed using the pGEM-T Easy Vector System. The cloned gene was sequenced for bacterial identification. The identified microbiota in burong mustasa at different stages of fermentation include lactic acid bacteria and several uncultured bacteria (initial up to the final stages); acetic acid bacteria (middle stage); and Streptobacillus and Fusobacterium species (initial stage). The potential probiotic bacteria found in burong mustasa are Weissella and Lactobacillus. PMID:22146686

  19. Phylogenetic Analysis of Bacteroidales 16S rRNA Genes Unveils Sequences Specific to Diverse Swine Fecal Sources

    EPA Science Inventory

    Two of the currently available methods to assess swine fecal pollution (Bac1 and PF163) target Bacteroidales 16S rRNA genes. However, these assays have been shown to exhibit poor host-specificity and low detection limits in environmental waters, in part due to the limited number...

  20. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING BACTEROIDETES 16S RDNA-BASED ASSAYS

    EPA Science Inventory

    Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate between ruminant and human fecal pollution. These assays are rapid and relatively inexpensive but have been used in a limited number of studies. In this study, we evaluated the efficacy o...

  1. Methanogen diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

    PubMed

    Singh, K M; Tripathi, A K; Pandya, P R; Parnerkar, S; Rank, D N; Kothari, R K; Joshi, C G

    2012-06-01

    The methanogenic communities in buffalo rumen were characterized using a culture-independent approach of a pooled sample of rumen fluid from three adult Surti buffaloes. Buffalo rumen is likely to include species of various methanogens, so 16S rDNA sequences were amplified and cloned from the sample. A total of 171 clones were sequenced to examine 16S rDNA sequence similarity. About 52.63% sequences (90 clones) had ≥ 90% similarity, whereas, 46.78% of the sequences (81 clones) were 75-89% similar to 16S rDNA database sequences, respectively. Phylogenetic analyses were also used to infer the makeup of methanogenic communities in the rumen of Surti buffalo. As a result, we distinguished 23 operational taxonomic units (OTUs) based on unique 16S rDNA sequences: 12 OTUs (52.17%) affiliated to Methanomicrobiales order, 10 OTUs (43.47%) of the order Methanobacteriales and one OTU (4.34%) of Methanosarcina barkeri like clone, respectively. In addition, the population of Methanomicrobiales and Methabacteriales orders were also observed, accounting 4% and 2.17% of total archea. This study has revealed the largest assortment of hydrogenotrophic methanogens phylotypes ever identified from rumen of Surti buffaloes. PMID:21507441

  2. Ureaplasma urealyticum continuous ambulatory peritoneal dialysis-associated peritonitis diagnosed by 16S rRNA gene PCR.

    PubMed

    Yager, Jessica E; Ford, Emily S; Boas, Zachary P; Haseley, Leah A; Cookson, Brad T; Sengupta, Dhruba J; Fang, Ferric C; Gottlieb, Geoffrey S

    2010-11-01

    In some patients with peritonitis complicating continuous ambulatory peritoneal dialysis (CAPD), a causative organism is never identified. We report a case of Ureaplasma urealyticum CAPD-associated peritonitis diagnosed by 16S rRNA gene PCR. Ureaplasma may be an underrecognized cause of peritonitis because it cannot be recovered using routine culture methods. PMID:20739488

  3. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    EPA Science Inventory

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  4. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING PCR AND PHYLOGENETIC ANALYSES OF BACTEROIDETES 16S RDNA

    EPA Science Inventory

    Traditional methods for assessing fecal pollution in environmental systems, such as monitoring for fecal coliforms are not capable of discriminating between different sources fecal pollution. Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate betw...

  5. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis.

    PubMed

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2016-08-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes. PMID:27245411

  6. A Web-Hosted R Workflow to Simplify and Automate the Analysis of 16S NGS Data

    EPA Science Inventory

    Next-Generation Sequencing (NGS) produces large data sets that include tens-of-thousands of sequence reads per sample. For analysis of bacterial diversity, 16S NGS sequences are typically analyzed in a workflow that containing best-of-breed bioinformatics packages that may levera...

  7. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

  8. Nuclease mapping of the secondary structure of the 49-nucleotide 3' terminal cloacin fragment of Escherichia coli 16s RNA and its interactions with initiation factor 3.

