The iscS gene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

Abstract

Three NifS-like proteins, IscS, CSD, and CsdB, from Escherichia coli catalyze the removal of sulfur and selenium from l-cysteine and l-selenocysteine, respectively, to form l-alanine. These enzymes are proposed to function as sulfur-delivery proteins for iron-sulfur cluster, thiamin, 4-thiouridine, biotin, and molybdopterin. Recently, it was reported that selenium mobilized from free selenocysteine is incorporated specifically into a selenoprotein and tRNA in vivo, supporting the involvement of the NifS-like proteins in selenium metabolism. We here report evidence that a strain lacking IscS is incapable of synthesizing 5-methylaminomethyl-2-selenouridine and its precursor 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) in tRNA, suggesting that the sulfur atom released from l-cysteine by the action of IscS is incorporated into mnm⁵s²U. In contrast, neither CSD nor CsdB was essential for production of mnm⁵s²U and 5-methylaminomethyl-2-selenouridine. The lack of IscS also caused a significant loss of the selenium-containing polypeptide of formate dehydrogenase H. Together, these results suggest a dual function of IscS in sulfur and selenium metabolism.

Sulfur and selenium are similar in many of their chemical properties. This similarity allows many enzymes of sulfur metabolism to catalyze the analogous reactions with selenium-containing substrates (1–3). Nevertheless, several enzymes, such as selenophosphate synthetase (4), selenocysteine methyltransferase (5), and selenocysteine lyase (6, 7), strictly distinguish these two elements. The enzymatic discrimination between selenium-containing substrates and the corresponding sulfur analogs is important for cells to metabolize selenium-containing biomolecules without disturbing sulfur metabolism. The specific incorporation of selenium into several proteins and tRNAs has been identified (1, 8). Selenium is present as an essential selenocysteine residue in the polypeptide chains of various selenoproteins including bacterial formate dehydrogenase (9) and mammalian glutathione peroxidase (10). Selenium-containing tRNAs found in prokaryotes contain 5-methylaminomethyl-2-selenouridine (mnm⁵se²U) in the wobble position of the anticodons in glutamate, lysine, and glutamine isoaccepting tRNAs (11). The biosynthetic pathway for mnm⁵se²U has been partly characterized as shown in Fig. 1. The 2-thiolation step was shown to require the mnmA gene product, but neither the specific function of the gene product nor the mechanism of the thiolation reaction is clarified (12). The replacement of sulfur by selenium requires monoselenophosphate (13). The selD gene product, selenophosphate synthetase, catalyzes the synthesis of monoselenophosphate from ATP and selenide. Monoselenophosphate serves as a selenium donor for both mnm⁵se²U in tRNA and a specific selenocysteine residue of selenoproteins (14). Lacourciere et al. (15) showed that selenium derived from l-selenocysteine by the action of Escherichia coli NifS-like proteins can be used as the substrate for selenophosphate synthetase in vitro. More recently, it has been demonstrated that l-selenocysteine provided in the growth medium is effectively used as a selenium source for in vivo selenophosphate-dependent biosynthesis (16). These results suggest that the NifS-like proteins function as components of a selenium delivery system for the biosynthesis of selenoproteins and selenium-containing tRNAs.

Figure 1.

Proposed pathway for the biosynthesis of mnm⁵se²U in tRNA. s²U, mnm⁵U, and mnm⁵s²U denote 2-thiouridine, 5-methylaminomethyluridine, and 5-methylaminomethyl-2-thiouridine, respectively. The asterisk denotes one or more additional gene products that are not identified.

