Sequencing of the resultant PCR products revealed that BR1 contained an insertion within a gene similar to phoR from E. coli. A further PCR using chromosomal DNA from the BR9 mutant with primers PHORL and PHORR (homologous to phoR FG-4592 molecular weight 5′ and 3′ ends) and primers KML and KMR demonstrated that BR9 contained an insertion within a gene with similarity to phoB from E. coli. To further confirm the phoBR sequence, PCR products of phoB and phoR were generated with primer pairs PF154/PF155 and PF180/PF182 respectively and sequenced on both strands from independent products. Construction of a plasmid (pTA74) that expresses native PhoB A construct that
enabled expression of native, untagged PhoB was created as outlined below. The phoB gene was amplified by PCR, using primers PF154 and PF155, which contain EcoRI and HindIII restriction sites, respectively. Additionally, primer PF154 contains a consensus ribosome-binding site (RBS, AGGAGGA). The PCR fragment of phoB was cloned into pQE-80L, previously digested with the enzymes EcoRI and HindIII. The resulting plasmid, pTA74, was learn more confirmed selleck chemical by DNA sequencing. Expression of plasmid pTA74 in E. coli was induced with 1 mM IPTG. Construction of promoter::lacZ fusions and assay conditions Plasmid pTA15 was constructed as described previously .
The rap and smaI promoter regions were cloned into the promoterless lacZ plasmid pRW50  to give the plasmid constructs pTA14 and pTG27, respectively. Plasmid pTG27 was constructed by cloning an EcoRI/HindIII digested PCR product (generated using
forward primer OTG124 and reverse primers OTG125) into EcoRI/HindIII digested pRW50. Plasmid pTA14 was constructed by cloning an EcoRI/HindIII digested PCR product (generated using forward primer PF43 and reverse primer PF42) into EcoRI/HindIII digested pRW50. All constructs were confirmed by DNA sequencing. Promoter activity assays were performed in E. coli DH5α cells as described in . Briefly, DH5α cells were transformed with the promoter::lacZ construct (pTA14, pTA15 or pTG27) and either pTA74 (encoding native PhoB) or the empty vector control, pQE-80L. The resulting strains were grown in LB containing Ap, Tc and 1 mM isopropyl-β-D-thiogalactopyranoside Janus kinase (JAK) (IPTG). At late exponential phase, 1 ml samples were assayed for β-galactosidase activity. Prodigiosin, carbapenem, AHL, β-galactosidase, β-glucuronidase and alkaline phosphatase assays The assays for Pig and Car were performed as described previously . Pig production was plotted as (A534 ml-1 OD600 -1). Detection of AHLs was performed using the Serratia LIS bioassay described in . β-Galactosidase activity was determined as described previously  and was represented as (ΔA420 min-1 ml-1 OD600 -1).