Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron–sulfur clusters

Biological assembly of iron–sulfur (Fe–S) clusters is mediated by complex systems consisting of multiple proteins. Escherichia coli possesses two distinct systems called the ISC and SUF machineries encoded by iscSUA‐hscBA‐fdx‐iscX and sufABCDSE respectively. Deletion of both pathways results in absence of the biosynthetic apparatus for Fe–S clusters, and consequent lethality, which has hampered detailed genetic studies. Here we report that modification of the isoprenoid biosynthetic pathway can offset the indispensability of the Fe–S cluster biosynthetic systems and show that the resulting Δisc Δsuf double mutants can grow without detectable Fe–S cluster‐containing proteins. We also constructed a series of mutants in which each isc gene was disrupted in the deletion background of sufABCDSE. Phenotypic analysis of the mutants revealed that Fdx, an essential electron‐transfer Fe–S protein in the ISC machinery, is dispensable under anaerobic conditions, which is similar to the situation with IscA. Furthermore, we found that several suppressor mutations in IscU, an Fe–S scaffold protein responsible for the de novo Fe–S cluster assembly, could bypass the essential role of the chaperone system HscA and HscB. These findings pave the way toward a detailed molecular analysis to understand the mechanisms involved in Fe–S cluster biosynthesis.     

The iron‐binding CyaY and IscX proteins assist the ISC‐catalyzed Fe‐S biogenesis in Escherichia coli

In eukaryotes, frataxin deficiency (FXN) causes severe phenotypes including loss of iron‐sulfur (Fe‐S) cluster protein activity, accumulation of mitochondrial iron and leads to the neurodegenerative disease Friedreich's ataxia. In contrast, in prokaryotes, deficiency in the FXN homolog, CyaY, was reported not to cause any significant phenotype, questioning both its importance and its actual contribution to Fe‐S cluster biogenesis. Because FXN is conserved between eukaryotes and prokaryotes, this surprising discrepancy prompted us to reinvestigate the role of CyaY in Escherichia coli. We report that CyaY (i) potentiates E. coli fitness, (ii) belongs to the ISC pathway catalyzing the maturation of Fe‐S cluster‐containing proteins and (iii) requires iron‐rich conditions for its contribution to be significant. A genetic interaction was discovered between cyaY and iscX, the last gene of the isc operon. Deletion of both genes showed an additive effect on Fe‐S cluster protein maturation, which led, among others, to increased resistance to aminoglycosides and increased sensitivity to lambda phage infection. Together, these in vivo results establish the importance of CyaY as a member of the ISC‐mediated Fe‐S cluster biogenesis pathway in E. coli, like it does in eukaryotes, and validate IscX as a new bona fide Fe‐S cluster biogenesis factor.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

Nfu facilitates the maturation of iron‐sulfur proteins and participates in virulence in Staphylococcus aureus

The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron‐sulfur (Fe‐S) clusters, which are required for functional Fe‐S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe‐S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe‐S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as an Fe‐S cluster carrier, which aids in the maturation of Fe‐S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non‐incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe‐S cluster metabolism as an attractive antimicrobial target.     

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na⁺ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na⁺ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na⁺/H⁺ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs. Received: August 30, 1998 / Accepted: November 13, 1998     

Cross-genomic analysis of the translational systems of various organisms

We have characterized the genes encoding ribosomal proteins (r-proteins) as well as other translation-related factors of 15 eubacteria and four archaebacteria, and the genes for the mitochondrial r-proteins of Saccharomyces cerevisiae by using the complete genomic nucleotide sequence data of these organisms. In eubacteria, including two species of Mycoplasma, the operon structure of the r-protein genes is well conserved, while their relative orientation and chromosomal location are quite divergent. The operon structure of the r-protein genes in archaebacteria, on the other hand, is quite different from eubacteria and also among themselves. In addition, many archaebacterial r-proteins show similarity to rat cytoplasmic r-proteins. Nonetheless, characteristic features of several genes encoding proteins of functional importance are well conserved throughout the bacterial species including archaebacteria, as well as in S. cerevisiae. We searched for the genes encoding mitochondrial r-proteins in yeast by combining informatics and genetic experiments. Furthermore, we characterized some of the r-proteins genes by exchanging portions between Escherichia coli and S. cerevisiae and performed functional analysis of some of the genes from different evolutionary points of view. Our work may be extended towards phylogenetic analysis of organisms producing secondary metabolites of various sorts. Journal of Industrial Microbiology & Biotechnology (2001) 27, 163–169. Received 21 September 1999/ Accepted in revised form 22 September 2000     

Oxidant-responsive induction of the suf operon, encoding a Fe-S assembly system, through Fur and IscR in Escherichia coli

The suf operon encoding a Fe-S assembly system is induced by peroxides through activators OxyR and IscR in Escherichia coli. For apo-IscR to bind, oxidation-mediated dissociation of Fur is required. Therefore, a peroxide-responsive signal is transduced through OxyR, IscR, and Fur to achieve oxidation-sensitive and maximal induction of this operon.     

