Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

Abstract

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

Figure 1.

(A) Immunoblot analysis of the fur mutant strain using a polyclonal anti-Fur antiserum. Wt, wild-type strain NA1000; fur, SP0057 strain; fur+, SP0057(pMRFur). (B) Growth curve of strains NA1000 (white square), SP0057 (white triangle) or SP0057(pMRFur) (black triangle). The figure shows one representative experiment of three independent replicates.

Figure 2.

Survival test of strains NA1000 (white square), SP0057(white triangle) or SP0057(pMRFur) (black triangle) after addition of 5 mM paraquat (A) or 5 mM tert-butyl hydropheroxide (B). Cultures grown at 30°C in PYE medium up to midlog phase and the oxidative agents were added at time zero. At the indicated time points, aliquots were removed and plated for counting colonies. Survival was determined relative to the colony number at time 0. The results shown are the average of three independent experiments.

Figure 3.

Fur binding to promoter regions was assessed by gel electrophoresis mobility shift assay. (A) DNA fragments corresponding to each promoter was ³²P-labeled and incubated or not (0) with increasing concentrations of purified Fur protein (50, 200 and 500 nM, respectively). As controls, the binding of Fur (250 nM) was carried out in the presence of 30-fold of unlabeled fragments of the same region (S) or the fur coding region (N) as competitors. The last panel shows a control experiment using as probe the fur coding region, and the DNA was incubated with the following Fur concentrations: 50-, 100-, 250-, 500- and 1000-nM protein, respectively. (B) Fur-binding sites at the promoter regions of operons sdh and nuo were mutagenized (indicated by an asterisk) substituting the conserved TGCGA motif by the sequences in bold type. The fragments were incubated or not (0) with increasing concentrations of purified Fur protein (50, 200, 500 and 1000 nM, respectively).

Figure 4.

DNase I footprinting assays of Fur in the sdhC and CC2194 promoter regions. Probes containing each promoter region were end-labeled and incubated in the presence or absence of increasing concentrations of purified Fur (50, 100, 250, 500 and 1000 nM, respectively). The DNA–protein complexes were treated with DNase I as described in ‘Materials and Methods’ section. The protected regions are boxed in the respective sequences. A minus sign indicates no protein. Asterisks indicate hypersensitive sites.

Figure 5.

Analysis of expression of selected genes in response to iron and Fur. (A) Expression was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of either 100 μM FeSO₄ (Fe) or 100 μM 2′-dipyridyl (DDP). The last panel shows as a control the expression of CC2194 in NA1000, the fur mutant (fur) and fur complemented with the gene in trans (fur⁺), indicating that the presence of Fur restores the wild-type expression. (B) Expression driven by the nuoA and sdhC promoters was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of 100 μM FeSO₄. The assays were carried out using either the wild-type promoters (P) or the mutagenized promoters (P*) as described in Figure 3, introduced into the NA1000 or SP0057 strains. β-Galactosidase activity is expressed in Miller units (38) and is the average of at least three independent assays.

Figure 6.

Promoter sequences of selected genes, indicating the Fur-binding sites (shaded) predicted in silico or experimentally demonstrated (sdhC and CC2194). Boxes indicate conserved –35 and –10 sequences of Caulobacter σ⁷⁰ promoters (TTGAC-16 bp-^G/_CCTANA) and previously identified transcription start sites (39) are indicated in bold. Annotated start codons are also indicated in bold letters, except for the putative start codon of CC0028, which was proposed in this work.

Figure 7.

DNA sequence logos representing C. crescentus Fur-binding site. Experimentally validated Fur-binding sites (11 sites) (A) and computationally predicted Fur binding sites (62 sites with score >7.5 shown in Table S2) (B) were used to create the logos with the WebLogo generator (http://weblogo.berkeley.edu/).