Frédéric Barras

SufC: an unorthodox cytoplasmic ABC/ATPase required for [Fe—S] biogenesis under oxidative stress

Proteins containing [Fe—S] clusters perform essential functions in all domains of life. Previously, we identified the sufABCDSE operon as being necessary for virulence of the plant pathogen Erwinia chrysanthemi. In addition, we collected preliminary evidence that the sufABCDSE operon might be involved in the assembly of [Fe—S] clusters. Of particular interest are the sufB, sufC and sufD genes, which are conserved among Eubacteria, Archaea, plants and parasites. The present study establishes SufC as an unorthodox ATPase of the ABC superfamily that is located in the cytosol, wherein it interacts with both SufB and SufD. Moreover, under oxidative stress conditions, SufC was found to be necessary for the activity of enzymes containing oxygen‐labile [Fe—S] clusters, but dispensable for glutamate synthase, which contains an oxidatively stable [Fe—S] cluster. Lastly, we have shown SufBCD to be essential for iron acquisition via chrysobactin, a siderophore of major importance in virulence. We discuss a model wherein the SufBCD proteins contribute to bacterial pathogenicity via their role in the assembly of [Fe—S] clusters under oxidative stress and iron limitation.

Building Fe–S proteins: bacterial strategies

The broad range of cellular activities carried out by Fe–S proteins means that they have a central role in the life of most organisms. At the interface between biology and chemistry, studies of bacterial Fe–S protein biogenesis have taken advantage of the specific approaches of each field and have begun to reveal the molecular mechanisms involved. The multiprotein systems that are required to build Fe–S proteins have been identified, but the in vivo roles of some of the components remain to be clarified. The way in which cellular Fe–S cluster trafficking pathways are organized remains a key issue for future studies.

Biogenesis of Fe-S Cluster by the Bacterial Suf System SufS AND SufE FORM A NEW TYPE OF CYSTEINE DESULFURASE

Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems. Recently, we described the SUF system of Escherichia coli and Erwinia chrysanthemi as being important for Fe-S biogenesis under stressful conditions. The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet. Here we demonstrate that: (i) SufE and SufS are both cystosolic as all members of the SUF system; (ii) SufE is a homodimeric protein; (iii) SufE forms a complex with SufS as shown by the yeast two-hybrid system and by affinity chromatography; (iv) binding of SufE to SufS is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS. This is the first example of a two-component cysteine desulfurase enzyme.

Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity.

Iron/sulfur centers are key cofactors of proteins intervening in multiple conserved cellular processes, such as gene expression, DNA repair, RNA modification, central metabolism and respiration. Mechanisms allowing Fe/S centers to be assembled, and inserted into polypeptides have attracted much attention in the last decade, both in eukaryotes and prokaryotes. Basic principles and recent advances in our understanding of the prokaryotic Fe/S biogenesis ISC and SUF systems are reviewed in the present communication. Most studies covered stem from investigations in Escherichia coli and Azotobacter vinelandii. Remarkable insights were brought about by complementary structural, spectroscopic, biochemical and genetic studies. Highlights of the recent years include scaffold mediated assembly of Fe/S cluster, A-type carriers mediated delivery of clusters and regulatory control of Fe/S homeostasis via a set of interconnected genetic regulatory circuits. Also, the importance of Fe/S biosynthesis systems in mediating soft metal toxicity was documented. A brief account of the Fe/S biosynthesis systems diversity as present in current databases is given here. Moreover, Fe/S biosynthesis factors have themselves been the object of molecular tailoring during evolution and some examples are discussed here. An effort was made to provide, based on the E. coli system, a general classification associating a given domain with a given function such as to help next search and annotation of genomes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

SoxR-dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase.

Erwinia chrysanthemi causes soft‐rot disease in a great variety of plants. In addition to the depolymerizing activity of plant cell wall‐degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. Here, we studied the pin10 locus originally thought to encode new virulence factors. The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes. Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe–S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter. The reverse transcription–polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon. Expression of a sufB::uidA fusion was found to be induced in iron‐deficient growth conditions and to be repressed by the iron‐sensing Fur repressor. Each of the six suf genes was inactivated by the insertion of a cassette generating a non‐polar mutation. The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron‐activated antibiotic streptonigrin. In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat. Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha. The E. coli sufC homologue was inactivated by reverse genetic. This mutation was found to modify the soxR‐dependent induction of soxS gene expression. We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe–S cluster‐assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe–S ABC exporter. Overall, these results stress the importance of the connection between iron metabolism and oxidative stress during the early steps of infection by E. chrysanthemi.

