Direct Monitoring of Membrane Fatty Acid Changes and Effects on the Isoleucine/Valine Pathways in an ndgR Deletion Mutant of Streptomyces coelicolor

Abstract

NdgR, a global regulator in soil-dwelling and antibiotic-producing Streptomyces, is known to regulate branched-chain amino acid metabolism by binding to the upstream region of synthetic genes. However, its numerous and complex roles are not yet fully understood. To more fully reveal the function of NdgR, phospholipid fatty acid (PLFA) analysis with gas chromatography-mass spectrometry (GC-MS) was used to assess the effects of an ndgR deletion mutant of Streptomyces coelicolor. The deletion of ndgR was found to decrease the levels of isoleucine- and leucine-related fatty acids but increase those of valine-related fatty acids. Furthermore, the defects in leucine and isoleucine metabolism caused by the deletion impaired the growth of Streptomyces at low temperatures. Supplementation of leucine and isoleucine, however, could complement this defect under cold shock condition. NdgR was thus shown to be involved in the control of branched-chain amino acids and consequently affected the membrane fatty acid composition in Streptomyces. While isoleucine and valine could be synthesized by the same enzymes (IlvB/N, IlvC, IlvD, and IlvE), ndgR deletion did not affect them in the same way. This suggests that NdgR is involved in the upper isoleucine and valine pathways, or that its control over them differs in some respect.

Keywords: Streptomyces coelicolor A3(2); gas chromatography-mass spectrometry; ndgR; phospholipid fatty acid.

Fig. 1. Pathway from branched-chain amino acids (isoleucine, valine and leucine) to branched-chain fatty acids related to membrane phospholipid and involved enzymes.

Among the enzymes, bright yellow-colored enzymes are regulated by NdgR.

Fig. 2. Phospholipid fatty acid peaks detected using gas chromatography-mass spectrometry (GC-MS) for the Streptomyces coelicolor A3(2) wild-type strain (A), ΔleuCD mutant (B), ΔndgR mutant (C), and ΔndgR::ndgR complementary sample (D).

Fatty acids with red box mean increased in portion when compared to wild type and blue box means decreased in portion.

Fig. 3. Phospholipid fatty acid composition changes in the Streptomyces coelicolor A3(2) wild-type strain, ΔleuCD, ΔndgR, and ΔndgR::ndgR after 48 h and 72 h cultivation.

Branched-chain fatty acids directly related to each branched-chain amino, valine, leucine and lsoleucine are marked with black, red and blue colored line, respectively.

Fig. 4. Overall changes to the fatty acid fluxes in the ΔleuCD (A) and ΔndgR (B) mutants.

The wide blue arrows indicate an increase in the fatty acid composition due to the deletion of genes and the narrow red arrows indicate a decrease in the fatty acid composition.

Fig. 5. Comparison of growth at 30°C (A) and 20°C (B) with the addition of branched-chain amino acids.

At 30°C, both Streptomyces coelicolor A3(2) wild-type strain and ΔndgR mutant grew well with and without branched-chain amino acids supplementation. However, at 20°C only isoleucine and leucine supplied ΔndgR mutant could grow.

Fig. 6. Phospholipid fatty acid analysis results for the Streptomyces coelicolor A3(2) wild-type strain (A) and ΔndgR (B) with and without branched-chain amino acid supplement.

ILE, VAL, LEU, and Others means each branched-chain related fatty acid, respectively.

Fig. 7. Phospholipid fatty acid analysis results for the Streptomyces coelicolor A3(2) wild-type strain (A) and ΔleuCD mutant (B) in supplemented with isoleucine and the wild-type strain (C) and ΔndgR (D) supplemented with valine.

ILE, VAL, LEU, and Others means each branched-chain related fatty acid, respectively.