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Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P(trc)) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P(trc)-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In stimulating effector functions of mononuclear phagocytes, IFN-gamma is of pivotal importance in host defense against intramacrophage pathogens including salmonellae. As the activity of IFN-gamma is modulated by iron and since a sufficient availability of iron is essential for the growth of pathogens, we investigated the regulatory effects of IFN-gamma on iron homeostasis and immune function in murine macrophages infected with Salmonella typhimurium. In Salmonella-infected phagocytes, IFN-gamma caused a significant reduction of iron uptake via transferrin receptor 1 and resulted in an increased iron efflux caused by an enhanced expression of the iron exporter ferroportin 1. Moreover, the expression of haem oxygenase 1 and of the siderophore-capturing antimicrobial peptide lipocalin 2 was markedly elevated following bacterial invasion, with IFN-gamma exerting a super-inducing effect. This observed regulatory impact of IFN-gamma reduced the intracellular iron pools within infected phagocytes, thus restricting the acquisition of iron by engulfed Salmonella typhimurium while concomitantly promoting NO and TNF-alpha production. Our data suggest that the modulation of crucial pathways of macrophage iron metabolism in response to IFN-gamma concordantly aims at withdrawing iron from intracellular Salmonella and at strengthening macrophage immune response functions. These regulations are thus consistent with the principles of nutritional immunity.

The cytoplasmic membrane protein FeoB of Escherichia coli, Helicobacter pylori, Legionella pneumophila and Synechocystis sp. strain PCC 6803 is necessary for Fe(2+) uptake. The C-terminal part of FeoB is predicted to contain 8-12 membrane-spanning helices. The N-terminal domain shows much similarity to eukaryotic and prokaryotic G proteins and, indeed, GTPase activity is necessary for Fe(2+) transport. Four of the five characteristic conserved G protein motifs have been identified in FeoB proteins. Whether FeoB is involved directly, via its Me(2+) binding site, or indirectly in Fe(2+) transport, remains to be investigated.

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-MSMS 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.

The regulator Fur represses with Fe2+ as cofactor iron uptake genes. The fhuF gene reacts very sensitive to minor changes of Fe2+ and Fur. It is assumed that FhuF helps in the mobilisation of iron out of the hydroxamate siderophores transported into the cell. Analysis of the protein revealed an unusual [2Fe-2S] cluster bound to a Cys-Cys-X10-Cys-X2-Cys motif in FhuF. suf genes responsible for the synthesis of the iron sulfur center were identified. The Zur protein shows 27% identity to the Fur protein of E. coli. It regulates as a repressor the high affinity uptake system znuACB. Only two additional Zur binding sites in the promoter region of genes with unknown function were found. Properties of Zur and Fur proteins from different bacteria are compared.

The uptake of Mn(2+), a cofactor for several enzymes in Escherichia coli, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe(2+) with lower affinity. MntH belongs to the NRAMP family of eukaryotic Fe(2+) and Mn(2+) transporters. In E. coli strains with chromosomal mntH-lacZ fusions, mntH was partially repressed by both Mn(2+) and Fe(2+). Inactivation of fur resulted in the loss of Fe(2+)-dependent repression of mntH transcription, demonstrating that Fe(2+) repression depends on the global iron regulator Fur. However, these fur mutants still showed Mn(2+)-dependent repression of mntH. The Mn(2+)-responsive transcriptional regulator of mntH was identified as the gene product of o155 (renamed MntR). mntR mutants were impaired in Mn(2+) but not Fe(2+) repression of mntH transcription. Binding of purified MntR to the mntH operator was manganese dependent. The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome. The Fur binding site, localized within 22 nucleotides of the mntH operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.

In Escherichia coli, the iron regulator Fur is regulated by two oxidative-stress response regulators. The generation of dangerous radicals by oxygen and iron is the basis for this dual regulation, which is also found in eukaryotes. The binding of iron-regulated transcripts to apo-aconitase and results of mRNA half-life studies indicate that there is post-transcriptional iron regulation in bacteria, as in eukaryotes. Fur contains two metal-binding sites, one for Zn2+ and one for Fe2+. Zinc uptake systems are regulated by members of the Fur protein family, and zinc is a cofactor. DtxR and related proteins constitute another family of iron regulators, first found in Gram-positive organisms with a high GC content. In organisms with Fur-dependent iron regulation, members of the DtxR family regulate manganese transport.

