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J B Neilands

Publications and source records attributed to J B Neilands.

At least 37 records · Page 2Linked to original sources
PubMed1988

Metal ion regulation of gene expression. Fur repressor-operator interaction at the promoter region of the aerobactin system of pColV-K30.

Transcription of the iron-controlled aerobactin operon of the enterobacterial plasmid pColV-K30 is negatively regulated through the interaction of a Fe2+-binding repressor (the Fur protein) with operator sequences within the promoter region of the operon. The DNA sequences essential for interaction with the repressor were located by site-directed mutagenesis of specific regions within the 31 base-pair protected by the repressor from DNase I nicking. Occupation of two contiguous repressor-binding sites appears to be required for the complete repression of the system. Contacts of the Fur repressor with the corresponding operator sequences were analyzed with hydroxyl radical footprint and methylation protection experiments. These indicate that DNA-protein contacts approach a symmetrical mode and take place at all sides of the DNA helix.

DNA, Bacterial↗
PubMed1988

Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli.

A fusion between the fur (ferric uptake regulation) gene, known to mediate negative regulation of iron absorption in Escherichia coli, and lacZ was constructed in vitro. beta-Galactosidase levels of cells harboring this fusion were under the control of sequences contained in a 185-bp DNA fragment located upstream of the fur structural gene. The fusion was prepared in multicopy (pVLN102 plasmid) and low-copy-number states, the latter constructed as a lambda phage lysogen carrying a fur'-'lacZ insert. DNase I footprinting experiments with purified Fur protein, performed on a 250-bp restriction fragment carrying the promoter region of the fusion, showed the presence of a single Fur-protected site overlapping the -10 region of a potential promoter sequence. Examination of the DNA sequences located upstream of the fur gene revealed a possible binding site for the catabolite-activator protein (CAP). beta-Galactosidase synthesis of E. coli cells harboring the fusion were measured in fur, crp and cya genetic backgrounds and compared with the corresponding levels in wild-type strains. The data obtained indicate a moderate autoregulation of fur expression by its gene product and also a significant stimulation by the cAMP-CAP system. Transcription start sites were mapped by primer-extension experiments with total RNA obtained in vivo from cells harboring pVLN102. The results show that transcription of the fur gene is initiated from at least two different sites separated by 6 bp, which appear to originate from two overlapping promoters sensitive to catabolic activation.

Bacterial Proteins↗
PubMed1988

IS1-mediated mobility of the aerobactin system of pColV-K30 in Escherichia coli.

Genes determining the high affinity iron transport system mediated by the siderophore aerobactin are flanked in the enterobacterial plasmid pColV-K30 by inverted repeats of IS1 sequences, suggesting that the aerobactin genes are part of a transposon. To study this possibility, the entire region between the two IS1 sequences was cloned as an 18 kb HindIII-BamHI restriction fragment in pUC8 giving plasmid pMO1. A number of derivatives of pMO1, in which aerobactin genes were tagged with a kanamycin resistance gene, were prepared in order to assess the ability of both IS1s to promote the formation of cointegrates with pCJ105, an F derivative devoid of insertion sequences. Mating-out assays indicated that both flanking IS1s were active in cointegrate formation at detectable frequencies. In some cases, the cointegrates could be resolved, the final result being a transposition-like event for the entire aerobactin system.

DNA Transposable Elements↗
PubMed1988

Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12.

The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid. The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose. The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue. All four cysteines were shown to be present as thiols. Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis. Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M. A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions.

Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway. The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced. E. coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence. Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA). Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane. The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides.

Amino Acid Sequence↗
PubMed1987

Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli.

The Fur (ferric uptake regulation) protein is a negative regulator of the aerobactin operon and of several other siderophore-mediated, high-affinity iron transport systems in Escherichia coli. The purified Fur protein and a plasmid containing a lacZ fusion to the aerobactin operon were used in conjunction with an in vitro coupled transcription/translation system to demonstrate that the Fur protein requires Fe(II) or certain other divalent metals as a cofactor to negatively regulate expression of the aerobactin operon. In a second set of experiments, using a restriction site protection assay, Fur was shown to bind to and block the aerobactin promoter in a metal-dependent fashion. It is concluded that Fur acts as a classical negative repressor that, under in vivo conditions, uses ionic Fe(II) as a corepressor. Our results support the hypothesis [Williams, R.J.P. (1982) FEBS Lett. 140, 3-10] that prokaryotic cells may contain a standing pool of free or loosely bound Fe(II) that is capable of acting in a regulatory capacity.

