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      Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110

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          Abstract

          Small open reading frames (sORFs) and genes for non-coding RNAs are poorly investigated components of most genomes. Our analysis of 1391 ORFs recently annotated in the soybean symbiont Bradyrhizobium japonicum USDA 110 revealed that 78% of them contain less than 80 codons. Twenty-one of these sORFs are conserved in or outside Alphaproteobacteria and most of them are similar to genes found in transposable elements, in line with their broad distribution. Stabilizing selection was demonstrated for sORFs with proteomic evidence and bll1319_ISGA which is conserved at the nucleotide level in 16 alphaproteobacterial species, 79 species from other taxa and 49 other Proteobacteria. Further we used Northern blot hybridization to validate ten small RNAs (BjsR1 to BjsR10) belonging to new RNA families. We found that BjsR1 and BjsR3 have homologs outside the genus Bradyrhizobium, and BjsR5, BjsR6, BjsR7, and BjsR10 have up to four imperfect copies in Bradyrhizobium genomes. BjsR8, BjsR9, and BjsR10 are present exclusively in nodules, while the other sRNAs are also expressed in liquid cultures. We also found that the level of BjsR4 decreases after exposure to tellurite and iron, and this down-regulation contributes to survival under high iron conditions. Analysis of additional small RNAs overlapping with 3’-UTRs revealed two new repetitive elements named Br-REP1 and Br-REP2. These REP elements may play roles in the genomic plasticity and gene regulation and could be useful for strain identification by PCR-fingerprinting. Furthermore, we studied two potential toxin genes in the symbiotic island and confirmed toxicity of the yhaV homolog bll1687 but not of the newly annotated higB homolog blr0229_ISGA in E. coli. Finally, we revealed transcription interference resulting in an antisense RNA complementary to blr1853, a gene induced in symbiosis. The presented results expand our knowledge on sORFs, non-coding RNAs and repetitive elements in B. japonicum and related bacteria.

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          Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.

          Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.
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            The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae.

            Quorum-sensing bacteria communicate with extracellular signal molecules called autoinducers. This process allows community-wide synchronization of gene expression. A screen for additional components of the Vibrio harveyi and Vibrio cholerae quorum-sensing circuits revealed the protein Hfq. Hfq mediates interactions between small, regulatory RNAs (sRNAs) and specific messenger RNA (mRNA) targets. These interactions typically alter the stability of the target transcripts. We show that Hfq mediates the destabilization of the mRNA encoding the quorum-sensing master regulators LuxR (V. harveyi) and HapR (V. cholerae), implicating an sRNA in the circuit. Using a bioinformatics approach to identify putative sRNAs, we identified four candidate sRNAs in V. cholerae. The simultaneous deletion of all four sRNAs is required to stabilize hapR mRNA. We propose that Hfq, together with these sRNAs, creates an ultrasensitive regulatory switch that controls the critical transition into the high cell density, quorum-sensing mode.
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              Proteogenomics: concepts, applications and computational strategies.

              Proteogenomics is an area of research at the interface of proteomics and genomics. In this approach, customized protein sequence databases generated using genomic and transcriptomic information are used to help identify novel peptides (not present in reference protein sequence databases) from mass spectrometry-based proteomic data; in turn, the proteomic data can be used to provide protein-level evidence of gene expression and to help refine gene models. In recent years, owing to the emergence of new sequencing technologies such as RNA-seq and dramatic improvements in the depth and throughput of mass spectrometry-based proteomics, the pace of proteogenomic research has greatly accelerated. Here I review the current state of proteogenomic methods and applications, including computational strategies for building and using customized protein sequence databases. I also draw attention to the challenge of false positive identifications in proteogenomics and provide guidelines for analyzing the data and reporting the results of proteogenomic studies.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                27 October 2016
                2016
                : 11
                : 10
                : e0165429
                Affiliations
                [1 ]Institute of Microbiology and Molecular Biology, Justus-Liebig-University, Giessen, Germany
                [2 ]A. A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Bolshoi Karetny Ln. 19, Moscow, 127051, Russia
                [3 ]ETH, Institute of Molecular Systems Biology, Zürich, Switzerland
                [4 ]Skolkovo Institute of Science and Technology, Nobel Str. 3, Moscow, 143026, Russia
                [5 ]Faculty of Bioengineering and Bioinformatics, M. V. Lomonosov Moscow State University, Vorobyevy Gory 1–73, Moscow, 119234, Russia
                [6 ]Faculty of Computer Science, Higher School of Economics, Kochnovsky Dr. 3, Moscow, 125319, Russia
                INSERM U869, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: EEH MSG.

                • Data curation: OVT.

                • Formal analysis: EEH JČ JH OVT ST.

                • Funding acquisition: EEH MSG.

                • Investigation: JČ JH OVT ST.

                • Methodology: EEH JH MSG OVT ST.

                • Software: OVT.

                • Supervision: EEH MSG.

                • Validation: JH ST.

                • Visualization: JČ JH OVT ST.

                • Writing – original draft: EEH JH OVT.

                • Writing – review & editing: EEH MSG.

                Author information
                http://orcid.org/0000-0001-7270-3168
                Article
                PONE-D-16-11375
                10.1371/journal.pone.0165429
                5082802
                27788207
                63ec95bb-4ac7-42f6-8cdc-5e9fc8de6b67
                © 2016 Hahn et al

                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 author and source are credited.

                History
                : 18 March 2016
                : 11 October 2016
                Page count
                Figures: 10, Tables: 1, Pages: 24
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: EV42/4-2
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: IRTG 1384
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100006769, Russian Science Foundation;
                Award ID: 14-24-00155
                Award Recipient :
                This work was supported by Deutsche Forschungsgemeinschaft (grant DFG EV42/4-2 to E. E.-H.) and Russian Science Foundation (O.V.T. and M.S.G., grant 14-24-00155). J.H. is a member of IRTG 1384 “Enzymes and multienzyme complexes acting on nucleic acids” supported by Deutsche Forschungsgemeinschaft. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Bacteria
                Bradyrhizobium
                Biology and Life Sciences
                Biochemistry
                Proteomics
                Biology and life sciences
                Genetics
                DNA
                Forms of DNA
                Complementary DNA
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                Forms of DNA
                Complementary DNA
                Biology and Life Sciences
                Molecular Biology
                Macromolecular Structure Analysis
                Protein Structure
                Protein Structure Comparison
                Biology and Life Sciences
                Biochemistry
                Proteins
                Protein Structure
                Protein Structure Comparison
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Messenger RNA
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Molecular probe techniques
                Probe hybridization
                RNA hybridization
                Research and analysis methods
                Molecular biology techniques
                Molecular probe techniques
                Probe hybridization
                RNA hybridization
                Biology and Life Sciences
                Species Interactions
                Symbiosis
                Research and Analysis Methods
                Electrophoretic Techniques
                Gel Electrophoresis
                Electrophoretic Blotting
                Northern Blot
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Probe Techniques
                Electrophoretic Blotting
                Northern Blot
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Probe Techniques
                Electrophoretic Blotting
                Northern Blot
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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