A highly sensitive genetic screen for the detection of cloned genes coding for iron-regulated and iron-storage/binding proteins was developed. The Fur titration assay (FURTA) enabled identification of cloned iron-regulated genes from different Gram-positive and Gram-negative bacteria such as: Bacillus subtilis, Escherichia coli, Pantoea agglomerans, Pseudomonas putida, Salmonella typhimurium, Serratia marcescens and Yersinia enterocolitica. An ordered E. coli cosmid library was screened for either new genes containing Fur-box nucleotide sequences or genes coding for iron-storage/binding proteins. Among 150 cosmids covering approximately 85% of the E. coli genome, 24 cosmids were identified as positive by FURTA. Nine of them contained new E. coli Fur-regulated genes and/or iron-storage/binding genes since they mapped at loci different to any of the known Fur-box containing genes. A new E. coli gene encoding a 8.7 kDa high-potential iron-sulfur-like protein was identified, cloned and sequenced. The Fur titration assay was also used to probe in vivo interaction between Fur repressor and different synthetic plasmid-located Fur-boxes. Non-optimal base-pairs in one half of the Fur-box nucleotide sequence led to a dramatic decrease of Fur repressor affinity. A synthetic Fur-box with changes in both Fur-box halves was no longer bound by the Fur repressor complex in vivo.

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 of feoAB contained a binding site for the regulatory protein Fur, which acts with iron(II) as a corepressor in all known iron transport systems of E. 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 or feoB gene, respectively.

Iron assimilation systems have been shown to be important for virulence in Yersinia enterocolitica. In principle, iron transport in Y. enterocolitica is similarly organized as in Escherichia coli although some differences exist in the siderophores used. A TonB function was identified which in a tonB mutant was complemented by the E. coli tonB gene. Three outer membrane receptors for siderophores were cloned and sequenced: FoxA for ferrioxamine B and E, FcuA for ferrichrome and HemR for heme uptake. In addition, two receptors were identified by mutants: CccA for catechol cephalosporins and FyuA for yersiniabactin, the siderophore produced by virulent yersiniae. In addition, the FyuA protein is assumed to be the pesticin receptor.

Iron-repressible outer membrane proteins (Irp) and siderophore production of Yersinia enterocolitica, serotype 08, were subjected to analysis. Here four Irps of apparent molecular weights of 62,000, 65,000, 74,000 and 75,000 could be identified which were expressed constitutively by a fur mutant. Production of a novel catechol-containing siderophore (denoted yersiniabactin) was detected by siderophore-indicator agar (chrome azurol S) and feeding experiments. Growth support by yersiniabactin under iron-restricted conditions was TonB- and Irp65-dependent and correlated with pesticin-sensitivity of Yersinia enterocolitica and Escherichia coli O. From these results we conclude that Irp65 of Y. enterocolitica functions as yersiniabactin receptor (FyuA) and as pesticin receptor. By immunoblotting using rabbit antibodies against Irp65 and chrome azurol S-agar, we were able to demonstrate that all tested mouse-lethal Y. enterocolitica and Yersinia pseudotuberculosis strains of different serotypes express siderophores and Irp65. Moreover, the anti-Irp65 rabbit serum did not cross-react with the known iron-repressible high-molecular-weight proteins (HMWPs). Evidently, the mouse lethality trait in enteropathogenic Yersinia spp. is closely associated with a novel iron-uptake system, comprising the production of a siderophore and a siderophore receptor of apparent molecular mass 65,000 Da.

The streptomycin-treated mouse colonization model was used to investigate the role of the Fe2+ uptake system (Feo) of Escherichia coli K12 in the colonization of the mouse intestine. Mutants impaired in the uptake of Fe2+ ions were shown to be deficient also in their colonization ability. Both enterochelin-producing and enterochelin-nonproducing Escherichia coli feo mutants were unable to colonize the mouse intestine. These results demonstrated that Fe(II) is an essential source of iron for E. coli grown in the intestine.

