Nitric oxide produced by activated macrophages plays a key role as one of the immune system's weapons against pathogens. Because the lifetime of nitric oxide is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased as in the infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions. In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the helix-turn-helix LysRtype (YidZ). The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide is shown to cause a significant alteration of the global E. coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulfur cluster assembly systems, DNA-repairing enzymes, and stress response regulators.Macrophages are important weapons of host innate immunity, exhibiting a panoply of concerted strategies for microbial elimination. These strategies include the creation of an environment hostile to bacteria proliferation caused by, among other factors, the production of small diffusible reactive molecules such as nitric oxide (NO). 1 The NO released by eukaryotes is a product of the enzymatic oxidation of L-arginine by NO synthases, and it regulates a plethora of important processes such as signaling, neuronal communication, vasodilatation, smooth muscle relaxation, and inhibition of platelet aggregation. However, these functions are achieved by using low amounts of NO and, once the concentration of NO rises above micromolar levels, the molecule becomes harmful and causes serious deleterious effects, namely tissue inflammation, chronic infection, malignant transformations, and degenerative diseases. Nevertheless, high concentrations of NO are used to fight invading prokaryotic pathogens and parasites. The NO released by macrophages is not the only source of NO that microbes need to deal with, because this compound is also produced abiotically (e.g. by decomposition of nitrite) and biotically by denitrifiers/ammonifiers or other bacteria (1).Like all living organisms, bacteria have the ability to respond to aggression by developing a series of not yet fully understood mechanisms that include damage repair, eliciting the SOS system, resistance increase, and use of virulence as a counteracting tactic. Apart from the microbial membranebound heme-iron NO reductases, the cytoplasmatic globins, the flavodiiron NO reductases, and the multiheme nitrite reductases (2) are also proposed to metabolize NO. Escherichia coli contains these three proteins: (i) the flavorubredoxin NorV, a flavodiiron NO reductase (3), the discovery of which allowed the identification of similar enzymes in a large number of prokaryotic and protozoan genomes (4); (ii) the flavohemoglobin HmpA (5)...

Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H 2 O 2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H 2 O 2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

Our previous analysis of the transcriptome of Escherichia coli under nitrosative stress showed that the ytfE gene was one of the highest induced genes. Furthermore, the E. coli strain mutated on the ytfE gene was found to be more sensitive to nitric oxide than the wild-type strain. In the present work, we show that the mutation of the ytfE gene in E. coli yielded a strain that grows poorly under anaerobic respiratory conditions and that has an increased sensitivity to iron starvation. Furthermore, all examined iron-sulphur proteins have decreased activity levels in the strain lacking ytfE. Altogether, the results suggest a role for ytfE in iron-sulphur cluster biogenesis. YtfE was overexpressed in E. coli and it is shown to contain a di-iron centre of the histidine-carboxylate family.

DNA microarray experiments showed that the expression of the Escherichia coli ytfE gene is highly increased upon exposure to nitric oxide. We also reported that deletion of ytfE significantly alters the phenotype of E. coli, generating a strain with enhanced susceptibility to nitrosative stress and defective in the activity of several iron-sulfur-containing proteins. In this work, it is shown that the E. coli ytfE confers protection against oxidative stress. Furthermore, we found that the damage of the [4Fe-4S] 2؉ clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild type E. coli once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to the mutant cell extracts, the enzymatic activities of fumarase and aconitase are fully recovered and at rates similar to the wild type strain. We concluded that YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions. Iron-sulfur ([Fe-S]) clusters are very simple, almost ubiquitous, and evolutionary ancient prosthetic groups that are essential for the function of proteins involved in a wide range of biological processes, including electron transfer chains, redox and nonredox catalysis of reactions that underpin metabolic pathways, and gene regulation, and as environmental sensors (1-3). Although in many cases in vitro assembly of [Fe-S] clusters and incorporation into apoproteins can occur spontaneously under reducing conditions and in the presence of ferrous iron and sulfide salts, in other cases [Fe-S] cluster biogenesis requires specific accessory proteins (2, 4 -6). Furthermore, proteins that contain iron-sulfur clusters are one of the major targets of nitrosative and oxidative compounds that cause displacement of the iron atoms of the cluster and consequent malfunction of the protein/enzyme (7-10). In addition, the release of iron may further potentiate the effects of oxidative stress due to formation of the hydroxyl radical by the Fenton reaction (11). Under these conditions, enzymes of the dehydratase family that contain 2ϩ catalytic centers (e.g. aconitase and fumarase) are rapidly inactivated (12).In Escherichia coli, three different machineries for iron-sulfur cluster biosynthesis were identified: the ISC (iron sulfur cluster) operon, the SUF (sulfur assimilation) operon, and the recently discovered CSD operon (reviewed in Refs. 2, 5, and 6). These systems are widely spread in nature, with the ISC system and their homologs present in bacteria and most eukaryotes and the SUF system present in bacteria, archaea, plants, and parasites. Despite the similarity of the biochemical activity exhibited by some of the proteins encoded by the isc and suf operons, which might suggest overlapping functions, the two systems seem to be adapted for different purposes. Regulation and phenotyp...

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