The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli Christopher J. Schwartz*, Ouliana Djaman†, James A. Imlay†, and Patricia J. Kiley*‡ *Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706; and †Department of Microbiology, University of Illinois, Urbana, IL 61801 Communicated by Helmut Beinert, University of Wisconsin, Madison, WI, June 7, 2000 (received for review May 8, 2000) The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster cluster assembly in vitro, their role in in vivo Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation formation has not yet been established. In addition, Flint et al. has yet to be established. To study the in vivo function of IscS in (11) also showed by using E. coli cell extracts that other proteins Escherichia coli, a strain lacking IscS was constructed and charac- can function to donate sulfur for Fe-S cluster assembly, leaving terized. Using this iscS deletion strain, we have observed decreased open the question as to which protein is the physiologically specific activities for proteins containing [4Fe-4S] clusters from relevant sulfur donor. Thus, the goal of this study was to soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate investigate the role of IscS in donating sulfur for in vivo Fe-S synthase, fumarase A, and FNR) and membrane-bound proteins cluster assembly. (NADH dehydrogenase I and succinate dehydrogenase). A specific Fe-S cluster assembly is important for the de novo synthesis of role for IscS in in vivo Fe-S cluster assembly was demonstrated by a broad spectrum of proteins and also the repair of certain Fe-S showing that an Fe-S cluster independent mutant of FNR is unaf- proteins that are damaged during oxidative stress. A family of fected by the lack of IscS. These data support the conclusion that, dehydratases, including the major fumarase and aconitase via its cysteine desulfurase activity, IscS provides the sulfur that isozymes, use [4Fe-4S]2ϩ clusters as Lewis acids during catalysis. subsequently becomes incorporated during in vivo Fe-S cluster These solvent-exposed clusters can be rapidly oxidized by su- synthesis. We also have characterized a growth phenotype asso- ciated with the loss of IscS. Under aerobic conditions the deletion peroxide, and the oxidized clusters are unstable, losing one and of IscS caused an auxotrophy for thiamine and nicotinic acid, perhaps more iron atoms (12). The inactivation of these enzymes whereas under anaerobic conditions, only nicotinic acid was re- is responsible for many of the characteristic deficiencies of quired. The lack of IscS also had a general effect on the growth of superoxide-stressed cells. Work in several labs has shown that E. coli because the iscS deletion strain grew at half the rate of wild these clusters are reassembled in vivo, using iron drawn from type in many types of media even when the auxotrophies were ferritin and a presumptive electron donor that has not been satisfied. identified (13, 14). In fact, the fully demetallated form of one such enzyme, dihydroxyacid dehydratase, was used as a substrate NifS ͉ FNR ͉ oxidative stress repair ͉ sulfur metabolism during the purification of the E. coli IscS protein (10). However, it is not clear whether cluster disintegration in vivo progresses to ron–sulfur proteins are ubiquitous in nature and participate in the point where sulfur atoms are lost, so that the role of IscS in Imany vital cellular functions such as DNA repair, transcrip- cluster repair is unclear. tional regulation, nucleotide and amino acid biosynthesis, and An attempt to delete iscS from A. vinelandii was unsuccessful, energy metabolism. Although many studies have focused on the apparently because the gene is essential (5). In addition, studies activities of Fe-S proteins, little is known about how Fe-S clusters in yeast have demonstrated that the IscS homologue (Nfs1) is are assembled and inserted into these proteins in vivo. Under also essential in this organism (6). In this study, we report the reducing conditions in vitro, such clusters are slowly assembled successful disruption of iscS from E. coli, allowing us to analyze into proteins when only Fe2ϩ and S2- are provided (1). However, the function of IscS in vivo. We have found that the lack of IscS studies of the N2-fixation (nif) gene cluster from Azotobacter leads to an overall decrease in the activity of a number of Fe-S vinelandii suggest that cells contain activities that increase the proteins and results in a general growth defect. efficiency of Fe-S cluster assembly (2). In particular, the char- acterization of NifS led to the discovery that this protein is a Materials and Methods pyridoxal 5Ј-phosphate-containing cysteine desulfurase that Growth Experiments. Cultures were grown aerobically overnight at transfers the sulfur from cysteine to an active site