The iscS Gene in Escherichia coli Is Required for the Biosynthesis of 4-Thiouridine, Thiamin, and NAD

Abstract

IscS, a cysteine desulfurase implicated in the repair of Fe-S clusters, was recently shown to act as a sulfurtransferase in the biosynthesis of 4-thiouridine (s4U) in tRNA (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561–16568). In frame deletion of the iscS gene in Escherichia coli results in a mutant strain that lacks s4U in its tRNA. Assays of cell-free extracts isolated from the iscS −strain confirm the complete loss of tRNA sulfurtransferase activity. In addition to lacking s4U, the iscS −strain requires thiamin and nicotinic acid for growth in minimal media. The thiamin requirement can be relieved by the addition of the thiamin precursor 5-hydroxyethyl-4-methylthiazole, indicating thatiscS is required specifically for thiazole biosynthesis. The growth rate of the iscS − strain is half that of the parent strain in rich medium. When theiscS− strain is switched from rich to minimal medium containing thiamin and nicotinate, growth is preceded by a considerable lag period relative to the parent strain. Addition of isoleucine results in a significant reduction in the duration of this lag phase. To examine the thiazole requirement, we have reconstituted the in vitro biosynthesis of ThiS thiocarboxylate, the ultimate sulfur donor in thiazole biosynthesis, and we show that IscS mobilizes sulfur for transfer to the C-terminal carboxylate of ThiS. ThiI, a known factor involved in both thiazole and s4U synthesis, stimulates this sulfur transfer step by 7-fold. Extracts from the iscS − strain show significantly reduced activity in the in vitro synthesis of ThiS thiocarboxylate. Transformation of the iscS −strain with an iscS expression plasmid complemented all of the observed phenotypic effects of the deletion mutant. Of the remaining two nifS-like genes in E. coli,neither can complement loss of iscS when each is overexpressed in the iscS − strain. Thus, IscS plays a significant and specific role at the top of a potentially broad sulfur transfer cascade that is required for the biosynthesis of thiamin, NAD, Fe-S clusters, and thionucleosides. IscS, a cysteine desulfurase implicated in the repair of Fe-S clusters, was recently shown to act as a sulfurtransferase in the biosynthesis of 4-thiouridine (s4U) in tRNA (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561–16568). In frame deletion of the iscS gene in Escherichia coli results in a mutant strain that lacks s4U in its tRNA. Assays of cell-free extracts isolated from the iscS −strain confirm the complete loss of tRNA sulfurtransferase activity. In addition to lacking s4U, the iscS −strain requires thiamin and nicotinic acid for growth in minimal media. The thiamin requirement can be relieved by the addition of the thiamin precursor 5-hydroxyethyl-4-methylthiazole, indicating thatiscS is required specifically for thiazole biosynthesis. The growth rate of the iscS − strain is half that of the parent strain in rich medium. When theiscS− strain is switched from rich to minimal medium containing thiamin and nicotinate, growth is preceded by a considerable lag period relative to the parent strain. Addition of isoleucine results in a significant reduction in the duration of this lag phase. To examine the thiazole requirement, we have reconstituted the in vitro biosynthesis of ThiS thiocarboxylate, the ultimate sulfur donor in thiazole biosynthesis, and we show that IscS mobilizes sulfur for transfer to the C-terminal carboxylate of ThiS. ThiI, a known factor involved in both thiazole and s4U synthesis, stimulates this sulfur transfer step by 7-fold. Extracts from the iscS − strain show significantly reduced activity in the in vitro synthesis of ThiS thiocarboxylate. Transformation of the iscS −strain with an iscS expression plasmid complemented all of the observed phenotypic effects of the deletion mutant. Of the remaining two nifS-like genes in E. coli,neither can complement loss of iscS when each is overexpressed in the iscS − strain. Thus, IscS plays a significant and specific role at the top of a potentially broad sulfur transfer cascade that is required for the biosynthesis of thiamin, NAD, Fe-S clusters, and thionucleosides. 4-thiouridine ThiS thiocarboxylate sulfane sulfur polyacrylamide gel electrophoresis kilobase pair polymerase chain reaction phenylmethylsulfonyl fluoride high pressure liquid chromatography base pair isopropyl-1-thio-β-d-galactopyranoside Cysteine has been shown to be the source of sulfur for the biosynthesis of a variety of cofactors such as biotin, lipoic acid, and thiamin (1.Begley T.P. Xi J. Kinsland C. Taylor S. McLafferty F. Curr. Opin. Chem. Biol. 1999; 3: 623-629Crossref PubMed Scopus (84) Google Scholar), as well as metallosulfur clusters in proteins (2.Sticht H. Rosch P. Prog. Biophys. Mol. Biol. 1998; 70: 95-136Crossref PubMed Scopus (109) Google Scholar) and thionucleotides in tRNA (3.Ajitkumar P. Cherayil J.D. Microbiol. Rev. 1988; 52: 103-113Crossref PubMed Google Scholar). Despite identification of the source, the biochemical pathways for sulfur incorporation into these molecules remain elusive. A major advance in this area was the report by Dean and co-workers (4.Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Nat. