Developmentally regulated cleavage of tRNAs in the bacterium Streptomyces coelicolor

Abstract

The ability to sense and respond to environmental and physiological signals is critical for the survival of the soil-dwelling Gram-positive bacterium Streptomyces coelicolor. Nutrient deprivation triggers the onset of a complex morphological differentiation process that involves the raising of aerial hyphae and formation of spore chains, and coincides with the production of a diverse array of clinically relevant antibiotics and other secondary metabolites. These processes are tightly regulated; however, the genes and signals involved have not been fully elucidated. Here, we report a novel tRNA cleavage event that follows the same temporal regulation as morphological and physiological differentiation, and is growth medium dependent. All tRNAs appear to be susceptible to cleavage; however, there appears to be a bias towards increased cleavage of those tRNAs that specify highly utilized codons. In contrast to what has been observed in eukaryotes, accumulation of tRNA halves in S. coelicolor is not significantly affected by amino acid starvation, and is also not affected by induction of the stringent response or inhibition of ribosome function. Mutants defective in aerial development and antibiotic production exhibit altered tRNA cleavage profiles relative to wild-type strains.

Figure 1.

(a) A novel ∼30–35 nt RNA species is detectable on MS, but not rich media. Total RNA harvested from MS and rich (R2YE) media after 24 or 48 h was labeled with pCp. Samples were separated on a 12% denaturing acrylamide gel and exposed to X-ray film. Distinct 30–35 nt RNA species were detectable only in samples isolated from MS medium-grown cultures. M: Decade marker (Ambion); MS: soy flour-mannitol medium, RM: Rich (R2YE) medium. (b) The appearance of a ∼30–35 nt RNA species is temporally correlated with aerial development. A time course of 3′ end-labeled RNA harvested from MS medium was separated on a 12% denaturing acrylamide gel. Shown in the top panel is full length tRNAs, together with the 30–35 nt region of the acrylamide gel, and in the bottom panel is the 5S RNA region of the gel, which was used as a control for RNA loading and RNA integrity.

Figure 2.

Northern blot analysis for the 5′ and 3′ halves of histidine tRNA in a wild-type background. Total RNA samples harvested from MS medium were separated on 12% polyacrylamide gels and were subjected to northern blotting using probes complementary to the 5′ and 3′ halves of the histidine tRNA. Blots were exposed to a phosphorimager.

Figure 3.

Northern blot analysis of tRNAs isolated from MS medium grown cultures. Total RNA was isolated from MS medium-grown cultures after 12, 24, 48 and 72 h of growth. The RNA was separated on 12% polyacrylamide gels and was subjected to northern blotting using probes that were complementary to the indicated tRNA half before being exposed to X-ray film.

Figure 4.

Northern blot analysis of tRNAs isolated from bld mutants. Total RNA from bldA, bldB, bldC and bldH mutant strains was isolated from MS-grown cultures after 24, 48, 72 and 96 h of growth, as indicated. The RNA was separated on 12% polyacrylamide gels, and was subjected to northern blotting using probes complementary to the 5′ half of Met (left panels) or His (right panels) tRNA.

Figure 5.

Translational inhibition and stringent response assay. Cultures were grown for ∼40 h in liquid MM at 30°C before the addition of the indicated compound (shx = 25 mM serine hydroxamate; spec = 200 μg/ml spectinomycin; hyg = 50 μg/ml hygromycin; thio = 50 μg/ml thiostrepton) and incubation for an additional 60 min. Total RNA was then harvested and tRNA halves were detected using northern blot hybridization. The change in the ratio of tRNA halves in treated samples to untreated controls was determined by quantifying the appropriate bands after normalizing for background signal. The data shown are the average of results from three independent experiments. Error bars indicate one standard deviation from the mean.