Figure 5 Analysis of BsaN box requirements for transcription activation by BsaN/BicA. The ability of BsaN/BicA to directly activate the expression of truncated promoters #Nutlin3a randurls[1|1|,|CHEM1|]# was examined by providing regulatory genes in trans and measuring β-galactosidase activities arising from the expression of transcriptional promoter-lacZ fusions in E. coli DH5α. The top sequence of each gene includes the intact promoter region; sequence 1 is

deleted up to the BsaN box; sequence 2 also includes a 6 nucleotide deletion of the BsaN box. Effect of BsaN/BicA on the expression of A. PbicA-lacZ fusion, B. PvirA-lacZ fusion and C. Ps1518-lacZ fusion; Ps1518 denotes the promoter region of BPSS1518. Effect of BsaN/BicA on the expression of D. PbprD-lacZ fusion and E. PbopA-lacZ fusion. BprP directly activates bsaN and bsaM In the hierarchical control of T3SS3 and T6SS1 expression, BspR was suggested to activate the expression of bprP [14]. Previously, BprP was shown to bind sequences

upstream of bsaM and bsaN (refer to Figure 2C for gene location) [14], suggesting that it directly activates their transcription. bsaN is the first orf of the putative operon that encodes structural components of T3SS3 (Figure 2C) and is divergently transcribed from bsaM. To better understand how bsaN expression itself is controlled, we examined the relationships to its upstream regulators BspR and BprP using the LacZ fusion assay as described previously [8]. Plasmids with either bspR or bprP under find more arabinose induction control were introduced into E. coli containing plasmids with either a bsaN-lacZ fusion or a bsaM-lacZ fusion. A bprP-lacZ fusion served as control for BspR regulation. The ability of BspR and BprP to directly activate bsaN-lacZ, bsaM-lacZ and bprP-lacZ expression was determined by measuring β-galactosidase activity. As

expected, BprP activated both the bsaM and bsaN promoters click here in E. coli (Figure 6A, B). The presence of bprQ, a gene immediate downstream from bprP, had no effect on the activity of BprP. Furthermore, BprP did not activate its own promoter in E. coli (data not shown). However, BspR was not able to activate the promoter of bprP demonstrating that this regulator is not active in E. coli or that additional factors are required for activation (Figure 6C). Figure 6 Activation of bsaM and bsaN promoters by BprP in E. coli. The ability of BprP to directly activate the expression of promoters in the presence and absence of BprQ was examined by providing the bprP and bprQ genes in trans and measuring β-galactosidase activities arising from the expression of transcriptional promoter-lacZ fusions in E. coli DH5α. A. Effect of BprP and BprQ on the expression of PbsaN-lacZ fusion. B. Effect of BprP and BprQ on the expression of PbsaM-lacZ fusion. C. Effect of BspR on the expression of PbprP-lacZ fusion. *p < 0.05.