Regulation of nitrate reductase activity in Mycobacterium tuberculosis by oxygen and nitric oxide. The supernatant was collected every 5 days and secreted succinate was analyzed for 13C incorporation. bis Favorite Answer. Separation was carried out using an ion exclusion column (Aminex, Bio-Rad) HPX-87H, 7.8 mm-300 mm, with guard column, heated to 40°C, with an isocratic flow rate of 0.8 ml/min of 0.1% (v/v) formic acid in water. The efflux of succinate is electrogenic and bioenergetic studies in E. coli have shown that H+/succinate symport is very efficient [41]. The notion that glycolytic intermediates feed into the reductive branch of the TCA cycle by a reverse gluconeogenic processes is particularly appealing considering the environment of the lung where CO2 concentrations are more than 10-fold higher than atmospheric CO2 concentrations [53]. Anonymous. The absence of a phenotype in the frdA knockout mutant during infection of mice is perhaps not surprising since typical strains of mice do not form the hypoxic granulomas associated with human tuberculosis disease [6]. Expression levels of 15 days NRP cultures were expressed as ratios compared with aerobic culture and normalized to the levels of sigA. The MSD used electrospray ionization in negative ion mode with selective ion monitoring (SIMES-). The combination of severely limited oxygen availability coupled to the presence of reactive nitrogen intermediates, which inactivate respiratory cytochromes, creates a metabolic bottleneck as unrespired electrons become trapped in the form of an abundance of reduced cofactors. Citation: Watanabe S, Zimmermann M, Goodwin MB, Sauer U, Barry CE 3rd, Boshoff HI (2011) Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis. Electroporation and generation of double crossover strains was performed as previously described [25], [56]. Each time point represents the median CFU and standard error of 5 mice per group. For continuous cultures, M. tuberculosis H37Rv was grown in fermentors under defined dissolved oxygen tensions, 50.0 mmHg, 8.16 mmHg, 1.85 mmHg, 1.44 mmHg and 0.42 mmHg. In this organism the TCA cycle is complete but also bifurcated so that the oxidative and reductive branches can be engaged independently of one another. No, Is the Subject Area "Respiratory infections" applicable to this article? https://doi.org/10.1371/journal.ppat.1002287, Editor: Vojo Deretic, University of New Mexico, United States of America, Received: November 30, 2010; Accepted: August 12, 2011; Published: October 6, 2011. Analysis of gene expression levels of M. tuberculosis during survival under oxygen tensions that do not support replication previously revealed upregulation of phosphoenolpyruvate carboxykinase and the malic enzyme which catalyzes the oxidative decarboxylation of malate coupled with the reduction of NAD(P)+ producing pyruvate and CO2 [42]. Eventually, two clones, named IW1 and IW2, were proven to have the expected deletion of the narG gene. To rule out that the observed succinate was accumulating in response to nitrogen assimilation from asparagine we repeated the analysis with media in which asparagine was replaced with aspartate and observed the same robust accumulation of succinate. (A) Growth curve of H37Rv in fermentor culture maintained at various dissolved oxygen tensions.
Biochemical characteristics among Mycobacterium bovis BCG substrains. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to … Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland. For more information about PLOS Subject Areas, click It takes 4-6 weeks to get visual colonies. Published by Elsevier Ltd. International Journal of Mycobacteriology, https://doi.org/10.1016/j.ijmyco.2014.11.066. Gene expression levels of 8, 2, 1, 0.5 and 0.1 mmHg culture grown with or without 5% carbon dioxide were presented as ratios compared with 50 mmHg culture; (A) frdA, (B)sdhA, (C) Rv0248c, (D) mez, (E), pckA, (F) pca, (G) aceE, (H) pdhA, (l) lldD1, (J) citA, (K) acn, (L) icd1, (M) icd2, (N)kgd, (O) korA, (P) fum, (Q) icl1, (R) ilvB2, (S) hsaG, (T) accA1, (U) glpD1, (V) Rv3837c, (W) tgs1, and (X) hspX. In the complete absence of an external electron acceptor, oxidation of cholesterol or fatty acids would not lead to ATP generation since metabolism of these substrates does not produce enough high energy intermediates through substrate level phosphorylation. We observed a slight increase in lactic acid secretion in our anaerobic cultures over time (Figure 3A). Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor.

In contrast to anaerobic nitrate reductase activity, it has been known for many years that M. tuberculosis is strongly positive for nitrate reductase activity under aerobic conditions whereas M. bovis and M. bovis BCG are not, and this is now a frequently used method for differentiating between the species (Virtanen, 1960; Hedgecock and Costello, 1962; Ratledge, …

Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username, I have read and accept the Wiley Online Library Terms and Conditions of Use, Inherited interleukin 12 deficiency in a child with bacille Calmette‐Guerin and, The persistence of bacille Calmette‐Guerin in the bladder after intravesical treatment for bladder cancer, Idiopathic disseminated bacillus Calmette‐Guerin infection: a French national retrospective study, Correlation of granuloma structure with clinical outcome defines two types of idiopathic disseminated BCG infection, Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays, Utilization of nitrate by pathogenic and saprophytic mycobacteria, Interferon‐γ‐receptor deficiency in an infant with fatal bacille Calmette‐Guerin infection, Incidence and treatment of complications of bacillus Calmette‐Guerin intravesical therapy in superficial bladder cancer, A mutation in the interferon‐γ‐receptor gene and susceptibility to mycobacterial infection, Mycobacterial virulence.

NADH/NAD+ and ATP concentrations were measured in chemostat cultures with a constant growth rate of 0.0077 h−1 under 50.0, 8.19, 1.85, 1.44 and 0.42 mmHg of dissolved oxygen tensions. Dissolved oxygen concentration was measured by InPro 6800 D.O. These studies show that fermentation plays an important role in the maintenance of tuberculosis infection under oxygen tensions proven relevant in granulomatous infections in higher vertebrates, a better understanding of this process may lead to the rational selection of targets for latent infection as well as holding the potential for shortening the duration of tuberculosis chemotherapy. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. System control and data analysis were performed using Agilent Chemstation version A.09.03. Consistent with our finding that lactate also slightly accumulated during anaerobic non-replicating persistence (Figure 3A; vide infra), lactate dehydrogenase, was upregulated by reduced oxygen concentrations (Figure S1 and Figure S2). NnrA Is Required for Full Virulence and Regulates Several Brucella melitensis Denitrification Genes. Schematic illustration of glycolysis and the TCA cycle and graphs of isotopomer distribution during metabolism of U-13C glucose. This corresponds to the end state of non-replicating persistence in Wayne model cells. Inspection of previously published microarray data on Wayne-model adapted cells implicated the upregulation of several enzymes of the reductive branch of the TCA cycle. Although the M. tuberculosis nitrate reductase genetically contains all the elements of a proton pumping respiratory enzyme, nitrate appears not to support growth of this organism and the function of this enzyme may be limited to a role in nitrogen assimilation [24].

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