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J B Neilands

Publications and source records attributed to J B Neilands.

At least 19 recordsLinked to original sources
PubMed1992

Iron(III) complexes of chrysobactin, the siderophore of Erwinia chrysanthemi.

The phytopathogenic bacterium Erwinia chrysanthemi produces the monocatecholate siderophore chrysobactin under conditions of iron deprivation. Only the catecholate hydroxyl groups participate in metal coordination, and chrysobactin is therefore unable to provide full 1:1 coordination of Fe(III). The stoichiometry in aqueous solution is a variable dependent on pH and metal/ligand ratio, in addition to being concentration dependent. At neutral pH and concentrations of about 0.1 mM, ferric chrysobactin exists as a mixture of bis and tris complexes. Chrysobactin and its isomers form optically active tris complexes. The dominant configuration depends on the chirality of the amino acid to which the catecholate moiety is attached.

Dickeya chrysanthemi↗
PubMed1992

Negative transcriptional control of iron transport in Erwinia chrysanthemi involves an iron-responsive two-factor system.

Systemic virulence of the phytopathogen Erwinia chrysanthemi 3937 requires a functional iron assimilation system which, in this enterobacterium, is mediated by the siderophore chrysobactin and the outer membrane transport protein Fct. We investigated the regulation of this system by iron. No direct similarity with the Escherichia coli fur gene was found. Insertional mutagenesis allowed isolation of a regulatory mutant which expressed chrysobactin and two other high-affinity iron transport systems previously characterized in strain 3937, regardless of the iron level. RNA/DNA hybridization analysis established that regulation of chrysobactin by iron occurs at the transcriptional level. From a wild-type gene library, a recombinant cosmid able to restore normal regulation in the mutant strain was isolated. By generating a series of subclones and mini-Mulac insertions, we identified a regulatory locus (cbr) extending beyond c. 2.5kb which encodes two polypeptides, CbrA and CbrB, with molecular weights of 34,000 and 55,000 respectively. Functional analysis of the locus suggests that the cognate genes cbrA and cbrB are clustered within an operon. Their expression was studied through chromosomal lac gene fusions, in the presence of plasmid-borne wild-type constructions, under high- and low-iron conditions. In summary, the data show that in the presence of iron, cbr negatively regulates the chrysobactin biosynthetic and transport genes, while under conditions of depletion, cbr is subject to negative autogeneous regulation.

Biological Transport↗
PubMed1992

Identification of 2,3-dihydroxybenzoic acid as a Brucella abortus siderophore.

Brucella abortus grown in low-iron medium or in the presence of iron chelators [ethylenediamine-di(o-hydroxyphenylacetic acid) and 2,2-dipyridyl] showed reduced cell yields and released a material positive in chemical and biological assays for catechols. This material was purified from culture fluids of B. abortus 2308 by chromatography on agarose-iminodiacetic acid-Fe3+ and identified as 2,3-dihydroxybenzoic acid (2,3-DHBA) by thin-layer chromatography, paper electrophoresis, and UV-visible nuclear magnetic resonance and mass spectroscopy. No other major catechols were observed at different stages of growth, and 2,3-DHBA was also produced upon iron limitation by representative strains of B. abortus biotypes 1, 5, 6, and 9. Both synthetic 2,3-DHBA and the natural catechol relieved the growth inhibition of B. abortus 2308 by ethylenediamine-di(o-hydroxyphenylacetic acid), and 2,3-DHBA promoted 55Fe uptake by B. abortus 2308 by an energy-dependent mechanism. Two other monocatechols tested, 2,3-dihydroxybenzoyl-Ser and 2,3-dihydroxybenzoyl-Gly, also promoted 55Fe uptake. More complex catechol siderophores (agrobactin and enterobactin), hydroxamate siderophores (aerobactin, ferrichrome, and deferriferrioxamine mesylate [Desferal]), and an EDTA-related siderophore (rhizobactin) failed to mediate 55Fe uptake. B. abortus cells grown in low-iron medium or in medium with iron had similar rates of iron uptake when supplied with 55Fe-2,3-DHBA, and the release of 2,3-DHBA under iron starvation was not associated with the expression of new outer membrane proteins. These results suggest an uptake system in which only the synthesis of the siderophore is regulated by the iron available for growth.

