Measurements of promoter activity with lacZ transcriptional fusions were performed as described previously (Miller, 1972). The complete coding region of C. crescentus katG was amplified by PCR from genomic DNA of strain NA1000 using primers KatG6 (5′-ATGAAGCTTCAAAATGAGTGGATTC-3′) and KatG10 (5′-TCGAATTCATGGAAACACCTGCGCGGAG-3′), and the 2.33-kb EcoRI/HindIII fragment was cloned into vector pProEX HT (Gibco BRL). The recombinant His-KatG protein was purified from E. coli DH5α by chromatography on a nickel column (Qiagen), according
to the manufacturer’s instructions. The immune serum was obtained in New Zealand rabbits after two subcutaneous injections of 0.7 mg of purified protein in Freund’s adjuvant. Sera were collected from the ear vein using Telazol as an anesthetic according to the Biomedical Sciences Entinostat supplier Institute Ethics Committee procedures. Immunoblots were performed essentially as described (Towbin et al., 1979), using a 1 : 1000 dilution of the antiserum and a secondary anti-rabbit-alkaline phosphatase conjugate
(1 : 30 000 dilution). An anti-Fur polyclonal antiserum (da Silva Neto et al., 2009) was used as a control of the amount of protein loaded. The isolation of a partially functional truncated mutant of Rho in a Tn5 mutagenesis screen of C. crescentus represents a new opportunity for studying the physiological functions see more of Rho. The mutant strain SP3710 does not show pleiotropic deficiencies as do some other rho mutants (Das et al., 1976), in that it exhibits wild-type motility and cell cycling with a doubling time in a rich medium of 200 min compared with 140 min for the wild type. Moreover, the total mRNA half-life in strain SP3710 is similar to the wild type (V.C.S. Italiani & M.V. Marques, unpublished data). Although it is wild type in its sensitivity to other stresses, strain SP3710 is extremely sensitive to Cisplatin research buy H2O2 (Italiani et al., 2002), and in this work, we investigate the
biochemical reasons for this defect in SP3710 oxidative stress response. Because of the severe sensitivity of rho mutant strain SP3710 to exogenously added H2O2 (Italiani et al., 2002), the response of SP3710 to other reactive oxygen species (ROS) was also tested. Increased sensitivity of SP3710 to tert-butyl hydroperoxide was demonstrated by larger zones of inhibition compared with strain NA1000 in a plate assay (Table 1). This difference was evident for both exponential- and stationary-phase cells, and was not observed for the katG mutant strain SGC111. The response to superoxide was analyzed by determining cell viability in the presence of paraquat, which generates superoxide intracellularly.