Concerted Actions of a Thermo-labile Regulator and a Unique Intergenic RNA Thermosensor Control Yersinia Virulence
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{"title"=>"Concerted actions of a thermo-labile regulator and a unique intergenic RNA thermosensor control Yersinia virulence", "type"=>"journal", "authors"=>[{"first_name"=>"Katja", "last_name"=>"Böhme", "scopus_author_id"=>"23472214300"}, {"first_name"=>"Rebekka", "last_name"=>"Steinmann", "scopus_author_id"=>"55215111800"}, {"first_name"=>"Jens", "last_name"=>"Kortmann", "scopus_author_id"=>"23135042000"}, {"first_name"=>"Stephanie", "last_name"=>"Seekircher", "scopus_author_id"=>"36163722400"}, {"first_name"=>"Ann Kathrin", "last_name"=>"Heroven", "scopus_author_id"=>"6506667327"}, {"first_name"=>"Evelin", "last_name"=>"Berger", "scopus_author_id"=>"57198319630"}, {"first_name"=>"Fabio", "last_name"=>"Pisano", "scopus_author_id"=>"35933525700"}, {"first_name"=>"Tanja", "last_name"=>"Thiermann", "scopus_author_id"=>"55043246200"}, {"first_name"=>"Hans", "last_name"=>"Wolf-Watz", "scopus_author_id"=>"7005842190"}, {"first_name"=>"Franz", "last_name"=>"Narberhaus", "scopus_author_id"=>"7003831342"}, {"first_name"=>"Petra", "last_name"=>"Dersch", "scopus_author_id"=>"6701882734"}], "year"=>2012, "source"=>"PLoS Pathogens", "identifiers"=>{"pmid"=>"22359501", "scopus"=>"2-s2.0-84871714811", "pui"=>"364799336", "issn"=>"15537366", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "doi"=>"10.1371/journal.ppat.1002518", "sgr"=>"84871714811"}, "id"=>"2697268d-f3b2-3f77-a9c9-42ec75f32cd3", "abstract"=>"Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.", "link"=>"http://www.mendeley.com/research/concerted-actions-thermolabile-regulator-unique-intergenic-rna-thermosensor-control-yersinia-virulen", "reader_count"=>94, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>5, "Researcher"=>21, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>31, "Student > Postgraduate"=>5, "Student > Master"=>12, "Student > Bachelor"=>10, "Lecturer > Senior Lecturer"=>1, "Professor"=>4}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>5, "Researcher"=>21, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>31, "Student > Postgraduate"=>5, "Student > Master"=>12, "Student > Bachelor"=>10, "Lecturer > Senior Lecturer"=>1, "Professor"=>4}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>10, "Agricultural and Biological Sciences"=>69, "Medicine and Dentistry"=>1, "Arts and Humanities"=>1, "Veterinary Science and Veterinary Medicine"=>1, "Chemistry"=>1, "Immunology and Microbiology"=>7}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>7}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>69}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}, "Unspecified"=>{"Unspecified"=>3}, "Arts and Humanities"=>{"Arts and Humanities"=>1}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"Iran"=>1, "United States"=>2, "Germany"=>1}, "group_count"=>3}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/347815", "https://ndownloader.figshare.com/files/347868", "https://ndownloader.figshare.com/files/347921", "https://ndownloader.figshare.com/files/347957", "https://ndownloader.figshare.com/files/348004", "https://ndownloader.figshare.com/files/348051", "https://ndownloader.figshare.com/files/348097", "https://ndownloader.figshare.com/files/348182", "https://ndownloader.figshare.com/files/348246"], "description"=>"<div><p>Expression of all <em>Yersinia</em> pathogenicity factors encoded on the virulence plasmid, including the <em>yop</em> effector and the <em>ysc</em> type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of <em>lcrF</em> is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the <em>yscW</em>-<em>lcrF</em> operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique <em>cis</em>-acting RNA element located within the intergenic region of the <em>yscW-lcrF</em> transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the <em>lcrF</em> ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different <em>Y. pseudotuberculosis</em> patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, <em>Yersinia</em> strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.