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{"title"=>"Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice", "type"=>"journal", "authors"=>[{"first_name"=>"Carlos A.", "last_name"=>"Santiviago", "scopus_author_id"=>"6507214143"}, {"first_name"=>"M. Megan", "last_name"=>"Reynolds", "scopus_author_id"=>"57195268668"}, {"first_name"=>"Steffen", "last_name"=>"Porwollik", "scopus_author_id"=>"6603054994"}, {"first_name"=>"Sang Ho", "last_name"=>"Choi", "scopus_author_id"=>"57192713489"}, {"first_name"=>"Fred", "last_name"=>"Long", "scopus_author_id"=>"24463663900"}, {"first_name"=>"Helene L.", "last_name"=>"Andrews-Polymenis", "scopus_author_id"=>"9737261400"}, {"first_name"=>"Michael", "last_name"=>"McClelland", "scopus_author_id"=>"7103047213"}], "year"=>2009, "source"=>"PLoS Pathogens", "identifiers"=>{"pui"=>"355237372", "sgr"=>"69349099557", "issn"=>"15537366", "pmid"=>"19578432", "scopus"=>"2-s2.0-69349099557", "doi"=>"10.1371/journal.ppat.1000477", "isbn"=>"1553-7366"}, "id"=>"de4d5f06-7871-3ea0-b99f-759f3e48e7d7", "abstract"=>"Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS).", "link"=>"http://www.mendeley.com/research/analysis-pools-targeted-salmonella-deletion-mutants-identifies-novel-genes-affecting-fitness-during", "reader_count"=>94, "reader_count_by_academic_status"=>{"Unspecified"=>4, "Professor > Associate Professor"=>6, "Researcher"=>19, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>37, "Student > Postgraduate"=>4, "Student > Master"=>12, "Student > Bachelor"=>3, "Lecturer"=>2, "Lecturer > Senior Lecturer"=>2, "Professor"=>4}, "reader_count_by_user_role"=>{"Unspecified"=>4, "Professor > Associate Professor"=>6, "Researcher"=>19, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>37, "Student > Postgraduate"=>4, "Student > Master"=>12, "Student > Bachelor"=>3, "Lecturer"=>2, "Lecturer > Senior Lecturer"=>2, "Professor"=>4}, "reader_count_by_subject_area"=>{"Engineering"=>3, "Unspecified"=>4, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>13, "Agricultural and Biological Sciences"=>61, "Medicine and Dentistry"=>1, "Philosophy"=>1, "Veterinary Science and Veterinary Medicine"=>1, "Chemistry"=>1, "Immunology and Microbiology"=>8}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>3}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>8}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>61}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>13}, "Unspecified"=>{"Unspecified"=>4}, "Environmental Science"=>{"Environmental Science"=>1}, "Philosophy"=>{"Philosophy"=>1}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"United States"=>2, "Brazil"=>1, "Indonesia"=>1}, "group_count"=>4}

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/892772"], "description"=>"<p>10⁶ cfu of 933 mutants (pool) or a 1∶1 mixture of individual mutants versus wild-type ATCC14028 Δ<i>phoN</i>::<i>kan</i> were injected into 5–6 mice and recovered from spleen one or two days post infection. Statistical significance was determined using a Student's 2-tailed <i>t</i> test, and asterisks indicate that normalized output ratios were significantly statistically different from the equivalent ratio in the inoculum. ** P value of <0.001, * P value of <0.05. Strains marked with (FRT) have undergone FLP-mediated recombination to reduce or eliminate polar effects. A. Confirmation of mutants, identified by ABACUS, in competitive infections in BALB/c mice. ABACUS prediction for selected mutants (black bars) and confirmation data from competitive infections in BALB/c mice (dark blue bars are from spleen, lighter blue are from liver and cecum) of infected animals. B. Complementation of <i>ΔSTM2303</i>::FRT and <i>ΔSTM3121</i>::FRT mutants <i>in trans</i>. The predictions from ABACUS are indicated as black bars. For complementation experiments animals were infected with either a 1∶1 mixture of the mutant bearing pWSK29 vs. ATCC14028 Δ<i>phoN</i>::<i>kan</i> (negative control, blue bars) or the mutant bearing the corresponding complementing gene on pWSK29 vs. the wild-type ATCC14028Δ <i>phoN</i>::<i>kan</i> (complemented, grey bars).