Structural Alterations of the Cysteine Desulfurase IscS of Salmonella enterica Serovar Typhimurium Reveal Substrate Specificity of IscS in t
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《细菌学杂志》
Department of Molecular Biology, Ume University, S-901 87 Ume, Sweden
ABSTRACT
The cysteine desulfurase IscS in Salmonella enterica serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s4U) and 5-methylaminomethyl-2-thiouridine (mnm5s2U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s2C) and N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm5s2U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms2io6A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms2io6A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.
INTRODUCTION
Many nucleic acids contain, besides the four major nucleosides adenosine (A), guanosine (G), uridine (U), and cytidine (C), a variety of modified nucleosides, which are derivatives of these major nucleosides (reviewed in reference 5). One group of such modified nucleosides is the thiolated nucleosides, which are ubiquitously present in tRNAs from the three phylogenetic domains Bacteria, Eucarya, and Archaea (2, 30). tRNA from Salmonella enterica serovar Typhimurium contains six thiolated nucleosides: 2-thiocytidine (s2C), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 4-thiouridine (s4U), N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), and N6-(isopentenyl)-2-thioadenosine (ms2i6A). The locations of these thiolated nucleosides and their structures are shown in Fig. 1. All thiolated nucleosides except s4U8 are present in the anticodon loop, consistent with their importance in the fidelity and efficiency of decoding of the message (reviewed in reference 5).
The synthesis of all thiolated nucleosides in tRNA depends on IscS (23, 34). The iscS structural gene is part of the isc operon, which consists of eight phylogenetically conserved genes (iscR, iscS, iscU, iscA, hscA, hscB, fdx, and orf3) (43, 49). The IscR protein senses the status of the iron-sulfur cluster (designated the [Fe-S] cluster irrespective of which type of iron-sulfur cluster is present in a protein) in the cell and thereby regulates the expression of the isc operon (40). IscS is a pyridoxal-phosphate-dependent enzyme that mobilizes sulfur through desulfurization of cysteine to yield alanine and an IscS-bound persulfide. IscU, which is a scaffold protein in the [Fe-S] cluster assembly, accepts sulfur from IscS and in turn delivers it as an [Fe-S] cluster to the target apoprotein (1, 12, 42). IscA, which is an alternative scaffold protein in the [Fe-S] cluster assembly, may provide iron for the [Fe-S] cluster formed in IscU (9). HscA and HscB (for heat shock cognate) are chaperons apparently associated only with IscS-directed [Fe-S] cluster assembly (15). Fdx (ferredoxin), which is an iron-sulfur protein, is involved in electron transfer and complexes with IscA (35; for recent reviews of the Isc system, see references 3 and 18).
The sulfur relay in the synthesis of thiolated nucleosides is summarized in Fig. 2. ThiI transfers sulfur directly from IscS to the target nucleoside (U8) in tRNA (19, 33). A similar sulfur transfer reaction was suggested for the synthesis of the s2 group of (c)mnm5s2U34 (mnm5s2U and cmnm5s2U), in which IscS transfers the sulfur to TusA, which in turn delivers it to TusD in the TusBCD complex. TusE may accept sulfur from TusD and deliver it to MnmA, which transfers it to U8 in tRNA (16, 21). Thus, in these two cases, the IscS persulfide donates its sulfur directly to a protein [ThiI in the synthesis of s4U8 and TusA in the case of (c)mnm5s2U34], and no [Fe-S] protein is thought to participate in the synthesis of these two thiolated nucleosides (designated the [Fe-S] protein-independent pathways). TtcA is required for the synthesis of s2C32 (17), and since it contains several cysteines it may be an [Fe-S] protein, which has recently been demonstrated (Marc Fontecave and Mohamed Atta [Grenoble, France], personal communication). The MiaB protein, which belongs to the SAM-radical family of enzymes, is an [Fe-S] protein (36), and apparently it can alone catalyze the formation of the ms2 group of ms2i(o)6A37 (ms2i6A and ms2io6A), albeit at a low efficiency (37). The presence of IscU, HscA, or Fdx is required in the synthesis of s2C32 and ms2i(o)6A37 but not in the synthesis of s4U8 and (c)mnm5s2U34 (26). Therefore, it was suggested that in the synthesis of s2C32 and ms2i(o)6A37, an [Fe-S] protein(s) participates. Thus, two distinct pathways for