SummaryLike other bacterial species, Mycobacterium tuberculosis has multiple sigma (s) factors encoded in its genome. In previously published work, we and others have shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and, in some cases, cause attenuated virulence phenotypes. In this paper, we characterize a M. tuberculosis mutant lacking the ECF s factor s H . This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show decreased ability to grow inside macrophages. Using quantitative reverse transcription-PCR and microarray technology, we have started to define the s H regulon and its involvement in the global regulation of the response to heat shock and the thiol-specific oxidizing agent diamide. We identified 48 genes whose expression increased after exposure of M. tuberculosis to diamide; out of these, 39 were not induced in the sigH mutant, showing their direct or indirect dependence on s H . Some of these genes encode proteins whose predicted function is related to thiol metabolism, such as thioredoxin, thioredoxin reductase and enzymes involved in cysteine and molybdopterine biosynthesis. Other genes under s H control encode transcriptional regulators such as sigB, sigE, and sigH itself.

The numerous sigma () factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis B regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.factors are components of RNA polymerases that bind to the enzyme's core subunits and give promoter specificity. The presence of 13 factors in Mycobacterium tuberculosis reflects the ability of this microorganism to adapt to various stress conditions that are likely encountered during host infection and that make M. tuberculosis a successful pathogen.B is closely related to the primary sigma factor A in terms of amino acid sequence (4). sigB, the structural gene for B , is induced by different stresses (12) and is positively regulated by three extracytoplasmic function sigma factors, E , H , and L (3,13,14). In vitro transcription studies showed that isolated mycobacterial RNA polymerases containing E , H , and L can transcribe sigB using the same transcription start site (3).F -containing RNA polymerase was also shown to transcribe sigB in these studies by using a different transcriptional start site. However, transcriptome studies with sigF mutants or strains overexpressing F show no changes in sigB expression (7, 11), suggesting that the F RNA polymerase transcription of sigB observed in biochemical experiments may not be physiological. The autoregulation of B has also been recently determined by primer extension and reverse transcription-PCR (RT-PCR) (11).In M. tuberculosis, the response to cell envelope is regulated by E and the response to oxidative stress and heat shock is regulated by H , and B is a component of both regulons (13,14). The fact that sigB expression is controlled by many regulatory pathways suggests that B plays a central role in the M. tuberculosis stress response. In this report, we describe the in vitro phenotype of an M. tuberculosis sigB mutant exposed to stress conditions related to the activation of E and H . To analyze the extent to which the response to stress of E and H is transmitted through B , we studied the B regulon activated by cell envelope and oxidative stress in vitro. We also evaluated the growth of the M. tuberculosis sigB mutant strain in THP-1 macrophage-like cells and in vivo in the mouse and guinea pig models of infection. MATERIALS AND METHODSBacterial strains, media, and growth conditions. Escherichia coli strains JM109 and GM161 were grown in Luria broth (LB) (Difco) at 37°C. Antibiotic concentrations used to isolate selectable markers in E. coli were as follows: kanamycin, 50 g/ml; streptomycin, 20 g/ml; and hygromycin, 50 g/ml. The M. tuberculosis strains created in this study were derivatives of M. tuberculosis H37Rv. All M. tuberculosis culture c...

SummarysigA encodes a factor of the 70 family, A , that is found in all mycobacterial species. As A shows high similarity to the primary factor in Streptomyces coelicolor, it was postulated that A has the same role in mycobacteria. However, a point mutation in sigA, resulting in the replacement of arginine 522 by histidine, was found responsible for the attenuated virulence of the Mycobacterium bovis strain ATCC 35721. This raised the possibility that A was an alternative factor specifically required for virulence gene expression. In this work, we show that sigA can not be disrupted in Mycobacterium smegmatis unless an extra copy of the gene is provided at another chromosomal site, which demonstrates that sigA is essential. To characterize the pattern of sigA expression during exponential and stationary phase in M. smegmatis, we measured the ␤-galactosidase activity in a strain carrying a sigA-lacZ transcriptional fusion and monitored A levels using Western blotting. Our results indicate that sigA is expressed throughout the growth of the culture. The essential character of sigA and its pattern of expression corroborate the hypothesis that sigA codes for the primary factor in M. smegmatis and, most likely, in all mycobacteria.

Proteins secreted by Mycobacterium tuberculosis are usually targets of immune responses in the infected host. Here we describe a search for secreted proteins that combined the use of bioinformatics and phoA' fusion technology. The 3,924 proteins deduced from the M. tuberculosis genome were analyzed with several computer programs. We identified 52 proteins carrying an NH(2)-terminal secretory signal peptide but lacking additional membrane-anchoring moieties. Of these 52 proteins-the TM1 subgroup-only 7 had been previously reported to be secreted proteins. Our predictions were confirmed in 9 of 10 TM1 genes that were fused to Escherichia coli phoA', a marker of subcellular localization. These findings demonstrate that the systematic computer search described in this work identified secreted proteins of M. tuberculosis with high efficiency and 90% accuracy.

Exochelin is the primary extracellular siderophore ofMycobacterium smegmatis, and the iron-regulatedfxbA gene encodes a putative formyltransferase, an essential enzyme in the exochelin biosynthetic pathway (E. H. Fiss, Y. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557–569, 1994). We investigated the regulation of fxbA by the mycobacterial IdeR, a homolog of the Corynebacterium diphtheriae iron regulator DtxR (M. P. Schmitt, M. Predich, L. Doukhan, I. Smith, and R. K. Holmes, Infect. Immun. 63:4284–4289, 1995). Gel mobility shift experiments showed that IdeR binds to the fxbA regulatory region in the presence of divalent metals. DNase I footprinting assays indicated that IdeR binding protects a 28-bp region containing a palindromic sequence of the fxbA promoter that was identified in primer extension assays. fxbA regulation was measured in M. smegmatis wild-type and ideR mutant strains containing fxbA promoter-lacZ fusions. These experiments confirmed that fxbA expression is negatively regulated by iron and showed that inactivation of ideRresults in iron-independent expression of fxbA. However, the levels of its expression in the ideR mutant were approximately 50% lower than those in the wild-type strain under iron limitation, indicating an undefined positive role of IdeR in the regulation of fxbA.