The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2)

Abstract

Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development.

Figure 1. Determination of the transcriptional architecture of the S. coelicolor genome.

(a) Example of a dRNA-seq profile mapped onto the S. coelicolor genome. For TSS determination, total RNA samples from 44 growth conditions were pooled and two sequencing libraries were constructed, one from TEX-treated (TEX+) and the other from untreated total RNA (TEX−); TEX, terminator-5′-phosphate-dependent exonuclease. The criteria for assigning TSS are detailed in the Methods. (b) A total of 3,570 TSSs were identified and classified according to their positions relative to adjacent open reading frames (ORFs). TSSs located from 500 bp upstream to 150 bp downstream of the respective annotated start codon of each ORF were classified as the primary (P) or secondary (S). TSSs located within an annotated ORF or on the opposite strand were classified as internal (I) or antisense (A), respectively. TSSs not included in any of these categories were classified as intergenic (N). (c) Mapped TSSs in relation to those reported from previous studies. I, this study; II, ref. .; III, ref. . (d) TSSs associated with secondary metabolic gene clusters; prodiginine (left), bacteriocin (middle) and siderophore (right). (e) Proportion of each nucleotide at TSS (+1) and 2 nt upstream and downstream of the TSS.

Figure 2. Genome-scale analysis of promoter sequences.

(a) −10 motif (5′-TANNNT) and −35 motif (5′-NTGACC) were identified relative to TSS position (+1). The analysis showed three identical positions to the −10 motif of the E. coli promoter (that is, 5′-TATAAT) recognized by its housekeeping sigma factor (σ⁷⁰). It has been suggested that the well-conserved TTG motif commonly found in the 5′ half of the E. coli −35 motif (that is, TTGACA) is located in the 5′ half of the S. coelicolor −35 motif. Although S. coelicolor has a lower level of conservation of the TTG motif this analysis clearly identifies the motif at the same position. The bottom panel shows the position distribution of the −10 motif (red) and −35 motif (blue) relative to the TSS. (b) Distribution of 5′-UTR lengths reveals a dual peak distribution at 30–39 nt and 0–9 nt; the latter group are considered to produce leaderless mRNAs (lmRNAs). (c) The same −10 and −35 consensus sequences are observed upstream of lmRNAs. The motif around the TSS (+1) is also indicated. The third motif found at the +1 position clearly shows the translation initiation codon, indicating that lmRNAs are translated without 5′-UTR-mediated recognition. (d) Start codon usage of all open reading frames (ORFs; Total), primary TSS-identified ORFs (TSS), 5′-UTR-associated genes (umRNA) and leaderless genes (lmRNA).

Figure 3. Transcriptome dynamics at different growth phases.

(a) All transcriptional regulatory genes are clustered by their expression patterns where almost half of the regulatory genes are differentially expressed at different growth phases (Ι), whereas the other half showed no changes, or expression levels lower than the cutoff (ΙΙ). Bold black letters indicate sigma factors, and bold red letters indicate regulators of secondary metabolic gene clusters. (b) Differential expression of umRNAs, lmRNAs and sRNAs at different growth phases. Clusters are labelled U-I, U-II, L-I, L-II, S-I and S-II and the genes comprising each cluster are listed in Supplementary Data 6. M, mid-exponential phase; T, transition phase; L, late exponential phase; S, stationary phase.

Figure 4. Determination of the translatome of S. coelicolor.

(a) An example of visualization of transcription start sites (TSS), mRNA expression profiles (RNA) and ribosome-protected fragment profiles (RPF) at genomic region between 4,924,959 and 4,969,730. (b) RPF read data for the first and the second gene in the operon were compared at four growth phases: M, mid-exponential phase; T, transition phase; L, late exponential phase; S, stationary phase. (c) RPF data for ribosomal proteins RplJ and RplL (stoichiometry=1:4) and ATP synthase operon encoding AtpB, AtpE, AtpF, AtpH, AtpA, AtpG, AtpD and AtpC (stoichiometry=1:10:2:1:3:1:3:1) show proportional relationships with their subunit stoichiometry.

Figure 5. Translational buffering revealed by comparison of changes in mRNA and ribosome-protected fragment abundances.

(a) Distribution of mRNA fold-change and RPF fold-change of total genes, primary metabolic genes and secondary metabolic genes; ***P<0.001 (Wilcoxon signed-rank test); T, fold-change between mid-exponential and transition phases; L, fold-change between mid- and late exponential phases; S, fold-change between mid-exponential and stationary phases. (b) Negative correlation between changes in mRNA levels and translational efficiency (TE) becomes higher at later growth phases. Red dots indicate secondary metabolic genes. (c) TE change distributions of umRNAs and lmRNAs across growth phases. *P<0.05; ***P<0.001 (Wilcoxon rank-sum test). (d) G+C content of translation initiation regions (TIR: 20 nt sequence upstream of start codon); high, genes with high TE (upper 20%); total, all coding sequences; low, genes with low TE (lower 20%). (e) Correlation between free energy of TIR and TE. (f) Conserved ribosome-binding sequences for umRNAs were observed at 8–12 bp upstream region of start codon; lmRNAs, 5′-UTR length=0. TIR of genes with high TE (High) shows more conserved polypurine (G>A) motif than genes with low TE (Low).

Figure 6. Translational landscape of secondary metabolic genes.

(a) Ribosome-protected fragment (RPF) levels and translation efficiency (TE) fold-changes of the 221 known secondary metabolic genes in S. coelicolor. Leftmost numbers are the SCO gene numbers for each gene cluster. The respective known chemical structures are indicated for each secondary metabolite structure. 1, Mid-exponential phase; 2, transition phase; 3, late exponential phase; 4, stationary phase; T, fold-change between mid-exponential and transition phases; L, fold-change between mid- and late exponential phases; S, fold-change between mid-exponential and stationary phases. (b) mRNA and RPF levels of cluster-situated regulator-encoding transcripts for SCO3217 (CdaR), SCO5085 (ActΙΙ-ORF4), SCO5877 (RedD) and SCO5881 (RedZ) across the four growth phases. (c) mRNA and RPF levels of transcripts for SCO5803 (LexA), SCO3226 (AbsA2), SCO6265 (ScbR) and SCO4230, which represent other regulators of secondary metabolism. (d) Changes in mRNA level and TE. Red dots indicate genes encoding the cluster-situated regulators CdaR, ActΙΙ-ORF4, RedD and RedZ. Blue dots indicate other known regulators of secondary metabolism: SCO2792 (AdpA), SCO5803 (LexA), SCO3226 (AbsA2), SCO5231, SCO3063, SCO4907 (AfsQ1), SCO5260 (AtrA), SCO6008, SCO4159 (GlnR), SCO0310, SCO3932, SCO5405, SCO6265 (ScbR) and SCO4230 (PhoP). Grey dots and dark grey dots indicate, respectively, all genes and secondary metabolic genes. T, fold-change between mid-exponential and transition phases; L, fold-change between mid- and late exponential phases; S, fold-change between mid-exponential and stationary phases.