In E. coli, KsgA serves as a gate-keeper to prevent improperly assembled pre-30S subunits from entering the translation Apoptosis Compound Library molecular weight cycle [3]. Under normal conditions, KsgA only provides modest benefit to 30S maturation and function. However, KsgA’s importance becomes clear under stress conditions, such as growth at cold temperature. In this

work, we sought to define the importance of KsgA to the survivability of the human pathogen S. aureus and to compare our results to those in the model organism E. coli. Somewhat surprisingly, we found that S. aureus has a lesser reliance on KsgA under the conditions tested. In E. coli, overexpression of KsgA rescued the cold-sensitive phenotype of ΔksgA cells at low temperature but was deleterious for cell growth at 37°C in both

knockout and parental cells. Overexpression of a catalytically inactive mutant of KsgA, E66A, was deleterious in both strains at both temperatures, even in the presence of endogenous CA3 chemical structure WT protein [3]. We showed that in S. aureus the ksgA knock-out strain displayed a slow growth phenotype at low temperature when compared to the parental strain, similar to results in E. coli. However, unlike in E. coli, catalytic inactivation of KsgA’s enzymatic function has only mild phenotypic effects, and these effects are not dominant in the presence of WT KsgA. It is noteworthy that the negative growth effect was seen at 37°C but not at 25°C. This result was unexpected, both because ksgA knockout led to cold sensitivity and because negative effects in E. coli were exacerbated at low temperature; however, it is possible that growth at the lower temperature results in lower expression of the mutant protein and therefore a CX-5461 clinical trial smaller negative effect. Ribonucleotide reductase In S.

aureus, KsgA also appears to be less critical for the assembly of mature ribosomes. Experiments in E. coli showed that loss or inactivation of KsgA had obvious effects on ribosome biogenesis even under conditions where a growth phenotype was not apparent [3]. In other words, ribosome biogenesis is sensitive to disruptions in KsgA function that don’t affect overall cell growth. We did not see this effect in S. aureus; knockout or inactivation of KsgA resulted in, at most, slight disruption of polysome profiles even under conditions where cell growth was slowed. On the basis of the data presented here, it would appear that in S. aureus KsgA holds less promise as a drug target than in E. coli. However, we did observe that knockout of ksgA rendered S. aureus marginally more sensitive to clinically used aminoglycoside antibiotics, similar to results seen in E. coli.

TPC runs were made with a PID-regulated tubular oven, into which a U-tube quartz reactor with the catalytic bed had been inserted. The temperature rose till 750°C at 5°C/minute, while 100 ml/min of 10% O2 (obtained by dilution of air with N2) was made to flow through a fixed bed of 5 mg of Printex-U synthetic soot (Degussa, Essen, Germany), 45 mg of catalyst and 200 mg of silica, according to the standard operating procedure described in [11], with the only difference being an increased amount Akt inhibitor of silica in the catalytic bed, to achieve a better temperature homogeneity. The

CO/CO2 concentration in the outlet gas was measured via NDIR analyzers (by ABB). Each test was repeated three times to ensure reproducibility of the obtained results. The peak temperature, T p, in the TPC plot of the outlet CO2 concentration was taken as an index of the catalytic activity. The onset (T 10%) combustion temperature, defined as the temperature at which 10% of the initial soot is OSI-027 concentration converted, was also considered in order to better discriminate

between the intrinsic catalytic activities of the prepared catalysts. The half conversion temperature (T 50%) was also taken into account. The onset temperature is important to rank the catalysts, according to the selleckchem catalytic reaction; other phenomena (such as mass transfer or diffusion limitations) may in fact influence the performances of catalysts at higher Digestive enzyme conversion stages. The modification to the inert silica content in the bed composition led to slightly different oxidation temperatures for the materials tested in [11], especially as far as the onset temperature was concerned. In fact, the higher dilution heat capacity of the here adopted silica bed was relevant, especially at the reaction onset, i.e. when the heat released by soot oxidation was not able to self-sustain the reaction, and therefore had most impact on the reaction rate itself. However, the catalyst ranking in loose and tight contact conditions obtained in [11] has here been confirmed, and it has been shown that the SA stars offer a major improvement over the other ceria morphologies

