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Enterococcus faecalis 24FS is a bacteriocin-producing, multiply antibiotic-resistant, and potentially virulent bacterium isolated from healthy infant feces. The draft 2.9-Mb genome sequence revealed 2,968 protein-encoding genes; 11 antibiotic resistance, 8 virulence, and 3 bacteriocin genes; and 2 plasmids, 4 prophages, 30 insertion sequence (IS) elements, 1 transposon, and 1 integron.

The RIC (repair of iron clusters) protein of Escherichia coli is a di-iron hemerythrin-like protein that has a proposed function in repairing stress-damaged iron-sulfur clusters. In this work, we performed a bacterial two-hybrid screening to search for RIC-protein interaction partners in E. coli. As a result, the DNA-binding protein from starved cells (Dps) was identified, and its potential interaction with RIC was tested by bacterial adenylate cyclase-based two-hybrid (BACTH) system, bimolecular fluorescence complementation, and pulldown assays. Using the activity of two Fe-S-containing enzymes as indicators of cellular Fe-S cluster damage, we observed that strains with single deletions of ric or dps have significantly lower aconitase and fumarase activities. In contrast, the ric dps double mutant strain displayed no loss of aconitase and fumarase activity with respect to that of the wild type. Additionally, while complementation of the ric dps double mutant with ric led to a severe loss of aconitase activity, this effect was no longer observed when a gene encoding a di-iron site variant of the RIC protein was employed. The dps mutant exhibited a large increase in reactive oxygen species (ROS) levels, but this increase was eliminated when ric was also inactivated. Absence of other iron storage proteins, or of peroxidase and catalases, had no impact on RIC-mediated redox stress induction. Hence, we show that RIC interacts with Dps in a manner that serves to protect E. coli from RIC protein-induced ROS. IMPORTANCE The mammalian immune system produces reactive oxygen and nitrogen species that kill bacterial pathogens by damaging key cellular components, such as lipids, DNA, and proteins. However, bacteria possess detoxifying and repair systems that mitigate these deleterious effects. The Escherichia coli RIC (repair of iron clusters) protein is a di-iron hemerythrin-like protein that repairs stress-damaged iron-sulfur clusters. E. coli Dps is an iron storage protein of the ferritin superfamily with DNA-binding capacity that protects cells from oxidative stress. This work shows that the E. coli RIC and Dps proteins interact in a fashion that counters RIC protein-induced reactive oxygen species (ROS). Altogether, we provide evidence for the formation of a new bacterial protein complex and reveal a novel contribution for Dps in bacterial redox stress protection.

Lactobacillus plantarum 10CH is a bacteriocin-producing potential probiotic lactic acid bacterium (LAB) strain isolated from cheese. Its complete nucleotide sequence shows a single circular chromosome of 3.3 Mb, with a G+C content of 44.51%, a 25-gene plantaricin bacteriocin gene cluster, and the absence of recognized virulence factors.

The health benefits of garlic have been proven by epidemiological and experimental studies. Diallyl disulphide (DADS), the major organosulfur compound found in garlic oil, is known to lower the incidence of breast cancer both in vitro and in vivo. The studies reported here demonstrate that DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G0 population and substantially halts DNA synthesis. DADS also induces phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3. Further studies revealed that DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL and Bcl-w in a dose-dependent manner, suggesting the involvement of Bcl-2 family proteins in DADS induced apoptosis. Histone deacetylation inhibitors (HDACi) are known to suppress cancer growth and induce apoptosis in cancer cells. Here it is shown that DADS has HDACi properties in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation. The data thus indicate that the HDACi properties of DADS may be responsible for the induction of apoptosis in breast cancer cells.

Ribonucleotide reductases supply cells with their deoxyribonucleotides. Three enzyme types are known, classes I, II and III. Class II enzymes are anaerobic whereas class I enzymes are aerobic, and so class I and II enzymes are often produced by the same organism under opposing oxygen regimes. Escherichia coli contains two types of class I enzyme (Ia and Ib) with the Fe-dependent Ia enzyme (NrdAB) performing the major role aerobically, leaving the purpose of the Ib enzyme (NrdEF) unclear. Several papers have recently focused on the class Ib enzymes showing that they are Mn (rather than Fe) dependent and suggesting that the E. coli NrdEF may function under redox-stress conditions. A paper published in this issue of Molecular Microbiology from James Imlay's group confirms that this unexplained NrdEF Ib enzyme is Mn-dependent, but shows that it does not substitute for NrdAB during redox stress. Instead, a role during iron restriction is demonstrated. Thus, the purpose of NrdEF (and possibly other class Ib enzymes) is to enhance growth under aerobic, low-iron conditions, and to functionally replace the Fe-dependent NrdAB when iron is unavailable. This finding reveals a new mechanism by which bacteria adjust to life under iron deprivation.

