title: Serratia marcescens arn, a PhoP-regulated locus necessary for polymyxin B resistance.
abstract: Polymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly against Serratia marcescens. To investigate the underlying mechanisms, Tn5 mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5 inserted into the arnB and arnC genes. In other bacteria, arnB and arnC belong to the seven-gene arn operon, which is involved in lipopolysaccharide (LPS) modification. LPSs of arn mutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility in S. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression of phoP and arn in the wild-type strain but not in the phoP mutant. Complementation of the phoP mutant with the full-length phoP gene restored the PB MIC and induction by PB and low Mg2+ levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to the arn promoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+ levels protected S. marcescens from a PB challenge in the wild-type strain but not in the phoP mutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression of ugd, a gene required for LPS modification, in S. marcescens through a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression of arnA upon exposure to PB than did susceptible isolates. This is the first report to describe the role of S. marcescens arn in PB resistance and its modulation by PB and Mg2+ through the PhoP protein.
authors: Department and Graduate Institute of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
authors: Quei Yen
authors: QY
authors: Lin
authors: Department and Graduate Institute of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
authors: Yi-Lin
authors: YL
authors: Tsai
authors: Department and Graduate Institute of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
authors: Ming-Che
authors: MC
authors: Liu
authors: Department and Graduate Institute of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
authors: Wei-Cheng
authors: WC
authors: Lin
authors: Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China.
authors: Po-Ren
authors: PR
authors: Hsueh
authors: Department and Graduate Institute of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China sjliaw@ntu.edu.tw.
authors: Shwu-Jen
authors: SJ
authors: Liaw
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000235:genetics; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009876:Operon / Q000235:genetics; D011112:Polymyxin B / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012706:Serratia marcescens / Q000187:drug effects* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C061064:PhoP protein, Bacteria; D011112:Polymyxin B