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...

The slow-growing Mycobacterium celatum is known to have two different 16S rRNA gene sequences. This study confirms the presence of two rrn operons and describes their organization. One operon (rrnA) was found to be located downstream from murA and the other (rrnB) was found downstream from tyrS. The promoter regions were sequenced, and also the intergenic transcribed spacer (ITS1 and ITS2) regions separating the 16S rRNA, 23S rRNA and 5S rRNA gene coding regions. Analysis of the RNA fraction revealed that rrnA is regulated by two (P1 and PCL1) promoters and rrnB is regulated by one (P1). These data show that the two rrn operons of M. celatum are organized in the same way as the two rrn operons of classical fast-growing mycobacteria. This information was incorporated into a phylogenetic analysis of the genus based on both 16S rRNA gene sequences and (where possible) the number of rrn operons per genome. The results suggest that the ancestral Mycobacterium possessed two (rrnA and rrnB) operons per genome and that subsequently, on two separate occasions, an operon (rrnB) was lost, leading to two clusters of species having a single operon (rrnA); one cluster includes the classical pathogens and the other includes Mycobacterium abscessus and Mycobacterium chelonae.