</p> </div>", "links"=>[], "tags"=>["concerted", "actions", "thermo-labile", "intergenic", "rna", "thermosensor", "virulence"], "article_id"=>128773, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.s001", "https://dx.doi.org/10.1371/journal.ppat.1002518.s002", "https://dx.doi.org/10.1371/journal.ppat.1002518.s003", "https://dx.doi.org/10.1371/journal.ppat.1002518.s004", "https://dx.doi.org/10.1371/journal.ppat.1002518.s005", "https://dx.doi.org/10.1371/journal.ppat.1002518.s006", "https://dx.doi.org/10.1371/journal.ppat.1002518.s007", "https://dx.doi.org/10.1371/journal.ppat.1002518.s008", "https://dx.doi.org/10.1371/journal.ppat.1002518.s009"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Concerted_Actions_of_a_Thermo_labile_Regulator_and_a_Unique_Intergenic_RNA_Thermosensor_Control_Yersinia_Virulence/128773", "title"=>"Concerted Actions of a Thermo-labile Regulator and a Unique Intergenic RNA Thermosensor Control <em>Yersinia</em> Virulence", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-02-16 02:26:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/682085"], "description"=>"<p>(<b>A</b>) Schematic presentation of the <i>yscW-lacZ</i> and <i>yscW-lcrF-lacZ</i> fusion plasmids. Numbers given in brackets represent the nucleotide positions of the 5′-end of the <i>yscW</i> regulatory region of the fusion constructs with respect to the start codon of <i>yscW</i>. The <i>yscW</i> gene is indicated in grey, the 5′-portion of the <i>lcrF</i> gene is given in black and the <i>lacZ</i> reporter gene is illustrated by a white arrow. (<b>B</b>) Strains YPIII and YP50 (Δ<i>ymoA</i>) harboring the <i>yscW-lacZ</i> (pKB10) or the <i>yscW-lcrF-lacZ</i> (pSF3 and pSF4) fusion plasmids ± pAKH71 (<i>ymoA</i><sup>+</sup>) were grown overnight in LB medium at 25°C or 37°C. β-Galactosidase activity from overnight cultures was determined and is given in µmol min<sup>−1</sup> mg<sup>−1</sup> for comparison. The data represent the average ± SD from at least three different experiments each done in duplicate. Data were analyzed by the Student's t test. Stars indicate the results that differed significantly from those of YPIII at the same temperature with ** (P<0.01), and *** (P<0.001). The activity of all reporter constructs differed significantly between 25°C and 37°C with P<0.001 (not shown). (<b>C</b>) Whole-cell extracts from overnight cultures of <i>Y. pseudotuberculosis</i> wild type and the mutant strains YP66 (Δ<i>lcrF</i>) and YP50 (Δ<i>ymoA</i>) grown at 25°C or 37°C were prepared and analysed by Western blotting with a polyclonal antibody directed against LcrF. A molecular weight marker is loaded on the left. A higher molecular weight protein (c) that reacted with the polyclonal antiserum was used as loading control.</p>", "links"=>[], "tags"=>["operon"], "article_id"=>352564, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_the_yscW_lcrF_operon_in_response_to_temperature_/352564", "title"=>"Expression of the <i>yscW-lcrF</i> operon in response to temperature.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:42:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/682161"], "description"=>"<p>(<b>A</b>) Schematic presentation of the <i>yscW-lcrF</i> operon, the <i>yscW-lcrF</i> mRNA and the <i>lcrF</i> probe used for Northern Blot analysis shown below. (<b>B</b>) Total RNA of YPIII, YP50 and YP66 was prepared, separated on a 1.2% agarose gel, transferred onto a Nylon membrane and probed with Digoxigenin-labelled PCR fragment encoding the <i>lcrF</i> gene. The 16S and 23S rRNAs are shown as RNA loading control. A RNA marker is loaded on the left.</p>", "links"=>[], "tags"=>["mrna", "wildtype", "mutant"], "article_id"=>352641, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_the_yscW_lcrF_mRNA_in_wildtype_and_the_ymoA_mutant_strain_/352641", "title"=>"Analysis of the <i>yscW-lcrF</i> mRNA in wildtype and the <i>ymoA</i> mutant strain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:44:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/682266"], "description"=>"<p>(<b>A</b>) The 5′-ends of the reverse transcription products are indicated by vertical arrows on the schematic presentation of the <i>yscW-lcrF</i> mRNA. The numbers indicate the position of the nucleotide of the 5′-ends with respect to the start codon of <i>yscW</i>. Location of the annealed primer used for primer extension is indicted by a horizontal arrow. (<b>B</b>) Total RNA of <i>Y. pseudotuberculosis</i> YPIII was prepared and used for primer extension analysis. Primers specific for the <i>yscW</i> coding sequence and 20 µg template RNA were applied for primer extension and obtained products were separated on a denaturing 6% polyacrylamide/urea gel. Sequencing reactions performed with the same primer are shown on the left. The sequence of the promoter region is shown on the right and identified 5′-end of the detected primer extension products are given in bold. (<b>C</b>) The regulatory region of the <i>yscW-lcrF</i> operon is shown. The broken arrows indicate the 5′-end points of the promoter deletion constructs and straight arrows show the 5′-end of the degradation products. The −35 and −10 region of the identified promoter is underlined, the transcriptional start site is given in bold, and the Shine-Dalgarno sequence (SD) is indicated.