The R. capsulatus rbaV and rbaW genes are in a predicted two-gene operon (Figure 1) with the start of rbaW overlapping rbaV, suggesting possible translational coupling of the two genes. No predicted σ factor-encoding gene could be found near these genes [14]. An analysis of orthologous neighbourhood GDC 0449 regions using the IMG database (http://​img.​jgi.​doe.​gov/​cgi-bin/​w/​main.​cgi; [59]) showed that this

is different than what is found outside of the Rhodobacterales order (Figure 1). Some species, such as Rhodopseudomonas palustris, also have an rsbY homologue in a predicted 3-gene operon with rsbV and rsbW homologues (Figure 1), whereas gram-positive Bacillus (Figure 1) and Staphylococcus[15] species have other genes associated with rsbVW, including sigB that encodes the VEGFR inhibitor cognate sigma factor. rba mutant phenotypes Insertional disruptions of the rba genes in R. capsulatus demonstrated that loss of the proteins encoded by these genes affected RcGTA production. The rbaW mutant showed an increase in RcGTA gene transfer activity of 2.85-fold relative to SB1003 (Figure 2A), which agreed with an increase in RcGTA capsid protein levels inside and outside the cells (Figure 2B). This mutant had no observable differences in viable cell number or colony morphology relative to SB1003 (Figures 3

and 4). Nec-1s price complementation with wild type rbaW alone did not restore RcGTA activity or capsid levels (Figure 2), but complementation with the complete predicted transcriptional unit of rbaV and rbaW resulted in wild type RcGTA gene transfer activity (Figure 2), possibly indicating translational coupling between rbaV and rbaW is important

for normal expression of rbaW. However, we do believe rbaW is expressed to some degree from pW because it restores flagellar motility to the rbaW mutant, which is non-motile (Mercer and Lang, unpublished). Figure 2 Effects of rba mutations and in trans expression of rba genes on RcGTA gene transfer activity and protein levels. A. The ratio of gene transfer activity for each indicated strain relative to the parental strain, SB1003. The gene transfer activity was determined as an average relative to SB1003 in 3 replicate bioassays and the error bars represent the standard deviation. RcGTA production levels Erythromycin that differed significantly from the wild type were identified by analysis of variance (ANOVA) and are indicated by an asterisk (*; p < 0.05) or two asterisks (**; p < 0.1). B. Western blot detection of the RcGTA major capsid protein in the cells and culture supernatants of indicated strains. Blots were performed on all replicate gene transfer bioassay cultures (in A) and one representative set of blots is shown. Figure 3 Effects of rba mutations and in trans expression of rba genes on R. capsulatus colony forming unit numbers in stationary phase.