    PubMed Central

    Wickstrom, E

    1983-01-01

    Escherichia coli translational initiation factor 3 (IF3) may be crosslinked to the 3' end of 16S RNA in 30S ribosomal subunits. In order to determine the sequence to which IF3 may bind in vivo, samples of 5'-32P labelled 3' terminal 49-nucleotide fragment of 16S RNA were incubated 5 min. at 37 degrees in 40 mM Tris-HOAc, pH 7.4, 100 mM NaCl, 1 mM Mg (OAc)2, 1 mM ZnSO4, with or without IF3, then reacted a further 5 min with nuclease S1, RNase T1, or RNase A. Base pairing between the 5' and 3' legs of the fragment occurs in the absence of IF3, but is disrupted by IF3 binding. IF3 appears to protect some residues near the 5' end of the fragment (U1495, A1499, A1500, A1502, and A1503) from nuclease S1, and potentiates S1 attack on others (G1494, G1497, C1501, G1504, G1505, U1506, G1517, G1529, G1530, and C1533). A series of equimolar reactions at increasing dilution imply an association constant range of 1.4-7.0 X 10(7) M-1. Images PMID:6340066

  9. Microbial Diversity of the Brine-Seawater Interface of the Kebrit Deep, Red Sea, Studied via 16S rRNA Gene Sequences and Cultivation Methods

    PubMed Central

    Eder, Wolfgang; Jahnke, Linda L.; Schmidt, Mark; Huber, Robert

    2001-01-01

    The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other Halanaerobium representatives, showing unusually large amounts of Δ7 and Δ11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus Halanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface. PMID:11425725

  10. Prevalence of 16S rRNA methylase genes among β-lactamase-producing Enterobacteriaceae clinical isolates in Saudi Arabia

    PubMed Central

    Al Sheikh, Yazeed A.; Marie, Mohammed Ali M.; John, James; Krishnappa, Lakshmana Gowda; Dabwab, Khaled Homoud M.

    2014-01-01

    Background Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Results Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were bla TEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced β-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. Conclusion β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae. PMID:25005152

  11. Broadly tunable picosecond ir source

    DOEpatents

    Campillo, A.J.; Hyer, R.C.; Shapiro, S.L.

    1980-04-23

    A picosecond traveling-wave parametric device capable of controlled spectral bandwidth and wavelength in the infrared is reported. Intense 1.064 ..mu..m picosecond pulses (1) pass through a 4.5 cm long LiNbO/sub 3/ optical parametric oscillator crystal (2) set at its degeneracy angle. A broad band emerges, and a simple grating (3) and mirror (4) arrangement is used to inject a selected narrow-band into a 2 cm long LiNbO/sub 3/ optical parametric amplifier crystal (5) along a second pump line. Typical input energies at 1.064 ..mu..m along both pump lines are 6 to 8 mJ for the oscillator and 10 mJ for the amplifier. This yields 1 mJ of tunable output in the range 1.98 to 2.38 ..mu..m which when down-converted in a 1 cm long CdSe crystal mixer (6) gives 2 ..mu..J of tunable radiation over the 14.8 to 18.5 ..mu..m region. The bandwidth and wavelength of both the 2 and 16 ..mu..m radiation output are controlled solely by the diffraction grating.

  12. Ghost imaging with broad distance

    NASA Astrophysics Data System (ADS)

    Duan, De-Yang; Zhang, Lu; Du, Shao-Jiang; Xia, Yun-Jie

    2015-10-01

    We present a scheme that is able to achieve the ghost imaging with broad distance. The physical nature of our scheme is that the different wavelength beams are separated in free space by an optical media according to the slow light or dispersion principle. Meanwhile, the equality of the optical distance of the two light arms is not violated. The photon correlation is achieved by the rotating ground glass plate (RGGP) and spatial light modulator (SLM), respectively. Our work shows that a monochromic ghost image can be obtained in the case of RGGP. More importantly, the position (or distance) of the object can be ascertained by the color of the image. Thus, the imaging and ranging processes are combined as one process for the first time to the best of our knowledge. In the case of SLM, we can obtain a colored image regardless of where the object is. Project supported by the National Natural Science Foundation of China (Grant Nos. 61178012, 11204156, 11304179, and 11247240), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant Nos. 20133705110001 and 20123705120002), the Scientific Research Foundation for Outstanding Young Scientists of Shandong Province, China (Grant No. BS2013DX034), and the Natural Science Foundation of Shandong Province, China (Grant No. ZR2012FQ024).

  13. Broadly tunable picosecond IR source

    DOEpatents

    Campillo, Anthony J.; Hyer, Ronald C.; Shapiro, Stanley J.

    1982-01-01

    A picosecond traveling-wave parametric device capable of controlled spectral bandwidth and wavelength in the infrared is reported. Intense 1.064 .mu.m picosecond pulses (1) pass through a 4.5 cm long LiNbO.sub.3 optical parametric oscillator crystal (2) set at its degeneracy angle. A broad band emerges, and a simple grating (3) and mirror (4) arrangement is used to inject a selected narrow-band into a 2 cm long LiNbO.sub.3 optical parametric amplifier crystal (5) along a second pump line. Typical input energies at 1.064 .mu.m along both pump lines are 6-8 mJ for the oscillator and 10 mJ for the amplifier. This yields 1 mJ of tunable output in the range 1.98 to 2.38 .mu.m which when down-converted in a 1 cm long CdSe crystal mixer (6) gives 2 .mu.J of tunable radiation over the 14.8 to 18.5 .mu.m region. The bandwidth and wavelength of both the 2 and 16 .mu.m radiation output are controlled solely by the diffraction grating.