E. coli NifS-like proteins are pyridoxal 5′-phosphate-dependent enzymes that catalyze the removal of sulfur and selenium from cysteine and selenocysteine, respectively. E. coli encodes three nifS-like genes called iscS, csdA, and csdB (17–19). Specific functions have been suggested for CSD (the csdA gene product) as a sulfur donor for molybdopterin biosynthesis (20) and for CsdB as an enzyme involved in selenium (18) or iron (21) metabolism. Recent studies showed that IscS provides sulfur for the biosynthesis of iron-sulfur clusters, thiamin, biotin, and 4-thiouridine (s⁴U) in tRNA (22–26). In addition to IscS, s⁴U synthesis requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and l-cysteine as the sulfur donor. Kambampati and Lauhon (27) have shown that the sulfane sulfur generated by IscS is first transferred to ThiI and then to tRNA during the in vitro synthesis of s⁴U. Considering that the transfer of sulfur from l-cysteine to a variety of sulfur-containing molecules involves the NifS-like proteins, these enzymes may provide sulfur for the biosynthesis of thionucleotides such as 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) and 2-(methylthio-N⁶-isopentenyl)adenosine (28) in tRNA. To determine whether the three NifS-like proteins in E. coli were involved in the biosynthesis of mnm⁵se²U and mnm⁵s²U, we analyzed ⁷⁵Se- and ³⁵S-labeled tRNA nucleosides isolated from individual strains lacking iscS, csdA, or csdB. The effect of the iscS deletion on the biosynthesis of a selenoprotein, formate dehydrogenase H (FDH_H), also was investigated.

Experimental Procedures

Media and Reagents.

In general, the rich medium used was LB medium supplemented with 0.5% glucose and selenite where indicated. l-Selenocysteine was synthesized as described (29). [⁷⁵Se]Selenite was purchased from the University of Missouri Research Reactor Facility, Columbia, MO. l-[³⁵S]Cysteine was purchased from Amersham Pharmacia.

Plasmids and Bacterial Strains.

Bacterial strains and plasmids used in this study are listed in Table 1. Plasmid pBEN66 was a gift from Y. Yamamoto (Hyogo College of Medicine). A csdB-disrupted strain, H1489, was a gift from K. Hantke, Universität Tübingen (21). An iscS-deleted strain, iscS∷neo23, was constructed by Tokumoto and Takahashi (26). An mnmA⁻ strain, TH159, was a gift from T. Hagervall, Umeå University (30). A csdA-disrupted strain was constructed as follows. A 527-bp fragment of csdA lacking the start and stop codons was amplified by PCR with primers 5′-CTCGTCCCCTGGCTGATG-3′ and 5′-CTTTCGGCCTGGTTGATA-3′, blunt-ended, and inserted into the EcoRV site of pBEN66. The resulting plasmid, pBENifS2, was digested with NotI to yield a 1,917-bp fragment containing the 527-bp csdA fragment and a kanamycin-resistance (kan) gene. The fragment was self-ligated and introduced into W3110. The self-ligated circular product cannot replicate because of the lack of its own ori. A mutant strain, HM75, with a csdA gene disrupted by the insertion of kan was selected on solid LB medium containing 25 μg/ml of kanamycin. Disruption of the gene was confirmed by PCR and Southern blot analysis. P1 phages were generated from iscS∷neo23, HM75, and H1489. The ΔiscS∷kan, csdA∷kan, and sufS∷MudI(Ap^r lac) markers were transduced into MC4100 by selection on LB plates containing an appropriate antibiotic, generating strains SK23, SK75, and SK100, respectively. Standard methods were used for genetic manipulation according to Miller (31) and Sambrook et al. (32).

Table 1.

Strains and plasmids used in this work

	Genotype/property	Ref./source
Strains
MC4100	F−araD139 Δ(argF-lac)U169 ptsF25 deoC1 relA1 flbB5301 rpsL150λ−	NIG collection
iscS∷neo23	F− ΔiscS∷kan	26
H1489	aroB rpsL sufS∷MudI	21
W3110	F−IN(rrnD-rrnE)1	NIG collection
HM75	W3110 csdA∷kan	This work
TH159	F−ara Δ(lac-proB) nalA argEAm rif thi metB supB val(R) mnmE fadR13∷Tn10 mnmA107	30
WL400	MC4100 selD204∷cam	35
SK23	MC4100 ΔiscS∷kan	This work
SK100	MC4100 sufS∷MudI	This work
SK75	MC4100 csdA∷kan	This work
Plasmids
pBEN66	Gene disruption plasmid, Kmr	Y. Yamamoto
pBENifS2	pBEN66 containing csdA disruption construct, Kmr	This work

NIG, National Institute of Genetics. 

Polyacrylamide Gel Electrophoretic Analysis of ⁷⁵Se-Labeled Protein and tRNA.

