Fur-like proteins: Beyond the ferric uptake regulator (Fur) paralog.

Proteins belonging to the FUR (ferric uptake regulator) family are the cornerstone of metalloregulation in most prokaryotes. Although numerous reviews have been devoted to these proteins, these reports are mainly focused on the Fur paralog that gives name to the family. In the last years, the increasing knowledge on the other, less ubiquitous members of this family has evidenced their importance in bacterial metabolism. As the Fur paralog, the major regulator of iron homeostasis, Zur, Irr, BosR and PerR are tightly related to stress defenses and host-pathogen interaction being in many cases essential for virulence. Furthermore, the Nur and Mur paralogs largely contribute to control nickel and manganese homeostasis, which are cofactors of pivotal proteins for host colonization and bacterial redox homeostasis. The present review highlights the main features of FUR proteins that differ to the canonical Fur paralog either in the coregulatory metal, such as Zur, Nur and Mur, or in the action mechanism to control target genes, such as PerR, Irr and BosR.

MRSA infections among patients in the emergency department: a European multicentre study.

BACKGROUND: MRSA is a therapeutic concern worldwide, and a major agent of community-acquired skin and soft tissue infections (CA-SSTIs). While the US epidemiology of MRSA in CA-SSTIs is well described and reports the high prevalence of the USA300 clone, data on the European situation are lacking. OBJECTIVES: To determine the prevalence and clonal characteristics of MRSA in CA-SSTIs in seven European emergency departments. PATIENTS AND METHODS: From April to June 2015, patients presenting to the tertiary hospital emergency department with a Staphylococcus aureus CA-SSTI were prospectively enrolled. S. aureus isolates were characterized by antimicrobial susceptibility testing, detection of Panton-Valentine leucocidin encoding genes and spa-typing, MLST and/or DNA microarray. RESULTS: Two-hundred and five cases of S. aureus-associated CA-SSTIs were included, comprising folliculitis, furuncles, abscesses, paronychia, impetigo, carbuncles and cellulitis. Of the 205 cases, we report an MRSA prevalence rate of 15.1%, with a north (0%) to south (29%) increasing gradient. Fifty-one isolates were Panton-Valentine leucocidin-positive (24.9%), whether MSSA or MRSA, with a heterogeneous distribution between countries. Clonal distribution of MSSA and MRSA showed high diversity, with no predominant circulating clone and no archetypical USA300 CA-MRSA clone. CONCLUSIONS: This original prospective multicentre study highlights stark differences in European MRSA epidemiology compared with the USA, and that the USA300 CA-MRSA clone is not predominant among community-infected patients in Europe.

Evaluation of the R-Biopharm RIDA®GENE Panton-Valentine leukocidin (PVL) kit for the detection of Staphylococcus aureus PVL from pus samples.

Staphylococcus aureus Panton-Valentine leukocidin (PVL) is associated with primary skin and soft-tissue infections (SSTI). We aimed to divert the RIDA®GENE PVL kit (RBiopharm) from its intended use on cultures to the detection of PVL-encoding genes directly from pus samples. Performance was compared with that of the in-house PCR method developed by the French National Reference Centre for Staphylococci. From June 2013 to May 2014, pus samples from S. aureus SSTI were tested. Our in-house PCR was performed on parallel cultures as the gold standard, while the RIDA®GENE PVL assay was used directly on pus samples from the sterile container, or a swab or an Eswab previously dipped in the pus. The kit specificity was also evaluated with pus samples that grew Streptococcus pyogenes. S. aureus reference strains harboring PVL-encoding genes, including known polymorphisms, were also tested. A total of 56 S. aureus-containing pus samples (28 PVL + and 28 PVL-) were collected and analyzed. Sensitivity and specificity of the commercial kit were 96.4 % and 100 % respectively, with equal performance whether tested directly from the sterile container or the Eswab. Sensitivity was lower (67.9 %) when the test was performed from a regular SSTI swab. None of the Streptococcus pyogenes pus samples scored positive (n = 5). Specificity was assessed using reference strains (n = 14); in all strains the PVL gene was correctly detected. This study identified the RIDA®GENE PVL kit as an efficient, sensitive, and specific tool for the rapid detection of PVL-encoding genes in pus samples.

