Isolation and characterization of a gene, pmrD, from Salmonella typhimurium that confers resistance to polymyxin when expressed in multiple copies

We have isolated from Salmonella typhimurium a gene, designated pmrD, that confers resistance to the membrane-damaging drug, polymyxin B when expressed from the medium-copy-number plasmid pHSG576. The gene maps to 46 min on the standard genetic map, near the menB gene, and is therefore distinct from the previously described pmrA locus. We have mapped the polymyxin resistance activity to a 1.3-kb ClaI-PvaII fragment which contains a small open reading frame that could encode an 85-amino-acid peptide. When an Ω-Tet insertion was made into the putative pmrD open reading frame (pmrD2::Ω-Tet), the resulting plasmid no longer conferred polymyxin resistance, whereas an Ω- Tet insertion into vector sequences had no effect. Maxicell analysis confirmed that a protein of the expected size is made in vivo. The PmrD protein shows no significant homology to any known protein, but it does show limited homology across the active site of the p15 acid protease from Rous sarcoma virus, indicating that the protein may have proteolytic activity. However, changing the aspartic acid residue at the putative active site to alanine reduced but did not eliminate polymyxin resistance. When pmrD2::Ω- Tet replaced the chromosomal copy of pmrD, the resulting strain showed wild- type sensitivity to polymyxin and could be complemented to resistance by a plasmid that carried pmrD. The pmrA505 allele confers resistance to polymyxin when present in single copy on the chromosome or when present on a plasmid in pmrA^- pmrD⁺ cells. In combination with the pmrD2::Ω-Tet mutation, the effect of the pmrA505 allele on polymyxin resistance was reduced, whether pmrA505 was present on the chromosome or on a plasmid. Conversely, a strain carrying an insertion in pmrA could be complemented to polymyxin resistance by a plasmid carrying the pmrA505 allele but not by a plasmid carrying pmrD. On the basis of these results, we suggest that polymyxin resistance is mediated by an interaction between PmrA or a PmrA-regulated gene product and PmrD.