Identification and characterization of Pseudomonas membrane transporters necessary for utilization of the siderophore pyridine-2,6-bis(thiocarboxylic acid) (PDTC)

• Published in: Microbiology (Society for General Microbiology) 2006-10, Vol.152 (10), p.3157-3166
• Main Authors: Leach, Lynne H, Lewis, Thomas A
• Format: Article
• Language: English
• Subjects: Artificial Gene Fusion; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacteriology; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Reporter; Genetics; Iron - metabolism; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Microbiology; Pseudomonas; Pseudomonas putida; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Pyridines - metabolism; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Transcription, Genetic
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.29116-0

Department of Microbiology and Molecular Genetics, University of Vermont, 95 Carrigan Drive, Burlington, VT 05405, USA Correspondence Thomas A. Lewis talewis{at}uvm.edu The compound pyridine-2,6-bis(thiocarboxylic acid) (PDTC) is known to be produced and excreted by three strains of Pseudomonas . Its reactivity includes the complete dechlorination of the environmental contaminant carbon tetrachloride. PDTC functions as a siderophore; however, roles as a ferric reductant and antimicrobial agent have also been proposed. PDTC function and regulation were further explored by characterizing the phenotypes of mutants in predicted membrane transporter genes. The functions of a predicted outer-membrane transporter (PdtK) and a predicted inner-membrane permease (PdtE) were examined in Pseudomonas putida DSM 3601. Uptake of iron from 55 Fe(III):PDTC, and bioutilization of PDTC in a chelated medium, were dependent upon PdtK and PdtE. Another strain of P. putida (KT2440), which lacks pdt orthologues, showed growth inhibition by PDTC that could be relieved by introducing a plasmid containing pdt KCPE. Transcriptional activation in response to exogenously added PDTC (25 µM) was unaltered by the pdt K or pdt E mutations; each mutant showed activation of a pdt transcriptional reporter, indistinguishable from an isogenic PDTC utilization-proficient strain. The data demonstrate that PdtK and PdtE constitute a bipartite outer-membrane/inner-membrane transport system for iron acquisition from Fe(III):PDTC. Disruptions in this portion of the P. putida DSM 3601 pdt gene cluster do not abolish PDTC-dependent transcriptional signalling. Abbreviations: Cm, chloramphenicol; CT, carbon tetrachloride; Fur, ferric uptake regulator; Gm, gentamicin; Km, kanamycin; o-p, 1,10-phenanthroline; PDTC, pyridine-2,6-bis(thiocarboxylic acid); Tc, tetracycline