, 2005; Miot & Betton, 2007). CpxP has no obligatory function for the induction of the Cpx response (Raivio et al., 1999; DiGiuseppe & Silhavy, 2003). However, the cpxP gene was identified as a CpxR target involved in inhibiting the expression of toxic envelope proteins, including misfolded pilus subunits of P-pili that are crucial for uropathogenic E. coli (UPEC) during kidney colonization (Jones et al., 1997; Danese et al., 1998; Hung et al., 2001; Isaac et al., 2005). In agreement with its function www.selleckchem.com/products/dinaciclib-sch727965.html in quality control for subunits of surface appendages, CpxP is also involved in the early steps of biofilm

formation (Beloin et al., 2004; Yang et al., 2008). Biochemical analysis of the reconstituted CpxAR phosphorylation cascade demonstrated that CpxP, incorporated into the lumen of the proteoliposomes, inhibits the autophosphorylation of CpxA (Fleischer et al., 2007). As the reconstituted system excludes the involvement of other factors, this finding indicates a direct protein–protein interaction between CpxP and CpxA (Fleischer et al., 2007; Zhou et al., 2011). In support of this, peptide library

screens showed that the purified PSD of CpxA directly interacts with CpxP (Zhou et al., Ku-0059436 molecular weight 2011). Interestingly, the interaction of purified CpxP with peptides derived from the PSD of CpxA depends on negative charges within this domain (Zhou et al., 2011). The crystal structure of CpxP gave further insight into this interaction (Thede et al., 2011; Zhou et al., 2011). CpxP consists of a dimer, the monomers of which are interwined like ‘left hands’ (Thede et al., 2011; Zhou et al., 2011). Thereby, each monomer is strengthened by double hydrogen bonds between two highly conserved LTxxQ repeat motifs. Based on the structural and biochemical analysis, CpxP-mediated Cpx inhibition results from

an interaction between the concave polar surface of CpxP and the negatively charged sensor domain of CpxA (Fig. 3a; Zhou et al., 2011). The CpxP dimer acts as a patch to shield the CpxA sensor domain from inducing signals, maintaining cAMP the SK in an ‘off’ mode. Moreover, the structure of CpxP provides explanations of how CpxP might act as a sensor for salt (Zhou et al., 2011), pH (Thede et al., 2011) and misfolded pilus subunits (Zhou et al., 2011) for the Cpx system. Physicochemical and chemical stimuli inducing the Cpx response include alkaline pH, salt (Raivio & Silhavy, 1997), depletion of the major lipid phosphatidylethanolamine (Mileykovskaya & Dowhan, 1997), attachment to hydrophobic surfaces (Otto & Silhavy, 2002), intermediates of the acetyl-CoA pathway (Wolfe et al., 2008; Lima et al., 2011), low cAMP levels (Strozen et al., 2005), carbon monoxide (Davidge et al., 2009), metals (Lee et al., 2005; Yamamoto & Ishihama, 2006), indole (Raffa & Raivio, 2002), alcohols, acetone and the anaesthetics procaine and phenethyl alcohol (Clarke & Voigt, 2011; Table 1).

2b). Comparisons with known lipopolysaccharide profiles from other gram-negative bacteria suggests that the LMW band corresponds to the rough lipopolysaccharide (lipid A plus core) and the HMW bands to the smooth lipopolysaccharide (complete lipopolysaccharide molecules with different number of attached O-antigen units) (Choudhury et al., 2005; Vilches et al., 2007). The mutant did not produce the smooth lipopolysaccharide bands and showed faint LMW lipopolysaccharide bands with

different electrophoretic mobility from the parental bands (Fig. 2b). The results indicated that BM07-59 was damaged in the production of normal lipopolysaccharide. These Selleck Sirolimus results were not unexpected, as the galU mutants in Pseudomonas aeruginosa and Aeromonas hydrophila produced truncated lipopolysaccharide core and lacked the O-antigen (Choudhury et al., 2005; Vilches et al., 2007). UDP-glucose formed through the GalU catalyzed reaction can serve as glucose donor for core and O-antigen polysaccharide biosynthesis in the production of lipopolysaccharide (Dean & Goldberg, 2002). To determine why the O-antigen is missing in BM07-59, we analyzed the composition of lipopolysaccharide from wild-type and mutant strains grown in M1 medium containing 70 mM fructose at 30 °C. Purified lipopolysaccharide from wild type and BM07-59 is predominantly

