A High-Throughput Method to Examine Protein-Nucleotide Interactions Identifies Targets of the Bacterial Transcriptional Regulatory Protein Fur

<div><p>The <u>F</u>erric <u>u</u>ptake <u>r</u>egulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, <u>I</u>nterferometric <u>R</u>eflectance <u>I</u>maging <u>S</u>ensor (IRIS), to rapidly characterize Fur-DNA interactions <i>in vitro</i> with predicted Fur binding sequences in the genome of <i>Neisseria gonorrhoeae</i>, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur <i>in vitro</i> with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal <i>fur</i> mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.</p></div

Fur binding to the predicted Fur boxes. (A) Cold competition assay for the specificity of Fur binding to the predicted Fur boxes.

<p>³²P-labeled ∼50 bp dsDNAs were analyzed after incubation with Fur and unlabeled dsDNA probes (cold probes). Two types of cold competitor probes were used: <i>fur</i>, which contains a Fur box, and <i>rmp</i>, which does not bind to Fur. When the unlabeled probes <i>fur</i> compete out binding of labeled probes, but the unlabeled <i>rmp</i> cannot, the binding of Fur to the predicted Fur box was considered specific. Lane 1, free ³²P-labeled DNA; Lane 2 through Lane 10 contained gonococcal Fur. For the <i>fur</i> and NGO0073 Fur box, Fur protein was added at a concentration of 100 nM and for the NGO0101 Fur box, 400 nM of Fur protein was added. The fold excess of the cold probes was increased from Lane 3 to Lane 6 and Lane 7 to Lane 10 ranging from 50 fold, 500 fold to 1000 fold (indicated by the triangles). <b>(B) Fur binding affinities determined by EMSA.</b> The ³²P labeled dsDNA probes were incubated with a gradient of concentrations of purified gonococcal Fur protein (Lane 1 to Lane 10, 0 nM, 5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 600 nM, 800 nM and 1000 nM, respectively). Arrows indicate the shift of Fur-bound probes. The apparent binding affinities (K_D) were calculated using GraphPad Prism and were represented as mean ± standard error. The gene designations of <i>N. gonorrhoeae</i> F62 were assigned according to their homologues in <i>N. gonorrhoeae</i> FA1090.</p

Identification of Fur binding to predicted Fur boxes by label-free IRIS screening.

<p>A set of ∼50 bp dsDNA probes containing the predicted Fur box in the middle of the probes were designed for IRIS screening (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832.s006" target="_blank">Table S4</a></b>). Each dsDNA probe was immobilized on a chip to produce a spot with a diameter of approximately 100 µm. Three concentrations of Fur protein, 200 nM (red bars), 400 nM (blue bars) and 800 nM (orange bars), were incubated with the individually prepared arrays (in addition to a 0 nM control incubation (black bars) and the binding of Fur protein to dsDNA spots was measured. A mass increase for each spot was represented as a differential spot height (DSH). The known Fur boxes in <i>fur</i>, <i>norB</i> and <i>nspA</i> promoter regions were used as positive controls, and the <i>aniA</i> promoter region was used as negative control in this IRIS assay. (<b>A</b>) Differential spot height for each dsDNA probe after incubation with 0 nM, 200 nM, 400 nM and 800 nM Fur protein, respectively. (<b>B</b>) The number of Fur dimers bound per dsDNA molecule of each probe as calculated from initial and post-incubation mass density measurements. The gene designations of <i>N. gonorrhoeae</i> F62 were assigned according to their homologues in <i>N. gonorrhoeae</i> FA1090.</p

Transcriptional regulation patterns of genes determined by quantitative real-time PCR.

<p>The RNA samples were purified from cultures of the wild-type (WT) and <i>fur</i> mutant and <i>fur</i> complemented strains under iron-replete (+Fe, grey bars) or iron-deplete (-Fe, white bars) conditions 1 h after addition of 100 µM iron or 150 µM desferal. The mRNA levels of <i>fbpA</i> and <i>norB</i>, genes that were repressed and activated by iron-bound Fur respectively, were used as controls for iron and Fur regulation in <i>N. gonorrhoeae</i>. The mRNA levels observed for the five conditions (WT strain under −Fe conditions, <i>fur</i> mutant strain under +Fe and −Fe conditions, and <i>fur</i> complemented strain under +Fe and −Fe conditions) were compared to the value of WT strain under +Fe conditions. The final results were represented as mean ± standard deviation. A * indicates significantly different compared to the mRNA level of WT+Fe. A ** indicates significantly different compared to the mRNA level of WT-Fe. The gene designations of <i>N. gonorrhoeae</i> F62 were assigned according to their homologues in <i>N. gonorrhoeae</i> FA1090.</p

Predicted Fur binding sequences within the promoter regions of <i>N. gonorrhoeae</i> iron-induced genes.¹

1<p>Iron regulation as previously determined by microarray analyses of <i>N. gonorrhoeae</i> grown in defined medium CDM (-Fe) or CDM with 10 µM ferric nitrate (+Fe) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Ducey1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Jackson1" target="_blank">[51]</a>.</p>2<p>Fur regulation was determined by comparing mRNA levels in <i>N. gonorrhoeae</i> WT, <i>fur</i> mutant and <i>fur</i> complemented strains grown under iron-replete and iron-deplete conditions at 1h after addition of iron and desferal using qRT-PCR. NR; not regulated by Fur. Activated; mRNA level of the gene was decreased in the <i>fur</i> mutant strain under either iron-replete or iron-deplete conditions compared to that in the wild type strain. Repressed; mRNA level of the gene was increased in the <i>fur</i> mutant strain under either iron-replete or iron-deplete conditions compared to that in the wild type strain. NE, not examined; transcriptional regulation was not tested in this study.</p>3<p>Fur regulation of the genes as previously determined <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Yu1" target="_blank">[8]</a>.</p>4<p><i>In vitro</i> Fur binding to the 500 bp upstream sequence of ATG of these genes was determined using EMSA and foot printing in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Yu1" target="_blank">[8]</a>.</p

Schematic depicting Fur mediated control mechanisms in <i>N. gonorrhoeae</i> as revealed by IRIS.

<p>Solid lines with arrowheads indicate direct activation of transcription via Fur binding to promoter regions of gonococcal genes. Solid lines with bars indicate direct repression of transcription via binding of Fur to promoter regions of gonococcal genes. Ellipsoids indicate transcriptional regulatory proteins other than Fur. Transcriptional repression of NGO0641, encoding a methyltransferase (triangle), results in alteration of DNA methylation (*) and subsequently alters transcription of a subset of genes. Transcriptional repression of NGO0155, encoding a putative transcriptional regulator, results in alteration of transcription of NGO0155 target genes. A subset of genes contain a Fur box in their putative promoter regions but were not regulated by Fur under the used growth conditions in this study are termed silent Fur binding.</p