The Largest Open Reading Frame (pks12) in the Mycobacterium tuberculosis Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis

Tuberculosis accounts for more than one-fourth of all preventable adult deaths in the world (Global tuberculosis control, World Health Organization report, http://www.who.int/gtb/publications/glovrep01/, 2001). Mycobacterium tuberculosis, the causative agent, can evade the defense mechanisms of the host and grow in macrophages that normally phagocytose and destroy most other bacterial pathogens. It has been widely recognized that the unusually complex cell wall of the organism plays a major role in the exceptional ability of the pathogen to be a successful pathogen (3, 8). The cell wall is rich (50 to 60%) in unusual lipids that are highly hydrophobic, and some of the cell wall components may also help the pathogen to enter macrophages (24) and suppress the defense mechanisms of the host (18, 22). Therefore, it is not surprising that cell wall synthesis has proved to be the target of some of the most successful antimycobacterial drugs. However, the pathogen has acquired resistance to several frontline antimycobacterial drugs, and the multidrug-resistant M. tuberculosis has been classified as a class C organism by the Centers for Disease Control and Prevention. The potential for such multidrug-resistant pathogens to cause major public health problems in highly populated areas makes identification of new targets for antimycobacterial therapy a critical need. The presence of very-long-chain fatty acids with multiple methyl branches at alternate positions near the carboxyl end is a unique feature of mycobacterial cell wall lipids (17). Derivatives of such acids are virulence factors. For example, it was suggested that dimycocerosyl phthiocerol (DIM), composed of mycocerosic acids (2,4,6,8-tetramethyl C32 fatty acid and homologues) esterified to the long-chain diol phthiocerol, is a virulence factor because mutants that lack this compound were attenuated in human monocytes and in the murine lung (4, 7, 25). We cloned the mycocerosic acid synthase (MAS) gene, mas (19), and proved it to be the one responsible for the production of mycocerosic acids by gene disruption (1). The mycobacterial genome contains many polyketide synthase (PKS) genes (pks) (6), including seven mas-like (msl) genes (25). These genes usually encode one full complement of catalytic domains required to catalyze the synthesis of a fully saturated fatty acid (one module). These include the acyl transferase (AT) that transfers the reactants to the synthase ketoacyl synthase (KS), which catalyzes the condensation of the reactants to form the carbon-carbon bond generating the ketoacyl derivative ketoreductase, which reduces the carbonyl to the secondary hydroxyl dehydratase, which eliminates water to generate the unsaturated acyl group and enoyl reductase, which reduces the olefin to a fully saturated moiety. All of these reactions happen while the growing chain is still attached to the phosphopantetheine of the acyl carrier protein (ACP) domain of the synthase (17, 19). One of the msls (msl6, pks12), the largest open reading frame (ORF) in the mycobacterial genome (6), encodes two modules that can catalyze the synthesis of a saturated acid. Such large ORFs encoding multiple sets of modules have been previously found only in antibiotic-producing organisms (15, 16, 20). Whether this gene is expressed in M. tuberculosis and, if it is, what the nature of the product and its biological function are and whether gene expression contributes to virulence remain unknown. In this paper we report that this largest mycobacterial ORF is expressed in M. tuberculosis and we identify the protein product by showing that the amino acid sequences of 54 peptides distributed throughout the 430-kDa protein in M. tuberculosis H37Rv matches with the sequences predicted from the nucleotide sequence of the gene. We also report disruption of this gene in M. tuberculosis and show that the msl6 mutant does not produce the 430-kDa protein. The msl6-disrupted mutant is defective in DIM synthesis and is highly attenuated.