Fate of Labeled Hydroxamates During Iron Transport from Hydroxamate-Iron Chelates

Double radioactive label transport assays with iron, chromium, and gallium chelates were used to investigate the mechanism of iron uptake by Ustilago sphaerogena. In iron-deficient cells, ferrichrome A iron was taken up without appreciable uptake of the ligand. Iron-sufficient cells partially accumulated the ligand with the metal. The chromiumand gallium-containing analogs of ferrichrome A were transported as intact chelates. Ferrichrome A iron uptake was inhibited by dipyridyl. The data suggest that the intact ferrichrome A chelate binds to a specific receptor, the iron is then separated from the ligand at the membrane by reduction, and the metal is released to the inside of the cell while the ligand is released to the exterior. The reduction step is not transport rate limiting. Iron chelated to citrate was taken up by an energy-dependent process. The citrate ligand was not taken up with the metal. Uptake was sensitive to dipyridyl and ferrozine. Chromic ion chelated to citrate was not transported, suggesting that the iron, rather than the chelate, is recognized by the receptor or that reduction of the metal is required for transport.Under iron-deficient growth conditions, many microorganisms excrete low-molecular-weight iron-chelating agents (known as siderophores) for the purpose of sotubilizing extracellular iron. After complexing iron, the ferric siderophores supply the metal to the cell by binding to specific receptors in the cell membrane (14,15). Siderophore-mediated iron transport has been investigated primarily by following the fate of radioactively labeled iron in transport assays, although recently investigators have followed iron transport by electron paramagnetic resonance and Mossbauer spectroscopy (3,13).Double-label experiments with both the iron and ligand labeled have revealed two basic mechanisms for iron uptake from ferric siderophores. Ferrichrome uptake in Ustilago sphaerogena is the prototype for the first mechanism, known as the iron shuttle mechanism, in which the iron and ligand are taken up at identical rates (6). After the iron is removed from the ligand inside the cell, the free ligand reappears in the medium and may serve for another round of iron transport. In the second mechanism, uptake of iron occurs without uptake of ligand. In this case, the ferric siderophore binds to the cell membrane where the iron is removed from the ligand; the iron is taken into the cell, and the ligand remains extracellular. This is known as t Present address:

Section: Downloaded Fromsupporting

confidence: 84%

Iron uptake from ferrichrome A and iron citrate in Ustilago sphaerogena

Ecker¹,

Emery²

1983

“…The sedimentation of 92% of the total iron in sonically disrupted meningococci was not surprising, since one could expect a large proportion of iron proteins to be membrane associated; however, such sedimentation does reduce the possibility in meningococcus for a large low-molecular-weight iron "pool" of the type suggested by Arceneaux et al (1,2) for schizokinen-Fe in Bacillus megaterium.…”

Section: Resultsmentioning

confidence: 95%

Iron in Neisseria meningitidis: minimum requirements, effects of limitation, and characteristics of uptake

Archibald¹,

DeVoe²

1978

A simple defined medium (neisseria defined medium) was devised that does not require iron extraction to produce iron-limited growth of Neisseria meningitidis (SDIC). Comparison of this medium to Mueller-Hinton broth and agar showed nearly identical growth rates and yields. The defined medium was used in batch cultures to determine the disappearance of iron from the medium and its uptake by cells. To avoid a number of problems inherent in batch culture, continuous culture, in which iron and dissolved oxygen were varied independently, was used. Most of the cellular iron was found to be nonheme and associated with the particulate fraction in sonically disrupted cells. Nonheme and catalase-heme iron were reduced by iron starvation far more than cytochromes b and c and

“…In addition, enterobactin has a relatively low water solubility (37) and is chemically unstable, and its highly aromatic character may cause it to adhere to proteins as a haptene, explaining the presence of specific antibodies in human serum (30,31). Aerobactin, however, is a much more water-soluble, stable, and even recyclable molecule (1,7). In summary, the properties and the distribution of aerobactin support our present view (24, 32) that enterobactin, although having the largest known stability constant of any iron chelate (18), is inferior to other siderophores, such as aerobactin, which evolved structural and functional qualities adapted for iron acquisition in serum.…”

mentioning

confidence: 99%

Aerobactin genes in clinical isolates of Escherichia coli

Bindereif¹,

Neilands²

1985

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase Hindlll fragment upstream and adjacent to the 16.3-kilobase HindlIl fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS], in inverted orientation to the IS] element downstream from the aerobactin operon. The upstream and downstream sequences of IS] appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS) elements. * Corresponding author.Recently we reported a survey of a large number of E. coli clinical isolates for aerobactin synthesis (J. Z. Montgomerie, A. Bindereif, J. B. Neilands, G. Kalmanson, and L. B. Guze, submitted for publication). A high incidence of aerobactin-positive strains (39%) was found. In this paper we determined the genetic location, viz., whether plasmid coded or chromosomal, of the aerobactin biosynthesis and transport determinants in eight aerobactin-positive clinical isolates of E. coli and compare the particular organization of the aerobactin gene complex in these strains. MATERIALS AND METHODSClinical isolates of E. coli. A collection of 54 clinical isolates of E. coli was obtained from J. Z. Montgomerie, Rancho Los Amigos Hospital, Downey, Calif. In addition, the survey included E. coli 0111, a clinical isolate from the Centers for Disease Control, Atlanta, Ga. Recombinant DNA. Restriction enzymes, SI nuclease, DNA polymerase I, and T4 DNA ligase were purchased from Bethesda Research Laboratories (Bethesda, Md.). The large fragment of DNA polymerase I was from New England Biolabs (Beverly, Mass.) The conditions recommended by the supplier were used for restriction enzyme digestions and ligations. The protocols given by Maniatis et al. (26) were followed for most standard recombinant DNA procedures.The cloning vector for the construction of pABN20 was pUC8, 2.7 kilobases (kb) (49). E. coli JM83 [ara A(lac-pro) thi rpsL 4)80dlacZzAM15I was the host for pUC8-derived recombinant plasmids. E. coli JM83 was screened for plasmids by a quick minilysate procedure, which was also used for large-scale isolations of small plasmids (20). For largescale isolations of large plasmids (pColV-K30 and total plasmid DNA from E. coli isolates) the Portnoy procedure was employed (38). Cells from 11 cultures were harvested after 2 to 3 days of growth in Tris-buffered medium (41) with sodium succinate (30 ...