Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence

GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK 1 London School of Hygiene & Tropical Medicine, Keppel St, London WC1E 7HT, UK 2 Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 3 Author for correspondence: Ken Duncan. Tel: +...

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Published in:Microbiology (Society for General Microbiology) 2002-10, Vol.148 (10), p.2975-2986
Main Authors: McAdam, Ruth A, Quan, Selwyn, Smith, Debbie A, Bardarov, Stoyan, Betts, Joanna C, Cook, Fiona C, Hooker, Elizabeth U, Lewis, Alan P, Woollard, Peter, Everett, Martin J, Lukey, Pauline T, Bancroft, Gregory J, Jacobs, William R., Jr, Duncan, Ken
Format: Article
Language:English
Subjects:
Animals
Disease Models, Animal
DNA Transposable Elements - genetics
Gene Library
Humans
Mice
Mice, SCID
Mutagenesis, Insertional
Mutation
Mycobacterium tuberculosis
Mycobacterium tuberculosis - genetics
Mycobacterium tuberculosis - pathogenicity
Open Reading Frames - genetics
transposon Tn5370
Tuberculosis, Pulmonary - microbiology
Virulence
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Summary:GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK 1 London School of Hygiene & Tropical Medicine, Keppel St, London WC1E 7HT, UK 2 Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 3 Author for correspondence: Ken Duncan. Tel: +44 1438 763841. Fax: +44 1438 764799. e-mail: kd9430{at}gsk.com A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn 5370 . The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine. Keywords: gene disruption, severe combined immune deficiency (SCID) mouse, attenuation Abbreviations: BCG, Bacille Calmette–Guérin; SCID, severe combined immune deficiency b The precise locations of all of the insertions examined in this study can be found as supplementary data in Microbiology Online (http://mic.sgmjournals.org). a Present address: Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111, USA.
ISSN:1350-0872
1465-2080
DOI:10.1099/00221287-148-10-2975