composed of a lipid, with 10.7% and 3.5% of the lipopolysaccharide find more composed of carbohydrate, respectively. The carbohydrate fraction of lipopolysaccharide from wild-type strain contained rhamnose, xylose, mannose, glucose, N-acetyl glucosamine and 3-deoxy-d-manno-oct-2-ulsonic acid (KDO) in a mole ratio of 31.8 : 1.7 : 0.3 : 50.2 : 14.9 : 1.1, respectively, whereas the carbohydrate fraction of lipopolysaccharide

from BM07-59 contained rhamnose, glucose, N-acetyl glucosamine and KDO in a mole ratio of 3.9 : 11.2 : 30.8 : 54.1, respectively. Thus, in comparison with the wild-type lipopolysaccharide, the lipopolysaccharide from BM07-59 contained a much smaller molar amount of rhamnose and glucose but a much larger (50-fold) molar amount of KDO was detected in the mutant lipopolysaccharide. Janus kinase (JAK) This significant sugar compositional difference of lipopolysaccharide between wild-type and mutant strains clearly reflects the fact that BM07-59 is unable to supply UDP-glucose for O-antigen and core lipopolysaccharide synthesis. To further confirm that galU gene is involved in lipopolysaccharide and exobiopolymer production, a complementary assay was performed. Plasmid pBBR-KT galU harboring galU gene from P. putida KT2440 was introduced into BM07-59 to recover GalU activity. As expected, the complement BM07-59 (KT GalU) restored the parental phenotype for colony morphology (Fig. 1a), autoagglutination phenotype (Fig. 2a), exobiopolymer production (Fig. 1b) and lipopolysaccharide synthesis (Fig. 2b). These results indicated that the expression of galU gene from P.

The global analysis of transcription within a bacterial biofilm is an appealing technique to identify genes and specialized gene expression patterns associated with biofilm formation, without the need for extensive and time-consuming studies of individual genes (Beloin & Ghigo, 2005; An & Parsek, 2007). The reduction in the cost of genome sequencing and the availability of custom microarrays has resulted in an increase in studies using microarrays to investigate gene

expression in biofilms of their bacteria of interest. However, INK 128 mw interpretation of results from these studies is problematic because RNA is extracted from cells throughout a biofilm, which are in a wide range of metabolic states. To obtain enough biofilm material for transcriptional profiling, the entire biofilm is normally collected for RNA extraction. This is a major problem, because cells with a range of different physiological and phenotypic states are used for comparison against a homogeneous planktonic culture. Small differences between experimental setups can thus lead to large differences in results. This has been highlighted by the comparison of three independent microarray-based studies of the Pseudomonas aeruginosa quorum-sensing regulon

(An & Parsek, 2007). The independent studies contained as many differences as similarities even when a fivefold change was used as threshold. While reproducibility may have been an early major concern for microarray studies, this issue highlights the importance for researchers to consider what is actually being compared. In microbial fuel cells, there LDK378 are a number of processes that can

occur within the biofilm. Put simply, expression of individual genes may play a role in the process of biofilm formation, in the process of extracellular electron transfer, or in both. To understand these processes in a current-producing Geobacter sulfurreducens biofilm, microarrays have been used to compare gene expression in electrical biofilms, to both planktonic cells and nonelectrical biofilms. These microarrays were designed to examine genes important for biofilm formation and/or genes important for extracellular electron transfer in a biofilm. In these Ribose-5-phosphate isomerase cases, many targets have been identified. However, their importance could only be confirmed through mutational analysis, which identified important features such as nanowire production and extracellular cytochromes for power production, and/or biofilm formation. This highlights an important consideration: how are transcriptome data to be used? Typically, a quantitative reverse transcriptase-PCR reaction is used to corroborate the microarray results. Although useful, this process provides no spatial information about expression within the biofilm. This is a very challenging aspect of biofilm studies.

