Abstract
The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or H2O2, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to H2O2, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.
Highlights
The glyoxylate shunt (GS), a carbon metabolic process from isocitrate to malate via glyoxylate, is a well-known TCA variant during acetate, alkane, and fatty acid metabolism
Metabolomic approaches have demonstrated that isoniazid, rifampicin, and streptomycin commonly activate ICL in M. tuberculosis and an ICL-deficient strain is significantly susceptible to those three antibiotics
Www.nature.com/scientificreports pathway is essential for defense against antibiotic action in M. tuberculosis, and antibiotic-induced oxidative stress could be protected against using an antioxidant in GS-deficient cells[7]
Summary
Comparative growth of WT and GS null-strains in the presence of acetate and Hex. Previously, it was shown that GS-related genes in DR1 are highly upregulated when cells degrade Hex and TRI16,17. Expression of GS-participating genes and growth under high and low concentrations of NaAc and Hex. To confirm the importance of the GS during acetate and Hex metabolism, the expression of aceA and glcB was quantified using a Northern blot assay. Upstream genes (citrate to succinyl CoA) of the TCA cycle (gltA, acnB, icd, and sucAB) including GS-participating genes (aceA, and glcB) in WT strain were upregulated in the NaAc-supplemented media compared to that in NaSc-supplemented media, confirming that acetate metabolism occurs actively through the GS and upstream of TCA cycles (Fig. S4). The expression level of aarC in the ΔaceA mutant strain is slightly lower than that of the WT in the presence of NaAc, resulting in the retarded growth of the aceA-null mutant compared to that of the WT strain (Figs 2 and S5A). The GS pathway is a main carbon flux in DR1, an alternative pathway via lactate dehydrogenase exists to enable survival under several dynamic environments
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