Salmonella enterica serovar typhimurium resistance to bile: identification and characterization of the tolQRA cluster

Abstract

Salmonella enterica serovar Typhimurium is resistant to the action of bile salts, and resistance to bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated. To identify genes necessary for bile resistance, MudJ transposon mutagenesis was performed on a strain containing a phoP mutation that results in constitutive expression of PhoP-activated genes. After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-bile plates, 14 bile-sensitive mutants were identified. Of these 14 mutants, 3 were found to retain the bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide. Southern hybridization experiments showed that all three strains contained unique insertions. DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative Salmonella orf1-tolQRA operon, with insertions in tolQ, orf1, and a gene upstream of the orf1-tolQRA operon not previously associated with Tol function (orfX). Through the use of transcriptional fusions, none of the putative tol (or tol-associated) genes were shown to be regulated by PhoPQ, bile, or the RcsC-RcsB two-component system; however, all of the genes (orfX, orf1, tolQRA) are predicted to be cotranscribed. This is the first identification of Salmonella serovar Typhimurium Tol homologs and the first demonstration of their role in bile resistance in this organism. In addition, the observed regulation, operon arrangement, and phenotypes associated with these tol genes demonstrate significant differences from their Escherichia coli homologs.

FIG. 1.

Analysis of LPS in MudJ-mutagenized bile-sensitive strains. Samples were prepared from stationary-phase cultures, electrophoresed on an SDS-15% PAGE gel, and detected by silver staining. Lanes: 1, JSG854; 2, JSG855; 3, JSG856; 4, JSG857; 5, JSG858; 6, JSG861; 7, JSG862; 8, JSG871; 9, JSG872; 10, JSG872; 11, JSG878; 12, JSG879; 13, JSG880; 14, JSG881; C−, LB5010; C+, Salmonella serovar Typhimurium PhoP^c. The strains are designated smooth (presence of the typical O-antigen ladder, as seen for the parental Salmonella serovar Typhimurium PhoP^c strain) (lane C+) or rough (absence of the typical O-antigen ladder, as seen for the galE mutant LB5010) (lane C−). Rough strains (JSG871, JSG880, and JSG881) were subsequently discarded from further characterization of the bile sensitivity phenotype.

FIG. 2.

Differentiation of unique bile-sensitive strains by Southern blot analysis. DNAs were digested with EcoRI or SalI (each cuts once within MudJ) as indicated below the gel, and blots were probed with DNA specific for the aph (kanamycin) gene of the transposon. Lanes: 1, JSG854; 2, JSG856; 3, JSG858; 4, JSG861; 5, JSG862; 6, JSG872; 7, JSG873; 8, JSG879; 9, JSG878; C, Salmonella serovar Typhimurium 14028s. The strains in lanes 5 and 8 appear to be similar, with that in lanes 5 exhibiting multiple insertions. Lanes 5 (JSG862) were not considered to contain a unique strain due to its similarity to the strain with the single insertion in lanes 8 (JSG879), an insertion which is alone responsible for the bile sensitivity phenotype. Although the strains in lanes 4 (JSG861) and 6 (JSG872) show similar banding patterns, both were still considered unique due to marked differences in the MICs for them.

FIG. 3.

Chromosomal map of the orf1-tolQRA chromosomal region showing the locations of MudJ transposon insertions. Solid triangles above the map show the locations of the MudJ insertions that mapped to this region.

FIG. 4.

Effects of bile and RcsCB on the transcription of orf1 and tolQ. Strains were grown in the presence (+) or absence (−) of a functional RcsCB two-component system. Transcription was also measured after growth in the presence (+) or absence (−) of 3% bile.

FIG. 5.

Effects of insertion mutations in upstream genes on the transcription of tolR. Mutations in orf1, orfX, and tolQ were individually placed on the chromosome upstream of a tolR::luc reporter. The level of luciferase activity (measured in relative light units [RLU]) was determined for each strain, which demonstrated the cotranscription of the entire orfX-orf1-tolQRA cluster.