Supplemental Material for. Figure S1. Identification of TetR responsive promoters in F. novicida and E. coli.
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1 Supplemental Material for Synthetic promoters functional in Francisella novicida and Escherichia coli Ralph L. McWhinnie and Francis E. Nano Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C. Canada Figure S1. Identification of TetR responsive promoters in F. novicida and E. coli. Figure S2. Immunoblot detection of TetR controlled CAT expression in F. novicida clones with synthetic promoters. Figure S3. Immunoblot detection of TetR controlled CAT expression in F. novicida clones with synthetic promoters: overexposed to show non-specific bands. Table S1. Ratio of intensity of CAT band to the intensity of non-specific band in Figure S3. Figure S4. Immunoblot detection of VgrG and non-specific bands in F. novicida. Table S2. Ratio of intensity of CAT band to the intensity of non-specific band in Figure S4. Table S3. Ratio of intensity of VgrG band to the intensity of non-specific band in Figure S4. Figure S5. TetR control in F. novicida of virulence factor DotU associated with inducible promoter, P40, or constitutive promoter, P146. Figure S6. Partial restoration of intramacrophage growth of F. novicida vgrg mutant by expressing plasmid borne vgrg with promoter P40. Figure S7. Plasmid borne vgrg lacking a promoter does not rescue growth of F. novicida in macrophages. Figure S8. Sequences of five synthetic constitutive promoter regions that function in F. novicida. Figure S9. β-galactosidase expression of E. coli synthetic promoters in F. novicida expressing TetR. Figure S10. Sequences of synthetic teto-containing promoters selected for function in E. coli. Figure S11. Sequences of F. novicida minimal promoters. 1
2 A B C D 2
3 Supplemental Figure S1. Identification of TetR responsive promoters in F. novicida (Panels A and B) and E. coli (Panels C and D) through a screen of Cm R clones for approximate β- galactosidase levels as determined by X-gal hydrolysis. Panels A and B, synthetic F. novicida promoters. Top half of each image is with ATc, bottom is without ATc. Plates were grown overnight then overlaid with X-gal-soaked filter paper and color was allowed to develop for 12 h at 30 C. The filter paper was then removed and imaged. Panel A is plate 1, Panel B is plate 2. The DNA sequences of each of the promoters represented in these images are provided in Data set S1. The names of the promoters correspond to the plate number and grid location. For example, promoter 1-A2 corresponds to the promoter in the recombinant clone shown on plate 1, position A2. Note that some promoter regions appear to have resulted from the ligation of two synthetic oligonucleotides. Panels C and D. E. coli strains with synthetic promoters isolated in E. coli (separate experiment from promoter selection in F. novicida). X-gal was included in the agar plates and images are of plates. Panel C plate does not have ATc; Panel D plate includes ATC. The DNA sequences of each of the promoters represented in these images are provided in Data set S2. The names of the promoters correspond to the plate number and grid location. Figure S2. Immunoblot detection of TetR controlled CAT expression in F. novicida clones with synthetic promoters. Data corresponds to that presented in Figure 3. Samples here are heavily loaded, revealing some examples of leaky expression. The samples used are the same as those in Figures 3 and S3, but the blotting procedure was performed independently. 3
4 Figure S3. Immunoblot detection of TetR controlled CAT expression in F. novicida clones with synthetic promoters: overexposed to show non-specific bands. Digital over-exposure of immunoblots shown in Figure 3 reveals reactivity of antibody with non-specific bands in all of the lanes containing bacterial cell extract; reactivity with CAT appears in some lanes. The relatively even intensity of the non-specific bands in all of the lanes with bacterial extract show that the amount of protein loaded was approximately equal for all of these lanes. See ratio of band intensities (CAT/non-specific band) in Table S1. 4
5 Table S1. Ratio of intensity of CAT band to the intensity of non-specific band in Figure S3. Band intensity was determined using Image Studio Lite software (Li-Cor). pmp829-cat/lacz + ATc was defined as zero intensity and P40-cat + ATc as 100 with the others reported as a percentage of P40-cat + ATc Strain (all are TetR + ) CAT intensity Non-specific intensity pmp pmp829-cat/lacz + ATc Pbfr-cat P40-cat P40-cat + ATc P94-cat P94-cat + ATc P135-cat P135-cat + ATc PZ P39-cat P39-cat + ATc P20-cat P20-cat + ATc P142-cat P142-cat + ATc P146-cat P165-cat CAT relative intensity normalized to non-specific intensity* *Band intensity was determined using Image Studio Lite software (Li-Cor). To find relative intensity normalized to non-specific band the intensity of the CAT band was divided by the intensity of the non-specific band. Background was then subtracted out by defining the promoterless control pmp829-cat/lacz with ATc as zero CAT/non-specific intensity and subtracting this value from that of each other strain. The values where then scaled to be presented as a percentage of P40-cat + ATc 5
6 Figure S4. Immunoblot detection of CAT (upper panel), VgrG (lower panel) and non-specific bands. Digital over-exposure of immunoblots shown in Figure 4 reveals reactivity of antibody with non-specific bands in all of the lanes containing bacterial cell extract; reactivity with CAT or VgrG appears in some lanes. The relatively even intensity of the nonspecific bands in all of the lanes with bacterial extract show that the amount of protein loaded was approximately equal for all of these lanes. See ratio of band intensities (CAT or VgrG/non-specific band) in Tables S2 and S3. 6