  14. The Identification of Discriminating Patterns from 16S rRNA Gene to Generate Signature for Bacillus Genus.

    PubMed

    More, Ravi P; Purohit, Hemant J

    2016-08-01

    The 16S ribosomal RNA (16S rRNA) gene has been widely used for the taxonomic classification of bacteria. A molecular signature is a set of nucleotide patterns, which constitute a regular expression that is specific to each particular taxon. Our main goal was to identify discriminating nucleotide patterns in 16S rRNA gene and then to generate signatures for taxonomic classification. To demonstrate our approach, we used the phylum Firmicutes as a model using representative taxa Bacilli (class), Bacillales (order), Bacillaceae (family), and Bacillus (genus), according to their dominance at each hierarchical taxonomic level. We applied combined composite vector and multiple sequence alignment approaches to generate gene-specific signatures. Further, we mapped all the patterns into the different hypervariable regions of 16S rRNA gene and confirmed the most appropriate distinguishing region as V3-V4 for targeted taxa. We also examined the evolution in discriminating patterns of signatures across taxonomic levels. We assessed the comparative classification accuracy of signatures with other methods (i.e., RDP Classifier, KNN, and SINA). Results revealed that the signatures for taxa Bacilli, Bacillales, Bacillaceae, and Bacillus could correctly classify isolate sequences with sensitivity of 0.99, 0.97, 0.94, and 0.89, respectively, and specificity close to 0.99. We developed signature-based software DNA Barcode Identification (DNA BarID) for taxonomic classification that is available at website http://www.neeri.res.in/DNA_BarID.htm . This pattern-based study provides a deeper understanding of taxon-specific discriminating patterns in 16S rRNA gene with respect to taxonomic classification. PMID:27104769

  15. A framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates.

    PubMed

    Helbling, Damian E; Johnson, David R; Lee, Tae Kwon; Scheidegger, Andreas; Fenner, Kathrin

    2015-03-01

    The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific

  16. Plastid 16S rRNA Gene Diversity among Eukaryotic Picophytoplankton Sorted by Flow Cytometry from the South Pacific Ocean

    PubMed Central

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J.; Vaulot, Daniel

    2011-01-01

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image. PMID:21552558

  17. Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

    PubMed Central

    Wise, M G; McArthur, J V; Shimkets, L J

    1997-01-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. PMID:9097448

  18. Bacterial diversity of a Carolina Bay as determined by 16S rRNA gene analysis: Confirmation of novel taxa

    SciTech Connect

    Wise, M.G.; Shimkets, L.J.; McArthur, J.V.

    1997-04-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhabit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow Bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project`s taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. 50 refs., 7 figs., 1 tab.

  19. Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

    PubMed

    Wise, M G; McArthur, J V; Shimkets, L J

    1997-04-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. PMID:9097448

  20. The phylogeny of intestinal porcine spirochetes (Serpulina species) based on sequence analysis of the 16S rRNA gene.

    PubMed Central

    Pettersson, B; Fellström, C; Andersson, A; Uhlén, M; Gunnarsson, A; Johansson, K E

    1996-01-01

    Four type or reference strains and twenty-two field strains of intestinal spirochetes isolated from Swedish pig herds were subjected to phylogenetic analysis based on 16S rRNA sequences. Almost complete (>95%) 16S rRNA sequences were obtained by solid-phase DNA sequencing of in vitro-amplified rRNA genes. The genotypic patterns were compared with a previously proposed biochemical classification scheme, comprising beta-hemolysis, indole production, hippurate hydrolysis, and alpha-galactosidase, alpha-glucosidase, and beta-glucosidase activities. Comparison of the small-subunit rRNA sequences showed that the strains of the genus Serpulina were closely related. Phylogenetic trees were constructed, and three clusters were observed. This was also confirmed by signature nucleotide analysis of the serpulinas. The indole-producing strains, including the strains of S. hyodysenteriae and some weakly beta-hemolytic Serpulina strains, formed one cluster. A second cluster comprised weakly beta-hemolytic strains that showed beta-galactosidase activity but lacked indole production and hippurate-hydrolyzing capacity. The second cluster contained two subclusters with similar phenotypic profiles. A third cluster involved strains that possessed a hippurate-hydrolyzing capacity which was distinct from that of the former two clusters, because of 17 unique nucleotide positions of the 16S rRNA gene. Interestingly, the strains of this third cluster were found likely to have a 16S rRNA structure in the V2 region of the molecule different from that of the serpulinas belonging to the other clusters. As a consequence of these findings, we propose that the intestinal spirochetes of this phenotype (i.e., P43/6/78-like strains) should be regarded as a separate Serpulina species. Furthermore, this cluster was found to be by far the most homogeneous one. In conclusion, the biochemical classification of porcine intestinal spirochetes was comparable to that by phylogenetic analysis based on 16S r

