FliZ Regulates Expression of the Salmonella Pathogenicity Island 1 Invasion Locus by Controlling HilD Protein Activity in Salmonella enterica Serovar Typhimurium

FliZ activates hilA through hilD. β-Galactosidase (β-Gal) activity was examined in strains containing hilA-lacZ transcriptional fusions and the indicated plasmids and/or mutations. The strains were grown under SPI1-inducing conditions. β-Galactosidase activity units are defined as (micromoles of ONP formed per minute × 10³)/(OD₆₀₀ × milliliter of cell suspension) and are reported as means ± standard deviations (error bars) for four replicate samples relative to the results for the wild-type (WT) strain. The strains used were JS749, JS778, JS946, JS798 to JS807, JS947, and JS948.

FliZ activation of hilA is dependent on HilD. (A) β-Galactosidase activity in strains containing a hilA-lacZ transcriptional fusion and the indicated mutations after growth under SPI1-inducing conditions. (B) β-Galactosidase activity of strains containing a hilA-lacZ transcriptional fusion and indicated mutations with rtsA under the control of a tetracycline-regulated promoter. The strains were grown under SPI1-inducing conditions with the indicated concentrations of tetracycline (Tet). The strains used were JS749 and JS950 to JS956. β-Galactosidase activity units are defined as (μmol of ONP formed per min × 10³)/(OD₆₀₀ × ml of cell suspension) and are reported as means ± standard deviations (n = 4).

FliZ acts at the level of HilD protein. (A) β-Galactosidase activity in strains containing either a hilD-lacZ transcriptional or translational fusion and the indicated plasmids. The fusion joints of the two constructs are identical (14). The strains were grown under SPI1-inducing conditions with 10 mM arabinose. Arabinose is required for induction of pHilC but was included in all cultures. The strains used were JS883, JS957, JS958, JS892, JS959, and JS960. (B) β-Galactosidase activity in strains containing a hilA-lacZ transcriptional fusion and the indicated mutations. The strains were grown under SPI1-inducing conditions (left panel) or in LB medium (0.5% NaCl) with the indicated tetracycline concentrations and with shaking (right panel). The strains used were JS749, JS778, JS633, JS961, JS962, and JS963. β-Galactosidase activity units are defined as (μmol of ONP formed per min × 10³)/(OD₆₀₀ × ml of cell suspension) and are reported as means ± standard deviations (n = 4).

FliZ regulates hilA independently of HilE and RpoS. (A) β-Galactosidase activity in strains containing a hilA-lacZ transcriptional fusion and the indicated mutations after growth under SPI1-inducing conditions. The strains used were JS749, JS576, JS577, JS579, JS633 to JS636, and JS964 to JS967. (B) β-Galactosidase activity of strains containing sodCII, katE, or hilA transcriptional fusions in otherwise wild-type or rpoS backgrounds with or without pFliZ. The strains were grown under SPI1-inducing conditions. The strains used were JS749, JS968, JS969, JS970, JS909, JS910, JS971, JS972, JS531, JS541, JS973, and JS974. β-Galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10³/(OD₆₀₀ × ml of cell suspension) and are reported as means ± standard deviations (n = 4).

HilD protein levels in relation to FliZ and HilE. The hilD-3×FLAG construct is under tetRA control, and all strains contained a hilA-lacZ transcriptional fusion and the indicated mutations or plasmids. (A) HilD protein levels in stationary-phase cells. The strains were grown under SPI1-inducing conditions with 0.4 μg/ml tetracycline. The cultures were divided to determine β-galactosidase activity and to perform the Western blot analysis to detect FLAG-tagged HilD. Extracts from equal concentrations of cells were loaded on the gel. The intensities of the bands were quantified using ImageJ and are presented above the gel relative to the wild-type strain (set at 1). Note that the doublets seen are artifacts of this particular gel. The strains used were JS975 to JS979. (B) HilD protein stability in cells in late log phase. The genotypes for lon and fliZ strains are indicated to the left of the gels (++ indicates overproduction [pFliZ]). The cells were induced with 0.8 μg/ml tetracycline and grown in LB medium (0.5% NaCl) with shaking to late log phase, and antibiotics were added to stop transcription and translation. β-Galactosidase activity produced from the hilA-lacZ fusion in the samples shown on these gels was determined from each sample taken at time zero. ImageJ was used for half-life analysis. The half-life was calculated from 2 (lon) or 3 replicates of the experiments. The mean half-life ± SEM is listed for each background. The strains used were JS975, JS976, JS977, JS980, and JS981.