The multi-proteins Isc and Suf systems catalyse the biogenesis of [Fe-S] proteins. Here we investigate how NsrR and IscR, transcriptional regulators that sense NO and [Fe-S] homeostasis, acquire their [Fe-S] clusters under both normal and iron limitation conditions. Clusters directed at the apo-NsrR and apo-IscR proteins are built on either of the two scaffolds, IscU or SufB. However, differences arise in [Fe-S] delivery steps. In the case of NsrR, scaffolds deliver clusters to either one of the two ATCs, IscA and SufA, and, subsequently, to the non-Isc non-Suf ATC, ErpA. Nevertheless, a high level of SufA can bypass the requirement for ErpA. In the case of IscR, several routes occur. One does not include assistance of any ATC. Others implicate ATCs IscA or ErpA, but, surprisingly, SufA was totally absent from any IscR maturation pathways. Both IscR and NsrR have the intrinsic capacity to sense iron limitation. However, NsrR appeared to be efficiently matured by Isc and Suf, thereby preventing NsrR to act as a physiologically relevant iron sensor. This work emphasizes that different maturation pathways arise as a function of the apo-target considered, possibly in relation with the type of cluster, [2Fe-2S] versus [4Fe-4S], it binds.
Nickel is a virulence determinant for the human gastric pathogen Helicobacter pylori. Indeed, H. pylori possesses two nickel-enzymes that are essential for in vivo colonization, [NiFe] hydrogenase and urease, an abundant virulence factor that contains 24 nickel ions per active complex. Because of these two enzymes, survival of H. pylori relies on an important supply of nickel, implying a tight control of its distribution and storage. In this review, we will present the pathways of activation of the nickel enzymes as well as original mechanisms found in H. pylori for the uptake, trafficking and distribution of nickel between the two enzymes. These include (i) an outer-membrane nickel uptake system, the FrpB4 TonB-dependent transporter, (ii) overlapping protein complexes and interaction networks involved in nickel trafficking and distribution between urease and hydrogenase and, (iii) Helicobacter specific nickel-binding proteins that are involved in nickel storage and can play the role of metallo-chaperones. Finally, we will discuss the implication of the nickel trafficking partners in virulence and propose them as novel therapeutic targets for treatments against H. pylori infection.
It is well known that ppGpp and DksA interact with bacterial RNA polymerase (RNAP) to alter promoter activity. This study suggests that GreA plays a major role and GreB plays a minor role in the ppGpp-DksA regulatory network. We present evidence that DksA and GreA/GreB are redundant and/or share similar functions: (i) on minimal medium GreA overproduction suppresses the growth defects of a dksA mutant; (ii) GreA and DksA overexpression partially suppresses the auxotrophy of a ppGpp-deficient strain; (iii) microarrays show that many genes are regulated similarly by GreA and DksA. We also find instances where GreA and DksA seem to act in opposition: (i) complete suppression of auxotrophy occurs by overexpression of GreA or DksA only in the absence of the other protein; (ii) PgadA and PgadE promoter fusions, along with many other genes, are dramatically affected in vivo by GreA overproduction only when DksA is absent; (iii) GreA and DksA show opposite regulation of a subset of genes. Mutations in key acidic residues of GreA and DksA suggest that properties seen here probably are not explained by known biochemical activities of these proteins. Our results indicate that the general pattern of gene expression and, in turn, the ability of Escherichia coli to grow under a defined condition are the result of a complex interplay between GreA, GreB, and DksA that also involves mutual control of their gene expression, competition for RNA polymerase binding, and similar or opposite action on RNA polymerase activity.
CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.
NifS-like cysteine desulfurases are widespread enzymes involved in the mobilization of sulfur from cysteine. The genome of the filamentous diazotrophic cyanobacterium Anabaena PCC 7120 contains four open reading frames potentially encoding NifS-like proteins. One of them, alr2505, belongs to the pkn22 operon, which enables Anabaena to cope with oxidative stress. The Alr2505 protein was purified and found to share all the features characteristic of cysteine desufurases. This is the first NifS-like enzyme to be functionally characterized in this bacterium. On the basis of the transcriptional profiling of all nifS-like genes in Anabaena, it is concluded that alr2505 is the only cysteine desulfurase-encoding gene induced by oxidative stress. The function of Alr2505, which was termed OsiS, is discussed.
