Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis
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{"title"=>"Phenotype enhancement screen of a regulatory Spx mutant unveils a role for the ytpQ gene in the control of Iron homeostasis", "type"=>"journal", "authors"=>[{"first_name"=>"Peter", "last_name"=>"Zuber", "scopus_author_id"=>"7004893453"}, {"first_name"=>"Shefali", "last_name"=>"Chauhan", "scopus_author_id"=>"53163111500"}, {"first_name"=>"Praseeda", "last_name"=>"Pilaka", "scopus_author_id"=>"50661860700"}, {"first_name"=>"Michiko M.", "last_name"=>"Nakano", "scopus_author_id"=>"7403427725"}, {"first_name"=>"Sairam", "last_name"=>"Gurumoorthy", "scopus_author_id"=>"53163815300"}, {"first_name"=>"Ann A.", "last_name"=>"Lin", "scopus_author_id"=>"36017109900"}, {"first_name"=>"Skye M.", "last_name"=>"Barendt", "scopus_author_id"=>"26535516300"}, {"first_name"=>"Bui Khanh", "last_name"=>"Chi", "scopus_author_id"=>"36450231100"}, {"first_name"=>"Haike", "last_name"=>"Antelmann", "scopus_author_id"=>"6603456358"}, {"first_name"=>"Ulrike", "last_name"=>"Mäder", "scopus_author_id"=>"7003437511"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"362585440", "doi"=>"10.1371/journal.pone.0025066", "sgr"=>"80052930551", "scopus"=>"2-s2.0-80052930551", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"21949854"}, "id"=>"183d02c4-d8c9-3dad-88c3-f4a0ab3136cd", "abstract"=>"Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. The regulon that it governs is comprised of over 120 genes based on microarray analysis, although it is not known how many of these are under direct Spx control. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products that are conserved in low %GC Gram-positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as poor growth in minimal medium and sensitivity to methyglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methyglyoxal. The ytpQ mutant showed elevated oxidative protein damage when exposed to methylglyoxal, and reduced growth rate in liquid culture. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons of B. subtilis. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in iron homeostasis. The ytpQ mutant phenotype mimics that of a fur mutation, suggesting a condition of low cellular iron. In vitro transcription analysis indicated that Spx stimulates transcription from the ytpPQR operon within which the ytpQ gene resides. The work uncovers a link between Spx and control of iron homeostasis.", "link"=>"http://www.mendeley.com/research/phenotype-enhancement-screen-regulatory-spx-mutant-unveils-role-ytpq-gene-control-iron-homeostasis", "reader_count"=>12, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Student > Doctoral Student"=>2, "Researcher"=>1, "Student > Ph. D. Student"=>2, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Student > Doctoral Student"=>2, "Researcher"=>1, "Student > Ph. D. Student"=>2, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>7}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>7}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"United States"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/733708"], "description"=>"<p>Horizontal line denotes genotype and vertical arrows represent level of expression of each gene. SRG is Spx-regulated gene. The X on the horizontal line is a null mutation, eliminating expression. The SRG mutation yields little observable phenotype with respect to electrophile stress. The <i>spx</i> mutation reduces overall SRG expression, thus reducing effect of genetic buffering and functional redundancy. The <i>srg spx</i> double mutant is now found to be hypersensitive to electrophile (methylgyoxal), or shows other defects such as reduced growth rate.</p>", "links"=>[], "tags"=>["phenotype", "enhancement"], "article_id"=>404058, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025066.g001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Rationale_for_phenotype_enhancement_screen_/404058", "title"=>"Rationale for phenotype enhancement screen.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-20 01:07:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/371138", "https://ndownloader.figshare.com/files/371175", "https://ndownloader.figshare.com/files/371213", "https://ndownloader.figshare.com/files/371252", "https://ndownloader.figshare.com/files/371284", "https://ndownloader.figshare.com/files/371341", "https://ndownloader.figshare.com/files/371375", "https://ndownloader.figshare.com/files/371400"], "description"=>"<div><p>Spx is a global regulator of genes that are induced by disulfide stress in <em>Bacillus subtilis</em>. The regulon that it governs is comprised of over 120 genes based on microarray analysis, although it is not known how many of these are under direct Spx control. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products that are conserved in low %GC Gram-positive bacteria. Using a gene-disruption library of <em>B. subtilis</em> genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as poor growth in minimal medium and sensitivity to methyglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an <em>spx</em> mutant background to determine which mutant SRG allele would enhance the <em>spx</em> mutant phenotype. One of the SRGs, <em>ytpQ</em> was the site of a mutation that, when combined with an <em>spx</em> null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methyglyoxal. The <em>ytpQ</em> mutant showed elevated oxidative protein damage when exposed to methylglyoxal, and reduced growth rate in liquid culture. Proteomic and transcriptomic data indicated that the <em>ytpQ</em> mutation caused the derepression of the Fur and PerR regulons of <em>B. subtilis</em>. Our study suggests that the <em>ytpQ</em> gene, encoding a conserved DUF1444 protein, functions directly or indirectly in iron homeostasis. The <em>ytpQ</em> mutant phenotype mimics that of a <em>fur</em> mutation, suggesting a condition of low cellular iron. In vitro transcription analysis indicated that Spx stimulates transcription from the <em>ytpPQR</em> operon within which the <em>ytpQ</em> gene resides. The work uncovers a link between Spx and control of iron homeostasis.</p> </div>", "links"=>[], "tags"=>["phenotype", "enhancement", "mutant", "unveils", "homeostasis"], "article_id"=>133320, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0025066.s001", "https://dx.doi.org/10.1371/journal.pone.0025066.s002", "https://dx.doi.org/10.1371/journal.pone.0025066.s003", "https://dx.doi.org/10.1371/journal.pone.0025066.s004", "https://dx.doi.org/10.1371/journal.pone.0025066.s005", "https://dx.doi.org/10.1371/journal.pone.0025066.s006", "https://dx.doi.org/10.1371/journal.pone.0025066.s007", "https://dx.doi.org/10.1371/journal.pone.0025066.s008"], "stats"=>{"downloads"=>3, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Phenotype_Enhancement_Screen_of_a_Regulatory_spx_Mutant_Unveils_a_Role_for_the_ytpQ_Gene_in_the_Control_of_Iron_Homeostasis/133320", "title"=>"Phenotype Enhancement Screen of a Regulatory <em>spx</em> Mutant Unveils a Role for the <em>ytpQ</em> Gene in the Control of Iron Homeostasis", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-09-20 00:55:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/733786"], "description"=>"<p>A. Chromosomal organization of <i>ytoQ ytpQ</i> locus. Arrows represent location and orientation of coding sequences and lollipop figure denotes location of putative transcriptional terminator. B. Phenotype of <i>ytpP</i> and <i>ytpQ</i> mutations in the wild-type and <i>spx</i> mutant backgrounds. Cultures were grown to late log phase and serially diluted 10-fold. Ten µl were spotted on TSS minimal medium plates with and without MG. C. Minimal TSS plate onto which JH642 (wild-type parent), the <i>spx</i> mutant, the <i>ytpQ</i>::pMUTIN mutant, and the double mutant <i>spx ytpQ</i>::pMUTIN were streaked. D and E. Growth curves of <i>ytoQ</i> and <i>ytpQ</i> mutants in wild-type and <i>spx</i> mutant backgrounds. Open circles: JH642. Open squares: <i>spx</i> mutant. Closed triangles <i>ytoQ</i> mutant (D), and <i>ytpQ</i> mutant (E). Closed squares: <i>ytoQ spx</i> mutant (D) and <i>ytpQ spx</i> mutant (E).</p>", "links"=>[], "tags"=>["mutations", "mutant"], "article_id"=>404134, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025066.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phenotype_of_srg_mutations_in_spx_mutant_background_/404134", "title"=>"Phenotype of <i>srg</i> mutations in <i>spx</i> mutant background.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-20 01:08:54"}
  • {"files"=>["https://ndownloader.figshare.com/files/734435"], "description"=>"<p>Cytoplasmic proteins were separated by 2D PAGE as described in Materials and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025066#s3\" target=\"_blank\">Methods</a>. Image analysis was performed using the Decodon Delta 2D software. Proteins with increased levels in the mutants in at least two independent experiments are indicated by white labels.</p>", "links"=>[], "tags"=>["images", "amounts", "compared", "mutants"], "article_id"=>404783, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025066.g007", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dual_channel_images_of_the_protein_amounts_in_B_subtilis_wild_type_green_image_compared_to_the_ytpQ_left_and_spx_mutants_right_red_images_under_control_conditions_/404783", "title"=>"Dual-channel images of the protein amounts in <i>B. subtilis</i> wild type (green image) compared to the <i>ytpQ</i> (left) and <i>spx</i> mutants (right) (red images) under control conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-20 01:19:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/733885"], "description"=>"<p>A. The nucleotide sequence of the <i>ytpPQR</i> promoter region is shown, The bold plain text indicates the oligonucleotide primers used to generate the linear DNA promoter template fragment for the in vitro transcription reaction. Also the region underlined and in italics shows the putative