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.     

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.     

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.     

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.     

The selC-associated SHI-2 pathogenicity island of Shigella flexneri

Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non-pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI-2 (Shigella island 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI-2. SHI-2-hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI-2 may allow Shigella survival in stressful environments, such as those encountered during infection.     

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.     

Colicins produced by the Escherichia fergusonii strains closely resemble colicins encoded by Escherichia coli

Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and the colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of colicin E1, one of colicin Ib, and one of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

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.     

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.     

Specific detection of Escherichia coli and Shigella species using fragments of genes coding for β-glucuronidase

Abstract The occurrence of β-glucuronidase activity, a main characteristic of Escherichia coli and the presence of the uid chromosomal region of E. coli , coding for this enzyme, were tested on representative members of enteric bacteria. DNA hybridization techniques using uid probes and ampplification experiments of uidA gene by the polymerase chain reaction (PCR) confirmed the specificity of uid genes fro E. coli and Shigella spp. (i.e., S. boydii, S. dysenteriae, S. flexneri and S. sonnei ), independent of the β-glucuronidase phenotype of bacterial strains. This specificity seemed to be conserved when studies were extended to a wide range of bacteria. It was not possible to distinguish E. coli from Shigella spp. The detection sensitivity using double stranded DNA radiolabeled probes was 3 × 10⁴ bacteria and could be brought down to 8 bacteria by PCR. Thus, the uid genes appeared to be ideal candidates for DNA probes technology to detect E. coli-Shigella species.     

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)     

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.     

The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes.

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS+ lysU+) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS+ delta lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginose and B, megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.     

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.     

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.     

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4.7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.     

Nucleotide sequence of the ipaBCD structural genes of Shigella dysenteriae

A 9 kb EcoRI and two PstI fragments from the virulence plasmid of Shigella dysenteriae CG097 were shown to contain all ipa genes by probing with Shigella flexneri ipaB, -C, -D and -A gene probes. The DNA sequences of S. dysenteriae ipaBC genes were very similar to those of S. flexneri M90T and S. flexneri YSH6000, but ipaD differed by 22 codons from that of S. flexneri. The differences in ipaD may account for the different in vitro host specificities shown by S. dysenteriae and S. flexneri. The nucleotide composition of ipa genes revealed an unusually large number of codons that are rarely used in Escherichia coli chromosomal genes, indicating a different origin.