E. coli cells were grown anaerobically overnight at 37°C in the LB medium supplemented with 0.1 μM Na₂SeO₃ and 20 μCi of Na₂⁷⁵SeO₃ per 15 ml of culture. The cells were harvested by centrifugation (10,000 × g), resuspended in 100 mM potassium phosphate buffer (pH 7.0), and lysed by freeze-thawing in liquid nitrogen. Aliquots of supernatants containing 6–8 μg of protein were analyzed for ⁷⁵Se-containing tRNA by SDS/PAGE (12%) and PhosphorImager detection of radioactivity.

tRNA Digestion and Nucleoside Analysis.

E. coli cells were grown in LB medium and labeled with 20 μCi of Na₂⁷⁵SeO₃ or 10 μCi of l-[³⁵S]cysteine, and ⁷⁵Se- or ³⁵S-labeled tRNA was isolated as described (11, 16). Nuclease digestion of tRNA was performed as described by Gehrke et al. (33) with minor modifications. About 150 μg of tRNA in 25 μl of water was heated at 100°C for 2 min, cooled on ice, and then digested by incubation at 37°C for 4 h in a reaction mixture containing 0.65 mM ZnSO₄, 6 units of P1 nuclease, and 0.16 mg/ml of potato phosphatase in a total volume of 62 μl. The mixture was heated at 100°C for 5 min and centrifuged (10,000 × g). The supernatant was diluted 7-fold with 170 mM Tris⋅HCl (pH 7.9) and analyzed by RP-HPLC. Aliquots of the digested tRNA were loaded onto an analytical C₁₈ HPLC column (Vydac, Hesperia, CA) and eluted with a linear gradient of 0–3.5% methanol in 10 mM ammonium acetate (pH 5.3) for 0–25 min and that of 3.5–15% methanol for 25–35 min, followed by washing the column with 100% methanol. Nucleosides were detected by UV absorbance at 257 and 313 nm and liquid scintillation counting with Radioflow Detector LB508 (EG & G, Berthold, Bundoora, Australia).

Other Methods.

The FDH_H activity was detected by the benzyl viologen agar overlay method (34). Proteins were assayed with a Bio-Rad protein assay kit, using BSA as a standard.

Results and Discussion

An iscS Mutant Strain Was Defective in the Biosynthesis of mnm⁵se²U in tRNAs.

It has been established that E. coli grown under anaerobic conditions in media supplemented with [⁷⁵Se]selenite incorporates ⁷⁵Se into the 79-kDa FDH_H polypeptide and tRNA nucleosides (8, 16). To investigate whether mutant E. coli strains, which are unable to produce active CSD, CsdB, or IscS, can incorporate selenium into tRNAs, we analyzed extracts from each strain for the presence of ⁷⁵Se-labeled tRNA by SDS/PAGE and PhosphorImager detection. Radioactive bands that migrated to positions expected for seleno-tRNAs were observed on 12% SDS/polyacrylamide gels for all strains except the iscS mutant (Fig. 2). To confirm that the radioactive bands were attributed to seleno-tRNA, RNaseA was added to the extracts from all strains. The ⁷⁵Se-labeled bands corresponding to the seleno-tRNA disappeared because of degradation of tRNA by RNaseA (Fig. 2). These results show that the iscS mutant is unable to incorporate ⁷⁵Se into the tRNA nucleosides and in this respect resembles the E. coli selD mutant strain MB08 (35). The selD⁻ strain cannot produce FDH_H or selenium-containing tRNAs because it is defective in the formation of monoselenophosphate. In contrast, the csdA and csdB mutations did not affect the incorporation pattern of ⁷⁵Se into tRNAs (Fig. 2), indicating their lack of involvement in the formation of the selenium-containing tRNAs. The inability of the iscS⁻ strain to form seleno-tRNA nucleosides also was confirmed by HPLC analysis. Bulk tRNA isolated from wild-type E. coli grown in the presence of [⁷⁵Se]selenite was digested with P1 nuclease and phosphatase. The resulting nucleosides were resolved by RP-HPLC on a C₁₈ column. The [⁷⁵Se]mnm⁵se²U eluted with a retention time of 3.7 min (Fig. 3). However, no [⁷⁵Se]mnm⁵se²U was detected in digests of bulk tRNA isolated from the iscS⁻ strain. The previous work by Veres and Stadtman (36) indicated that the conversion of mnm⁵s²U in tRNAs to mnm⁵se²U requires selenophosphate and at least one protein termed tRNA 2-thiouridine synthase in Salmonella typhimurium. Therefore, it is possible that IscS is involved either in the conversion of mnm⁵s²U to mnm⁵se²U or the formation of the precursor mnm⁵s²U itself.

