Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer
Identifieur interne : 002775 ( Pmc/Curation ); précédent : 002774; suivant : 002776Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer
Auteurs : Nicholas J. Tobias [Australie] ; Torsten Seemann [Australie] ; Sacha J. Pidot [Australie] ; Jessica L. Porter [Australie] ; Laurent Marsollier [France] ; Estelle Marion [France] ; Franck Letournel [France] ; Tasnim Zakir [Australie] ; Joseph Azuolas [Australie] ; John R. Wallace [États-Unis] ; Hui Hong [Royaume-Uni] ; John K. Davies [Australie] ; Benjamin P. Howden [Australie] ; Paul D. R. Johnson [Australie] ; Grant A. Jenkin [Australie] ; Timothy P. Stinear [Australie]Source :
- PLoS Neglected Tropical Diseases [ 1935-2727 ] ; 2009.
Abstract
Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans.
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DOI: 10.1371/journal.pntd.0000553
PubMed: 19936295
PubMed Central: 2775157
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Tobias</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Seemann, Torsten" sort="Seemann, Torsten" uniqKey="Seemann T" first="Torsten" last="Seemann">Torsten Seemann</name><affiliation wicri:level="1"><nlm:aff id="aff3"><addr-line>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Pidot, Sacha J" sort="Pidot, Sacha J" uniqKey="Pidot S" first="Sacha J." last="Pidot">Sacha J. 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Tobias</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Seemann, Torsten" sort="Seemann, Torsten" uniqKey="Seemann T" first="Torsten" last="Seemann">Torsten Seemann</name><affiliation wicri:level="1"><nlm:aff id="aff3"><addr-line>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Pidot, Sacha J" sort="Pidot, Sacha J" uniqKey="Pidot S" first="Sacha J." last="Pidot">Sacha J. Pidot</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Porter, Jessica L" sort="Porter, Jessica L" uniqKey="Porter J" first="Jessica L." last="Porter">Jessica L. Porter</name><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Marsollier, Laurent" sort="Marsollier, Laurent" uniqKey="Marsollier L" first="Laurent" last="Marsollier">Laurent Marsollier</name><affiliation wicri:level="1"><nlm:aff id="aff4"><addr-line>Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, Université d'Angers, Angers, France</addr-line></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, Université d'Angers, Angers</wicri:regionArea></affiliation></author><author><name sortKey="Marion, Estelle" sort="Marion, Estelle" uniqKey="Marion E" first="Estelle" last="Marion">Estelle Marion</name><affiliation wicri:level="1"><nlm:aff id="aff4"><addr-line>Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, Université d'Angers, Angers, France</addr-line></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, Université d'Angers, Angers</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff5"><addr-line>Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Angers, France</addr-line></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Angers</wicri:regionArea></affiliation></author><author><name sortKey="Letournel, Franck" sort="Letournel, Franck" uniqKey="Letournel F" first="Franck" last="Letournel">Franck Letournel</name><affiliation wicri:level="1"><nlm:aff id="aff6"><addr-line>Laboratoire de Neurobiologie et Transgénèse, UPRES-EA 3143, Université d'Angers, Angers, France</addr-line></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratoire de Neurobiologie et Transgénèse, UPRES-EA 3143, Université d'Angers, Angers</wicri:regionArea></affiliation></author><author><name sortKey="Zakir, Tasnim" sort="Zakir, Tasnim" uniqKey="Zakir T" first="Tasnim" last="Zakir">Tasnim Zakir</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Azuolas, Joseph" sort="Azuolas, Joseph" uniqKey="Azuolas J" first="Joseph" last="Azuolas">Joseph Azuolas</name><affiliation wicri:level="1"><nlm:aff id="aff7"><addr-line>Department of Primary Industries, Mickleham Road, Attwood, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Primary Industries, Mickleham Road, Attwood, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Wallace, John R" sort="Wallace, John R" uniqKey="Wallace J" first="John R." last="Wallace">John R. Wallace</name><affiliation wicri:level="1"><nlm:aff id="aff8"><addr-line>Department of Biology, Millersville University, Millersville, Pennsylvania, United States of America</addr-line></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biology, Millersville University, Millersville, Pennsylvania</wicri:regionArea></affiliation></author><author><name sortKey="Hong, Hui" sort="Hong, Hui" uniqKey="Hong H" first="Hui" last="Hong">Hui