ISOLATION AND CHARACTERIZATION OF A GENE, PMRD, FROM SALMONELLA-TYPHIMURIUM THAT CONFERS RESISTANCE TO POLYMYXIN WHEN EXPRESSED IN MULTIPLECOPIES

We have isolated from Salmonella typhimurium a gene, designated pmrD, that confers resistance to the membrane-damaging drug, polymyxin B whe n 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 the refore distinct from the previously described pmrA locus. We have mapp ed the polymyxin resistance activity to a 1.3-kb ClaI-PvuII fragment w hich contains a small open reading frame that could encode an 85-amino -acid peptide. When an Omega-Tet insertion was made into the putative pmrD open reading frame (pmrD2::Omega-Tet), the resulting plasmid no l onger conferred polymyxin resistance, whereas an Omega-Tet insertion i nto 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 sa rcoma virus, indicating that the protein may have proteolytic activity . However, changing the aspartic acid residue at the putative active s ite to alanine reduced but did not eliminate polymyxin resistance. Whe n pmrD2::Omega-Tet replaced the chromosomal copy of pmrD, the resultin g strain showed wild-type sensitivity to polymyxin and could be comple mented to resistance by a plasmid that carried pmrD. The pmrA505 allel e confers resistance to polymyxin when present in single copy on the c hromosome or when present on a plasmid in pmrA(+) pmrD(+) cells. In co mbination with the pmrD2::Omega-Tet mutation, the effect of the pmrA50 5 allele on polymyxin resistance was reduced, whether pmrA505 was pres ent on the chromosome or on a plasmid. Conversely, a strain carrying a n insertion in pmrA could be complemented to polymyxin resistance by a plasmid carrying the pmrA505 allele but not by a plasmid carrying pmr D. On the basis of these results, we suggest that polymyxin resistance is mediated by an interaction between PmrA or a PmrA-regulated gene p roduct and PmrD.