developed in this work. Results and discussion Characterization The SEM analysis revealed the achievement of the desired morphologies sought for ceria. Figure  1 depicts the nanofiber ceria morphology, which shows a filamentous shape of the obtained structures, and a high aspect ratio, as already found in [9, 11]. The three-dimensional network that is formed by the fibers has a high open porosity and is able to effectively come into contact with the soot particles in large number of points. Figure  2 reports the morphology of the nanopowders obtained by means of the SCS technique, which shows the rather uncontrolled shape of these catalysts. In this case, the aspect ratio is much smaller, and thus the maximum soot coverage of the particle, based on the catalyst weight, is lower.

Colonies were counted after 48 h incubation at 30°C. No further colonies appeared after that incubation period. Sensitivity to acetic acid Dropout tests were performed from mid-exponential YNB cultures containing approximately 1 × 106

cells/ml. Ten-fold serial dilutions were made, and 5 μl AP26113 mw of each suspension was applied on YNB medium BMN 673 in vivo supplemented with different acetic acid concentrations (50, 80 and 100 mM). Results were scored after 48 h incubation at 30°C. Acetic acid treatment Yeast strains were grown until exponential phase (OD600 = 0.5–0.6) on YNB medium. Then the cultures were collected and resuspended to a final concentration of 107 cells ml-1 in fresh YNB adjusted to pH 3.0 with HCl and containing 160 mM acetic acid. Incubation took place for 180 min at 30°C as previously

described [4, 72]. At determined time points, 40 μl from a 10−4 cell suspension were inoculated onto YPD agar plates and c.f.u. were counted after 48 h incubation at 30°C. The percentage of viable C646 purchase cells was estimated considering 100% survival the number of c.f.u. obtained in time 0. Apoptotic markers PI, Annexin V, DAPI and DiOC6 staining were performed both in cells treated with acetic acid and in aging cells as previously described, with some modifications [1, 3, 4, 37]. Membrane integrity was assessed by PI (Propidium Iodide) staining. Cells were harvested, washed and resuspended in PBS (137 mM NaCl; 2.7 mM KCl; 100 mM Na2HPO4; 2 mM KH2PO4; pH 7.4) containing PI (4 μg/ml) (Sigma). The samples were incubated for 10 min at room temperature in the dark and analyzed in an Epics® XL™ (Beckman Coulter) flow cytometer. At least 20,000 cells from each sample were analyzed. Phosphatidylserine exposure was detected by an FITC-coupled Annexin V reaction with the ApoAlertAnnexin V Apoptosis Kit (CLONTECH Laboratories). For that, cells were primarily harvested and washed in digesting

buffer (1.2 M sorbitol; 0.5 mM MgCl2; 35 mM K2HPO4; pH 6.8). To promote the drug course through cell wall, an incubation step with Zymolyase (20 T) Rutecarpine at 30°C was performed. Phase-contrast microscopy was used to monitor that step, preventing this way damage to the unfixed spheroplasts. Cells were subsequently centrifuged (10 min at 1500 rpm) and resuspended in 200 μl of binding buffer (1.2 M sorbitol; 10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM Cacl2). To 40 μl of this cell suspension, 2 μl Annexin V (1 μg/ml) and 1 μl PI (4 μg/ml) were added, and the mixture incubated for 20 min at room temperature in the dark. Finally, extra 400 μl of binding buffer were added to the mixture just prior to analysis in an Epics® XL™ (Beckman Coulter) flow cytometer. At least 20,000 cells from each sample were analyzed. For evaluation of mitochondrial potential the probe DiOC6 (3,3′dihexyloxacarbocyanine iodide) (Invitrogen) was used. Cells were harvested, washed, and resuspended in DiOC6 buffer (10 mM MES; 0.