  1. Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

    PubMed Central

    Shen, Li-dong; Liu, Shuai; Lou, Li-ping; Liu, Wei-ping; Xu, Xiang-yang; Zheng, Ping

    2013-01-01

    Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 104 ± 0.42 × 104 to 3.69 × 106 ± 0.25 × 106 copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils. PMID:23747706

  2. Development of a real-time PCR method for the detection of fossil 16S rDNA fragments of phototrophic sulfur bacteria in the sediments of Lake Cadagno.

    PubMed

    Ravasi, D F; Peduzzi, S; Guidi, V; Peduzzi, R; Wirth, S B; Gilli, A; Tonolla, M

    2012-05-01

    Lake Cadagno is a crenogenic meromictic lake situated in the southern range of the Swiss Alps characterized by a compact chemocline that has been the object of many ecological studies. The population dynamics of phototrophic sulfur bacteria in the chemocline has been monitored since 1994 with molecular methods such as 16S rRNA gene clone library analysis. To reconstruct paleo-microbial community dynamics, we developed a quantitative real-time PCR methodology for specific detection of 16S rRNA gene sequences of purple and green sulfur bacteria populations from sediment samples. We detected fossil 16S rDNA of nine populations of phototrophic sulfur bacteria down to 9-m sediment depth, corresponding to about 9500 years of the lake's biogeological history. These results provide the first evidence for the presence of 16S rDNA of anoxygenic phototrophic bacteria in Holocene sediments of an alpine meromictic lake and indicate that the water column stratification and the bacterial plume were already present in Lake Cadagno thousands of years ago. The finding of Chlorobium clathratiforme remains in all the samples analyzed shows that this population, identified in the water column only in 2001, was already a part of the lake's biota in the past. PMID:22433067

  3. Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages.

    PubMed

    Blaiotta, Giuseppe; Pennacchia, Carmelina; Ercolini, Danilo; Moschetti, Giancarlo; Villani, Francesco

    2003-09-01

    Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains. PMID:14529185

  4. Broad-spectrum antiviral agents

    PubMed Central

    Zhu, Jun-Da; Meng, Wen; Wang, Xiao-Jia; Wang, Hwa-Chain R.

    2015-01-01

    Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents. PMID:26052325

  5. CHARACTERIZATION OF BACTERIAL BIOMASS IN MARINE SEDIMENTS BENEATH THE ROSS ICE SHEET, ANTARCTICA BY PHOSPHOLIPIDS ANALYSIS AND 16S RRNA GENE SEQUENCING

    NASA Astrophysics Data System (ADS)

    Carr, S. A.; Glossner, A. W.; Dunbar, R. B.; Vogel, S. W.; Brandes, J.; Sahl, J. W.; Pepe-Ranney, C.; Spear, J. R.; Naish, T.; Powell, R. D.; Mandernack, K. W.

    2009-12-01

    As concerns regarding climate change increase, so does the importance of understanding the biogeochemical cycling of elements such as carbon. In the marine sediments of the Ross Sea, Antarctica, the in situ microbial community plays a significant role in the decomposition, mineralization and recycling of both organic and inorganic carbon. In this study, viable biomass for the top 155 cm below seafloor of sediment cores in the Ross Sea were estimated based on microbial phospholipid concentrations and Acridine Orange direct cell counts (AODC). Results for the biomass estimates suggest that both methods are able to accurately estimate viable biomass. Structural and isotopic analyses of phospholipid fatty acids (PLFAs) and phospholipid ether lipids (PELs), as well as isotopic analyses of carbon sources within sediment porewaters were used to identify changes in microbial metabolic pathways. The δ13C values of dissolved inorganic carbon (DIC) in porewaters ranged from -2.52‰ to -3.72‰ while corresponding δ13C values for sedimentary organic carbon (OC) varied from -26.25‰ to -23.12‰ in the surface and 155cm porewaters, respectively. The δ13C values of PLFAs are slightly lighter than the δ13C values of the organic carbon, ranging between -29‰ to -35‰ throughout the sediment core. 16S ribosomal RNA gene sequencing was preformed to classify the microbial species present at various depths. 16S sequences revealed that members of this microbial community include α, δ, ɛ, and γ proteobacteria, acitobacteria, acidobacteria, and flavobacteria, all of which have been previously sequenced from other Antarctic continental shelf sediments. Archaea represent 1 to 3% of the microbial community which is similar to comparable studies. Amongst the sequenced organisms, many have been reported to utilize organic carbon sources such as amino acids, oligosaccharides, and lactose. These heterotropic organisms compliment the constant lipid isotope values and suggest that

  6. Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

    PubMed Central

    Tapprich, W E; Goss, D J; Dahlberg, A E

    1989-01-01

    A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis. PMID:2662189

  7. Isolation and 16S DNA characterization of soil microorganisms from tropical soils capable of utilizing the herbicides hexazinone and tebuthiuron.

