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. 2022 Nov 15;13(6):e02541-22. doi: 10.1128/mbio.02541-22

FIG 3.

FIG 3

The Pa ORF (prpC) encoding 2-methylcitrate synthase is essential for growth on propionate. (A) Wild-type Pa (PAO1) and the ΔacsA, ΔaceA, and ΔprpC mutants all grow comparably on MOPS agar containing glucose (20 mM) or succinate (30 mM) as a sole carbon source. The ΔacsA mutant has a growth defect during growth on MOPS-acetate (40 mM) and MOPS-propionate (40 mM). The ΔprpC mutant cannot grow on MOPS propionate. The plates were photographed after 24 h of incubation. (B) Wild-type PAO1 and the ΔacsA, ΔaceA, and ΔprpC mutants were cultured on LB agar containing an increasing concentration of propionate (0, 5, 10, and 20 mM, as indicated). The ΔprpC mutant displays a pronounced growth defect in the presence of propionate concentrations of >10 mM. The plates were photographed after 24 h of incubation. (C) Intracellular propionyl-CoA concentration in wild-type Pa (PAO1) and in the ΔprpC mutant following a 3-h exposure to propionate (5 mM) during growth in succinate (unpaired t test with Welch’s correction, P = 0.0026). The experiment was performed using biological triplicates. (D) Illustration of the interwoven reactions for propionate and acetate activation in Pa, feeding into the 2-methylcitrate cycle and TCA cycle, respectively. Following uptake, acetate and propionate are activated by AcsA. The resulting propionyl-CoA (PrCoA) is condensed with oxaloacetate (OAA) in a PrpC-catalyzed reaction to form 2-methylcitrate (2-MC), whereas the acetyl-CoA (AcCoA) is condensed with oxaloacetate in a GltA-catalyzed reaction to form citrate (CIT). (E) Growth of the Pa glyoxylate shunt mutants ΔaceA and ΔglcB is blocked on MOPS agar plates containing a combination of high acetate concentration (40 mM) and low propionate concentration (5 mM) as the carbon source. However, this growth inhibition is partially overcome by increasing the propionate concentration to 20 mM (left to right in the figure). The plates were photographed after 48 h of growth. The data are representative of two independent experiments, each performed in triplicate.