​timone.​univ-mrs.​fr/​MST_​YPestis/​mst. We observed no growth over 7 days for any of the Y. pestis isolates being studied after ethanol inactivation. MALDI-TOF protein profiles for the three main biotypes following 70% ethanol inactivation, including Y. pestis Antiqua (Y. pestis Nairobi-rattus), Medievalis (Y. pestis 14-47), and Orientalis (Y. pestis 6/69M) are shown in Figure 1. Figure 2 contains a pseudo-gel representing the protein profile for the three Y. pestis biotypes. Figure 1 Protein profile of the major Y. pestis biotypes generated by MALDI-TOF-MS. a.i., arbitrary intensity given by the software. Figure 2 Pseudo-gel representing the protein profile obtained after

MALDI-TOF-MS analysis of Y. pestis organisms representative of the Antiqua, Medievalis and Orientalis biotypes. arb.u., arbitrary unit – transcription for arbitrary intensity Vemurafenib concentration in the Bruker software; selleck compound sp# is the numbers of the spectrum. MALDI-TOF-MS identification of Yersinia organisms For the Y. pestis

isolates, default identification against the Bruker database resulted in a false result of Y. pseudotuberculosis with an identification score > 2 in two of two cases. When the identification was performed using our local updated database, the isolates were correctly matched as Y. pestis in two of two cases with an identification score > 2.7, effectively identifying the isolates at the species level. The 11 Y. enterocolitica isolates were correctly identified as Y. enterocolitica with an identification score Edoxaban > 2. Further analysis of the Y. pestis isolates using ClinPro Tools software allowed us to assign them to a biotype, with the exception of the Y. pestis JHUPRI strain for which the unique MALDI-TOF profile did not match any of the three biotypes. Reproducibility of MALDI-TOF-MS identification We obtained a unique MALDI-TOF profile for each

of the 39 Yersinia isolates being studied: for each isolate, the 12 MALDI-TOF profiles derived from triplicate analysis were similar and yielded identical, accurate identification. A list of m/z values characteristic for Y. pestis is given in additional file 1. Discussion Given that the MALDI BioTyper™ database contained 42 Yersinia profiles derived from 11 species but lacked the major pathogen Y. pestis, as well as the recently described species Y. massiliensis [17], we aimed to complete this database by deriving a MALDI-TOF profile for 12 species currently included in the Yersinia genus [17]. We obtained a unique MALDI-TOF profile for each of the Yersinia species used in this study. In each case, the species-specific profile did not match any of the 3,000 non-Yersinia profiles deposited in the MALDI BioTyper™ database, including those for closely-related enteric bacteria.

2) for 20 min and then labelled with [35 S]-methionine for 20 min. Proteins were Gefitinib research buy separated by their isoelectric point (pH 4–7) and then by their molecular weight on a 10%–20% Tris–HCl gel. The gel was scanned and only proteins, with incorporated [35 S]-methionine, were visible. Arrows point at induced proteins: 19 kDa periplasmic protein (p19), alkyl hydroperoxide reductase (AhpC), Superoxide dismutase (Fe) (SodB), Thioredoxin-disulfide reductase (TrxB), hypothetical protein (Cj0706), and molybdenum cofactor biosynthesis protein (MogA).

Quantitative RT-PCR Transcriptomic analysis using qRT-PCR technique was performed to determine if the proteins induced during acid stress were induced at transcription level. Figure  4 illustrates the transcription profiles represented by fold change relative to control of dps, cj0706, sodB, trxB, ahpC, mogA, p19 and fur during HCl and acetic acid stress for strain NCTC 11168. Interestingly, the transcriptomic data did not correspond completely with the

proteomic data (Figure  4). The increased gene expression of trxB (P HCl = 0.009) and p19 (P HCl, Ac < 0.05) during acid stress corresponded well with enhanced protein production. Especially noteworthy is the high acid stress response of p19 gene compared with the other genes. Proteins such as SodB and AhpC, which were not significantly induced in NCTC 11168, were, however, over-expressed at transcription level during acetic acid exposure (P sodB, Ac = 0.03, Buparlisib cell line P ahpC, Ac = 0.000). The regulator 5-FU clinical trial Fur was included in the qRT-PCR study because a search of putative Fur-regulated genes indicated that genes involved in iron-transport genes such as p19, cj0178, ceuB, cfrA, chuA, exbB, feoB and cfhuA and the iron-storage genes such as dps, ferritin (cft) and cj0241 all contained Fur box promoters [37]. Fur was not induced in the proteomic study, but there was a tendency, however not significant, that fur was over-expressed during acetic acid stress (P fur, Ac = 0.06). Figure 4 Relative change in transcription level during