    PubMed

    Mostafa, Fadwa I Y; Helling, Charles S

    2003-11-01

    Six non-fermentative bacteria were isolated from Colombian (South America) and Hawaiian (USA) soils after enrichment with minimal medium supplemented with two herbicides, hexazinone (Hex) and tebuthiuron (Teb). Microscopic examination and physiological tests were followed by partial 16S DNA sequence analysis, using the first 527 bp of the 16S rRNA gene for bacterial identification. The isolated microorganisms (and in brackets, the herbicide that each degraded) were identified as: from Colombia. Methylobacterium organophilum [Teb], Paenibacillus pabuli [Teb], and Micrmbacterium foliorum [Hex]; and from Hawaii, Methylobacterium radiotolerans [Teb], Paenibacillus illinoisensis [Hex], and Rhodococcus equi [Hex]. The findings further explain how these herbicides, which have potential for illicit coca (Erythroxylum sp.) control, dissipate following their application to tropical soils. PMID:14649709

  8. Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

    PubMed

    Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

    2015-09-01

    Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds. PMID:26189016

  9. Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis

    PubMed Central

    Ennahar, Saïd; Cai, Yimin; Fujita, Yasuhito

    2003-01-01

    A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Weissella. Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality. PMID:12514026

  10. Short-Read Assembly of Full-Length 16S Amplicons Reveals Bacterial Diversity in Subsurface Sediments

    PubMed Central

    Miller, Christopher S.; Handley, Kim M.; Wrighton, Kelly C.; Frischkorn, Kyle R.; Thomas, Brian C.; Banfield, Jillian F.

    2013-01-01

    In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the “long tail” of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change. PMID:23405248

  11. Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.

    PubMed Central

    Krueger, D M; Cavanaugh, C M

    1997-01-01

    The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins. PMID:8979342

  12. Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6

    SciTech Connect

    Allison E. Ray; Stephanie A. Connon; Peter P. Sheridan; Jeremy Gilbreath; Malcolm S. Shields; Deborah T. Newby; Yoshiko Fujita; Timothy S. Magnuson

    2010-06-01

    The determination of variation in 16S rRNA gene sequences is perhaps the most common method for assessing microbial community diversity. However, the occurrence of multiple copies of 16S rRNA genes within some organisms can bias estimates of microbial diversity. During phylogenetic characterization of a metal-transforming, fermentative bacterium (strain UFO1) isolated from the Field Research Center (FRC) in Oak Ridge, TN, we detected an apparent 16S rRNA pseudogene. The putative 16S rRNA pseudogene was first detected in clone libraries constructed with 16S rRNA genes amplified from UFO1 genomic DNA. Sequencing revealed two distinct 16S rRNA genes, with one differing from the other by a 100 bp insert near the 5’ end. Ribosomal RNA was extracted from strain UFO1 and analyzed by RT-qPCR with insert and non-insert specific primers; however, only the non-insert 16S rRNA sequence was expressed. Reverse-transcribed rRNA from strain UFO1 was also used to construct a cDNA library. Of 190 clones screened by PCR, none contained the 16S rRNA gene with the 100 bp insert. Examination of GenBank 16S rRNA gene sequences revealed that the same insert sequence was present in other clones, including those from an environmental library constructed from FRC enrichments. These findings demonstrate the existence of widely disparate copies of the 16S rRNA gene in the same species and a putative 16S rRNA pseudogene, which may confound 16S rRNA-based methods for assessments of microbial diversity in environmental samples.

  13. Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

    PubMed Central

    Nübel, Ulrich; Garcia-Pichel, Ferran; Kühl, Michael; Muyzer, Gerard

    1999-01-01

    We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied. PMID:9925563

  14. Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences.

