A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

Chunxiao Yu,M Selim Ünlü,George G Daaboul,Han Hu,Carlos A Lopez,Caroline Attardo Genco,David S Freedman,Alexander P Reddington,Yu Xia,Szabolcs Semsey

doi:10.1371/journal.pone.0096832

Chunxiao Yu, M Selim Ünlü + Show 8 more

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https://doi.org/10.1371/journal.pone.0096832

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Journal: PloS one	Publication Date: May 8, 2014
Citations: 56	License type: CC BY 4.0

Affiliation: Boston University

Abstract

The Ferric uptake regulatory 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, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, 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 in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

Highlights

The Interferometric Reflectance Imaging Sensor (IRIS) is a photometric biosensing technology, which is designed on the principles of interferometry and has been applied as a microarraybased screening technique for studying the interactions of biomacromolecules [1,2,3,4]
Neisseria Ferric Uptake Regulator protein (Fur) boxes previously defined by DNase I foot printing studies were utilized to avoid the mismatches caused by the species-specific nucleotides in Fur boxes of different bacteria (Table S1)
Due to the fact that transcription of Fur regulated genes responds to iron availability, we focused on the iron-regulated genes identified via microarray analyses [50,51] of N. gonorrhoeae cultured under iron-replete and iron-deplete conditions

Summary

Introduction

The Interferometric Reflectance Imaging Sensor (IRIS) is a photometric biosensing technology, which is designed on the principles of interferometry and has been applied as a microarraybased screening technique for studying the interactions of biomacromolecules [1,2,3,4]. IRIS can be broadly applied to characterize many types of biomolecular interactions, such as DNA-DNA hybridization, protein-protein binding, and proteinDNA interaction in a quantitative format [1,2,3,4] This technique has previously demonstrated utility for probing transcription factor interactions with arrayed oligonucleotides [5]. Bacterial transcription factors control tight regulation of gene expression in response to host specific environmental niches in a number of human pathogenic bacteria. Gonococcal colonization of mucosal surfaces requires tight regulation of gene expression in response to host specific environmental niches including iron-limited host environments This adaptation is mainly achieved by the up-regulation of ironacquisition components, which are repressed by a ubiquitous bacterial regulatory protein, the Ferric Uptake Regulator protein (Fur) [6,7,8]. Fur influences secondary regulatory components, such as small RNAs and histone-like nucleotide binding protein (H-NS), to regulate a subset of genes indirectly [25,26,27,28,29,30,31, 32,33]

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