Figure 2.

Effect of csdA, csdB, and iscS deletions on the formation of ⁷⁵Se-labeled tRNAs. Cell extracts from E. coli MC4100 (WT), SK75 (csdA⁻), SK100 (csdB⁻), and SK23 (iscS⁻), grown anaerobically in LB containing 0.1 μM Na₂SeO₃ and 20 μCi of Na₂⁷⁵SeO₃, were analyzed by SDS/PAGE and PhosphorImager detection. Lanes indicated by “+” denote samples treated with RNaseA (5 μg/ml) at 37°C for 1 h.

Figure 3.

HPLC analysis of nucleosides from ⁷⁵Se-labeled bulk tRNA form E. coli MC4100 (thin line) and SK23 (thick line). Elution positions of the four major nucleosides are indicated by arrows. tRNA labeled with ⁷⁵Se was digested to nucleosides and analyzed by HPLC at a flow rate of 1 ml/min as described.

IscS Is Involved in the Biosynthesis of both mnm⁵s²U and s⁴U.

Bulk tRNA isolated from ³⁵S-labeled cells was analyzed to determine whether the iscS deletion affects the biosynthesis of mnm⁵s²U. HPLC analysis of radiolabeled tRNA digests showed that two major ³⁵S-labeled nucleosides corresponding to mnm⁵s²U that eluted at 8.3 min and s⁴U that eluted at 27.1 min were present in the wild-type, csdA⁻, csdB⁻, and selD⁻ strains (Fig. 4), whereas the tRNA isolated from the iscS⁻ strain contained neither [³⁵S]mnm⁵s²U nor [³⁵S]s⁴U. Analysis of bulk tRNA isolated from a strain containing an mnmA deletion (TH159) revealed the presence of only one ³⁵S-labeled nucleoside, s⁴U (data not shown). Taken together, these results indicate that IscS acts as a sulfur donor for both s⁴U and mnm⁵s²U biosynthesis. The mnmA gene product is also needed for the formation of mnm⁵s²U probably for the 2-thiolation reaction on the unmodified uridine. It has been shown that the mnmE mutant contains s²U, indicating that this thiolation reaction does not require an initial modification in position 5 (37). Sullivan et al. (12) have shown that tRNA from an mnmA mutant contains mnm⁵U, and thus this modification at position 5 can occur without dependence on thiolation at the 2 position. The requirement of IscS and the mnmA gene product for 2-thiouridine biosynthesis suggests the process may be similar to the biosynthesis of s⁴U. In the formation of s⁴U, IscS removes sulfur from l-cysteine to generate an IscS-derived cysteine persulfide with transfer of sulfur from the cysteine persulfide to ThiI (27). A mechanism involving the participation of cysteine residues of ThiI has been proposed for the formation of s⁴U (38). The mnmA gene product contains several cysteine residues that are highly conserved among its putative homologs (H.M. and N.E., unpublished observation). It is likely that one or more cysteine residues are involved in the 2-thiolation reaction.

Figure 4.

HPLC analysis of nucleosides from ³⁵S-labeled bulk tRNA from wild-type E. coli MC4100 (thin line) and mutant SK23 (thick line). Elution positions of the four major nucleosides are indicated by arrows. tRNA labeled with ³⁵S was digested to nucleosides and analyzed by HPLC at a flow rate of 0.5 ml/min as described.

It has been shown that mnm⁵s²U localized in the anticodon loop at position 34 of tRNAs is important for modulation of codon recognition during mRNA translation in E. coli (30, 39). 2-Thiouridine has also been found in eukaryotes (40, 41). The iscS and mnmA genes are ubiquitous and are found in genomic sequences of bacteria, archaea, and eukaryotes (H.M. and N.E., unpublished observation). Recently, the defect in 2-thiolation in the wobble base of the human mitochondrial tRNA^Lys anticodon was proposed to disturb codon–anticodon interaction, leading to the onset of a mitochondrial disease, myoclonus epilepsy associated with ragged-red fibers (42). Although little is known about the modification system in eukaryotes, our finding that IscS is essential for 2-thiolation of tRNA in the wobble position of the anticodon suggests that eukaryotic NifS homologs may function as sulfur-transfer proteins for 2-thiouridine biosynthesis.