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (498) Google Scholar) that the NifS protein from Azotobacter vinelandii is a cysteine desulfurase that is required for the maintenance of the metallosulfur clusters in nitrogenase. A similar enzyme in Escherichia coli, termed IscS, has been shown to mobilize sulfur from cysteine for the repair of the [4Fe-4S] cluster in apo-dihydroxy-acid dehydratase (5.Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Zheng et al. (6.Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar) have reported that many organisms contain iscShomologs in a gene cluster thought to be involved in the maintenance of [Fe-S] proteins. Unsuccessful attempts to delete iscS inA. vinelandii suggest that it is required for viability in that organism (6.Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). We recently reported that in E. coli, IscS also functions as a sulfurtransferase for the in vitro biosynthesis of 4-thiouridine in tRNA (8.Kambampati R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar). Lipsett and co-workers (9.Lipsett M.N. J. Biol. Chem. 1972; 247: 1458-1461Abstract Full Text PDF PubMed Google Scholar, 10.Abrell J.W. Kaufman E.E. Lipsett M.N. J. Biol. Chem. 1971; 246: 294-301Abstract Full Text PDF PubMed Google Scholar, 11.Lipsett M.N. J. Bacteriol. 1978; 135: 993-997Crossref PubMed Google Scholar) reported that s4U1 was synthesized by two factors, A and C, that were the products of thenuvA and nuvC genes, respectively. Interestingly, the nuvC mutant, which lacked s4U in its tRNA, also required thiamin for growth in minimal media (12.Ryals J. Hsu R.-Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar). ThethiI gene was recently identified as a requirement for both s4U (13.Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (85) Google Scholar) and thiamin synthesis (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) in E. coliand for thiazole biosynthesis in Salmonella typhimurium(14.Webb E. Class K. Downs D.M. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar). Begley and co-workers (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have elegantly shown that in E. coli the ultimate sulfur donor in thiamin biosynthesis is a C-terminal thiocarboxylate of the ThiS protein. Studies with athiI − strain revealed that thiI is required for ThiS-COSH synthesis in vivo (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The shared pathway for s4U and thiamin biosynthesis suggested that ThiI was a sulfurtransferase. However, the factor that initiates the mobilization of sulfur from cysteine for ThiS-COSH synthesis remained unknown (7.Deleted in proofGoogle Scholar). that IscS was to complete s4U biosynthesis in vitro in suggested that it also to sulfur transfer in the synthesis of In the we have reconstituted ThiS thiocarboxylate and we that IscS can sulfur in the synthesis of E. coli genes, with to Thus, it is that the in vitro observed for IscS of its role in In to for vivo of iscS in s4U, thiamin, and Fe-S cluster biosynthesis, we the in E. E. coli strain was from the E. coli E. coli was from the E. coli iscS gene into as (8.Kambampati R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar). media was to with media were with and in addition to the as nicotinic acid, and isoleucine were at a of respectively. and its were in containing was isolated by the J. Nucleic Acids Res. PubMed Scopus Google Scholar). was isolated a was with a a by the specific for E. were a of the were by the 1978; PubMed Scopus Google Scholar). ThiI was from an strain as (8.Kambampati R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar). E. was by in vitro of plasmid (8.Kambampati R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar) and by electrophoresis gel as of E. coli each of the and the iscS In of the iscS the plasmid a by the of for the of deletion in E. coli J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar). The deletion was to the reported J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar). were as suggested by of the E. coli with the of the which was to to the which we and (5.Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar, K. 1997; PubMed Scopus Google Scholar) have identified by of E. coli as a were the and and the the and The of the was the were in to for the of each reaction was in a containing the and and the at was a gel by with a The was and gel was into the of and by into E. and in this from et al. containing iscS deletion containing E. coli and Lauhon and the is shown in and in a and the is shown in and A of the by and co-workers P. J. Bacteriol. PubMed Google Scholar) and and co-workers J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar) was to of mutant that in rich media. of plasmid into E. coli the were with of medium and to with at for A of of the were to and and were two at were observed the many the The was to be The two were in for by into media. The were and at to was isolated from of a of and were to have the gene the indicating a gene of these were into of and for plasmid A of the was an to that plasmid and were were in of for of of the was that the iscS of − were as A of the iscS was in of to A was into of and was by at in minimal media was in the as when from rich to minimal media were by of the parent in medium