Glycosylated p-hydroxybenzoic acid methyl esters and structurally related phenolphthiocerol glycolipids are important virulence factors of Mycobacterium tuberculosis. Although both types of molecules are thought to be derived from p-hydroxybenzoic acid, the origin of this putative biosynthetic precursor in mycobacteria remained to be established. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives. The transposon was found to be inserted in Rv2949c, a gene located in the vicinity of the polyketide synthase gene pks15/1, involved in the elongation of p-hydroxybenzoate to phenolphthiocerol in phenolic glycolipidproducing strains. A recombinant form of the Rv2949c enzyme was produced in the fast-growing non-pathogenic Mycobacterium smegmatis and purified to near homogeneity. The chorismate pathway, present only in bacteria, fungi, and plants, provides a wealth of compounds with diverse biological functions, including aromatic amino acids, folate cofactors, menaquinones, ubiquinones, pigments, and iron-chelating siderophores. Mycobacterium tuberculosis, the etiological agent of tuberculosis in humans, is no exception to the rule, and chorismate in this species is the probable common precursor for the biosynthesis of a series of products (see Fig. 1) that are important both in the physiology and in the pathogenicity of the bacterium. In addition to folate and aromatic amino acids (phenylalanine, tyrosine, and tryptophan), M. tuberculosis produces salicylic acid-derived siderophores known as mycobactins (1), isoprenoid quinones of the naphthalene series (menaquinones) (2), and glycosylated p-hydroxybenzoic acid methyl esters (p-HBAD 4 s) (3). A few strains of M. tuberculosis also produce species-specific phenolic glycolipids (PGL), complex lipids of the cell envelope that share with p-HBADs the same glycosylated aromatic nucleus. These lipids and their counterparts found in Mycobacterium leprae have been largely associated with pathogenicity (4 -9). Likewise, preliminary data indicate that p-HBADs, which are essentially recovered from the secretion products of the tubercle bacillus, modulate the secretion of pro-inflammatory cytokines by murine macrophages (10) and are required for virulence in immunodeficient mice (11).Because of the important roles played by PGL and p-HBADs in the pathogenesis of mycobacterial infections, their biosynthesis has stimulated some interest, and, during the last decade, several genes involved in the synthesis of the lipid core of PGL and in the methylation and glycosylation of PGL and p-HBADs have been described (3,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Most of these genes are clustered on a 70-kilobase region of the chromosome. The synthesis of both p-HBADs and PGL is thought to proceed from p-hydroxybenzoic acid. Comparison of the chemical structures of these compounds suggested that, in the biosynthetic route of p-HBADs, p-hydroxybenzoic acid would be first methylated to p-hydr...

Using signature-tagged transposon mutagenesis, we isolated 23 Mycobacterium tuberculosis mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells.Parasitism of host macrophages (Ms) by pathogenic mycobacteria, including Mycobacterium tuberculosis, the agent of tuberculosis in humans, is a key feature of mycobacterial virulence that needs to be further understood (7,11,25). M infection by the bacillus involves adhesion to and phagocytosismediated entry inside the host cell, as well as resistance to phagosome-lysosome fusion and to free radicals (25). A better comprehension of the microbial molecular determinants involved in these processes might help not only to better understand the cell biology of mycobacterial infections but also to design novel targets for new antimycobacterials and possibly better vaccine candidates. Various approaches have been used in the past to identify mycobacterial genes involved in macrophage infection. These approaches include promoter fusion to reporter genes in order to identify genes induced intracellularly (6, 31), substractive hybridization techniques (8, 21), and transcriptome (27) and proteome (12, 16) analyses, as well as screening of mutant libraries (4,18,20,28). Here we took advantage of the negative selection technique signature-tagged transposon mutagenesis (STM) (9) to identify virulence genes required for M. tuberculosis parasitism of human Ms.Screening of an M. tuberculosis STM library in human Ms. Mycobacteria were grown at 37°C in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC; Difco Laboratories) and 0.05% Tween 80 (Sigma-Aldrich, Corp., St. Louis, MO). THP-1 (ATCC TIB-202) cells were grown in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Dutscher, Brumath, France). THP-1 monocytes were differentiated into Ms by incubation with 10 ng of phorbol 12-myristate 13-acetate (Sigma)/ml for 48 h. An STM library of 1,410 members was constructed with M. tuberculosis MT103 as a parental strain (4) and was used to infect THP-1 Ms for 7 days (input pools). Bacteria were extracted from infected cells and used in a new 7-day infection round in order to enrich in attenuated mutants. Transposon-mediated M. tuberculosis mutants were cultured in the presence of kanamycin (20 g/ml; Sigma). To dislocate clumps, bacteria were passaged through a needle and mildly centrifuged before infection, as described previously (24,29). Infected cells were lysed after each infection round, and bacteria were recovered by plating the material onto selective agar medium. Mutants were recovered 3 weeks after culture (output pools), and the chromosomal DNA was extracted as described previously (17). Tags from input and output pools were amplified and hybridized as described previously (4...

In Drosophila, widely-used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the Twin Spot Generator technique (TSG) and demonstrate that through mitotic recombination, TSG generates green and red twin spots in internal fly tissues, visible even as single cells. We discuss the wide applications of TSG to lineage and genetic mosaic studies.

BACKGROUND AND PURPOSEThe level of cell surface expression of the meningococcal vaccine antigen, Factor H binding protein (FHbp) varies between and within strains and this limits the breadth of strains that can be targeted by FHbp-based vaccines. The molecular pathway controlling expression of FHbp at the cell surface, including its lipidation, sorting to the outer membrane and export, and the potential regulation of this pathway have not been investigated until now. This knowledge will aid our evaluation of FHbp vaccines. EXPERIMENTAL APPROACHA meningococcal transposon library was screened by whole cell immuno-dot blotting using an anti-FHbp antibody to identify a mutant with reduced binding and the disrupted gene was determined. KEY RESULTSIn a mutant with markedly reduced binding, the transposon was located in the lnt gene which encodes apolipoprotein N-acyl transferase, Lnt, responsible for the addition of the third fatty acid to apolipoproteins prior to their sorting to the outer membrane. We provide data indicating that in the Lnt mutant, FHbp is diacylated and its expression within the cell is reduced 10 fold, partly due to inhibition of transcription. Furthermore the Lnt mutant showed 64 fold and 16 fold increase in susceptibility to rifampicin and ciprofloxacin respectively. CONCLUSION AND IMPLICATIONSWe speculate that the inefficient sorting of diacylated FHbp in the meningococcus results in its accumulation in the periplasm inducing an envelope stress response to down-regulate its expression. We propose Lnt as a potential novel drug target for combination therapy with antibiotics. LINKED ARTICLESThis article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit