Figure 1.
The growth and survival of Mycobacterium smegmatis in the presence and absences of hydrogenases.
(A) Growth of M. smegmatis mc2155 and hyd mutants into carbon-limitation. (B) Survival of M. smegmatis mc2155 and hyd mutants during carbon-limitation. The strains were grown in aerated conical flasks on HdB minimal medium supplemented with 22 mM glycerol. Growth is shown in OD600. Survival is shown in percentage colony forming units relative to day four. Legend: Blue circles/bars = Wild-type; Red squares/bars = Δhyd123; Orange point-up triangles/bars = Δhyd1; and Purple point-down triangles/bars = Δhyd2. Error bars show standard deviations from biological triplicates. * = p<0.05, ** = p<0.01, *** = p<0.001 difference relative to wild-type bars (Student’s T-test, unpaired, two-tailed).
Figure 2.
Observation, complementation, and recovery of mutant growth phenotypes.
Strains were grown on HdB minimal medium. (A) Growth on 5.5 mM glycerol. (B) Complementation on 5.5 mM glycerol in the presence of 50 µg mL−1 hygromycin. (C) Growth on 12.5 mM acetate. (D) Partial complementation on 12.5 mM acetate in the presence of 50 µg mL−1 hygromycin. (E) Growth on 12.5 mM acetate in serum vials injected with 10% pure N2. (F) Growth on 12.5 mM acetate in serum vials injected with 10% pure H2. Legend: Blue circles = Wild-type (or wild-type with empty pOLYG vector for complementation); Red squares = Δhyd123; Orange point-up triangles = Δhyd1; Purple point-down triangles = Δhyd2 (or Δhyd2 with empty pOLYG vector for complementation); and Grey diamonds = Δhyd2 with pOLYG vector expressing MSMEG_2720-2719. Error bars show standard deviations from biological triplicates.
Table 1.
Energetic parameters of wild-type and Δhyd2 cells two hours following the induction of stationary phase.
Figure 3.
Genes with significant changes in expression in Δhyd2 vs. wild-type cells.
Both strains were grown synchronously on HdB minimal medium supplemented with 22>2.0, p value≤0.05. The genes were classified as significantly downregulated if expression ratio <0.5, p value≤0.05. The number of genes affected are listed by functional category. The full list of genes in each category is shown in Table S4, Table S5, and Dataset S1.
Figure 4.
Profiles of intracellular metabolites in Δhyd2 vs. wild-type cells.
Metabolites were detected by gas chromatography-mass spectrometry (GC/MS). The values show the relative abundance of each metabolite detected in the samples (arbitrary units) on a logarithmic scale. Legend: Green = Wild-type, Yellow = Δhyd2. Means were calculated from three biological replicates and five technical replicates for each strain. p values were determined using a Student’s T-test. * = p<0.05, ** = p<0.01 difference relative to wild-type bars.
Figure 5.
Altered balance of catabolic/anabolic carbon metabolism in Δhyd2 cells.
Cells lacking Hyd2 compensate for the loss of electrons derived from H2 by increasing oxidation of organic carbon sources. There is an increased flux though the tricarboxylic acid cycle due to upregulation of enzymes involved in oxidative decarboxylation (e.g. ketoglutarate-ferredoxin oxidoreductase) (highlighted in yellow) and downregulation of those involved in anaplerosis (i.e. isocitrate lyase) (highlighted in blue). We model that loss of CO2 through oxidative decarboxylation reactions is principally responsible for the decreased biomass of Δhyd2 cells. Oxidative pathways are depicted with green arrows, whereas reductive pathways are represented with blue arrows. The red text shows the expression ratios of the significantly upregulated or downregulated genes in Δhyd2 vs. wild-type cells.