acid stress of selected genes: dps , cj0706 , sodB , trxB , ahpC , mogA , p19 and fur analyzed by qRT-PCR. C. jejuni strain NCTC 11168 was grown to 1 × 10 8 CFU/ml and exposed to HCl (pH 5.2) and acetic acid (pH 5.7). The expression level of acid stressed for a specific gene was compared with unstressed cells and the horizontal line illustrates the fold change at 1.0 for the reference genes (rpoA and lpxC). Fold changes and standard deviations were calculated from the outcome of qRT-PCR runs from three microbiological independent experiments. Genes marked with an asterisk are significantly over-expressed compared with genes from non-stressed cells. Discussion Proteome analysis for Campylobacter during acid stress revealed different protein profiles between the strains and the type of acid used.

1 (Lighthouse data). The annotation of S. aureus N315 was used for protein identification and denotation. Peptide mixtures that yielded at least twice a Mowes score of at least 50 and a sequence coverage

of at least 30% were regarded as positive identifications. Proteins that failed to exceed the 30% sequence coverage cut-off were subjected to MALDI-MS/MS [73]. Database searches were performed using the Mascot search engine with the protein databases of S. aureus strain N315. Protein quantitation approaches The 2D gel image analysis was performed with the software “”Delta2D”" (DECODON GmbH, Greifswald, Germany). Three different data sets were analyzed in order to screen for differences in the amount of cytoplasmic proteins identified Selleckchem Everolimus on 2D gels. Detection of glucose, acetate and lactate EPZ015666 mw The concentrations of glucose, acetate and lactate in the supernatants were determined using commercially available

kits (Boehringer) according to the manufacturer’s instructions. Urease assay McFarland 0.5-standard cell suspensions were diluted 100-fold in urea medium [74] and incubated in 12-well plates at 37° for 24 hours. In parallel, colony forming units (cfu/ml) were determined. Acknowledgements This study was supported by the Swiss National Science Foundation grants 310000-117707 (to BBB), 3100A0-112370/1 (to JS), and 3100A0-116075/1 (to PF) and the Deutsche Forschungsgemeinschaft (grant Bi 1350/1-1 to MB). Electronic supplementary material Additional file 1: Genes with lower expression in wild-type versus Δ ccpA mutant. The table represents genes

showing a lower gene expression in the from wild-type than the ΔccpA mutant (wt/mutant ratio ≤ 0.5). Cells were grown in LB, without glucose addition. (DOC 236 KB) Additional file 2: Genes with higher expression in wild-type versus Δ ccpA mutant. The table represents genes showing a higher gene expression in the wild-type than the ΔccpA mutant (wt/mutant ratio ≥ 2.0). Cells were grown in LB, without glucose addition. (DOC 210 KB) Additional file 3: CcpA-dependent down-regulation by glucose. The table shows genes found to be subject to down-regulation by glucose in a CcpA-dependent manner (with/without glucose ratio of 0.5 or lower in wild-type, with/without glucose ratio of approximately 1, but below 2 in the mutant). (DOC 284 KB) Additional file 4: CcpA-dependent up-regulation by glucose. The table shows genes found to be subject to up-regulation by glucose in a CcpA-dependent manner (with/without glucose ratio of 2 or higher in wild-type, with/without glucose ratio of approximately 1, but below 2 in the mutant). (DOC 258 KB) Additional file 5: Primers used for the construction of DIG-labelled DNA probes. (DOC 36 KB) References 1. Fujita Y: Carbon catabolite control of the metabolic network in Bacillus subtilis. Bioscience, Biotechnology, and Biochemistry 2009,73(2):245–259.CrossRefPubMed 2.

