Gene expression of the flagellar system is tightly controlled by external stimuli or intracellular signals. the operon, encoding two proteins which form the heterotetrameric positive transcriptional regulator of the class II genes. Global regulators, such as the cyclic AMP receptor protein, DnaA, and the nucleoid-associated protein H-NS, influence the expression TAE684 supplier level of this operon and consequently the formation of flagella (18, 21, 23). The expression of class II genes is dependent on the RNA polymerase-?70 holoenzyme (E?70) and FlhD-FlhC. Proteins involved in the formation of the hook and the basal body complex (HBB), as well as the regulatory proteins FlgM and FliA, belong to this class (11). FliA is a specific sigma factor (?28) required for the expression of class III genes, while FlgM is an anti-sigma factor that inhibits FliA TAE684 supplier activity. The release of FliA from the inhibitory action of FlgM occurs when the HBB structure is completed, allowing FlgM export out of the cell. FliA is then free to associate with the RNA polymerase core enzyme in order to transcribe class III genes (10, 15). The flagellar genetic system of is poorly understood. Detailed analyses of some structural components of the flagellum have been described, but nothing is known about the factors that regulate gene expression. Recently, genetic evidence has suggested the location of functional flagellar promoters in this organism. Complementation studies have indicated the presence of promoters at the intercistronic region (7), upstream of the operon (T. Ballado, L. Camarena, B. Gonzlez-Pedrajo, E. Silva-Herzog, and G. Dreyfus, unpublished data) and upstream of (6). However, no physical evidence supporting these results has been reported. In this work we show evidence that a ?54 promoter is located at the intercistronic region and is responsible for the transcription of the operon. In addition, primer extension experiments revealed transcription start sites upstream of and fliKgenes did not affect the expression of other flagellar genes dependent on ?54. In contrast, mRNA was reduced in or strains. These results allow us to propose a regulatory hierarchy controlling the expression of the flagellar genes in cells were grown in Sistrom’s succinate-basal salt medium at 30C (20). Heterotrophic growth conditions were achieved by growing 10-ml cultures in 250-ml Erlenmeyer flasks with strong shaking (300 rpm) in the dark. Phototrophic conditions were achieved by growing cultures in completely filled screw-cap tubes under continuous illumination. Cultures were harvested at an optical density at 600 nm of 0.5 0.05 (mean standard deviation). When required, spectinomycin (15 g/ml), kanamycin (25 g/ml), or tetracycline (1 g/ml) was added to the culture medium. strains were grown aerobically at 37C Rabbit polyclonal to Receptor Estrogen beta.Nuclear hormone receptor.Binds estrogens with an affinity similar to that of ESR1, and activates expression of reporter genes containing estrogen response elements (ERE) in an estrogen-dependent manner.Isoform beta-cx lacks ligand binding ability and ha on Luria-Bertani medium. Antibiotics were added at the following concentrations: ampicillin, 100 g/ml; tetracycline, 10 g/ml; and kanamycin, 50 g/ml. Recombinant DNA techniques. Routine genetic manipulations were performed as described elsewhere (2). Restriction enzymes, alkaline phosphatase, T4 ligase, and T4 polynucleotide kinase were purchased from GIBCO-BRL. Plasmid DNA was isolated from using Qiagen columns and procedures. Sequencing was carried out using a Thermosequenase kit (Amersham) on single-stranded DNA. Conjugal mating. Plasmid DNA was mobilized into cells by conjugation according to procedures previously reported (5). Site-directed mutagenesis. Site-directed mutagenesis was performed according to the method TAE684 supplier of Kunkel (14) with a uracil-containing single-stranded DNA as the template. The oligonucleotides used for mutagenesis were 5-CTGCAACATCCGTGACGCCCGCCCGCG-3, 5-CTGCAACATCCGTGCTGCCCGCCCGCG-3, 5-GTCCCCCTCCGCTACAACATCCGTGCCG-3, and 5-GTCCCCCTCCGCAACAACATCCGTGCCG-3. RNA isolation and Northern blot analysis. Total RNA was isolated from heterotrophic cultures as described previously (24). For Northern blotting, 20 g of each RNA sample was separated electrophoretically on agarose-formaldehyde gels and transferred by capillary action onto nylon membranes with a pore size of 0.45 m. Filter hybridizations were performed as described previously (2). The DNA probe used was a 1.1-kb and reactions and 100 g for reactions) was annealed with a specific primer at 42C in the presence of 50% formamide. Oligonucleotides used as primers for cDNA synthesis were 5 end labeled with T4 polynucleotide kinase and 20 Ci of [-32P]ATP at 37C for 30 min. Unincorporated nucleotides were removed by chromatography. The primer elongation reactions were carried out with avian myeloblastosis virus reverse transcriptase (Promega). Unlabeled primers were used to generate a nucleotide sequence ladder. -Glucuronidase activity assay. -Glucuronidase assays employed 4-methylumbelliferyl–d-glucuronide as a substrate along with sonicated cell extracts as described previously (12). Samples of 100 l were taken at three time points between 10 and 40 min and then mixed with 0.9 ml of stop buffer.