Genetic basis of iron assimilation in pathogenic Escherichia coli.
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Animals↗
Biomedical subjects
A Bindereif
Publications and source records attributed to A Bindereif.
Aerobactin genes in clinical isolates of Escherichia coli.
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 HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII 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 IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.
Base Sequence↗
Promoter mapping and transcriptional regulation of the iron assimilation system of plasmid ColV-K30 in Escherichia coli K-12.
The promoter of the high-affinity iron assimilation system coded in an approximately 8-kilobase-pair segment of the large Escherichia coli plasmid ColV-K30 was localized to a 0.7-kilobase HindIII-SalI fragment by in vitro runoff transcription. By an S1 nuclease protection assay, with in vitro-transcribed RNA and total in vivo-synthesized RNA, the major start site for transcription was mapped within this fragment and found to be identical in vitro and in vivo. A minor initiation site was located about 50 base pairs upstream from the major site. DNA sequencing of the HindIII-SalI fragment revealed the presence of two promoter-like structures within an extremely AT-rich region with transcriptional initiation sites at 30 and about 80 base pairs upstream from the initiation codon for the first structural gene. Numerous potential secondary structures were found in the DNA sequence around the major promoter. The major transcriptional start site was determined precisely by sequencing the 5' end of in vitro-transcribed RNA. The effect of iron on both the level of specific RNA, as determined by a quantitative S1 nuclease mapping assay, and on beta-galactosidase activity in a iucA'-'lacZ protein fusion, showed that the aerobactin operon is regulated at the transcriptional level. The iron-regulatory sequences are contained within a 152-base-pair Sau3A fragment of the promoter region.
Base Sequence↗
Lack of homology between the iron transport regions of two virulence-linked bacterial plasmids.
Two plasmids involved in bacterial virulence, the Escherichia coli plasmid pColV-K30 and the Vibrio anguillarum plasmid pJM1, have been studied with respect to the iron sequestering systems mediated by these two plasmids. Bioassay results show that the two systems are not related functionally because specific iron uptake-deficient mutants in each system cannot be cross-fed by the heterologous bacteria using culture supernatants from iron-proficient strains containing wild-type plasmids. DNA hybridization studies show an extensive lack of homology between regions involved in iron sequestration in both plasmids.
Biological Transport, Active↗
Association of hydroxamate siderophore (aerobactin) with Escherichia coli isolated from patients with bacteremia.
Clinical isolates of Escherichia coli were examined for the presence of hydroxamate siderophore (aerobactin). The incidence of aerobactin-positive strains of E. coli from the blood was greater than the incidence of these strains isolated from other sites. The presence of aerobactin and the virulence of strains of E. coli in urinary tract infection were also examined in mice. The presence of aerobactin in the strains of E. coli correlated with virulence as measured by proportion of deaths but not with renal infection. These results suggest that the presence of aerobactin may be a significant factor in the invasion of the blood stream.
Animals↗
Cloning of the aerobactin-mediated iron assimilation system of plasmid ColV.
The high-affinity iron assimilation system of plasmid ColV-K30 was cloned on the vector plasmid pPlac. Plasmid pABN1 was isolated by means of sensitivity to cloacin, a bacteriocin using the same outer membrane receptor as ferric aerobactin. Restriction maps were determined for this plasmid and for a subclone, pABN5. Plasmid pABN1 codes for the complete gene complex, whereas plasmid pABN5 encodes only the biosynthetic genes for aerobactin. Regulation of the uptake system by iron is retained in cloned sequences of pABN1.
Bacterial Outer Membrane Proteins↗
Aerobactin-mediated utilization of transferrin iron.
Aerobactin and enterobactin, hydroxamate- and catechol-type siderophores, respectively, were found capable of removing iron (III) from transferrin in buffered solution. Although under these conditions aerobactin displaced the iron much more slowly than did enterobactin, the rate for the former could be accelerated by addition of pyrophosphate as mediator. Transfer of iron (III) from transferrin to aerobactin appeared to proceed via a ternary complex. Cells of Escherichia coli BN 3040 NalR iuc containing transport systems for both enterobactin and aerobactin, the genetic determinants for the latter specified on a ColV-type plasmid, took up iron from [55Fe]transferrin in minimal medium. In this case aerobactin was effective at a much lower concentration, although enterobactin still displayed superior ability to transfer the iron. In serum, however, the rate measured with aerobactin exceeded that found with enterobactin. The results indicate that aerobactin, in spite of its relatively unimpressive affinity for iron (III) as a siderophore, is nonetheless equipped with structural features or properties that enhance its ability to remove the metal ion from transferrin, especially when receptor-bearing cells of E. coli are present to act as a thermodynamic sink for the iron. These attributes of the aerobactin system of iron assimilation may account for its status as a virulence determinant in hospital isolates of E. coli.
Diphosphates↗
The cloacin receptor of ColV-bearing Escherichia coli is part of the Fe3+-aerobactin transport system.
Escherichia coli strains which contain the Fe3+-aerobactin transport system specified by the ColV plasmid became deficient in aerobactin-dependent iron transport when they were converted to cloacin-resistant derivatives. An outer membrane protein with a molecular mass of 74,000 daltons was overproduced under iron-limiting growth conditions and was absent in cloacin-resistant mutants. Fe3+-aerobactin protected cells against cloacin. These results suggest that the cloacin receptor protein, controlled by the colV plasmid, also participates in Fe3+-aerobactin transport.
Bacterial Outer Membrane Proteins↗
ColV plasmid-specific aerobactin synthesis by invasive strains of Escherichia coli.
Certain strains of Escherichia coli associated with bacteremia of humans and domestic animals harbor plasmids that promote efficient iron uptake. The mechanism, which is an important component of the virulence of invasive strains, is independent of the enterobactin system for iron uptake. Plasmid-specified siderophore was assayed by its ability to support the growth of a chelator-deficient mutant in conditions of iron deprivation. The chelator, which was chemically determined to be a hydroxamate compound, was identical on the basis of field desorption mass spectrometry with aerobactin, a siderophore synthesized by Aerobacter aerogenes. In conditions of iron stress, aerobactin is secreted into the culture medium of plasmid-bearing E. coli strains. Reconstruction experiments involving a chelator-deficient mutant growing with exogenous chelator suggested that association of a small fraction of the total siderophore synthesized with cellular material is due to transient binding of aerobactin to membrane receptors during active bacterial growth.
Bacteriocin Plasmids↗
A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing.
A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 x 10(7) mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class II promoters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous for biochemical analysis of transcription and RNA processing in mammalian cells.
DNA-Binding Proteins↗