Cysteine desulphurases are primary sources of sulphur that can eventually be used for Fe/S biogenesis or thiolation of various cofactors and tRNA. Escherichia coli contains three such enzymes, IscS, SufS and CsdA. The importance of IscS and SufS in Fe/S biogenesis is well established. The physiological role of CsdA in contrast remains uncertain. We provide here additional evidences for a functional redundancy between the three cysteine desulphurases in vivo. In particular, we show that a deficiency in isoprenoid biosynthesis is the unique cause of the lethality of the iscS sufS mutant. Moreover, we show that CsdA is engaged in two separate sulphur transfer pathways. In one pathway, CsdA interacts functionally with SufE-SufBCD proteins to assist Fe/S biogenesis. In another pathway, CsdA interacts with CsdE and a newly discovered protein, which we called CsdL, resembling E1-like proteins found in ubiquitin-like modification systems. We propose this new pathway to allow synthesis of an as yet to be discovered thiolated compound.
Iron sulfur (Fe/S) proteins are ubiquitous and participate in multiple biological processes, from photosynthesis to DNA repair. Iron and sulfur are highly reactive chemical species, and the mechanisms allowing the multiprotein systems ISC and SUF to assist Fe/S cluster formation in vivo have attracted considerable attention. Here, A-Type components of these systems (ATCs for A-Type Carriers) are studied by phylogenomic and genetic analyses. ATCs that have emerged in the last common ancestor of bacteria were conserved in most bacteria and were acquired by eukaryotes and few archaea via horizontal gene transfers. Many bacteria contain multiple ATCs, as a result of gene duplication and/or horizontal gene transfer events. Based on evolutionary considerations, we could define three subfamilies: ATC-I, -II and -III. Escherichia coli, which has one ATC-I (ErpA) and two ATC-IIs (IscA and SufA), was used as a model to investigate functional redundancy between ATCs in vivo. Genetic analyses revealed that, under aerobiosis, E. coli IscA and SufA are functionally redundant carriers, as both are potentially able to receive an Fe/S cluster from IscU or the SufBCD complex and transfer it to ErpA. In contrast, under anaerobiosis, redundancy occurs between ErpA and IscA, which are both potentially able to receive Fe/S clusters from IscU and transfer them to an apotarget. Our combined phylogenomic and genetic study indicates that ATCs play a crucial role in conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets. We propose a model wherein the conserved biochemical function of ATCs provides multiple paths for supplying Fe/S clusters to apotargets. This model predicts the occurrence of a dynamic network, the structure and composition of which vary with the growth conditions. As an illustration, we depict three ways for a given protein to be matured, which appears to be dependent on the demand for Fe/S biogenesis.
Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.
Biosynthesis of iron-sulphur (Fe-S) proteins is catalysed by multi-protein systems, ISC and SUF. However, non-ISC, non-SUF Fe-S biosynthesis factors have been described, both in prokaryotes and eukaryotes. Here we report in vitro and in vivo investigations of such a non-ISC, non SUF component, the Nfu proteins. Phylogenomic analysis allowed us to define four subfamilies. Escherichia coli NfuA is within subfamily II. Most members of this subfamily have a Nfu domain fused to a degenerate A-type carrier domain (ATC*) lacking Fe-S cluster co-ordinating Cys ligands. The Nfu domain binds a [4Fe-4S] cluster while the ATC* domain interacts with NuoG (a complex I subunit) and aconitase B (AcnB). In vitro, holo-NfuA promotes maturation of AcnB. In vivo, NfuA is necessary for full activity of complex I under aerobic growth conditions, and of AcnB in the presence of superoxide. NfuA receives Fe-S clusters from IscU/HscBA and SufBCD scaffolds and eventually transfers them to the ATCs IscA and SufA. This study provides significant information on one of the Fe-S biogenesis factors that has been often used as a building block by ISC and/or SUF synthesizing organisms, including bacteria, plants and animals.
Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei, its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasites cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress.
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