Identification and genetic characterization of PmrA-regulated genes and genes involved in polymyxin B resistance in Salmonella enterica serovar typhimurium

Abstract

Salmonella enterica serovar Typhimurium encounters antimicrobial peptides (AP) within the phagosomes of professional phagocytes and at intestinal mucosal surfaces. Salmonella serovar Typhimurium utilizes the two-component regulatory system PmrA-PmrB, which is activated in response to the environmental conditions encountered in vivo, to regulate resistance to several AP, including polymyxin B (PM). Random MudJ transposon mutagenesis was used to identify PmrA-PmrB-regulated genes, as well as genetic loci necessary for PM resistance. Three different phenotypic classes of genes were identified: those necessary for PM resistance and regulated by PmrA, those necessary for PM resistance and not regulated by PmrA, and PmrA-regulated genes not required for PM resistance. Loci identified as necessary for PM resistance showed between 6- and 192-fold increased sensitivities to PM, and transposon insertion sites include surA, tolB, and gnd. PmrA-regulated loci identified included dgoA and yibD and demonstrated 500- and 2,500-fold activation by PmrA, respectively. The role of the identified loci in aminoarabinose modification of lipid A was determined by paper chromatography. The gnd mutant demonstrated a loss of aminoarabinose from lipid A, which was suggested to be due to a polar effect on the downstream gene pmrE. The remaining PM(s) mutants (surA and tolB), as well as the two PmrA-regulated gene (yibD and dgoA) mutants, retained aminoarabinose on lipid A. yibD, dgoA, and gnd (likely affecting pmrE) played no role in PmrA-regulated resistance to high iron concentrations, while surA and tolB mutations grew poorly on high iron media. All PM(s) mutants identified in this study demonstrated a defect in virulence compared to wild-type Salmonella serovar Typhimurium when administered orally to mice, while the PmrA-regulated gene (yibD and dgoA) mutants showed normal virulence in mice. These data broaden our understanding of in vivo gene regulation, lipopolysaccharide modification, and mechanisms of resistance to AP in enteric bacteria.

FIG. 1.

Schematic of the strategy used to mutagenize JSG435 (PmrA^c) with MudJ in order to obtain mutants in PmrA-regulated genes and/or genes involved in PM resistance. The MudJ transposon encodes genes involved in β-galactosidase production, as well as a kanamycin resistance cassette. The SalI site used in cloning the MudJ-chromosomal DNA junction for sequencing is shown. Black circles represent blue colonies resulting from active fusion of lacZ in the MudJ transposon to an ORF. White circles symbolize Kan^r colonies that contain inactive fusions. Light grey colonies represent a light blue phenotype corresponding to low-level transcription of lacZ. Abbreviations: Kan, kanamycin; Tet, tetracycline; Cam, chloramphenicol; XG, X-Gal.

FIG. 2.

Regulation of yibD and dgoA by PmrA. Transcription of the loci was measured by the production of β-galactosidase from the lacZ gene in the MudJ transposon. Strains compared were yibD::MudJ in a PmrA^c and a PmrA-null background and dgoA::MudJ in a PmrA^c and a PmrA-null background. Loci were transcribed effectively in a PmrA^c background (grey bars); transcription of the loci was essentially eliminated in a PmrA-null background (black bars, barely visible).

FIG. 3.

Promoter analysis of PmrA-regulated loci identified in screen. The consensus PmrA-binding sites shown for pmrE (ugd) and pmrCAB were identified previously by Aguirre and colleagues (1). The YTTAAK direct repeats are marked in bold and underlined. The predicted −10 regions are italicized and marked in bold. Transcriptional start sites are labeled with arrows for pmrCAB and pmrE (56). For yibD, the putative PmrA consensus binding site begins 67 bp upstream of the translational start site. Ambiguous codes used are as follows: Y, C/T; K, G/T.

FIG. 4.

Analysis of lipid A for the presence of aminoarabinose. Paper chromatography was performed on lipid A from PmrA^c and PmrA-null Salmonella serovar Typhimurium, as well as lipid A from the four PM^s mutants and two PmrA-regulated gene mutants identified in this study. The chromatograms were run with a solvent system of isopropanol-ethyl acetate-water (7:1:2). Yellow color upon development with ninhydrin is characteristic of 4-aminoarabinose (yellow color was converted to grey).