The levels of antibodies to Panton-Valentine leukocidin (PVL) vary with PVL prevalence along a north-to-south gradient.

Recent research on Staphylococcus aureus vaccine development has focused on active immunization against Panton-Valentine leukocidin (PVL), a potent leukotoxin associated with both superficial and severe deep-seated infections. PVL prevalence is highly variable worldwide, but it is unknown to what extent immunity to PVL varies between patients from geographic areas with different PVL-positive S. aureus prevalences. We conducted a retrospective multicentric study of anti-PVL and anti-alpha-toxin (Hla) antibody levels in uninfected adult patients from France (low PVL prevalence; n = 200), Algeria (moderate prevalence; n = 143), and Senegal (high prevalence; n = 228). The antibody levels were quantified by an enzyme-linked immunosorbent assay (ELISA) procedure. Because Hla is present in virtually all S. aureus strains, its corresponding antibody levels were considered to reflect population exposure to S. aureus. Compared with French participants, the average anti-PVL antibody levels were 2.5-fold and 8.2-fold higher in Algerian and Senegalese participants, respectively (p < 0.001). Conversely, anti-Hla antibody levels did not differ between participants from the three countries, suggesting that the observed differences in anti-PVL antibody levels were not biased by variations in population exposure to S. aureus. Hence, anti-PVL antibody levels in the general populations of France, Algeria, and Senegal vary widely and match variations in PVL-positive S. aureus strain prevalence, with an increasing north-to-south gradient. To conclude, immunity to PVL in a given population correlates with local PVL prevalence. This finding can help to inform PVL vaccine strategies.

Detection of methicillin-resistant Staphylococcus aureus carrying the mecC gene in human samples in Slovenia.

SUMMARY Following the recognition of a mecC MRSA isolate from a patient hospitalized in the northeastern region of Slovenia, a national collection of 395 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates from 2006 to 2013 was screened. An additional six mecC MRSA strains were found and characterized as spa types t843, t9397 and t10009, and multilocus sequence type ST130. The low oxacillin minimum inhibitory concentrations and absence of the mecA gene make recognition of these MRSA strains problematical for diagnostic laboratories. In such strains the presence of mecC should be determined.

Community-acquired infections due to Staphylococcus argenteus lineage isolates harbouring the Panton-Valentine leucocidin, France, 2014.

We describe two cases of human infections caused by Staphylococcus aureus clonal complex (CC) 75, also called Staphylococcus argenteus, harbouring the Panton-Valentine leucocidin (PVL). These two sporadic cases were community-acquired, and identified in France in 2014. Both had an epidemiological link with Mayotte, an overseas department of France located in the Indian Ocean off the south-eastern African coast. This report illustrates that, contrary to previous descriptions, S. argenteus can acquire important virulence factors and be responsible for severe infections.

Early predictors of antiviral treatment response in liver transplant recipients with recurrent hepatitis C genotype 1.

The success of current antiviral treatment for hepatitis C virus (HCV) recurrence in liver transplant (LT) recipients remains limited. We aimed at evaluating the value of IL28B genotype and early viral kinetics to predict response to standard treatment in the transplant setting. We retrospectively evaluated 104 LT recipients treated for HCV genotype 1 recurrence between 2001 and 2010. Baseline variables, including IL28B genotype, and early viral kinetics were compared among patients who did or did not achieve a sustained virological response (SVR). Logistic regression analyses of candidate variables were conducted to generate a reliable predictive model based on the minimum set of variables. Twenty-nine (28%) achieved an SVR. On multivariate analysis, the magnitude of HCV RNA decline at 4 weeks (OR: 3.74, 95% CI: 1.64-9.39; P = 0.003) and treatment compliance (OR: 35.27, 95% CI: 3.35-365.54; P = 0.003) were the only independent predictors of SVR. Favourable recipient IL28B genotype significantly correlates with virological response at week 4 (OR 3.23; 95% CI, 1.12-9.15; P = 0.03). By logistic regression analysis, a model including donor age, recipient rs12979860 genotype and viral load at 4 weeks showed the best predictive value for SVR with an area under the receiver operating curve of 0.861. Favourable recipient IL28B genotype strongly correlates with the viral response at week 4 which is the strongest predictor of response. The combination of recipient IL28B genotype and donor age with the week 4 response reliably estimates the probability of SVR early on-treatment and may facilitate therapeutic strategies incorporating new antiviral agents.

