Evidence for a copper-dependent iron transport system in the marine, magnetotactic bacterium strain MV-1

Abstract

Cells of the magnetotactic marine vibrio, strain MV-1, produce magnetite-containing magnetosomes when grown anaerobically or microaerobically. Stable, spontaneous, non-magnetotactic mutants were regularly observed when cells of MV-1 were cultured on solid media incubated under anaerobic or microaerobic conditions. Randomly amplified polymorphic DNA analysis showed that these mutants are not all genetically identical. Cellular iron content of one non-magnetotactic mutant strain, designated MV-1nm1, grown anaerobically, was~20-to 80-fold less than the iron content of wild-type (wt) MV-1 for the same iron concentrations, indicating that MV-1nm1 is deficient in some form of iron uptake. Comparative protein profiles of the two strains showed that MV-1nm1 did not produce several proteins produced by wt MV-1. To understand the potential roles of these proteins in iron transport better, one of these proteins was purified and characterized. This protein, a homodimer with an apparent subunit mass of about 19 kDa, was an iron-regulated, periplasmic protein (p19). Two potential 'copper-handling' motifs (MXM/MX 2 M) are present in the amino acid sequence of p19, and the native protein binds copper in a 1 : 1 ratio. The structural gene for p19, chpA (copper handling protein) and two other putative genes upstream of chpA were cloned and sequenced. These putative genes encode a protein similar to the iron permease, Ftr1, from the yeast Saccharomyces cerevisiae, and a ferredoxin-like protein of unknown function. A periplasmic, copper-containing, iron(II) oxidase was also purified from wt MV-1 and MV-1nm1. This enzyme, like p19, was regulated by media iron concentration and contained four copper atoms per molecule of enzyme. It is hypothesized that ChpA, the iron permease and the iron(II) oxidase might have analogous functions for the three components of the S. cerevisiae copper-dependent high-affinity iron uptake system (Ctr1, Ftr1 and Fet3, respectively), and that strain MV-1 may have a similar iron uptake system. However, iron(II) oxidase purified from both wt MV-1 and MV-1nm1 displayed comparable iron oxidase activities using O 2 as the electron acceptor, indicating that ChpA does not supply the multi-copper iron(II) oxidase with copper.

Ecology, Diversity, and Evolution of Magnetotactic Bacteria

“…Therefore, it seems very likely that this genomic region represents a large MAI that appears to be present with variations in other cultured and uncultured MTB (195,196,202,215). More evidence comes from the loss of magnetosome formation and, thus, magnetotaxis by some cultivated MTB relatively easily in culture following the loss of the MAI (68,170).…”

“…How iron is taken up by MTB is unknown, but it would seem that there would be multiple mechanisms for this in a single bacterium, as has been found for other nonmagnetotactic bacteria. Thus far, siderophores, low-molecular-weight ligands produced by the cell that chelate and solubilize Fe(III) (178,179), have been implicated in iron uptake by MTB (68,180,181) as well as in a putative copper-dependent iron uptake system similar to that found in the yeast Saccharomyces cerevisiae (68).…”

“…Both oxygen concentration gradient and anaerobic shake tubes can be made by using air or oxygen-free gas in the headspace, respectively. Using either agar plates or shake tubes, colonies of MTB are usually brown or black due to the formation of magnetite (65,68). For those organisms that do not form colonies either on plates or in shake tubes, pure cultures can be obtained by a repeated series of dilutions to extinction in media as long as the dominant bacterium present in the original culture is the one targeted for isolation (26,28,49,53).…”

“…In addition, genomic islands are thought to undergo frequent gene rearrangements (216). Gene rearrangements, gene deletions, and duplications may be the reason for the frequent development of spontaneous nonmagnetotactic mutants of various strains (68,170). Spontaneous deletions that lead to a loss of the magnetotactic phenotype with a frequency of 10 Ϫ2 were observed under starvation condi-tions in late-stationary-phase cultures of Ms. gryphiswaldense and most likely were caused by RecA-dependent homologous recombination between numerous repeats present in the MAI (197,217).…”

“…Activated charcoal is known to scavenge and decompose toxic free oxygen radicals and peroxides thought to inhibit the growth of many microaerophiles (66,67). Once inoculated, ACA plates are incubated under microaerobic or anaerobic conditions in special gas mixtures (e.g., 1% oxygen in nitro-gen) or oxygen-free gases, depending upon the organism (65,68). A second method for obtaining individual colonies is through the use of solid medium in shake tubes (8,69).…”