</p>", "links"=>[], "tags"=>["experiments", "mutants", "wild-type", "intraperitoneal"], "article_id"=>563223, "categories"=>["Cell Biology", "Genetics", "Microbiology", "Infectious Diseases", "Biotechnology"], "users"=>["Carlos A. Santiviago", "M. Megan Reynolds", "Steffen Porwollik", "Sang-Ho Choi", "Fred Long", "Helene L. Andrews-Polymenis", "Michael McClelland"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000477.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Competitive_Index_experiments_with_individual_mutants_versus_wild_type_after_intraperitoneal_delivery_and_recovery_from_the_spleen_liver_and_cecum_/563223", "title"=>"Competitive Index experiments with individual mutants versus wild-type after intraperitoneal delivery and recovery from the spleen, liver, and cecum.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-03 00:53:43"}
• {"files"=>["https://ndownloader.figshare.com/files/892680"], "description"=>"<p>Twelve array hybridizations were performed using two each for six mice. WebArray analysis results using quantile normalization for all oligos representing candidate deletions in the pool are shown. Data with P<0.0005 are depicted in red, those with P>0.0005 in grey. The X-axis plots the data for each of the mutants in the order in which those mutants occur in the <i>Salmonella</i> genome. Mutants confirmed individually in a competitive assay with wild-type are labeled in blue. Examples of important genetic elements are marked in black.</p>", "links"=>[], "tags"=>["defined", "mutants", "intraperitoneal", "mice", "spleen", "48"], "article_id"=>563130, "categories"=>["Cell Biology", "Genetics", "Microbiology", "Infectious Diseases", "Biotechnology"], "users"=>["Carlos A. Santiviago", "M. Megan Reynolds", "Steffen Porwollik", "Sang-Ho Choi", "Fred Long", "Helene L. Andrews-Polymenis", "Michael McClelland"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000477.g003", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loss_of_defined_mutants_from_the_input_pool_after_intraperitoneal_delivery_to_six_BALB_c_mice_and_recovery_from_spleen_at_48_h_post_infection_/563130", "title"=>"Loss of defined mutants from the input pool after intraperitoneal delivery to six BALB/c mice and recovery from spleen at 48 h post-infection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-03 00:52:10"}
• {"files"=>["https://ndownloader.figshare.com/files/892613"], "description"=>"<p>Labeling technique for detection of mutants in a pool by arrays.</p>", "links"=>[], "tags"=>["detection", "mutants"], "article_id"=>563067, "categories"=>["Cell Biology", "Genetics", "Microbiology", "Infectious Diseases", "Biotechnology"], "users"=>["Carlos A. Santiviago", "M. Megan Reynolds", "Steffen Porwollik", "Sang-Ho Choi", "Fred Long", "Helene L. Andrews-Polymenis", "Michael McClelland"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000477.g002", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Labeling_technique_for_detection_of_mutants_in_a_pool_by_arrays_/563067", "title"=>"Labeling technique for detection of mutants in a pool by arrays.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-03 00:51:07"}