Functional characterization of Fur from the strict anaerobe Clostridioides difficile provides insight into its redox-driven regulatory capacity.

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur-mediated mechanism independent of iron, which affects Fur DNA-binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur-regulated genes in vivo, is specific and does not require co-regulator metal under reducing conditions. Fur treatment with H2O2 produces dose-dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH-thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection.

FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in Anabaena sp. PCC 7120.

Knowledge on the regulatory mechanisms controlling iron homeostasis in cyanobacteria is limited. In Anabaena sp. PCC 7120, the ferric uptake regulator FurA is a constitutive and essential protein whose expression is induced under iron deprivation. Our previous analyses have shown that this protein acts as a global transcriptional regulator, controlling the expression of several genes belonging to different functional categories, including schT, a gene coding for a TonB-dependent schizokinen transporter. In the present study we analysed the impact of FurA overexpression and iron availability on the transcriptional modulation of a broad range of Anabaena iron uptake, transport, storage and cellular iron utilization mechanisms, including enzymes involved in siderophore biosynthesis, TonB-dependent siderophore outer membrane transporters, siderophore periplasmic binding proteins, ABC inner membrane permeases, ferritin Dps family proteins, and enzymes involved in tetrapyrrole biosynthesis. By combining reverse transcription-PCR analyses, electrophoretic mobility shift assays and DNase I footprinting experiments, we defined a variety of novel direct iron-dependent transcriptional targets of this metalloregulator, including genes encoding at least five enzymes involved in the tetrapyrrole biosynthesis pathway. The results unravel the role of FurA as the master regulator of iron homeostasis in Anabaena sp. PCC 7120, providing new insights into the Fur regulons in cyanobacteria.

Reversal of nonstructural protein 3-specific CD4(+) T cell dysfunction in patients with persistent hepatitis C virus infection.

Hepatitis C virus (HCV)-specific T cell responses are essential for HCV control, and chronic infection is characterized by functionally altered antigen-specific T cells. It has been proposed that the early inactivation of specific CD4(+) T cell responses may be involved in establishment of HCV persistence. We have investigated whether HCV-specific CD4(+) T cells dysfunction can be reversed in vitro. Nonstructural protein 3 (NS3) and core-specific CD4(+) T cells from eight chronically infected and eight spontaneously resolved HCV individuals were selected through transient CD154 (CD40 ligand) expression, and their functional profile (IFN-γ, IL-2, TNF-α, IL-10 and IL-4 production by enzyme-linked immunospot assay, cytometric bead array and intracellular cytokine staining, and proliferation by carboxy-fluorescein diacetate succinimidyl ester dilution assay) was determined both ex vivo and after in vitro expansion of sorted CD154-expressing cells in the absence of specific antigen in IL-7/IL-15-supplemented medium. Ex vivo bulk CD4(+) T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11%vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-γ. Antigen-free in vitro expansion of NS3-specific CD154(+) cells from chronic patients restored IFN-γ and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4(+) T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.

IL28B genetic variation and hepatitis C virus-specific CD4(+) T-cell responses in anti-HCV-positive blood donors.

Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome-wide association studies have shown a strong correlation between single-nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B) gene and spontaneous or treatment-induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV-specific T-cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti-HCV-positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV-specific CD4(+) T-cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon-γ ELISpot assay. The rs12979860-CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2%vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4-25.3, P < 0.001). HCV-specific CD4(+) T-cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T-cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T-cell responses were only observed among those with non-CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4(+) T-cell responses towards NS3 were only evident among those with non-CC haplotypes.

An active photosynthetic electron transfer chain required for mcyD transcription and microcystin synthesis in Microcystis aeruginosa PCC7806.

In this study, quantitative real time RT-PCR has been used to monitor changes in the levels of transcripts encoding mcyD in Microcystis aeruginosa PCC7806 under oxidative agents and different conditions of light intensity. Microcystin content has also been determined in the same stressed cell aliquots. Our results corroborate the fact that changes in light intensities are able to induce mcyD gene transcription, but our data show that this is an early and short-term event. mcyD transcription requires an active photosynthetic electron transfer chain and the increased transcript level as a consequence of light is not related to oxidative stress. Indeed, oxidative stress leads to a general trend of a decrease of mcyD trancript. Microcystin amount found in the cells follows a tendency consistent with the mcyD transcript level. In summary, the data indicate that the synthesis of microcystin is dependent on photosynthesis, and also show that oxidative stress decreases the microcystin synthesis in toxigenic Microcystis.

Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation?

Metal and redox homeostasis in cyanobacteria is tightly controlled to preserve the photosynthetic machinery from mismetallation and minimize cell damage. This control is mainly taken by FUR (ferric uptake regulation) proteins. FurC works as the PerR (peroxide response) paralog in Anabaena sp. PCC7120. Despite its importance, this regulator remained poorly characterized. Although FurC lacks the typical CXXC motifs present in FUR proteins, it contains a tightly bound zinc per subunit. FurC: Zn stoichiometrically binds zinc and manganese in a second site, manganese being more efficient in the binding of FurC: Zn to its DNA target PprxA. Oligomerization analyses of FurC: Zn evidence the occurrence of different aggregates ranging from dimers to octamers. Notably, intermolecular disulfide bonds are not involved in FurC: Zn dimerization, dimer being the most reduced form of the protein. Oligomerization of dimers occurs upon oxidation of thiols by H2O2 or diamide and can be reversed by 1,4-Dithiothreitol (DTT). Irreversible inactivation of the regulator occurs by metal catalyzed oxidation promoted by ferrous iron. However, inactivation upon oxidation with H2O2 in the absence of iron was reverted by addition of DTT. Comparison of models for FurC: Zn dimers and tetramers obtained using AlphaFold Colab and SWISS-MODEL allowed to infer the residues forming both metal-binding sites and to propose the involvement of Cys86 in reversible tetramer formation. Our results decipher the existence of two levels of inactivation of FurC: Zn of Anabaena sp. PCC7120, a reversible one through disulfide-formed FurC: Zn tetramers and the irreversible metal catalyzed oxidation. This additional reversible regulation may be specific of cyanobacteria.

Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Staphylococcal species, notably, coagulase-negative staphylococci (CoNS), are frequently misidentified using phenotypic methods. The partial nucleotide sequences of the tuf and gap genes were determined in 47 reference strains to assess their suitability, practicability, and discriminatory power as target molecules for staphylococcal identification. The partial tuf gene sequence was selected and further assessed with a collection of 186 strains, including 35 species and subspecies. Then, to evaluate the efficacy of this genotyping method versus the technology of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the 186 strains were identified using MALDI-TOF-MS (Axima® Shimadzu) coupled to the SARAMIS® database (AnagnosTec). The French National Reference Center for Staphylococci identification method was used as a reference. One hundred and eighty-four strains (98.9%) were correctly identified by tuf gene sequencing. Only one strain was misidentified and one was unidentified. MALDI-TOF-MS identified correctly 138 isolates (74.2%). Four strains were misidentified, 39 were unidentified, five were identified at the group (hominis/warneri) level, and one strain was identified at the genus level. These results confirm the value of MALDI-TOF-MS identification for common species in clinical laboratory practice and the value of the partial tuf gene sequence for the identification of all staphylococcal species as required in a reference laboratory.

