Figure 1.
Growth and antibiotic production by S. coelicolor M145 and S. coelicolor ΔargR.
(A) Undecylprodigiosin production. (B) Actinorhodin production. Antibiotic production in MG medium by S. coelicolor M145 (white circles) and S. coelicolor ΔargR (white squares) and in MG supplemented with 25 mM arginine (black circles and squares). Vertical bars show standard deviation of three replicates.
Figure 2.
Scheme showing the number of genes differentially expressed in the five contrasts.
S. coelicolor M145 and S. coelicolor ΔargR were grown in absence (A and C) or in presence of 25 mM arginine (B and D). The arrow orientation refers to the comparison between two conditions. Differential transcription values were obtained by subtracting the Mg values of the first condition from those of the second condition (corresponding to the arrowheads). The small vertical arrows indicate the number of genes up-expressed (up-oriented arrows) or down-expressed (down-oriented arrows) in each of the contrasts. Contrast 5 is the interaction contrast that reflects the differential response to the arginine supplementation of the two strains.
Figure 3.
Genes differentially expressed in MG cultures of S. coelicolor M145 (A); S. coelicolor M145 supplemented with arginine (B); S. coelicolor ΔargR (C) and S. coelicolor ΔargR supplemented with arginine (D). The genes have been grouped in types and subtypes according with their expression profile. Each grey line corresponds to the change in transcription of a given gene. The thicker line is the transcription mean profile for all the genes in the proposed group.
Figure 4.
(A) Cluster of genes for arginine biosynthesis (black arrows) in S. coelicolor M145 (above) and the S. coelicolor ΔargR mutant (below). SCO1581 is indicated with broken lane since it might not be a real gene. (B) Arginine biosynthesis pathway showing its relation to proline and to pyrimidine biosynthesis via carbamoyl-phosphate incorporation. (C) Expression of the arginine biosynthesis genes in A, B, C and D conditions as indicated in Fig. 3. (D) RT-PCR amplification of the argC, argG, argH and argR genes in the same conditions and strains as above. M, Markers.
Figure 5.
Differential expression of nucleotides biosynthesis genes.
(A) Expression of genes related to pyrimidine biosynthesis. A scheme of the gene cluster and biosynthesis pathway is shown below. Genes marked as black arrows were differentially expressed. (B) Expression of genes related to deoxyribonucleotides and cobalamin biosynthesis. A, B, C and D correspond to the conditions indicated in Fig. 3. Notice that nrdX is not a real gene but a transcribed regulatory intergenic region.
Figure 6.
Differential expression of genes related to morphology and secondary metabolism.
(A) Transcriptomic of genes for rodlins and chaplins. The cluster chp–rdl is shown above with differentially expressed genes in black. (B) Transcriptomic of the genes for CPK biosynthesis. The cpk cluster is shown above. Genes with differential expression are indicated in black. Included are the genes for scbR, for a butyrolactone receptor protein, and scF, for a putative secreted FAD-binding protein. A, B, C and D correspond to the conditions indicated in Fig. 3.
Figure 7.
Detailed view of 2D-SDS-PAGE gels showing differences on the proteomes of S. coelicolor M145 (left panels) and S. coelicolor ΔargR (right panels). Arrows indicate the protein spots differentially represented corresponding from left to right in the upper panels to CpkI (SCO6282), ArgC (SCO1580), and Gap2 (SCO7511), and in the lower panels to TktA1 (SCO1935), PurH (SCO4814) and InhA (SCO1814).
Table 1.
Proteins differentially represented in S. coelicolor and S. coelicolor ΔargR.
Table 2.
Validation assays. Summary of EMSA, promoter probe assays or RT-PCR.
Figure 8.
Validation experiments by RT-PCR and EMSA.
(A) RT-PCR amplification of mRNA corresponding to the rdlA, cpkJ, (upper panels), SCO1485, scbR (medium panels), glnA and glnII (lower panels) genes. A, B, C and D correspond to the conditions indicated in Fig. 3. M, Marker. (B) EMSA of 6-FAM labelled argH (199 bp) and arcB (251 bp) promoters using Strep-ArgR protein. P indicates the free probe and B the binding reaction.
Figure 9.
Validation experiments by heterologous gene expression.
Luciferase activity of luxAB-fused promoters transformed in the adequate S. coelicolor strain and grown in the A, B, C and D conditions indicated in Fig. 3. Activity corresponds to the promoter of argH, arcB, leuA, pyrB (upper panels) pyrR, whiB, nrdA and pyk1 (lower panels). On top of each panel is shown the expression profile of the corresponding gene in the transcriptomic studies. The luciferase activity values correspond to 32-h cultures. Vertical bars show standard deviation of two biological replicates measured twice.
Figure 10.
Functional analysis of ARG boxes by EMSA.
(A) Transcriptomic expression of the tested genes in the A, B, C and D conditions indicated in Fig. 3. (B) EMSA of PCR-amplified DNA fragments containing putative ARG boxes located upstream of the indicated genes. In all cases, P indicates the free probe and B binding reaction using ArgR (0.8 µM protein). The size of the probes is indicated in the panels. (C) Sequence of the putative ARG box present upstream of the indicated gene and Ri value determined using the ARG box model represented on the right. The numbers in parenthesis indicate the nucleotides to the translation start codon. The argH, argC, argG and arcB ARG boxes are included for comparison.