Several studies have reported the usefulness of phage-display applications for mapping epitopes of flaviviruses [[22–25]]. The aim of our study was to identify WNV-specific and/or JEV serocomplex-specific B-cell epitopes in NS1 using phage display technology. The information provided by this study will facilitate the development of diagnostic tools for the specific serological diagnosis of WNV infection, and will contribute to selleck chemicals the rational design of vaccines by furthering understanding

of the antigenic structure of NS1. Results Production of recombinant NS1 Recombinant WNV NS1 was successfully expressed in E. coli TB1 cells, predominantly as soluble protein, after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was recognized by DAPT WNV-positive equine serum in Western blot (WB) (Figure 1, lane 1). Figure 1 WNV-positive equine sera PRIMA-1MET in vitro recognize recombinant NS1. Binding of antibodies from WNV-positive equine serum

to recombinant NS1 (lane 1) and MBP-tag (lane 2) by Western blot. M, PageRuler™ Prestained Protein Ladder (Fermentas, Canada). Production and characterization of NS1-specific mAbs Purified protein was used to immunize BALB/c mice. After cell fusion and screening, several hybridoma cell lines were obtained which produced NS1-specific mAbs. Among them two cell lines were selected for their strongest reactivity against recombinant NS1 using indirect ELISA (data not shown), WB (Figure 2a), and against native NS1 in IFA using WNV antigen slides (Figure 2b). Further characterization of the specificity of the two mAbs by IFA, demonstrated that the mAb 3C7 reacted with WNV, but did not react with JEV, DENV1-4, Yellow fever virus (YFV) and Tick-borne encephalitis virus (TBEV), whereas mAb 4D1 reacted with both WNV and JEV, but did not react

with other non-JEV serocomplex flaviviruses (Figure 2b). Figure 2 Reactivity of mAbs with recombinant NS1 and C6/36 cells infected with flaviviruses. (a) Western blot analysis of mAbs 3C7 (lanes 1, 2) and 4D1 (lanes 3, 4) against recombination NS1 (lane 1, 3) and MBP-tag (lane 2, 4). M, PageRuler™ Prestained Protein Ladder (Fermentas, Canada). (b) Pattern of immunofluorescence Thalidomide produced by anti-NS1 mAbs on antigen slides which were prepared on porous slides using C6/36 cells infected with different flaviviruses. Panels 1-8: reactivity of mAb 3C7 with cells infected with WNV (panel 1), JEV (panel 2), DENV1 (panel 3), DENV2 (panel 4), DENV3 (panel 5), DENV4 (panel 6), YFV (panel 7), and TBEV (panel 8). Panels 11-18: reactivity of mAb 4D1 with cells infected with WNV (panel 11), JEV (panel 12), DENV1 (panel 13), DENV2 (panel 14), DENV3 (panel 15), DENV4 (panel 16), YFV (panel 17), and TBEV (panel 18).

20 μM phospholipid substrates (10 μl) were reacted with an equal volume (10 μl) of various samples, and incubated at different conditions, as described for each experiment. For some experiments,

purified standard phospholipases were used: PLA2 (Sigma) from porcine pancreas, PLC (Sigma) from Clostridium perfringens, and PLD (Sigma) from cabbage. The reaction this website products were analyzed by thin-layer chromatography (TLC). Briefly, 20 μl of 1-butanol was added to the above reaction mixes (20 μl), followed by vigorous vortex mixing for 30 s and centrifugation (10,000 × g, 1 min). The upper lipid extract layer (5 μl) was CCI-779 solubility dmso loaded onto a plastic-backed silica gel G60 plate without fluorescent indicator (Sigma) and air-dried for 20 min. TLC Tariquidar was performed either with chloroform-methanol–water-acetic acid (45/45/10/1 by vol.) when BODIPY-PC was used as the substrate, or with chloroform-methanol-acetic acid (60/20/1 by vol.) when NBD-PE, NBD-PS, or NBD-SM used