As we discuss later, the IL12B region is also associated with TAK. These data strongly suggest that ustekinumab would be a very promising therapeutic option for patients with TAK. The only established genetic component associated with TAK has been HLA-B52. The association between HLA-B*52:01 and TAK has been repeatedly shown in different populations.[33-38] There are studies reporting the importance of other alleles, including HLA-DPB1

or HLA-DRB1 alleles.[39, 40] The recent genome-wide association study (GWAS) showed an independent association in the HLA-DQB1/DRB1 locus.[41] Although HLA-B51, a strong susceptibility allele to BYL719 Behçet disease,[42] shares large parts of amino acid sequencing with HLA-B*52:01, the association between HLA-B51 and TAK was negatively reported.[34] Our recent work might provide an answer to these observed different susceptibilities.[38] Our study indicates the importance of the 67th and 171st amino acid residues for TAK susceptibility where the 67th is one of the selleck kinase inhibitor two amino acid residues not shared between HLA-B*51:01 and *52:01. Furthermore, both the amino acid positions are located at peptide binding grooves,[43-45] suggesting that peptide binding at these positions would be very important for the predisposition of the two different autoimmune diseases. A previous Mexican study suggested

the involvement of the 63rd and 67th amino acids.[46] Thus, different studies suggest the importance of the 67th amino acid of the HLA-B protein. Although HLA-B39 was reported to be associated with severe complications of TAK as well as TAK onset in a previous study,[47] the association was not observed in a recent Japanese study, and it did not find a different association between

HLA-B67:01 and TAK clinical manifestations.[48] Our recent work confirmed this lack of association Chorioepithelioma of HLA-B39 and the positive association of HLA-B*67:01.[38] HLA-B*67:01 has not been reported in Turkey and Middle-East Asia. Although GCA shows association with HLA-DR4,[49] which is not a TAK susceptibility allele, meta-analysis of TAK and GCA would reveal similarity and differences between the two large vessel arterites. Recently, we reported the first GWAS results for this disease at the same time as a US/Turkish group.[41, 50] Both groups reported IL12B as a strong susceptibility locus to TAK. Our group also reported the MLX region in chromosome 17 and a US/Turkish group reported the FCGR2A/3A region as another susceptibility locus. The US/Turkish group also reported the PSMG1 region as a suggestive locus. Our data also showed that the polymorphism in the IL12B region is associated with high incidence of AR, severity of AR and higher time-averaged CRP level as a representative of disease activity. Furthermore, our data indicated that the polymorphism of the IL12B region displayed a synergistic effect on TAK susceptibility in combination with HLA-B*52:01.

cinnamomea were evaluated. Among them, α-terpineol (0.5 mg L−1) showed the greatest stimulatory effect on the triterpenoid content (23.31 mg g−1) and triterpenoid production (91.33 mg L−1) of A. cinnamomea. Results of LC–MS analysis showed that α-terpineol (0.5 mg L−1)

stimulated the syntheses of six triterpenoids in the mycelia of A. cinnamomea. This indicates that α-terpineol can act as an elicitor for triterpenoid biosynthesis in A. cinnamomea. “
“Root rot of poinsettia, caused by Pythium helicoides at high temperatures in hydroponic cultures, has become a serious problem in many parts of the world. We have developed a species-specific, loop-mediated isothermal amplification (LAMP) assay for the rapid Trichostatin A diagnosis of this pathogen. The primers were designed using the ribosomal DNA internal transcribed spacer sequence. Primer specificity was established using 40 Pythium species including P. helicoides, 11 Phytophthora species, and eight other soil-borne pathogens. A sensitivity test was carried out using genomic DNA extracted from P. helicoides,

and the detection limit was c. 100 fg which is comparable to that of the polymerase chain reaction (PCR). In addition, we tested the ease of pathogen detection in poinsettia roots. The LAMP results were consistent with those from the conventional plating method and showed more sensitivity than the PCR results. Consequently, the LAMP method developed in this study is effective for the rapid and easy detection AZD6244 mouse of P. helicoides. “
“Fusarium graminearum (teleomorph: Gibberella zeae), the dominant pathogen of Fusarium head blight (FHB) on wheat, can cause substantial economic losses. The Spt-Ada-Gcn5-acetyltransferase (SAGA) transcription coactivator plays multiple roles in regulating transcription because of the presence of functionally independent modules of subunits within the complex. The transcription factors spt3 and spt8 are components of the SAGA complex and they are important in yeasts and filamentous fungi including F. graminearum. In this study, we identified Fgspt3 and

Fgspt8, homologs of Saccharomyces cerevisiae spt3 and spt8 from F. graminearum using the blastp program. The aim of the present study was to investigate the functions of Fgspt3 and Fgspt8 in F. graminearum. The deletion mutants grew Epothilone B (EPO906, Patupilone) significantly more slowly than the wild-type parent and did not produce conidia. Expression of the sporulation-related genes FgFlbC and FgRen1 were significantly down-regulated in the mutants. The mutants exhibited no sexual reproduction on infected wheat kernels and a 90% decrease in virulence on wheat. Pigment formation was also greatly altered in the mutants. All of the defects were restored by genetic complementation of the mutant with wild-type Fgspt3 and Fgspt8 genes. Overall, Fgspt3 and Fgspt8 are essential genes in F. graminearum.

