The Role of SwrA, DegU and PD3 in fla/che Expression in B. subtilis
Publication Date
December 27, 2013
Journal
PLOS ONE
Authors
Serena Mordini, Cecilia Osera, Simone Marini, Francesco Scavone, et al
Volume
8
Issue
12
Pages
e85065
DOI
https://dx.plos.org/10.1371/journal.pone.0085065
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0085065
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/24386445
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874003
Europe PMC
http://europepmc.org/abstract/MED/24386445
Web of Science
000329117900121
Scopus
84893547455
Mendeley
http://www.mendeley.com/research/role-swra-degu-pd3-flache-expression-b-subtilis
Events
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Mendeley | Further Information

{"title"=>"The Role of SwrA, DegU and PD3 in fla/che Expression in B. subtilis", "type"=>"journal", "authors"=>[{"first_name"=>"Serena", "last_name"=>"Mordini"}, {"first_name"=>"Cecilia", "last_name"=>"Osera"}, {"first_name"=>"Simone", "last_name"=>"Marini"}, {"first_name"=>"Francesco", "last_name"=>"Scavone"}, {"first_name"=>"Riccardo", "last_name"=>"Bellazzi"}, {"first_name"=>"Alessandro", "last_name"=>"Galizzi"}, {"first_name"=>"Cinzia", "last_name"=>"Calvio"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24386445", "doi"=>"10.1371/journal.pone.0085065", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"1932-6203"}, "id"=>"ad61a2b6-91aa-3711-8d26-8d6d563037d2", "abstract"=>"In B. subtilis swarming and robust swimming motility require the positive trigger of SwrA on fla/che operon expression. Despite having an essential and specific activity, how SwrA executes this task has remained elusive thus far. We demonstrate here that SwrA acts at the main σ(A)-dependent fla/che promoter PA(fla/che) through DegU. Electrophoretic mobility shift assays (EMSA) reveal that SwrA forms a complex with the phosphorylated form of DegU (DegU~P) at PA(fla/che) while it is unable to do so with either unphosphorylated DegU or the DegU32(Hy) mutant protein. Motility assays show that a highly phosphorylated DegU is not detrimental for flagellar motility provided that SwrA is present; however, DegU~P represses PA(fla/che) in the absence of SwrA. Overall, our data support a model in which DegU~P is a dual regulator, acting either as a repressor when alone or as a positive regulator of PA(fla/che) when combined with SwrA. Finally, we demonstrate that the σ(D)-dependent PD3(fla/che) promoter plays an important role in motility, representing a contingent feedback loop necessary to maintain basal motility when swrA is switched to the non-functional swrA(-) status.", "link"=>"http://www.mendeley.com/research/role-swra-degu-pd3-flache-expression-b-subtilis", "reader_count"=>21, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Student > Doctoral Student"=>2, "Researcher"=>2, "Student > Ph. D. Student"=>5, "Student > Master"=>6, "Student > Bachelor"=>4}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Student > Doctoral Student"=>2, "Researcher"=>2, "Student > Ph. D. Student"=>5, "Student > Master"=>6, "Student > Bachelor"=>4}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>5, "Agricultural and Biological Sciences"=>11, "Computer Science"=>1, "Sports and Recreations"=>1}, "reader_count_by_subdiscipline"=>{"Sports and Recreations"=>{"Sports and Recreations"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>11}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Unspecified"=>{"Unspecified"=>3}}, "reader_count_by_country"=>{"Germany"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1330574"], "description"=>"<p>The effect of a P<sub>D3(fla/che)</sub> deletion on motility was evaluated in strains differing in the status of the <i>swrA</i> allele or of the σ<sup>D</sup>-dependent <i>swrA</i> promoter. The isogenic strains used are PB5392 [<i>swrA</i><sup>+</sup> P<sub>D3(fla/che)</sub>wt], PB5455 [<i>swrA</i><sup>+</sup> ΔP<sub>D3(fla/che)</sub>], PB5394 [P<sub><i>swrA</i></sub>D<sup>-</sup>-<i>swrA</i><sup>+</sup> P<sub>D3(fla/che)</sub>wt], PB5458 [P<sub><i>swrA</i></sub>D<sup>-</sup>-<i>swrA</i><sup>+</sup> ΔP<sub>D3(fla/che)</sub>], PB5396 [<i>swrA</i><sup>-</sup> P<sub>D3(fla/che)</sub>wt] and PB5466 [<i>swrA</i><sup>-</sup> ΔP<sub>D3(fla/che)</sub>]. (<b>A</b>) Swimming assays. Genotypes are indicated on the left and on top of each panel. (<b>B</b>) Swimming expansion measurements. Relevant genotypes are specified on the right. Results shown are an average of three independent experiments; error bars correspond to standard deviation. (<b>C</b>) Western blot of flagellin collected at T<sub>1</sub> from liquid cultures of the strains used in A and B, vertically aligned to the bullets identifying the strains in B. (<b>D</b>) Swimming assay for a Δ<i>sigD </i><i>swrA</i><sup>+</sup> strain (PB5427) shown as reference for non-motile behavior.