Sequencing and analysis of aMycoplasma gallisepticum A5969 chromosome region containing the S10 andrrn23-5 operons

A 20,115-nt region of theMycoplasma gallisepticum A5959 genome was sequenced (GenBank accession no. AF036708). The region contains therrn23-5 and S10 operons, the lactate dehydrogenase gene, and two open reading frames (ORF293 and ORF129/ORF171) coding for proteins of unknown function. Therrn23-5 operon includes genes for 23S and 5S rRNAs. The S10 operon includes genes for 20 ribosomal proteins, Sec Y transport protein, adenylate kinase, and methionine aminopeptidase, and lacks theinfA-rpl36-rps13-rpoA-rpl17 genes found in the S10 operon ofM. genitalium, M. pneumoniae, andBacillus subtilis. The product ofM. gallisepticum ldh is equally similar to the corresponding proteins of mycoplasmata andB. subtilis but contains only a part of the motif characteristic of the active center of lactate dehydrogenases. The chromosome region adjacent to the sequenced one containsuvrA,nrdE,nrdF, andptsI.     

Distinct roles for U‐type proteins in iron–sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon

The biosynthesis of iron–sulfur (Fe–S) clusters in Bacillus subtilis is mediated by the SUF‐like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe–S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe–S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe–S cluster assembly.     

Ferredoxin Competes with Bacterial Frataxin in Binding to the Desulfurase IscS

The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.     

Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.     