p.m. Different nucleosides

were assayed at 30 °C and pH 7. Reaction mixtures contained 1 × 1010 CFU, 2.5 mM 5-fluorouracil and 10 mM uridine, thymidine, 2′-deoxyuridine, 2′-deoxycytidine or 2′,3′-dideoxyuridine. Reactions were performed at different 5-fluorouracil and thymidine ratios (1 : 1, 4 : 1 and 1 : 4) at 30 °C, pH 7, and 200 r.p.m. All assays were performed selleck inhibitor three times in 1 mL of reaction medium. Subsequent to system characterization with 5-fluorouracil, the incorporation of other halogenated pyrimidine bases was tested using 5-chlorouracil and 5-bromouracil. Reactions were performed in a 1 : 4 ratio (2.5 mM halogenated base and 10 mM thymidine) in potassium phosphate buffer (30 mM, pH 7) at 30 °C. 1 × 1010 CFU were mixed with 3 mL of 1, 2, and 3% (w/v) agar or agarose. The mixture was then added dropwise to stirred sunflower oil (20 mL) at 25 °C. The resulting gel beads were cooled, filtered, washed with hexane and then with physiological solution to obtain solvent-free beads. 1 × 1010 CFU were mixed with 3 mL of phosphate buffer (30 mM, pH 7) containing 15, 20, and 25% (w/v) acrylamide/bis-acrylamide,

subsequently 50 μL of 10% (w/v) ammonium persulfate (APS) and 14 μL of N, N, N′, N′tetramethylethylenediamine (TEMED). The resulting gel was cut into small cubic pieces (1.0 × 1.0 × 0.2 cm). The biosynthesis of nucleoside analogues selleck chemicals llc was qualitatively evaluated by TLC Merck Silica gel 60 F254 in chloroform/methanol (80 : 20, v/v) as mobile phase. The quantitative analysis was performed by HPLC (Gilson) equipped with a UV detector (254 nm) using a Nucleodur 100-5 C18 column (5 μm, 125 × 5 mm). The isocratic mobile phase used was water/methanol (95 : 5, v/v) at room temperature and at a flow rate of 1.2 mL min−1. Retention times of substrates and products were as follows: Floxuridine biosynthesis: (1) uracil (1.0 min), 5-fluorouracil (1.4 min), 2′-deoxyuridine (2.0 min), floxuridine (3.0 min); (2) cytosine (1.1 min), 5-fluorouracil (1.4 min),

2′-deoxycytidine (2.2 min), floxuridine (3.0 min); (3) 5-fluorouracil (1.4 min), thymine (2.6 min), U0126 cell line floxuridine (3.0 min), thymidine (4.2 min). 5-fluorouridine biosynthesis: uracil (1.0 min), 5-fluorouracil (1.4 min), uridine (1.8 min), 5-fluorouridine (2.8 min). 5-chloro-2′-deoxyuridine biosynthesis: (1) uracil (1.0 min), 2′-deoxyuridine (2.0 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min); (2) cytosine (1.1 min), 2′-deoxycytidine (2.2 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min); (3) thymine (2.6 min), thymidine (4.2 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min). Product identification was performed by MS-HPLC (See Supporting Information, Data S1). 5-fluoro-2′-deoxyuridine biosynthesis from thymidine and 5-fluorouracil was used as reaction test for the screening (Fig. 1).

5) (Kaether et al., 2000; MacAskill & Kittler, 2010). For time-lapse

imaging with electrical field stimulation, neurons in Tyrode’s solution (119 mm NaCl, 2.5 mm KCl, 2 mm CaCl2, 2 mm MgCl2, CP-868596 price 25 mm HEPES and 30 mm glucose, pH 7.4) with 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione (Tocris, Ellisville, MO, USA) and 50 μm D(-)-2-amino-5-phosphonovaleric acid (Tocris) or in low-Ca2+ Tyrode’s solution (119 mm NaCl, 2.5 mm KCl, 0.1 mm CaCl2, 4 mm MgCl2, 25 mm HEPES and 30 mm glucose, pH 7.4) with 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione and 50 μm D(-)-2-amino-5-phosphonovaleric acid were placed on a heated stage (set at 37 °C) with a home-prepared acrylic box to prevent temperature fluctuation. Electrical field stimulation (1 ms duration, 400 stimuli,

