Figure 1.
S. coelicolor development stages and sample preparation.
(a) Cell-cycle features of Streptomyces development. Mycelial structures (MI, first compartmentalized mycelium; MII, second multinucleated mycelium). The classical nomenclature of substrate and aerial mycelium, and hydrophobic layers are indicated. (b) Confocal laser fluorescence micrographs of the different mycelia after fractioning and stained with SYTO9 and PI (see Methods). Notice the absence of dead cells (red staining). Mycelial types and developmental time points are indicated. See text for details.
Figure 2.
Schematic representation of biochemical pathways regulating Streptomyces differentiation.
Pathways involved in hydrophobic covers formation (“bld”, “sky”), sporulation (“whi”, “septation”) are illustrated. Pre-sporulation pathways (“MI/MII transition”) not contemplated in the classical developmental model are labelled in red. See text for details.
Table 1.
Primers used for qRT-PCR.
Figure 3.
Quantitative transcriptomic data analysis.
(a) Correlation of transcription abundance values (log2 ratio against chromosomal DNA) for biological replicates (three biological replicates compared in pairs) significantly quantified (p-value<0.05) (Table S1). Coefficients of regressions among replicates are shown. (b) Abundance values of the ORFs identified by proteomics [5] and transcriptomics (this work). Dashed lines indicate the limit for considering abundance variations as significant (log2 abundances greater than ±1).
Figure 4.
Quantitative transcriptomic data of genes involved in the synthesis of the most important secondary metabolites produced by S. coelicolor.
(a) CDA cluster, genes SCO3210–SCO3249. (b) ACT cluster, genes SCO5071–SCO5092. (c) RED cluster, genes SCO5877–5898. (d) Cpk cluster, genes SCO6273–SCO6288. Genes were grouped into graphs according to their predicted operons. Genes included in the most reliable transcripts (Figure 3 and Table S1) are highlighted in bold. The remaining transcripts (Roman letters) also had good reproducibility in their abundance values, albeit not enough to meet the rigorous criteria used in this work (Table S2).
Figure 5.
Abundance values (log2 MII/MI; averages from three biological replicates) of the genes significantly quantified (1901 in total) and grouped in functional categories.
Primary metabolism (DNA/RNA replication, aerobic and anaerobic energy production, glycolysis and glyconeogenesis, pentose phosphate pathway, amino acid metabolism, nucleotide metabolism, translation, protein folding, RNA/protein processing, nucleases/RM methylases); secondary metabolism (secondary metabolites synthesis); differentiation (TTA BldA targets, Bld and Whi proteins); transporters and secreted (ABC transporters, transporters and secreted proteins); catabolism and degradation; lipid metabolism; stress and defense proteins. Dashed lines indicate the limit for considering abundance variations significant (log2 abundance ±1).
Figure 6.
Gene abundance values (log2 MII/MI) of the genes grouped in functional categories (continuation).
Regulatory proteins (transcriptional regulators, kinases, other regulatory proteins); transposons - insertion sequences; conjugation, recombination, mutagenesis. Dashed lines indicate the limit for considering abundance variations significant (log2 abundance ±1).
Table 2.
Well characterized genes showing the greatest abundance differences between MI and MII (see Table S1).
Table 3.
Most differentially expressed genes among MI and MII with unknown function and conserved in the Streptomyces genus.
Table 4.
Genes putatively involved in secondary metabolism (according to Bentley et al [21]) (see Table S1 for details).