RNA was pelleted at 16 000 g for 20 min at 4 °C, washed once with 1 mL of 70% ethanol, repelleted and briefly air-dried before being resuspended in 100 μL of RNase-free water. The

resuspended RNA was then further purified using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The pure RNA was stored at −80 °C. RNA was DNase treated using the Ambion turbo-free DNA kit according to the manufacturer’s instructions. cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems). A total of ∼1.2−1.5 μg of RNA was used in a 20-μL reaction in all cases. cDNA was synthesized using a PCR cycle of 25 °C for 10 min, XL184 mw 37 °C for 120 min and 85 °C for 5 s. qRT-PCR was performed using the custom-made Taqman gene expression assays (Applied Biosystems). A total of 60 ng of cDNA was used in each 20 μL reaction. Reactions were performed in 20 μL containing 10 μL 2 × Taqman gene expression Mastermix (Applied Biosystems),

1 μL Taqman gene expression assay (Applied Biosystems) and 9 μL cDNA (60 ng). The real-time PCR cycle was carried out in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) (50 °C for 2 min, 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min). The fold change in the expression levels of each of the genes was calculated using the ΔΔCt method (Livak & Schmittgen, 2001). RNA was extracted from mid-log cultures of M. smegmatis as described above, and the 5′RACE system for the rapid amplification of cDNA ends check details (Version 2.0, Invitrogen) was used according to the manufacturer’s instructions, using the primers cpn60.1 gsp1, cpn60.1 gsp2 and cpn10 gsp2. cDNA was tailed at the 5′ ends using poly-cytosine and transcriptional start sites were identified by detection

of the junction of this poly-C tail in the sequenced cDNA. The promoterless lacZ E. coli–Mycobacterium shuttle vector pSD5B was used to analyse promoter activity (Jain et al., Tau-protein kinase 1997). Fragments of varying lengths upstream of the cpn60 or cpn10 genes were amplified with primers containing XbaI and SphI sites, or XbaI sites alone. The products were digested as appropriate and ligated into plasmid pSD5B. The resultant recombinant plasmids contained the various promoter regions just upstream of the lacZ gene (Table 1 and Fig. 1). Each of the pSD5B constructs containing a promoter region was electroporated into M. smegmatis mc2155 cells. The strains were grown in liquid media at 37 °C for 2 days, after which their absorbance at OD600 nm was measured. Each culture (100 μL) was added to 900 μL Z buffer (30 °C). A drop each of 0.1% sodium dodecyl sulphate and chloroform was then added to the tubes, which were vortexed to lyse the cells. The reaction was started by adding 200 μL ONPG (4 mg mL−1) and mixing well. When a significant yellow colour developed, the reaction was stopped by addition of 500 μL 0.

A minimum of 6 months is recommended between the third and fourth doses, the latter administered after the age of 4 years this website for optimal response. Regardless of the number of doses received, vaccine protection against polio decreases over time. Polio serology would be useful in guiding the need for booster doses, but as tests are not routinely available a reinforcing dose of IPV is recommended empirically for adolescents, especially before travel to endemic countries if the last dose was administered more than 10 years before. Recent guidance on immunizations for HIV-infected adults does not advise repeated boosters into adulthood [57]. A low prevalence

of measles, mumps and rubella infections is no longer assured even in developed countries, and recent outbreaks in nonimmune healthy children have been reported in a number of European countries [58-60]. HIV-positive children are susceptible to serious disease, so their immunity should be optimized. MMR vaccines contain live attenuated strains of the three viruses. There is good evidence of safety for measles-containing vaccines in children without severe immunocompromise [13], as summarized by the Global Advisory Committee

of the WHO in 2009 [61]. Those who are severely immunocompromised would probably derive little benefit from vaccination. Therefore, recommended CD4 cell count thresholds for MMR administration should be observed [62, 63] (Table 1). Most children receive MMR at 12–18 months of age http://www.selleckchem.com/products/Y-27632.html and a second dose after an interval ranging from 1 month to several years, based on national recommendations. However, there appears to be a marked decay in specific antibodies, even in children receiving effective HAART. In a study of children immunized before the initiation of HAART [33], fewer

than half (24 of 59) had antibodies against all three vaccine components. Individually, rubella antibodies were best preserved (89%) and measles antibodies least well preserved (60%), indicating impaired primary responses to vaccines. By comparison, in a study of 19 children on HAART, 79% of whom had achieved full virological suppression, only one had detectable measles antibody after routine MMR vaccination, but 15 Forskolin ic50 of the remaining 18 seroconverted after receiving a booster dose, the majority remaining seropositive at 1 year post-revaccination [54]. The majority of children on effective HAART are likely to be able to develop protective antibodies on revaccination [31, 64]. Measles and rubella antibodies should be measured routinely (this is not recommended for mumps because the assays available are poor), and if the patient is seronegative for any component, MMR revaccination should be encouraged, ideally following immune reconstitution on HAART. Frequency of testing is difficult to stipulate because of the lack of data; 3–5-yearly testing is advised empirically and annual consideration is encouraged where affordable.