as the substrates. For some experiments, an apolar solvent (n-hexane (70): diethyl ether (30): acetic aid (4)) was used. Fluorescence was detected and quantified using a Typhoon 9410 laser scanner. Subcellular fractionation V. anguillarum cells were fractionated as described previously [6] and the subcellular location of Plp determined. Briefly, 100 ml NSS-washed overnight grown bacterial cells were resuspended in 10 ml of ultrapure water for 20 min to cause osmotic shock and centrifuged (10,000 × g, 5°C, 10 min) to collect the periplasmic fraction (the supernatant). The remaining pellets were washed twice with ultrapure water and lysed by sonication (four cycles at 35% power for Idelalisib 20 s, then allowed to cool for 1 min). The sonicated cells were centrifuged (10,000 × g, 5°C, 20 min) to remove cell debris and any unlysed cells, and the supernatant cell lysate was separated by ultracentrifugation (200,000 × g, 1 h, 4°C) to yield the cytosolic (supernatant) and membrane (pellet) fractions. The membrane fraction was treated with 1% Sarkosyl to obtain Sarkosyl-soluble (inner

membrane) and -insoluble (outer membrane) fractions. Protein concentration in various fractions was measured using BCA protein determination kit (Pierce). Preparation of polyclonal antibody Truncated Plp protein was over-expressed and purified to serve as the antigen to create polyclonal antibody against Plp. Briefly, primer Pm212 and Pm213 (listed in Table 3) were used to amplify central portion of the plp gene, which encodes the truncated Plp protein (amino acid 93 to 293). PCR product was ligated into pQE30UA vector (QIAGEN), and transformed into E. coli M15 and transformants were selected on LB10 agar containing kanamycin and ampicillin. Plasmid DNA was purified and the sequence confirmed by DNA sequencing. Protein purification was performed under denaturing conditions according to the instructions of the manufacturer (QIAGEN, USA) and protein purity was determined by SDS-PAGE and Coomassie blue staining.

It is found in both developed and developing parts of the world [1, 2]. Clinical illness ranges from mild self-limiting, non-inflammatory diarrhea to severe inflammatory bloody diarrhoea that may be associated with pyrexia and bacteriaemia [1]. In addition, Campylobacter

enteritis has been associated with subsequent development of Guillain Barré syndrome, an acute inflammatory polyneuropathy [3]. Although various virulence factors such as adherence and invasive abilities and toxin production and motility have been implicated [4–8], the precise mechanism(s) involved in the pathogenesis is yet to be CP673451 mouse elucidated. The pathogenesis of C. jejuni is poorly understood, partly because of the lack of a suitable animal model and partly due to the difficulties in genetic manipulation [9]. Bacterial toxins have been considered important factors for the pathogenesis of Campylobacter infection. The best GSK2126458 characterized toxin of Campylobacter spp. is the cytolethal distending toxin (CDT). The C. jejuni cdt operon

consists of three adjacent genes, cdtA, cdtB and cdtC, that encode proteins with predicted molecular masses of 27, 29 and 20 kDa, respectively [10]. The effect of CDT was first described as an activity in culture supernatants of Campylobacter spp. and of certain enteropathogenic strains of Escherichia coli that caused eukaryotic cells to slowly distend over a period of 2-5 days, eventually leading to cell death [11]. CDT appears to be common in C. jejuni strains e.g. in one study of 117 isolates there was positive

Selumetinib nmr evidence for CDT in 114 of the isolates in Vero cell assays [12]. A study in Bahrain showed that among the 96 C. jejuni strains examined, 80 (83.0%) were cdtB positive and 16 (17.0%) were negative by PCR [13]. Recently, Jain et al described that the presence of the cdtB gene in C. jejuni was associated with increased adherence to, invasion of and cytotoxicity towards HeLa cells [14]. The significant pathological changes in the colons of mice treated with the supernatant containing C. jejuni CDT suggested that CDT is an important virulence attribute and that the colon is the major target of CDT. CDT belongs ID-8 to a family of bacterial protein toxins that affects the epithelial cell layer and interrupts the cell division process with resulting cell cycle arrest and cell death [10, 15]. CDT activity is not unique to E. coli and Campylobacter spp. but has been described in various other Gram-negative bacteria including Shigella spp., Helicobacter hepaticus, Haemophilus ducreyi, and Actinobacillus actinomycetemcomitans. [16]. It has been suggested that CDT is a tripartite “”AB2″” toxin in which CdtB is the active toxic unit; CdtA and CdtC make up the “”B2″” units required for CDT binding to target cells and for delivery of CdtB into the cell interior [17].

