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Regulation of the phototrophic iron oxidation (pio) genes in Rhodopseudomonas palustris TIE-1 is mediated by the global regulator, FixK.

Bose A, Newman DK - Mol. Microbiol. (2010)

Bottom Line: To test this possibility, a ΔfixK mutant of R. palustris TIE-1 was assessed for pioABC expression. pioABC expression decreased dramatically in ΔfixK versus WT during photoferrotrophic growth, implying that FixK positively regulates its expression; coincidently, the onset of iron oxidation was prolonged in this mutant.Mutant expression analysis revealed that FixK regulates anaerobic phototrophic expression of other target genes with FixK binding sites in their promoters.This study shows that FixK regulates key iron metabolism genes in an α-proteobacterium, pointing to a departure from the canonical Fur/Irr mode of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, 77 Massachusetts Ave., 68-380, Cambridge, MA 02139, USA.

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β-Galactosidase activity was determined for translational fusions with FixK I and FixK II boxes deleted. FW, fresh water minimal medium for anaerobic phototrophic growth; S, succinate; A, acetate; B, benzoate; HB, 4-hydroxybenzoate; H2, hydrogen; Fe-NTA, FeCl2 with nitrilotriacetic acid; Fe, FeCl2 alone. The grey-hashed boxes represent the FixK I and II boxes. The black box represents the −35 and the white box represents the −10. The black bent arrow represents the TSS. β-Galactosidase activity was determined using a modified Miller assay and normalized with total protein to get specific activity [milliUnits-mU (nmol min−1 mg protein−1)]. Values represent average of three independent triplicates ± standard error.
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fig03: β-Galactosidase activity was determined for translational fusions with FixK I and FixK II boxes deleted. FW, fresh water minimal medium for anaerobic phototrophic growth; S, succinate; A, acetate; B, benzoate; HB, 4-hydroxybenzoate; H2, hydrogen; Fe-NTA, FeCl2 with nitrilotriacetic acid; Fe, FeCl2 alone. The grey-hashed boxes represent the FixK I and II boxes. The black box represents the −35 and the white box represents the −10. The black bent arrow represents the TSS. β-Galactosidase activity was determined using a modified Miller assay and normalized with total protein to get specific activity [milliUnits-mU (nmol min−1 mg protein−1)]. Values represent average of three independent triplicates ± standard error.

Mentions: To assess the importance of the canonical FixK binding sites conserved in the pioABC promoter, we constructed DNA templates lacking either FixK I or FixK II boxes and fused them in frame with lacZ, forming translational fusions. These constructs were then integrated onto the chromosome of WT R. palustris TIE-1 and assayed for β-galactosidase activity on various growth substrates. Deletion of FixK I led to dramatic downregulation of β-galactosidase activity on all the substrates tested (Fig. 3). These data demonstrated that FixK I was important for activation of expression of the pioABC operon. Only a modest downregulation of β-galactosidase activity was observed when FixK II was deleted. This suggests that although the primary DNA site that influences pioABC expression is FixK I, FixK II does play a role in activating pioABC expression.


Regulation of the phototrophic iron oxidation (pio) genes in Rhodopseudomonas palustris TIE-1 is mediated by the global regulator, FixK.

Bose A, Newman DK - Mol. Microbiol. (2010)

β-Galactosidase activity was determined for translational fusions with FixK I and FixK II boxes deleted. FW, fresh water minimal medium for anaerobic phototrophic growth; S, succinate; A, acetate; B, benzoate; HB, 4-hydroxybenzoate; H2, hydrogen; Fe-NTA, FeCl2 with nitrilotriacetic acid; Fe, FeCl2 alone. The grey-hashed boxes represent the FixK I and II boxes. The black box represents the −35 and the white box represents the −10. The black bent arrow represents the TSS. β-Galactosidase activity was determined using a modified Miller assay and normalized with total protein to get specific activity [milliUnits-mU (nmol min−1 mg protein−1)]. Values represent average of three independent triplicates ± standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050613&req=5

fig03: β-Galactosidase activity was determined for translational fusions with FixK I and FixK II boxes deleted. FW, fresh water minimal medium for anaerobic phototrophic growth; S, succinate; A, acetate; B, benzoate; HB, 4-hydroxybenzoate; H2, hydrogen; Fe-NTA, FeCl2 with nitrilotriacetic acid; Fe, FeCl2 alone. The grey-hashed boxes represent the FixK I and II boxes. The black box represents the −35 and the white box represents the −10. The black bent arrow represents the TSS. β-Galactosidase activity was determined using a modified Miller assay and normalized with total protein to get specific activity [milliUnits-mU (nmol min−1 mg protein−1)]. Values represent average of three independent triplicates ± standard error.
Mentions: To assess the importance of the canonical FixK binding sites conserved in the pioABC promoter, we constructed DNA templates lacking either FixK I or FixK II boxes and fused them in frame with lacZ, forming translational fusions. These constructs were then integrated onto the chromosome of WT R. palustris TIE-1 and assayed for β-galactosidase activity on various growth substrates. Deletion of FixK I led to dramatic downregulation of β-galactosidase activity on all the substrates tested (Fig. 3). These data demonstrated that FixK I was important for activation of expression of the pioABC operon. Only a modest downregulation of β-galactosidase activity was observed when FixK II was deleted. This suggests that although the primary DNA site that influences pioABC expression is FixK I, FixK II does play a role in activating pioABC expression.

Bottom Line: To test this possibility, a ΔfixK mutant of R. palustris TIE-1 was assessed for pioABC expression. pioABC expression decreased dramatically in ΔfixK versus WT during photoferrotrophic growth, implying that FixK positively regulates its expression; coincidently, the onset of iron oxidation was prolonged in this mutant.Mutant expression analysis revealed that FixK regulates anaerobic phototrophic expression of other target genes with FixK binding sites in their promoters.This study shows that FixK regulates key iron metabolism genes in an α-proteobacterium, pointing to a departure from the canonical Fur/Irr mode of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, 77 Massachusetts Ave., 68-380, Cambridge, MA 02139, USA.

Show MeSH
Related in: MedlinePlus