Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species

Abstract

Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.

FIG. 1.

Induction of secondary-metabolite production by mycolic acid-containing bacteria in a coculture with S. lividans TK23. The coculture of T. pulmonis and S. lividans was performed to induce the production of secondary metabolites. (A) Coculture of T. pulmonis and S. lividans in Bennett's medium. S. lividans spores were overlaid, and T. pulmonis was inoculated at the center of the plate (left). S. lividans and T. pulmonis were inoculated in parallel on the medium (right). Sl, S. lividans; Tp, T. pulmonis. Red pigment production was found only at the site of cell-to-cell contact. The organisms were grown at 30°C for 3 days. (B) Liquid medium without bacteria (left), pure culture of S. lividans (center), and coculture (right). The organisms were grown at 30°C for 3 days in A-3M medium. (C) Coculture of T. pulmonis and S. lividans in a dialysis flask. T. pulmonis was grown in the left compartment and S. lividans in the right compartment at 30°C for 7 days. Red pigments were not detected in the broth. (D) Optical micrograph of S. lividans (left), coculture (center), and T. pulmonis (right) at 1,000-fold magnification.

FIG. 2.

Effect of mycolic acid on the induction of secondary metabolites in the coculture. (A) Coculture of S. lividans with wild-type C. glutamicum (left) and with Δpks13, the mycolic acid-deficient mutant of C. glutamicum (right). In this experiment, two Δpks13 mutants were used independently for coculture. (B) Effect of isoniazid on metabolite production. Shown are the coculture of S. lividans with T. pulmonis (left) and coculture with added isoniazid (right).

FIG. 3.

Effects of the coculture of Streptomyces isolated from soil. Metabolite profiles of pure and combined cultures of Streptomyces. (A) Flowchart of the coculture procedure. (B to F) The results of the comparison between the secondary-metabolite profiles of the pure and cocultures are shown in a Venn diagram. (B to D) Comparison between the HPLC profiles of a pure culture of Streptomyces and cocultures. Each profile was classified according to the increase/decrease in metabolite production, appearance of new metabolites, and disappearance of some secondary metabolites. The increase/decrease part is subclassified into increase, both increase and decrease, and decrease in the right panel. (E and F) Comparison of the HPLC profiles of cocultures of Streptomyces with T. pulmonis, R. erythropolis, or C. glutamicum. The numerals represent the number of strains. New, new secondary metabolites appeared in the coculture; increase/decrease, the secondary-metabolite levels in the coculture were higher/lower than the levels in the pure culture; lost, secondary-metabolite production was absent in the coculture; no change, there was no difference between the secondary-metabolite levels in the pure culture and cocultures.

FIG. 4.

Production of a novel antibiotic, alchivemycin A, in a combined culture of Streptomyces endus S-522 and T. pulmonis or C. glutamicum. Shown is the production of alchivemycin A in a combined culture of S. endus S-522 and T. pulmonis or C. glutamicum. (A to D) HPLC profiles of the secondary metabolites produced by S-522 cultured with or without T. pulmonis. Shown are the culture broths of the combined culture with T. pulmonis (A), combined culture with C. glutamicum (B), S-522 pure culture (C), and T. pulmonis pure culture (D). The arrow shows the peak for alchivemycin A. Elution was performed with a linear gradient, as indicated on the scale at the right. (E) Chemical structure of alchivemycin A.