0) and growth arrest at the OD600 of ~0.8 for iron-deficient conditions. Methods Bacterial strains

and culture conditions The Y. pestis strain KIM6+ used in this study is an avirulent derivative of the fully virulent KIM strain, which was cured of the pCD1 plasmid but retained the chromosomal pgm locus and the plasmids pMT1 and pPCP1 [36]. We used strain maintenance and cell growth procedures and verified the presence of the pgm locus on Congo Red agar as described previously [37]. Bacterial colonies were grown on tryptose blood agar at 30°C, Selleckchem TPCA-1 harvested after 48 h and stored at -80°C. Aliquots of these cell stocks were used to grow 5-10 mL cultures in chemically defined PMH2 medium [14] supplemented with 10 μM FeCl3, followed by dilution Selleck RO4929097 to an OD600 of ~0.05 with 0.3-1 L of PMH2. PMH2 was deferrated by incubation with Chelex-100 resin overnight at 4°C [14]. Two passages of cell

stocks in 10-30 mL of this medium were followed by dilution to an OD600 of ~0.05 with 0.3-1 L of deferrated PMH2. Overnight cell cultures (13-15 h) reached OD600s of ca. 1.8-2.5 and 0.6-0.9 for iron-rich and iron-deficient cells, respectively. Chelex-100 treatment was previously shown to reduce contaminating C188-9 chemical structure iron levels to 0.2-0.3 μM, and replenishment of this medium with 10 μM FeCl3 resulted in full recovery of the normal Y. pestis growth rate and yield. Chelex-100 treatment likely removes some other metal ions as well. However, in contrast to iron, addition of Mn, Zn

and Cu did not enhance the observed growth rate or yield. Cell pellets were harvested by centrifugation at 8,000 × g for 15 min at 4°C and washed with ca. 30 volumes of 33 mM K2HPO4 (pH 7.5). Subcellular fractionation of Y. pestis cells K2HPO4-washed Y. pestis cells were subjected to a lysozyme/EDTA spheroplasting method, followed by lysis of spheroplasts via sonication in a hypotonic buffer as previously described [38, 39]. Soluble periplasmic and cytoplasmic fractions were exchanged into buffer A (25 mM NH4HCO3, 1 mM Na-EDTA and 1 mM benzamidine) and concentrated to 2-5 mg/mL protein at 3,000 × g using membrane filtration units (NMWL ~10,000). Protein concentrations were measured with the bicinchoninic acid assay, unless stated otherwise. Mixed membrane pellets were isolated Adenosine from spheroplast lysates by centrifugation at 50,000 × g for 1 h at 4°C. These pellets were homogenized in 0.25 M sucrose, 150 mM NaCl, 10 mM Tris-OAc, pH 7.8, 5 mM Na-EDTA, 0.2 mM DTT, 10 μg/ml Leupeptin, 5 μg/ml Pepstatin, 10 μg/ml Nα-p-Tosyl-L-arginine methyl ester and 2 mM PMSF (ca. 10 mL/g pellet weight), and washed to remove most soluble protein contaminants. Sodium bromide (2.5 M final concentration) was added to the suspended membrane pellet, stirred for 1 h at 20°C and centrifuged at 50,000 × g for 1 h at 4°C. Insoluble pellets were then extracted with an ice-cold solution of 0.18 M Na2CO3, pH 11.

