Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure

Abstract The cloacin DF13/aerobactin receptor protein from Escherichia coli (pFS8) and from Klebsiella edwardsii were isolated by repeated Triton X-100 extractions and purified by affinity chromatography. Both receptor proteins ran as a single protein band on SDS-PAGE. Their apparent M_r values were 74 000 and 76 000, respectively. The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloacin DF13 were determined. Values of 2.0 × 10⁸ M⁻¹ and 1.0 × 10⁹ M⁻¹, respectively, were found.
The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced. The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues. The mature protein has an M_r of 77 345.     

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.     

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.     

Sensitivity of Escherichia coli to cloacin DF13 involves the major outer membrane protein OmpF

Fourteen spontaneous cloacin DF13-insensitive mutants of an Escherichia coli strain expressing the aerobactin-cloacin DF13 receptor protein IutA were isolated. The mutants fell into three classes on the basis of outer membrane profiles analyzed by electrophoresis in denaturing polyacrylamide gels. The most frequent class lacked the IutA protein and was unable to bind cloacin DF13 or aerobactin. A second class of mutants had lost protein species corresponding in size to the porin proteins OmpF and OmpC. To determine which porin was required for the bactericidal activity of cloacin DF13, defined strains with mutations at the ompB (ompR envZ) locus were transformed with a recombinant plasmid carrying the iutA gene and screened for cloacin DF13 sensitivity. OmpF- strains, whether OmpC+ or OmpC-, were insensitive to cloacin DF13, indicating involvement of the OmpF protein in cloacin DF13 killing. An OmpC- OmpF+ strain, on the other hand, was more sensitive than the wild-type parent strain, probably because of compensatory overexpression of OmpF. The third class of cloacin DF13-insensitive mutant had lost an outer membrane protein of approximately 31 kDa. The nature and function of this protein are not yet known, but it is not the protease OmpT. Mutants of classes 2 and 3 bound cloacin DF13 and aerobactin as effectively as the cloacin DF13-sensitive parental strain, indicating that they remained IutA+. We propose that these mutants (more accurately described as cloacin DF13 tolerant) are defective in translocation of the active portion of cloacin DF13 across the bacterial membranes.     

Subcloning of the cloacin DF13/aerobactin receptor protein and identification of a pColV-K30-determined polypeptide involved in ferric-aerobactin uptake.

A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin.     

Inhibition of biological activities of the aerobactin receptor protein in rough strains of Escherichia coli by polyclonal antiserum raised against native protein

The aerobactin iron-uptake system of plasmid ColV-K30, genetically isolated from other plasmid determinants by molecular cloning, was sufficient to restore full virulence in a mouse peritonitis model to a clinical Escherichia coli isolate, D551 (O78:H-), whose resident aerobactin-encoding ColV plasmid had been lost by curing. Antiserum was raised in rabbits against live E. coli K12 cells expressing the outer-membrane aerobactin receptor protein and absorbed with an isogenic strain lacking the receptor. This antiserum inhibited binding of aerobactin, cloacin DF13 and bacteriophage B74K to the native protein in whole E. coli K12 bacteria expressing the receptor, or in membranes prepared from such organisms. However, it did not react with the native receptor protein in several wild strains unless lipopolysaccharide was first removed by treatment with trichloroacetic acid, nor did it protect mice in experimental infections with strain D551. Antisera raised in rabbits against partially or fully denatured forms of the aerobactin receptor reacted only in assays involving denatured protein; they showed no inhibition of the biological activities of the native receptor.     

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.     

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.     

tolQ is required for cloacin DF13 susceptibility in Escherichia coli expressing the aerobactin/cloacin DF13 receptor IutA

We investigated the role of the tolQ gene in the import of cloacin DF13 across the outer membrane of Escherichia coli strains expressing the IutA receptor. The IutA outer-membrane protein is the receptor for the siderophore ferric aerobactin and also binds cloacin DF13, a bacteriocin produced by strains of Enterobacter aerogenes. In this report we present evidence that tolQ is required for the internalization of cloacin DF13 upon binding to IutA but it is not involved in the transport of ferric aerobactin.     

Regulation of the ColV plasmid-determined iron (III)-aerobactin transport system in Escherichia coli.

Regulation by iron was studied in Escherichia coli strains whose iron supply was entirely dependent on the iron(III)-aerobactin system determined by the ColV plasmid. By the insertion of phage Mu (Ap lac) into the ColV plasmid, mutants were selected that could no longer grow in iron-limited media. The inserted Mu (Ap lac) strongly reduced the amount of aerobactin and he cloacin receptor protein formed by the cells. Their production was no longer subject to regulation by iron. The Mu (Ap lac) insertion apparently led to a polar effect on the expression of the presumably closely linked genes that control the synthesis of aerobactin and the cloacin receptor protein. The expression of the beta-galactosidase gene on the inserted phage genome came under the control of the iron state of the cells. Under iron-limited growth conditions, the amount of beta-galactosidase synthesized was, depending on the strain studied, 6 to 30 times higher than under iron-sufficient growth conditions. In fur mutants with an impaired iron regulation of ll iron supply systems studied so far, high amounts of beta-galactosidase were synthesized independent of the cells' iron supply. The results demonstrate an iron-controlled promoter on the ColV plasmid which is subject to regulation by the chromosomal fur gene.     