</p>", "links"=>[], "tags"=>["transcription", "primer"], "article_id"=>352748, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mapping_of_the_lcrF_transcription_start_site_by_primer_extension_analysis_/352748", "title"=>"Mapping of the <i>lcrF</i> transcription start site by primer extension analysis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:45:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/682353"], "description"=>"<p>(<b>A</b>) Schematic presentation of the reporter gene fusion harboring the <i>yscW-lcrF</i> intergenic region and different portions of <i>yscW</i> under control of the P<i><sub>BAD</sub></i> promoter. (<b>B</b>) <i>E. coli</i> K-12 harboring the different P<i><sub>BAD</sub></i>::<i>yscW-lcrF-lacZ</i> reporter plasmids (pED10, pED11 and pKB14) or the P<i><sub>BAD</sub></i>::<i>gnd-lacZ</i> control plasmid (pED05) were grown overnight in LB medium at 25°C or 37°C in the presence of 0.05% arabinose. β-Galactosidase activity from overnight cultures was determined and is given in mmol min<sup>−1</sup> mg<sup>−1</sup> for comparison. The data represent the average ± SD from at least three different experiments each done in duplicate. Data were analyzed by the Student's t test. Stars indicate the reporter activity that differed significantly between 25°C and 37°C with ** (P<0.01), and *** (P<0.001).</p>", "links"=>[], "tags"=>["intergenic", "operon", "implicated", "lcrf"], "article_id"=>352832, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_intergenic_region_of_the_yscW_lcrF_operon_is_implicated_in_the_temperature_control_of_LcrF_production_/352832", "title"=>"The intergenic region of the <i>yscW-lcrF</i> operon is implicated in the temperature control of LcrF production.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:47:12"}
  • {"files"=>["https://ndownloader.figshare.com/files/682444"], "description"=>"<p>(<b>A</b>) The Mfold program <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002518#ppat.1002518-Zuker2\" target=\"_blank\">[81]</a> was used for the prediction of the secondary structure of the <i>yscW-lcrF</i> 124 nt intergenic region. The most probable prediction with the lowest free energy is shown. The blue dots represent base pairing. The start of the protein synthesis at the AUG start codon (START) and the ribosome binding site (RBS) paired with the fourU motif are labelled. Deletion of the hairpin I and II are indicated. Nucleotide exchanges leading to increased complementarity are marked in red, mutations impairing base pair formation are given in green. Numbers indicate the nucleotides relative to the <i>lcrF</i> start codon. (<b>B</b>) Strains YPIII harboring the P<i><sub>BAD</sub></i>::<i>lcrF</i>′ (−124)-‘<i>lacZ</i>, including the different hairpin deletions or nucleotide exchanges were grown overnight in LB medium at 25°C or 37°C supplemented with 0.05% arabinose. β-Galactosidase activity from overnight cultures was determined and is given in µmol min<sup>−1</sup> mg<sup>−1</sup> for comparison. The data represent the average ± SD from at least three different experiments each done in duplicate. Data were analyzed by the Student's t test. Stars indicate the results that differed significantly from those of the wildtype at the same temperature with * (P<0.05), ** (P<0.01), and *** (P<0.001). (<b>C</b>) <i>Y. pseudotuberculosis</i> strains harboring the deletion and nucleotide substitution illustrated in (<b>A</b>) in the pYV were grown at 25°C and 37°C. Whole cell extracts of equal amounts of the bacteria were prepared, separated on a 15% SDS polyacrylamide gel, transferred onto a Immobilon membrane and intracellular LcrF was visualized by Western blotting. A higher molecular weight protein (c) that reacted with the polyclonal antiserum was used as loading control.</p>", "links"=>[], "tags"=>["rna"], "article_id"=>352926, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Predicted_secondary_structure_of_the_lcrF_RNA_thermometer_/352926", "title"=>"Predicted secondary structure of the <i>lcrF</i> RNA thermometer.