Deciphering functional redundancy and energetics of malate oxidation in mycobacteria

Abstract

Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.

Keywords: Mycobacterium smegmatis; Mycobacterium tuberculosis; bacterial genetics; bacterial metabolism; bioenergetics; energy metabolism; malate dehydrogenase; malate oxidation; malate quinone oxidoreductase; mycobacteria.

Figure 1

Prevalence of malate dehydrogenase (Mdh) and malate quinone oxidoreductase (Mqo) enzymes in bacteria and Mycobacteria.A, Mqo and B, Mdh enzyme reactions. C, visualization of combination of Mqo and Mdh present in each UniProt reference bacterial proteomes with the absence (black) or presence of Mqo (blue) and Mdh (orange) indicated in surrounding colored coded rings created using GraPhlAn. Families and orders highlighted represent the top ten families/orders with the highest fraction of species with orphaned Mqo, except Mycobacteriaceae (minimum 20 species). D, phylogeny of mycobacteria taken from Fedrizzi et al. (42) and reconstructed using GraPhlAn to show the presence or absence of Mqo and Mdh indicated in surrounding colored coded rings. Colored clades represent different mycobacterial complexes.

Figure 2

Activity of Mqo in IMVs of M. smegmatis.A, succinate- and malate-driven proton translocation in IMVs of M. smegmatis. Quenching of ACMA fluorescence in IMVs was initiated with either 5 mM succinate or 5 mM malate (∗ final concentration), and at the indicated time points (arrows), the uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) at 50 μM was added to collapse the proton gradient (reversal of ACMA fluorescence). Experiments are representative of a technical triplicate. B, oxygen consumption rates of IMVs prepared from the WT (closed blue circles) and the Δmqo mutant (closed red triangles) with either NADH or malate as an electron donor (5 mM each). C, activity of Mqo in IMVs prepared from the WT (closed blue circles) and the Δmqo mutant (closed red triangles). Data in (B) and (C) are an average of biological triplicates with error bars representing standard deviation. ACMA, 9-amino-6-chloro-2-methoxyacridine; IMVs, inverted membrane vesicles; Mqo, malate quinone oxidoreductase.

Figure 3

M. smegmatis Δmqo mutant has delayed growth on fermentable carbon sources and is unable to grow on nonfermentable carbon sources.A, growth of the Δmqo mutant (solid red triangles) compared to WT (solid blue circles) on HdB minimal medium with the fermentable carbon sources glycerol (22 mM) or glucose (20 mM). B, regrowth of Δmqo mutant on HdB minimal medium with glycerol (22 mM) following adaptation on glycerol (solid black squares). C, malic enzyme (Mez) activity of cytoplasmic fractions prepared from the Δmqo mutant compared to WT. D, growth of the Δmqo mutant (solid red triangles) compared to WT (solid blue circles) on HdB minimal medium with the nonfermentable carbon sources malate (20 mM), succinate (20 mM), or acetate (20 mM). E, oxygen consumption rates of washed cell suspensions energized with either glycerol (5 mM) or succinate (5 mM). CCCP was added at 10 μM (final concentration): Δmqo mutant (solid red triangles), WT (solidblue circles). Two-way ANOVA with Sidaks multiple comparisons: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. F, growth (solid blue circle and solid red triangle) and external pH (open symbols) of the Δmqo mutant (red triangles) compared to WT (blue circles) grown on HdB minimal medium with glycerol (22 mM). G, concentration of malate in the cell-free supernatant of bacterial cultures (Δmqo mutant and WT) grown in panel F. Samples were taken from stationary phase cultures for malate concentration determinations. All reported measurements are an average of biological triplicates with error bars representing standard deviation. CCCP, carbonyl cyanide m-chlorophenyl hydrazine.

Figure 4

Metabolic consequences of impaired malate oxidation in M. smegmatis. Comparison of selected intracellular and extracellular metabolites between the Δmqo mutant and WT M. smegmatis after 24 h incubation of bacteria-laden filters on plates containing Middlebrook 7H9 liquid broth with 0.2% glycerol (see Experimental procedures). Schematic of TCA cycle components of mycobacteria. Data values shown represent log2-fold change of biological duplicate with technical triplicate with error bars representing 95% confidence interval. All intracellular metabolite abundances were first normalized to a single reference intracellular compound (glycerol 3-phosphate) before computing log2-fold changes relative to average WT strain abundances. When no extracellular metabolites were detected for a strain, log2-fold change analyzed was relative to 1. TCA, tricarboxylic acid.

Figure 5

Complementation of M. smegmatis Δmqo mutant with either pMqoMS or pMdhTB restores growth on nonfermentable carbon sources. Growth of the M. smegmatis Δmqo mutant complemented with pMind empty vector control (solid blue circles), pMqoMS (M. smegmatis Mqo, solid black squares), pMdhTB (M. tuberculosis Mdh, solid red triangles) compared to WT with pMind empty vector control (solid purple inverted triangles) on HdB minimal medium with the following carbon sources: A, glycerol (20 mM); B, glucose (20 mM); C, malate (20 mM); D, succinate (20 mM) as the sole carbon and energy source. In panels (C) and (D), nicotinamide (1 mg/l) was included in the growth medium (all open symbols). All growth (A₆₀₀) measurements are an average of biological triplicate with error bars representing standard deviation.

Figure 6

Mdh and Mqo both contribute to optimal malate oxidation in M. tuberculosis. Relative expression (mRNA levels) of mdh (A) and mqo (B) with different CRISPRi knockdown single guide RNA (sgRNA) molecules targeting mqo (blue circles), mdh (red squares) and a combination of both (black triangles). The mRNA levels of mdh and mqo are expressed relative to a scrambled nontargeted (NT) sgRNA control (purple inverted triangles). Gene knockdowns were initiated by the addition of 300 ng/ml (final concentration) anhydrotetracycline. RT-qPCR values are an average of a technical triplicate with error bars representing standard deviation. C and D, growth of M. tuberculosis strain mc²6230 on Middlebrook 7H9 base medium with either 0.2% glucose (C) or 30 mM succinate (D) as the sole carbon and energy source with sgRNA molecules targeting; mqo (blue circles), mdh (red squares), and a combination of both (black triangles). An mmpL3-targeting sgRNA (yellow diamonds) was used as a positive control and a nontargeted sgRNA (purple inverted triangles) as a negative control. E and F, specific growth rate (h⁻¹) of all strains determined from the A₆₀₀ values at days 0 and 6 with a one-way ANOVA with Dunnett’s multiple comparisons to the NT sgRNA. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Measurements are an average of biological triplicate with error bars representing standard deviation. Mdh, malate dehydrogenase; Mqo, malate quinone oxidoreductase.