Clinical manifestations and outcome of skin infections caused by the community-acquired methicillin-resistant Staphylococcus aureus clone ST80-IV.

BACKGROUND: Several Panton-Valentin leukocidin-positive clones of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are spreading worldwide. The European clone ST80-IV is the main CA-MRSA clone in Europe. There is no reported study of the specific clinical manifestations and outcome of skin infections caused by the clone ST80-IV, using strict definitions of skin diseases. METHODS: Single-centre observational prospective cohort of S. aureus skin infections caused by the clone ST80-IV. RESULTS: From November 1999 to October 2009, we diagnosed skin infections due to the clone ST80-IV in 20 patients (median age 28 years, median 27; range 1-66). All the isolates had all the following characteristics: lukPV, etd and edin gene-positive, agr 3 allele, spa-type t044 and ST80. All the isolates were resistant to beta-lactam agents, kanamycin, tetracycline and fusidic acid. During the study period, the 20 patients had the following manifestations: 19 primary abscesses (18 single abscess and one patient with two), eight furuncles, four folliculitis, one case of cellulitis, one wound infection and one felon. Surgical treatment and drainage was required for all the primary abscesses. The infections occurred mainly in the perineal area (50%). No secondary infections occurred in family members. Despite strict hygiene measures, systemic antibiotics and nasal mupirocine, four patients (20%) had recurrent skin infections over a period of a few months to 6 years. CONCLUSIONS: The CA-MRSA clone ST80-IV is responsible for suppurative skin infections such as furuncles and abscesses, which can recur over a period of several years.

[Detection of the first strain of glycopeptide intermediary Staphylococcus aureus in Tunis Rabta hospital]. / Détection de la première souche de Staphylococcus aureus de sensibilité diminuée aux glycopeptides à l'hôpital la Rabta de Tunis.

Since the advent of the first glycopeptide intermediately susceptible Staphylococcus aureus (GISA) and its heterogeneous variant hGISA in 1997, debate still ensues as their clinical significance. We report here the first case of GISA in Rabta hospital of Tunisia. Antimicrobial resistance was determined by the disk diffusion method in accordance with CA-SFM (Comity of Antibiogramm of French society of Microbiology). The MIC of vancomycin and teicoplanin was determined by E-test. The detection of mec A gene, virulence factors genes and agr groups (1-4) was performed by multiplex PCR. spa types were determined with the assistance of Ridom of Staph Type software (Ridom GmbH, Wurburg, Germany). The allelic profiles of MRSA were assigned on the basis of their MLST type using the eBURST program. A MRSA bacteraemia patient was treated with teicoplanin for 14 days. S. aureus isolated from patient's blood culture was identified as MRSA and GISA with teicoplanin MIC of 16 mg/l. The molecular study of this strain showed that it belongs to the clonal complex CC8 and is attached to the iberian clone (agr1, enterotoxin A, ST 247, spa type t052). Clinicians and laboratories alike are increasingly aware that patients on long-term vancomycin therapy may signal the presence and potential spread of hGISA/GISA strains. hGISA/GISA strains emerged from lineages with agr types I and II. The multiresitance of the Iberian clone ST247 could be explained by the presence of several resistance genes.

Repeated introduction and spread of the MRSA clone t304/ST6 in northern Europe.