Our aim was to study the role of

bacterial phosphatidylcholine in the Bradyrhizobium–peanut (Arachis hypogaea) symbiosis. Phospholipid N-methyltransferase (Pmt) and minor phosphatidylcholine synthase (Pcs) activities were detected in crude extracts of the peanut-nodulating strain Bradyrhizobium selleck inhibitor sp. SEMIA 6144. Our results suggest that phosphatidylcholine formation in Bradyrhizobium sp. SEMIA 6144 is mainly due to the phospholipid methylation pathway. Southern blot analysis using pmt- and pcs-probes of B. japonicum USDA 110 revealed a pcs and multiple pmt homologues in Bradyrhizobium sp. SEMIA 6144. A pmtA knockout mutant was constructed in Bradyrhizobium sp. SEMIA 6144 that showed a 50% decrease in the phosphatidylcholine content in comparison with the wild-type strain. The mutant was severely affected in motility and cell size, but formed wild-type-like nodules on its host plant. However, in coinoculation experiments, the pmtA-deficient mutant was less competitive than the wild type, suggesting that wild-type levels of phosphatidylcholine are required for full competitivity of Bradyrhizobium in symbiosis with learn more peanut

plants. Peanut (Arachis hypogaea L.) is an agriculturally valuable plant originally coming from South America and later disseminated to the rest of the world. China leads in the production of peanuts, having a share of about 37.5% of the overall world production, followed by India (19%) and Nigeria (11%). The United States of America, Argentina, Brazil, Mexico and Nicaragua are the major producers in the Americas (FAOSTAT, 2009). In symbiotic association with Bradyrhizobium sp. (Urtz & Elkan, 1996), peanut

plants can fix atmospheric nitrogen, reducing the need for expensive and environmentally damaging nitrogen fertilizers. During symbiosis, rhizobia are hosted intracellularly and a molecular dialogue between the two partners is required to coordinate the events leading to the symbiosis and to avoid host defence responses (Kistner & Parniske, 2002). The relevance of rhizobial cell surface components in the symbiotic interaction has been described in several studies (Perret et al., 2000; Fraysse et al., 2003). However, few studies have focused on the importance of membrane lipids of rhizobia (Minder et al., 2001; López-Lara et al., 2005; RVX-208 Vences-Guzmán et al., 2008). There is general agreement that phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and cardiolipin are the major phospholipids in rhizobia (Wilkinson, 1988). While phosphatidylethanolamine, phosphatidylglycerol and cardiolipin are common phospholipids in many bacteria, phosphatidylcholine is restricted to a limited number of genera, and seems to be more common in those that establish close interactions with eukaryotes (Sohlenkamp et al., 2003). It was speculated that phosphatidylcholine might serve some special function during host–pathogen/symbiont interactions.

2, and 29.8 min (Fig. 1), which correspond to palmitic acid (C16:0), a mixed peak of linoleic (C18:2) and oleic (C18:1) acids, GSK2126458 price and ergosterol, respectively. The ethanol extract obtained from W. sebi mycelia showed concentration-dependent lysis of bovine erythrocytes (Fig. 2a). The hemolysis rate (1/t50) of 0.1 min−1 was produced by 25 μg mL−1 of the extract TS obtained after the cultivation at 20% NaCl. If W. sebi was cultivated at the lower 5% NaCl, the same rate of hemolysis was observed only after the addition of approximately 200 μg mL−1 of the extract TS, making this eightfold less active. To further explore the nature of hemolytically active compounds, the most abundant fatty acids in the

extract (C18:1, C18:2, and C16:0) were also tested for their hemolytic potential (Fig. 2b), both separately or in an equimolar mixture. Their hemolytic activity was comparable to that of the W. sebi extract and was associated with the unsaturated forms (C18:1 and C18:2). Ergosterol, which was detected in considerable amounts in the extract (Fig. 1), was also tested for hemolysis and found inactive. AZD2281 research buy Exposure to 100 °C significantly affected this ethanolic extract activity, as there was almost total loss of hemolytic activity in comparison with the control (Fig. 3a). The same loss of the activity after heating