cysteinyl 37°C in Luria broth (15), pelleted, washed, resuspended in residue of NifS, forming an enzyme-bound persulfide (3). Upon glucose minimal medium (15), and used as an inoculum at a 0 reduction and addition of an iron source, the S can be released 1:1,000 dilution. Growth was monitored by measuring the OD600. and efficiently incorporated into the cluster of the Fe-S protein When indicated, the growth medium was supplemented with of nitrogenase (4). yeast extract (5 mg͞ml), thiamine (2 ␮g͞ml), nicotinic acid (12.5 Proteins similar to NifS have been discovered in non-N2-fixing ␮g͞ml), chloramphenicol (20 ␮g͞ml), ampicillin (50 ␮g͞ml), bacteria (5), eukaryotes and archaea (BLAST, National Center for tetracycline (10 ␮g͞ml), and kanamycin (Kn, 40 ␮g͞ml). Biotechnology Information), indicating that such cysteine des- ulfurases might serve a general role in Fe-S cluster formation. In BIOCHEMISTRY support of this notion, a homolog of NifS, called Nfs1, was Abbreviations: Kn, kanamycin; FNR, fumarate nitrate reduction; WT, wild type; SDH, identified in the yeast Saccharomyces cerevisiae, where it appears succinate dehydrogenase; DH, dehydrogenase. to play a role in mitochondrial Fe-S cluster assembly (6–8). In ‡To whom reprint requests should be addressed at: 1300 University Avenue, Department of addition, cysteine desulfurases homologous to NifS have been Biomolecular Chemistry, University of Wisconsin Medical School, Madison, WI 53706. E-mail: [email protected]. purified from A. vinelandii and Escherichia coli (9, 10). These The publication costs of this article were defrayed in part by page charge payment. This proteins have been named IscS for iron sulfur cluster to distin- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. guish them from their counterparts encoded within the N2- §1734 solely to indicate this fact. fixation gene clusters of N2-fixing bacteria (5). Although IscS Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.160261497. from E. coli and A. vinelandii have been shown to facilitate Fe-S Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.160261497 PNAS ͉ August 1, 2000 ͉ vol. 97 ͉ no. 16 ͉ 9009–9014 Downloaded by guest on September 26, 2021 from PK4326, and the ⌬iscS::KnR marker was transduced into RZ4500, PK3292, or PK3293 (Table 1) by selecting on Kn, generating strains PK4331, PK4357, and PK4359, respectively. Fe-S Protein Assays. Isocitrate and potassium cyanide were from Aldrich; all other reagents were from Sigma. Cultures for assays of enzymatic activities were grown to 0.4–0.7 OD in media that would ensure consistent, substantial synthesis of the relevant enzymes. In all cases except for that of fumarase, the media were chosen to avoid a dependence on Fe-S enzymes for central metabolism. NADH DH1 (Ndh1) and succinate dehydrogenase Fig. 1. Genomic organization of the isc region. Plasmids used in this study are (SDH) were assayed by using inverted membrane vesicles pre- indicated. The genotype of the iscS mutant is indicated with the iscS coding region having been replaced by the KnR gene. pared from cells grown in minimal A salts (15) containing 1% casein hydrolysate, 0.5% yeast extract, and 0.5% NaCl (pH 7), and NADH DH2 (Ndh2) was assayed in vesicles derived from Plasmid Construction. Plasmids pPK4071 and pPK4083 were made cells grown in LB medium containing 0.2% glucose. For acon- by digesting ␭430 (16) with NruI and cloning this 5-kb fragment itase assays cells were grown in LB medium; for 6-phosphoglu- containing iscRSUAhscB (17) into the SmaI site of pUC19 (18) conate dehydratase assays, in minimal A salts containing 0.2% and pAlter-1 (Promega), respectively. Plasmids pPK4196 and gluconate and 0.2% casein hydrolysate; for glutamate synthase, pPK4194 were generated by cloning PCR products containing in minimal A salts containing 0.2% glucose; and for fumarase only the ORFs for iscS and iscSUA into pET11a (19), respectively and isocitrate dehydrogenase, in minimal A salts containing 40 (Fig. 1). Plasmid pPK4344 was generated by cloning into pGem2 mM fumarate. (Promega), a PCR product containing sequence beginning To stabilize Ndh1 for assay, vesicles were prepared by soni- within the iscR ORF and ending within the iscA ORF (Fig. 1). cation in 50 mM MES, pH 6. Ndh1 activity was measured by the oxidation of deaminoNADH (23), which was prepared by the For DNA amplification by PCR from chromosomal or plasmid ϩ DNA, either Pfu DNA polymerase (Stratagene) or Taq DNA enzymatic reduction of deaminoNAD using the protocol for polymerase (Promega) was used. The sequences of oligonucle- NADH preparation (24). Ndh2 was assayed by the oxidation of otides (Operon Technologies, Alameda, CA) used in this work NADH after Ndh1 had been inactivated by the overnight are available on request. incubation of the vesicles in 50 mM KPi, pH 7.8, on ice.