Communication between Binding Sites Is Required for YqjI Regulation of Target Promoters within the yqjH-yqjI Intergenic Region

Abstract

The nickel-responsive transcription factor YqjI represses its own transcription and transcription of the divergent yqjH gene, which encodes a novel ferric siderophore reductase. The intergenic region between the two promoters is complex, with multiple sequence features that may impact YqjI-dependent regulation of its two target promoters. We utilized mutagenesis and DNase I footprinting to characterize YqjI regulation of the yqjH-yqjI intergenic region. The results show that YqjI binding results in an extended footprint at the yqjI promoter (site II) compared to the yqjH promoter (site I). Mutagenesis of in vivo gene reporter constructs revealed that the two YqjI binding sites, while separated by nearly 200 bp, appear to communicate in order to provide full YqjI-dependent regulation at the two target promoters. Thus, YqjI binding at both promoters is required for full repression of either promoter, suggesting that the two YqjI binding sites cooperate to control transcription from the divergent promoters. Furthermore, internal deletions that shorten the total length of the intergenic region disrupt the ability of YqjI to regulate the yqjH promoter. Finally, mutagenesis of the repetitive extragenic palindromic (REP) elements within the yqjH-yqjI intergenic region shows that these sequences are not required for YqjI regulation. These studies provide a complex picture of novel YqjI transcriptional regulation within the yqjH-yqjI intergenic region and suggest a possible model for communication between the YqjI binding sites at each target promoter. Metal ions play an important role in all organisms. It is estimated that about one-third of all proteins require a metal for proper function, with approximately half of (40%) all enzymes having metal bound to them as a cofactor (1). However, even essential transition metals can catalyze spurious side reactions if present in excess. Due to their dual nature, acquisition and intracellular trafficking of essential first-row transition metals, such as iron, zinc, copper, and nickel, are carefully controlled by metal homeostasis systems. Metal homeostasis systems consist of metal transporters, metal storage proteins, and metallochaperones that work in concert to maintain the proper cellular metal quota (2). Expression of metal homeostasis genes is often controlled by specialized transcription factors known as metalloregulatory proteins (3). Besides their functional DNA binding domains, metalloregulatory proteins often have an allosteric metal binding domain that is able to directly sense cellular metal changes (4). Binding or dissociation of the metal ion(s) at the metal binding domain switches the DNA binding domain to increase or decrease its affinity for target promoter(s). This metal-responsive allosteric switch coordinates expression of the genes encoding metal utilizing proteins and other metal homeostasis proteins with cellular metal levels.In a previous study, we discovered that the predicted transcription factor YqjI from Escherichia coli is a nickel-responsive transcr...

Structure and Mechanism of the Siderophore-Interacting Protein from the Fuscachelin Gene Cluster of Thermobifida fusca

“…41,42 Interestingly, the DNA binding of YqjI is dependent on Ni 2+ binding (not iron), and it is proposed that the gene product helps regulate iron acquisition under conditions that include high nickel ion concentrations, which can disrupt iron homeostasis. 43 A second class of specific intracellular reductases contain an Fe−S cluster as exemplified by E. coli FhuF [termed the ferric siderophore reductase (FSR) family]. FhuF is part of the siderophorereductase system with a sufficient redox potential to reduce ferric ferrioxamine.…”

“…A preference of NADPH for reduction of bound flavin has been demonstrated for YqjH, along with the ability to reduce diverse iron chelates, including ferric enterobactin . The ygjH gene is a part of the Fur regulon and under transcriptional control of yqjI , a winged helix transcriptional regulator. , Interestingly, the DNA binding of YqjI is dependent on Ni 2+ binding (not iron), and it is proposed that the gene product helps regulate iron acquisition under conditions that include high nickel ion concentrations, which can disrupt iron homeostasis . A second class of specific intracellular reductases contain an Fe–S cluster as exemplified by E. coli FhuF [termed the ferric siderophore reductase (FSR) family].…”

Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

“…6A ); this repression is relieved under excess nickel conditions (Wang, Wu and Outten 2011 ; Wang et al . 2014 ). High nickel levels can disrupt iron homeostasis in E. coli , leading to a longer lag phase due to reduced accumulation of iron (Rolfe et al .…”

Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

“…NfeR contributes to metal homeostasis in E. coli by repressing expression of a neighbouring ferric reductase gene, nfeF (Fig. 6A); this repression is relieved under excess nickel conditions (Wang, Wu and Outten 2011; Wang et al . 2014).…”

“…NfeR contributes to metal homeostasis in E. coli by repressing expression of a neighbouring ferric reductase gene, nfeF (Fig. 6A); this repression is relieved under excess nickel conditions (Wang, Wu and Outten 2011;Wang et al 2014). High nickel levels can disrupt iron homeostasis in E. coli, leading to a longer lag phase due to reduced accumulation of iron (Rolfe et al 2012;Washington-Hughes et al 2019).…”