</p>", "links"=>[], "tags"=>["motility", "masked"], "article_id"=>888084, "categories"=>["Biological Sciences"], "users"=>["Serena Mordini", "Cecilia Osera", "Simone Marini", "Francesco Scavone", "Riccardo Bellazzi", "Alessandro Galizzi", "Cinzia Calvio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085065.g006", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_contribution_of_P_D3_fla_che_to_motility_is_masked_by_SwrA_/888084", "title"=>"The contribution of P<sub>D3(fla/che)</sub> to motility is masked by SwrA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-27 03:28:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1330569"], "description"=>"<p>(<b>A</b>) The region of the <i>fla/che</i> promoter, including the σ<sup>A</sup> and σ<sup>D</sup>-dependent promoters, is drawn to scale. The coding sequences of the preceding gene (codY) and of the first gene of <i>fla/che</i> (flgB) are in black. The span of the P<sub>A(fla/che)</sub> probe, of the K probe used in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085065#pone.0085065.s001\" target=\"_blank\">Figure S1</a> and of the markerless P<sub>D3</sub> deletion are indicated. The bar corresponds to 50 nt. (<b>B</b>) EMSA with fluorescently labeled P<sub>A(fla/che)</sub> probe. DegU, DegS and SwrA (listed on the left above the gel) were added to reactions labeled with a + above the lane. In each 10 μL-reaction proteins had the following concentrations: 0.35 μM for DegU; 0.4 μM for DegS; 1 μM for SwrA. ATP was omitted from the reaction in lane 9. On the right of the gel, the arrowhead labeled with S points to the probe shifted by DegU~P (lane 3); the SS arrow marks the super-shifted complex formed upon SwrA addition (lane 8). (<b>C</b>) Competition reactions with an excess (20 ng) of unlabeled P<sub>A(fla/che)</sub> DNA or with the same amount of unlabeled non-specific (N-S) DNA are indicated above the gel. The EMSA probe was radioactively labeled. In each reaction, the proteins had following concentration: 0.2 μM DegU, 0.1 μM DegS and 0.2 μM SwrA. (<b>D</b>) A GST-tag fused to SwrA reduces the migration of the SS complex. Radioactively labeled P<sub>A(fla/che)</sub> was incubated with DegU (0.1 μM), DegS (0.1 μM), and either SwrA (lane 6), GST-SwrA (lane 7) or GST (lane 8), each at 1.5 μM. The grey arrow labeled with SS* points at the retarded band obtained with GTS-SwrA (lane 7).</p>", "links"=>[], "tags"=>["bound"], "article_id"=>888079, "categories"=>["Biological Sciences"], "users"=>["Serena Mordini", "Cecilia Osera", "Simone Marini", "Francesco Scavone", "Riccardo Bellazzi", "Alessandro Galizzi", "Cinzia Calvio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085065.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SwrA_is_bound_to_P_A_fla_che_through_DegU_P_/888079", "title"=>"SwrA is bound to P<sub>A(fla/che)</sub> through DegU~P.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-27 03:28:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1330570"], "description"=>"<p>Fluorescently labeled P<sub>A(fla/che)</sub> was challenged with the mutant DegU32(Hy) or DegS200(Hy) proteins in EMSA. DegS200(Hy) was used to phosphorylated wild-type DegU in lanes 10 and 11. DegU32(Hy) was phosphorylated by wild-type DegS in lanes 5 and 6. Protein concentrations were the following: 0.2 μM for DegS; 0.4 μM for DegS200(Hy); 0.36 μM for DegU and 1 μM for SwrA. Different concentrations of DegU32(Hy) were used: 0.3 μM in lane 2; 0.6 μM in lanes 3, 5 and 6; 1.8 μM in lane 4. Control reactions with wild-type proteins are shown in lanes 7, 8, 9. An asterisk marks the particular pattern of bands, bracketed on the left-hand side of the gel, produced by DegU32(Hy) and DegU32(Hy)<b>~</b>P. Other symbols are the same as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085065#pone-0085065-g002\" target=\"_blank\">Figure 