40 Hz) was applied by two parallel platinum wires (between wires approximately 6 mm and approximately 1 mm distance from cells; Sigma-Aldrich, Tokyo, Japan) that were mounted in a plastic lid (Gärtner & Staiger, 2002). The mCherry-OMP dynamics were imaged at intervals of 3 s for 50 min with 3 min interval electrical stimulation of 40 Hz for 10 s. After time-lapse imaging, changes of G-CaMP6 fluorescence intensity elucidated by the same electrical stimulation were measured at approximately 3 Hz at the same axonal region. For time-lapse imaging in low-Ca2+ Tyrode’s solution, the G-CaMP6 measurements were performed both before and after replacing the Tyrode’s solution with normal Ca2+ concentration. We set the excitation laser power Ganetespib to be minimal but sufficient to obtain images with enough dynamic range. During imaging periods, reduction of mitochondrial mobility and impairment of mitochondrial morphology or distribution were not observed (De Vos & Sheetz, 2007).

We classified the axonal mitochondria into two dynamic states, stationary and mobile (Fig. 1A). We defined mitochondria that remained for ≧ 30 min at the same axonal region as stationary states (SS), and others as mobile states. Mobile mitochondria showed saltatory movement, science including moving periods (M) and short pauses (SP) (temporary stops). The definition of a short pause is given in the following section. Image analysis and quantification were performed by using ImageJ (NIH, Bethesda, MD, USA) and custom-written software (Visual Studio; Microsoft, Seattle, WA, USA). For all images, the average background pixel intensity of the individual image was subtracted before image processing. In mCherry-OMP, EGFP-VAMP2, FM1-43(Δ) and APP-mCherry images, puncta were identified as local fluorescence increases, which were > 0.15 μm2 and three times higher fluorescence intensities than the background fluorescence of nearby axonal regions without obvious fluorescence clusters.

Thereafter, the plate was shaken carefully and absorbance was measured using a Labsystem Multiscan MCC/340 plate reader,

at 340 nm every 15 s over 10 min to monitor the oxidation of NADH. Aspartase activity was calculated according to: To validate the repeatability of the method, six randomly selected strains were grown independently in triplicate for protein extraction and aspartase activity, which were repeated three times for each cultivation (data not shown; maximum SD=±14.08%). Based on the results showing good repeatability of the technique, aspartase activity from the remaining strains was determined as the mean of three parallel assays. To compare aspartase activity determined in PD-0332991 mw single isolates it was important to fix the growth conditions, as the highest enzyme activity was encountered at the late log phase of growth (data not shown).

Given the nature of the assay, the lowest quantification limit of aspartase activity was set at 100 units. Therefore, strains displaying aspartase activity lower than 100 units are considered as one group without a precisely determined activity. Figure 2 shows the aspartase activity of the PAB strains analysed in this study as well as the percentage of ABT199 strains belonging to the chart segments representing aspartase activity levels of 0–25%, 25–50%, 50–75% and 75–100% with respect to the highest activity detected in this study. More than 70% of the strains tested belonged to the segment representing the lowest aspartase activity (0–25%). Of this group, the aspartase activity of 42 strains was assayed as being lower than Cytidine deaminase 100 units. Of the remaining strains, the percentage categorized to the segments representing higher aspartase activity was decreased in parallel to the increase in activity (19% in activity level group 25–50%, 5% in activity level

group 50–75% and 3% in activity level group 75–100%). Thus, low aspartase activity was a common characteristic of propionibacteria of Swiss-type cheese origin studied here. Some strains with high aspartase activity were found, but at a low proportion compared with the isolates with low activity. Although a wide range of aspartase activity was detected between different strains, the commonly used dairy P. freudenreichii ssp. freudenreichii and shermanii could not be differentiated on the basis of enzyme activity. The role of aspartase activity in Swiss-type cheese manufacture has been recognized to have considerable impact on the formation of eyes and flavour (Langsrud & Reinbold, 1973; Thierry et al., 2005). Yet, as shown here, aspartase activity is strain dependent and so each strain must be tested separately in order to be able to choose the most suitable starter culture for cheese production.