Bacterial Outer Membrane Proteins↗
PubMed1992

Ferric iron uptake in Erwinia chrysanthemi mediated by chrysobactin and related catechol-type compounds.

Erwinia chrysanthemi 3937 possesses a saturable, high-affinity transport system for the ferric complex of its native siderophore chrysobactin, [N-alpha-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine]. Uptake of 55Fe-labeled chrysobactin was completely inhibited by respiratory poison or low temperature and was significantly reduced in rich medium. The kinetics of chrysobactin-mediated iron transport were determined to have apparent Km and Vmax values of about 30 nM and of 90 pmol/mg.min, respectively. Isomers of chrysobactin and analogs with progressively shorter side chains mediated ferric iron transport as efficiently as the native siderophore, which indicates that the chrysobactin receptor primarily recognizes the catechol-iron center. Free ligand in excess only moderately reduced the accumulation of 55Fe. Chrysobactin may therefore be regarded as a true siderophore for E. chrysanthemi.

Catechols↗
PubMed1992

Mechanism and regulation of synthesis of aerobactin in Escherichia coli K12 (pColV-K30).

The aerobactin operon of the virulence plasmid pColV-K30 of Escherichia coli K12 consists of four genes for biosynthesis and one for transport of the siderophore. Regulation by iron occurs at the transcriptional level and is mediated by a ferrous iron binding protein designated Fur (ferric uptake regulation). The metallated Fur repressor binds at a palindromic dyad, the "iron box" operator, situated in the vicinity of the RNA polymerase attachment site of the promoter. Evidence suggests that the ferrous iron enters the C-terminal domain of Fur to cause a conformational change in the N-terminal part of the protein. This results in greatly enhanced affinity of the repressor for the operator.

Bacterial Proteins↗
PubMed1979

Agrobactin, a siderophore from Agrobacterium tumefaciens.

A siderophore (microbial iron transport compound) was isolated from low iron cultures of Agrobacterium tumefaciens B6. The substance was characterized as a threonyl peptide of spermidine acylated with 3 residues of 2,3-dihydroxybenzoic acid, the carbonyl group of 1 residue of the latter participating in an oxazoline ring with the beta-hydroxyl of the threonine moiety. The compound, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2,3-dihydroxybenzamido)butyl]-2-(2,3-dihydroxyphenyl)-trans-5-methyl-oxazoline-4-carboxamide, was given the trivial name agrobactin. Exposure to acid opened the oxazoline ring to afford agrobactin A. Ferric agrobactin A and agrobactin A itself, but not agrobactin or its ferric complex, had some capacity to feed iron to enterobactin-deficient strains of Escherichia coli and Salmonella typhimurium. Agrobactin was produced by A. tumefaciens in response to iron deficiency and was able to reverse the iron starvation in this organism precipitated by the presence of a ferric complexing agent not utilized by the cells.

Biological Transport↗
PubMed1978

Electronic and resonance Raman spectra of iron(III) complexes of enterobactin, catechol, and N-methyl-2,3-dihydroxybenzamide.

Resonance Raman electronic absorption and circular dichroism spectra and pH titration curves are reported for the trianionic ferric complexes of enterobactin, catechol, and N-methyl-2,3-dihydroxybenzamide (MDHB). The spectral signatures of the enterobactin and MDHB complexes are virtually identical and differ from those of the catechol complex in ways that reflect the influence of the amide group on the electronic structure. Excitation in either the visible charge-transfer bands or the near-ultraviolet pi-pi* bands enhances Raman bands associated with benzene ring modes, although the relative enhancements differ markedly in the two regions. The data stronly support a structural model in which iron is bound exclusively to the phenolate oxygen atoms in all three complexes.

Benzamides↗
PubMed1978

Ferric enterobactin transport system in Escherichia coli K-12. Extraction, assay, and specificity of the outer membrane receptor.