IscS Is Indispensable for Formation of FDH_H.

Monoselenophosphate formed by selenophosphate synthetase is the selenium donor required for specific selenocysteine incorporation in proteins and mnm⁵se²U. Because three NifS-like proteins from E. coli have the ability to generate a selenium substrate for selenophosphate biosynthesis (15), we examined whether an active selenocysteine-containing FDH_H is produced in the E. coli NifS-like mutant strains. Active FDH_H can be detected readily in vivo by the benzyl viologen agar overlay assay (34). Wild-type MC4100, csdA⁻, and csdB⁻ strains reduced benzyl violgen, indicating that they are able to produce an active selenocysteine-containing FDH_H, but the iscS⁻ strain, like selD⁻ strains, failed to reduce benzyl viologen (Fig. 5). From these results it is clear that the iscS gene product also is required for production of active FDH_H.

Figure 5.

Effect of csdA, csdB, and iscS deletions on the FDH_H activity. E. coli MC4100 (wild-type), SK75 (csdA⁻), SK100 (csdB⁻), SK23 (iscS⁻), and WL400 (selD⁻) were grown anaerobically at 37°C on an LB agar plate containing 0.5% glucose. After growth the plates were overlaid with a dye solution containing 0.75% agar, 1 mg/ml of benzyl viologen, 0.25 M sodium formate, and 25 mM KH₂PO₄.

Effect of the iscS Mutation on the Incorporation of Selenocysteine into FDH_H.

In addition to a selenocysteine residue, FDH_H requires an Fe₄S₄ cluster and a Mo atom coordinated to two molybdopterin guanine dinucleotide cofactors for catalytic activity. Therefore, the loss of FDH_H activity in the iscS⁻ strain could be ascribed to failure of the mutant to incorporate selenocysteine into the enzyme, or to defective sulfur provision for iron-sulfur cluster formation, or for molybdopterin synthesis. We examined the incorporation of ⁷⁵Se into FDH_H in the iscS⁻ strain. The crude extracts from wild-type and iscS⁻ strains, prepared from cells grown with [⁷⁵Se]selenite, were analyzed by SDS/PAGE and PhosphorImager detection. The extract from the wild-type strain exhibited a 79-kDa radioactive band corresponding to FDH_H (Fig. 6). Addition of l-selenocysteine to the wild-type culture medium markedly depressed ⁷⁵Se incorporation in FDH_H, suggesting that selenium derived from l-selenocysteine also is used for its biosynthesis. In contrast to the result obtained with wild-type E. coli, the amount of ⁷⁵Se-labeled FDH_H observed in the extract of the iscS⁻ strain was significantly reduced in the absence of l-selenocysteine (Fig. 6). The low amount of ⁷⁵Se-labeled FDH_H observed under these conditions may be the result of a lower level of expression of FDH_H in this mutant. However, a slight increase in radioactivity is observed in extract from cells that were grown in the presence of l-selenocysteine. This increase may be the result of additional nonspecific incorporation of ⁷⁵Se into FDH_H. These results demonstrate that the synthesis of a selenocysteine-containing polypeptide of FDH_H is affected by the deletion of the iscS gene. The inability of this mutant strain to effectively synthesize an active selenium-containing FDH_H supports an in vivo role for IscS in selenium metabolism.

Figure 6.

Incorporation of ⁷⁵Se into FDH_H polypeptide in the MC4100 (WT) and SK23 (iscS⁻) strains in the presence (+) or absence (−) of added l-selenocysteine (0.2 μM). Cells were grown anaerobically in LB media containing 0.1 μM Na₂SeO₃ and 20 μCi of Na₂⁷⁵SeO₃ in the presence or absence of l-selenocysteine and analyzed by SDS/PAGE and PhosphorImager detection.

In summary, we show here that the iscS gene is essential for the formation of mnm⁵s²U and the selenocysteine-containing FDH_H polypeptide. These results provide evidence for additional functions of IscS in sulfur and selenium metabolism.

Abbreviations

mnm⁵se²U

5-methylaminomethyl-2-selenouridine

mnm⁵s²U

5-methylaminomethyl-2-thiouridine

s⁴U

4-thiouridine

FDH_H

formate dehydrogenase H