TheMycobacterium tuberculosisSigma Factor σ^BIs Required for Full Response to Cell Envelope Stress and Hypoxia In Vitro, but It Is Dispensable for In Vivo Growth

Abstract

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...

PhoY2 of Mycobacteria Is Required for Metabolic Homeostasis and Stress Response

“…Wayne model (9,28). M. marinum standing cultures prepared in 15-ml polypropylene tubes were sealed and incubated at 32°C with slow shaking (40 rpm) for a long time (up to 60 days).…”

“…The hypoxic cultures were established according to a published protocol (28). M. marinum cells grown in 7H9 broth to early exponential phase were washed and resuspended in 10 ml of 7H9 broth to a final OD 600 of 0.2 and transferred to 15-ml tubes (Corning) in order to maintain an air/medium volume ratio of 1:2.…”

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

“…Expression of the M. tuberculosis factors H , E , and B can be triggered by either H or E (268,269). A growing body of literature indicates that these sigma factors are induced in response to several different in vitro conditions mimicking in vivo stresses faced by the pathogen during the various stages of infection and disease (263)(264)(265)(266)(267)270).…”

“…Sigma () factors are a class of transcription factors that control bacterial gene expression (262)(263)(264)(265)(266)(267). Typically, most eubacteria encode a principal and a variable number of alternate factors, which control responses to specific environmental stimuli and adaptation to stress.…”

Regulation of Central Metabolism Genes of Mycobacterium tuberculosis by Parallel Feed-Forward Loops Controlled by Sigma Factor E (σ E )

“…Moreover, the gltA1 operon is regulated by B when tubercle bacilli are subjected to diamide stress (7). Thus, it is likely that multiple accessory sigma factors are involved in the regulation of icl1 and gltA1.…”