promoter region along (in bold) the −10 region (<b><i>tatgat</i></b>) with extended TG and −35 region (<b><i>tggttt</i></b>) and the Spx cis element (<b><i>tgcatataa</i></b>) <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025066#pone.0025066-Nakano10\" target=\"_blank\">[77]</a> upstream from the −35 region. B. In vitro transcription from <i>ytpP</i> promoter. The <i>ytpP</i> promoter template (10 nM) was incubated with 25 nM σ<sup>A</sup>-depleted RNAP and 25 nM σ<sup>A</sup> in the absence or presence of 75 nM Spx. Samples were collected from the reactions at indicated times during incubation (2, 4, 8, and 12 min). Transcripts were resolved by gel electrophoresis, visualized and quantified as previously described <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025066#pone.0025066-Nakano10\" target=\"_blank\">[77]</a>. Marker transcripts were generated using Spx protein and purified RNA polymerase, along with DNA fragments containing the Spx-controlled <i>trxB</i> promoter <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025066#pone.0025066-Nakano1\" target=\"_blank\">[11]</a> and the <i>yrrT</i> promoter <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025066#pone.0025066-Choi1\" target=\"_blank\">[14]</a> in transcription reactions.</p>", "links"=>[], "tags"=>["transcription", "operon"], "article_id"=>404238, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025066.g003", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Spx_activated_transcription_from_the_ytpPQR_operon_promoter_/404238", "title"=>"Spx-activated transcription from the <i>ytpPQR</i> operon promoter.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-20 01:10:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/734079"], "description"=>"<p>Gene expression data were clustered based on the induction or repression ratios in the <i>spx</i> and <i>ytpQ</i> mutants leading to different nodes specific to regulons. Nodes enriched for genes that belong to the thiol-specific stress regulons (CymR, Spx, PerR, Fur, HxlR, CatR) are shown.</p>", "links"=>[], "tags"=>["clustering", "profiles", "up-", "downregulated"], "article_id"=>404427, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025066.g005", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hierarchical_clustering_analysis_of_gene_expression_profiles_up_and_downregulated_in_spx_and_ytpQ_mutants_/404427", "title"=>"Hierarchical clustering analysis of gene expression profiles up- and downregulated in <i>spx</i> and <i>ytpQ</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-20 01:13:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/734257"], "description"=>"<p>Gene expression data were clustered based on the induction or repression ratios in the <i>spx</i> and <i>ytpQ</i> mutants leading to different nodes specific to regulons. Nodes including regulons involved in motility, competence and sporulation are shown (Com, SigmaD, SigmaH, CodY, SinR, Spo0A, SigmaL, AhrC, Rok, SigmaE, F, G, K regulons). Red indicates induction and green repression in the <i>spx</i> or <i>ytpQ</i> mutants under control conditions, and MG stress.</p>", "links"=>[], "tags"=>["clustering", "profiles", "up-", "downregulated"], "article_id"=>404601, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025066.g006", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hierarchical_clustering_analysis_of_gene_expression_profiles_up_and_downregulated_in_spx_and_ytpQ_mutants_/404601", "title"=>"Hierarchical clustering analysis of gene expression profiles up- and downregulated in <i>spx</i> and <i>ytpQ</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-20 01:16:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/733981"], "description"=>"<p>Cells were incubated in 100 ml 2xYT cultures to an OD<sub>600</sub> of 0.6. Cultures were split and MG to 2 mM was added to one of the two cultures. Crude cell extracts were prepared for SDS-PAGE and oxyblot analysis was performed as described in Materials and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025066#s3\" target=\"_blank\">Methods</a>. The same amounts of protein were applied to the SDS-PAGE gel. WT denotes blot of JH642 culture cell extracts. MG – methylglyoxal treatment.</p>", "links"=>[], "tags"=>["strains", "methylglyoxal"], "article_id"=>404324, "categories"=>["Biochemistry", "Genetics", "Microbiology"], "users"=>["Peter Zuber", "Shefali Chauhan", "Praseeda Pilaka", "Michiko M. Nakano", "Sairam Gurumoorthy", "Ann A. Lin", "Skye M. Barendt", "Bui Khanh Chi", "Haike Antelmann", "Ulrike Mäder"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025066.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assay_of_protein_damage_in_wild_type_spx_and_ytpQ_strains_in_the_presence_and_absence_of_methylglyoxal_MG_/404324", "title"=>"Assay of protein damage in wild-type, <i>spx</i>, and <i>ytpQ</i> strains in the presence and absence of methylglyoxal (MG).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-20 01:12:04"}

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