No abstract

SummaryIn Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn 2þ . In the presence of 0.5 mM EGTA, only the parental strain was able to take up 65 Zn 2þ . This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli. At high Zn 2þ concentrations, the znu mutants took up more 65 Zn 2þ than the parental strain. The high-affinity 65 Zn 2þ uptake was repressed by growth in the presence of 10 mM Zn 2þ . A znuA-lacZ operon fusion was repressed by 5 mM Zn 2þ and showed a more than 20-fold increase in b-galactosidase activity when Zn 2þ was bound to 1.5 mM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn 2þ -dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. Highaffinity 65 Zn 2þ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.

Escherichia coli has an iron(II) transport system (feo) which may make an important contribution to the iron supply of the cell under anaerobic conditions. Cloning and sequencing of the iron(II) transport genes revealed an open reading frame (feoA) possibly coding for a small protein with 75 amino acids and a membrane protein with 773 amino acids (feoB). The upstream region offeoAB contained a binding site for the regulatory protein Fur, which acts with iron(ll) as a corepressor in all known iron transport systems ofE. coli. In addition, a Fnr binding site was identified in the promoter region. The FeoB protein had an apparent molecular mass of 70 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was localized in the cytoplasmic membrane.The sequence revealed regions of homology to ATPases, which indicates that ferrous iron uptake may be ATP driven. FeoA or FeoB mutants could be complemented by clones with the feoA orfeoB gene, respectively.Since iron(III) is practically insoluble at neutral pH, many aerobic microorganisms secrete siderophores, iron(III) chelating compounds, for their iron supply. Six different siderophore-iron(III) transport systems in Escherichia coli have been sequenced and analyzed, and many more in other gram-negative bacteria have been characterized (4). These transport systems share a common structure. A ferric siderophore-specific receptor in the outer membrane delivers its substrate in an energy-dependent mechanism to the periplasm. The energy is provided by the TonB-ExbB-ExbD complex (4). Even heme (33) and transferrin iron (6) are taken up in a TonB-dependent manner. With

The lac genes were inserted with phage Mu(Ap, lac) into the fhuA, fepA, cir and tonB genes which specify components of iron uptake systems. The expression of lac in all these operon fusions was controlled by the availability of iron to the cells, thereby facilitating a quick and simple measurement of the expression of the genes listed above. In an iron rich medium under anaerobic conditions all systems were strongly repressed. fhuA was depressed at higher iron concentration than was fepA or cir, and tonB was repressed only under anaerobic conditions and could be induced by iron limitation. Mutants constitutive for the expression of beta-galactosidase were selected in a fhuA-lac fusion strain. The outer membrane proteins Cir, FhuA, FecA, 76K and 83K were made constitutively in such mutant strains. Therefore, they were termed fur mutants. In these fur mutant strains, the synthesis of a 19K protein was reduced. Furthermore, it was found that transport of ferric enterochelin and ferrichrome was also constitutive in the fur mutant cells, and that ferric citrate uptake could be induced by only 10 microM citrate in the growth medium in contrast to wild-type cells in which at least 100 microM citrate was necessary. The fepA gene was concluded to be under an additional control, because it was not fully derepressed by the fur mutation.

