Siderophore production by Enterobacter cloacae and a common receptor protein for the uptake of aerobactin and cloacin DF13.

Iron-starved cultures of Enterobacter cloacae produced two siderophores, identified as enterochelin and aerobactin. The aerobactin was excreted in larger amounts than was enterochelin, and it was synthesized preferentially in the late logarithmic and stationary growth phases under iron-deficient conditions. Enterochelin was synthesized by cultures in the logarithmic phase of growth and preferentially in medium with 1 microM ferric chloride. Both siderophores appeared to be excreted immediately after their synthesis, since no intracellular aerobactin or enterochelin could be detected. The killing activity of the bacteriocin cloacin DF13 was inhibited by aerobactin. It was shown that aerobactin and cloacin DF13 bound to the same receptor sites located in the outer membrane. The synthesis of these receptor sites was induced by iron limitation. We conclude that the receptor for the uptake of aerobactin also functions as receptor for cloacin DF13.     

Novel aerobactin receptor in Klebsiella pneumoniae

Several Klebsiella pneumoniae strains which produced enterochelin but not aerobactin were nevertheless sensitive to cloacin DF13. In contrast, a strain of serotype K1:O1 which produced both siderophores was cloacin-resistant. Loss by mutation of the O1 but not K1 antigen rendered this strain cloacin-sensitive, indicating that the O1 antigen prevented access of cloacin to the cloacin/aerobactin receptor. Unlike the K1:O1 strain, the aerobactin-negative strains failed to hybridize in a colony blot assay with an aerobactin receptor gene probe prepared from pColV-K30. However, antisera raised against the 74 kDa pColV-K30 aerobactin receptor cross-reacted with a 76 kDa outer-membrane protein in each K. pneumoniae strain. In addition to the 76 kDa protein, the K1:O1 strain also produced a strongly cross-reacting 74 kDa protein. To determine whether these aerobactin-negative strains could use aerobactin, mutants unable to synthesize siderophores were isolated. Aerobactin promoted the growth of these mutants in iron-deficient media. The evidence presented suggests that some K. pneumoniae strains produce an aerobactin iron-uptake system without apparent production of aerobactin and which is probably based on a 76 kDa receptor, the gene for which does not hybridize with aerobactin receptor gene encoded on pColV-K30.     

In vitro growth of urinaryEscherichia coli related to siderophore production

Thirty seven strains ofEscherichia coli isolated from the urine of patients with acute symptomatic urinary tract infection were examined for siderophore production: hydroxamate (aerobactin) and phenolate (enterochelin). All the strains were found to produce varying amounts of enterochelin. With the chemical assay, 24.3% strains were aerobactin producers, while 43.2% were positive in the bio-assay. All the aerobactin producers carried the aerobactin receptor on their surface. Attempts to correlate siderophore production with growth in minimal and iron-depleted medium showed that there was a positive quantitative correlation between enterochelin production and growth of organisms under iron depletion. Aerobactin production failed to give an additional advantage of growth to strains producing enterochelin.     

Incidence of the aerobactin iron uptake system among Escherichia coli isolates from infections of farm animals

To assess the importance of aerobactin-mediated iron uptake as a bacterial virulence determinant in animal infections, a total of 576 strains of Escherichia coli isolated from cattle, chickens, sheep and pigs were screened by colony hybridization to determine the presence of the aerobactin genetic determinants, and by a bioassay to detect aerobactin secretion in iron-limited conditions. Results obtained by the two complementary methods correlated well. The incidence of the aerobactin system was very high among septicaemia isolates, particularly those from cattle and chickens, an observation that strongly suggests an important role for this mechanism of iron assimilation in pathogenesis. On the other hand, the incidence of the aerobactin system among mastitis strains was not significantly higher than among faecal isolates from healthy animals. No classical enterotoxigenic E. coli strains tested carried the aerobactin genetic determinants. Although most strains that produced aerobactin were also able to make colicin V, the fact that the two characteristics existed separately in a significant minority of isolates suggested that colicin testing alone could not be reliably used to determine the presence of the aerobactin system.     