An inner membrane platform in the type II secretion machinery of Gram-negative bacteria.

The type II secretion machinery allows most Gram‐negative bacteria to deliver virulence factors into their surroundings. We report that in Erwinia chrysanthemi, GspE (the putative NTPase), GspF, GspL and GspM constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery. The GspE–GspF–GspL–GspM complex was demonstrated by two methods: (i) co‐immunoprecipitation of GspE–GspF–GspL with antibodies raised against either GspE or GspF; (ii) interactions in the yeast two‐hybrid system between GspF and GspE, GspF and GspL, GspL and GspM. GspL was found to have an essential role in complex formation. We propose a model in which the GspE–GspF–GspL–GspM proteins constitute a building block within the secretion machinery on top of which another building block, referred to as a pseudopilus, assembles. By analogy, we predict that a similar platform is required for the biogenesis of the type IV pilus.

Iron-Sulfur (Fe/S) Protein Biogenesis: Phylogenomic and Genetic Studies of A-Type Carriers

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.

Fe-S Cluster Biosynthesis Controls Uptake of Aminoglycosides in a ROS-Less Death Pathway

Unreactive Death A controversial proposal that all bactericidal antibiotics kill by reactive oxygen species (ROS) and not by their primary cell target has recently attracted high-profile refutations. The ROS-death pathway implicated overstimulation of the electron transport in respiratory chains; a malfunction that leads to ROS releasing Fe from Fe-S clusters and causing cell death via Fenton chemistry. Ezraty et al. (p. 1583) show that electron transport chains and Fe-S clusters are key to killing by aminoglycoside antibiotics but not for the reasons envisioned in the ROS theory. Fe-S clusters are essential for killing because they mature the respiratory chains that produce the necessary proton motive force for the energized uptake of aminoglycosides. Consequently, iron chelators protect against aminoglycosides, not because they scavenge the iron from Fenton chemistry, but because they block aminoglycoside uptake. The respiratory chain is required for antibiotic entry to the target cell rather than for its killing. All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.

Cobalt Stress in Escherichia coli THE EFFECT ON THE IRON-SULFUR PROTEINS

Cobalt is toxic for cells, but mechanisms of this toxicity are largely unknown. The biochemical and genetic experiments reported here demonstrate that iron-sulfur proteins are greatly affected in cobalt-treated Escherichia coli cells. Exposure of a wild-type strain to intracellular cobalt results in the inactivation of three selected iron-sulfur enzymes, the tRNA methylthio-transferase, aconitase, and ferrichrome reductase. Consistently, mutant strains lacking the [Fe-S] cluster assembly SUF machinery are hypersensitive to cobalt. Last, expression of iron uptake genes is increased in cells treated with cobalt. In vitro studies demonstrated that cobalt does not react directly with fully assembled [Fe-S] clusters. In contrast, it reacts with labile ones present in scaffold proteins (IscU, SufA) involved in iron-sulfur cluster biosynthesis. We propose a model wherein cobalt competes out iron during synthesis of [Fe-S] clusters in metabolically essential proteins.

Redundant Hydrogen Peroxide Scavengers Contribute to Salmonella Virulence and Oxidative Stress Resistance

Salmonella enterica serovar Typhimurium is an intracellular pathogen that can survive and replicate within macrophages. One of the host defense mechanisms that Salmonella encounters during infection is the production of reactive oxygen species by the phagocyte NADPH oxidase. Among them, hydrogen peroxide (H(2)O(2)) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H(2)O(2) degrading enzymes: three catalases (KatE, KatG, and KatN) and two alkyl hydroperoxide reductases (AhpC and TsaA). Inactivation of the five cognate structural genes yielded the HpxF(-) mutant, which exhibited a high sensitivity to exogenous H(2)O(2) and a severe survival defect within macrophages. When the phagocyte NADPH oxidase was inhibited, its proliferation index increased 3.7-fold. Moreover, the overexpression of katG or tsaA in the HpxF(-) background was sufficient to confer a proliferation index similar to that of the wild type in macrophages and a resistance to millimolar H(2)O(2) in rich medium. The HpxF(-) mutant also showed an attenuated virulence in a mouse model. These data indicate that Salmonella catalases and alkyl hydroperoxide reductases are required to degrade H(2)O(2) and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.