    PubMed

    Distel, D L; Lane, D J; Olsen, G J; Giovannoni, S J; Pace, B; Pace, N R; Stahl, D A; Felbeck, H

    1988-06-01

    The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis [C. R. Woese, Microbiol. Rev. 51: 221-271, 1987]). Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species. PMID:3286609

  15. Microbial community of salt crystals processed from Mediterranean seawater based on 16S rRNA analysis.

    PubMed

    Baati, Houda; Guermazi, Sonda; Gharsallah, Neji; Sghir, Abdelghani; Ammar, Emna

    2010-01-01

    Phylogenetic analysis of 16S rRNA was used to investigate for the first time the structure of the microbial community that inhabits salt crystals retrieved from the bottom of a solar saltern, located in the coastal area of the Mediterranean Sea (Sfax, Tunisia). This community lives in an extremely salty environment of 250-310 g/L total dissolved salt. A total of 78 bacterial 16S rRNA clone sequences making up to 21 operational taxonomic units (OTUs), determined by the DOTUR program to 97% sequence similarity, was analyzed. These OTUs were affiliated to Bacteroidetes (71.4% of OTUs), and gamma-Proteobacteria and alpha-Proteobacteria (equally represented by 14.2% of the OTUs observed). The archaeal community composition appeared more diverse with 68 clones, resulting in 44 OTUs, all affiliated with the Euryarchaeota phylum. Of the bacterial and archaeal clones showing <97% 16S rRNA sequence identity with sequences in public databases, 47.6% and 84.1% respectively were novel clones. Both rarefaction curves and diversity measurements (Simpson, Shannon-Weaver, Chao) showed a more diverse archaeal than bacterial community at the Tunisian solar saltern pond. The analysis of an increasing clone's number may reveal additional local diversity. PMID:20130693

  16. Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA

    PubMed Central

    Gregory, Steven T.; Dahlberg, Albert E.

    2009-01-01

    Characterization of base substitutions in rRNAs has provided important insights into the mechanism of protein synthesis. Knowledge of the structural effects of such alterations is limited, and could be greatly expanded with the development of a genetic system based on an organism amenable to both genetics and structural biology. Here, we describe the genetic analysis of base substitutions in 16S ribosomal RNA of the extreme thermophile Thermus thermophilus, and an analysis of the conformational effects of these substitutions by structure probing with base-specific modifying agents. Gene replacement methods were used to construct a derivative of strain HB8 carrying a single 16S rRNA gene, allowing the isolation of spontaneous streptomycin-resistant mutants and subsequent genetic mapping of mutations by recombination. The residues altered to give streptomycin resistance reside within the central pseudoknot structure of 16S rRNA comprised of helices 1 and 27, and participate in the U13–U20–A915 base triple, the G21–A914 type II sheared G–A base pair, or the G885–C912 Watson–Crick base pair closing helix 27. Substitutions at any of the three residues engaged in the base triple were found to confer resistance. Results from structure probing of the pseudoknot are consistent with perturbation of RNA conformation by these substitutions, potentially explaining their streptomycin-resistance phenotypes. PMID:19144908

  17. Identification of probiotic lactobacilli used for animal feeds on the basis of 16S ribosomal RNA gene sequence.

    PubMed

    Higuchi, Wataru; Muramatsu, Mineo; Dohmae, Soshi; Takano, Tomomi; Isobe, Hirokazu; Yabe, Shizuka; Da, Shi; Baranovich, Tatiana; Yamamoto, Tatsuo

    2008-11-01

    The use of probiotics such as Lactobacillus in animal feeds has gained popularity in recent years. In this study the 16S rRNA gene sequence of L. acidophilus in two commercial agents which have been used in animal feeds, LAB-MOS and Ghenisson 22, was determined. Phylogenetic tree analysis revealed that the two agents, strain MNFLM01 in LAB-MOS and strain GAL-2 in Ghenisson 22, belonged to L. rhamnosus (a member of the L. casei group) and L. johnsonii (a member of the L. acidophilus group), respectively. Biochemical tests assigned the two as L. rhamnosus and ambiguously as L. acidophilus. The data suggest that 16S rRNA gene sequence analysis provides more accurate identification of Lactobacillus species than biochemical tests and would allow quality assurance of relevant commercial products. The 16S rRNA gene sequences of strains MNFLM01 and GAL-2 determined in this study have been submitted to the DDBJ/EMBL/GenBank accession numbers under accession numbers AB288235 and AB295648, respectively. PMID:19090836

  18. Molecular Diagnosis of Periprosthetic Joint Infection by Quantitative RT-PCR of Bacterial 16S Ribosomal RNA

    PubMed Central

    Lee, Mel S.; Chang, Wen-Hsin; Chen, Su-Chin; Hsieh, Pang-Hsin; Shih, Hsin-Nung; Ueng, Steve W. N.; Lee, Gwo-Bin