Bacterial Proteins↗
PubMed1987

Universal chemical assay for the detection and determination of siderophores.

A universal method to detect and determine siderophores was developed by using their high affinity for iron(III). The ternary complex chrome azurol S/iron(III)/hexadecyltrimethylammonium bromide, with an extinction coefficient of approximately 100,000 M-1 cm-1 at 630 nm, serves as an indicator. When a strong chelator removes the iron from the dye, its color turns from blue to orange. Because of the high sensitivity, determination of siderophores in solution and their characterization by paper electrophoresis chromatography can be performed directly on supernatants of culture fluids. The method is also applicable to agar plates. Orange halos around the colonies on blue agar are indicative of siderophore excretion. It was demonstrated with Escherichia coli strains that biosynthetic, transport, and regulatory mutations in the enterobactin system are clearly distinguishable. The method was successfully used to screen mutants in the iron uptake system of two Rhizobium meliloti strains, DM5 and 1021.

Colorimetry↗
PubMed1987

Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor.

The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion. A 78-base-pair (bp) region containing the main (P1) promoter retained the character of inducibility under iron starvation. A 250-bp fragment carrying this sequence was examined for protection against DNase I by the Fur protein, the product of a gene (fur) required for negative control of several iron-regulated functions. The DNase I footprints, in the presence of various divalent heavy-metal ions added as corepressors, revealed two contiguous binding sites with different lengths and affinities for Fur. Increased concentrations of the protein appeared to elicit formation of repressor oligomers which bind to the upstream and downstream regions of the P1 promoter in a metal-dependent fashion, but with a presently undefined stoichiometry. The primary site for Fur binding spans 31 bp and contains two overlapping symmetry dyads which share the sequence 5'-TCATT-3'. It also contains extensive homology with a 19-bp consensus sequence for iron-regulated genes as deduced from comparison with the fhuA and fepA putative promoter sequences.

Bacterial Proteins↗
PubMed1986

Isolation and properties of N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase from Escherichia coli pABN11.

The enzyme N epsilon-hydroxylysine acetylase has been isolated from Escherichia coli 294 carrying recombinant plasmid ABN11. Activity of the enzyme was followed by measurement of the rate of appearance of 2-nitro-5-thiobenzoate, the product of cleavage of 5,5'-dithiobis(2-nitrobenzoate) by free coenzyme A released from its acetyl derivative. The enzyme bound firmly to Reactive Blue 2-Sepharose CL-6B and was eluated with 1.5 M KCl. The protein gave a single band, corresponding to a Mr of 33,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In contrast, gel filtration of the native enzyme gave a Mr of 150,000-200,000. A sequence analysis of the DNA at the junction of the first and second genes in the aerobactin operon, considered in conjunction with the N-terminal amino acid sequence of the isolated protein, enabled the conclusion that the acetylase is specified by the second gene in the complex. The enzyme transfers the acetyl moiety from acetyl coenzyme A to a variety of hydroxylamines, with N epsilon-hydroxylysine as the preferred substrate. In agreement with the results found by affinity chromatography, Coomassie Blue was observed to act as a potent inhibitor.

Acetyltransferases↗
PubMed1986

Aerobactin biosynthesis and transport genes of plasmid ColV-K30 in Escherichia coli K-12.

