ABSTRACT
BACKGROUND: Zra belongs to the envelope stress response (ESR) two-component systems (TCS). It is atypical because of its third periplasmic repressor partner (ZraP), in addition to its histidine kinase sensor protein (ZraS) and its response regulator (ZraR) components. Furthermore, although it is activated by Zn2+, it is not involved in zinc homeostasis or protection against zinc toxicity. Here, we mainly focus on ZraS but also provide information on ZraP. METHODS: The purified periplasmic domain of ZraS and ZraP were characterized using biophysical and biochemical technics: multi-angle laser light scattering (MALLS), circular dichroism (CD), differential scanning fluorescence (DSF), inductively coupled plasma atomic emission spectroscopy (ICP-AES), cross-linking and small-angle X-ray scattering (SAXS). In-vivo experiments were carried out to determine the redox state of the cysteine residue in ZraP and the consequences for the cell of an over-activation of the Zra system. RESULTS: We show that ZraS binds one Zn2+ molecule with high affinity resulting in conformational changes of the periplasmic domain, consistent with a triggering function of the metal ion. We also demonstrate that, in the periplasm, the only cysteine residue of ZraP is at least partially reduced. Using SAXS, we conclude that the previously determined X-ray structure is different from the structure in solution. CONCLUSION: Our results allow us to propose a general mechanism for the Zra system activation and to compare it to the homologous Cpx system. GENERAL SIGNIFICANCE: We bring new input on the so far poorly described Zra system and notably on ZraS.
Subject(s)
Arabinose/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Trans-Activators/chemistry , Zinc/chemistry , Amino Acid Sequence , Arabinose/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Models, Molecular , Periplasm/genetics , Periplasm/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Trans-Activators/genetics , Trans-Activators/metabolism , Zinc/metabolismABSTRACT
During their lifecycle, bacteria are exposed to continuous changes in their environment, some of which are stressful and can be harmful. The cell envelope is the first line of defense against a hostile environment, but it is also the first target for damage. To deal with this problem, bacteria have evolved systems collectively called "envelope stress response," or ESR, dedicated to the detection and repair of damaged components. Here we decided to investigate whether the atypical two-component system ZraP-SR is a novel ESR. Based on the screening of more than 240 drugs using the Biolog technology, we show that the deletion of zraP or zraR confers increased susceptibility to five classes of antibiotics and to some environmental stress targeting the envelope. Using a microscopy approach, we also establish that ZraP and ZraR are required to maintain envelope integrity. So far, the ZraR regulator was only known to activate the transcription of zraP and zraSR. Using chromatin immunoprecipitation followed by sequencing and RT-qPCR, we have now identified 25 additional genes regulated by ZraR, the majority of which are involved in the response against stress. Taken together, our results demonstrate that ZraP-SR is a novel ESR.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Trans-Activators/genetics , Chromatin Immunoprecipitation , Drug Resistance, Bacterial , Escherichia coli/drug effects , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial , Sequence Analysis, RNA , Stress, Physiological , Trans-Activators/metabolismABSTRACT
Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress.
