Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine

Abstract

Pseudomonas aeruginosa strain K437 is defective in the production of a 90 kDa ferripyoverdine receptor and is unable to grow in an iron‐deficient medium in the presence of the non‐metabolizable iron chelator 2,2′‐dípyrídyl (0.25mM). An attempt to clone the ferripyoverdine receptor gene was made by complementing this growth defect. A number of clones restoring growth of K437 on dipyridyl‐containing medium were obtained and several of these restored moderate expression of the 90 kDa receptor. A 5.5 kb Xhol‐HindIII fragment derived from one of these clones was similarly capable of complementing the dipyridyl growth defect although it failed to restore expression of the 90 kDa ferripyoverdine receptor. Nucleotide sequencing of the 5.5 kb fragment revealed two large open reading frames (ORFs), designated ORFA and ORFB, which appeared to form an operon and were capable of encoding products of 41 kDa and 112 kDa, respectively. Using a phage T7‐based expression system, products of 42 kDa and c. 108 kDa were produced from the cloned DNA, confirming that the ORFs were, indeed, expressed. The cloned ORFAB operon was inducible under conditions of iron limitation in both P. aeruginosa and Escherichia coli. In addition, mutants expressing ORFAB constitutively were constitutive for pyoverdine and ferripyoverdine receptor production suggesting that components of the pyoverdine‐mediated iron‐transport system are co‐regulated with ORFAB. The predicted products of ORFA and ORFB showed significant homology to the Escherichia coli EnvC and EnvD polypeptides which are reportedly involved in septum formation. In addition, the ORFB product showed moderate homology to the CzcA polypeptide identified as a component of a membrane‐associated plasmid‐encoded cation efflux system in Alcaligenes eutrophus. Using in vitro mutagenesis and gene replacement, ORFA‐ and ORFB‐deficient mutants of K372, the parent strain of K437, were constructed. These mutants were unable to grow on iron‐deficient minimal medium containing 0.25 mM dipyridyl although they expressed the ferripyoverdine receptor and were proficient in pyoverdine‐mediated iron uptake. Despite the homology of the ORFA and ORFB products to EnvC and EnvD, respectively, the ORFA‐ORFB‐deficient mutants were not defective in septum formation. Introduction of an ORFB mutation into the pyoverdine‐producing strain ML5087 resulted in poor growth of the mutant in an iron‐limited medium. The same mutation in a pyoverdine‐deficient derivative of ML5087 did not adversely affect growth. These data are discussed in the context of a possible role in pyoverdine secretion for the ORFAB products.