The ability of Mycobacterium tuberculosis to adapt to different environments in the infected host is essential for its pathogenicity. Consequently, this organism must be able to modulate gene expression to respond to the changing conditions it encounters during infection. In this paper we begin a comprehensive study of M. tuberculosis gene regulation, characterizing the transcript levels of 10 of its 13 putative sigma factor genes. We developed a real‐time RT‐PCR assay using a family of novel fluorescent probes called molecular beacons to quantitatively measure the different mRNAs. Three sigma factor genes were identified that have increased mRNA levels after heat shock, two of which also responded to detergent stress. In addition, we also identified a sigma factor gene whose mRNA increased after mild cold shock and a second that responded to conditions of low aeration.

Summary: Like other bacterial species, Mycobacterium tuberculosis has multiple sigma (σ) 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 σ factor σ^H. This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show de‐creased ability to grow inside macrophages. Using quantitative reverse transcription‐PCR and microarray technology, we have started to define the σ^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 σ^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 σ^H control encode transcriptional regulators such as sigB, sigE, and sigH itself.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. TheMycobacterium tuberculosisgenome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zuroperon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated azurmutant inM. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in thezurmutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response toM. tuberculosisinfection.

The nucleotide sequence of comC, the gene encoding the 17-residue competence-stimulating peptide (CSP) of Streptococcus pneumoniae (L. S. Havarstein, G. Coomaraswamy, and D. A. Morrison, Proc. Natl. Acad. Sci. USA 92:11140-11144, 1995) was determined with 42 encapsulated strains of different serotypes. A new allele, comC2, was found in 13 strains, including the type 3 Avery strain, A66, while all others carried a gene (now termed comC1) identical to that originally described for strain Rx1. The predicted mature product of comC2 is also a heptadecapeptide but differs from that of comC1 at eight residues. Both CSP-1 and CSP-2 synthetic peptides were used to induce competence in the 42 strains; 48% of the strains became competent after the addition of the synthetic peptide, whereas none were transformable without the added peptides.

Pathogenetic processes that facilitate the entry, replication, and persistence ofMycobacterium tuberculosis(MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressedin vivowould provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited. In this study, the levels of selected MTB mRNAs were quantifiedin vitroin axenic culture,in vivoin the lungs of mice, and in lung specimens obtained from TB patients with active disease. We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL/6 mice as compared with bacteria grownin vitro. Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.