Comparative proteomic analysis of Streptomyces lividans Wild-Type and ppk mutant strains reveals the importance of storage lipids for antibiotic biosynthesis

Abstract

Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

Fig 1

Representative 2D gels of intracellular proteins extracted from wt Streptomyces lividans TK24 (A) and the ppk mutant derived from it (B) grown for 96 h on solid R2YE medium limited in phosphate.

Fig 2

Schematic representation of central metabolic pathways of S. lividans TK24 and its ppk mutant grown for 96 h on solid R2YE medium limited in phosphate. Green, proteins upregulated in the ppk mutant; red, proteins downregulated in the ppk mutant. Dashed arrows indicate multistep links.

Fig 3

(A) From top to bottom, pictures of mycelial lawns of wt S. lividans TK24, its ppk mutant, and S. coelicolor M145 grown for 96 h on solid R2YE with no P_i added. On the left of the plates, the numbers indicate the amount of ACT excreted by the strains in nmol · mg⁻¹ of dry biomass. ND, not determined. (B) Transmission electron microscopy images (magnification, ×11,000) of mycelial fragments of S. lividans TK24, its ppk mutant, and S. coelicolor M145 grown under the same conditions as for panel A. The scale bar corresponds to 1 μm. (C) Thin-layer chromatography analysis of the total lipid contents of the three strains of interest. Lipids were extracted from two independent biological replicates (indicated as 1 and 2). The ppk mutant of S. lividans and S. coelicolor M145 contain 30% and 50% less TAG than the wild-type strain S. lividans TK24, respectively. SE, steryl esters; ME, methyl esters; FFA, free fatty acids.