Iron transport systems of Serratia marcescens.

Serratia marcescens W225 expresses an unconventional iron(III) transport system. Uptake of Fe3+ occurs in the absence of an iron(III)-solubilizing siderophore, of an outer membrane receptor protein, and of the TonB and ExbBD proteins involved in outer membrane transport. The three SfuABC proteins found to catalyze iron(III) transport exhibit the typical features of periplasmic binding-protein-dependent systems for transport across the cytoplasmic membrane. In support of these conclusions, the periplasmic SfuA protein bound iron chloride and iron citrate but not ferrichrome, as shown by protection experiments against degradation by added V8 protease. The cloned sfuABC genes conferred upon an Escherichia coli aroB mutant unable to synthesize its own enterochelin siderophore the ability to grow under iron-limiting conditions (in the presence of 0.2 mM 2.2'-dipyridyl). Under extreme iron deficiency (0.4 mM 2.2'-dipyridyl), however, the entry rate of iron across the outer membrane was no longer sufficient for growth. Citrate had to be added in order for iron(III) to be translocated as an iron citrate complex in a FecA- and TonB-dependent manner through the outer membrane and via SfuABC across the cytoplasmic membrane. FecA- and TonB-dependent iron transport across the outer membrane could be clearly correlated with a very low concentration of iron in the medium. Expression of the sfuABC genes in E. coli was controlled by the Fur iron repressor gene. S. marcescens W225 was able to synthesize enterochelin and take up iron(III) enterochelin. It contained an iron(III) aerobactin transport system but lacked aerobactin synthesis. This strain was able to utilize the hydroxamate siderophores ferrichrome, coprogen, ferrioxamine B, rhodotorulic acid, and schizokinen as sole iron sources and grew on iron citrate as well. In contrast to E. coli K-12, S. marcescens could utilize heme. DNA fragments of the E. coli fhuA, iut, exbB, and fur genes hybridized with chromosomal S. marcescens DNA fragments, whereas no hybridization was obtained between S. marcescens chromosomal DNA and E. coli fecA, fhuE, and tonB gene fragments. The presence of multiple iron transport systems was also indicated by the increased synthesis of at least five outer membrane proteins (in the molecular weight range of 72,000 to 87,000) after growth in low-iron media. Serratia liquefaciens and Serratia ficaria produced aerobactin, showing that this siderophore also occurs in the genus Serratia.     

High-affinity iron uptake systems present in Erwinia carotovora subsp. carotovora include the hydroxamate siderophore aerobactin.

The phytopathogenic bacterium Erwinia carotovora subsp. carotovora W3C105 produced the hydroxamate siderophore aerobactin under iron-limiting conditions. A survey of 22 diverse strains of E. carotovora revealed that strain W3C105 alone produced aerobactin. The ferric-aerobactin receptor of strain W3C105 was an 80-kDa protein, identified by immunoblots of Sarkosyl-soluble proteins obtained from E. carotovora cells grown in iron-depleted medium and probed with antiserum raised against the 74-kDa ferric-aerobactin receptor encoded by the pColV-K30 plasmid of Escherichia coli. Genes determining aerobactin biosynthesis and uptake were localized to an 11.3-kb EcoRI-HindIII chromosomal fragment of strain W3C105. A 10-kb subclone of the fragment conferred on E. coli DH5 alpha both aerobactin biosynthesis and uptake, determined by cloacin DF13 sensitivity, the presence of the 80-kDa receptor protein, and iron-independent growth of E. coli clones. The aerobactin biosynthesis genes of E. carotovora W3C105 hybridized to those of the pColV-K30 plasmid of E. coli, but the restriction patterns of the aerobactin regions of E. coli and E. carotovora differed. Although the aerobactin region of enteric bacteria is commonly flanked by IS1-like sequences, IS1 sequences were not detected in the genomic DNA or the cloned aerobactin region of E. carotovora. E. coli DH5 alpha cells harboring cloned aerobactin biosynthesis genes from E. carotovora W3C105 produced greater quantities of aerobactin and the 80-kDa ferric-aerobactin receptor when grown in iron-limited than in iron-replete medium. Strain W3C105 grew on an iron-limited medium, whereas derivatives that lacked a functional aerobactin iron acquisition system did not grow on the medium. These results provide evidence for the occurrence and heterogeneity of aerobactin as a high-affinity iron uptake system of both clinical and phytopathogenic species of the Enterobacteriaceae. Although future studies may reveal a role for aerobactin in the virulence or ecology of strain W3C105, a functional aerobactin iron acquisition system is not necessary for the pathogenicity of E. carotovora.     

