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.     

Cloning and characterization of the iutA gene which encodes ferric aerobactin receptor from marine Vibrio species

The iutA gene from marine Vibrio species SD004, which encoded a ferric aerobactin receptor for the uptake of iron(III), was cloned onto a multicopy plasmid, pUC 18, in Escherichia coli. Identification of the positive clone was achieved on the basis of its deferrization activity and was detected as a halo formation on the chrome azurol S (CAS)-containing selective plate. Nucleotide sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) which encoded a polypeptide of 706 amino acid residues, and the deduced molecular mass of this polypeptide was 77.906 kD. The amino acid sequence showed a 41% homology with that of the lutA protein from E. coli. The cloned gene was iutA, which encoded the ferric aerobactin receptor. Another incomplete ORF was found 100 bp upstream of the iutA gene, which was homologous (31 out of 49 amino acids) with the C-terminal region of the luc D protein of E. coli. It is suggested that aerobactin biosynthesis and the transport genes are located tandemly on the Vibrio chromosome and may form an aerobactin operon.     

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster.

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of two contiguous operons required for aerobactin transport and biosynthesis in Vibrio mimicus

In response to iron deprivation, Vibrio mimicus produces aerobactin as a major siderophore. Application of the Fur titration assay to a V. mimicus genomic DNA library followed by further cloning of the surrounding regions led to the identification of two adjacent, iron-regulated operons. One contains three genes encoding homologs of the Escherichia coli FhuCDB and the other, five genes encoding homologs of the E. coli IucABCD IutA. Construction of the V. mimicus polar disruptants in the respective operons allowed us to confirm their functions. The genetic arrangement of the aerobactin-mediated iron acquisition system in V. mimicus is unique in that the aerobactin operon (iucABCD iutA ) is contiguous to the operon (matCDB ) encoding components of an ATP-binding cassette transport system for ferric aerobactin. This is the first report demonstrating that aerobactin transport and biosynthesis genes are present in a species outside the family Enterobacteriaceae.     

Transport of ferric-aerobactin into the periplasm and cytoplasm of Escherichia coli K12: role of envelope-associated proteins and effect of endogenous siderophores.

Purified [14C]aerobactin, supplied exogenously to non-growing bacteria, was translocated via the periplasm into the cytoplasm of Escherichia coli K12 strains expressing the aerobactin receptor protein IutA. No significant uptake was observed into either compartment of strains lacking the iutA gene or specifically defective in tonB. Uptake into both compartments was markedly reduced, but not abolished, in an exb mutant. Accumulation of [14C]aerobactin in the periplasm of fhuD, fhuB or fhuC mutant strains was not significantly lower than in the wild-type strain, but entry into the cytoplasm was greatly reduced in all cases. Uptake of aerobactin by strains wild-type for all transport functions occurred most efficiently in strains either lacking or specifically defective in the genetic determinants for aerobactin biosynthesis; significantly lower levels of exogenous 14C-labelled siderophore were observed in both compartments of strains producing aerobactin. Aerobactin-mediated 59Fe uptake, however, was not inhibited by the presence of endogenous aerobactin. Endogenous enterochelin did not affect aerobactin uptake.     

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.     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

The<Emphasis Type="Italic">Escherichia fergusonii iucABCD iutA</Emphasis> genes are located within a larger chromosomal region similar to pathogenicity islands

Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.     

Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus

Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis. DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus. This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.     

New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae.

Unlike the great majority of the aerobactin-producing enteric bacteria documented in the literature, Enterobacter cloacae EK33, isolated from a case of human neonatal meningitis, did not show any homology at the DNA level with the prototype aerobactin system encoded by the ColV-K30 plasmid. However, both the nuclear magnetic resonance spectrum and fast-atom bombardment mass spectrometry of the siderophore purified from EK33 confirmed its identity with aerobactin. Bioassay screening of a gene library of total DNA of EK33 led to the isolation of several aerobactin-positive clones. Under conditions of iron limitation, these clones expressed in Escherichia coli a protein of 72 kilodaltons that reacted with antiserum raised against the pColV-K30 74-kilodalton aerobactin receptor, while the original E. cloacae strain synthesized an 85-kilodalton protein which also cross-reacted with the antiserum. Restriction endonuclease analysis of the cloned DNA confirmed the structural differences between the two aerobactin genetic systems.     

Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids

Summary Iron acquisition via aerobactin enhances the virulence of Escherichia coli. Genes that specify functions for aerobactin synthesis and iron(III)-aerobactin transport have been identified on several ColV plasmids. Previously, we cloned the locus for aerobactin synthesis from pColV-K311 an dassigned to three loci termed AeA, aerB, and areC the functions for hydroxylation of lysine, acetylation of the 6-amino group of 6-hydroxy-lysine and coupling of N-acetyl-N-hydroxy-lysine with citrate, respectively (Gross et al. 1984). In this paper we show that aerA and aerB determine polypeptides with molecular weights of 50,000 and 35,000, respectively. We identified a fourth gene designated aerD that codes for a polypeptide with a molecular weight of 60,000, and which is required for the linkage of one residue of N-acetyl-N-hydroxy-lysine to citrate. The aerC gene product completes aerobactin synthesis by coupling the secod N-acetyl-N-hydroxy-lysine to the monoacylated derivative citrate. The order of the genes in the operon was found to be aerD-aerB-areC-aerA.     

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.     

Identification of rhtX and fptX, novel genes encoding proteins that show homology and function in the utilization of the siderophores rhizobactin 1021 by Sinorhizobium meliloti and pyochelin by Pseudomonas aeruginosa, respectively

Rhizobactin 1021 is a hydroxymate siderophore produced by the soil bacterium Sinorhizobium meliloti 2011. A regulon comprising rhtA, encoding the outer membrane receptor protein for the ferrisiderophore; the biosynthesis operon rhbABCDEF; and rhrA, the Ara-C-like regulator of the receptor and biosynthesis genes has been previously described. We report the discovery of a gene, located upstream of rhbA and named rhtX (for "rhizobactin transport"), which is required, in addition to rhtA, to confer the ability to utilize rhizobactin 1021 on a strain of S. meliloti that does not naturally utilize the siderophore. Rhizobactin 1021 is structurally similar to aerobactin, which is transported in Escherichia coli via the IutA outer membrane receptor and the FhuCDB inner membrane transport system. E. coli expressing iutA and fhuCDB was found to also transport rhizobactin 1021. We demonstrated that RhtX alone could substitute for FhuCDB to transport rhizobactin 1021 in E. coli. RhtX shows similarity to a number of uncharacterized proteins which are encoded proximal to genes that are either known to be or predicted to be involved in iron acquisition. Among these is PA4218 of Pseudomonas aeruginosa, which is located close to the gene cluster that functions in pyochelin biosynthesis and outer membrane transport. PA4218 was mutated by allelic replacement, and the mutant was found to have a pyochelin utilization-defective phenotype. It is proposed that PA4218 be named fptX (for "ferripyochelin transport"). RhtX and FptX appear to be members of a novel family of permeases that function as single-subunit transporters of siderophores.