• {"files"=>["https://ndownloader.figshare.com/files/442033", "https://ndownloader.figshare.com/files/442142", "https://ndownloader.figshare.com/files/442192", "https://ndownloader.figshare.com/files/442218", "https://ndownloader.figshare.com/files/442256"], "description"=>"<div><p>Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 <em>Salmonella enterica</em> serovar Typhimurium genes, including almost all genes found in <em>Salmonella</em> but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in <em>Salmonella</em>. We refer to the method as <u>A</u>rray-<u>b</u>ased <u>a</u>nalysis of <u>c</u>istrons <u>u</u>nder <u>s</u>election (ABACUS).</p></div>", "links"=>[], "tags"=>["pools", "targeted", "deletion", "mutants", "identifies", "genes", "affecting", "mice"], "article_id"=>147138, "categories"=>["Cell Biology", "Genetics", "Microbiology", "Cancer", "Biotechnology"], "users"=>["Carlos A. Santiviago", "M. Megan Reynolds", "Steffen Porwollik", "Sang-Ho Choi", "Fred Long", "Helene L. Andrews-Polymenis", "Michael McClelland"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1000477.s001", "https://dx.doi.org/10.1371/journal.ppat.1000477.s002", "https://dx.doi.org/10.1371/journal.ppat.1000477.s003", "https://dx.doi.org/10.1371/journal.ppat.1000477.s004", "https://dx.doi.org/10.1371/journal.ppat.1000477.s005"], "stats"=>{"downloads"=>30, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Analysis_of_Pools_of_Targeted_Salmonella_Deletion_Mutants_Identifies_Novel_Genes_Affecting_Fitness_during_Competitive_Infection_in_Mice/147138", "title"=>"Analysis of Pools of Targeted <em>Salmonella</em> Deletion Mutants Identifies Novel Genes Affecting Fitness during Competitive Infection in Mice", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2009-07-03 01:58:58"}
• {"files"=>["https://ndownloader.figshare.com/files/892520"], "description"=>"<p>Our procedure is identical to the Red-swap described in reference <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000477#ppat.1000477-Datsenko1\" target=\"_blank\">[6]</a> with the exception that we re-engineered the original insert to include an in-frame T7 RNA polymerase promoter (P_T7), and the sequences used for recombination are longer. A gene, identified in the schematic as <i>genE</i>, is targeted for deletion. Two 65mer primers (red) are used to amplify the region containing the antibiotic resistance cassette, the P_T7, and the FRT sites (among other elements) from the plasmid pCLF4. The resulting PCR product has 45 base sequences at each end that are homologous to sequences near the 5′ and 3′ ends of the <i>targeted ORF</i>. Transformation of these PCR products into ATCC14028 expressing lambda Red recombinase <i>in trans</i> leads to a recombination event resulting in the swap-in of the PCR product, and swap-out of the targeted gene. A ribosomal binding site (RBS) and a downstream ATG start codon near the 3′ end of the inserted sequence ensures that a 12 amino acid peptide is made from any RNA that is transcribed in this strand to reduce polar effects. Targeted mutants in many genes were pooled and used for <u>A</u>rray-<u>b</u>ased <u>a</u>nalysis of <u>c</u>istrons <u>u</u>nder <u>s</u>election (ABACUS). Finally, antibiotic resistance markers in targeted genes can be removed using the FLP recombinase resulting in a gene encoding a mini-protein of the first ten amino acids of GenE, 39 amino acids from the inserted DNA (called a “scar”), and the last nine amino acids of GenE, and retaining the P_T7.</p>", "links"=>[], "tags"=>["deletions", "serovar", "typhimurium"], "article_id"=>562973, "categories"=>["Cell Biology", "Genetics", "Microbiology", "Infectious Diseases", "Biotechnology"], "users"=>["Carlos A. Santiviago", "M. Megan Reynolds", "Steffen Porwollik", "Sang-Ho Choi", "Fred Long", "Helene L. Andrews-Polymenis", "Michael McClelland"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000477.g001", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_specific_deletions_in_S_enterica_serovar_Typhimurium_ATCC14028_/562973", "title"=>"Generation of specific deletions in <i>S. enterica</i> serovar Typhimurium ATCC14028.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-07-03 00:49:33"}

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Relative Metric

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F1000Prime