Responses of Mycobacterium tuberculosis to Growth in the Mouse Lung

Using a promoter trap, we have identified 56 Mycobacterium tuberculosis genes preferentially expressed in the mouse lung. Quantitative real-time PCR showed that RNA levels of several genes were higher from bacteria growing in mouse lungs than from broth cultures. These results support the current hypothesis that Mycobacterium tuberculosis utilizes fatty acids as a carbon source in the mouse lung

Cholesterol Is Not an Essential Source of Nutrition for Mycobacterium tuberculosis during Infection ▿ †

Rv1106c (hsd; 3β-hydroxysteroid dehydrogenase) is required by Mycobacterium tuberculosis for growth on cholesterol as a sole carbon source, whereas Rv3409c is not. Mutation of Rv1106c does not reduce Mycobacterium tuberculosis growth in infected macrophages or guinea pigs. We conclude that cholesterol is not required as a nutritional source during infection

Structure-Function Studies of the Bacillus subtilis Ric Proteins Identify the Fe-S Cluster-Ligating Residues and Their Roles in Development and RNA Processing

In Bacillus subtilis, the RicA (YmcA), RicF (YlbF), and RicT (YaaT) proteins accelerate the phosphorylation of the transcription factor Spo0A, contributing to genetic competence, sporulation, and biofilm formation, and are also essential for the correct maturation of several protein-encoding and riboswitch RNAs. These proteins form a stable complex (RicAFT) that carries two 4Fe-4S](+2) clusters. We show here that the complex is a 1:1:1 heterotrimer, and we present the X-ray crystal structures of a RicAF heterotetramer and of a RicA dimer. We also demonstrate that one of the Fe-S clusters (cluster 1) is ligated by cysteine residues donated exclusively by RicT and can be retained when the RicT monomer is purified by itself. Cluster 2 is ligated by C167 from RicT, by C134 and C146 located near the C terminus of RicF, and by C141 at the C terminus of RicA. These findings imply the following novel arrangement: adjacent RicT residues C166 and 167 ligate clusters 1 and 2, respectively, while cluster 2 is ligated by cysteine residues from RicT, RicA, and RicF. Thus, the two clusters must lie close to one another and at the interface of the RicAFT protomers. We also show that the cluster-ligating cysteine residues, and therefore most likely both Fe-S clusters, are essential for cggR-gapA mRNA maturation, for the regulation of ricF transcript stability, and for several Ric-associated developmental phenotypes, including competence for transformation, biofilm formation, and sporulation. Finally, we present evidence that RicAFT, RicAF, and RicA and the RicT monomer may play distinct regulatory roles in vivo. IMPORTANCE The RicA, RicF, and RicT proteins are widely conserved among the firmicute bacteria and play multiple roles in Bacillus subtilis. Among the phenotypes associated with the inactivation of these proteins are the inability to be genetically transformed or to form biofilms, a decrease in sporulation frequency, and changes in the stability and maturation of multiple RNA species. Despite their importance, the molecular mechanisms of Ric protein activities have not been elucidated and the roles of the two iron-sulfur clusters on the complex of the three proteins are not understood. To unravel the mechanisms of Ric action, molecular characterization of the complex and of its constituent proteins is essential. This report represents a major step toward understanding the structures of the Ric proteins, the arrangement and roles of the Fe-S clusters, and the phenotypes associated with Ric mutations

A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol▿ †

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipid-metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a β-ketoacyl-CoA thiolase function in cholesterol β-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol