Identification, isolation, and analysis of a gene cluster involved in iron acquisition by Pseudomonas mendocina ymp

Abstract

Microbial acquisition of iron from natural sources in aerobic environments is a little-studied process that may lead to mineral instability and trace metal mobilization. Pseudomonas mendocina ymp was isolated from the Yucca Mountain Site for long-term nuclear waste storage. Its ability to solubilize a variety of Fe-containing minerals under aerobic conditions has been previously investigated but its molecular and genetic potential remained uncharacterized. Here, we have shown that the organism produces a hydroxamate and not a catecholate-based siderophore that is synthesized via non-ribosomal peptide synthetases. Gene clustering patterns observed in other Pseudomonads suggested that hybridizing multiple probes to the same library could allow for the identification of one or more clusters of syntenic siderophore-associated genes. Using this approach, two independent clusters were identified. An unfinished draft genome sequence of P. mendocina ymp indicated that these mapped to two independent contigs. The sequenced clusters were investigated informatically and shown to contain respectively a potentially complete set of genes responsible for siderophore biosynthesis, uptake, and regulation, and an incomplete set of genes with low individual homology to siderophore-associated genes. A mutation in the cluster's pvdA homolog (pmhA) resulted in a siderophore-null phenotype, which could be reversed by complementation. The organism likely produces one siderophore with possibly different isoforms and a peptide backbone structure containing seven residues (predicted sequence: Acyl-Asp-DabSer-fOHOrn-Ser-fOHorn). A similar approach could be applied for discovery of Fe− and siderophore-associated genes in unsequenced or poorly annotated organisms.

Roles of Siderophores, Oxalate, and Ascorbate in Mobilization of Iron from Hematite by the Aerobic Bacterium Pseudomonas mendocina

“…The large amounts of siderophores secreted by P. mendocina and similar aerobic bacteria, even in the chemically and spatially defined confines of a shaker flask, remain perplexing. Like DFO-B, P. mendocina's siderophore is expected to be an amino acid-derived trishydroxamic acid (3). Only a low concentration of DFO-B, which one would expect to be metabolized less efficiently than P. mendocina's own siderophore, is needed to support WT-like growth of the siderophore Ϫ strain.…”

“…Fe(III)-siderophore stability constants can be as high as 10 52 (1,40), which is many orders of magnitude higher than the stability constants for low-molecular-weight organic acids, such as oxalic acid [for Fe(III) ϩ 3 oxalate % Fe(oxalate) 3 , K ϭ 10 18.6 ] (45). While their high affinity for Fe(III) is clearly important for helping siderophores mobilize Fe from Fe(III) (hydr)oxides in the aqueous phase, the mechanisms of Fe mobilization appear to be complex and are the subject of much recent study (14,17,18,26,28,49).…”

“…In particular, the role of siderophores in ligand-promoted dissolution mechanisms has undergone careful evaluation in vitro. The model is described simply here as follows for amorphous Fe(OH) 3 and has been described in detail by Kraemer (26): Fe(OH) 3 (39), suggests that [FeL] could in principle easily be micromolar or higher and could support vigorous bacterial growth. However, the trishydroxamate siderophores that have been studied most to date adsorb only weakly to Fe(III) (hydr)oxide minerals, likely due to steric constraints, although charge repulsion may also play a role for positively charged siderophores, such as desferrioxamine B (DFO-B) (6,26,41,42).…”

“…The amount of dissolved Fe in each sample was analyzed using a Perkin Elmer AAnalyst 800 GFAA spectrophotometer equipped with a built-in AS-800 autosampler. Each sample was sterile filtered and loaded into plastic sample cups that had been washed with trace metal-grade HNO 3 and Millipore ultrapure water. Each analysis included three replicate runs, and a standard was generated using a single-element Fe standard (CPI International).…”

“…However, in aerobic, circumneutral environments, Fe is bound primarily in scarcely soluble minerals and amorphous solids [e.g., the solubility product (K SP ) for amorphous Fe(OH) 3 …”