    2013-01-01

    The diagnosis of periprosthetic joint infection is sometimes straightforward with purulent discharge from the fistula tract communicating to the joint prosthesis. However it is often difficult to differentiate septic from aseptic loosening of prosthesis because of the high culture-negative rates in conventional microbiologic culture. This study used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to amplify bacterial 16S ribosomal RNA in vitro and in 11 clinical samples. The in vitro analysis demonstrated that the RT-qPCR method was highly sensitive with the detection limit of bacterial 16S rRNA being 0.148 pg/μl. Clinical specimens were analyzed using the same protocol. The RT-qPCR was positive for bacterial detection in 8 culture-positive cases (including aerobic, anaerobic, and mycobacteria) and 2 culture-negative cases. It was negative in one case that the final diagnosis was confirmed without infection. The molecular diagnosis of bacterial infection using RT-qPCR to detect bacterial 16S rRNA around a prosthesis correlated well with the clinical findings. Based on the promising clinical results, we were attempting to differentiate bacterial species or drug-resistant strains by using species-specific primers and to detect the persistence of bacteria during the interim period before the second stage reimplantation in a larger scale of clinical subjects. PMID:24453929

  19. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  20. Structure of ERA in Complex with the 3 End of 16s rRNBA Implications for Ribosome Biogenesis

    SciTech Connect

    Tu, C.; Zhou, X; Tropea, J; Austin, B; Waugh, D; Court, D; Ji, X

    2009-01-01

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3? end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  1. Identification and characterization of alkaline protease producing Bacillus firmus species EMBS023 by 16S rRNA gene sequencing.

    PubMed

    Wishard, Rohan; wishard, Rohan; Jaiswal, Mahak; Parveda, Maheshwari; Amareshwari, P; Bhadoriya, Sneha Singh; Rathore, Pragya; Yadav, Mukesh; Nayarisseri, Anuraj; Nair, Achuthsankar S

    2014-12-01

    Probiotic microorganisms are those which exert a positive exect on the growth of the host, when administered as a dietary mixture in an adequate amount. They form the best alternative to the use of antibiotics for controlling enteric diseases in poultry farm animals, especially in the light of the gruesome problems of development of antibiotic resistance in enteric pathogens and the contamination of poultry products with antibiotics. 16S rDNA sequencing which has gained wide popularity amongst microbiologists for the molecular characterization and identification of newly discovered isolates provides accurate identification of isolates down to the level of sub-species (strain). It's most important advantage over the traditional biochemical characterization methods are that it can provide an accurate identification of strains with atypical phenotypic characters as well. The following work is an application of 16S rRNA gene sequencing approach to identify a novel, alkaline protease producing bacteria, from poultry farm waste. The sample was collected from a local poultry farm in the Guntur district, Andhra Pradesh, India. Subsequently the sample was serially diluted and the aliquots were incubated for a suitable time period following which the suspected colony was subjected to 16S rDNA sequencing. The results showed the isolate to be a novel, high alkaline protease producing bacteria, which was named Bacillus firmus isolate EMBS023, after characterization the sequence of isolate was deposited in GenBank with accession number JN990980. PMID:25118655

  2. Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli.

    PubMed

    Luidalepp, Hannes; Berger, Stefan; Joss, Oliver; Tenson, Tanel; Polacek, Norbert

    2016-05-22

    Stationary-phase bacterial cells are characterized by vastly reduced metabolic activities yielding a dormant-like phenotype. Several hibernation programs ensure the establishment and maintenance of this resting growth state. Some of the stationary phase-specific modulations affect the ribosome and its translational activity directly. In stationary-phase Escherichia coli, we observed the appearance of a 16S rRNA fragmentation event at the tip of helix 6 within the small ribosomal subunit (30S). Stationary-phase 30S subunits showed markedly reduced activities in protein biosynthesis. On the other hand, the functional performance of stationary-phase large ribosomal subunits (50S) was indistinguishable from particles isolated from exponentially growing cells. Introduction of the 16S rRNA cut in vitro at helix 6 of exponential phase 30S subunits renders them less efficient in protein biosynthesis. This indicates that the helix 6 fragmentation is necessary and sufficient to attenuate translational activities of 30S ribosomal subunits. These results suggest that stationary phase-specific cleavage of 16S rRNA within the 30S subunit is an efficient means to reduce global translation activities under non-proliferating growth conditions. PMID:27067112

  3. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    PubMed

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  4. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  5. Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

    PubMed Central

    Stackebrandt, E; Witt, D; Kemmerling, C; Kroppenstedt, R; Liesack, W

    1991-01-01

    The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization. Images PMID:1854202

  6. Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences.

    PubMed

    Lu, Jingrang; Domingo, Jorge Santo

    2008-10-01

    The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the microbial composition of avian fecal microbial communities. PMID:18974945

  7. Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

    PubMed

    Lange, Carla C; Brito, Maria A V P; Reis, Daniele R L; Machado, Marco A; Guimarães, Alessandro S; Azevedo, Ana L S; Salles, Érica B; Alvim, Mariana C T; Silva, Fabiana S; Meurer, Igor R

    2015-04-17

    Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms. PMID:25704228

  8. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    PubMed

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions. PMID:22510214

  9. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples

    PubMed Central

    Barb, Jennifer J.; Oler, Andrew J.; Kim, Hyung-Suk; Chalmers, Natalia; Wallen, Gwenyth R.; Cashion, Ann; Munson, Peter J.; Ames, Nancy J.