Hong</name><affiliation wicri:level="1"><nlm:aff id="aff9"><addr-line>Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom</addr-line></nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Department of Biochemistry, University of Cambridge, Cambridge</wicri:regionArea></affiliation></author><author><name sortKey="Davies, John K" sort="Davies, John K" uniqKey="Davies J" first="John K." last="Davies">John K. Davies</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Howden, Benjamin P" sort="Howden, Benjamin P" uniqKey="Howden B" first="Benjamin P." last="Howden">Benjamin P. Howden</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff10"><addr-line>Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Infectious Diseases, Austin Health, Heidelberg, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Johnson, Paul D R" sort="Johnson, Paul D R" uniqKey="Johnson P" first="Paul D. R." last="Johnson">Paul D. R. Johnson</name><affiliation wicri:level="1"><nlm:aff id="aff10"><addr-line>Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Infectious Diseases, Austin Health, Heidelberg, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Jenkin, Grant A" sort="Jenkin, Grant A" uniqKey="Jenkin G" first="Grant A." last="Jenkin">Grant A. Jenkin</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author><author><name sortKey="Stinear, Timothy P" sort="Stinear, Timothy P" uniqKey="Stinear T" first="Timothy P." last="Stinear">Timothy P. Stinear</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Department of Microbiology, Monash University, Clayton, Victoria</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria</wicri:regionArea></affiliation></author></analytic><series><title level="j">PLoS Neglected Tropical Diseases</title><idno type="ISSN">1935-2727</idno><idno type="eISSN">1935-2735</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Amofah, G" uniqKey="Amofah G">G Amofah</name></author><author><name sortKey="Bonsu, F" uniqKey="Bonsu F">F Bonsu</name></author><author><name sortKey="Tetteh, C" uniqKey="Tetteh C">C Tetteh</name></author><author><name sortKey="Okrah, J" uniqKey="Okrah J">J Okrah</name></author><author><name sortKey="Asamoa, K" uniqKey="Asamoa K">K Asamoa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Debacker, M" uniqKey="Debacker M">M Debacker</name></author><author><name sortKey="Aguiar, J" uniqKey="Aguiar J">J Aguiar</name></author><author><name sortKey="Steunou, C" 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS Negl Trop Dis</journal-id><journal-id journal-id-type="iso-abbrev">PLoS Negl Trop Dis</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosntds</journal-id><journal-title-group><journal-title>PLoS Neglected Tropical Diseases</journal-title></journal-title-group><issn pub-type="ppub">1935-2727</issn><issn pub-type="epub">1935-2735</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19936295</article-id><article-id pub-id-type="pmc">2775157</article-id><article-id pub-id-type="publisher-id">09-PNTD-RA-0403R2</article-id><article-id pub-id-type="doi">10.1371/journal.pntd.0000553</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline"><subject>Genetics and Genomics/Gene Expression</subject></subj-group></article-categories><title-group><article-title>Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of <italic>Mycobacterium ulcerans</italic> and Buruli Ulcer</article-title><alt-title alt-title-type="running-head">Gene expression of the mycolactone locus</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Tobias</surname><given-names>Nicholas J.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Seemann</surname><given-names>Torsten</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Pidot</surname><given-names>Sacha J.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Porter</surname><given-names>Jessica L.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Marsollier</surname><given-names>Laurent</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Marion</surname><given-names>Estelle</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Letournel</surname><given-names>Franck</given-names></name><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zakir</surname><given-names>Tasnim</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Azuolas</surname><given-names>Joseph</given-names></name><xref ref-type="aff" rid="aff7"><sup>7</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wallace</surname><given-names>John R.</given-names></name><xref ref-type="aff" rid="aff8"><sup>8</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hong</surname><given-names>Hui</given-names></name><xref ref-type="aff" rid="aff9"><sup>9</sup></xref></contrib><contrib contrib-type="author"><name><surname>Davies</surname><given-names>John K.