Rcs and PhoPQ regulatory overlap in the control of Salmonella enterica virulence

Abstract

Genetic screens based on the use of MudJ-generated lac fusions permitted the identification of novel genes regulated by the Rcs signal transduction system in Salmonella enterica serovar Typhimurium. Besides genes that are also found in the Escherichia coli genome, our screens identified Salmonella-specific genes regulated by RcsB, including bapA, siiE, srfA, and srfB. Here we show that the srfABC operon is negatively regulated by RcsB and by PhoP. In vivo studies using mutants with constitutive activation of the Rcs and/or PhoPQ system suggested that there is an overlap between these regulatory systems in the control of Salmonella virulence.

FIG. 1.

Identification of IgaA- and RcsB-regulated genes. (A) Diagram of the genetic screen for identification of IgaA-regulated genes in S. enterica serovar Typhimurium. A plasmid carrying a wild-type igaA allele under the control of an arabinose-inducible promoter (P_BAD) was introduced into a strain harboring a null igaA mutation. igaA is expressed in the presence of arabinose as the sole carbon source but not in the presence of glucose. lac transcriptional fusions were generated using MudJ. The expression pattern of 10,000 independent fusions was monitored in glucose- and arabinose-containing LB medium plates supplemented with X-Gal. (B) Diagram of the genetic screen carried out to identify RcsB-regulated genes. A plasmid with a copy of the wild-type rcsB gene under P_BAD control was introduced into a strain harboring a null gmm mutation. rcsB is expressed in arabinose medium but not in glucose medium. lac transcriptional fusions were generated, as described above, with MudJ. The expression pattern of 20,000 independent fusions was monitored in glucose- and arabinose-containing LB medium plates supplemented with X-Gal. The gmm mutation was introduced to prevent mucoidy, thereby facilitating comparison of colony colors in different media. Ap, ampicillin; Km, kanamycin; Cm, chloramphenicol.

FIG. 2.

Regulation of srfA, sfrB, and srfC by RcsB at the protein level. Extracts from ATCC 14028 (wild-type strain) derivatives expressing 3×FLAG-tagged SrfA, SrfB, or SrfC were resolved by 10% SDS-PAGE. The same amount of protein was loaded in each lane. Immunoblotting was performed with a monoclonal anti-FLAG antibody. rcsC54 and igaA5 are mutants with constitutive activation of the Rcs system. wt, wild type.

FIG. 3.

Transcriptional repression of srfABC by PhoP. Expression levels of srfB (A) and prgH and ssaV (B) were monitored with lacZ transcriptional fusions. Strains carrying the indicated mutations were cultured in the following media: LB medium for SPI-1 induction; LPM for SPI-2 induction; and LB medium containing EDTA for Mg²⁺ chelation. The β-galactosidase activities shown were measured in stationary-phase cultures, but similar results were obtained in exponential cultures. The data represent the averages and standard deviations from two experiments. wt, wild type.

FIG. 4.

Regulation of SrfC-3×FLAG levels by PhoP. Strains carrying the indicated mutations were cultured in LB medium (for SPI-1 induction), LPM (for SPI-2 induction), and LB medium containing EDTA (for Mg²⁺ chelation). Protein extracts were resolved by 10% SDS-PAGE. The same amount of protein was loaded in each lane. Immunoblotting was performed with a monoclonal anti-FLAG antibody. wt, wild type.

FIG. 5.

Overlap of PhoPQ and Rcs systems in the control of virulence: analysis of the pho-24 igaA1 double mutant in mixed infections with either the pho-24 or igaA1 single mutant. The indices represented are CIs (single mutant versus wild type) and COIs (double mutant versus single mutant). The strains used in each mixed infection are represented by the relevant mutations, as indicated under the bars. COIs were compared to 1.0 and to the CI relevant in each case. COIs are significantly different from the corresponding CIs and from 1.0 (P < 0.05). wt, wild type.