into minimal medium containing as tRNA sulfurtransferase activity for s4U was as (8.Kambampati R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar). The reaction vitro E. coli of ThiI, and of cell-free from parent − strain. tRNA was for s4U by with and to et J. 1982; PubMed Scopus Google Scholar). containing tRNA, and of were at for To this for a was by of and for of tRNA was a and with a of in were at which is specific for the of Begley and co-workers (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have reported the of a expression plasmid and of these proteins as a We and ThiS by from E. coli for and ThiS in reaction of each of each and of of the was a gel and a The was and into and were into for the of plasmid for by the were into for protein were at to of and was to the were by and with in at for by with for at for the was to a with The was with of and the protein was with a of in of of the were to was by gel with were and at ThiS was to a and as The was by gel as ThiS were and a and at ThiS ThiI IscS and were as in the were in of in the of were for at of was to the at for and to electrophoresis polyacrylamide were by by The in vitro ThiS were as cysteine was were a liquid was The were acid in and in A from to rate was for The with an source was for were the The in the is We a for the in frame deletion of E. coli genes that was by and co-workers P. J. Bacteriol. PubMed Google Scholar) and recently by and co-workers J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar). The deletion was a which the of iscS with a that for an J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar). in frame is to effects genes the is it is known to be in a (6.Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). The iscS deletion was into plasmid J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar), which a of that the of with plasmid into the at the in the of of isolated at the in the of in which the plasmid has been from the also gene for gene is for growth in E. coli in the of high Thus, it is to for that have both and the plasmid from by at from the that in the of and have the deletion by can that contain the deletion in the However, we have that deletion that in relative to the parent strain be at this and the of many the mutant is was for the iscS − which in rich medium. We it to the by at for in and and the plasmid of for the of the have the deletion in of the and these a of the deletion of plasmid such required an to the shown in we the gene deletion by of with the iscS the of the parent strain and the for the iscS deletion strain In with iscS a that was from the parent strain. A the growth of − and E. coli parent in medium at The iscS − strain was significantly in rich medium. The of the strain is which is that of the parent strain strain in minimal media with strain is a of (12.Ryals J. Hsu R.-Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar, E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (85) Google Scholar, E. Class K. Downs D.M. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar) the s4U and thiamin biosynthesis and we that thiamin was also for However, addition of thiamin to the minimal medium was for addition of nicotinic acid and thiamin was growth A significant lag relative to the parent strain was observed when the iscS − strain was switched from rich to minimal medium containing thiamin and Flint (5.Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) has reported that IscS can repair the Fe-S clusters of apo-dihydroxy-acid an enzyme required for the biosynthesis of isoleucine and we the duration of the lag in the of these that the lag relative to the parent strain is reduced in the of isoleucine and is reduced in the of both isoleucine and that an enzyme involved in biosynthesis, is in the iscS − strain. acid Interestingly, the of growth the for both parent and mutant all minimal medium it be that this is a rate of growth in rich medium. Transformation of the iscS − with a IscS expression in of all of the observed phenotypic effects The growth rate of was to the parent in rich medium in the of indicating that the expression of iscS in the is gel of extracts from − strain in the of a at the relative of IscS was also complemented by a of that the E. coli iscS gene with the as in the deletion Extracts from the parent and iscS were for tRNA sulfurtransferase activity. − significantly reduced activity of parent strain tRNA isolated from theiscS− strain in was for the of The of tRNA coli a to of s4U at for the tRNA is of a in this with which the in this of the tRNA To confirm the of s4U, tRNA isolated from each strain was to as J. 1982; PubMed Scopus Google Scholar). of the tRNA that − strain is of s4U tRNA from the complemented strain a for s4U in the that is similar in to that of the parent strain results show thatiscS is required for the biosynthesis of proteins were as and a protein of as a we the of the of proteins as by were to the report by Begley and co-workers (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), we have and ThiS as by Begley and co-workers (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have shown that the ultimate sulfur donor in the biosynthesis of thiazole is reconstituted ThiS-COSH biosynthesis and and an factor in E. coli T.P. Downs D.M. McLafferty F.W. Taylor S. Chiu Kinsland C. Xi J. Microbiol. 