The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmid ColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, iron uptake chelate) and transport (iut, iron uptake transport) of the siderophore. The gene order iucABCD iutA was established. The genes were mapped within restriction nuclease fragments of a cloned 16.3-kilobase-pair HindIII fragment. Stepwise deletion and subsequent minicell analysis of the resulting plasmids allowed assignment of four of the five genes to polypeptides of molecular masses 63,000, 33,000 53,000, and 74,000 daltons, respectively. The 74-kilodalton protein, the product of gene iutA, is the outer membrane receptor for ferric aerobactin, whereas the remaining three proteins are involved in biosynthesis of aerobactin. The 33-kilodalton protein, the product of gene iucB, was identified as N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase (acetylase) by comparison of enzyme activity in extracts from various deletion mutants. The 53-kilodalton protein, the product of gene iucD, is required for oxygenation of lysine. The 63-kilodalton protein, the product of gene iucA, is assigned to the first step of the aerobactin synthetase reaction. The product of gene iucC, so far unidentified, performs the second and final step in this reaction. This is based on the chemical characterization of two precursor hydroxamic acids (N epsilon-acetyl-N epsilon-hydroxylysine and N alpha-citryl-N epsilon-acetyl-N epsilon-hydroxylysine) isolated from a strain carrying a 0.3-kilobase-pair deletion in the iucC gene. The results support the existence of a biosynthetic pathway in which aerobactin arises by oxygenation of lysine, acetylation of the N epsilon-hydroxy function, and condensation of 2 mol of the resulting aminohydroxamic acid with citric acid.

Acetyltransferases↗
PubMed1986

Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli.

A cloned 8.3-kilobase-pair DNA fragment carrying all the genes (iucABCD iutA) of the aerobactin iron transport system of plasmid pColV-K30 was subjected to in vitro mutagenesis to afford mutant genes iucA, iucC, and iucA iucC. Complementation analyses and identification of aerobactin precursors accumulated by Escherichia coli cells harboring the different constructions allowed assignment of the iucA and iucC genes to discrete steps in biosynthesis of the siderophore from N epsilon-acetyl-N epsilon-hydroxylysine and citrate. Plasmid pVLN10, a derivative carrying a DNA fragment complementing an iucC mutation, expressed in a minicell system a single 62,000-dalton protein as the product of this gene.

Bacteriocin Plasmids↗
PubMed1985

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12.

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.

Bacterial Outer Membrane Proteins↗
PubMed1985

Artificial siderophores. 1. Synthesis and microbial iron transport capabilities.

Several di- and trihydroxamate analogues of natural microbial iron chelators have been prepared. The syntheses involved linkage of core structural units, including pyridinedicarboxylic acid, benzenetricarboxylic acid, nitrilotriacetic acid, and tricarballylic acid, by amide bonds to 1-amino-omega-(hydroxyamino)alkanes to provide the polyhydroxamates 1-5. The required protected (hydroxyamino)alkanes 8, 16, and 21 were prepared by different routes. 1-Amino-3-[(benzyloxy)amino]propane di-p-toluenesulfonate (8) was prepared from the N-protected aminopropanol 6 by oxidation to the aldehyde, formation of the substituted oxime, and reduction with NaBH3CN followed by deprotection of the Boc group. The pentyl derivatives 16 and 21 were made by direct alkylation with either benzyl acetohydroxamate or N-carbobenzoxy-O-benzylhydroxylamine. In Escherichia coli RW193 most of the analogues behaved nutritionally as ferrichrome. However, in E. coli AN193, a mutant lacking the ferrichrome receptor, capacity to use other natural siderophores was retained while response to all analogues was lost.

Biological Transport↗
PubMed1985

Artificial siderophores. 2. Syntheses of trihydroxamate analogues of rhodotorulic acid and their biological iron transport capabilities in Escherichia coli.

Tris[(acetylhydroxyamino)alkyl] isocyanurates 2a-c were synthesized from alpha, omega-dibromoalkanes 5 in four steps. The alkylation of the bromides 5a-c with O-benzyl-N-[(trichloroethoxy)carbonyl]hydroxylamine in the presence of DBU gave N-alkylation products 7a-c. The (trichloroethoxy)cabronyl protecting group of 7a-c was easily removed by Zn dust in acetic acid. When the reaction was performed with acetic anhydride, the desired N-acetylated materials 10a-c were obtained. The alkylation of cyanuric acid with 12 in the presence of base provided the N-alkylated materials 13, which were hydrogenated to provide 2a-c. In order to determine the affect of structural modifications on biological activity, various chain lengths of the side arms were utilized and the retroanalogue 3 was prepared. Most of the compounds examined acted as ferrichrome in supporting the iron nutrition of Escherichia coli. However, tris[(acetylhydroxyamino)butyl] isocyanurate 2b, and to some extent its pentyl analogue, 2c, displayed the unique and remarkable property of supporting growth of fhuB mutants, the latter unresponsive to the other analogues and to all natural siderophores tested.

Escherichia coli↗