OBJECTIVES: During the last decades several methicillin-resistant Staphylococcus aureus (MRSA) clones with the capability of global spread have emerged in the community. Here, we have investigated a large collection of clinical isolates belonging to MRSA clone t304/ST6, which has emerged in many European countries over the last years, in order to retrace its phylogeny and its spread. METHODS: We characterized 466 ST6 isolates from Denmark (n = 354), France (n = 10), Norway (n = 24), Sweden (n = 27) and the UK (n = 51). All had spa-type t304 (n = 454) or t304-related spa-types (n = 12) and whole genome sequencing (WGS) was carried out on Illumina Miseq or Hiseq with 100-300 bp reads. cgMLST was performed using Ridom SeqSphere. RESULTS: A minimum spanning tree (MST) of all 466 isolates showed one large cluster including 182 isolates collected only from Denmark and related to a long-term neonatal outbreak in Copenhagen. This cluster contrasted with numerous small clusters, including the remaining Danish isolates and isolates from the other countries that interspersed throughout the tree. Most isolates were Panton-Valentine leukocidin (PVL) negative (95%) and harboured SCCmec IVa. One genome was closed using Oxford Nanopore technology and Illumina MiSeq. It contained a plasmid of 19.769 bp including the blaZ gene. A similar plasmid was found in 78% of all isolates. DISCUSSION: t304/ST6 is a successful emerging clone and the fact that isolates from five countries are interspersed throughout the MST indicates a common origin. This clone is commonly described in the Middle East and its emergence in Europe coincides with influx of refugees from the Syrian Civil War.

Overexpression of FurA in Anabaena sp. PCC 7120 reveals new targets for this regulator involved in photosynthesis, iron uptake and cellular morphology.

Previous genomic analyses of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 have identified three ferric uptake regulator (Fur) homologs with low sequence identities and probably different functions in the cell. FurA is a constitutive protein that shares the highest homology with Fur from heterotrophic bacteria and appears to be essential for in vitro growth. In this study, we have analysed the effects of FurA overexpression on the Anabaena sp. phenotype and investigated which of the observed alterations were directly operated by FurA. Overexpression of the regulator led to changes in cellular morphology, resulting in shorter filaments with rounded cells of different sizes. The furA-overexpressing strain showed a slower photoautotrophic growth and a marked decrease in the oxygen evolution rate. Overexpression of the regulator also decreased both catalase and superoxide dismutase activities, but did not lead to an increase in the levels of intracellular reactive oxygen species. By combining phenotypic studies, reverse transcription-PCR analyses and electrophoretic mobility shift assays, we identified three novel direct targets of FurA, including genes encoding a siderophore outer membrane transporter (schT), bacterial actins (mreBCD) and the PSII reaction center protein D1 (psbA). The affinity of FurA for these novel targets was markedly affected by the absence of divalent metal ions, confirming previous evidence of a critical role for the metal co-repressor in the function of the regulator in vivo. The results unravel new cellular processes modulated by FurA, supporting its role as a global transcriptional regulator in Anabaena sp. PCC 7120.

New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress.

Fur (ferric uptake regulator) is a prokaryotic transcriptional regulator that controls a large number of genes mainly related to iron metabolism. Several Fur homologues with different physiological roles are frequently found in the same organism. The genome of the filamentous cyanobacterium Anabaena (Nostoc) sp. PCC 7120 codes for three different fur genes. FurA is an essential protein involved in iron homoeostasis that also modulates dinitrogen fixation. FurA interacts with haem, impairing its DNA-binding ability. To explore functional differences between Fur homologues in Anabaena, factors affecting their regulation, as well as some biochemical characteristics, have been investigated. Although incubation of FurB with haem severely hinders its ability to interact with DNA, binding of haem to FurC could not be detected. Oxidative stress enhances the transcription of the three fur genes, especially that of furB and furC. In addition, overexpression of FurA and FurB in Escherichia coli increases survival when the cells are challenged with H(2)O(2) or Methyl Viologen (paraquat), a superoxide-anion-generating reagent. When present in saturating concentrations, FurB exhibits unspecific DNA-binding activity and protects DNA from cleavage produced by hydroxyl radicals or DNaseI. On the basis of these results, we suggest that, whereas at low concentrations FurB would act as a member of the Fur family, at saturating concentrations FurB protects DNA, showing a DNA-protection-during-starvation-like behaviour.