Primers used in the construction are listed in Table 2. A PCR product containing 637 bp proximal to the 5′ end of sigE was amplified from RB50 genomic DNA using primers SigEKO_LeftF and SigEKO_LeftR. A non-overlapping PCR product containing 534 bp proximal to the 3′ end of sigE was amplified with primers SigEKO_RightF and SigEKO_RightR. The two fragments were digested with BamHI and ligated. The resulting construct was amplified
with primers SigEKO_LeftF and SigEKO_RightR, cloned into the TopoTA vector (Invitrogen), and verified by sequencing to give plasmid pXQ002. In this deletion construct, the 528 bp central region of the sigE gene is deleted leaving 66 bp at the 5′ end and 6 bp at the 3′ end of the sigE gene. The deletion see more construct from pXQ002 was then cloned into the EcoRI site of the allelic exchange vector pSS3962 (Stibitz S., unpublished data) to generate pXQ003 and transformed into E. coli Lazertinib research buy strain DH5α. Tri-parental mating with wild-type
B. bronchiseptica Selleckchem NCT-501 strain RB50, E. coli strain DH5α harboring the pXQ003 vector (strain XQ003), and DH5α harboring the helper plasmid pSS1827 (strain SS1827) [69, 70] and selection of mutants were performed as previously described . The deletion strain was verified by PCR using primers SigEKO_LeftF and SigEKO_RightR and by Southern blot analysis. β-galactosidase assays Overnight cultures were diluted into fresh medium and grown to an OD600 of 0.1-0.2 at 30°C. Where indicated, IPTG was added to a final concentration of 1 mM. Samples were collected 2.5 hours later and β-galactosidase activity from the σE-dependent reporter was assayed as previously described [60, 71]. Complementation of E. coli ΔrpoE by B. bronchiseptica sigE The ability of B. bronchiseptica sigE to suppress
the lethality caused by deletion of rpoE in E. coli was determined using a cotransduction assay as described . The ΔrpoE::kan ΔnadB::Tn10 allele from strain SEA4114 was moved via P1 transdution into strain SEA5005, which carries sigE on the plasmid pSEB006. Tet-resistant (tetR) transductants were selected and then screened for kanamycin resistance (kanR). Although the nadB and rpoE alleles are tightly linked (>99%), cotransduction resulting in tetR kanR colonies will only occur if rpoE is no longer essential PD184352 (CI-1040) for viability. In transductions with E. coli expressing sigE (strain SEA5005) as the recipient strain, 31 out of 32 tetR transductants were also kanR. In contrast, none of the 39 tetR transductants were kanR when E. coli carrying the empty cloning vector (strain SEA008) was the recipient strain. Protein purification N-terminally His-tagged B. bronchiseptica SigE and E. coli σE were purified from strain XQZ001 and SEA5036, respectively, as previously described for E. coli σE. Briefly, cells were grown at 25°C to an OD600 of 0.5, at which point IPTG was added to induce protein production. Following 1.