1999; PubMed Scopus Google Scholar). We to the in vitro reaction by and IscS to the the and of to the for ThiS-COSH in Addition of IscS to results in the transfer of from to ThiS In the of this sulfur transfer was reduced by C, is in with the report that requires for of ThiS (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Interestingly, the of ThiI is required in ThiI is to ThiS-COSH Begley and co-workers also reported in to the in vivo of ThiI is required for the in vitro reaction T.P. Downs D.M. McLafferty F.W. Taylor S. Chiu Kinsland C. Xi J. Microbiol. 1999; PubMed Scopus Google Scholar), its addition its We also observed that both IscS and ThiI with the in of ThiS-COSH of ThiI that IscS mobilizes from and it to ThiI ThiS-COSH revealed that addition of ThiI to the in vitro reaction results in the loss of from IscS by with that ThiI stimulates reduction of IscS we have reported R. Lauhon C.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that addition of ThiI the cysteine desulfurase activity of IscS by that IscS initiates sulfur transfer for ThiS-COSH synthesis is by liquid A ThiS with observed of which is of the of (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). the of ThiS reaction with IscS, and the and A with of was which is with the for However, with of and were also the reaction in the of a with ThiS-COSH is observed which is the ThiS-COSH in with and also relative to the reaction containing ThiI the complete reaction in the of cysteine results in the of the with and that these products in with ThiS-COSH and by reaction with we have identified these However, the observed with and the transfer of from to ThiS shown in the gel that ThiS-COSH is Extracts of the parent and iscS E. coli were for the to ThiS-COSH in the of all the IscS shown in the parent strain transfer from to ThiS. − strain significantly reduced sulfurtransferase activity of the ThiS-COSH by − a of that of The cysteine of the NifS for sulfur and in the IscS, an NifS was isolated from E. coli was shown to cysteine into and sulfane sulfur in the of a cysteine in its (5.Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). nifS-like genes have been in to contain which in many organisms is to the gene has to E. coli iscS at the acid its the and at in to the P. C. R. J. 1999; PubMed Scopus Google Scholar, T. Mol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), which is the of many Fe-S in is required for viability and was recently shown to be required for the in vivo activity of many Fe-S proteins and for the of J. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). that IscS can sulfur for s4U biosynthesis to tRNA (8.Kambampati R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar, R. Lauhon C.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to it for a role in sulfur it has been shown that thiI is required for the biosynthesis of both s4U and thiazole (13.Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (85) Google Scholar, E. Class K. Downs D.M. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar, S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), we that iscS also be required for thiazole biosynthesis. an in frame gene deletion we have the iscS gene and confirm that iscS is required for both s4U and thiazole biosynthesis in To the biochemical step for the thiamin we an in vitro for ThiS-COSH biosynthesis, the transfer of from to ThiS. The minimal for activity IscS, and ThiI is required in we that it stimulates the of an that be for of ThiS-COSH in We have recently shown for the transfer of from IscS to ThiI and to tRNA the biosynthesis of s4U R. Lauhon C.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). shown in this suggest that a similar pathway is in the biosynthesis of However, significant the two ThiS-COSH synthesis requires by (16.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and ThiI to act as a sulfurtransferase. in s4U synthesis, ThiI sulfur from IscS and also the the tRNA and the sulfur to We have for the of s4U biosynthesis by have we observed transfer of to ThiS-COSH is that a is a the and is the results suggest that ThiI is the ultimate sulfurtransferase for both s4U and ThiS-COSH the two pathways the step is the mobilization of sulfur from by IscS, in the of an cysteine is from to ThiI to a ThiI is from and to the ThiS to the We have that can be by of the that is in the for addition to an We have shown that the acid can the tRNA sulfurtransferase activity of ThiI R. Lauhon C.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, of the cysteine in ThiI is required for s4U synthesis, which is with the in Flint (5.Flint D.H. J. Biol. 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PubMed Google Scholar), and which is required for the synthesis of the in J. Bacteriol. 1999; PubMed Google Scholar). We the role of iscS in the biosynthesis of each of these at of the of sulfur transfer as well as the of the transfer of this Palenchar et al. P.M. Buck C.J. H. E.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (136) Google Scholar) that of of ThiI tRNA sulfurtransferase activity and the synthesis of We have also recently of two that have isolated iscS and report that with and We of for We also Downs and for of results to