Figure 1.
Growth curves of WT WP3 and the fur mutant in liquid 2216E with or without 60 µM iron chelator under aerobic conditions.
(▪) WT WP3, (•) fur mutant, (▴) WT WP3 with 60 µM iron chelator, and (▾) fur mutant with 60 µM iron chelator. The data represent averages of triplicate cultures.
Figure 2.
Growth curves of the WT WP3, fur mutant, and fur complement strains with different electron acceptors under anaerobic conditions.
(a–c) Growth on 20 mM fumarate, 2 mM nitrate, and 20 mM DMSO as the electron acceptor, respectively. (d) Time course of Fe2+ concentration with 15 mM HFO as an electron acceptor. The following abbreviations are used for all of the panels: (▪) WT WP3, (•) fur mutant, (▴) fur complement strain. The data represent averages of triplicate cultures.
Figure 3.
Spectrum analysis of the cytochrome c content of WT and fur mutant strains under aerobic and anaerobic conditions.
The reduced-minus-oxidized difference spectra of equal amounts of total protein from the WT and fur mutant strains treated with sodium dithionite were recorded. The absorbance levels of the corresponding untreated strains were set as a control. Line 1 Absorbance of the aerobically grown WT strain. Line 2 Absorbance of the aerobically grown fur mutant strain. Line 3 Absorbance of the anaerobically grown WT strain. Line 4 Absorbance of the anaerobically grown fur mutant strain.
Table 1.
Number of differentially expressed genes in Δfur.
Table 2.
Fur-responsive modules for iron acquisition and storage systems with fumarate as the EA.
Table 3.
Fur-responsive modules for anaerobic electron transport with fumarate as the EA.
Figure 4.
Fur binding to selected promoters (omcA, Crp-like regulator gene and napD) by EMSA.
The binding assays were performed in the presence of 200(lanes 2–4, lanes 6–8, lanes 10–12) and 0.2 pmol DIG-labeled (lanes 1–12) promoter DNA. Non-specific competitor DNA (50 µg/ml Salmon Sperm DNA) was used in all these binding reactions to control for the presence of unspecific binding. Specific competitors (2 pmol and 20 pmol unlabeled probes) were added respectively in lane 3, 4; lane 7, 8; lane 11, 12 to verify the specificity of a band resulting from protein-binding to the labeled probe.
Figure 5.
A model of the Fur regulatory system in WP3.
In this model, Fur acts as both a direct and indirect regulator of iron homeostasis and anaerobic respiration. As a direct regulator, the Fur protein generally binds to a Fur Box region to down- or up-regulate the expression of genes. As an indirect regulator, the Fur protein represses an antisense non-coding regulatory RNA or controls secondary regulators, such as Crp, ArcA, the H-NS protein, and the TetR family regulator. These proteins are also capable of regulating genes in the anaerobic respiration system as well as those that encode iron using proteins. The genes regulated by Fur are involved in iron homeostasis, the anaerobic electron transport system, the heme biosynthesis and transport systems, and the CCM system. Direct regulation is depicted with a solid arrow, and indirect regulation is depicted with a dotted arrow.