Molecular characterization of the PmrA regulon

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The PmrA/PmrB two-component system in Salmonella enterica regulates lipopolysaccharide modifications, conferring resistance to polymyxin antibiotics.
• Understanding the regulatory mechanisms of polymyxin resistance is crucial for combating bacterial infections.

Purpose of the Study:

• To elucidate the transcriptional regulation of PmrA-controlled genes involved in polymyxin resistance.
• To identify and characterize the DNA binding sites of the PmrA regulator.

Main Methods:

• Mapping of transcription start sites for PmrA-regulated genes (pmrC, pmrG, pbgPE, ugd).
• Electrophoretic mobility shift assays (EMSA) using His-tagged PmrA protein.
• DNase I footprinting analysis to determine PmrA binding sites.

Main Results:

• A conserved binding site, a 16-bp imperfect inverted repeat (5'-TTAAKTTCTTAAKGTT-3'), was identified in the promoter regions of pmrC, pmrG, and pbgPE.
• PmrA binds to this conserved site in the intergenic region of the divergent pmrG and pbgPE promoters, suggesting PmrA dimer-mediated activation.
• The ugd gene appears to be indirectly regulated by the PmrA/PmrB system.

Conclusions:

• PmrA directly regulates the expression of pmrC, pmrG, and pbgPE by binding to specific promoter sequences.
• The PmrA/PmrB system activates pmrG and pbgPE transcription through a single binding site in their intergenic region.
• The regulation of ugd is likely indirect, indicating a complex regulatory network.