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.

FtnA is the major iron-storage protein of Escherichia coli accounting for < or = 50% of total cellular iron. The FtnA gene (ftnA) is induced by iron in an Fe(2+)-Fur-dependent fashion. This effect is reportedly mediated by RyhB, the Fe(2+)-Fur-repressed, small, regulatory RNA. However, results presented here show that ftnA iron induction is independent of RyhB and instead involves direct interaction of Fe(2+)-Fur with an 'extended' Fur binding site (containing five tandem Fur boxes) located upstream (-83) of the ftnA promoter. In addition, H-NS acts as a direct repressor of ftnA transcription by binding at multiple sites (I-VI) within, and upstream of, the ftnA promoter. Fur directly competes with H-NS binding at upstream sites (II-IV) and consequently displaces H-NS from the ftnA promoter (sites V-VI) which in turn leads to derepression of ftnA transcription. It is proposed that H-NS binding within the ftnA promoter is facilitated by H-NS occupation of the upstream sites through H-NS oligomerization-induced DNA looping. Consequently, Fur displacement of H-NS from the upstream sites prevents cooperative H-NS binding at the downstream sites within the promoter, thus allowing access to RNA polymerase. This direct activation of ftnA transcription by Fe(2+)-Fur through H-NS antisilencing represents a new mechanism for iron-induced gene expression.

Escherichia coli possesses iron transporters specific for either Fe2+ or Fe3+. Although Fe2+ is far more soluble than Fe3+, it rapidly oxidizes aerobically at pH >= 7. Thus, FeoAB, the major Fe2+ transporter of E. coli, operates anaerobically. However, Fe2+ remains stable aerobically under acidic conditions, although a low-pH Fe2+ importer has not been previously identified. Here we show that ycdNOB (efeUOB) specifies the first such transporter. efeUOB is repressed at high pH by CpxAR, and is Fe2+-Fur repressed. EfeU is homologous to the high-affinity iron permease, Ftr1p, of Saccharomyces cerevisiae and other fungi. EfeO is periplasmic with a cupredoxin N-terminal domain; EfeB is also periplasmic and is haem peroxidase-like. All three Efe proteins are required for Efe function. The efeU gene of E. coli K-12 is cryptic due to a frameshift mutation - repair of the single-base-pair deletion generates a functional EfeUOB system. In contrast, the efeUOB operon of the enterohaemorrhagic strain, O157:1147, lacks any frameshift and is functional. A 'wild-type' K-12 strain bearing a functional EfeUOB displays a major growth advantage under aerobic, low-pH, low-iron conditions when a competing metal is provided. Fe-55 transport assays confirm the ferrous iron specificity of EfeUOB.

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22(2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPDwe generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Microarray-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, like-wise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaC1 protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaC1) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaC1) proteins.

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C 4 -dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C 4 -dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [γ- 32 P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C 4 -dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes ( dctA , dcuB , and frdA ), with apparent K D s of 6 to 32 μM for untreated DcuR and ≤1 to 2 μM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB , dctA , and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron-rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of 55Fe-labeled E. coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.

The hyf locus ( hyfABCDEFGHIJ-hyfR-focB ) of Escherichia coli encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential σ 54 -dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated hyf expression by using a hyfA-lacZ transcriptional fusion. This work revealed that hyf is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was σ 54 dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus, hyf expression resembles that of the hyc operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the hyfA structural gene, with appropriately located −24 and −12 boxes indicative of a σ 54 -dependent promoter. No reverse transcriptase PCR product could be detected for hyfJ-hyfR , suggesting that hyfR-focB may be independently transcribed from the rest of the hyf operon. Expression of hyf was strongly induced (∼1,000-fold) in the presence of a multicopy plasmid expressing hyfR from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The hyfR- expressing plasmid increased fdhF-lacZ transcription just twofold but did not influence the expression of hycB-lacZ . Interestingly, inactivation of the chromosomal hyfR gene had no effect on hyfA-lacZ expression. Purified HyfR was found to specifically interact with the hyf promoter/operator region. Inactivation of the hyf operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.

