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. 2010 Mar;192(6):1662-70.
doi: 10.1128/JB.00926-09. Epub 2009 Dec 18.

The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy

Rachel L Leistikow  1 , Russell A Morton, Iona L Bartek, Isaac Frimpong, Karleen Wagner, Martin I Voskuil
Affiliations

The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy

Rachel L Leistikow et al. J Bacteriol. 2010 Mar.

Abstract

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.

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Figures

FIG. 1.
FIG. 1.
Growth and survival during anaerobic dormancy determined by recovery on solid and liquid media. Wild-type, DorKO mutant, and complemented bacteria were grown in glass tubes with stir bars and a culture-to-headspace ratio of 0.65. Cultures were stirred with magnetic stirrers. (A) CFU were counted at various times by plating samples from tubes harvested sacrificially. Squares, H37Rv; triangles, DorKO; circles, DorCO. (B) Viability assessed by serial dilution of dormant bacteria in DTA broth using the MPN technique and comparison to CFU values (39). Open bars, H37Rv on plates; bars with horizontal stripes, H37Rv in liquid medium; solid bars, DorKO on plates; checkered bars, DorKO in liquid medium; gray bars, DorCO on plates; stippled bars, DorCO in liquid medium. The values are the averages of three experiments.
FIG. 2.
FIG. 2.
Live-dead staining during dormancy. Viability was assessed using live-dead staining in combination with the DNA stain DAPI. Cultures from log phase, day 20, and day 100 were washed in HEPES with Tween, stained with fluorescein diacetate (FDA) and DAPI for 20 min, and then washed and visualized as wet mounts. The images are representative of multiple captured images.
FIG. 3.
FIG. 3.
Oxygen consumption (methylene blue decolorization) in cultures during adaptation to dormancy. Methylene blue (9 μg/ml) was added to dormancy model cultures before they were sealed. The optical density at 600 nm was measured every 2 h during the critical period between day 3 and day 6 after the start of the experiment. The values are the averages of four experiments. Squares, H37Rv; triangles, DorKO; circles, DorCO.
FIG. 4.
FIG. 4.
ATP measurement during early dormancy. Samples from RAD model cultures were harvested at various time points in an anaerobic chamber. ATP was extracted using a chloroform heat-based method and frozen until measurements could be obtained using the Promega Enliten ATP assay system. The values are the averages of three experiments. Open bars, H37Rv; black bars, DorKO; gray bars, DorCO. RLUs, relative light units.
FIG. 5.
FIG. 5.
NAD and NADH levels and NAD/NADH ratio during early dormancy. Samples from dormancy model cultures were harvested at various time points in an anaerobic chamber. Nucleotides were extracted using either acid or base combined with heat and frozen prior to measurement using an alcohol dehydrogenase-based NAD-NADH cycling assay. The values are the averages of three experiments. (A and B) Levels of NAD (A) and NADH (B). Open bars, H37Rv; black bars, DorKO; gray bars, DorCO. (C) Ratio of NAD to NADH. Squares, H37Rv; triangles, DorKO; circles, DorCO.
FIG. 6.
FIG. 6.
Growth rate during recovery from dormancy. After 10 or 40 days of dormancy, the wild-type, DorKO, and DorCO strains were exposed to oxygen and fresh medium and allowed to resume growth. Bacterial growth was measured every day for 5 days by plating on DTA agar plates. The growth rate was calculated by determining the fold increase in the number of CFU compared with the number of bacteria present immediately after the cultures were opened at day 10 or 40. The values are the averages of three experiments. (A) Cultures opened at day 10. (B) Cultures opened at day 40. Squares, H37Rv; triangles, DorKO; circles, DorCO.
FIG. 7.
FIG. 7.
DosR regulon and recovery from extended exposure to a low dose of NO. DETA-NO (100 μM) was added to aerobic growing H37Rv, DorKO, or DorCO cultures every 6 h over a 36-h period. Growth was followed by determining the OD600. Squares, strain H37Rv; triangles, DorKO; circles, DorCO; open symbols, control; filled symbols, NO added.
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