Phosphorylated PmrA interacts with the promoter region of ugd in Salmonella enterica serovar typhimurium

Abstract

The Salmonella PmrA-PmrB system controls the expression of genes necessary for polymyxin B resistance. Four loci were previously identified as part of the regulon, and interaction of PmrA with the promoter region of three of them was observed. Here we characterized the interaction of PmrA with the promoter region of ugd, previously suggested to be regulated indirectly by PmrA. Our results indicate that PmrA controls the expression of ugd by interacting with a specific sequence in the promoter region of this gene.

FIG. 1

PmrA interacts with the promoter region of ugd. The electrophoretic shift mobility assay was performed using the ³²P-, 3′-end-labeled 354-bp PCR fragment of the promoter region of ugd, incubated with purified PmrA-H6, in the absence or presence of different amounts of either salmon sperm DNA as a nonspecific competitor (nonsp. DNA) or the unlabeled promoter region of ugd (unlab. Pugd).

FIG. 2

Autokinase and phosphotransfer activities of H6-PmrBc. One picomole of the H6-PmrBc protein was incubated with 1 mM [γ-³²P]ATP in the presence of 30 pmol of PmrA-H6 in FTB buffer (without DTT) for 1, 5, and 20 min at 37°C. For the negative control (−), H6-PmrB was incubated under the same conditions for 20 min without the addition of PmrA-H6. The reactions were stopped by the addition of 0.5% SDS. The samples were loaded in an SDS–12% PAGE gel, transferred to nitrocellulose, and analyzed by autoradiography.

FIG. 3

Enhanced affinity of phosphorylated PmrA for the promoter region of ugd. The footprinting assay was performed on the coding strand of the promoter region of ugd, incubated with 0, 0.5, 1, 2.5, 5, 10, 15, 30, and 60 pmol of PmrA-H6 and 1 pmol of H6-PmrBc, in the absence or presence of 1 mM ATP and without addition of DTT. The solid line represents the PmrA-protected region.

FIG. 4

Alignment of the promoter regions of ugd, pmrCAB, and the pmrF operon. The sequences from the ugd, pmrCAB, and pmrF promoter regions were piled up from the transcriptional start site. The YTTAAK direct repeats are boxed, and the proposed −10 regions are marked in bold. The arrows indicate the transcription start sites identified previously (24).