to 100 °C could be observed with the equimolar mixture of three tested fatty acids (Fig. 2b). A significant increase in hemolytic activity of the W. sebi extract was observed

at pH above 8.5 (Fig. 3b), and the higher ionic strengths also induced significant increases, although small, in the hemolytic activity (Fig. 3c). As shown on Fig. 4a, the SUVs containing phosphocholine (i.e. those formed with DPPC, DOPC, and POPC) and/or sphingomyelin completely prevented lysis of the erythrocytes that otherwise occurred Rebamipide in first few minutes of assay. This suggests that the phospholipids with a choline headgroup in their structures can bind the hemolytically active compound(s) in the extract and thus diminished their activity toward the erythrocytes. Additionally, the fluorescence of the calcein released from the SUVs was measured after the addition of the extract. Here, the percentage of released calcein was highest in cholesterol-containing vesicles (Fig. 4b), indicating that membranes with a higher degree of fluidity are more susceptible to lysis induced by this W. sebi ethanolic extract. Wallemia sebi is an important pan-global contaminant of foods and feeds preserved with low aw. It can contaminate food not only as an airborne or soil-borne contaminant, but it can also be inoculated with the preservative itself (Butinar et al., 2011). Wallemia sebi can grow over a wide range of aw (0.997–0.690) in glucose/fructose media (Pitt & Hocking, 1997), but in media with NaCl as the major solute, the lowest aw for its growth was reported as 0.80 (Zalar et al., 2005; Plemenitaš et al., 2008), which corresponds to 4.5 M NaCl.

The first experiment compared PS5 and NorE5 expression levels and showed that not only was a band of the expected size detected but also its expression was significantly increased in NorE5 (Fig. 2). The second experiment compared

strains GC4468 and JTG936 and showed a similarly increased expression in the SoxS-overexpressing strain JTG936 (Fig. 2). Selleckchem PI3K inhibitor Thus, we conclude from these experiments that the ydbK and the ompN genes are cotranscribed. To further test whether SoxS induced the cotranscription of ydbK and ompN, a ydbK::lacZ fusion was constructed (strain M4458b; Fig. 1). This transcriptional fusion was activated 19-fold by treatment with PQ. A moderate effect was found for DIP treatment PD0325901 cell line (3.5-fold), but no significant activation was found for SAL (1.2-fold; Table 3). Moreover, to rule out any possible side effect of PQ treatment, strain M4458b was transformed with the plasmids pRGM9817, pJLR70, pRGM9818, pRGM489, and pRGM5009, which respectively correspond to the vector alone or carrying SoxS, MarA, Rob, and MarA E89A (a modified MarA protein with the substitution E89A which has been shown to act like SoxS in the preferential activation of the regulon genes (Martin & Rosner, 2011). Accordingly, the

results shown in Table 3 indicate that only SoxS or MarA E89A can activate the ydbK promoter (10- and 5-fold increments, respectively). As has been previously reported, all genes belonging to the SoxS regulon contain a 20 bp motif, termed soxbox, in their promoter where the regulator binds to activate their expression (Martin & Rosner, 2002; Fabrega et al., 2010). By Tryptophan synthase means of bioinformatic tools we tried to find a DNA fragment matching the consensus soxbox sequence but we could not identify such a motif, suggesting that the SoxS effect observed on the ydbK promoter could be indirect, that is, acting via an unknown regulator. Similarly, recent results have reported an indirect effect for SoxS (not MarA or Rob) in activating the znuACB genes involved in zinc uptake and growth in the absence or limitation of zinc (Warner & Levy, 2012). Mutants of the ompN and ydbK

genes were constructed in the wild-type background of GC4468 (strains M6131 and M6133, respectively) and in the multipump mutant strain M5950 (strains M6135 and M6137, respectively). As the YdbK function has been related to superoxide resistance and this two-gene operon is activated by SoxS and not by MarA, the mutants were tested for resistance to oxidative stress. These mutants were grown in LB and M9 agar plates in the absence and presence of several concentrations of the superoxide-generating agent PQ (10, 20, 30, and 40 μg mL−1). Results showed that only the ydbK mutant displayed a significant growth restriction phenotype when grown on M9 plates with a PQ concentration equal and higher than 30 μg mL−1 [similar to previous results (Eremina et al., 2010)].