Phenetic analysis confirmed that the

BoNT/G complex of proteins shared the most similarity with the/B serotype (Figure 3C-E), as previously reported [10, 23]. To determine the extent of/G’s homology to the/B toxin serotype, we completed an in-depth comparison of six/B subtypes, 22 different accession numbers (Figure 3B, additional files 2). The comparison of individual domains–translocation domain, binding domain NT, binding domain CT, and peptidase–revealed the area of the toxin in which/G shares the greatest (translocation domain) and least (binding domain CT) similarity. Overall, each domain compared, between the two toxins, is greater than 50% similar. This comparison helped to determine which substrate peptide would be optimal to test the activity of/G. FG4592 Although there are no direct indications that sequence similarity would imply overall identical functionality, similar sequences would allow similar crystal structures to form, suggesting similar functionality [24]. It is currently known that both BoNT/B and/G cleave the Synaptobrevin protein;/B cleaves a Gln76-Phe77 bond and/G an Ala81-Ala82 bond five amino acids downstream (Table 1). Because the cleavage

sites of both toxins are relatively near one another–thus the similarity of their binding domain sequences and therefore structures–the same peptide substrate currently used to test/B activity was used to test/G activity Topoisomerase inhibitor [19]. In order to confirm that the commercial BoNT/G complex was active and therefore ever could be considered analogous to the toxin complex found in clinical samples, various dilutions of the commercial toxin were tested using the Endopep-MS method previously described (Figure 6) [19]. In addition to confirming the toxin’s activity, the Endopep-MS experiments indicated a new optimum temperature for/G activity. When reactions were pulsed at 47°C for 10 min, followed by incubation at 42°C for at least eight hours–as opposed to 37°C for a minimum of 17 hr–an increase in activity and in the quality of mass spectra produced was observed. Other serotypes of BoNT (/C and/D) are often associated with botulism in animals,

avians, equines, and bovines, whose body temperatures are higher than those of humans. BoNT/G has yet to be associated with botulism in a particular organism; however, it is possible that/G would be more effective at causing disease in an organism with a higher body temperature than that of humans, similar to BoNT/C and/D. Figure 6 Endopep-MS method confirmation of commercial BoNT/G activity. This is a representative spectrum indicating BoNT/G activity on a specific substrate peptide. 1Intact substrate, 2C-Terminus product mass 1762.9, and 3N-Terminus product mass 2281.8. The sequences are listed in Table 1. *Indicates double charged ion of the intact substrate peptide. Proteomic strategies and analyses used in this study were important to help define the characteristics of proteins associated with the BoNT/G complex.

However, the recent sequencing of two strains of T. princeps from P. citri (PCIT and PCVAL) has shown that it is, in fact, the smallest (139 kb) and most simplified bacterial genome described to date [16, 19]. Functional analysis reveals that the genetic repertoire of T. princeps is unable to sustain cellular life, according to Gil et al. (2004) [20], and that it entirely depends on M. endobia for many essential functions. Even though most of its genome is occupied by ribosomal

genes and genes involved in the biosynthesis of essential amino acids, T. princeps likely depends on its symbiotic consortium partner to build its own ribosomes and for amino acid production [16, 19]. The work published by McCutcheon and von Dohlen [16] mainly focused selleck chemicals on the analysis of the T. princeps genome and detangling the amino acid biosynthetic pathways in which all three partners (T. princeps, M. endobia and the

host) appear to be involved. However, the characteristics and functionality of the M. endobia genome, as well as other possible modes of complementation between the two endosymbionts, have remained largely unexplored. In this work we present Selumetinib chemical structure a comprehensive analysis of the predicted consortium functional capabilities and interactions, thus offering new insights into how this bacterial consortium may function internally. Additionally, we have performed a comparative analysis of both endosymbiont genomes in two P. citri strains, PCIT [16] and PCVAL ([19] and this work). Our analysis suggests that both genomes have undergone reductive evolution, albeit with some unusual genomic http://www.selleck.co.jp/products/MDV3100.html features, probably as a consequence of their unprecedented compartmentalized organization. Results and discussion Main features and genomic variability between two

strains of P. citri nested endosymbionts The main molecular features of the genomes of T. princeps str. PCVAL [19] and PCIT [16], and M. endobia str. PCVAL (this work) and PCIT [16] are summarized in Table 1. It is worth mentioning that differences in CDS numbers and coding density between both strains are due to differences in the annotation criteria used, since the number of polymorphisms detected between the two sequenced strains of T. princeps and M. endobia is minimal (see Additional file 1 for a list of annotation differences in CDS and tRNA genes). Table 1 Main genomic features of the two strains of the P. citri endosymbiotic consortium already sequenced   T. princeps PCVAL T. princeps PCIT M. endobia PCVAL M.

