Iron Regulation of Ferric Iron Uptake in a Fluorescent Pseudomonad: Cloning of a Regulatory Gene

Abstract

Two iron-regulated lac gene fusion plasmids (pSP1 and pMSR1) were constructed that showed lacZ expression in Pseudomonas spp. strain M114 only during growth under low iron conditions. No expression of the reported lacZ gene was detected in Escherichia coli. Plasmid pMSR1 was exploited to screen a Tn5-induced Pseudomonas Fe3+ uptake regulatory mutant (strain M114FR1). lacZ expression from pMSR1 and pSP1 in strain M114FR1 was not subject to regulation by iron. This strain could produce siderophore when iron was present in the growth medium in contrast to the wild-type strain. The outer membrane of the wild-type strain M114 contains four iron-regulated polypeptides detected following sodium dodecyl sulfate polyacrylamide gel electrophoresis. However in mutant strain M114FR1, three of these proteins were synthesized in the presence of Fe3+ whereas the fourth (an 88-kDa protein) was partially deregulated. This strain showed inhibition of both bacterial and fungal indicator organisms under conditions in which the iron concentration was sufficiently high to render the wild-type strain ineffective. Complementation of strain M114FR1 witha pLAFR1-based gene bank derived from parent strain M114 resulted in the isolation of a cosmid clone pMS639 that restored normal regulation by Fe3+. This plasmid was shown to contain a 28-kilobase DNA insert containing the gene(s) responsible for the regulation of the Fe3+ uptake system of strain M114FR1 by iron.