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J B Neilands

Publications and source records attributed to J B Neilands.

At least 55 records · Page 3Linked to original sources
PubMed1985

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↗
PubMed1985

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↗
PubMed1984

Effect of serum albumin on siderophore-mediated utilization of transferrin iron.

The effect of serum and serum proteins on enterobactin- and aerobactin-mediated utilization of transferrin iron has been investigated. Serum was found to impede transfer of iron from iron transferrin to enterobactin and from [55Fe]ferric enterobactin to cells of Escherichia coli BN3040 Na 1R iuc . In contrast, serum had essentially no effect on the rate of these reactions mediated by aerobactin. Three purified serum proteins, human serum albumin, bovine serum albumin, and human immunoglobulin, were comparable to human serum in their selective ability to interfere with the transfer of 55Fe from [55Fe]ferric enterobactin to E. coli BN3040 Na 1R iuc . The inhibitory effect of human serum albumin on the enterobactin-mediated transfer of iron from [55Fe]transferrin was enhanced by preincubation of the protein with the siderophore. Pretreatment of the bacterial cells with human serum albumin did not affect the rate of utilization of siderophore iron. A linear, reciprocal relationship was found to hold for human albumin concentration vs. the first-order rate constant ( kobsd ) for the velocity of iron transfer from iron transferrin to enterobactin. Binding of serum albumin to enterobactin increased the intensity of the near-ultraviolet absorption band of the siderophore and shifted it to longer wavelengths. The stoichiometry of binding to human and bovine serum albumins was established as 1:1, and the binding constant for both enterobactin and ferric enterobactin was estimated to be in the range 1 X 10(4)-1.2 X 10(5) M-1. These results indicate that serum albumin may act synergistically with other factors in the serum, such as transferrin, to limit iron supply and in this way restrict the growth of invading microorganisms.

Enterobactin↗
PubMed1984

Vibriobactin, a siderophore from Vibrio cholerae.

A novel siderophore (microbial iron transport compound) has been isolated from low iron cultures of Vibrio cholerae. Belonging to the catecholamide family of chelators, it has been shown to contain three residues of 2,3-dihydroxybenzoic acid and two residues of threonine. Both threonine moieties are present in the form of oxazoline rings. Furthermore, the polyamine backbone of the molecule was proved to be not spermidine, but the rare N-(3-aminopropyl)-1,3-diaminopropane, norspermidine. The structure of the new siderophore has been determined to be N-[3-(2,3-dihydroxybenzamido)propyl]-1, 3-bis[2,3-dihydroxyphenyl)-trans-5-methyl-2-oxazoline-4-carboxamido]prop ane. The compound has been given the trivial name vibriobactin. Mutants defective in the synthesis and utilization of vibriobactin were isolated. In an iron-limited environment V. cholerae was found to respond more strongly to vibriobactin, agrobactin, and ferrichrome than to enterobactin.

Catechols↗
PubMed1984

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↗
PubMed1984

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↗
PubMed1984

Genetic and biochemical characterization of the Escherichia coli K-12 fhuB mutation.

The fhuB region of Escherichia coli K-12 was subcloned from pLC4-44 into pP lac to obtain pCPN1. Deletions of this recombinant plasmid were made, and a 1.4-kilobase PstI fragment was further subcloned into the vector plasmid pKK177-2 to obtain pCPN12. The response of tonA and tonB strains and fhuB strains containing the plasmids to 15 hydroxamate siderophores were assayed. Results showed that tonA strains were deficient only in the utilization of ferrichrome-type siderophores, whereas fhuB strains were deficient in the utilization of all hydroxamate-type siderophores. The response of the plasmid-containing fhuB strains to the siderophores showed that the fhuB gene resides on a 1.4-kilobase PstI fragment of DNA. The proteins synthesized by these plasmids were examined in maxicells of strain CSR603. Plasmid pCPN1 expressed five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700. By the use of deletions of pCPN1, the approximate order of the genes for these proteins was determined. Plasmid pCPN12 expressed no proteins other than the beta-lactamase proteins in maxicell strain CSR603. However, in maxicell strain BN660, a lon mutant, it expressed a 20,000-molecular-weight protein. Inner membrane vesicles made from tonB and fhuB strains were able to transport [55Fe]ferrichrome and [55Fe]rhodotorulate at rates similar to those obtained in vesicles from tonB+ and fhuB+ strains.

Bacterial Proteins↗
PubMed1984

Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion sequences flank operon in plasmid-mediated systems.

Large plasmids were detected in two aerobactin-producing enteric bacterial species (Aerobacter aerogenes 62-I, Salmonella arizona SA1, and S. arizona SL5301) and designated pSMN1, pSMN2, and pSMN3, respectively. Other Salmonella spp., namely, S. arizona SL5302, S. arizona SLS, Salmonella austin, and Salmonella memphis, formed aerobactin but contained no detectable large plasmids. S. arizona SL5283 made no aerobactin. A probe consisting of the aerobactin biosynthetic genes cloned on plasmid pABN5 hybridized to a HindIII digest of pSMN1 but not to digests of pSMN2 or pSMN3. A larger probe, the insert of pABN1 containing the complete aerobactin operon, hybridized to four fragments in HindIII digests of the parent plasmid, pColV-K30. A 2.0-kilobase PvuII fragment responsible for this multiple-hybridization pattern was cloned into vector pUC9 to form pSMN30. The latter was mapped and shown to correspond to either IS1 or to a closely related insertion sequence.