The high-resolution structure of the non-haem ferritin from Escherichia coli (EcFtnA) is presented together with those of its Fe3+ and Zn2+ derivatives, this being the first high-resolution X-ray analysis of the iron centres in any ferritin. The binding of both metals is accompanied by small changes in the amino acid ligand positions. Mean FeA3+-FeB3+ and ZnA2+-ZnB2+ distances are 3.24 A and 3.43 A, respectively. In both derivatives, metal ions at sites A and B are bridged by a glutamate side-chain (Glu50) in a syn-syn conformation. The Fe3+ derivative alone shows a third metal site (FeC3+) joined to FeB3+ by a long anti-anti bidentate bridge through Glu130 (mean FeB3+-FeC3+ distance 5.79 A). The third metal site is unique to the non-haem bacterial ferritins. The dinuclear site lies at the inner end of a hydrophobic channel connecting it to the outside surface of the protein shell, which may provide access for dioxygen and possibly for metal ions shielded by water. Models representing the possible binding mode of dioxygen to the dinuclear Fe3+ pair suggest that a gauche μ-1,2 mode may be preferred stereochemically. Like those of other ferritins, the 24 subunits of EcFtnA are folded as four-helix bundles that assemble into hollow shells and both metals bind at dinuclear centres in the middle of the bundles. The structural similarity of EcFtnA to the human H chain ferritin (HuHF) is remarkable (r.m.s. deviation of main-chain atoms 0.66 A) given the low amino acid sequence identity (22 %). Many of the conserved residues are clustered at the dinuclear centre but there is very little conservation of residues making inter-subunit interactions.

Tripartite ATP-independent periplasmic ('TRAP') transporters are a novel group of bacterial and archaeal secondary solute uptake systems which possess a periplasmic binding protein, but which are unrelated to ATP-binding cassette (ABC) systems. In addition to the binding protein, TRAP transporters contain two integral membrane proteins or domains, one of which is 40-50 kDa with 12 predicted transmembrane (TM) helices, thought to be the solute import protein, while the other is 20-30 kDa and of unknown function. Using a series of plasmid-encoded beta-lactamase fusions, we have determined the topology of DctQ, the smaller integral membrane protein from the high-affinity C4-dicarboxylate transporter of Rhodobacter capsulatus, which to date is the most extensively characterised TRAP transporter. DctQ was predicted by several topology prediction programmes to have four TM helices with the N- and C-termini located in the cytoplasm. The levels of ampicillin resistance conferred by the fusions when expressed in Escherichia coli were found to correlate with this predicted topology. The data have provided a topological model which can be used to test hypotheses concerning the function of the different regions of DctQ and which can be applied to other members of the DctQ family.

The dcuB gene of Escherichia coli encodes an anaerobic C 4 -dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition, dcuB expression is strongly induced by C 4 -dicarboxylates, suggesting the presence of a novel C 4 -dicarboxylate-responsive regulator in E. coli . This paper describes the isolation of a Tn 10 mutant in which the 160-fold induction of dcuB expression by C 4 -dicarboxylates is absent. The corresponding Tn 10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to the dcuB gene at ∼93.5 min in the E. coli chromosome. The yjdHG genes (redesignated dcuSR ) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the dcuSR operon restored the C 4 -dicarboxylate inducibility of dcuB expression in the dcuS mutant to levels exceeding those of the dcuS + strain by approximately 1.8-fold. The dcuS mutation affected the expression of other genes with roles in C 4 -dicarboxylate transport or metabolism. Expression of the fumarate reductase ( frdABCD ) operon and the aerobic C 4 -dicarboxylate transporter ( dctA ) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C 4 -dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal. However, under aerobic conditions with C 4 -dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant. Studies employing a dcuA dcuB dcuC triple mutant unable to transport C 4 -dicarboxylates anaerobically revealed that C 4 -dicarboxylate transport is not required for C 4 -dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ∼140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C 4 -dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae . DcuS is not closely related to the C 4 -dicarboxylate-sensing DctS or DctB protein of Rhodobacter capsulatus or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.

