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.     

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.     

Aerobactin iron uptake sequences in plasmid ColV-K30 are flanked by inverted IS1-like elements and replication regions.

By using Southern blot hybridization procedures, we found that a specific sequence within a 16.3-kilobase HindIII restriction fragment of pColV-K30 was also present in at least three other pColV-K30 HindIII fragments. Restriction endonuclease mapping of these HindIII fragments indicated that two of these repeated sequences, identified as IS1-like, occur in reverse orientation adjacent to the ends of the aerobactin iron uptake region, also included in the 16.3-kilobase HindIII fragment. There are two distinct replication regions enclosing the IS1-flanked aerobactin genes. A pColV-K30 BamHI restriction endonuclease fragment, carrying one of these replication regions, showed homology with the F plasmid EcoRI fragment f5, which carries the F replication sequences.     

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.     

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.     

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.     

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.     

Aerobactin-mediated iron uptake system in intestinal Escherichia coli

Abstract Escherichia coli strains isolated from children with diarrhea were studied to determine the incidence of the components of the aerobactin system. The production of aerobactin and the presence of the aerobactin receptor were demonstrated using bioassay techniques. The presence and location of the genes for the aerobactin system was determined by colony and Southern DNA-DNA hydridization. It was found that aerobactin and the aerobactin receptor could be coded by either chromosomal or plasmid genes. A high percentage of strains expressed the receptor in the absence of aerobactin. Of 19 enterotoxin-producing strains one was found to have the complete aerobactin system, while 11 expressed only the aerobactin receptor which was most frequently coded by a plasmid.     

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.     

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.     

Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.     

The SHI-3 Iron Transport Island of Shigella boydii 0-1392 Carries the Genes for Aerobactin Synthesis and Transport

In Shigella boydii 0-1392, genes encoding the synthesis and transport of the hydroxamate siderophore aerobactin are located within a 21-kb iron transport island between lysU and the pheU tRNA gene. DNA sequence analysis of the S. boydii 0-1392 island, designated SHI-3 for Shigella island 3, revealed a conserved aerobactin operon associated with a P4 prophage-like integrase gene and numerous insertion sequences (IS). SHI-3 is present at the pheU tRNA locus in some S. boydii isolates but not in others. The map locations of the aerobactin genes vary among closely related species. The association of the aerobactin operon with phage genes and mobile elements and its presence at different locations within the genomes of enteric pathogens suggest that these virulence-enhancing genes may have been acquired by bacteriophage integration or IS element-mediated transposition. An S. boydii aerobactin synthesis mutant, 0-1392 iucB, was constructed and was similar to the wild type in tissue culture assays of invasion and intercellular spread.     

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.     

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.     

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.     

Novel incompatibility and partition loci for the REPI replication region of plasmid ColV-K30.

The minimum pColV-K30 REPI region necessary for replication was located within a ca. 1.3-kilobase DNA segment. Adjacent to the essential replication sequences, there are two DNA regions that express incompatibility with plasmids containing the F secondary replicon of the F EcoRI fragment f7. One of these regions corresponds to incE, already described in that F plasmid fragment which expresses incompatibility with f7-containing plasmids. The other is a novel sequence that we designated incF, which confers incompatibility with REPI, P307, and f7 derivatives, cis-acting pColV-K30 sequences conferring stability to REPI-containing plasmids were also identified and localized noncontiguous to REPI, ca. 20 kilobases downstream from the aerobactin iron transport genes, which were thus flanked by REPI and its partition (par) sequences.     

The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.