Table 1.
Strains of Salmonella enterica serovar Typhimurium.
Table 2.
Plasmids constructed for this study.
Figure 1.
Diagram of the region containing STM2209-STM2208 on the Salmonella enterica chromosome.
The homologous regions of Salmonella bongori, E. coli, and Shigella flexneri are also shown. The STM2209-STM2208 operon is shown in yellow. Black arrows represent conserved genes. White arrows represent non conserved genes. Grey arrows represent genes found at a different chromosome location on the S. enterica chromosome.
Figure 2.
Regulation of STM2209-STM2208 by Dam methylation.
A. Levels of STM2209 and STM2208 mRNAs, measured by qRT-PCR (Dam+: white histograms; Dam−: black histograms). Level of STM2209 mRNA in Dam− background is considered 100%. Values are averages and standard deviations from 7 independent experiments. B. ß-galactosidase activity of transcriptional STM2209::lac and STM2208::lac fusions in Dam+ and Dam− backgrounds (white and black histograms, respectively). Values are averages and standard deviations from 3 independent experiments. C. ß-galactosidase activities of translational STM2209::lac and STM2208::lac fusions in Dam+ and Dam− backgrounds (white and black histograms, respectively). Values are averages and standard deviations from 3 independent experiments. D. Western blot analysis of STM2209-3xFLAG and STM2208-3xFLAG proteins in Dam+ and Dam− backgrounds.
Figure 3.
Identification of the transcription initiation site of STM2209-STM2208 by primer extension.
Putative -35 and -10 promoter modules and the +1 site are shown in boldface. The transcription initiation site is indicated by an arrow.
Figure 4.
Regulation of STM2209-STM2208 expression by Dam methylation and OxyR.
A. Effect of an oxyR null mutation on the ß-galactosidase activity of translational STM2209::lac and STM2208::lac fusions in Dam+ and Dam− backgrounds (white and black histograms, respectively). Values are averages and standard deviations from 3 independent experiments. B. Western blot analysis of the effect of an oxyR null mutation on the levels of STM2209-3xFLAG and STM2208-3xFLAG proteins in Dam+ and Dam− backgrounds.
Figure 5.
Visual observation of phase variation on LB + X-gal plates in strains carrying an STM2208::lac fusion in different backgrounds.
Strains in the upper row are SV5679 (Dam+ OxyR+), SV5683 (Dam− OxyR+), SV7031 (Dam+ OxyR+ mut. GATC) and SV7032 (Dam− OxyR+ mut. GATC). OxyR− derivatives (SV5989, SV5990, SV7232 and SV7233) are shown in the lower row.
Figure 6.
Effect of GATC mutations on STM2209-STM2208 expression.
A. Diagram of the promoter region of STM2209-STM2208, showing GATC sites (red squares), putative OxyR-binding-sites (orange bars), putative −35 and −10 modules (green boxes) and the transcription initation site (black arrow). B. Effect of eliminating the 4 GATC sites upstream the STM2209-STM2208 promoter on STM2209-STM2208 expression, monitored by comparing the ß-galactosidase activity of a translational STM2208::lac fusion in Dam+ and Dam− backgrounds (white and black histograms, respectively). Values are averages and standard deviations from 6 independent experiments. C. Effect of eliminating the 4 GATC sites upstream the STM2209-STM2208 promoter on STM2209-STM2208 expression, monitored by Western blot analysis of STM2208-3xFLAG levels in different backgrounds.
Figure 7.
Distribution of STM2209 and STM2208 proteins tagged with a 3xFLAG epitope in subcellular fractions of S. enterica serovar Typhimurium.
Anti-FLAG Western hybridization is shown for three fractions: cytoplasm, inner membrane, and outer membrane. The volume loaded for all fractions was normalized to the same number of bacteria (7×107 c.f.u.).
Figure 8.
Analysis of the in vivo interaction between STM2209 and STM2208 using the BACTH system.
The E. coli BTH101 strain was co-transformed with plasmids encoding fusion proteins or empty. The basal level of ß-galactosidase activity measured with empty vectors was approximately 90 Miller units. Values are averages and standard deviations from 3 independent experiments.
Figure 9.
Effect of constitutive expression of STM2209-STM2208 on adsorption of bacteriophage P22 to S. enterica.
The efficiency of P22 attachment to S. enterica is shown as the percentage of non adsorbed phages relative to the initial number. Strains are represented by black squares (wild type), white squares (SV6013, ΔSTM2209-STM2208), black circles (SV6401, mut. GATC) and white circles (SV6976, mut. GATC ΔSTM2209-STM2208). Values are averages and standard deviations from 6 independent experiments.
Figure 10.
Lipopolysaccharide profiles of the wild type strain (lane 1), SV6013 (ΔSTM2209-STM2208) (lane 2), SV6401 (mut. GATC) (lane 3) and SV6976 (mut. GATC ΔSTM2209-STM2208) (lane 4), as observed by electrophoresis and silver staining.
Figure 11.
Survival in presence of 30% guinea pig serum.
Strains are represented by black squares (wild type), white squares (SV6013, ΔSTM2209-STM2208), black circles (SV6401, mut. GATC) and white circles (SV6976, mut. GATC ΔSTM2209-STM2208). Values are averages and standard deviations from 5 independent experiments.
Figure 12.
Rate of intramacrophage proliferation for the wild type strain (lane 1), SV6013 (ΔSTM2209-STM2208) (lane 2), SV6401 (mut. GATC) (lane 3) and SV6976 (mut. GATC ΔSTM2209-STM2208) (lane 4).
Values are averages and standard deviations from 3 independent experiments.