2B</a>. </p>", "links"=>[], "tags"=>[], "article_id"=>888080, "categories"=>["Biological Sciences"], "users"=>["Serena Mordini", "Cecilia Osera", "Simone Marini", "Francesco Scavone", "Riccardo Bellazzi", "Alessandro Galizzi", "Cinzia Calvio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085065.g003", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DegU32_Hy_and_DegS200_Hy_behave_differently_in_EMSA_/888080", "title"=>"DegU32(Hy) and DegS200(Hy) behave differently in EMSA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-27 03:28:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1330583", "https://ndownloader.figshare.com/files/1330585", "https://ndownloader.figshare.com/files/1330586", "https://ndownloader.figshare.com/files/1330587", "https://ndownloader.figshare.com/files/1330588", "https://ndownloader.figshare.com/files/1330589"], "description"=>"<div><p>In <i>B. subtilis</i> swarming and robust swimming motility require the positive trigger of SwrA on <i>fla/che</i> operon expression. Despite having an essential and specific activity, how SwrA executes this task has remained elusive thus far. We demonstrate here that SwrA acts at the main σ<sup>A</sup>-dependent <i>fla/che</i> promoter P<sub>A(<i>fla/che</i>)</sub> through DegU. Electrophoretic mobility shift assays (EMSA) reveal that SwrA forms a complex with the phosphorylated form of DegU (DegU~P) at P<sub>A(<i>fla/che</i>)</sub> while it is unable to do so with either unphosphorylated DegU or the DegU32(Hy) mutant protein. Motility assays show that a highly phosphorylated DegU is not detrimental for flagellar motility provided that SwrA is present; however, DegU~P represses P<sub>A(<i>fla/che</i>)</sub> in the absence of SwrA. Overall, our data support a model in which DegU~P is a dual regulator, acting either as a repressor when alone or as a positive regulator of P<sub>A(<i>fla/che</i>)</sub> when combined with SwrA. Finally, we demonstrate that the σ<sup>D</sup>-dependent P<sub>D3(<i>fla/che</i>)</sub> promoter plays an important role in motility, representing a contingent feedback loop necessary to maintain basal motility when <i>swrA</i> is switched to the non-functional <i>swrA</i><sup>-</sup> status. </p> </div>", "links"=>[], "tags"=>["degu"], "article_id"=>888093, "categories"=>["Biological Sciences"], "users"=>["Serena Mordini", "Cecilia Osera", "Simone Marini", "Francesco Scavone", "Riccardo Bellazzi", "Alessandro Galizzi", "Cinzia Calvio"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085065.s001", "https://dx.doi.org/10.1371/journal.pone.0085065.s002", "https://dx.doi.org/10.1371/journal.pone.0085065.s003", "https://dx.doi.org/10.1371/journal.pone.0085065.s004", "https://dx.doi.org/10.1371/journal.pone.0085065.s005", "https://dx.doi.org/10.1371/journal.pone.0085065.s006"], "stats"=>{"downloads"=>9, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_Role_of_SwrA_DegU_and_P_D3_in_fla_che_Expression_in_B_subtilis_/888093", "title"=>"The Role of SwrA, DegU and P<sub>D3</sub> in <i>fla/che</i> Expression in <i>B. subtilis</i>", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-12-27 03:28:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1330568"], "description"=>"<p>The chromosomal regions of <i>fla/che</i> and <i>swrA</i> are depicted (not to scale); each locus is preceded by its own σ<sup>A</sup> (P<sub>A</sub>) and σ<sup>D</sup>-dependent (P<sub>D</sub> or P<sub>D3</sub>) promoter indicated by bent black arrows. <i>sigD</i>, the penultimate gene of <i>fla/che</i>, is highlighted. Black arrows indicate direct positive effects. Line thickness is proportional to the strength of the effect of each element. A wavy line represents transcripts originating from each <i>fla/che</i> promoter. Dashed orange arrows mark the autoregulatory loops that can be predicted, which are identified by numbers in parenthesis. (<b>A</b>) In <i>swrA</i><sup>+</sup> strains an extremely efficient loop connects SwrA with <i>fla/che</i> expression. It starts with <i>sigD</i> basal transcription from the P<sub>A(fla/che)</sub> promoter (1); SigD allows transcription of <i>swrA</i> through stimulation of the P<sub>D(swrA)</sub> promoter (2a). SwrA enhances transcription from P<sub>A(fla/che)</sub> (3a) closing the circuitry [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085065#B6\" target=\"_blank\">6</a>, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085065#B8\" target=\"_blank\">8</a>]. (<b>B</b>) In <i>swrA</i><sup>-</sup> strains the closure of the SwrA-based loop is prevented; in these conditions an ancillary and weaker feedback loop takes over. It starts again with <i>sigD</i> transcription P<sub>A(fla/che)</sub> (1); SigD directly activates the weak P<sub>D3(fla/che)</sub> promoter (4) that transcribes <i>fla/che</i> in a positive feedback circuitry. The effect of the weak P<sub>D3(fla/che)</sub>-based loop can be appreciated only in <i>swrA</i><sup>-</sup> strains, although it is also active in <i>swrA</i><sup>+</sup> strains (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085065#pone-0085065-g006\" target=\"_blank\">Fig. 6</a> and text for details).</p>", "links"=>[], "tags"=>["operon"], "article_id"=>888078, "categories"=>["Biological Sciences"], "users"=>["Serena Mordini", "Cecilia Osera", "Simone Marini", "Francesco Scavone", "Riccardo Bellazzi", "Alessandro Galizzi", "Cinzia Calvio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085065.g001", "stats"=>{"downloads"=>3, "page_views"=>45, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_model_for_the_fla_che_operon_double_autoregulation_/888078", "title"=>"Schematic model for the <i>fla/che</i> operon double-autoregulation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-27 03:28:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1330573"], "description"=>"<p>In the A and B panels swimming (left) and swarming (right) performances of <i>swrA</i><sup>-</sup> or <i>swrA</i><sup>- </sup><i>degU32</i>(Hy) strains containing the <i>dhsA6</i> mutation is respectively shown. The single nucleotide <i>dhsA6</i> mutation in P<sub>A(fla/che)</sub> improves similarity to the σ<sup>A</sup> consensus sequence [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085065#B16\" target=\"_blank\">16</a>] and thus the predicted promoter strength. Motility of the parental <i>swrA</i><sup>-</sup> and <i>swrA</i><sup>- </sup><i>degU32</i>(Hy) strains is shown in C and D respectively. Relevant genotypes are indicated on top of each panel. Strains used to generate the images are: PB5452 (A); PB5447 (B); PB5370 (C); PB5384 (D). </p>", "links"=>[], "tags"=>["defects", "caused", "alleles", "suppressed", "optimized"], "article_id"=>888083, "categories"=>["Biological Sciences"], "users"=>["Serena Mordini", "Cecilia Osera", "Simone Marini", "Francesco Scavone", "Riccardo Bellazzi", "Alessandro Galizzi", "Cinzia Calvio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085065.g005", "stats"=>{"downloads"=>0, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Motility_defects_caused_by_the_swrA_and_degU32_Hy_alleles_are_suppressed_by_an_optimized_P_A_fla_che_/888083", "title"=>"Motility defects caused by the <i>swrA</i><sup>-</sup> and <i>degU32</i>(Hy) alleles are suppressed by an optimized P<sub>A(fla/che)</sub>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-27 03:28:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1330572"], "description"=>"<p>Swimming (on the left of each panel) and swarming (on the right) motility assays for strains differing in the status of the <i>swrA</i> allele (indicated on top of each panel) and of the <i>degS/degU</i> alleles (indicated on the left). Each strain was inoculated at the center of motility plates and images were taken after 13 hr of incubation. Strains are: PB5384 (A) and PB5383 (B) for <i>degU32</i>(Hy); PB5391 (C) and PB5390 (D) for <i>degS200</i>(Hy); PB5370 (E) and PB5249 (F) for wild-type <i>degS/degU</i>.</p>", "links"=>[], "tags"=>["repressed"], "article_id"=>888082, "categories"=>["Biological Sciences"], "users"=>["Serena Mordini", "Cecilia Osera", "Simone Marini", "Francesco Scavone", "Riccardo Bellazzi", "Alessandro Galizzi", "Cinzia Calvio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085065.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Motility_is_not_repressed_by_high_levels_of_DegU_P_in_the_presence_of_SwrA_/888082", "title"=>"Motility is not repressed by high levels of DegU~P in the presence of SwrA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-27 03:28:29"}

PMC Usage Stats | Further Information

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Relative Metric

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