RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium

Abstract

Salmonella enterica serovar Typhimurium encounters numerous host environments and defense mechanisms during the infection process. The bacterium responds by tightly regulating the expression of virulence genes. We identified two regulatory proteins, termed RtsA and RtsB, which are encoded in an operon located on an island integrated at tRNA(PheU) in S. enterica serovar Typhimurium. RtsA belongs to the AraC/XylS family of regulators, and RtsB is a helix-turn-helix DNA binding protein. In a random screen, we identified five RtsA-regulated fusions, all belonging to the Salmonella pathogenicity island 1 (SPI1) regulon, which encodes a type III secretion system (TTSS) required for invasion of epithelial cells. We show that RtsA increases expression of the invasion genes by inducing hilA expression. RtsA also induces expression of hilD, hilC, and the invF operon. However, induction of hilA is independent of HilC and HilD and is mediated by direct binding of RtsA to the hilA promoter. The phenotype of an rtsA null mutation is similar to the phenotype of a hilC mutation, both of which decrease expression of SPI1 genes approximately twofold. We also show that RtsA can induce expression of a SPI1 TTSS effector, slrP, independent of any SPI1 regulatory protein. RtsB represses expression of the flagellar genes by binding to the flhDC promoter region. Repression of the positive activators flhDC decreases expression of the entire flagellar regulon. We propose that RtsA and RtsB coordinate induction of invasion and repression of motility in the small intestine.

FIG. 1.

Effect of RtsA and RtsB on secretion of proteins into culture supernatants. The strains are ΔrtsAB7 and contain the plasmid and mutations specified. Overnight cultures were subcultured into LB (without salt)-ampicillin-0.2% l-arabinose and grown with aeration to an OD₆₀₀ of ∼0.8. Culture supernatants were prepared as described in Materials and Methods. An equivalent amount of sample from the supernatant of each strain was separated by SDS-12.5% PAGE. The gel was stained for total protein with GelCode Blue. Proteins of greater than ∼25 kDa are shown. The strains used were plasmid-containing derivatives of JS250, JS270, JS271, and JS272.

FIG. 2.

Effect of RtsA, RtsB, or RtsAB on the expression of the SPI1 regulatory genes (A) and the flagellar genes (B). The strains are ΔrtsAB7 and contain pBAD30, pRtsA, pRtsB, or pRtsAB and a lacZ transcriptional fusion to the specified gene. Overnight cultures were subcultured into LB (without salt)-ampicillin-0.2% l-arabinose and grown to an OD₆₀₀ of ∼0.6. β-Galactosidase activity units are defined as (micromoles of ONP formed minute⁻¹) × 10³/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. The strains used were plasmid-containing derivatives of JS273 through JS278.

FIG. 3.

Effect of ΔrtsA5, ΔrtsB6, or ΔrtsAB7 mutations on the expression of RtsA-regulated genes. Stationary-phase cultures were subcultured 1/100 into LB-1% NaCl and grown statically overnight at 37°C, at which point β-galactosidase activities were determined. β-Galactosidase activity units are defined as (micromoles of ONP formed minute⁻¹) × 10³/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. The strains used were plasmid-containing derivatives of JS271, JS275, JS276, and JS279 through JS299. WT, wild type.

FIG. 4.

Effect of mutations in SPI1 regulatory genes on RtsA-induced expression of hilA-lac⁺ (A) and invF-lac⁺ (B) fusions. The hilA-lac⁺ or invF-lac⁺ transcriptional fusion strains are ΔrtsAB; contain pBAD30, pRtsA, pRtsB, or pRtsAB; and have the indicated regulatory gene(s) deleted. The hilC, hilD, and hilA mutations are simple deletion-insertions of a chloramphenicol cassette. The ΔhilC-D mutation deletes hilC to hilD, including the prgHIJK operon, and the ΔhilC-A deletion removes hilC, prgHIJK, hilD, and hilA. Overnight cultures were subcultured into LB (without salt)-ampicillin-0.2% l-arabinose and grown to an OD₆₀₀ of ∼0.6 before assay of β-galactosidase. β-Galactosidase activity units are defined as (micromoles of ONP formed minute⁻¹) × 10³/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 2. The strains used were plasmid-containing derivatives of JS275, JS276, and JS300 through JS305. WT, wild type.

FIG. 5.

Gel shift analysis of RtsA binding to the hilA promoter region. Binding reaction mixtures contained ∼0.1 ng of ³²P-labeled hilA promoter region DNA corresponding to −189 to +19 bp from the start site of transcription. Increasing amounts of the Myc-RtsA whole-cell extract from 2 to 1,000 ng were included in the indicated reaction mixtures. The hilA DNA lane has no extract. The negative control (Neg Cntrl) lane contains 1,000 ng of whole-cell extract of the vector control strain. One hundred nanograms of anti-Myc antibody (Ab) was added to the indicated reaction mixtures. The strain used was MG1655 with either pBAD or pCE81.

FIG. 6.

Effect of invF and hilA mutations on RtsA induction of sopA, sopB, invC, and icgA fusions. The strains are ΔrtsAB7; contain pBAD30, pRtsA, pRtsB, or pRtsAB; and are ΔhilA::Cm or ΔinvF::Cm. The stains contain a lacZ transcriptional fusion to the gene specified. Overnight cultures were subcultured into LB (without salt)-ampicillin-0.2% l-arabinose and grown to an OD₆₀₀ of ∼0.6 before assay of β-galactosidase. β-Galactosidase activity units are defined as (micromoles of ONP formed minute⁻¹) × 10³/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 2. The strains used were plasmid-containing derivatives of JS306 through JS317. WT, wild type.

FIG. 7.

Effect of invF, hilA, and hilC-D mutations on RtsA induction of slrP (A) and ability of HilC, HilD, and RtsA to induce expression of hilA (B) and slrP (C). (A) The strains are ΔrtsAB7 and contain pBAD30 or pRtsA; ΔhilC-D::Cm, ΔhilA::Cm, or ΔinvF::Cm; and an slrP-lac fusion. (B and C) The strains contain a lac transcriptional fusion with hilA and slrP, respectively, and the chromosomal ΔrtsA ΔhilC-D::Cm mutations along with the plasmid specified. pHilD is pLS118, and pHilC is pLS119 (72). Overnight cultures were subcultured into LB (without salt)-ampicillin-0.2% l-arabinose and grown to an OD₆₀₀ of ∼0.6 before assay of β-galactosidase. β-Galactosidase activity units are defined as (micromoles of ONP formed minute⁻¹) × 10³/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. The strains used were plasmid-containing derivatives of JS318 through JS323. WT, wild type.

FIG. 8.

Effect of RtsB production on motility. The strains are ΔrtsAB7 (JS250) and contain pBAD30 or pRtsB. Motility assays were performed in plates with 0.3% agar supplemented with either 0.2% glucose or 0.2% l-arabinose.

FIG. 9.

Gel shift analysis of RtsB binding to the flhDC promoter region. Binding reaction mixtures contained ∼0.1 ng of ³²P-labeled flhDC promoter region DNA corresponding to −4 to +106 bp from the start site of transcription. Increasing amounts of the Myc-RtsB whole-cell extract from 2 to 1,000 ng were included in the indicated reaction mixtures. The flhDC DNA lane has no extract. The negative control (Neg Cntrl) lane contains 1,000 ng of whole-cell extract of the vector control strain. One hundred nanograms of anti-Myc antibody (Ab) was added to the indicated reaction mixtures. The strain used was MG1655 with either pBAD or pCE82.