Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells
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{"title"=>"Genome-scale analysis of Mycoplasma agalactiae loci involved in interaction with host cells", "type"=>"journal", "authors"=>[{"first_name"=>"Agnès", "last_name"=>"Skapski", "scopus_author_id"=>"36022955700"}, {"first_name"=>"Marie Claude", "last_name"=>"Hygonenq", "scopus_author_id"=>"26040759800"}, {"first_name"=>"Eveline", "last_name"=>"Sagné", "scopus_author_id"=>"26658699400"}, {"first_name"=>"Sébastien", "last_name"=>"Guiral", "scopus_author_id"=>"6506767924"}, {"first_name"=>"Christine", "last_name"=>"Citti", "scopus_author_id"=>"6701372760"}, {"first_name"=>"Eric", "last_name"=>"Baranowski", "scopus_author_id"=>"57195256550"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-80053112373", "doi"=>"10.1371/journal.pone.0025291", "pui"=>"362619284", "issn"=>"19326203", "pmid"=>"21966487", "isbn"=>"1932-6203", "sgr"=>"80053112373"}, "id"=>"93575e2b-00ff-3673-881c-0cecc38f2cc2", "abstract"=>"Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of \"minimal bacteria\" with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, \"transport and metabolism\" was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions.", "link"=>"http://www.mendeley.com/research/genomescale-analysis-mycoplasma-agalactiae-loci-involved-interaction-host-cells", "reader_count"=>12, "reader_count_by_academic_status"=>{"Researcher"=>2, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>3, "Student > Master"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>2, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>3, "Student > Master"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>5, "Medicine and Dentistry"=>2, "Chemistry"=>1, "Social Sciences"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>1}, "Social Sciences"=>{"Social Sciences"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}}, "reader_count_by_country"=>{"Canada"=>1, "United Kingdom"=>2}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/731975"], "description"=>"a<p>The cut-off value is the number of colonies counted on solid medium following 3 days co-cultivation of <i>M. agalactiae</i> knockout mutants with cells. Titers in parentheses indicate the predicted titers in the co-culture.</p>b<p>Number of <i>M. agalactiae</i> growth deficient mutants selected on goat mammary epithelial cells (TIGMEC), goat embryonic fibroblast cells (TIGEF), and HeLa cells (HeLa).</p>c<p>Percentage of growth deficient mutants selected from the mutant library.</p>", "links"=>[], "tags"=>["detection", "mutants", "cultured"], "article_id"=>402331, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025291.t002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_High_throughput_detection_of_M_agalactiae_mutants_unable_to_grow_on_cultured_cells_/402331", "title"=>"High-throughput detection of <i>M. agalactiae</i> mutants unable to grow on cultured cells.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-09-23 00:38:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/731948"], "description"=>"a<p>CDS found disrupted in <i>M. agalactiae</i> growth-deficient mutants <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone.0025291-SirandPugnet2\" target=\"_blank\">[6]</a>.</p>b<p>Letters E and F indicate that the <i>M. agalactiae</i> growth-deficient mutants were selected on TIGMEC or TIGEF cells, respectively. Asterisks (*) indicate mutants that were also selected during high-throughput screening on HeLa cells.</p>c<p>For each CDS, the number of mutants identified during the screening on caprine cell lines is indicated, as well as the number of different mini-Tn insertion sites.</p>d<p>For each CDS, the relative position and the orientation of the inserted transposon are indicated. Mini-Tn insertion sites were determined by direct sequencing of genomic DNA, and their positions were defined based on the published genome sequence (NC_009497).</p>e<p>Hypothetical proteins (HP) have no homolog outside the species <i>M. agalactiae</i>. Conserved hypothetical proteins (CHP) share sequence similarity with proteins of unknown function identified in other <i>Mollicutes</i> or other bacteria. COG categories of encoded proteins are indicated in parentheses <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone.0025291-Tatusov1\" target=\"_blank\">[38]</a>.</p>f<p>Protein localization was predicted using TMHMM <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone.0025291-Krogh1\" target=\"_blank\">[41]</a>, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone.0025291-Barr1\" target=\"_blank\">[42]</a>; membrane (M), cytosolic (C), or indirectly linked to the membrane (IM).</p>g<p>Proteins with peptides detected during proteomic analysis of gene products expressed by <i>M. agalactiae</i> strain PG2 in axenic culture are identified by a plus sign (+), while proteins not detected are identified by a minus sign (−) <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone.0025291-Nouvel1\" target=\"_blank\">[32]</a>.