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1.

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.     

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.     

DNA environment of the aerobactin iron uptake system genes in prototypic ColV plasmids.

The aerobactin iron uptake system genes in the prototypic plasmid pColV-K30 are flanked by inverted copies of insertion sequence IS1 and by two distinct replication regions. To address the question of how these flanking regions may facilitate the maintenance and spread of the aerobactin system among the plasmids and chromosomes of enteric species, we investigated the DNA environment of 12 ColV plasmids. We found that the aerobactin system-specific genes are conserved in every plasmid phenotypically positive for the aerobactin system. The upstream IS1 and its overlapping replication region (REPI) are also conserved. This replication region was cloned from several ColV plasmids and found to be functional by transforming these cloned derivatives into a polA bacterial host. In contrast, the downstream flanking region is variable. This includes the downstream copy of IS1 and the downstream replication region (REPII). We infer from these results that sequences in addition to the two flanking copies of IS1, in particular the upstream region including REPI, have been instrumental in the preservation and possible spread of aerobactin genes among ColV plasmids and other members of the FI incompatibility group.     

Identification of the siderophores from Vibrio hollisae and Vibrio mimicus as aerobactin

Abstract Hydroxamate siderophores were purified from low-iron cultures of Vibrio hollisae ATCC 33564 and Vibrio mimicus ATCC 33653. By a combination of ¹H and ¹³C NMR spectroscopy, fast atom bombardment mass spectrometry, and compositional analysis, both of the siderophores were identified as aerobactin, a citrate-based dihydroxamate siderophore, which is highly prevalent in species of the family Enterobacteriaceae . Four other clinical strains belonging to these species also produced aerobactin. In response to iron limitation, all strains expressed two high molecular mass outer membrane proteins. The protein with an apparent molecular mass of 77 kDa, which was common to all strains examined, may be the ferric aerobactin receptor.     

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

Summary 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.     

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.     

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.     

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.     

Divergence of the aerobactin iron uptake systems encoded by plasmids pColV-K30 in Escherichia coli K-12 and pSMN1 in Aerobacter aerogenes 62-1.

Although the aerobactin-mediated iron uptake system has been characterized genetically in Escherichia coli, the siderophore aerobactin was chemically characterized after purification from culture supernatants of Aerobacter aerogenes 62-1, a member of the Klebsielleae. We have cloned and mapped the genes encoding the aerobactin system genes of A. aerogenes 62-1 and begun characterization of the relevant proteins and enzymatic activities of this plasmid-mediated aerobactin system. Published chemical data indicate that the siderophore aerobactin of E. coli is the same molecule as the aerobactin of Aerobacter aerogenes 62-1, but we have found that both the genes and the complement of proteins making up the biosynthetic enzymes in the two systems have diverged. In contrast, the outer membrane receptors for ferric aerobactin of the two systems showed immunologic cross-reactivity, were of the same molecular size (74 kilodaltons), and were encoded by homologous DNA sequences.     

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.     

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.