SufU Is an Essential Iron-Sulfur Cluster Scaffold Protein in Bacillus subtilis

Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacterium''s genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to ∼1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.Iron-sulfur (Fe/S) clusters are one of the most ubiquitous and versatile cofactors employed by nature for catalyzing a variety of redox reactions or for serving as redox sensors in a broad range of regulatory processes (10). Iron and sulfide are toxic for the cells in concentrations needed for spontaneous chemical Fe/S protein maturation. Hence, cells have developed complex biosynthesis machineries which are essential in vivo to assemble Fe/S proteins. Three phylogenetically distinct biosynthesis systems have been found in bacteria: ISC (iron-sulfur cluster), SUF (sulfur mobilization), and NIF (nitrogen fixation) (9, 11, 24). The ISC machinery is the most widely distributed bacterial Fe/S cluster biogenesis system and is also present in eukaryotes (31). In Escherichia coli a second system for Fe/S cluster assembly, SUF, is induced under conditions of iron limitation and/or oxidative stress, thus replacing the housekeeping ISC system for assembly of Fe/S proteins. In contrast, SUF was found as the exclusive Fe/S biogenesis system in mycobacteria (22) and Enterococcus faecalis (39) and hence may also serve as a constitutive system. Furthermore, SUF is present in plastids of green plants, resembling the situation found in their cyanobacterial ancestors (25, 53). The NIF system is responsible for the dedicated maturation of the complex Fe/S protein nitrogenase involved in nitrogen fixation, e.g., in Azotobacter vinelandii. Some NIF genes are associated with anaerobic or microaerobic growth in Helicobacter pylori and Entamoeba histolytica (24).Common principles for Fe/S protein assembly in each system have been defined (30). The de novo assembly of an Fe/S cluster occurs on scaffold proteins which transiently bind the Fe/S cluster before transfer to target apoproteins. Cysteine desulfurases such as IscS and SufS serve as sulfur donors, which acquire sulfur from free l-cysteine by pyridoxal-5′-phosphate-dependent desulfuration. The sulfur is transiently bound in the form of a persulfide to an active-site cysteine of the desulfurase and is subsequently transferred to the scaffold protein. Several SUF systems contain SufE, which specifically forms a complex with the cysteine desulfurase SufS (36, 42). SufE enhances SufS activity significantly and assists the sulfur transfer to scaffold proteins. In this case the persulfide is transiently bound to SufE and not to the desulfurase. Recent studies show that the E. coli cysteine desulfurase CsdA is able to complement the SUF system and interacts with SufE if SufS is inactivated (50). A general iron donor involved in Fe/S cluster assembly is not known yet; however, frataxin homologs in prokaryotes and eukaryotes are postulated to deliver iron to the scaffold protein IscU in the ISC system (5, 9, 31, 34).Several components have been suggested to act as scaffold proteins. U-type scaffold proteins such as bacterial NifU, IscU, and eukaryotic Isu1 preferentially bind [2Fe-2S] clusters. However, the assembly of [4Fe-4S] clusters was described to proceed by reductive coupling of two [2Fe-2S] clusters that bind successively to an IscU dimer (1). A-type scaffolds like bacterial SufA or IscA can bind [2Fe-2S] clusters in their monomeric state and were found to be involved in the maturation of [4Fe-4S] proteins such as aconitase (17, 32, 47). Overproduced IscA was also shown to bind mononuclear iron which could be used for Fe/S cluster assembly on IscU in vitro.The ISC system characteristically contains the molecular chaperone pair HscA and HscB that are involved in Fe/S cluster transfer from the IscU scaffold protein to the target proteins. The Hsp70-type HscA specifically binds to a highly conserved LPPVK motif located near the third strictly conserved cluster-binding cysteine in IscU. Specific IscU-HscA complex formation was found to be necessary and sufficient to stimulate the ATPase activity of HscA (12, 21, 48). The SUF system, in contrast, does not contain HscA and HscB chaperones in the suf gene cluster. Instead it comprises the SufBCD proteins. The SufC protein has intrinsic ATPase activity and forms a complex with SufB, a putative scaffold protein, and SufD (29). The precise molecular function of the ATP-hydrolyzing SufBCD complex is not yet clear.The best-characterized SUF system from E. coli contains the gene cluster sufABCDSE (5). However, many bacteria, in particular members of the phylum Firmicutes, contain a different suf gene cluster encoding sufCDSUB, which has been studied so far only by bioinformatic approaches (39, 46). While SufS and SufBCD in the two gene clusters appear to be similar proteins, SufE is lacking in most Firmicutes and also in the mycobacterial and Thermotoga maritima SUF machineries (22, 24). The additionally present SufU shares similarities with IscU of the ISC system (39). The protein contains all three conserved cysteine residues involved in Fe/S cluster association and yet characteristically lacks the LPPVK motif, consistent with the absence of HscA and HscB proteins in sufCDSUB species.In this study, we made use of the Gram-positive bacterium Bacillus subtilis to initiate functional analysis of the sufCDSUB genes in Fe/S cluster biosynthesis by genetic and biochemical approaches. In particular, since no functional information is available for SufU, we tested its putative role as an Fe/S scaffold protein. SufU was found to be crucial for cell viability and for Fe/S-dependent enzyme activities in crude cell lysates. In vitro cluster reconstitution with recombinant SufU indicated that SufU binds a labile Fe/S cluster which can be transferred to apo-Leu1 in a fast and efficient way, fully activating its catalytic function as a [4Fe-4S] cluster-dependent isopropylmalate isomerase. The B. subtilis SufU was found to activate the desulfurase activity of purified B. subtilis SufS. Our results suggest that SufS and the SufU scaffold protein closely act together in the Fe/S cluster biogenesis in B. subtilis.     

The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc ⁻ strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O₂-stable [4Fe-4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by ⁵⁵Fe-labeling experiments. Using [³⁵S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O₂-inactivated apo-FNR into [4Fe-4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.     

Identification of Three <Emphasis Type="Italic">Superoxide dismutase</Emphasis> Genes from a <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

We report the characterization of three Superoxide dismutase (sod) genes isolated from a bacterium in the Geobacillus genus. We isolated the bacterium from high-temperature pond mud and used 16S rRNA gene sequence to confirm its identity in the Geobacillus genus. The three genes Mn-sod, Fe/Mn-sod, and Cu/Zn-sod were cloned and analyzed. Their open reading frames are Mn-sod: 615 bp encoding a 204 amino acid protein; Fe/Mn-sod: 1,236 bp encoding a 411 amino acid protein; Cu/Zn-sod: 522 bp encoding a 173 amino acid protein. When these sod genes were expressed in Escherichia coli, only Mn-SOD was able to be purified. The activity of the purified Mn-SOD we got was about 2,730 U/mg. Studies of this Mn-SOD showed that it was thermostable at 60°, had 70% activity at 80° after 2.5 h, and still had 30% activity at 90° after 2.5 h. Mn-SOD activity required the ion Mn²⁺. Based on gel electrophoresis, we deduced that this Mn-SOD was a homotetramer. No activity was detected after the other two genes (Fe/Mn-sod, Cu/Zn-sod) were expressed in Escherichia coli, but activities were detected when expressed in Pichia pastoris.