Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer.
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 mega...
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ftdoajarticles:oai:doaj.org/article:f5ca91bc94794b68aef17ebbecdb2553 2023-05-15T15:14:49+02:00 Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. Nicholas J Tobias Torsten Seemann Sacha J Pidot Jessica L Porter Laurent Marsollier Estelle Marion Franck Letournel Tasnim Zakir Joseph Azuolas John R Wallace Hui Hong John K Davies Benjamin P Howden Paul D R Johnson Grant A Jenkin Timothy P Stinear 2009-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0000553 https://doaj.org/article/f5ca91bc94794b68aef17ebbecdb2553 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC2775157?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0000553 https://doaj.org/article/f5ca91bc94794b68aef17ebbecdb2553 PLoS Neglected Tropical Diseases, Vol 3, Iss 11, p e553 (2009) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2009 ftdoajarticles https://doi.org/10.1371/journal.pntd.0000553 2022-12-31T07:18:01Z 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. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 3 11 e553 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Nicholas J Tobias Torsten Seemann Sacha J Pidot Jessica L Porter Laurent Marsollier Estelle Marion Franck Letournel Tasnim Zakir Joseph Azuolas John R Wallace Hui Hong John K Davies Benjamin P Howden Paul D R Johnson Grant A Jenkin Timothy P Stinear Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
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. |
format |
Article in Journal/Newspaper |
author |
Nicholas J Tobias Torsten Seemann Sacha J Pidot Jessica L Porter Laurent Marsollier Estelle Marion Franck Letournel Tasnim Zakir Joseph Azuolas John R Wallace Hui Hong John K Davies Benjamin P Howden Paul D R Johnson Grant A Jenkin Timothy P Stinear |
author_facet |
Nicholas J Tobias Torsten Seemann Sacha J Pidot Jessica L Porter Laurent Marsollier Estelle Marion Franck Letournel Tasnim Zakir Joseph Azuolas John R Wallace Hui Hong John K Davies Benjamin P Howden Paul D R Johnson Grant A Jenkin Timothy P Stinear |
author_sort |
Nicholas J Tobias |
title |
Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. |
title_short |
Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. |
title_full |
Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. |
title_fullStr |
Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. |
title_full_unstemmed |
Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. |
title_sort |
mycolactone gene expression is controlled by strong siga-like promoters with utility in studies of mycobacterium ulcerans and buruli ulcer. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2009 |
url |
https://doi.org/10.1371/journal.pntd.0000553 https://doaj.org/article/f5ca91bc94794b68aef17ebbecdb2553 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 3, Iss 11, p e553 (2009) |
op_relation |
http://europepmc.org/articles/PMC2775157?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0000553 https://doaj.org/article/f5ca91bc94794b68aef17ebbecdb2553 |
op_doi |
https://doi.org/10.1371/journal.pntd.0000553 |
container_title |
PLoS Neglected Tropical Diseases |
container_volume |
3 |
container_issue |
11 |
container_start_page |
e553 |
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1766345226752884736 |