L

L., Robins-Browne R. (12), and ToxT from (13), are required for the expression of factors essential for intestinal colonization and virulence. To study the regulation of virulence gene expression by AraC-like proteins, we have used the prototypical AraC-like regulator, RegA, from your EPEC-like mouse pathogen, (14). Like its human-specific counterparts, enterohemorrhagic (EHEC) and EPEC, carries a pathogenicity island known as the locus for enterocyte effacement (LEE), which is required for the romantic attachment of bacteria to intestinal epithelial cells and the formation of characteristic attaching and effacing lesions (15). RegA is usually a global regulator that controls the expression of 60 genes and facilitates colonization of the intestine by (16, 17). In the presence of bicarbonate, which functions as a small intestinal-specific environmental trigger, Maackiain RegA coordinately activates transcription of virulence determinants and represses the expression of various housekeeping genes (16). Among the most strongly activated genes by RegA are the and operons, which encode a putative K99-like pilus and Aap, an anti-aggregation protein, respectively (16). RegA also indirectly enhances the expression of the LEE by stimulating the GrlA-Ler regulatory cascade and thus activating the transcription of the to operons (18). The crucial importance of RegA in virulence and the relevance of the ICC169Spontaneous NalR derivative of wild-type ICC168, NalR(19)????EMH1ICC169 E22EPEC serotype O103:H2(20)????”type”:”entrez-nucleotide”,”attrs”:”text”:”H10407″,”term_id”:”875229″H10407ETEC serotype O78:H11, LT+, ST+(21)????042EAEC serotype O44:H18, SmR, TetR, CmR(22)????395Serogroup O1, biotype Classical(23)????MC4100F?JP8042XL1-Reddish(pEH6)in pACYC184, CmR(17)????pACYC177-(pMU1065)in pACYC177, KnR(25)????(pMU2385-promoter region in pMU2385, TpR(27)????(pMU3190)promoter region in pMU2385, TpR(28)????promoter region in pMU2385, TpRThis study????promoter region in pMU2385, TpRThis study????promoter region Rabbit Polyclonal to KITH_VZV7 in pMU2385, TpRThis study????promoter region in pMU2385, TpRThis study????pACYC184-in pACYC184, CmRThis study????pACYC184-in pACYC184, CmRThis study????pACYC184-in pACYC184, CmRThis study????pACYC184-in pACYC184, CmRThis study Open in a separate window TABLE 2 Primers used in this study TAATCJY-8R223A mutationpromoterpromotergenegenepromoterpromotergenegenepromoterpromotergenegenepromoterpromotergenegeneJP8042(strains MC4100(promoter and TyrR-mediated transcriptional activation of the promoter. The compound in E11 represents a specific inhibitor of RegA (true positive) as it inhibits only RegA-mediated activation of the reporter. Cytotoxicity Assay The Cytotox 96 Non-radioactive Cytotoxicity Assay (Promega) was used to measure lactate dehydrogenase release, an indication of cell death, in the culture supernatants. The assay was performed as per the manufacturer’s protocol. Briefly, HeLa cells in DMEM supplemented with FBS (10%, v/v) were seeded at 0.8 104 cells per well. After overnight incubation, regacin suspended in DMSO was Maackiain added to a final concentration of 100 m and incubated at 37 C with 5% CO2 for 6 h. Controls included cells incubated in the absence or presence of DMSO (1%, v/v). A substrate mix was added to each well, and plates were incubated for 30 min in the dark at room heat before the enzymatic reaction was halted. Absorbance (at 450 nm) from each well was measured using the FLUOStar Omega plate reader. Percentage of cell death (cytotoxicity) was determined by dividing the lactate dehydrogenase release from test samples with the maximum lactate dehydrogenase release from cells treated with Triton X-100. -Galactosidase Assay Bacterial cells were produced in the presence or absence of chemical inhibitors to mid-log phase (fragment, the primer JY-1 (Table 2) was labeled with 32P at its 5 end by using [-32P]ATP and T4 polynucleotide kinase. The promoter fragment was generated by PCR using primer pairs (32P-JY-1 and JY-2) with plasmid as template. The fragment was generated using primer pairs (32P-JY-9 and JY-10) with the chromosomal DNA of K-12 strain MC4100 as template. The end-labeled and fragments were incubated with 200 nm purified MBP::RegA or TyrR, respectively, in the presence of varying amounts of regacin at 30 C for 20 min in binding buffer (10 mm Tris-HCl (pH 7.4), 50 mm KCl, 1 mm DTT, 100 g/ml BSA, 5 ng/l poly(dI-dC), and 45 mm NaHCO3). Glycerol was added to a final concentration of 6.5% (v/v). Samples were separated by electrophoresis on 5% native polyacrylamide gels (37.5:1) at 4 C for 12 h at 10 V/cm and analyzed by using by FLA3000 phosphorimaging (Fujifilm). Random and Site-directed Mutagenesis Random Maackiain mutations in the gene were generated by transforming plasmid pACYC184-into qualified cells of XL1-Red (Stratagene) according to the manufacturer’s instructions. Individual chloramphenicol-resistant colonies were cultured overnight in LB broth, subcultured, and reincubated overnight. These cultures were used to prepare a strain Maackiain MC4100(gene was performed by using the QuikChange mutagenesis kit (Stratagene). Plasmid pACYC184-was used as the template. Experiments.