Roland K L, Esther C R, Spitznagel J K
Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, USA.
J Bacteriol. 1994 Jun;176(12):3589-97. doi: 10.1128/jb.176.12.3589-3597.1994.
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-PvuII fragment which 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 longer conferred polymyxin resistance, whereas an omega-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::omega-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 pmrD(2)::-Tet mutation, the effect o the pmrA505 allele on polymyxin resistance was reduced, whether pmrA505 was present in 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.
我们从鼠伤寒沙门氏菌中分离出一个名为pmrD的基因，当该基因从中等拷贝数质粒pHSG576表达时，可赋予对膜损伤药物多粘菌素B的抗性。该基因在标准遗传图谱上位于46分钟处，靠近menB基因，因此与先前描述的pmrA位点不同。我们已将多粘菌素抗性活性定位到一个1.3kb的ClaI - PvuII片段，该片段包含一个小的开放阅读框，可能编码一个85个氨基酸的肽。当在假定的pmrD开放阅读框中进行ω - Tet插入（pmrD2::ω - Tet）时，所得质粒不再赋予多粘菌素抗性，而在载体序列中进行ω - Tet插入则没有影响。最大细胞分析证实，体内产生了预期大小蛋白质。PmrD蛋白与任何已知蛋白均无明显同源性，但在来自劳氏肉瘤病毒的p15酸性蛋白酶的活性位点上显示出有限的同源性，这表明该蛋白可能具有蛋白水解活性。然而，将假定活性位点处的天冬氨酸残基突变为丙氨酸会降低但并未消除多粘菌素抗性。当pmrD2::ω - Tet取代pmrD的染色体拷贝时，所得菌株对多粘菌素表现出野生型敏感性，并且可以被携带pmrD的质粒互补为抗性。pmrA505等位基因当以单拷贝存在于染色体上或存在于pmrA + pmrD +细胞的质粒上时，可赋予对多粘菌素的抗性。与pmrD(2)::-Tet突变相结合，无论pmrA505存在于染色体上还是质粒上，pmrA505等位基因对多粘菌素抗性的影响都会降低。相反，携带pmrA插入的菌株可以被携带pmrA505等位基因的质粒互补为多粘菌素抗性，但不能被携带pmrD的质粒互补。基于这些结果，我们认为多粘菌素抗性是由PmrA或PmrA调控的基因产物与PmrD之间的相互作用介导的。