The dcuA and dcuB genes of Escherichia coli encode homologous proteins that appear to function as independent and mutually redundant C 4 -dicarboxylate transporters during anaerobiosis. The dcuA gene is 117 bp downstream of, and has the same polarity as, the aspartase gene ( aspA ), while dcuB is 77 bp upstream of, and has the same polarity as, the anaerobic fumarase gene ( fumB ). To learn more about the respective roles of the dcu genes, the environmental and regulatory factors influencing their expression were investigated by generating and analyzing single-copy dcuA - and dcuB-lacZ transcriptional fusions. The results show that dcuA is constitutively expressed whereas dcuB expression is highly regulated. The dcuB gene is strongly activated anaerobically by FNR, repressed in the presence of nitrate by NarL, and subject to cyclic AMP receptor protein (CRP)-mediated catabolite repression. In addition, dcuB is strongly induced by C 4 -dicarboxylates, suggesting that dcuB is under the control of an uncharacterized C 4 -dicarboxylate-responsive gene regulator. Northern blotting confirmed that dcuA (and aspA ) is expressed under both aerobic and anaerobic conditions and that dcuB (and fumB ) is induced anaerobically. Major monocistronic transcripts were identified for aspA and dcuA , as well as a minor species possibly corresponding to an aspA-dcuA cotranscript. Five major transcripts were observed for dcuB and fumB : monocistronic transcripts for both fumB and dcuB ; a dcuB-fumB cotranscript; and two transcripts, possibly corresponding to dcuB-fumB and fumB mRNA degradation products. Primer extension analysis revealed independent promoters for aspA , dcuA , and dcuB , but surprisingly no primer extension product could be detected for fumB . The expression of dcuB is entirely consistent with a primary role for DcuB in mediating C 4 -dicarboxylate transport during anaerobic fumarate respiration. The precise physiological purpose of DcuA remains unclear.

Escherichia coli can utilize C4-dicarboxylates as sole carbon and energy sources under both aerobic and anaerobic conditions (7). Aerobically, uptake of C4-dicarboxylates (fumarate, malate, and succinate) and l-aspartate is mediated by a secondary transporter, designated DctA (14, 18). The corresponding gene, dctA, has been sequenced, and the role of its product in the utilization of C4-dicarboxylates (and the cyclic monocarboxylate orotate) has been established by complementation studies in Salmonella typhimurium dctA or outA mutants (2, 28). Uptake, exchange, and efflux of C4-dicarboxylic acids under anaerobic conditions is mediated by the Dcu systems (Km for fumarate uptake = 51 μM), which are genetically distinct from the aerobic Dct system (7, 8, 34). Measurements of both C4-dicarboxylate uptake and exchange have suggested that the Dcu systems are exclusively expressed under anaerobic conditions, activated by the anaerobic activator protein FNR, and repressed in the presence of nitrate. Three independent Dcu systems have been identified, DcuA, DcuB, and DcuC (27, 34). DcuA and DcuB are homologous proteins (36% identical), whereas DcuC is only 22 to 24% identical to DcuA and DcuB. Growth tests and transport studies with dcuA, dcuB, and dcuC single, double, and triple mutants showed that DcuA, DcuB, and DcuC each mediate exchange as well as uptake (27, 34). The triple mutants were almost completely devoid of Dcu activity. The single mutants exhibited no phenotype, but the dcuA-dcuB double mutant displayed marked deficiencies in C4-dicarboxylate transport and growth by fumarate respiration, suggesting that DcuA and DcuB have analogous and mutually complementary transport functions in the anaerobic uptake of C4-dicarboxylates (27, 34). The affinities of DcuA and DcuB for C4-dicarboxylates are similar, except for the lower affinity of DcuA for malate (27). DcuA and DcuB have 433 and 446 amino acid residues, respectively, and their sequences suggest that they are highly hydrophobic and lack N-terminal signal sequences, which together indicate that they are polytopic inner-membrane proteins (27). A combination of SOAP, Helixmem, hydropathy plot, and “von Heijne positive-inside rule” analyses were used to predict that DcuA and DcuB have either 12 or 14 transmembrane spanning helices and that their N and C termini are located in the cytoplasm or periplasm (27). The reasons for having three independent Dcu systems in E. coli and their specific roles in anaerobic C4-dicarboxylate transport are unknown. In particular, the presence of the homologous and apparently mutually redundant DcuA and DcuB systems remains to be explained. Homologues of DcuA and DcuB are present in Serratia marcescens, Haemophilus influenzae (two homologues), Wolinella succinogenes, Helicobacter pylori, and Salmonella typhimurium, suggesting that the DcuA-like transporters are widespread among the proteobacteria. However, members of the DcuA family have no significant sequence similarity to members of other transporter families, indicating that the DcuA-like proteins are a distinct group (1). In order to better understand the roles and properties of DcuA-like transporters and to provide a robust topological model that would facilitate more accurate comparisons between this group and other transporter families, the topological organization of DcuA within the inner membrane has been analyzed with in-frame translational fusions between a series of progressively truncated forms of the dcuA gene and a downstream “reporter gene” (blaM, encoding β-lactamase). The resulting topological model differs from the predicted models and indicates that the DcuA-like transport proteins represent a unique subgroup of the “duo-decimal transporters” (DDTs).

