No abstract

Original genome annotations need to be regularly updated if the information they contain is to remain accurate and relevant. Here the complete reannotation of the genome sequence of Mycobacterium tuberculosis strain H37Rv is presented almost 4 years after the first submission. Eighty-two new protein-coding sequences (CDS) have been included and 22 of these have a predicted function. The majority were identified by manual or automated reanalysis of the genome and most of them were shorter than the 100 codon cutoff used in the initial genome analysis. The functional classification of 643 CDS has been changed based principally on recent sequence comparisons and new experimental data from the literature. More than 300 gene names and over 1000 targeted citations have been added and the lengths of 60 genes have been modified. Presently, it is possible to assign a function to 2058 proteins (52 % of the 3995 proteins predicted) and only 376 putative proteins share no homology with known proteins and thus could be unique to M. tuberculosis.

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.A single Buruli ulcer, which can cover Ͼ15% of a person's skin surface, contains huge numbers of extracellular bacteria. Despite their abundance and extensive tissue damage, there is a remarkable absence of an acute inflammatory response to the bacteria, and the lesions are often painless (1). This unique pathology is attributed to mycolactone, a macrolide toxin consisting of a polyketide side chain attached to a 12-membered core that seems to have cytotoxic, analgesic, and immunosuppressive activities. Its mode of action is unclear, but, in a guinea pig model of the disease, purified mycolactone injected s.c. reproduces the natural pathology, and mycolactone negative variants are avirulent, implying a key role for the toxin in pathogenesis (2).Mycobacterium ulcerans (MU) and Mycobacterium marinum (MM) share over 98% DNA sequence identity, they occupy aquatic environments, and both cause cutaneous infections (3). However, MM produces a granulomatous intracellular lesion, typical for pathogenic mycobacteria and totally distinct from Buruli ulcer in which MU are mainly found extracellularly. The fact that MM does not produce mycolactone suggested that it might be possible to identify genes for mycolactone synthesis by performing genomic subtraction experiments between MU and MM. Fragments of MU-specific polyketide synthase (PKS) genes were identified from these experiments (4). The subsequent investigation of these sequences led to the discovery of the MU virulence plasmid pMUM001 and the extraordinary PKS locus it encodes. Plasmid Sequence Determination....