“
“Chronic arthritis Selleckchem Raf inhibitor (CA) is a common clinical entity associated with persistent pain and limited response to opioid analgesic therapy. However, it is unknown whether these features of CA change depending on its stage of evolution. To address this, in a well-established animal model of CA we studied the time course of electromyographic responses to electrical stimulation of C fibers (C-reflex), pain-like behavior as a response to mechanical nociceptive stimulation, and the inhibition of both responses by a prototypic opioid analgesic, morphine. To induce CA, rats received a single injection of complete Freund’s adjuvant into the ankle joint and the

C-reflex responses to electrical stimuli or the nociceptive response to paw pressure test were studied 2, 4 or 6 weeks later. The C-reflexes evoked by threshold and supra-threshold electrical stimulation exhibited progressive increases together with enhancement of the nociceptive behavior to mechanical stimulation during induction of monoarthritis. Notably, while systemic morphine produced antinociceptive effects upon both experimental approaches, the effects were markedly reduced during the early stages of CA but enhanced at later stages. These data indicate

that C-reflex and pain-like responses evolve in parallel, and are inhibited by morphine in a stage-dependent manner through the induction of CA. The present results may contribute to explain the enhanced pain response and variable selleck inhibitor analgesic efficacy of opioids that characterize arthritic pain in humans. “
“In this work, functional changes in the sensorimotor cortex following unilateral hand immobilisation were investigated in 11 healthy volunteers. Sensory and motor function of both hands was also assessed. Cortical activation was monitored with functional magnetic resonance imaging at 3 T. All examinations were performed prior to and directly after 72 h of immobilisation of the dominant hand and wrist. Following

unilateral immobilisation, cortical activation increased substantially during tactile stimulation of the non-immobilised hand. This was particularly evident in the ipsilateral somatosensory cortex. Urease Additionally, a redistribution of hemispheric dominance towards zero lateralisation was seen. A bilateral cortical activation increase was also seen during performance of a finger-tapping task by the non-immobilised hand, although this increase was less prominent than during tactile stimulation. In contrast, performance of the finger-tapping task with the immobilised hand resulted in an activation decrease, predominantly in the ipsilateral sensorimotor cortex. This site was anatomically close to the regional activation increase of the non-immobilised hand. These functional changes were associated with reduced grip strength, dexterity and tactile discrimination of the immobilised hand, and simultaneously improved tactile discrimination of the non-immobilised hand.

6 mA footstock; inter-trial interval 20–180 s). Four CS–US pairings were used in one solely behavioral experiment to determine if the extinction impairment of PN-1 KO mice depended on the number of pairings. Five pairings were used for all other experiments to ensure that WT mice showed strong freezing responses at the beginning of extinction trials. The onset of the US coincided with the offset of the CS. To score freezing behavior, we used an automatic infrared beam-detection system placed on the bottom of the experimental chambers (Coulbourn Whitehall, PA, USA). The mice were considered to be freezing if no movement was detected for 2 s. Freezing Selleck Olaparib was sampled for

2 min before the CS presentation to establish baseline activity and during the 30-s CS presentations. The fear conditioning context differed from the extinction context in shape, smell and

light intensity. Conditioning, early extinction and late extinction sessions took place on three consecutive days. The extinction group selleck inhibitor (ext.) was conditioned in one context, and on the following 2 days underwent early and late extinction training sessions consisting of 16 CS presentations in a different context in order to eliminate contextual conditioning effects. The no extinction group (no ext.) was conditioned as the extinction group, but only exposed to four CS presentations on the following 2 days. The freezing response to the first two CS presentations in early extinction sessions was used as the measure of fear retrieval. The CS-only group (CS-only) underwent the same regime as the no extinction group except that they were never exposed to a foot shock. Naïve control