The significance of intracellular iron levels of Escherichia coli on the expression of the fumarate reductase operon (frd), which is regulated by the transcriptional activator FNR, was studied in vivo. The iron contents of aerobically and anaerobically grown E. coli were determined and related to the expression of frd and of genes (fiu, fepA, fhuF) which are regulated by the iron uptake regulatory protein Fur. The iron contents varied from 1.6 to 6.9 mumol Fe/g protein with no significant difference in aerobic and anaerobic bacteria. Expression of frd was not related to the different iron levels, but to oxygen supply. Only severe iron limitation in iron-depleted medium, which caused lower iron contents (0.8 to 1.6 mumol/g), reduced the expression of frd under anaerobic conditions. On the other hand, expression of fiu, fepA and fhuF clearly responded to iron supply and cellular content, but only slightly to changed O2 supply. Generally, expression of frd responded only to much stricter iron limitation, than expression of Fur regulated genes. It is concluded that the functional state of FNR during aerobic/anaerobic switch is not regulated by iron content and reversible binding of Fe2+ under physiological conditions. Therefore FNR does not communicate with the iron pool regulating the Fur protein.

The TonB protein is involved in energ-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and phi 80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, or tonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in the fhuA gene are suppressed by point mutations in the tonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron is an essential growth factor for procaryotes as well as for eucaryotes. Microorganisms have developed specific iron-uptake systems by producing low-molecular weight iron-chelating compounds (siderophores). We have examined the effect of the siderophores desferrioxamine (DFO), desferrichrome (DFC), desferriaerobactin (DFAB) and desferrienterobactin (DFEA) on the mitogen-stimulated activation and proliferation of mouse T cells. The hydroxamate siderophores DFO, DFC and DFAB cause an immunosuppressive effect on T cells which is related to the iron complexation constant of the siderophores and can be reversed by equimolar ferric iron. In contrast, the catecholate siderophore DFEB and its ferrated derivative turned out to be cytotoxic for T cells. These results suggest a dual role of siderophores in the infectious process, i.e., growth enhancement of the invading pathogen and inhibition of the host immune defense.

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.

In Escherichia coli the iron uptake systems are regulated by the fur gene product. The synthesis of the outer membrane proteins fiu, fepA, fecA, fhuA, fhuE and cir is derepressed at low iron concentrations in the medium or constitutive in a fur mutant. The fur gene region cloned into pACYC184 was analysed by restriction analysis, Tn1000 mutagenesis and complementation studies. The presence of fur+ plasmids repressed synthesis of the proteins fepA, fecA, fhuE and cir in a chromosomal fur mutant. More quantitatively, the repression to wild-type levels was shown with lac fusions to the genes fiu, fepA and cir. In minicells an 18,000 dalton protein was identified as the fur gene product. Correlated with the fur protein a slightly smaller protein, possibly a degradation product, was observed. The gene fur was mapped on the E. coli chromosome near nagA at about 15.5 min.

Escherichia coli strains which contain the Fe3+-aerobactin transport system specified by the ColV plasmid became deficient in aerobactin-dependent iron transport when they were converted to cloacin-resistant derivatives. An outer membrane protein with a molecular mass of 74,000 daltons was overproduced under iron-limiting growth conditions and was absent in cloacin-resistant mutants. Fe3+-aerobactin protected cells against cloacin. These results suggest that the cloacin receptor protein, controlled by the colV plasmid, also participates in Fe3+-aerobactin transport.

Streptonigrin was used to select mutants impaired in the citrate-dependent iron transport system of Escherichia coli K-12. Mutants in fecA and fecB could not transport iron via citrate. fecA-lac and fecB-lac operon fusions were constructed with the aid of phage Mu dl(Ap lac). Strains deficient in ferric dicitrate transport which were mutated in fecB were as inducible as transport-active strains. They expressed the FecA outer membrane protein and beta-galactosidase of the fecB-lac operon fusions. In contrast, all fecA::lac mutants and fecA mutants induced with N-methyl-N'-nitro-N-nitrosoguanidine did not respond to ferric dicitrate supplied in the growth medium. tonB fecB mutants which were lacking all tonB-related functions were not inducible. We conclude that binding of iron in the presence of citrate to the outer membrane receptor protein is required for induction of the transport system. In addition, the tonB gene has to be active. However, iron and citrate must not be transported into the cytoplasm for the induction process. These data support our previous conclusion of an exogenous induction mechanism. Mutants in fur expressed the transport system nearly constitutively. In wild-type cells limiting the iron concentration in the medium enhanced the expression of the transport system. Thus, the citrate-dependent iron transport system shares regulatory devices with the other iron transport systems in E. coli and, in addition, requires ferric dicitrate for induction.