Chromosomes, Bacterial↗
PubMed1984

Siderophores of bacteria and fungi.

Siderophore-mediated iron assimilation systems are widely distributed in bacteria and fungi. The systems are comprised of low molecular weight ferric-ion specific ligands (siderophores) and cognate cell-bound receptor and utilization components. Most siderophores are classed chemically as hydroxamic acids or catechols; expression of both types is regulated by iron. Of the various siderophore systems present in E. coli, the enterobactin and aerobactin genes, encoded on the chromosome and pColV respectively, have been the most intensively studied to date as regards their molecular genetics.

Bacteria↗
PubMed1983

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↗
PubMed1983

Absence of siderophore activity in Legionella species grown in iron-deficient media.

Growth of Legionella species in a defined medium deficient in iron did not result in the production of phenolic or hydroxamate siderophores which could be detected by chemical or biological assay methods. Growth of a variety of other gram-negative organisms under the same conditions resulted in the production of both hydroxamate and phenolate siderophores. The iron-deficient medium limited growth of the Legionella species more severely than it did the growth of the other gram-negative organisms. We have concluded that Legionella species do not make the commonly recognized siderophores, probably because they are restricted in their growth to those environments in which inorganic iron is readily available or is supplied in a form bound to an unknown carrier.

Biological Assay↗
PubMed1983

Siderophores.

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Animals↗
PubMed1982

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↗
PubMed1982

Properties and proteolysis of ferric enterobactin outer membrane receptor in Escherichia coli K12.

A protein with a relative subunit molecular weight of 81000 (81K) has been isolated in virtually pure form from the outer membrane of low iron grown cells of Escherichia coli K12. The 81K protein, which is part of the receptor complex for translocation of the siderophore ferric enterobactin, displays activity in vitro for binding both ferric enterobactin and colicin B. The dissociation constant for the 81K-ferric enterobactin compound at 4 degrees C in 2% Triton-0.1 M Tris, pH 7, was determined to be 10 nM. The N-terminal amino acid was identified as phenylalanine, and the amino acid composition was shown to be similar to that published for the ferric aerobactin-cloacin receptor of Enterobacter cloacae. A plasmid-bearing strain of E. coli was employed to confirm that degradation of 81K to a slightly smaller, inactive form (81K) is performed by a second outer membrane component, protein a. The endoproteolytic action of protein a was verified by the finding of alanine as the N-terminal residue of 81K. A survey of enteric species suggests that the 81K-protein a interaction is confined to the K12 strain of E. coli.

Amino Acids↗
PubMed1982

Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli.

Using biological iron chelators to control specifically iron availability to Escherichia coli K-12 in conjunction with radioactive pulse-labels, we examined the biosynthesis of six iron-regulated membrane proteins. Iron deprivation induced the synthesis of five proteins, which had molecular weights of 83,000 (83K), 81K (Fep), 78K (TonA), 74K (Cir), and 25K. The kinetics of induction were the same in entA and entA(+) strains, but were affected by the initial iron availability in the media. Iron-poor cells induced rapidly (half-time, 10 min), whereas iron-rich cells began induction after a lag and showed a slower induction half-time (30 min). Within this general pattern of induction after iron deprivation, several different kinetic patterns were apparent. The 83K, 81K, and 74K proteins were coordinately controlled under all of the conditions examined. The 78K and 25K proteins were regulated differently. The synthesis of a previously unrecognized 90K inner membrane protein was inhibited by iron deprivation and stimulated by iron repletion. Both ferrichrome and ferric enterobactin completely repressed 81K and 74K synthesis when the siderophores were supplied at concentrations of 5 muM in vivo (half-time, 2.5 min). At concentrations less than 5 muM, however, both siderophores repressed synthesis only temporarily; the duration of repression was proportional to the amount of ferric siderophore added. The half-lives of the 81K and 74K mRNAs, as measured by rifampin treatment, were 1.2 and 1.6 min, respectively. The results of this study suggest that enteric bacteria are capable of instantaneously detecting and reacting to fluctuations in the extracellular iron concentration and that they store iron during periods of iron repletion for utilization during periods of iron stress. Neither iron storage nor iron regulation of envelope protein synthesis is dependent on the ability of the bacteria to form heme.

Bacterial Proteins↗
PubMed1981

Stereospecificity of the ferric enterobactin receptor of Escherichia coli K-12.

Synthetic enterobactin and enantioenterobactin (D-seryl enterobactin) have been examined for the ability to transport iron in Escherichia coli. Failure of the unnatural, D-serine-derived material to support growth of E. coli mutants indicates outer membrane receptor specificity for the naturally occurring complex having an L-seryl backbone and the delta-cis configuration of the Fe(III).catecholate center. Enantioenterobactin was markedly less effective in protecting cells against colicin B compared to synthetic or natural enterobactin.

Bacterial Outer Membrane Proteins↗