The rpoD and rpoHI σ factor-encoding genes were amplified using rpoD-F/rpoD-R and rpoH-AF/rpoH-AR, respectively. Putative ECF σ factor-encoding genes rcc02637 and rcc00699 were amplified using 2637-AF and 2637-AR, and 699-AF and 699-AR, respectively. All amplicons were cloned as KpnI fragments into all 4 BACTH vectors: pKNT25, pKT25, pUT18 and pUT18c (Additional file 2). All pair-wise combinations of bait (rbaW) and prey (rbaV, rpoD, rpoHI, rcc02637 and rcc00699) recombinant vectors were co-transformed into cya – E. coli BTH101 and plated on

LB agar supplemented with ampicillin, kanamycin, 40 μg ml-1 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside OSI-906 supplier (X-Gal) and 0.5 mM IPTG. Positive control plasmids encoding interacting fragments of a leucine zipper protein, pKT25-zip and pUT18C-zip (Additional file 2), were also co-transformed. Plates were incubated for 48 hours eFT508 at 30°C. For quantitative determination of β-galactosidase activity, 3 replicate co-transformants were picked for each interaction to inoculate fresh LB broth containing antibiotics and 0.5 mM IPTG. Cultures

were grown overnight at 37°C and then diluted 1:5 in LB broth and the OD600 was determined. The cells were permeabilized with one drop of 0.1% SDS and 2 drops of chloroform and then mixed in a 1:1 ratio with PM2 buffer (70 mM Na2HPO4, 30 mM NaH2PO4, 1 mM MgSO4, 0.2 mM MnSO4; pH 7) containing 100 mM 2-mercaptoethanol. The cells were incubated for 5 minutes at 28°C and one volume of 0.4% ο-nitrophenol-β-D-galactopyranoside (ONPG) substrate in PM2 buffer was added to 4 volumes of cell suspension. After sufficient colour development, the reaction was stopped by addition of 2 volumes of 1 M NaHCO3. The OD420 and OD550 were obtained for each sample and β-galactosidase activity was calculated as units mg-1 dry weight bacteria [55]. Results Identification, sequence

characteristics, and genomic contexts of rsb homologues in R. capsulatus In addition to genes rcc03323 and rcc03324 encoding putative RsbV and RsbW orthologues, respectively, previously identified as affected by loss of CtrA [8], Akt inhibitor searching the R. capsulatus genome sequence by BLAST [57] for other Rsb-related sequences identified a gene (rcc00181) encoding a putative orthologue of the B. cereus RsbY. PAK5 This gene also had lower transcript levels in the ctrA mutant [8]. We propose to rename these genes as rbaV, rbaW and rbaY, where Rba is the 3-letter abbreviation for Rhodobacter[58]. The RbaV and RbaW protein sequences contain conserved STAS and HATPase domains, respectively, and the RbaY protein possesses an N-terminal phosphorelay REC domain and a C-terminal PP2C phosphatase domain. The RbaV, RbaW and RbaY sequences were the reciprocal best BLAST matches with the respective B. cereus RsbV, RsbW and RsbY proteins. A BLAST search of the NCBI GenBank database revealed that highly similar homologues of the R.