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:48:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/682555"], "description"=>"<p>(<b>A</b>) Enzymatic hydrolysis of the <i>yscW-lcrF</i> wildtype sequence with endonucleases T1 (0.001 U/µl) and V (0.0002 U/µl) performed on the 5′-end labelled intergenic region between the <i>yscW</i> and <i>lcrF</i> gene on the pYV at 25°C and 37°C. The G nucleotides in single stranded regions are indicated. (<b>B</b>) Magnification of the enzymatic probing pattern of the fourU/Shine-Dalgarno region. (<b>C</b>) Enzymatic probing of the <i>yscW-lcrF</i> of the repressed UU-28/-27CC variant, and (<b>D</b>) enzymatic probing of the <i>yscW-lcrF</i> of the derepressed GUU-30/-28AAA variant. The RNA fragments were separated on 8% polyacrylamide gels. Lane L: alkaline ladder; lane C: controls without RNase. The Shine-Dalgarno sequence and the nucleotide exchanges are indicated.</p>", "links"=>[], "tags"=>["probing", "intergenic"], "article_id"=>353037, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Enzymatic_probing_of_the_yscW_lcrF_intergenic_region_variants_/353037", "title"=>"Enzymatic probing of the <i>yscW-lcrF</i> intergenic region variants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:50:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/682665"], "description"=>"<p>Toeprinting analysis was performed with the wildtype, the repressed (UU-28/-27CC) and derepressed (GUU-30/-28AAA) variants as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002518#s4\" target=\"_blank\">material and methods</a>. The presence (+) and absence (−) of the 30S ribosomal subunits are indicated. The terminated primer extension products (toeprints) are marked. The sequencing ladder (ACGU) generated with the same <i>lcrF</i>-specific primer is loaded on the left. The positions of the fourU motif, the Shine-Dalgarno sequence and the start codon AUG are indicated.</p>", "links"=>[], "tags"=>["binding", "ribosomes", "intergenic"], "article_id"=>353146, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Temperature_dependent_binding_of_ribosomes_to_the_yscW_lcrF_intergenic_region_/353146", "title"=>"Temperature-dependent binding of ribosomes to the <i>yscW-lcrF</i> intergenic region.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:52:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/682751"], "description"=>"<p>(<b>A</b>) Strains YPIII (wildtype), YP66 (Δ<i>lcrF</i>) and the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) harboring the <i>yadA-lacZ</i> fusion plasmid pSF1 were grown in LB medium at 25°C or 37°C. β-Galactosidase activity from overnight cultures was determined and is given in µmol min<sup>−1</sup> mg<sup>−1</sup> for comparison. The data represent the average ± SD from at least three different experiments each done in duplicate. Data were analyzed by the Student's t test. Stars indicate the results that differed significantly from those of the wildtype at the same temperature with ** (P<0.01), and *** (P<0.001). Whole-cell extracts from overnight cultures of <i>Y. pseudotuberculosis</i> YPIII (wildtype) and the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) grown at 25°C or 37°C were prepared, and analysed by Western blotting with a polyclonal antibody directed against LcrF and YadA. A higher molecular weight protein (c) was used as control the protein content of the cell extracts. (<b>B</b>) Strains YPIII (wildtype) and the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) harboring the <i>yopE-luxCDABE</i> plasmid pWO34 were grown in LB medium at 25°C and 37°C. Bioluminescence emitted by the cultures was monitored and is given as relative luminescence units (RLU) and represents the mean of three independent experiments done in triplicate. Data were analyzed by the Student's t test. Stars indicate the results that differed significantly from those of the wildtype at the same temperature with ** (P<0.01), and *** (P<0.001). The panel below shows TCA-precipitated supernatants of YPIII (wildtype), the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) grown at 25°C and 37°C in the presence (+) or absence (−) of Ca<sup>2+</sup>. The secreted Yop proteins are indicated.