    2016-01-01

    Objectives There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology. Methods This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9) processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY). Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies) and their sequencing data is subjected to a novel analytical pipeline. Results Results are presented at family and genus level. The Kullback-Leibler divergence (DKL), a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst) average DKL while the V4 gave the lowest (best) average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria. Conclusions The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at

  10. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    PubMed

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings. PMID:25179393

  11. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    SciTech Connect

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  12. 16S rRNA and As-Related Functional Diversity: Contrasting Fingerprints in Arsenic-Rich Sediments from an Acid Mine Drainage.

    PubMed

    Fahy, Anne; Giloteaux, Ludovic; Bertin, Philippe; Le Paslier, Denis; Médigue, Claudine; Weissenbach, Jean; Duran, Robert; Lauga, Béatrice

    2015-07-01

    To gain an in-depth insight into the diversity and the distribution of genes under the particular evolutionary pressure of an arsenic-rich acid mine drainage (AMD), the genes involved in bacterial arsenic detoxification (arsB, ACR3) and arsenite oxidation (aioA) were investigated in sediment from Carnoulès (France), in parallel to the diversity and global distribution of the metabolically active bacteria. The metabolically active bacteria were affiliated mainly to AMD specialists, i.e., organisms detected in or isolated from AMDs throughout the world. They included mainly Acidobacteria and the non-affiliated "Candidatus Fodinabacter communificans," as well as Thiomonas and Acidithiobacillus spp., Actinobacteria, and unclassified Gammaproteobacteria. The distribution range of these organisms suggested that they show niche conservatism. Sixteen types of deduced protein sequences of arsenite transporters (5 ArsB and 11 Acr3p) were detected, whereas a single type of arsenite oxidase (AioA) was found. Our data suggested that at Carnoulès, the aioA gene was more recent than those encoding arsenite transporters and subjected to a different molecular evolution. In contrast to the 16S ribosomal RNA (16S rRNA) genes associated with AMD environments worldwide, the functional genes aioA, ACR3, and to a lesser extent arsB, were either novel or specific to Carnoulès, raising the question as to whether these functional genes are specific to high concentrations of arsenic, AMD-specific, or site-specific. PMID:25592635

  13. New Degenerate Cytophaga-Flexibacter-Bacteroides-Specific 16S Ribosomal DNA-Targeted Oligonucleotide Probes Reveal High Bacterial Diversity in River Taff Epilithon

    PubMed Central

    O’Sullivan, Louise A.; Weightman, Andrew J.; Fry, John C.

    2002-01-01

    River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors. In this study the bacterial diversity of River Taff epilithon in South Wales was investigated. A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones. Members of the Cytophaga-Flexibacter-Bacteroides division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the α subdivision of the division Proteobacteria (α-Proteobacteria), γ-Proteobacteria, gram-positive bacteria, Cyanobacteria, β-Proteobacteria, δ-Proteobacteria, and the Prosthecobacter group. This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376. The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division. CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b. The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced. They affiliated with the RDP-designated groups Cytophaga, Sphingobacterium, Lewinella, and Cytophaga aurantiaca. CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use. Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance. PMID:11772628

  14. Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplificatio...

  15. 16S rRNA gene-based identification of microbiota associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga (Diptera, Psychodidae) from central Amazon, Brazil

    PubMed Central

    de Sousa, Katianne Barbosa Alves; da Silva, Túllio Romão Ribeiro; Alencar, Ronildo Baiatone; Baton, Luke Anthony; Naveca, Felipe Gomes; Shimabukuro, Paloma Helena Fernandes

    2013-01-01

    Bacteria associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga were characterized by sequencing cloned 16S rDNA PCR products. Eleven novel partial 16S rDNA sequences, with varying degrees of similarity to Actinobacteria, were identified. None of the sequences identified had homology to those known from parthenogenesis-inducing bacteria. PMID:24159323

  16. Anaplasma phagocytophilum in Questing Ixodes ricinus Ticks: Comparison of Prevalences and Partial 16S rRNA Gene Variants in Urban, Pasture, and Natural Habitats

    PubMed Central

    Pfister, Kurt; Thiel, Claudia; Herb, Ingrid; Mahling, Monia; Silaghi, Cornelia

    2013-01-01

    Urban, natural, and pasture areas were investigated for prevalences and 16S rRNA gene variants of Anaplasma phagocytophilum in questing Ixodes ricinus ticks. The prevalences differed significantly between habitat types, and year-to-year variations in prevalence and habitat-dependent occurrence of 16S rRNA gene variants were detected. PMID:23263964

  17. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...