The bacterioferritin (BFR) of Escherichia coliconsists of 24 identical subunits, each containing a dinuclear metal-binding site consisting of two histidines and four carboxylic acid residues. Earlier studies showed that the characterization of iron binding to BFR could be aided by EPR analysis of iron–nitrosyl species resulting from the addition of NO to the protein [Le Brun, Cheesman, Andrews, Harrison, Guest, Moore and Thomson (1993) FEBS Lett. 323, 261–266]. We now report data from gas chromatographic head space analysis combined with EPR spectroscopy to show that NO is not an inert probe: iron(II)–BFR catalyses the reduction of NO to N2O, resulting in oxidation of iron(II) at the dinuclear centre and the subsequent detection of mononuclear iron(III). In the presence of excess reductant (sodium ascorbate), iron(II)–BFR also catalyses the reduction of NO to N2O, giving rise to three mononuclear iron–nitrosyl species which are detectable by EPR. One of these, a dinitrosyl–iron complex of S = ½, present at a maximum of one per subunit, is shown by EPR studies of site-directed variants of BFR not to be located at the dinuclear centre. This is consistent with a proposal that the diferric form of the centre is unstable and breaks down to form mononuclear iron species.

The b acterioferritin-associated f erre d oxin (Bfd) of Escherichia coli is a 64-residue polypeptide encoded by the bfd gene located upstream of the gene ( bfr ) encoding the iron-storage haemoprotein, bacterioferritin. The Bfd sequence resembles those of the ∼60-residue domains found in NifU proteins (required for metallocluster assembly), nitrite reductases, and Klebsiella pneumoniae nitrate reductase. These related-domains contain four well-conserved cysteine residues, which are thought to function as ligands to a [2Fe-2S] cluster. The Bfd protein was over-produced, purified, and characterised. Bfd was found to be a positively-charged monomer containing two iron atoms and two labile sulphides. Ultraviolet-visible, EPR, variable-temperature magnetic-circular dichroism and resonance Raman spectroscopies, together with cyclic voltogram measurements, revealed the presence of a [2Fe-2S] 2+,+ centre (E 1/2 = −254 mV) having remarkably similar properties to the Fe-S cluster of NifU. Bfd may thus be a 2Fe ferredoxin participating either in release/delivery of iron from/to bacterioferritin (or other iron complexes), or in iron-dependent regulation of bfr expression.

The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.

The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake.

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

Recent studies have indicated that Escherichia coli possesses at least two iron-storage proteins, the haem-containing bacterioferritin and ferritin. The ferritin protein has been amplified 600-fold to 11-14% of total cell protein in a bfr mutant and purified to homogeneity with an overall yield of 13%. The cellular ferritin content remained relatively constant throughout the growth cycle and amplification was accompanied by a 2.5-fold increase in cellular iron content. The isolated ferritin contained 5-20 non-haem iron atoms/holomer and resembled the eukaryotic ferritins rather than the prokaryotic bacterioferritins in containing no haem. The 24 subunits of this ferritin (M(r) 19,400) assemble into a spherical protein shell (12 +/- 1 nm diameter, M(r) 465,000) which sequesters at least 2000 iron atoms in vitro to form an electron-dense iron core of 7.9 +/- 1 nm diameter. Electron-microscopic and Mössbauer spectroscopic studies with iron-loaded ferritin showed that the core can be either crystalline (ferrihydrite) or amorphous, depending on the absence or presence of phosphate, respectively. Mössbauer spectroscopy with intact E. coli revealed a novel-high spin Fe(II) component which is enhanced in bacteria amplified for ferritin but not in the parental strain. Western blotting showed that ferritin and bacterioferritin are immunologically distinct proteins. E. coli is thus an organism containing both a ferritin and a bacterioferritin and the relative roles of the two iron-storage proteins are discussed in this study.