A dye-sensitized solar cell is composed of three main structures: (1) a dye sensitizer whose function is to harvest solar energy and generate excitons [7, 8], (2) a nanostructured metal oxide to transport electrons efficiently [9, 10], and (3) a redox electrolyte or hole-transporting material [11, 12]. The key element in a DSSC is the photoanode, which is composed of a thin film of TiO2 NPs. Though the nanoparticle thin film has a high specific surface area, electron percolation is hindered by limited interconnected NPs resulting in photoelectron loss due to recombination between the photoelectrons and the oxidized

dye molecules or electron-accepting species in the electrolyte. To solve this issue, mechanical compression of the photoanode thin film was adopted to increase the Histone Methyltransferase inhibitor effective interconnection between NPs. The optimal

thickness of the mechanically compressed TiO2 nanoparticle thin film was reported. www.selleckchem.com/products/rxdx-106-cep-40783.html Methods Experimental details Deposition of TiO2 thin film as photoanode TiO2 paste (10 wt%) was prepared by mixing nanocrystalline TiO2 nanoparticles (TG-P25, Degussa, Shinjuku, Tokyo, Japan; the average nanoparticle diameter was about 25 to 30 nm) with tert-butyl alcohol and deionized water. The TiO2 paste was then scraped on a transparent fluorine-doped tin oxide (FTO) glass of 8-Ω/sq resistivity by doctor blading method. The films were mechanically compressed with a pressure of 420 kg/cm2. After the compression, the films were annealed in air by two consecutive steps: 150°C for 90 min and 500°C for 30 min. The 150°C annealing is to decompose residual organic compounds, and the 500°C annealing is to assist the interconnection of TiO2 NPs. DSSC fabrication Figure 1 shows the structure of the dye-sensitized solar cell with

TiO2 NP thin film as photoanode. The compressed TiO2 NP thin films were immersed in 0.3 mM N3 dye (cis-bis(dithiocyanato)-bis(4,4′-dicarboxylic acid-2,2′-bipyridine) ruthenium(II)) for 5 h. Subsequently, they were rinsed in acetonitrile for a few seconds to wash out unbound dyes and then dried in the oven at 45°C. The Pt thin film as counter electrode was grown on an indium tin oxide (ITO) glass by an electroplating process. The those FTO substrate with deposited compressed TiO2 NP thin film with adsorbed dyes was then bonded to the ITO glass with Pt counter electrode using a 50-μm-thick hot-melt polymer spacer. Sealing was accomplished by pressing the two electrodes together at about 115°C for a few seconds. The redox electrolyte, consisting of 0.5 M LiI, 0.05 M I2, 0.5 M 4-tert-butylpyridine (TBP), and 1 M 1-propy1-2,3-dimethylimidazolium (DMPII) mixed into 3-methoxypropionitrile (MPN), was injected into the cell by capillary forces through an injecting hole, previously made in the counter electrode using a drilling machine. Finally, the hole was covered and sealed with a piece of hot-melt polymer, preventing the leakage of the fluid-type electrolyte.

e., dissolution-reprecipitation mechanism (Figure 5d) [58]. The constitutional α-Fe2O3 subcrystals grew into larger NPs, with 1D assembly behavior disappeared largely. Figure 5 Formation mechanism

of the hierarchical mesoporous pod-like hematite nanoarchitectures. https://www.selleckchem.com/products/poziotinib-hm781-36b.html It is notable, however, that the boric acid played a significant role in the formation of the present mesoporous pod-like α-Fe2O3 nanoarchitectures with uniform morphology and size, confirmed by the above experimental results (Figures 1 and 2). Also, as confirmed to improve the uniformity, the amount of boric acid or molar ratio of FeCl3/H3BO3/NaOH should be tuned within a certain composition range. As known, as a weak acid, H3BO3 could form sodium borate (i.e., borax) after the introduction of NaOH, giving rise to the buffer solution. This could tune the release of hydroxyl ions and further control the mild formation of amorphous Fe(OH)3 gel, leading to subsequent β-FeOOH fibrils with relatively uniform size.

This was believed to contribute to the further formation of the peanut-like β-FeOOH/α-Fe2O3 assemblies and ultimate occurrence of the pod-like α-Fe2O3 nanoarchitectures. Optical absorbance analysis Hematite NPs have been widely https://www.selleckchem.com/products/ldk378.html used as ultraviolet absorbents for their broad absorption in the ultraviolet region from the electron transmission of Fe-O. Figure 6 shows the