mice for Fos immunohistological staining were handled as above, but kept in their home cage and never exposed to the CS. Unless stated otherwise, behavioral data were analysed by two-way repeated measure anova and Bonferroni post hoc tests (GraphPad Prism4 software, San Diego, CA, USA), and shown as mean ± SEM total freezing time. Student’s t-test analysis was performed using GraphPad Prism4 software. For immunohistochemical and immunoblotting Chlormezanone experiments, mice were killed 2 h after the start of Day 3 trials. Mice were deeply anesthetized using ketamine (ml/kg, i.p.) and perfused transcardially with 50 mL 0.1 m ice cold phosphate-buffered saline, pH 7.4 and 80 mL 4% paraformaldehyde in said buffer (unless specified otherwise, reagents were from FLUKA). The brains were dissected and postfixed for 24 h. Samples for cryostat sectioning were cryoprotected in 25% sucrose for 2 days, embedded in Shandon M-1 Embedding Matrix (#1310 Thermo Electron Corporation, Thermo Fisher Scientific) and frozen in −40°C isopentane. Sections were collected either on slides (12 or 25 μm thick) or as free-floating sections (40 or 60 μm thick) in sterile 0.05 m TRIS-buffered saline-filled wells, pH 7.4 (24-well plates) and stored at 4°C until use. Free-floating sections were stained three per well.

, 2003). Our discussion therefore mainly focuses on the induced genes in our dataset. A complete list of all genes differentially expressed by BC treatment can be found in Table S2. To validate the microarray data, QRT-PCR assays were performed with the same cDNA preparations used in array Ixazomib concentration hybridizations. Overall, nine genes of interest were selected. A strong positive correlation (Pearson’s correlation coefficient R=0.97) between the microarray and QRT-PCR data

was observed (Fig. 3), indicating that the microarray data obtained from the present work were reliable. In the present study, most of the key elements involved in replication initiation, such as DnaA, subunits of DNA polymerase III, DnaG and DnaC, were induced by BC. Of these proteins, DnaA (encoded by dnaA) binds to the origin of replication, oriC, resulting in the initiation of chromosome replication. Evidence suggests that transcription from the promoters of gid operon and mioC are involved in the initiation control of the replication

of the whole E. coli chromosome when oriC is under suboptimal conditions (Ogawa & Okazaki, 1991; Bates et al., 1997). As a result, the induction 5-FU mw of gidAB, mioC and dnaA in our study strongly suggests that berberine may influence dnaA and oriC function. It has been established that berberine binds strongly to DNA predominantly by intercalation with a preference for the AT sequence (Pilch et al., 1997). Generally, replication origins contain AT-rich sequences. Sf301 has an E. coli-like origin with an AT-content of 59% (Sugimoto et al., 1979), which is much higher than the average value (49%) of Sf301 whole genome. Therefore, it is likely that BC may

be able to inhibit the initiation of replication through interaction with the origin region of Sf301. MreB has been shown to be necessary for the segregation of origin-proximal chromosome. During a state associated with inhibition of cell division, the expression of mreB was generally upregulated (Chiu et al., 2008). We found that mreB was induced Ribociclib cost by BC, which was further confirmed by QRT-PCR. It has been reported that excessive copies of the mreB gene led to filamentous cells, a reflection of cell division inhibition (Wachi & Matsuhashi, 1989). In the present study, microscopic examination of Sf301 treated with 160 μg mL−1 of BC for 30 min revealed an increase in the percentage of elongated cells (Fig. S1). These results indicate that chromosome segregation may be inhibited. It has been shown that inactivation of DnaA dramatically inhibited segregation of the oriC region in E. coli (Kruse et al., 2006). Therefore, it is likely that the drug may inhibit cell segregation through its influence on dnaA and oriC function. Additionally, a set of DNA repair genes –hepA, recJ, xseA, recQ, nfo, lig, SF2540, nei and dead– were also induced by BC, indicating that the drug may cause certain DNA damages.

, 1989; Pandey et al., 1994). Accordingly, both nonpathogenic as well as pathogenic bacteria (Ratledge & Dover, 2000) and fungi (Howard, 2004) require Fe for growth in the various environments in which they proliferate. Previous work has demonstrated the potential effectiveness of iron and other trace metal withdrawal for the inhibition of Saccharomyces cerevisiae growth (Feng et al., 1997a).

In this work, a trace iron methodology was developed and applied in order to study the Trametinib mouse effect of iron removal on microbial growth in a chemically defined medium. In addition to using media with trace iron concentrations, microbial inhibition by the natural host defence Fe chelator, lactoferrin, clinically used chelators, such as desferrioxamine and deferiprone, and other strong chelators, such as bathophenanthroline sulphonate (BPS), EDTA and a novel carried chelator with hydroxypyridinone-like Fe-ligand functionality, DIBI, was also studied. The organisms chosen for this study were the well-known opportunistic pathogen Candida ZD1839 cost albicans (McCullough et al., 1996) and Candida vini (Barnett et al., 1983),