It has been recently estimated to be 37.1 per 100 000 population [1]. Furthermore, road traffic collisions (RTC) account for more than 75% of unintentional injury deaths in the UAE [2]. The behavior of drivers and compliance with safety measures in the UAE are completely different from those in developed countries [3, 4]. In a recent report; only 25% of drivers who were involved in RTC used seatbelts [4]. We have recently

shown that severity of head injury was the most significant factor affecting mortality in patients involved with RTC in our community indicating low compliance CUDC-907 datasheet with use of seatbelts [5]. Hypotension on arrival was another significant factor affecting RTC mortality [5]. Vascular injuries can be life-threatening and their prompt diagnosis is essential for favorite outcome. The incidence, detailed mechanism, and nature of vascular injuries following road SGC-CBP30 manufacturer traffic collisions including

their anatomical distribution are not well studied in the Middle East. We aimed to prospectively study the incidence, detailed mechanism and anatomical distribution of hospitalized vascular trauma patients following road traffic collisions in a high-income developing country. Patients and methods Data from the RTC Injury Registry of Al-Ain City were collected prospectively from April 2006 to October 2007. The registry involved the two main hospitals in the city (Tawam and Al-Ain Hospitals). Al-Ain City, which is the largest city in the Eastern District of Abu-Dhabi and one of the four largest in the country, had a population of 463,000 inhabitants at the time of the study [6]. The Local Ethics Committee of Al-Ain Health District Area has approved data collection for all road traffic collision trauma patients who were Pregnenolone admitted to Al-Ain and Tawam Hospitals or who have died in the Emergency Department. The data collected included the patient’s age,

gender and other personal details. In Selleckchem MDV3100 addition it included the type of vehicle (s) involved, the exact mechanism of crash, the use of safety measures, vascular injuries, other injuries, the Injury Severity Score (ISS), the procedures required and the final outcome. The ISS was used as a global measure of injury severity. ISS was calculated manually using the Abbreviated Injury Scale handbook [7, 8]. A web-based database was used to enter the data. Data were analyzed with the Statistical Package for the Social Sciences (version 15, SPSS Inc.). Univariate analysis to compare patients with vascular injuries and those without them was done using Mann-Whitney U test for continuous or ordinal data and Fisher’s exact test for categorical data. Patients who died were excluded when total hospital stay was calculated. Statistical significance was set at 0.05. Results Out of the 1008 patients who were studied, there were 13 patients with vascular injuries (1.29%). The median age was 26 years (range 2-45). There were 12 males and one female.

The results of this earlier study were confirmed in a large, pivotal, multicenter, randomized, placebo-controlled study of GXR adjunctive to psychostimulants [15]. Despite these earlier investigations, the potential for pharmacokinetic

drug–drug interactions (DDIs) between GXR and LDX has not been thoroughly MGCD0103 ic50 evaluated. Pharmacokinetic DDIs can occur when two see more medications are coadministered, resulting in a change in the metabolism, absorption, tissue and/or plasma binding, distribution, or elimination of one or both medications [16]. Although guanfacine is known to be metabolized by cytochrome P450 (CYP) 3A4 [5], LDX is absorbed as the intact prodrug and is converted via enzymatic hydrolysis to l-lysine and therapeutically active d-amphetamine primarily in the blood by red blood cells [17]. Although intact LDX is not metabolized by the CYP system and is neither an inducer nor an inhibitor of the system, the metabolism of d-amphetamine has not been fully characterized [13, 18]. It is therefore prudent to study the pharmacokinetics of GXR coadministered with LDX to confirm the lack of metabolic interactions between these two therapies. Although there is a lack of pharmacokinetic LY3023414 price data on coadministration

of GXR and LDX, pharmacokinetic studies of each medication administered alone have been published [19–24]. An open-label, dose-escalation, pharmacokinetic study of GXR in children (aged 6–12 years) and adolescents (aged 13–17 years) with ADHD showed that GXR exhibits a linear pharmacokinetic profile [19]. A linear pharmacokinetic profile of GXR was also observed in an open-label crossover study examining single doses of GXR 1-, 2-, and 4-mg tablets in healthy adults aged 18–55 years [20]. Maximum guanfacine concentrations of 0.98, 1.57, and 3.58 ng/mL were attained at 6 h for the 1- and 2-mg doses and very at 5.5 h for

