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      Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge

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          Abstract

          Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates—ciliates frequently found in anoxic ecosystems—on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages ( i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates ( i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885–3,190 and 2,387–2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.

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          The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions.

          Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplification products was evaluated by comparisons with sequences of previously reported rRNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.
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            GuiTope: an application for mapping random-sequence peptides to protein sequences

            Background Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task. Results GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance. Conclusions GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC) at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.
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              Anaerolinea thermolimosa sp. nov., Levilinea saccharolytica gen. nov., sp. nov. and Leptolinea tardivitalis gen. nov., sp. nov., novel filamentous anaerobes, and description of the new classes Anaerolineae classis nov. and Caldilineae classis nov. in the bacterial phylum Chloroflexi.

              One thermophilic (strain IMO-1(T)) and two mesophilic (strains KIBI-1(T) and YMTK-2(T)) non-spore-forming, non-motile, Gram-negative, multicellular filamentous micro-organisms, which were previously isolated as members of the tentatively named class 'Anaerolineae' of the phylum Chloroflexi, were characterized. All isolates were strictly anaerobic micro-organisms. The length of the three filamentous isolates was greater than 100 microm and the width was 0.3-0.4 microm for strain IMO-1(T), 0.4-0.5 microm for strain KIBI-1(T) and thinner than 0.2 microm for strain YMTK-2(T). Strain IMO-1(T) could grow at pH 6.0-7.5 (optimum growth at pH 7.0). The optimal temperature for growth of strain IMO-1(T) was around 50 degrees C (growth occurred between 42 and 55 degrees C). Growth of the mesophilic strains KIBI-1(T) and YMTK-2(T) occurred at pH 6.0-7.2 with optimal growth at pH 7.0. Both of the mesophilic strains were able to grow in a temperature range of 25-50 degrees C with optimal growth at around 37 degrees C. Yeast extract was required for growth of all three strains. All the strains could grow with a number of carbohydrates in the presence of yeast extract. The G + C contents of the DNA of strains IMO-1(T), KIBI-1(T) and YMTK-2(T) were respectively 53.3, 59.5 and 48.2 mol%. Major fatty acids for thermophilic strain IMO-1(T) were anteiso-C(17 : 0), iso-C(15 : 0), C(16 : 0) and anteiso-C(15 : 0), whereas those for mesophilic strains KIBI-1(T) and YMTK-2(T) were branched C(14 : 0), iso-C(15 : 0), C(16 : 0) and branched C(17 : 0), and branched C(17 : 0), C(16 : 0), C(14 : 0) and C(17 : 0), respectively. Detailed phylogenetic analyses based on their 16S rRNA gene sequences indicated that the isolates belong to the class-level taxon 'Anaerolineae' of the bacterial phylum Chloroflexi, which for a long time had been considered as a typical uncultured clone cluster. Their morphological, physiological, chemotaxonomic and genetic traits strongly support the conclusion that these strains should be described as three novel independent taxa in the phylum Chloroflexi. Here, Anaerolinea thermolimosa sp. nov. (type strain IMO-1(T) = CM 12577(T) = DSM 16554(T)), Levilinea saccharolytica gen. nov., sp. nov. (type strain KIBI-1(T) = JCM 12578(T) = DSM 16555(T)) and Leptolinea tardivitalis gen. nov., sp. nov. (type strain YMTK-2(T) = JCM 12579(T) = DSM 16556(T)) are proposed. In addition, we formally propose to subdivide the tentative class-level taxon 'Anaerolineae' into Anaerolineae classis nov. and Caldilineae classis nov. We also propose the subordinate taxa Anaerolineales ord. nov., Caldilineales ord. nov., Anaerolineaceae fam. nov. and Caldilineaceae fam. nov.
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                Author and article information

                Journal
                Microbes Environ
                Microbes Environ
                Microbes and Environments
                the Japanese Society of Microbial Ecology (JSME)/the Japanese Society of Soil Microbiology (JSSM)/the Taiwan Society of Microbial Ecology (TSME)/the Japanese Society of Plant Microbe Interactions (JSPMI)
                1342-6311
                1347-4405
                September 2016
                12 July 2016
                : 31
                : 3
                : 279-287
                Affiliations
                [1 ]Department of Civil Engineering, National Institute of Technology, Nagaoka College Nagaoka, NiigataJapan
                [2 ]Department of Science of Technology Innovation, Nagaoka University of Technology Nagaoka, NiigataJapan
                [3 ]Department of Environmental Systems Engineering, Nagaoka University of Technology Nagaoka, NiigataJapan
                [4 ]Department of Civil and Environmental Engineering, Tohoku University Sendai, MiyagiJapan
                [5 ]New Industry Creation Hatchery Center (NICHe), Tohoku University Sendai, MiyagiJapan
                Author notes
                [* ]Corresponding author. E-mail: oshiki@ 123456nagaoka-ct.ac.jp ; Tel: +81–258–34–9277; Fax: +81–258–34–9284.
                Article
                31_279
                10.1264/jsme2.ME16067
                5017804
                27431197
                3c82e66c-fdeb-404b-814c-06a9a43462b8
                Copyright © 2016 by Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions.

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 04 April 2016
                : 20 May 2016
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                anaerobic protozoa,up-flow anaerobic sludge blanket (uasb) reactor,anaerobic granular sludge

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