a related, but lesser-known nonpathogenic spoilage yeast. Candida albicans (ATCC 10231) and C. vini (ATCC 20217) were obtained from the Microbiology Laboratory Culture Collection at the Department of Food Science, University of Guelph, Canada. Desferrioxamine (Desferal) was donated by Ciba Geigy, now Novartis, Basel, Switzerland. Deferiprone, EDTA, BPS and bovine lactoferrin were obtained from Flavopiridol (Alvocidib) Sigma-Aldrich. The developmental compounds DIBI and FEC-1 were donated by Chelation Partners. Apo-lactoferrin (i.e. Fe depleted) was prepared according to Holbein (1981). The other chelators were dissolved directly in the medium. The iron-binding capacity of the DIBI was determined

to be 800 μmol dry weight g−1 DIBI by adding varying amounts of Fe-citrate (1 : 3 molar ratio) to aqueous DIBI samples of known mass and then reading the Fe complex A530 nm, the main visible range absorption peak for the DIBI chelate as determined by an absorption scan. Throughout the work, the aerobic growth version of the chemically defined glucose-phosphate-proline (GPP) medium (pH 4.5) of Dumitru et al. (2004) was used with one modification: the mineral concentrate was prepared without the inclusion of FeSO4. Trace iron GPP was prepared by removing iron contaminations with the Fe-specific resin, FEC-1 (Feng et al., 1997b). For this, 5 g of hydrated and washed FEC-1 resin were batch contacted by shaking overnight with 1 L of complete GPP medium in a flask. After removal of the resin by filtration, the Fe-extracted medium was filter sterilized (0.22-μm nylon filter, Millipore) and stored in sterile plastic bottles at 4 °C. Typical trace iron concentrations attained using this method were 1.2 μg L−1. Different known iron concentrations were adjusted in the trace iron GPP by addition of appropriate amounts of a 0.

, 2010). These complex structures are composed of several protein subunits, all of which require tight control of their synthesis, export, folding

and assembly process for final functional structure formation (Ruiz & Silhavy, 2005). Stresses that interfere with these processes activate the Cpx-envelope stress system (Fig. 1; reviewed in MacRitchie et al., 2008), which responds not only by regulating the expression of folding factors and proteases in the envelope to deal with the misfolded proteins but also by inhibiting the expression of the surface Bioactive Compound Library high throughput structures (Dorel et al., 2006; Vogt et al., 2010). Because these surface structures include important virulence determinants such as adhesins and secretion systems, the Cpx system contributes to virulence in several Gram-negative species (Raivio, 2005; Rowley et al., 2006). The Cpx system belongs to the group of two-component signal transduction systems (TCSs) and is made up of the senor kinase (SK) CpxA, the

response regulator (RR) CpxR and the periplasmic accessory inhibitor CpxP (Fig. 1; Ruiz & Silhavy, 2005; Buelow & Raivio, 2010), which provides response to additional stimuli (Buelow & Raivio, 2010; Heermann & Jung, 2010; Krell et al., 2010). Three phosphotransfer reactions are involved in controlling the functional state of the Cpx-TCS: (1) the autophosphorylation Autophagy inhibitor of a conserved histidine of the SK CpxA, (2) the transphosphorylation of a conserved aspartate of the RR CpxR and (3) the dephosphorylation of phosphorylated RR to return the system back to the prestimulated resting state (Gao & Stock, 2009). Importantly, the balance between phosphorylated and dephosphorylated RRs is crucial not only for the initiation of a specific genetic response to the external stimulus but also for its duration

(Stock et al., 2000; Dorel et al., 2006). It has been suggested that all inducing cues involve misfolded envelope proteins as the actual common stimulus for the Cpx-TCS (Raivio & Silhavy, 2001) and/or dissociation of the inhibitory CpxP from CpxA (Rowley et al., 2006), as well as signal specificity for the Cpx response (DiGiuseppe & Silhavy, 2003; Hunke & Betton, 2003; Ruiz & Silhavy, 2005). However, where and how the independent FER entry points for this signalling system take place has to be addressed. The pivotal factor of the Cpx-TCS is CpxA with its central function as a sensor kinase. Sequence alignments revealed that CpxA belongs to class I SK (Grebe & Stock, 1998; Dutta et al., 1999), typically consisting of two transmembrane domains (TMDs) integrating a large periplasmic domain and a cytoplasmic, highly conserved kinase core that acts as a transmitter domain (Fig. 2). The cytosolic domain includes a HAMP domain, which links the second TMD of CpxA with its kinase core (Appleman et al.