</p>", "links"=>[], "tags"=>["cdss", "high-throughput", "reduced", "capacities", "cultured"], "article_id"=>402298, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025291.t003", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_M_agalactiae_CDSs_identified_by_high_throughput_screening_for_their_reduced_growth_capacities_on_cultured_cells_/402298", "title"=>"<i>M. agalactiae</i> CDSs identified by high-throughput screening for their reduced growth capacities on cultured cells.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-09-23 00:38:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/731837"], "description"=>"<p>Map of the 62 genomic regions found to be disrupted in <i>M. agalactiae</i> mutants selected on TIGMEC and/or TIGEF cells, produced using the Artemis genome browser and annotation tool <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone.0025291-Rutherford1\" target=\"_blank\">[39]</a>. Insertion sites found within CDS regions are indicated by a blue bar, while intergenic regions are designated by a letter code and a purple bar. Short CDS clusters are designated by capital letters and a schematic illustration of the locus, in which insertion sites are indicated by a filled circle on top of a blue bar. Genes are labeled. CDSs for hypothetical proteins of unknown function are labeled with their gene number. Genomic distances are indicated in kbp. <i>M. agalactiae</i> CDSs are colored in green (positive) or red (negative) on the chromosome to indicate their orientation. Non-coding RNAs are colored in black.</p>", "links"=>[], "tags"=>["loci", "carrying", "transposon", "insertions", "mutants", "displaying", "reduced", "capacities"], "article_id"=>402184, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025291.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Genomic_loci_carrying_transposon_insertions_in_M_agalactiae_mutants_displaying_reduced_growth_capacities_in_cell_culture_/402184", "title"=>"Genomic loci carrying transposon insertions in <i>M. agalactiae</i> mutants displaying reduced growth capacities in cell culture.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-23 00:36:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/731587"], "description"=>"<p>(A) Number of mutants selected on goat mammary epithelial cells (TIGMEC), goat embryo fibroblasts (TIGEF) and/or HeLa cells. (B) Number of unique transposon insertion sites identified in mutants selected for their inability to grow on TIGMEC and/or TIGEF. Some mutants had the same insertion site. (C) Distribution in COG categories of the 46 CDSs found disrupted in growth deficient mutants selected on caprine cells (see panel B) <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone.0025291-Tatusov1\" target=\"_blank\">[38]</a>. Multiple COG proteins include MAG1490 and MAG2120, which fall into several COG categories (energy production and conversion, C-COG, coenzyme transport and metabolism, H-COG and general function prediction only, R-COG for MAG1490; general function prediction only, R-COG, signal transduction mechanisms, T-COG, transcription, K-COG and replication, recombination and repair, L-COG, for MAG2120).</p>", "links"=>[], "tags"=>["deficient", "mutants", "co-cultivation"], "article_id"=>401937, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025291.g001", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overall_distribution_of_M_agalactiae_growth_deficient_mutants_selected_after_co_cultivation_with_host_cells_/401937", "title"=>"Overall distribution of <i>M. agalactiae</i> growth deficient mutants selected after co-cultivation with host cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-23 00:32:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/731682"], "description"=>"<p>(A) Schematic representation of the 4 kbp co-linear gene cluster <i>pip-nifS-nifU-mucB</i> indicating the four regions amplified by RT-PCR. Intergenic distances in nucleotides are indicated below. Transposon insertion sites are indicated by filled circles using a different color code for each mutant. RNA regions amplified by RT-PCRs I to IV are indicated by black bars. The promoter identified in the 3′ end of <i>pip</i> is indicated by a black arrow. (B) Bar graph indicating the titers obtained, after cultivation in axenic media or in the presence of TIGMEC, of <i>M. agalactiae</i> strain PG2 or mutants NifS1, NifS2, Pip and MucB. Titers were determined after 48 h cultivation in axenic medium and after 72 h co-cultivation with TIGMEC. The bars indicate the means of three independent assays, with standard deviations indicated by error bars. (C) Identification of promoter sequences in <i>M. agalactiae</i> using a new reporter system. The surface antigen P40-knockout mutant (mutant P40 minus) was complemented using plasmid constructs containing the P40 coding sequence preceded by its own promoter (a), without a promoter (b), or preceded by the 3′ end of <i>pip</i> (c). The expression of the P40 antigen