We are grateful to K. Maillard for providing the MBE193 and MBE194 strains and to E. Capron and N. Tanqueray for technical assistance. F.D and L.H. contributed equally to this work. Table S1.Rhodococcus equi strains used in this study. Table S2. Summary of the annotation

of pVAPA116 compared with that of pVAP1037. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Endospores are metabolically dormant, multi-layered GKT137831 datasheet cellular structures formed by Gram-positive bacteria belonging to the genera Bacillus, Clostridium and related organisms. click here Their external layers are composed of proteins which in part play a role in the resistance behaviour of spores to varied chemical and environmental assaults. Thus, protein analysis is of major interest in spore biology. Spore proteomic studies have been carried out previously but these studies have

focused on the soluble coat protein fraction. Using gel-based techniques, protein identification and analysis were performed. Mass spectrometry-driven proteomics has opened new avenues to resolve in particular the insoluble part of the spore layer proteomes. Mass spectrometry-based qualitative and quantitative proteomics PLEKHM2 methods expand the knowledge about both the actual composition and the amount of proteins in their various layers. The techniques can also be used to study the integrity of the layers as well as spore biology in general. This notion is explored concisely in this mini-review. “
“Immune system malfunctions cause many of the most severe human diseases. The immune system has evolved primarily to control bacterial, viral, fungal, and parasitic infections. In turn, over millions of years of coevolution, microbial pathogens have evolved various mechanisms to control and modulate the host immune system for their own benefit and survival. For example,

many bacterial pathogens use virulence proteins to modulate and exploit target cell mechanisms. Our understanding of these bacterial strategies opens novel possibilities to exploit ‘microbial knowledge’ to control excessive immune reactions. Gaining access to strategies of microbial pathogens could lead to potentially huge benefits for the therapy of inflammatory diseases. Most work on bacterial pathogen effector proteins has the long-term aim of neutralizing the infectious capabilities of the pathogen. However, attenuated pathogens and microbial products have been used for over a century with overwhelming success in the form of vaccines to induce specific immune responses that protect against the respective infectious diseases.

1a). Moreover, when STM4538 was expressed from its own promoter find more in the low-copy plasmid pMW118, the YK5009 strain showed an LDC-positive phenotype (Fig. 1a). However, the phenotype of the yfhK::Tn10dCm insertion was a false negative because this transposon insertion had no influence on LDC activity. We further compared the expression of a chromosomal cadA–lacZ fusion in strains JF3068 (wild-type), YK5007 (STM4538::Tn10dCm) and YK5011 (ΔSTM4538) using β-galactosidase assays. Following 30 min of acid stress, the level of cadA expression in the STM4538 mutants was approximately twofold lower than that in the wild-type (Fig. 1b). Together, these data suggest

that the PTS permease STM4538 is positively involved in the control of cadBA expression. To assess the potential role of STM4538 in the proteolytic activation of CadC, we performed an immunoblot selleck analysis of total protein extracts from the S. Typhimurium wild-type and ΔSTM4538 strains harboring pACYC184-HA-CadC. N-terminally HA-tagged CadC (HA-CadC) was expressed under the control of its own promoter in the low-copy plasmid pACYC184. The cells were grown in E glucose medium to an OD600 nm of 0.6 and subjected to acid stress. As shown in Fig. 2, HA-CadC levels rapidly decreased in the wild-type background, as previously reported (Lee et al., 2008).

However, despite wild-type levels of cadC transcription (data not shown), HA-CadC levels were slightly increased in the ΔSTM4538 null mutant after acid stress, indicating impaired proteolytic processing Oxymatrine of CadC. These results suggest that the PTS permease STM4538

is required for the proteolytic activation of CadC signaling in S. Typhimurium. To gain further insight into the signaling mechanism of CadC, which undergoes rapid proteolytic cleavage in response to low pH and lysine signals (Lee et al., 2008), we examined whether both signals are required for this proteolytic event. Immunoblot analysis was conducted on total protein prepared from the YK5005 (cadA::lacZ ΔcadC) strain harboring pACYC184-HA-CadC. Cells were grown in E glucose medium to an OD600 nm of 0.6 and exposed to three different types of signals. The samples were collected at the indicated times and immunoblotted with anti-HA antibodies. As shown in Fig. 3(a), proteolysis of CadC occurs strictly in response to a pH shift regardless of the lysine signal. On the other hand, the lysine signal is insufficient on its own to stimulate proteolysis. To further confirm the concomitant effects of CadC proteolysis on cadBA transcription, the β-galactosidase activity from a cadA-lacZ transcription fusion was measured 30 min after each treatment. As expected, cadA transcription was induced only when cells respond to both low pH and lysine signals (Fig. 3b). These results suggest that proteolytic processing is a necessary but not sufficient step for CadC activation.