optical absorbance spectra of the α-Fe2O3 particles with the photon wavelength in the range of 350 to 650 nm. For sample a1, it revealed two absorption edges around 380 to 450 and 540 to 560 nm, which were consistent with the reported hematite NPs [59–61]. When the α-Fe2O3 clustered into samples b1 and c1, the size of α-Fe2O3 agglomerates was around 500 to 800 nm. The absorbance spectra showed two absorption peaks around 520 to 570 and 600 to 640 nm. The change Fossariinae in the degree of transition depended on the shape and size of the particles. When the hematite particles aggregated to pod-like nanoarchitectures, the size became larger, and then the scattering of visible light was superimposed on the absorption of as-prepared architectures. Figure 6 Optical absorbance spectra (a 1 -c 1 ) of the α-Fe 2 O 3 with different morphologies (a 2 -c 2 ). Time (h) = 12.0; Temperature (°C) = 120 (a1, a2, b1, b2), 150 (c1, c2); FeCl3/H3BO3/NaOH = 2:3:6 (a1, a2), 2:3:4 (b1, b2, c1, c2). It was well illustrated that three types of electronic transitions occurred in the optical absorption spectra of Fe3+ substances: (a) the Fe3+ ligand field transition or the d d transitions, (b) the ligand to metal charge-transfer transitions, and (c) the pair excitations resulting from the simultaneous excitations of two neighboring Fe3+ cations that are magnetically coupled.

Nature 1993, 362:446–447.PubMedCrossRef 39. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1987. Authors’ contributions Experiments were carried out Tanespimycin solubility dmso by YD, AL, JW, TZ, SC, JL, YHD. Data analysis was finished by YD and LHZ. The study was designed by YD and LHZ, who also drafted the manuscript. All authors read and approved the final manuscript.”
“Background Members of the genus Bifidobacterium are Gram-positive, obligate anaerobic, non-motile, non-spore forming bacteria [1], and are the most important constituents of human and animal intestinal microbiota [2, 3]. Recently,

news species of bifidobacteria have been described [4–6] and now more than 30 species have been included in this genus. Bifidobacterium spp. can be detected in various ecological environments, such as intestines of different vertebrates and invertebrates, dairy products, dental caries and sewage. Considering the increasing application of Bifidobacterium spp. as protective and probiotic cultures [7–9], and the fast enlargement of the genus, easy identification tools to discriminate new isolates are essential. Moreover, their correct taxonomic identification is of outmost importance for their use as probiotics [2]. Conventional identification and classification of Bifidobacterium species have been based on phenotypic PI3K inhibitor and biochemical features, such as cell morphology, carbohydrate

fermentation profiles, and polyacrylamide gel electrophoresis analysis of soluble cellular proteins [10]. In the last years several molecular techniques have been proposed in order to identify bifidobacteria. Most available bifidobacterial identification tools are

based on 16S rRNA gene sequence analysis, such as ARDRA [11, 12], DGGE [13] and PCR with the use of species-specific primers [14–16]. However, 16S rDNA of Bifidobacterium spp. has a high similarity, ranging from 87.7 to 99.5% and bifidobacterial closely related species (e.g. B. catenulatum and B. pseudocatenulatum) or subspecies (e.g. B. longum and B. animalis subspecies) even possess identical 16S selleck compound rRNA gene sequences [17, 18]. For this reason different molecular approaches have been tested based on repetitive genome sequences amplification, such as ERIC-PCR [19, 20], BOX-PCR [21, 22] or RAPD fingerprinting analysis [23]. These fingerprinting methods have the disadvantage of a low reproducibility, and they need strict standardization of PCR conditions. The use of different polymerases, DNA/primer ratios or different annealing temperatures may lead to a discrepancy in the results obtained in different laboratories [24]. In recent years alternative molecular markers have been proposed for bifidobacteria identification (e.g. hsp60, recA, tuf, atpD, dnaK) and Ventura et al. [18] developed a multilocus approach, based on sequencing results, for the analysis of bifidobacteria evolution.

However, ingested carnosine is rapidly degraded by two forms of carnosinase (CN1, EC 3.4.13.20; and CN2, EC 3.4.13.18) [18]. In humans, the CN1 gene is expressed in liver and brain tissue, and the protein is found in serum and brain tissue. Since the human CN1 specifically degrades both carnosine and homocarnosine, carnosine is absent in human blood. Whereas, CN1 in other mammals such as rodents is localized in the kidney, and a considerable amount of carnosine is contained in the blood [19]. CN2, which is also a cytosolic non-specific