EPR studies of bacterioferritin (BFR), an iron-storage protein of Escherichia coli [1993, Biochem. J. 292, 47-56.], have revealed the presence of non-haem iron (III) (NHI) sites within the protein coat which may be involved in iron uptake and release. When nitric oxide was used as an EPR spin probe of the Fe(II) state of the NHI sites, two distinct mononuclear NHI species were found. Under certain conditions, an iron dimer was also observed. The reaction of phosphate with NHI species has been investigated. Results point to a function for this anion in core nucleation.

The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal 'Peptidase_M75' domain of 251 residues. The C-terminal domain contains a highly conserved 'HXXE' motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or 'Psyr_3370') encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M(W) 27,772Da). Circular dichroism spectroscopy of EfeM indicated a mainly alpha-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6A. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.

Background The Ferritins are part of the extensive ‘Ferritin-like superfamily’ which have diverse functions but are linked by the presence of a common four-helical bundle domain. The role performed by Ferritins as the cellular repository of excess iron is unique. In many ways Ferritins act as tiny organelles in their ability to secrete iron away from the delicate machinery of the cell, and then to release it again in a controlled fashion avoiding toxicity. The Ferritins are ancient proteins, being common in all three domains of life. This ubiquity reflects the key contribution that Ferritins provide in achieving iron homeostasis. Scope of the review This review compares the features of the different Ferritins and considers how they, and other members of the Ferritin-like superfamily, have evolved. It also considers relevant features of the eleven other known families within the Ferritin-like superfamily, particularly the highly diverse rubrerythrins. Major conclusions The Ferritins have travelled a considerable evolutionary journey, being derived from far more simplistic rubrerythrin-like molecules which play roles in defence against toxic oxygen species. The forces of evolution have moulded such molecules into three distinct types of iron storing (or detoxifying) protein: the classical and universal 24-meric ferritins; the haem-containing 24-meric bacterioferritins of prokaryotes; and the prokaryotic 12-meric Dps proteins. These three Ferritin types are similar, but also possess unique properties that distinguish them and enable then to achieve their specific physiological purposes. General significance A wide range of biological functions have evolved from a relatively simple structural unit.

Three soluble ferrisiderophore reductases (FsrA, FsrB and FsrC) were detected in Escherichia coli. FsrB was purified and identified as the haemoglobin-like protein (HMP) by size and N-terminal sequence analyses. HMP was previously isolated as a dihydropteridine reductase and is now shown to have ferrisiderophore reductase activity. Database searches revealed that the C-terminal region of HMP (FsrB) is homologous to members of a family of flavoprotein oxidoreductases which includes ferredoxin NADP+ reductase (FNR). The combination of FNR-like and haemoglobin-like regions in HMP (FsrB) represents a novel pairing of functionally and structurally distinct domains. Structure—function properties of other FNR-like proteins, including LuxG and VanB, are also discussed.

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was approximately 10-fold lower than in the wild type (with 0.5 microM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 microM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.

The gene ( dctA ) encoding the aerobic C 4 -dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and the o157a-o424-o328 (or orfQMP ) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti and Rhizobium leguminosarum H + /C 4 -dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C 4 -dicarboxylate transport system (DctQ, DctM, and DctP) of Rhodobacter capsulatus . To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactivated by inserting Sp r or Ap r cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was ∼10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428 -containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C 4 -dicarboxylate transport. Regulation studies with a dctA-lacZ ( f428-lacZ ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C 4 -dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive with respect to C 4 -dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