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Howden</surname><given-names>Benjamin P.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff10"><sup>10</sup></xref></contrib><contrib contrib-type="author"><name><surname>Johnson</surname><given-names>Paul D. R.</given-names></name><xref ref-type="aff" rid="aff10"><sup>10</sup></xref></contrib><contrib contrib-type="author"><name><surname>Jenkin</surname><given-names>Grant A.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Stinear</surname><given-names>Timothy P.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia</addr-line></aff><aff id="aff3"><label>3</label><addr-line>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia</addr-line></aff><aff id="aff4"><label>4</label><addr-line>Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, Université d'Angers, Angers, France</addr-line></aff><aff id="aff5"><label>5</label><addr-line>Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Angers, France</addr-line></aff><aff id="aff6"><label>6</label><addr-line>Laboratoire de Neurobiologie et Transgénèse, UPRES-EA 3143, Université d'Angers, Angers, France</addr-line></aff><aff id="aff7"><label>7</label><addr-line>Department of Primary Industries, Mickleham Road, Attwood, Victoria, Australia</addr-line></aff><aff id="aff8"><label>8</label><addr-line>Department of Biology, Millersville University, Millersville, Pennsylvania, United States of America</addr-line></aff><aff id="aff9"><label>9</label><addr-line>Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom</addr-line></aff><aff id="aff10"><label>10</label><addr-line>Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Picardeau</surname><given-names>Mathieu</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">Institut Pasteur, France</aff><author-notes><corresp id="cor1">* E-mail: <email>tstinear@unimelb.edu.au</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: NJT LM JA JRW JKD PDRJ GAJ TPS. Performed the experiments: NJT TS JLP LM EM FL TZ HH GAJ TPS. Analyzed the data: NJT TS SJP LM EM TZ JA JRW HH JKD BPH PDRJ GAJ TPS. Contributed reagents/materials/analysis tools: NJT TS SJP JLP LM EM FL TZ JA HH BPH PDRJ GAJ. Wrote the paper: NJT JRW TPS.</p></fn></author-notes><pub-date pub-type="collection"><month>11</month><year>2009</year></pub-date><pub-date pub-type="epub"><day>24</day><month>11</month><year>2009</year></pub-date><volume>3</volume><issue>11</issue><elocation-id>e553</elocation-id><history><date date-type="received"><day>10</day><month>8</month><year>2009</year></date><date date-type="accepted"><day>19</day><month>10</month><year>2009</year></date></history><permissions><copyright-statement>Tobias et al.</copyright-statement><copyright-year>2009</copyright-year><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</license-p></license></permissions><abstract><p>Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans.</p></abstract><abstract abstract-type="summary"><title>Author Summary</title><p>Buruli ulcer (BU) is a serious skin infection of humans predominantly occurring in West and Central Africa. The disease is caused by infection with <italic>Mycobacterium ulcerans</italic>, a bacterium that produces an unusual toxin called mycolactone. There are many unanswered questions surrounding BU, particularly regarding the role of mycolactone in disease and how <italic>M. ulcerans</italic> is transmitted to humans. Here, we have partly addressed these questions by identifying genetic factors controlling the transcription of the mycolactone genes. Using a variety of experimental approaches, including green fluorescent protein (GFP) as a reporter of gene expression, we have identified strong promoters that drive transcription of the mycolactone genes in <italic>M. ulcerans</italic>. We then used our GFP reporters to produce highly fluorescent <italic>M. ulcerans-</italic>GFP that were readily visualized by microscopy. Mosquitoes have been proposed as a potential vector of <italic>M. ulcerans</italic> so we used <italic>M. ulcerans</italic>-GFP in feeding experiments with mosquito larvae. <italic>M. ulcerans</italic>-GFP accumulated within the insects, whereas other mycobacteria did not. This is the first report of the mycolactone gene promoters, and we have used our findings to develop <italic>M. ulcerans</italic>-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a powerful tool for studying Buruli ulcer.</p></abstract><counts><page-count count="10"></page-count></counts></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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