SufC: an unorthodox cytoplasmic ABC/ATPase required for [Fe-S] biogenesis under oxidative stress

Proteins containing [Fe-S] clusters perform essential functions in all domains of life. Previously, we identified the sufABCDSE operon as being necessary for virulence of the plant pathogen Erwinia chrysanthemi. In addition, we collected preliminary evidence that the sufABCDSE operon might be involved in the assembly of [Fe-S] clusters. Of particular interest are the sufB, sufC and sufD genes, which are conserved among Eubacteria, Archaea, plants and parasites. The present study establishes SufC as an unorthodox ATPase of the ABC superfamily that is located in the cytosol, wherein it interacts with both SufB and SufD. Moreover, under oxidative stress conditions, SufC was found to be necessary for the activity of enzymes containing oxygen-labile [Fe-S] clusters, but dispensable for glutamate synthase, which contains an oxidatively stable [Fe-S] cluster. Lastly, we have shown SufBCD to be essential for iron acquisition via chrysobactin, a siderophore of major importance in virulence. We discuss a model wherein the SufBCD proteins contribute to bacterial pathogenicity via their role in the assembly of [Fe-S] clusters under oxidative stress and iron limitation.     

Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient

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 M?ssbauer 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.     

SufS Is a NifS-Like Protein, and SufD Is Necessary for Stability of the [2Fe-2S] FhuF Protein in Escherichia coli

Escherichia coli fhuF mutants, a sufS::MudI mutant, and a sufD::MudI mutant were found to have the same phenotype: the inability to use ferrioxamine B as an iron source in a plate assay. In addition, the sufS and sufD genes were shown to be regulated by the iron-dependent Fur repressor. Sequence analysis revealed that the sufS open reading frame corresponds to orf f406. The protein SufS belongs to the family of NifS-like proteins, which supply sulfur for [Fe-S] centers. The protein FhuF contains a [2Fe-2S] center. A mutation in the upstream sufD gene (orf f423) caused the same phenotype. The T7 expression system and a His tag allow the isolation in good yield of the FhuF protein from a wild-type strain. In contrast, overproduction of the protein in a DeltasufD strain failed. Radioactive labeling of N-His-FhuF with [35S]methionine showed that the protein was unstable in the DeltasufD mutant.     

Chrysobactin-dependent iron acquisition in Erwinia chrysanthemi. Functional study of a homolog of the Escherichia coli ferric enterobactin esterase.

Under iron limitation, the plant pathogen Erwinia chrysanthemi produces the catechol-type siderophore chrysobactin, which acts as a virulence factor. It can also use enterobactin as a xenosiderophore. We began this work by sequencing the 5'-upstream region of the fct-cbsCEBA operon, which encodes the ferric chrysobactin receptor and proteins involved in synthesis of the catechol moiety. We identified a new iron-regulated gene (cbsH) transcribed divergently relative to the fct gene, the translated sequence of which is 45.6% identical to that of Escherichia coli ferric enterobactin esterase. Insertions within this gene interrupt the chrysobactin biosynthetic pathway by exerting a polar effect on a downstream gene with some sequence identity to the E. coli enterobactin synthase gene. These mutations had no effect on the ability of the bacterium to obtain iron from enterobactin, showing that a functional cbsH gene is not required for iron removal from ferric enterobactin in E. chrysanthemi. The cbsH-negative mutants were less able to utilize ferric chrysobactin, and this effect was not caused by a defect in transport per se. In a nonpolar cbsH-negative mutant, chrysobactin accumulated intracellularly. These defects were rescued by the cbsH gene supplied on a plasmid. The amino acid sequence of the CbsH protein revealed characteristics of the S9 prolyl oligopeptidase family. Ferric chrysobactin hydrolysis was detected in cell extracts from a cbsH-positive strain that was inhibited by diisopropyl fluorophosphate. These data are consistent with the fact that chrysobactin is a d-lysyl-l-serine derivative. M?ssbauer spectroscopy of whole cells at various states of (57)Fe-labeled chrysobactin uptake showed that this enzyme is not required for iron removal from chrysobactin in vivo. The CbsH protein may therefore be regarded as a peptidase that prevents the bacterial cells from being intracellularly iron-depleted by chrysobactin.     

Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12.

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.     

Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity

In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3' of the anticodon (position 37) in seven tRNA species, two of which are tRNA(Pro)CGG and tRN(Pro)GGG. These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3' of m1G37. The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs. We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell. Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6tRNA. However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase. Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme. We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA. Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.     

The role of ferritins in the physiology of Salmonella enterica sv. Typhimurium: a unique role for ferritin B in iron-sulphur cluster repair and virulence

Ferritins are ubiquitous iron (Fe) storage proteins that play a fundamental role in cellular Fe homeostasis. The enteric pathogen Salmonella enterica serovar Typhimurium possesses four ferritins: bacterioferritin, ferritin A, ferritin B and Dps. The haem-containing bacterioferritin (Bfr) accounts for the majority of stored Fe, followed by ferritin A (FtnA). Inactivation of bfr elevates the intracellular free Fe concentration and enhances susceptibility to H2O2 stress. The DNA-binding Dps protein provides protection from oxidative damage without affecting the steady-state intracellular free Fe concentration. FtnB appears to be particularly important for the repair of oxidatively damaged Fe-sulphur clusters of aconitase and, in contrast to Bfr and FtnA, is required for Salmonella virulence in mice. Moreover, ftnB and dps are repressed by the Fe-responsive regulator Fur and induced under conditions of Fe limitation, whereas bfr and ftnA are maximally expressed when Fe is abundant. The absence of a conserved ferroxidase domain and the potentiation of oxidative stress by FtnB in some strains lacking Dps suggest that FtnB serves as a facile cellular reservoir of Fe2+.     

Nifs and Sufs in malaria

This review assembles data from three bodies of literature (bacterial genetics, plastid biogenesis and parasitology) that seldom have much direct cross-talk. After overcoming terminological complications to sort out microbial nifS from sufS genes, we connect a bacterial operon, recently found to be involved in iron metabolism, the formation of [Fe-S] clusters and oxidative stress to a potentially important gene (sufB) carried on the degenerate plastid genome of malaria and related parasites.     

Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus

Abstract The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene ( bfr ) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18 174 Da. The molecular mass of the purified protein was estimated to be 18 176.06 ± 0.80 Da by electrospray mass spectrometry. The bfr gene was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli . The amino acids which are involved in haem ligation, and those which provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conserved in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.     

Ferric iron uptake in Erwinia chrysanthemi mediated by chrysobactin and related catechol-type compounds.

Erwinia chrysanthemi 3937 possesses a saturable, high-affinity transport system for the ferric complex of its native siderophore chrysobactin, [N-alpha-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine]. Uptake of 55Fe-labeled chrysobactin was completely inhibited by respiratory poison or low temperature and was significantly reduced in rich medium. The kinetics of chrysobactin-mediated iron transport were determined to have apparent Km and Vmax values of about 30 nM and of 90 pmol/mg.min, respectively. Isomers of chrysobactin and analogs with progressively shorter side chains mediated ferric iron transport as efficiently as the native siderophore, which indicates that the chrysobactin receptor primarily recognizes the catechol-iron center. Free ligand in excess only moderately reduced the accumulation of 55Fe. Chrysobactin may therefore be regarded as a true siderophore for E. chrysanthemi.     

Bacterial iron homeostasis

Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. In many cases, this involves the secretion and internalisation of extracellular ferric chelators called siderophores. Ferrous iron can also be directly imported by the G protein-like transporter, FeoB. For pathogens, host-iron complexes (transferrin, lactoferrin, haem, haemoglobin) are directly used as iron sources. Bacterial iron storage proteins (ferritin, bacterioferritin) provide intracellular iron reserves for use when external supplies are restricted, and iron detoxification proteins (Dps) are employed to protect the chromosome from iron-induced free radical damage. There is evidence that bacteria control their iron requirements in response to iron availability by down-regulating the expression of iron proteins during iron-restricted growth. And finally, the expression of the iron homeostatic machinery is subject to iron-dependent global control ensuring that iron acquisition, storage and consumption are geared to iron availability and that intracellular levels of free iron do not reach toxic levels.     

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.