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 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.     

Siderophore production by Salmonella species isolated from different sources

A total of 230 Salmonella strains were screened for enterobactin and aerobactin production, sensitivity to bacteriocins and resistance to antibiotics. All the isolates produced the phenolate siderophore enterobactin. Amongst these, 74 strains, most belonging to S. enteritidis, were sensitive to colicin B. Only 26 isolates, all belonging to S. wien, produced an additional iron chelator, i.e. the siderophore aerobactin, and 22 out of these were sensitive to cloacin DF13. Analysis of iron repressible outer membrane proteins and plasmid profiles in S. wien strains showed that the expression of a 74-kDa iron-repressible outer membrane protein and the presence of large plasmids were associated with multiple antibiotic resistance, aerobactin production and sensitivity to cloacin DF13. The incidence of aerobactin-producing strains among S. wien isolates was higher during years 1974-1985; the epidemiological implications of these results are discussed.     

Plasmid-determined cloacin DF13-susceptibility inEnterobacter cloacae andKlebsiella edwardsii; identification of the cloacin DF13/aerobactin outer membrane receptor proteins

BothEnterobacter cloacae H478 andKlebsiella edwardsii S15 were shown to harbour a relatively large conjugative plasmid that coded for cloacin DF13-susceptibility and the production and uptake of a hydroxamate iron chelator, most probably aerobactin. Protein-blotting experiments with antiserum raised against the purified cloacin DF13/aerobactin receptor protein fromEscherichia coli (Co1V-K30) revealed that the corresponding outer membrane receptor proteins ofEnt. cloacae H478 andK. edwardsii S15 had apparent mol wts of 85 000 and 76000, respectively.E. coli transconjugants harbouring either the plasmid fromEnt. cloacae H478 orK. edwardsii S15 expressed a cloacin DF13/aerobactin outer membrane receptor protein with a mol wt of 74000. The receptor protein encoded by theEnt. cloacae andK. edwardsii plasmids were immunologically more related to each other than to the pCo1V-K30-encoded receptor protein.     

Cloning and Characterization of Aerobactin Biosynthesis Genes of the Biological Control Agent Enterobacter cloacae

Five strains of Enterobacter cloacae that are biological control agents of Pythium damping-off diseases produced the hydroxamate siderophore aerobactin under iron-limiting conditions. Genes determining aerobactin biosynthesis of the biocontrol strain E. cloacae EcCT-501 were localized to a 12.3-kb region, which conferred aerobactin production to Escherichia coli DH5α. The aerobactin biosynthesis genes of E. cloacae hybridized to those of the pColV-K30 plasmid of E. coli, but restriction patterns of the aerobactin regions of pColV-K30 and E. cloacae differed. A derivative strain with a deletion in the aerobactin biosynthesis locus was as effective as strain EcCT-501 in biological control of Pythium damping-off of cucumber. Thus, aerobactin production did not contribute significantly to the biological control activity of EcCT-501 under the conditions of this study.     

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of hydroxamate and phenolate siderophores by Shigella flexneri.

Shigella flexneri strains were assayed for the ability to synthesize and utilize phenolate and hydroxamate siderophores. The hydroxamate aerobactin was synthesized by all isolates tested, whereas phenolates were only rarely produced. Expression of aerobactin was accompanied by production of a single iron-regulated outer membrane protein (Mr = 74,000). This protein was not produced by a mutant defective in aerobactin utilization and may serve as the aerobactin receptor. Phenolate (enterobactin)-producing strains synthesized three additional outer membrane proteins (Mr = 74,000, 81,000, and 83,000) in response to iron starvation. These proteins are the same apparent size as those produced by Escherichia coli K-12 strains. Ent sequences are apparently present in strains which do not synthesize this compound. Although normally silent, ent genes can be activated in Ent- strains to produce Ent+ variants. These laboratory variants are phenotypically indistinguishable from clinical Ent+ isolates.     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins.

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion, but not overall synthesis, of catecholate siderophores contributes to virulence of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) use siderophores to sequester iron during infection. Enterobactin and salmochelins are catecholate siderophores produced by some ExPEC strains and other pathogenic enterobacteria. Siderophore export and synthesis mutants of avian ExPEC strain χ7122 were tested in a chicken infection model. In single-strain infections, siderophore-negative (ΔentDΔiuc), ΔentS and ΔentSΔiroC export mutants were attenuated in tissues and blood, whereas the ΔiroC export mutant was only attenuated in blood. Interestingly, the ΔentD mutant, producing only aerobactin, retained full virulence, and loss of entD in the ΔentSΔiroC mutant restored virulence. LC-MS/MS quantification of siderophores in export mutants demonstrated that loss of entS impaired enterobactin and mono-glucosylated enterobactin secretion, whereas loss of iroC impaired di- and tri-glucosylated enterobactin secretion. Loss of entS and/or iroC resulted in intracellular accumulation and increased secretion of siderophore monomers. Catecholate siderophore export mutants also demonstrated decreased fitness in a co-challenge infection model. By contrast, catecholate siderophore synthesis mutants (ΔentD and ΔiroB) competed as well as the wild-type strain. Results establish that EntS and IroC mediate specific export of catecholate siderophores and the role of these exporters for ExPEC virulence is contingent on enterobactin synthesis, which is not required when other siderophores like aerobactin are functional.