Members of a family of catecholate siderophores, called salmochelins, were isolated by reversed-phase HPLC from Salmonella enterica serotype Typhimurium and structurally characterized by Fourier transform ion cyclotron resonance-MS͞MS and GC-MS. The tentative structure of salmochelin 1 contained two 2,3-dihydroxybenzoylserine moieties bridged by a glucose residue, bound to the serine hydroxyl group of one moiety and the carboxylate of the second moiety. Salmochelin 2 contained in addition a second glucose residue linked to a third 2,3-dihydroxybenzoylserine moiety. Salmochelins were not produced by an iroBC mutant, which indicated that the IroB protein might be responsible for the glucosyl transfer predicted by sequence similarities to known glycosyltransferases. Uptake experiments with radiolabeled 55 Fe-salmochelin and growth promotion tests with salmochelins showed that the IroN outer membrane receptor, encoded in the iroA locus of S. enterica and uropathogenic Escherichia coli strains, was the main receptor for ferric salmochelin transport.I n iron-poor environments, many bacteria secrete ironcomplexing agents called siderophores to satisfy their iron needs. For some pathogenic bacteria, siderophores are important virulence factors because iron is bound to transferrin and lactoferrin in body fluids. These proteins reduce the free Fe 3ϩ concentration to about one molecule per liter. Enterobacteria, including Escherichia coli and Salmonella enterica, often produce the catecholate siderophore enterochelin (also called enterobactin) (1, 2). It has been postulated that enterochelin is an inferior siderophore in serum because it adsorbs to hydrophobic sites in serum proteins, such as albumin (3). This fact has always been puzzling because iron supply for many pathogens plays a decisive role in the infection process, and enterochelin, a major siderophore synthesized by S. enterica, does not seem to enhance pathogenicity.Recently, the iroA locus, consisting of the two convergent operons iroN and iroBCDE, has been defined in S. enterica serotype Typhi and also in most other S. enterica serotypes (4, 5). The outer membrane siderophore receptor, IroN, is involved in the transport of several catecholate siderophores in S. enterica (5, 6). In the present investigation, we show that the presence of the iro gene cluster in S. enterica leads to glycosylation of the enterochelin building block 2,3-dihydroxybenzoylserine (DHBS), which makes the hydrophobic enterochelin molecules more hydrophilic, thereby possibly contributing to the observed pathogenicity of Salmonella strains. This siderophore was named salmochelin because it appears to be a characteristic siderophore of Salmonella strains. Interestingly, certain E. coli strains, e.g., the uropathogenic E. coli 563, also possess a very similar iro gene cluster on pathogenicity island III (7). The production and the tentative structural elucidation of salmochelins and their specific uptake via the outer membrane receptor IroN are described. In addition, it is shown that salmo...

The hemin receptor HemR of Yersinia enterocolitica was identified as a 78 kDa iron regulated outer membrane protein. Cells devoid of the HemR receptor as well as cells mutated in the tonB gene were unable to take up hemin as an iron source. The hemin uptake operon from Y. enterocolitica was cloned in Escherichia coli K12 and was shown to encode four proteins: HemP (6.5 kDa), HemR (78 kDa), HemS (42 kDa) and HemT (27 kDa). When expressed in E.coli hemA aroB, a plasmid carrying genes for HemP and HemR allowed growth on hemin as a porphyrin source. Presence of genes for HemP, HemR and HemS was necessary to allow E.coli hemA aroB cells to use hemin as an iron source. The nucleotide sequence of the hemR gene and its promoter region was determined and the amino acid sequence of the HemR receptor deduced. HemR has a signal peptide of 28 amino acids and a typical TonB box at its amino‐terminus. Upstream of the first gene in the operon (hemP), a well conserved Fur box was found which is in accordance with the iron‐regulated expression of HemR.

No abstract

The Yersinia enterocolitica O:8 periplasmic binding-protein-dependent transport (PBT) system for haemin was cloned and characterized. It consisted of four proteins: the periplasmic haemin-binding protein HemT, the haemin permease protein HemU, the ATP-binding hydrophilic protein HemV and the putative haemin-degrading protein HemS. Y. enterocolitica strains mutated in hemU or hemV genes were unable to use haemin as an iron source whereas those mutated in the hemT gene were able to use haemin as an iron source. As Escherichia coli strains expressing only the haemin outer membrane receptor protein HemR from Y. enterocolitica were capable of using haemin as an iron source the existence of an E. coli K-12 haemin-specific PBT system is postulated. The first gene in the Y. enterocolitica haemin-specific PBT system encoded a protein, HemS, which is probably involved in the degradation of haemin in the cytoplasm. The presence of the hemS gene was necessary to prevent haemin toxicity in E. coli strains that accumulate large amounts of haemin in the cytoplasm. We propose a model of haemin utilization in Y. enterocolitica in which HemT, HemU and HemV proteins transport haemin into the cytoplasm where it is degraded by HemS thereby liberating the iron.