With the lac operon fusion technique, mutants were isolated in two genes that specify two outer membrane proteins designated FhuE (76 K) and Fiu (83 K). The synthesis of both proteins was increased under low iron growth conditions. The FhuE-protein was shown to be necessary for iron uptake via coprogen, an iron chelator produced by certain fungi, e.g. Neurospora crassa. In addition to fhueE the genes fhuCDB, tonB and exbB were necessary for iron coprogen uptake. The gene fhuE was mapped between kdp and gltA near 16 min on the genetic map of E. coli K12, while gene fiu was mapped near 18 min between chlA and chlE. Nor iron transport system could be assigned as yet to the Fiu protein.

It was shown that feuB mutants (defective in ferric enterochelin uptake) were unable to adsorb colicin B. In addition, they were missing one of the three outer-membrane proteins which are over produced in strains grown in iron-deficient, extracted medium. Thus this protein (the feuB protein) is probably the receptor for colicin B and functions in enterochelin-mediated iron transport. The feuB gene was located by P1 transduction at approximately 72.5 min on the Escherichia coli K-12 genetic map and thus maps separately from the other genes concerned with the enterochelin system. The outer membranes of various strains grown in the presence of 1 mM citrate contained a high level of a protein which was present in very small amounts when citrate was absent from the growth medium. This protein was most easily observed in feuB mutants grown in the presence of citrate, since on polyacrylamide gels it ran in a similar position to the feuB protein, which is missing in these mutants. The relationship of this citrate-inducible protein to the inducible citrate-dependent iron uptake system is discussed.

Induction of the citrate-dependent iron transport system of Escherichia coli K-12 required 0.1 mM citrate and 0.1 micrometer iron in the growth medium. Five--ten-times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate-dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (FecA), belonging to the transport system, was induced in fecB mutants which were devoid of citrate-dependent iron transport. The intracellular citrate and iron concentrations were 10--100-times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm. The Tn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate-dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with the fecA and fecB markers suggested the gene order fecA fecB Tn10.

The functional interaction of outer membrane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 and ø80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all 3 colicins. The interaction of the ton A, ton B, and feu functions apparently permits quite different "substrates" to overcome the permeability barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of 3H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferri-ferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source was used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: 1) ton A; 2) ton B mutants; 3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; 4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; 5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000-83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene...

A selection procedure using Mn2+ is described. A high percentage of the Mn2+ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::lambda placMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of beta-galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur+ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%-70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.

The mutant, generated by a Mud1 insertion, formed long non-viable filaments in the presence of iron and air. Under anaerobic conditions normal growth in the presence of iron was observed. The mutation was mapped by P1 transductions at 48 min on the genetic map of Escherichia coli. By Southern blotting the insertion point was determined to be in nrdB, the structural gene for the ribonucleotide reductase subunit B2. The mutation could be complemented by the cloned nrdB gene. Up to now it was assumed that E. coli possesses only one enzyme for the synthesis of deoxyribonucleotides and only conditional lethal (temperature sensitive) mutants were isolated in nrdB. The insertion of Mud1 in nrdB should lead to a complete loss of the essential B2 subunit. Since the strain was able to grow under anaerobic conditions on minimal medium lacking deoxyribonucleotides and additional pathway for the synthesis of deoxyribonucleotides is postulated.

The fur gene of Escherichia coli is involved in all iron-regulated transcriptions hitherto studied. The nucleotide sequence of an 868 basepair fragment containing the fur gene was determined. There was only a single longer reading frame. The amino acid sequence derived from the nucleotide sequence comprised 148 amino acids that together made a polypeptide of 16,795 daltons. The amino acid sequence was confirmed by determination of the amino acid composition, the carboxy-terminal lysine residue and the internal Lys-Lys and Lys-Arg sequences of the isolated Fur protein. The nucleotide sequence contains typical initiation and termination sites for transcription and translation.