Figure 1.
AbrA1 HK signal response in NMMP.
ACT production (blue colour) of the parent strain (M145) and the mutant ΔabrA1/A2 (ΔabrA) in NMMP complete medium and different modified versions lacking (w/o) one or several compounds of the original recipe. All plates were inoculated as a lawn with 7.5×106 spores of the corresponding strains and the images correspond to backward sections of the plates after four days at 30°C.
Figure 2.
Auto-regulation of AbrA2 and dependence on Fe.
A) Scheme of plasmids for xylanase gene expression (xysA) used as reporter. Plasmid pNA4: xysA under the control of abrA promoter (abrAp) was used, and plasmid pNX4: xysA under the control of its own promoter xysAp was used as a control. B) Top left panel: xysA expression triggered by pNA4 (abrAp) in the parent strain (M145) and the ΔabrA1/A2 mutant (ΔabrA), respectively, is shown. Top right panel, control expression by xysAp (pNX4) in both strains is shown. C) Western blot showing xysA expression under abrAp (plasmid pNA4), or xysAp, (plasmid pNX4) in the parent strain M145 in different medium conditions: complete NMMP, low-Fe or low-Mg media. The xylanase signal corresponds to western blot assays using anti-xysA against supernatants of 7-day cultures in NMMP medium (100 µL of pNA4 and 2 µL of pNX4). In the bottom panels of B and C the amount of protein loaded in each lane is shown with a Coomassie blue stain. Two independent cultures of each construction are shown.
Figure 3.
A) Colony morphology of S. coelicolor M145 and S. coelicolor ΔabrA1/A2 strains transformed with different multicopy plasmids: pIJ702 (control), pTXabrA2 (expressing the AbrA2 RR under the control of xysAp), and its derivatives pTXabrA2-DA (D55A) and pTXabrA2-DADE (D10A, D55E). The photographs correspond to four-day cultures on R2(YE) medium. Bar: 1 mm. B) Phenotypes of AbrA2 overexpression in S. coelicolor M145 compared to the mutant ΔabrA1/A2 phenotype. The phosphoablative version (pTXabrA2-DA) was included in the study. Upper part: production of ACT in NMMP medium plates (72 h); middle part: CDA bioassay against B. subtilis at 48 h; lower part, RED production at 48 h.
Figure 4.
Phosphorylation assays with small phospho-donors.
Phosphorylation of DrrBN (A), AbrA2 (B) or AbrA2N (C) proteins with acetylphosphate (AP), carbamoylphosphate (CP) or phosphoramidate (PA) visualized with the Phos-tag stain in SDS-PAGE (“-” means no phospho-donor added). In the bottom part of each panel the amount of protein used is shown with a Coomassie blue stain.
Figure 5.
Antibiotic production and morphological differentiation of different SCO1742-45 operon mutants.
Production of ACT on NMMP at six days of growth; CDA at two days of growth in NA medium; RED at two days in PGA, and morphological differentiation at six days of growth in MSA.
Figure 6.
Oviedomycin production of M145 and ΔabrA1/A2 strains carrying the cosmid CosAB4, containing the oviedomycin cluster, or an empty control pKC505. A) Solid NMMP medium at 4 days of growth. B) Antibiotic activity against M. luteus of acetone-methanol (solvents) extracts from the different strains grown for 6 days on solid NMMP. C) Supernatant of liquid NMMP cultures at 24 and 120 hours of growth.