7 Table S2. Ratio of intensity of CAT band to the intensity of non-specific band in Figure S4. CAT relative intensity CAT Non-specific normalized to nonspecific intensity* Strain intensity intensity WT (pmp829) ΔvgrG tetr + (pmp829) ΔvgrG tetr + (829::cat/vgrG) ΔvgrG (829::P40-cat/vgrG) ΔvgrG tetr + (829::P40-cat/vgrG) ΔvgrG tetr + (829::P40-cat/vgrG) + ATc ΔvgrG (829::P18-cat/vgrG) ΔvgrG tetr + (829::P18-cat/vgrG) ΔvgrG tetr + (829::P18-cat/vgrG) + ATc *Band intensity was determined using Image Studio Lite software (Li-Cor). To find relative intensity normalized to non-specific band the intensity of the CAT band was divided by the intensity of the nonspecific band. Background was then subtracted out by defining the promoterless control ΔvgrG tetr + (829::cat/vgrG) as zero CAT/non-specific intensity and subtracting this value from that of each other strain. The values where then scaled to be presented as a percentage of ΔvgrG tetr + (829::P40-cat/vgrG) + ATc. Table S3. Ratio of intensity of VgrG band to the intensity of non-specific band in Figure S4. Nonspecific intensity VgrG relative intensity normalized to nonspecific intensity* Strain VgrG intensity WT (pmp829) ΔvgrG tetr+ (pmp829) ΔvgrG tetr + (829::cat/vgrG) ΔvgrG (829::P40-cat/vgrG) ΔvgrG tetr + (829::P40-cat/vgrG) ΔvgrG tetr + (829::P40-cat/vgrG) + ATc ΔvgrG (829::P18-cat/vgrG) ΔvgrG tetr + (829::P18-cat/vgrG) ΔvgrG tetr + (829::P18-cat/vgrG) + ATc *Band intensity was determined using Image Studio Lite software (Li-Cor). To find relative intensity normalized to non-specific band the intensity of the VgrG band was divided by the intensity of the nonspecific band. Background was then subtracted out by defining the no VrgG control ΔvgrG tetr + (pmp829) as zero CAT/non-specific intensity and subtracting this value from that of each other strain. The values where then scaled to be presented as a percentage of ΔvgrG tetr + (829::P40-cat/vgrG) + ATc. Strain ΔvgrG tetr + (pmp829) was defined as zero rather than the promoterless control strain, ΔvgrG tetr + (829::cat/vgrG). This was done because ΔvgrG tetr + (829::cat/vgrG) has high background intensity in this blot resulting from what appears to be spill-over of signal from the neighboring lane. 7
8 Figure S5. TetR control in F. novicida of virulence factor DotU associated with inducible promoter, P40, or constitutive promoter, P146. The absence of any promoter (lanes 2 and 3) results in no DotU being produced. In the absence of TetR the P40 promoter drives DotU expression (lane4). With P40 in front of DotU, and in the presence of TetR, the addition of ATc greatly increases the expression of DotU (lanes 5 and 6). Figure S6. Partial restoration of intramacrophage growth of F. novicida vgrg mutant by expressing plasmid borne vgrg with promoter P40. Nearly full recovery of the intramacrophage growth phenotype is dependent on relieving repression of P40 by the addition of ATc (uninduced versus induced, p<0.0001; induced versus WT, p=0.08). Partial intramacrophage growth is seen in a TetR + strain with P40 driving vgrg even in the absence of ATc (growth versus growth of ΔvgrG, p=0.008). Comparisons were done by two-way ANOVA. Figure S7. Plasmid borne vgrg lacking a promoter does not rescue growth of F. novicida in macrophages. This control demonstrates that there are no plasmid sequences found in pmp329 acting as a promoter to drive the expression of vgrg. The data for the wild type and the ΔvgrG strain are the same as that shown in Figure 5. Error bars represent S.E.M. The ΔvgrG tetr + (829::vgrG) growth is not statistically different from the growth of the ΔvgrG strain (p=0.67). Comparisons were done by two-way ANOVA. 8
9 Figure S8. Sequences of five synthetic constitutive promoters regions that function in F. novicida. All of these promoters had the same organization of teto relative to the -10 and -35 regions. The promoters P142, 143, 146, 139, and 165 had 11, 12, 10, 10, and 19 bp between the - 35 region and teto, respectively. 9
10 Figure S9. β-galactosidase expression of E. coli synthetic promoters in F. novicida expressing TetR. Plasmids containing promoters selected in E. coli ( PE ) were introduced into F. novicida and their activity was measured by assaying β-galactosidase activity. For comparison, the LacZ activity driven by promoters isolated in F. novicida, P20, P40 and P146, or the natural promoter, Pbfr, are shown. Values on the y-axis are arbitrary luminosity units. Error bars represent S.E.M. Figure S10. Organization of synthetic teto-containing promoters selected for function in E. coli. The format is as in Fig. 6. All promoters presented here are tightly controlled by TetR in E. coli, with exception of P132 which generates reduced, but significant, expression in the absence of ATc. Flanking regions for each promoter can be found in the corresponding GenBank files (see Materials and Methods). The DNA sequences of 88 synthetic E. coli promoters are available in Supplemental Data Set 2. 10
11 Figure S11. Sequences of F. novicida minimal promoters. A. Sequences of minimal-length promoters derived from constitutive promoters isolated in F. novicida. B. The upstream randomly generated sequence and the downstream plasmid sequences the flank the minimal promoters. The same flanking sequences are present in all three promoters. 11
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