The membranes were washed again in TBST

and the bands were detected by chemiluminescence using the SuperSignal West Femto Reagent Kit (Thermo Fisher Scientific, Ottawa, Canada). Images were captured on an Alpha Innotech U400 camera, and then inverted and adjusted for brightness and contrast with image processing software. Viable cell counts Each culture used for gene transfer assays and western blotting was also assayed for viable cells as previously described [6]. Serial dilutions were plated RG-7388 and colony-forming units (cfu) were calculated for the 3 biological replicates to determine the number of viable cells. The data were converted to a ratio relative to the parental strain. Statistically significant differences in viable cell numbers were identified by one-way ANOVA in R [52]. β-galactosidase reporter fusions In-frame fusions of RcGTA orfg2 to the E. coli lacZ gene were constructed using PstI/BamHI fragments cloned into the promoter probe mTOR inhibitor vector pXCA601 vector [54]. Fragments 2 (pX2) and 2NP (pX2NP) were amplified by PCR using primers GTA-F1 and GTA-R1, and GTA-F2 and GTA-R1, respectively. Fragments 2.1 and 2.2 were amplified using primers GTA-F1

and GTA-DP-R, and GTA-DP-F and GTA-R1, respectively. Fragment g2Δp (pX2Δp) was created by ligating 2.1 and 2.2 via a primer-embedded KpnI restriction Pevonedistat cell line site, resulting in a deletion of the sequence from -129 to -100 5’ of RcGTA orfg1 (Additional file 2). Fragments 2.3 and very 2.4 were amplified using GTA-F1

and GTA-DS-R, and GTA-DS-F and GTA-R1, respectively. The fragment g2Δs was made by combining 2.3 and 2.4 via a primer-embedded KpnI restriction site, resulting in a deletion of the sequence from -73 to -46 5’ of orfg1 (Additional file 2). All fusions were confirmed to be in-frame by sequencing, and the plasmids were transferred into R. capsulatus strains by conjugation using E. coli S17-1 [50]. Strains of R. capsulatus containing the fusion constructs listed in Additional file 2 were grown in conditions identical to those for RcGTA activity assays. Cells were permeabilized for 15 minutes using 15% (v/v) isopropyl alcohol and washed using Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 1 mM MgSO4, 10 mM KCl, 50 mM β-mercaptoethanol; pH 7) [55]. The cells were resuspended in Z buffer and substrate, fluorescein di-β-D-galactopyranoside (FDG) (Sigma-Aldrich) dissolved in H2O:DMSO:ethanol (8:1:1), was added at a final concentration of 0.1 mg ml-1. The cells were then incubated for 1 hour at room temperature and diluted 1:200 in Z buffer before analysis by flow cytometry with recording of 105 events. The mean sample fluorescence was obtained from gated cells from two biological replicates. Expression and purification of recombinant proteins from E.