thiolation of tRNA exist. In the [Fe-S] protein-independent pathway, which is responsible for the synthesis of s4U8 and (c)mnm5s2U34 (pathway II in Fig. 2), there is a direct transfer of sulfur from IscS to a protein which in turn transfers the sulfur to the target nucleoside in tRNA for the synthesis of s4U or to another protein in the sulfur relay for the synthesis of (c)mnm5s2U. In the [Fe-S] protein-dependent pathway, which is responsible for the synthesis of s2C32 and ms2i(o)6A37 (pathway III in Fig. 2), an [Fe-S] protein(s) participates, and its formation and maintenance are dependent on IscS (26). In the latter two cases, the sulfur is transferred from IscS to IscU, and IscA, HscA, HscB, and Fdx participate in a complex way, not so far unraveled in detail, to form [Fe-S] cluster-loaded IscU, which then transfers the cluster to the receiving apoprotein (18) (Fig. 2). A lack of any of these participating proteins except IscA almost abolishes the synthesis of s2C32 and ms2io6A37. Apparently, to optimize the formation of ms2io6A, the presence of IscA is required, since the lack of it reduces the formation of only this thiolated nucleoside (26). Since this model of two distinct pathways in the thiolation of tRNA was based on the analysis of deletions of various genes involved in thiolation of tRNA, a more elaborate pathway may exist if proteins interact in a specific manner through various protein-protein complex formations. This paper addresses this question by analyzing mutants with point mutations in iscS selected for deficiency in the synthesis of one of the end products, (c)mnm5s2U34 or ms2i(o)6A37, of the two pathways. We found that some amino acid substitutions in IscS affected the synthesis of only a subset of the thiolated nucleosides, suggesting substrate specificity of IscS in the thiolation of tRNA (Fig. 2).
MATERIALS AND METHODS
Bacteria and growth conditions. The bacterial strains used were either derivatives of S. enterica serovar Typhimurium (Table 1) or Escherichia coli. Bacteria were grown in Luria-Bertani (LB) rich liquid medium (4) or on LA plates (LB medium with 15 g/liter agar added). The minimal media were made from the basal medium E (47) supplemented with 0.2% glucose and required amino acids or vitamins at concentrations recommended previously (8). Minimal medium E agar plates were made by adding 15 g of agar per liter of culture.
Frameshift mutants used for selecting frameshift suppressors. We used two different constructs to measure the effect of hypomodification on frameshifting. Both constructs contain the hisO1242 mutation, which increases the expression of the his operon and thereby enhances the synthesis of the frameshifting product, making the system very sensitive to detect low degree of frameshifting. The first construct contains the hisC3737 mutation, which is a C insertion in the hisC gene, resulting in the sequence CCC-CAA-UAA (the inserted C underlined). The insertion results in a shift in the reading frame. To be able to make a functional HisC protein, a +1 frameshift must occur before the newly created UAA stop codon in the zero reading frame, thus enabling bypassing of the stop codon and reentrance into the zero reading frame. Such suppression of the hisC3737 mutation results in a functional HisC and growth without externally added histidine. The frameshift window in the hisC3737 mutant is fairly large (GUA-GAG-CGC-CGG-ACG-GUU-CCC-GCG-CUU-GAA-AAC-UGG-CAG- CUG-GAU-CUA-CAG-GGG-AUU-UCC-GAC-AAC-CUU-GAC-GGC-ACA-AAA-GUG-UUC-GUU-UGU-AGC-CCC-CAA-UAA; the underlined UAG is the stop codon in the +1 frame, and the underlined CCC-CAA-UAA is the potential frameshift site with the UAA stop codon in the zero reading frame). The second construct contains, in addition to the hisO1242 mutation, the hisD10114 mutation, which is a +1 frameshift mutation in the hisD gene which must be suppressed for the strain to grow without externally added histidine. The potential frameshifting site is UUU-UAU-UAA, and the frameshifting window is only eight codons (CUG-AUU-GAC-UGG-AAC-AGC-UGU-AGC-UUU-UAU-UAA; the underlined UGA is the stop codon in the +1 frame, and the underlined UUU-UAU-UAA is the potential frameshifting site constructed with the UAA stop codon in the zero frame).
Genetic procedures. Transduction with phage P22 HT105/1 (int-201) (39) was performed as described previously (8). Mutagenesis was performed using overexpression of the DinB protein (22, 48) regulated by an araC promoter harbored on a plasmid kindly supplied by Dan Andersson, Uppsala, Sweden (28).