The bacterioferritin (BFR) of Escherichia coli is an iron-storage protein containing 24 identical subunits and between three and 11 protohaem IX groups per molecule. Titration with additional haem gave a maximum loading of 12-14 haems per molecule. The e.p.r. spectra and magnetic c.d. spectra of the protein-bound haem show it to be low-spin Fe(III), and coordinated by two methionine residues as previously reported for BFRs isolated from Pseudomonas aeruginosa and Azotobacter vinelandii [Cheesman, Thomson, Greenwood, Moore and Kadir, Nature (London) (1990) 346, 771-773]. A recent sequence alignment indicated that BFR may be structurally related to ferritin. The molecular model proposed for E. coli BFR has a four-alpha-helix-bundle subunit conformation and a quaternary structure similar to those of mammalian ferritins. In this model there are two types of hydrophobic pocket within which two methionine residues are correctly disposed to bind haem. The e.p.r. spectra also reveal a monomeric non-haem Fe(III) species with spin, S = 5/2. On the basis of sequence comparisons, a ferroxidase centre has recently been proposed to be present in BFR [Andrews, Smith, Yewdall, Guest and Harrison (1991) FEBS Lett. 293, 164-168] and the possibility that this Fe(III) ion may reside at or near the ferroxidase centre is discussed.

The 70-amino-acid-residue N-terminal sequence of the bacterioferritin (BFR) of Azotobacter vinelandii was determined and shown to be highly similar to the N-terminal sequences of the Escherichia coli and Nitrobacter winogradskyi bacterioferritins. Electrophoretic and immunological analyses further indicate that the bacterioferritins of E. coli, A. vinelandii and Pseudomonas aeruginosa are closely related. A novel, two-subunit assembly state that predominates over the 24-subunit form of BFR at low pH was demonstrated. The results indicate that the bacterioferritins form a family of proteins that are distinct from the ferritins of plants and animals.

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, ‘a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype’ in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin.

X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron-binding sites per subunit. Two of these form a di-iron site in the centre of the subunit as has been proposed for the ‘ferroxidase centres’ of human ferritin H chains. This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN.

The mechanism by which iron-storage proteins take up and oxidise iron(II) is not understood. We show by rapid-kinetic and EPR measurements that iron uptake, in vitro, by a bacterial iron-storage protein, bacterioferritin, involves at least three kinetically distinguishable phases: phase 1, the binding of Fe(II) ions, probably at a dimeric iron ferroxidase centre; phase 2, oxidation of the Fe(II) dimer and production of mononuclear Fe(III); and phase 3, iron core formation.

Iron-storage proteins can be divided into two classes; the bacterioferritins and ferritins. In spite of many apparent structural and functional analogies, no significant amino acid sequence similarity has been detected previously. This report now reveals a distant evolutionary relationship between bacterioferritins and ferritins derived by ‘Profile Analysis’. Optimum alignment of bacterioferritin and ferritin sequences suggests that key residues of the ferroxidase centres of ferritins are conserved in bacterioferritins.

(1) The nucleotide sequence of a 1991 bp segment of DNA that expresses the GMP reductase (guaC) gene of Escherichia coli K12 was determined. (2) This gene comprises 1038 bp, 346 codons (including the initiation codon but excluding the termination codon), and it encodes a polypeptide of Mr 37,437 which is in good agreement with previous maxicell studies. (3) The sequence contains a putative promoter 102 bp upstream of the translational start codon, and this is immediately followed by a (G + C)-rich discriminator sequence suggesting that guaC expression may be under stringent control (4) The GMP reductase exhibits a high degree of sequence identity (34%) with IMP dehydrogenase (the guaB gene product) indicative of a close evolutionary relationship between the salvage pathway and the biosynthetic enzymes, GMP reductase and IMP dehydrogenase, respectively. (5) A single conserved cysteine residue, possibly involved in IMP binding to IMP dehydrogenase, was located within a region that possesses some of the features of a nucleotide binding site. (6) The IMP dehydrogenase polypeptide contains an internal segment of 123 amino acid residues that has no counterpart in GMP reductase and may represent an independent folding domain flanked by (alanine + glycine)-rich interdomain linkers.

The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA).

Ferritin present within siderosomes of iron-loaded rats has a faster anodal mobility than that of cytosolic ferritin from the same rats. A 19-amino-acid-residue peptide was isolated from this fast ferritin and shown to be derived from the C-terminal end of its L-subunit. A 17.3 kDa peptide seen on electrophoresis in denaturing gels of this ferritin accounts for the major portion of the original 182-residue subunit. The two peptides arise from cleavage within the ‘insertion region’ of the L-subunit sequence that occurs between the D and E helices and lies on the outside of the assembled molecule. This cleavage is present in about 80% of the L-subunits of siderosomal ferritin but nevertheless leaves the molecular structure otherwise intact. It gives rise to an apparent decrease in molecular size, accounting for the faster anodal mobility on native gels. Hence a new form of heterogeneity in ferritin preparations has been explained.

