Fig 1.
Construction of the coaR-PcoaT::furA fusion strain AGcoaRFurA.
(A) The normal promoter region of the furA gene in the Anabaena sp. PCC 7120 chromosome was replaced with the divergent cobalt/zinc-inducible coaT promoter (PcoaT) and the coding region of the transcriptional activator CoaR from Synechocystis sp. PCC 6803. (B) Levels of FurA protein in wild-type Anabaena sp. strain PCC 7120, the furA-overexpressing strain AG2770FurA and the coaR-PcoaT::furA fusion strain AGcoaRFurA, revealed by Western blotting. Total cell extracts from filaments grown in Co2+/Zn2+ deprived medium (BG-11-Co/Zn) were separated in duplicate by SDS-PAGE, electrotransferred, and challenged with anti-FurA antiserum. Molecular weight is indicated. (C) furA expression in mid-log phase cultures of wild-type and AGcoaRFurA strains growing with and without Co2+/Zn2+, according to sqRT-PCR. Expression of the housekeeping gene rnpB is also shown.
Fig 2.
Expression of FurA results essential to the growth of Anabaena sp. under standard culture conditions.
Growth curves of the coaR-PcoaT::furA fusion strain Anabaena sp. AGcoaRFurA and the wild-type strain Anabaena sp. PCC 7120 in the presence or absence of Co2+/Zn2+.
Fig 3.
Distribution of known-function genes which exhibited differential expression after turning off furA in the coaR-PcoaT::furA fusion strain AGcoaRFurA.
Blue columns represent the percent of genes down-regulated of the total (n = 1155) genes with defined function, distributed according to their functional category. Red columns represent the percent of genes up-regulated. The numbers over the columns indicate the total amount of genes by functional category showing differential expression.
Table 1.
Subset of selected genes showing a ≥2-fold change increase of expression in the furA-turning off strain AGcoaRFurA.
Table 2.
Subset of selected genes showing a ≥2-fold change decrease of expression in the furA-turning off strain AGcoaRFurA.
Fig 4.
Electrophoretic mobility shift assays showing the ability of FurA to bind in vitro the promoter regions of novel direct target genes.
DNA fragments free (1) or mixed with recombinant FurA protein at concentration of 300 nM (2), 500 nM (3) and 700 nM (4) in the presence of Mn2+ and DTT were separated on a 4% PAGE. The impact of the metal co-regulator (removing Mn2+/adding EDTA) and reducing conditions (removing DTT) on the in vitro affinity of FurA (700 nM) to each target are also showed. The promoter region of nifJ gene was used as non-specific competitor DNA in all assays. Binding of FurA (700 nM) to its own promoter was included as positive controls, while promoter regions of superoxide dismutases genes sodA and sodB were used as negative controls.
Fig 5.
Semi-quantitative RT-PCR analyses showing the impact of FurA depletion, FurA overexpression and iron deprivation on the transcriptional pattern of novel FurA targets.
(A) Total RNA from the wild-type strain PCC 7120 (WT) and the coaR-PcoaT::furA fusion strain AGcoaRFurA (FurA-) were isolated from cells grown in Co2+/Zn2+ deprived medium (BG-11-Co/Zn). (B) Total RNA from the wild-type strain PCC 7120 (WT) and the furA overexpressing strain AG2770FurA (FurA+) were isolated from cells grown in standard BG-11 medium (+Fe2+) or iron deprived medium BG-11-Fe (-Fe2+). Housekeeping gene rnpB was used as control. Determinations for each gene were performed in the early exponential phase of PCR. Expression analyses of genes furA and isiA were included as controls of experimental conditions. All determinations were performed three times with independent biological samples, and the relevant portion of a representative gel is shown for each gene.