Role of Salmonella enterica serovar Typhimurium two-component system PreA/PreB in modulating PmrA-regulated gene transcription

Abstract

The PmrA/PmrB two-component system encoded by the pmrCAB operon regulates the modification of Salmonella enterica serovar Typhimurium lipopolysaccharide leading to polymyxin B resistance. PmrA and PhoP are the only known activators of pmrCAB. A transposon mutagenesis screen for additional regulators of a pmrC::MudJ fusion led to the identification of a two-component system, termed PreA/PreB (pmrCAB regulators A and B), that controls the transcription of the pmrCAB operon in response to unknown signals. The initial observations indicated that insertions in, or a deletion of, the preB sensor, but not the preA response regulator, caused upregulation of pmrCAB. Interestingly, the expression of pmrCAB was not upregulated in a preAB mutant grown in LB broth, implicating PreA in the increased expression of pmrCAB in the preB strain. This was confirmed by overexpression of preA(+) in preAB or preB backgrounds, which resulted in significant upregulation or further upregulation of pmrCAB. No such effect was observed in any tested preB(+) backgrounds. Additionally, an ectopic construct expressing a preA[D51A] allele also failed to upregulate pmrC in any of the pre backgrounds tested, which implies that there is a need for phosphorylation in the activation of the target genes. The observed upregulation of pmrCAB occurred independently of the response regulators PmrA and PhoP. Although a preB mutation led to increased transcription of pmrCAB, this did not result in a measurable effect on polymyxin B resistance. Our genetic data support a model of regulation whereby, in response to unknown signals, the PreB sensor activates PreA, which in turn indirectly upregulates pmrCAB transcription.

FIG. 1.

Map of preAB genes, mutations, and plasmids. Boxes represent open reading frames. The solid circle indicates a Tn10d insertion. Locations of the preA and preB deletions are noted below the gene map, and a solid line delimiting the region cloned into the expression plasmids pJSG2581 and pJSG2558 is shown.

FIG. 2.

Effect of preA or preB mutations on pmrCAB transcription. Salmonella strains were grown in LB medium to an OD₆₀₀ of 0.6 before β-galactosidase assays were performed. Activities are expressed in picomoles of 4-methylumbelliferone (MU) per minute per OD unit. Error bars indicate the standard deviations. The following strains were used: JSG215 (pmrC-lacZ), JSG1039 (preB::Tn10d pmrC-lacZ), JSG2003 (ΔpreA pmrC-lacZ), JSG2115 (ΔpreB pmrC-lacZ), and JSG2624 (ΔpreAB pmrC-lacZ). wt, wildtype.

FIG. 3.

Ectopic expression of complementing and suppressing regulatory genes. (A) Complementation of preB mutant. (B) Ectopic expression of preA⁺. (C) Ectopic expression of preA[D51A]. (D) Western blot analysis of Salmonella whole-cell lysates using polyclonal anti-His₆-PreA (α PreA). Lane 1, molecular mass marker; lane 2, JSG1998/pBAD18; lane 3, JSG1998/pJSG2558; lane 4, JSG1998/pJSG2700. Salmonella strains were grown in LB medium with l-arabinose (0.2%) to induce expression of the complementing/suppressing genes. β-Galactosidase assays were performed as described in Materials and Methods. Activities are expressed in Miller units (colorimetric protocol) or picomoles of 4-methylumbelliferone (MU) per minute per OD (fluorometric protocol). Error bars indicate the standard deviations. The following strains were used: JSG215 (pmrC-lacZ), JSG2115 (ΔpreB pmrC-lacZ), JSG2624 (ΔpreAB pmrC-lacZ), JSG2003 (ΔpreA pmrC-lacZ), JSG2115 (ΔpreB pmrC-lacZ), and JSG1998 (ΔpreA). Plasmid pJSG2581 is preAB expressed from P_BAD, pQseBC is qseBC expressed from P_BAD (pBAD18 vector), plasmid pJSG2558 is preA expressed from pBAD18, and plasmid pJSG2700 expresses preA[D51A]. wt, wild type.

FIG. 4.

Effects of pmrA, phoP, and the PreB putative autophosphorylation site on PreB-mediated regulation of pmrCAB. Salmonella strains were grown in LB medium, and β-galactosidase assays were performed as described in Materials and Methods. Activities are expressed in Miller units. Error bars indicate the standard deviations. The following strains were used: JSG215 (pmrC-lacZ), JSG1039 (preB pmrC-lacZ), JSG2422 (preB[H246G] pmrC-lacZ), JSG420 (pmrA pmrC-lacZ), JSG2366 (ΔpreB pmrA::Tn10d pmrC-lacZ), JSG2499 (pmrA[D51A] pmrC-lacZ), JSG2498 (preB pmrA[D51A] pmrC-lacZ), JSG1060 (phoP pmrC-lacZ), and JSG2365 (ΔpreB phoP pmrC-lacZ). WT, wild type.

FIG. 5.

Effect of preB mutations on the expression of pmrA regulon promoters. Salmonella strains were grown in LB medium, and β-galactosidase assays were performed as described in Materials and Methods. Activities are expressed in Miller units. Error bars indicate the standard deviations. Data are from a representative experiment with two replicates. The following strains were used: JSG1051 (pmrHFI-lacZ), JSG1058 (preB pmrI-lacZ), JSG214 (pmrE-lacZ), JSG1040 (preB pmrE-lacZ), JSG1525 (yibD-lacZ), JSG1527 (preB yibD-lacZ), JSG2527 (ΔpreB pmrA yibD::lacZ), JSG2420 (ΔpreA yibD-lacZ), and JSG2523 (ΔpreA pmrA yibD-lacZ).

FIG. 6.

Working model for PreA/PreB regulation of pmrCAB. (A) The genes pmrCAB and yibD are part of the PmrA regulon. Extracellular signals that affect the PreB sensor are unknown, but growth in LB medium activates its phosphatase activity, maintaining the PreA response regulator in the unphosphorylated conformation. In a PreB[H246G] background, PreA is also inactivated by the phosphatase activity of the mutant protein. (B) Under PreA/PreB-inducing conditions not yet identified, PreA indirectly regulates pmrCAB and yibD. (C) Ectopic PreA, but not PreA[D51A], can activate pmrCAB in a ΔpreB mutant even when grown in LB medium, presumably because of signal-independent cellular cross talk, which may become unmasked and more important due to the absence of the cognate sensor protein.