Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the pathogen's survival - PubMed (original) (raw)

Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the pathogen's survival

Gaëlle Huet et al. J Bacteriol. 2005 Sep.

Abstract

The worldwide recrudescence of tuberculosis and widespread antibiotic resistance have strengthened the need for the rapid development of new antituberculous drugs targeting essential functions of its etiologic agent, Mycobacterium tuberculosis. In our search for new targets, we found that the M. tuberculosis pps1 gene, which contains an intein coding sequence, belongs to a conserved locus of seven open reading frames. In silico analyses indicated that the mature Pps1 protein is orthologous to the SufB protein of many organisms, a highly conserved component of the [Fe-S] cluster assembly and repair SUF (mobilization of sulfur) machinery. We showed that the mycobacterial pps1 locus constitutes an operon which encodes Suf-like proteins. Interactions between these proteins were demonstrated, supporting the functionality of the M. tuberculosis SUF system. The noticeable absence of any alternative [Fe-S] cluster assembly systems in mycobacteria is in agreement with the apparent essentiality of the suf operon in Mycobacterium smegmatis. Altogether, these results establish that Pps1, as a central element of the SUF system, could play an essential function for M. tuberculosis survival virtually through its implication in the bacterial resistance to iron limitation and oxidative stress. As such, Pps1 may represent an interesting molecular target for new antituberculous drugs.

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Figures

FIG. 1.

FIG. 1.

Organization of the suf locus from different bacterial species. (A) Alignment of the suf loci from E. chrysanthemi and M. tuberculosis. Sizes of the genes and of intergenic regions are indicated in parentheses. (B) Organization of M. leprae (ORF numbered ML0592 to ML0598 according to the Leproma web site [

http://genolist.pasteur.fr/Leproma/

]) and M. smegmatis (ORF that we numbered Ms1460 to Ms1466 by homology with M. tuberculosis genome) loci. Arrows indicate the orientations of the genes. The intein sequence within M. tuberculosis and M. leprae pps1 gene appears as a gray box.

FIG. 2.

FIG. 2.

RT-PCR analysis of the mycobacterial suf operon. (A) Schematic representation of the RT-PCR experiments. Open arrowheads represent the RT primers, and black arrowheads represent the PCR primers. (B) PCR amplification of a fragment of the Ms1461 gene using primers LF 1461 (L1) and RG 1461 (R1) and different DNA templates, as follows: H2O, nonretrotranscribed M. smegmatis RNA, M. smegmatis genomic DNA, and cDNA obtained by retrotranscription of RNA using the different RT primers RT 1461 (1), RT 1462 (2), RT 1463 (3), RT 1464 (4), RT 1465 (5), and RT 1466 (6). (C) PCR amplification of a fragment of the Ms1464 gene using primers LF 1464 (L4) and RG 1464 (R4) and different DNA templates, as follows: H2O, nonretrotranscribed M. smegmatis RNA, M. smegmatis genomic DNA, and cDNA obtained by retrotranscription of RNA using the different RT primers RT 1463 (3), RT 1464 (4), RT 1465 (5), and RT 1466 (6).

FIG. 3.

FIG. 3.

The suicide vector strategy used in M. smegmatis. (A) pJQ200-pps1::hyg suicide vector. (B) Integration of the suicide vector by homologous recombination in the 5′ part of the pps1 gene (Ms1461). (C) Integration of the suicide vector by homologous recombination in the 3′ part of the pps1 gene. (D) Integration of the hygromycin resistance gene in the pps1 gene by double recombination. Ms1461′ and Ms1462′ represent 3′-truncated Ms1461 and Ms1462 genes, ′Ms1460, ′Ms1461, and ′Ms1462 represent the 5′-truncated genes. Arrows indicate the orientations of the genes. The original pJQ200 plasmid DNA is represented by a thick black line in panels B and C.

FIG. 4.

FIG. 4.

Generation time of the wild-type (white bars) and mutant (black bars) M. smegmatis in the presence of different concentrations of DIP.

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