Abstract
Iron-sulfur ([Fe-S]) cluster proteins have essential functions in many biological processes. [Fe-S] homeostasis is crucial for bacterial survival under a wide range of environmental conditions. IscR is a global transcriptional regulator in Pseudomonas aeruginosa; it has been shown to regulate genes involved in [Fe-S] cluster biosynthesis, iron homeostasis, resistance to oxidants, and pathogenicity. Many aspects of the IscR transcriptional regulatory mechanism differ from those of other well-studied systems. This study demonstrates the mechanisms of IscR Type-1 binding to its target sites that mediate the repression of gene expression at the isc operon, nfuA, and tpx. The analysis of IscR binding to multiple binding sites in the promoter region of the isc operon reveals that IscR first binds to the high-affinity site B followed by binding to the low-affinity site A. The results of in vitro IscR binding assays and in vivo analysis of IscR-mediated repression of gene expression support the role of site B as the primary site, while site A has only a minor role in the efficiency of IscR repression of gene expression. Ligation of an [Fe-S] cluster to IscR is required for the binding of IscR to target sites and in vivo repression and stress-induced gene expression. Analysis of Type-1 sites in many bacteria, including P. aeruginosa, indicates that the first and the last three AT-rich bases were among the most highly conserved bases within all analyzed Type-1 sites. Herein, we first propose the putative sequence of P. aeruginosa IscR Type-1 binding motif as 5’AWWSSYRMNNWWWTNNNWSGGNYWW3’. This can benefit further studies in the identification of novel genes under the IscR regulon and the regulatory mechanism model of P. aeruginosa IscR as it contributes to the roles of an [Fe-S] cluster in several biologically important cellular activities.
Highlights
Pseudomonas aeruginosa is an important environmental and human pathogenic Gram-negative bacterium
In our previous gene expression study, we determined that IscR functions as a transcriptional repressor on its own promoter, which supports the regulation of the in the ΔiscR (iscR) and the isc operon [38]
In P. aeruginosa, [2Fe-2S] cluster-ligated IscR is the active form of the regulator required for binding the Type-1 binding sites, resulting in repression of gene expression
Summary
Pseudomonas aeruginosa is an important environmental and human pathogenic Gram-negative bacterium. P. aeruginosa contains a plethora of antioxidant enzymes/proteins and reactive oxygen species (ROS)-sequestering compounds that contribute to the protection against oxidative stress and are important for bacterial proliferation and successful infections [1,2,3,4,5,6]. [Fe-S] cluster-containing proteins are required for many biological processes, including biosynthesis pathway activities, respiration, central metabolism, photosynthesis, nitrogen fixation, DNA repair, RNA modification, and gene regulation, across all domains of life [9, 10]. Active [Fe-S] clusters are rapidly damaged by univalent oxidants, and the loss of an electron destabilizes the cluster, causing it to release its catalytic iron atom and initially converting the cluster to an inactive [3Fe-4S]1+ form, often leading to defective proteins [11, 12]. Continued exposure to oxidants leads to further oxidation and iron release
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