was detected by Western blotting. The surface antigen P80 was used as control. (D) Chromatogram obtained after primer extension of total RNA extracted from a <i>M. agalactiae</i> PG2 culture with primer 5′-6FAM-NifS1 (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#pone-0025291-t001\" target=\"_blank\">Table 1</a>) as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025291#s2\" target=\"_blank\">Materials and Methods</a>. Red peaks correspond to the GS-400HD ROX internal lane standards and the shaded blue peaks to the primer extension product. The size is indicated on the X-axis in base pairs. The peak height indicates the fluorescence intensity (arbitrary units) with GS-400HD ROX fluorescence measured on the right Y-axis and FAM fluorescence on the left Y-axis. The position of the transcriptional start point in CDS MAG0710 (<i>pip</i>) identified by primer extension experiments is indicated. Putative −10 and −35 regions are underlined. The transcriptional start points are indicated in bold.</p>", "links"=>[], "tags"=>["locus", "promoter-less", "cds", "inserted", "transcriptionally"], "article_id"=>402031, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025291.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_nif_locus_in_M_agalactiae_is_a_promoter_less_CDS_cluster_inserted_within_a_transcriptionally_active_region_/402031", "title"=>"The <i>nif</i> locus in <i>M. agalactiae</i> is a promoter-less CDS cluster inserted within a transcriptionally active region.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-09-23 00:33:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/369592", "https://ndownloader.figshare.com/files/369634", "https://ndownloader.figshare.com/files/369673", "https://ndownloader.figshare.com/files/369715"], "description"=>"<div><p><em>Mycoplasma agalactiae</em> is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a <em>M. agalactiae</em> knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the <em>M. agalactiae</em> chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions.</p> </div>", "links"=>[], "tags"=>["genome-scale", "loci", "cells"], "article_id"=>133034, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0025291.s001", "https://dx.doi.org/10.1371/journal.pone.0025291.s002", "https://dx.doi.org/10.1371/journal.pone.0025291.s003", "https://dx.doi.org/10.1371/journal.pone.0025291.s004"], "stats"=>{"downloads"=>4, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Genome_Scale_Analysis_of_Mycoplasma_agalactiae_Loci_Involved_in_Interaction_with_Host_Cells/133034", "title"=>"Genome-Scale Analysis of <em>Mycoplasma agalactiae</em> Loci Involved in Interaction with Host Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-09-23 00:50:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/732020"], "description"=>"a<p>Oligonucleotide labeled by a 6-carboxyfluorescein (FAM) 5′-modification.</p>", "links"=>[], "tags"=>["genetics and genomics", "microbiology", "cell biology"], "article_id"=>402372, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025291.t001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Oligonucleotides_used_in_this_study_/402372", "title"=>"Oligonucleotides used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-09-23 00:39:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/732050"], "description"=>"a<p>NCRs were labeled with a letter based on their position in the genome of <i>M. agalactiae</i>.</p>b<p>Letters E and F indicate NCRs carrying insertions in mutants with reduced growth capacities on TIGMEC and/or TIGEF, respectively. Asterisks (*) indicate mutants that were also selected during high-throughput screening on HeLa cells.</p>c<p>For each NCR, the number of mutants identified during the screening with caprine cells is indicated, as well as the number of insertion sites.</p>d<p>Transposon insertion sites were determined by direct sequencing of genomic DNA and their positions were defined based on the published genome sequence (NC_009497). The orientation of the transposon is indicated in parentheses.</p>e<p>Surrounding CDS and mini-Tn disrupted AMIGene CDS predictions; CDS names are given in parenthesis; vestige of ICEA indicates an NCR located within a 20 kb locus containing a vestige of an integrative conjugative element (ICEA).</p>", "links"=>[], "tags"=>["ncrs", "high-throughput"], "article_id"=>402401, "categories"=>["Cell Biology", "Genetics", "Microbiology"], "users"=>["Agnès Skapski", "Marie-Claude Hygonenq", "Eveline Sagné", "Sébastien Guiral", "Christine Citti", "Eric Baranowski"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0025291.t004", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_M_agalactiae_NCRs_identified_by_high_throughput_screening_with_host_cells_/402401", "title"=>"<i>M. agalactiae</i> NCRs identified by high-throughput screening with host cells.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-09-23 00:40:01"}

PMC Usage Stats | Further Information

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