4-mg doses. When administered alone, LDX has demonstrated a linear dose-proportional pharmacokinetic profile in both children and adults [21, 22]. Maximum mean d-amphetamine concentrations of 53.2, 93.3, and 134 ng/mL were attained in children with ADHD at 3.5 h for the 30-, 50-, and 70-mg doses, respectively [21]. In healthy adults, maximum mean d-amphetamine concentrations of 44.6, 84.6, and 126.6 ng/mL were attained at 4 h for the 50-, 100-, and 150-mg doses. For the 200- and 250-mg doses, maximum mean concentrations of 168.8 and 246.3 ng/mL, respectively, were attained at 6 h [22]. Two studies that assessed the pharmacokinetics of LDX 70 mg in healthy adults found maximum mean d-amphetamine concentrations of 80.3 and 90.1 ng/mL at 3 h [23, 24]. The safety profiles of GXR and LDX have been examined in previous studies.

Discussion This work has shown that the Fnr protein of B. cereus is homodimeric and can bind one [4Fe-4 S] MLN2238 iron-sulfur cluster per monomer. Our first challenge was to accurately assemble the Fe-S cluster via an enzymatic system since all our attempts to purify holoFnr under anaerobiosis failed. We demonstrated that CsdA from E. coli was capable of assembling the B. cereus Fnr Fe-S cluster. Interestingly, B. cereus synthesizes [13]one pyridoxal 5-phosphate-containing enzyme (NP_834652) [13] that might be involved in Fe-S cluster biogenesis. When anaerobically reconstituted B. cereus Fnr was exposed to O2, we observed

a rapid loss of the Fe-S cluster, demonstrating that Fnr functions as an oxygen sensor via its Fe-S cluster. Importantly, the cluster of the reconstituted B. cereus Fnr appeared extremely unstable, judging from its fast destruction on exposure to air. In this respect, the B. subtilis holoFnr, which is the closest homolog of B. cereus Fnr [14] displayed greater stability [8]. Sequence comparison of the B. cereus and B. subtilis Fnr revealed a significant variation in the amino acid

residues around the three C-terminal cysteine residues (C219-X 2-C222-X4-C227) that serve as ligands for the cluster (Additional file 3) [7]. These observations GS-4997 order imply that the occurrence of certain amino acid residues close to the cluster ligands may affect the stability of the B. cereus holoFnr, thus providing a possible explanation for its high susceptibility to oxygen damage [15]. As a result, B. cereus Fnr might sense subtle changes in the redox status of the cells, a property that would reflect an adaptation of the pathogenic strain to the environment of its

human host. We proposed previously that B. cereus apoFnr binds promoter regions of enterotoxins only through the monomer pathway. In other words, we proposed that apoFnr was active as a DNA-binding protein only under its monomeric form [9]. Here we showed that, when produced in a GSK2399872A in vitro tag-less form, apoFnr is active as a DNA binding protein under its dimeric form. In addition, Selleck CHIR99021 we showed that dimeric apoFnr-DNA complexes were stable in contrast to what we observed previously [9]. We conclude that (i) in our previous studies, tags fused at the N-terminus and C-terminus of Fnr introduced steric hindrance that affected its oligomeric structure and/or DNA binding activity and (ii) B. cereus apoFnr may bind DNA both through the dimer and the monomer pathway under aerobiosis unlike its homologues of B. subtilis and E. coli[8]. There are probably many variables affecting the choice for a monomer or dimer recognition pathway in vivo. Among them, there is the redox state of the cell that may impact directly the ratio of monomeric to dimeric apoFnr since we observed that the addition of reductant (DTT) affected the dimerization state of apoFnr in solution.