</p>", "links"=>[], "tags"=>["thermosensor-dependent"], "article_id"=>353236, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g008"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_lcrF_thermosensor_dependent_expression_of_the_yadA_and_yopE_genes_/353236", "title"=>"<i>lcrF</i> thermosensor-dependent expression of the <i>yadA</i> and <i>yopE</i> genes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:53:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/682833"], "description"=>"<p>Strains YPIII (wildtype) and the <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) were infected intragastrically (5·10<sup>8</sup> CFU/mice) into BALB/c mice (n = 12/strain). After three days of infection, mice were sacrificed and the number of bacteria in homogenized host tissues and organs was determined by plating. Solid lines indicate the means. The statistical significances between the wildtype and the repressed and derepressed <i>lcrF</i> RNA thermometer variants were determined by Student's t test. P-values: *: <0.05; ***: <0.001.</p>", "links"=>[], "tags"=>["rna", "thermometer", "colonization"], "article_id"=>353314, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g009"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Influence_of_the_lcrF_RNA_thermometer_on_tissue_colonization_by_Y_pseudotuberculosis_/353314", "title"=>"Influence of the <i>lcrF</i> RNA thermometer on tissue colonization by <i>Y. pseudotuberculosis</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:55:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/682902"], "description"=>"<p>(<b>A</b>) 2·10<sup>9</sup> CFU of <i>Y. pseudotuberculosis</i> YPIII (wildtype), the <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA), and YP66 (Δ<i>lcrF</i>) were used to orally infect BALB/c mice (n = 10/strain). (<b>B</b>) 1·10<sup>10</sup> CFU of <i>Y. pseudotuberculosis</i> IP32953 (wildtype), the <i>yscW-lcrF</i> variants YPIP01 (UU-28/-27CC) and YPIP02 (GUU-30/-28AAA) were used to orally infect BALB/c mice (n = 10/strain). Survival of the mice was monitored up to 14 days.</p>", "links"=>[], "tags"=>["rna", "thermometer", "variants", "infected"], "article_id"=>353378, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g010"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_lcrF_RNA_thermometer_variants_affect_survival_of_Y_pseudotuberculosis_infected_mice_/353378", "title"=>"<i>lcrF</i> RNA thermometer variants affect survival of <i>Y. pseudotuberculosis</i> infected mice.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:56:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/682991"], "description"=>"<p>At moderate growth temperature, transcription of the <i>yscW-lcrF</i> operon is repressed by the regulatory protein YmoA through sequences located downstream of the transcription initiation site. In addition, translation of the <i>lcrF</i> transcript is blocked through the formation of a two-stemloop structure within the intergenic region which sequesters the RBS and prevents access of the ribosomes. After a sudden temperature upshift upon host entry, YmoA is rapidly degraded by the ClpP and Lon proteases, leading to an enhanced transcription of the <i>yscW-lcrF</i> operon. Furthermore, thermally-induced conformational changes allow access of ribosomes and translation of the <i>lcrF</i> transcript leading to LcrF synthesis and induction of all LcrF-dependent virulence genes of <i>Yersinia</i>.</p>", "links"=>[], "tags"=>["thermoregulated", "lcrf"], "article_id"=>353471, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.g011"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Model_of_thermoregulated_expression_of_LcrF_synthesis_/353471", "title"=>"Model of thermoregulated expression of LcrF synthesis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-16 00:57:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/683062"], "description"=>"a<p>The number indicates the nucleotide of <i>lcrF</i>, relative to the <i>lcrF</i> start codon.</p>b<p>The number indicates the nucleotide of <i>yscW</i>, relative to the <i>yscW</i> transcriptional start site.</p>", "links"=>[], "tags"=>["strains"], "article_id"=>353543, "categories"=>["Microbiology"], "users"=>["Katja Böhme", "Rebekka Steinmann", "Jens Kortmann", "Stephanie Seekircher", "Ann Kathrin Heroven", "Evelin Berger", "Fabio Pisano", "Tanja Thiermann", "Hans Wolf-Watz", "Franz Narberhaus", "Petra Dersch"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002518.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bacterial_strains_and_plasmids_/353543", "title"=>"Bacterial strains and plasmids.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-02-16 00:59:03"}

PMC Usage Stats | Further Information

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Relative Metric

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