An outer membrane preparation from cells of Escherichia coli K-12 grown in low iron medium was found to retain ferric enterobactin binding activity following solubilization in a Tris-HCl, Na2EDTA buffer containing Triton X-100. Activity was measured by means of a DEAE-cellulose column which separated free and receptor bound ferric enterobactin. The binding activity was greatly reduced in preparations obtained from cells grown in iron rich media or from cells of a colicin B resistant mutant grown in either high or low iron media. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis enabled correlation of this lack of activity to a single band missing in the outer membrane profile of the colicin B mutant. Evidence was obtained for in vitro competition between ferric enterobactin and colicin B for the extracted receptor. The binding specificity of the extracted receptor was examined by competition between ferric enterobactin and several iron chelates including a carbocyclic analogue of enterobactin, cis-1,5,9-tris(2,3-dihydroxybenzamido)cyclododecane. The ferric form of the latter compound supported growth of siderophore auxotrophs, apparently without hydrolysis to dihydroxybenzoic acid and resynthesis into enterobactin. These data may require revision of the accepted mechanism of enterobactin mediated iron utilization.

Cell Membrane↗
PubMed1976

The structure of two alanine containing ferrichromes: sequence determination by proton magnetic resonance.

Metal coordination confers an extraordinary structural stability to the ferrichromes which, independent of their variable amino acid composition, results in a basically unperturbed conformation for all the homologous peptides in the series. The proton magnetic resonance (pmr) characteristics for Al3+ analogues (alumichromes) reflect this conformational isomorphism in usual solvents so that single site substitutions are clearly recognized in the pmr spectra. Thus, the substitution of glycine by L-alanine or L-serine introduce new resonances characteristic of the sidechains and alter the pattern of the amide NH pmr region in that doublets substitute for glycyl triplets at the same site. Since for glycine- and L-serine containing alumichromes the resonances have already been identified, it is possible to unequivocally establish the primary structure of the two L-alanyl homologues ferrichrome C (see article) and sake colorant A (see article) on the basis of the comparative pmr spectra of their Al3+ analogues, namely, alumichrome C and alumisake. The resonance assignment, and hence the site occupancy, is substantiated by the temperature coefficients of the NH chemical shifts, rates of 1H-2H exchange and homonuclear proton spin decoupling experiments centered on the NH spectral region. Occupancy of site 1 by a glycine residue is observed for all known ferrichromes, which serves to conserve a "hairpin" turn. This method of obtaining sequence information should prove of general use for other systems of homologous polypeptides, provided their conformations are not affected by the residue substitutions.

Alanine↗
PubMed1976

Siderophore protection against colicins M, B, V, and Ia in Escherichia coli.

A variety of natural and synthetic siderophores capable of supporting the growth of Escherichia coli K-12 on iron-limited media also protect strain RW193+ (tonA+ ent-) from the killing action of colicins B, V, and Ia. Protective activity falls into two categories. The first, characteristic of enterobactin protection against colicin B and ferrichrome protection against colicin M, has properties of a specific receptor competition between the siderophore and the colicin. Thus, enterobactin specifically protects against colicin B in fes- mutants (able to accumulate but unable to utilize enterobactin) as predicted by our proposal that the colicin B receptor functions in the specific binding for uptake of enterobactin (Wayne and Neilands, 1975). Similarly ferrichrome specifically protects against colicin M in SidA mutants (defective in hydroxamate siderophore utilization). The second category of protective response, characteristic of the more general siderophore inhibition of colicins B, V, and Ia, requires the availability or metabolism of siderophore iron. Thus, enterobactin protects against colicins V and Ia, but only when the colicin indicator strain is fes+, and hydroxamate siderophores inhibit colicins B, V, and Ia, but only when the colicin indicator strain is SidA+. Moreover, ferrichrome inhibits colicins B, V, and Ia, yet chromium (III) deferriferrichrome is inactive, and ferrichrome itself does not prevent adsorption of colicin Ia receptor material in vitro. Although the nonspecific protection against colicins B, V, and Ia requires iron, the availability of siderophore iron for cell growth is not sufficient to bring about protection. None of the siderophores tested protect cells against the killing action of colicin E1 or K, or against the energy poisons azide, 2, 4-dinitrophenol, and carbonylcyanide m-chlorophenylhydrazone. We suggest that nonspecific siderophore protection against colicins B, V, and Ia may be due either to an induction of membrane alterations in response to siderophore iron metabolism or to a direct interference by siderophore iron with some unknown step in colicin action subsequent to adsorption.

Azides↗