dipeptidase, does not degrade homocarnosine, and exhibits a rather broad specificity towards various dipeptides. That is, ingestion ITF2357 concentration of ß-alanine or carnosine that was degraded by these carnosinases, was increased muscle carnosine and the increase of muscle carnosine may be involved in carnosine synthase. However, the details were not revealed. Recently, carnosine synthase was purified from chicken pectoral muscle and identified as an ATP-grasp domain-containing protein 1 (ATPGD1) [20]. It has been reported that ATPGD1 synthesizes carnosine using ATP, and the substrate specificity toward ß-alanine (carnosine) in the presence of ATP and L-histidine is 14-fold higher than that of γ-aminobutyrate (homocarnosine). To verify that ATPGD1 acts as a carnosine synthase in vivo, we investigated

the tissue distribution of ATPGD1 mRNA, and ATPGD1 and CN1 expression profiles in response to carnosine or ß-alanine administration using quantitative PCR analysis. Methods Oral administration study in mice Raf inhibitor Animal experiments Thiamet G were performed in accordance with the guidelines for Animal Experiments at Nippon Meat Packers Inc. and using minimum number of mice that dictated by an ethics committee ( n = 6 or 8). Male SPF-bred ddY (6-week-old) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). The mice were maintained under specifically

controlled environmental conditions, namely, a 12-h light–dark cycle, a temperature of 23°C, and a relative humidity of 50%. At 7 weeks of age, the mice were randomly assigned by body weight into three groups (pre-administration group, n = 8, body weight of 32.5 g; water administration group, n = 6, body weight of 33.4 g; carnosine administration group, n = 6 or 8, body weight of 33.2 g; ß-alanine administration group, n = 6, body weight of 34.0 g) and were orally given 2 g/kg body weight of carnosine (Hamari Chemicals Ltd., Osaka, Japan), ß-alanine (Wako Pure Chemical Industries, Ltd., Osaka, Japan), or water (control). After 15, 30, 60, 120, 180, or 360 min of treatment, the mice were anesthetized with Forane (Abbott Japan Co. Ltd., Japan) and then the brain, blood, liver, kidneys, olfactory bulbs, soleus muscles and vastus lateralis muscles were collected. The collected tissues were weighed, rapidly frozen with liquid nitrogen, and stored at −80°C until analysis.

coli and Salmonella[17].

The periplasmic chaperone CpxP binds to both the CpxA periplasmic domain and to certain misfolded proteins, which are degraded by the periplasmic protease DegP, therefore integrating information about their turnover status to the kinase activity of CpxA [18–20]. The outer membrane lipoprotein NlpE activates the CpxA protein upon its overexpression [21] and is required for CpxA protein activation Dorsomorphin concentration after adhering to hydrophobic surfaces [22]. Additional upstream components have been proposed to integrate other stresses in a process that is independent of the CpxP and NlpE pathways [17, 23]. For example, the CpxR/CpxA system confers a copper resistance phenotype even in CpxP and NlpE mutants [24]. Notably, nlpE (cutF or STM0241) is a pseudogene in Salmonella[25]. Here, we aimed to identify candidate connector genes that may integrate the signals of other systems. We identified a small protein as a novel connector-like factor from screening high copy plasmid learn more clones that could affect the CpxR/CpxA system status. Results Identification of a plasmid clone that activates cpxP transcription To

conduct a genetic screen for novel connector proteins acting on the CpxR/CpxA system, we constructed a strain harboring a cpxP lac transcriptional fusion in Salmonella. The cpxP gene was chosen as a readout of the activation status of the CpxR/CpxA system because it is likely directly regulated exclusively by this system, unlike other CpxR-activated genes that are also controlled by envelope stress-responsive systems [26–28]. The lacZY genes were inserted after the cpxP stop codon to ensure that the CpxP protein retained the ability to repress the CpxR/CpxA system. Then, Salmonella chromosomal DNA was partially digested with Sau3AI and ligated with the high-copy-number plasmid pUC19 (digested with BamHI) to generate a DNA fragment library. Of approximately 10,000 cpxP-lac Salmonella

transformants, a plasmid clone termed pWN1 yielded stable blue colonies on LB plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) and ampicillin and Farnesyltransferase was isolated four times. The blue color of the pWN1 strain was due to elevated cpxP-lac fusion expression. We demonstrated that this strain exhibit ~8-fold higher β-galactosidase activity than the same strain harboring the vector control or the plasmid clone pUC19-R1 that was randomly selected during the screening as a negative control (Figure 1A). Sequence analysis revealed that pWN1 harbors only the intact STM1852 open reading frame (ORF), which appeared to encode a 63-amino acid protein with no homology to any protein of known function, as well as the 3’ region of STM1851 and the 5’ region of pphA (Figure 1B).