Prodiginines Postpone the Onset of Sporulation in Streptomyces coelicolor

Abstract

Bioactive natural products are typically secreted by the producer strain. Besides that, this allows the targeting of competitors, also filling a protective role, reducing the chance of self-killing. Surprisingly, DNA-degrading and membrane damaging prodiginines (PdGs) are only produced intracellularly, and are required for the onset of the second round of programmed cell death (PCD) in Streptomyces coelicolor. In this work, we investigated the influence of PdGs on the timing of the morphological differentiation of S. coelicolor. The deletion of the transcriptional activator gene redD that activates the red cluster for PdGs or nutrient-mediated reduction of PdG synthesis both resulted in the precocious appearance of mature spore chains. Transcriptional analysis revealed an accelerated expression of key developmental genes in the redD null mutant, including bldN for the developmental σ factor BldN which is essential for aerial mycelium formation. In contrast, PdG overproduction due to the enhanced copy number of redD resulted in a delay or block in sporulation. In addition, confocal fluorescence microscopy revealed that the earliest aerial hyphae do not produce PdGs. This suggests that filaments that eventually differentiate into spore chains and are hence required for survival of the colony, are excluded from the second round of PCD induced by PdGs. We propose that one of the roles of PdGs would be to delay the entrance of S. coelicolor into the dormancy state (sporulation) by inducing the leakage of the intracellular content of dying filaments thereby providing nutrients for the survivors.

Keywords: bacterial development; cell differentiation; morphogenesis; programmed cell death; role of antibiotics; streptorubin; undecylprodigiosin.

Figure 1

Effect of the redD inactivation on morphological differentiation of S. coelicolor. (a) Pictures of R2YE plates inoculated with S. coelicolor M145 (WT, wild-type) and its redD mutant M510. Note the precocious appearance of white-pigmented aerial hyphae. (b) Scanning electron micrographs showing precocious sporulation of the S. coelicolor redD null mutant. In (a,b) (48 h) we can see in the WT the very first aerial hyphae emerging from the dense network of vegetative filaments while in strain M510 (∆redD; c,d) many aerial hyphae have already been produced with occasionally fully matured spore chains (d). In (e,f) (72 h) the redD mutant has fully differentiated into mature spore chains whereas the WT only displays erect aerial hyphae. Bars = 10 µm (a,c); = 3 µm (b,d); = 5 µm (e,f).

Figure 2

Multiple copies of redD results in higher PdG production, thereby blocking sporulation of S. coelicolor. (a) Phenotypes of 65 h old confluent lawns and single colonies of S. coelicolor M145 (wild-type, parental strain), M510 (redD deletion mutant), and strain ET003 (redD on the low-copy-number plasmid pIJ2587). (b) Semi-quantitative measurement of PdG synthesis in the three studied S. coelicolor strains revealing the overproduction of PdGs in ET003. Note that thiostreptone was not included in the medium for optimal comparison of strain ET003 with strains M145 and M510 that do not possess the tsr resistant gene present in pELT003.

Figure 3

Sugar phosphates reduce PdG production and accelerate sporulation of S. coelicolor. A suspension containing 10⁷ viable spores of S. coelicolor M145 was plated on R2YE solid medium. After 20 h of growth, wells (5 mm diameter) were made into the agar and 100 mM of glucosamine (GlcN), glucose (Glc), glucosamine-6-phosphate (GlcN-6P), and glucose-6-phosphate (Glc-6P) were deposited into them. Dotted circles highlight the zone around the source of phosphorylated sugars with reduced PdG production. Note how zones with reduced production of PdGs at 48 h coincides with the zones with precocious erection of aerial hyphae at 96 h.

Figure 4

Comparison of actinorhodin production between S. coelicolor M145 (WT) and the ∆redD mutant. (a) Production of the blue-pigmented actinorhodin on isolated colonies. Pictures show colonies from the reverse side of the plates. (b) Semi-quantitative assessment of actinorhodin production between S. coelicolor M145 (WT) and the ∆redD mutant grown in R2YE agar plates.

Figure 5

Transcription of developmental genes in S. coelicolor wild-type (M145) and its ∆redD mutant (M510). Gene names are shown at right of heat map; color legend for Log₂ expression (fold change) is shown above. Genes were considered significantly regulated if they had p values ≤ 0.02 in Student’s T-test (three replicates) when compared between wild-type and mutant strains. Genes meeting this criterion are noted with an asterisk (*). Colonies of S. coelicolor M145 (WT) and M510 (ΔredD) were around 5 mm in diameter.

Figure 6

Aerial growth of PdG non-producing filaments in S. coelicolor. Fluorescence confocal micrographs (3D reconstruction) of a transverse section of a S. coelicolor culture grown for 40 h on R2YE agar plates. Red autofluorescence (RAF) associated with PdG production (RAF, red-colored), and Syto9 staining of PdG non-producing filaments (green-colored, SYTO9). Note that Syto9 stains the DNA of both living and dying filaments unless PdG production is too abundant, which leads to DNA destruction thereby preventing intercalation of dyes). (a) Note the very rare occurrence of Syto9 stained filaments in the zone that massively produces PdGs. Close up and 3D reconstruction of a filament that does not produce PdGs. (b,c) Aerial hyphae emerging from the surface of the vegetative mycelium after 70 h (b) and 90 h (c) of growth. Filaments emanating from the lysing vegetative mycelium do not display RAF, suggesting that hyphae surviving the death round preceding morphological differentiation are those that did not produce PdGs.