The complete amino acid sequence of bacterioferritin (cytochrome b1) from Escherichia coli-K12 has been derived from the nucleotide sequence of the cloned gene. It comprises 158 amino acid residues giving an Mr of 18,495. The identity of the gene product was confirmed by an 87 residue N-terminal sequence obtained from the purified protein, but it differs significantly from much of the previously published partial amino acid sequence (1). Secondary structure prediction indicates a high alpha-helical content consistent with a 4-helix-bundle conformation. The fully assembled bacterioferritin molecule comprising 24 identical subunits and 12 haem moieties is a tetracosamer with an Mr of approximately 452,000.

Haemosiderin has been isolated from siderosomes and ferritin from the cytosol of livers of rats iron-loaded by intraperitoneal injections of iron-dextran. Siderosomal haermosiderin, like ferritin, was shown by electron diffraction to contain iron mainly in the form of small particles of ferrihydrite (5Fe2O3.9H2O), with average particle diameter of 5.36 +/- 1.31 nm (SD), less than that of ferritin iron-cores (6.14 +/- 1.18 nm). Mössbauer spectra of both iron-storage complexes are also similar, except that the blocking temperature, TB, for haemosiderin (23 K) is lower than that of ferritin (35 K). These values are consistent with their differences in particle volumes assuming identical magnetic anisotropy constants. Measurements of P/Fe ratios by electron probe microanalysis showed the presence of phosphorus in rat liver haemosiderin, but much of it was lost on extensive dialysis. The presence of peptides reacting with anti-ferritin antisera and the similarities in the structures of their iron components are consistent with the view that rat liver haemosiderin arises by degradation of ferritin polypeptides, but its peptide pattern is different from that found in human beta-thalassaemia haemosiderin. The blocking temperature, 35 K, for rat liver ferritin is near to that reported, 40 K, for human beta-thalassaemia spleen ferritin. However, the haemosiderin isolated from this tissue, in contrast to that from rat liver, had a TB higher than that of ferritin. The iron availability of haemosiderins from rat liver and human beta-thalassaemic spleen to a hydroxypyridinone chelator also differed. That from rat liver was equal to or greater, and that from human spleen was markedly less, than the iron availability from either of the associated ferritins, which were equivalent. The differences in properties of the two types of haemosiderin may reflect their origins from primary or secondary iron overload and differences in the duration of the overload.

Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes ( ftnA and bfr ) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants ( ftnA::spc and bfr::kan ) and a double mutant ( ftnA::spc bfr::kan ) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation ( fur ) mutants also had very low iron contents (2.5-fold less iron than Fur + strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of “reactive ferrous iron.” It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.

The bacterioferritin (BFR) of Escherichia coli K-12 is an iron-storage hemoprotein, previously identified as cytochrome b1. The bacterioferritin gene (bfr) has been cloned, sequenced, and located in the E. coli linkage map. Initially a gene fusion encoding a BFR-lambda hybrid protein (Mr 21,000) was detected by immunoscreening a lambda gene bank containing Sau3A restriction fragments of E. coli DNA. The bfr gene was mapped to 73 min (the str-spc region) in the physical map of the E. coli chromosome by probing Southern blots of restriction digests of E. coli DNA with a fragment of the bfr gene. The intact bfr gene was then subcloned from the corresponding lambda phage from the gene library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The bfr gene comprises 474 base pairs and 158 amino acid codons (including the start codon), and it encodes a polypeptide having essentially the same size (Mr 18,495) and N-terminal sequence as the purified protein. A potential promoter sequence was detected in the 5' noncoding region, but it was not associated with an "iron box" sequence (i.e., a binding site for the iron-dependent Fur repressor protein). BFR was amplified to 14% of the total protein in a bfr plasmid-containing strain. An additional unidentified gene (gen-64), encoding a relatively basic 64-residue polypeptide and having the same polarity as bfr, was detected upstream of the bfr gene.

Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores. Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation. Mössbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin. Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN). A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III). Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex. Mössbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins. Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components. This provides further evidence that the ferritin protein shell can influence the structure of its iron core.