NrdR Transcription Regulation: Global Proteome Analysis and Its Role in Escherichia coli Viability and Virulence.

Vankadari Naveen,Chwan-Deng Hsiao,Digby F Warner

doi:10.1371/journal.pone.0157165

Abstract

Bacterial ribonucleotide reductases (RNRs) play an important role in the synthesis of dNTPs and their expression is regulated by the transcription factors, NrdR and Fur. Recent transcriptomic studies using deletion mutants have indicated a role for NrdR in bacterial chemotaxis and in the maintenance of topoisomerase levels. However, NrdR deletion alone has no effect on bacterial growth or virulence in infected flies or in human blood cells. Furthermore, transcriptomic studies are limited to the deletion strain alone, and so are inadequate for drawing biological implications when the NrdR repressor is active or abundant. Therefore, further examination is warranted of changes in the cellular proteome in response to both NrdR overexpression, as well as deletion, to better understand its functional relevance as a bacterial transcription repressor. Here, we profile bacterial fate under conditions of overexpression and deletion of NrdR in E. coli. Biochemical assays show auxiliary zinc enhances the DNA binding activity of NrdR. We also demonstrate at the physiological level that increased nrdR expression causes a significant reduction in bacterial growth and fitness even at normal temperatures, and causes lethality at elevated temperatures. Corroborating these direct effects, global proteome analysis following NrdR overexpression showed a significant decrease in global protein expression. In parallel, studies on complementary expression of downregulated essential genes polA, eno and thiL showed partial rescue of the fitness defect caused by NrdR overexpression. Deletion of downregulated non-essential genes ygfK and trxA upon NrdR overexpression resulted in diminished bacterial growth and fitness suggesting an additional role for NrdR in regulating other genes. Moreover, in comparison with NrdR deletion, E. coli cells overexpressing NrdR showed significantly diminished adherence to human epithelial cells, reflecting decreased bacterial virulence. These results suggest that elevated expression of NrdR could be a suitable means to retard bacterial growth and virulence, as its elevated expression reduces bacterial fitness and impairs host cell adhesion.

Highlights

For all organisms, timely and temporal regulation of gene expression and its translation to protein level is crucial for cell proliferation
Our sequence alignment of NrdR from a broad range of species led to three important observations: 1) the alignment showed highest homology in the N-terminal domain, which is the primary DNA-binding region, and not in the well-known unique ATPcone domain; 2) previous reports have noted the importance of conserved N-terminal Cys residues in Zn+ binding [14], we observed that Cys residues were arranged in characteristic CPxC and CxxC motifs in all classes of bacterial species; and 3) we noticed that Arg residues (26–29) were highly conserved, exhibiting a unique arginine-motif (R4) that might be important for interactions with DNA substrate, as Arg residues are known to play a key role in DNAprotein interactions [38]
The transcription repressor NrdR has been implicated in the regulatory expression of various Ribonucleotide reductases (RNRs) and, recently, in bacterial chemotaxis through an unknown mechanism [8, 10]

Summary

Introduction

Timely and temporal regulation of gene expression and its translation to protein level is crucial for cell proliferation. These complex multistep events are controlled by various metabolic processes and their inter-regulation. Class 1a and 1c are both regulated by nrdAB genes, class 1c RNRs can be distinguished from class 1a RNRs by the protein radical that is generated through an Mn4-O-Fe3 center and are found in species like Chlamydia trachomatis [1, 4]. Most enterobacterial species like E. coli, Helicobactor and Klebsiella encode the two major classes of RNRs (Iab and III), but a few bacterial species such as Pseudomonas encode class II and lack RNRs of class Ib, which is a large known group [8, 9]

Methods

Results

Conclusion