Isolation of mutants. A crystal of nitrosoguanidine was placed on an LA rich agar plate seeded with strain GT6430 (STM2545-2525::Tn10dTc). The Tn10dTc insertion is only 2 kb away from the isc operon, and thus both the transposon and the isc operon can be present on the same DNA fragment of one P22 phage. After incubation overnight, a clear zone where no cells were growing appeared around the crystal. Cells from the area just outside the clear zone were collected, transferred to 1 ml of LB medium, and incubated for 2 h. Phage P22 was grown on such a culture and used to transduce either strain GT853 (hisC3737) or strain GT6942 (hisD10114). Five milliliters of overnight culture of the recipient cells was incubated with phage P22 for 30 min at 37°C. Cells were collected by centrifugation and washed twice with 0.9% NaCl containing 10 mM EGTA to remove unabsorbed phages before the culture was plated on selective plates. Tcr His+ transductants were selected on agar plates containing either medium E, 0.2% glucose, and 2.5 μg/ml tetracycline (Tc) or medium E, 0.2% glucose, thiamine, nicotinic acid, and 2.5 μg/ml Tc. Thiamine and nicotinic acid were included, since some mutations in iscS induce auxotrophy for these metabolites (24). Sometimes salt medium M9 (31) was used instead of medium E, with no discernible difference. The plates were incubated at 37°C for up to 7 days. The Tcr His+ transductants were purified on LA plates containing 10 mM EGTA. Phage P22 was grown on the purified transductants and backcrossed to the parent strain to obtain mutants, which did not contain mutations outside the isc region. The cross was performed on LA plates containing 15 μg/ml Tc and 10 mM EGTA, followed by replica printing on medium E plates containing 0.2% glucose with or without histidine. The cotransduction frequency between Tcr and His+ phenotypes was such that the His+ phenotype could be caused by a mutation in iscS (data not shown). One Tcr His+ transductant from each backcross was saved for further analysis. Mutational siblings were eliminated by comparing similarities in growth/suppression phenotype and/or high-pressure liquid chromatography (HPLC) analysis. Finally, mutant strains were complemented with the piscS plasmid, harboring only the wild-type allele of the iscS+ gene (41), and chromosomal sequencing of the iscS gene in the mutants was performed.
To isolate DinB-induced Tcr His+ mutants, an overnight culture of strain GT7172 (STM2545-2525::Tn10dTc/pSMP24; pSMP24 contains the dinB gene controlled by an arabinose promoter) (28) was diluted 1,000-fold in LB medium containing 0.2% arabinose and allowed to grow for 8 h, after which phage P22 was added to the culture and the phage-bacterium mixture was incubated overnight in a rotary shaker. The bacteria were removed by centrifugation, and the supernatant was treated with chloroform. Such phage stocks were used to select frameshift suppressors as described above.
DNA sequencing. Sequencing was performed on chromosomal DNA or PCR products as described in the manual for the Applied Biosystems ABI PRISM Cycle Sequencing Ready Reaction Big Dye Kit (Applied Biosystems, Foster City, California).
Analysis of modified nucleosides in tRNA. Bacterial strains were grown in LB medium (4) at 37°C to about 4 x 108 to 6 x 108 cells/ml. Cells were lysed, and total RNA was prepared (10). Total RNA was dissolved in buffer R200 (10 mM Tris-H3PO4, pH 6.3; 15% ethanol; 200 mM KCl) and applied to a Nucleobond column (AX500) equilibrated with the same buffer. tRNA was eluted with 7 ml of buffer R600 (R200 but containing 600 mM KCl), precipitated with 2.5 volumes of cold ethanol, washed twice with 80% ethanol, and dried. The dried tRNA was dissolved in water, and a portion of it was degraded to nucleosides by nuclease P1 followed by treatment with bacterial alkaline phosphatase (14). The hydrolysate was analyzed by HPLC (13) using a C18 HPLC column (250 by 4.6 mm; Phenomex Ltd., MacClesfield, Cheshire, United Kingdom).
Western blotting. Total protein was prepared from cells grown to a cell density of 5 x 108 cells/ml in LB medium by sonicating the cells using a VCX400 Vibracell sonicator (Sonics Inc). The cell debris was removed by centrifugation at 3,000 x g for 10 min. The supernatant was diluted with 1 ml of water, total protein was determined using a protein assay kit (Bio-Rad Inc., Hercules, California), and 25 μg was applied in each well on a 12% sodium dodecyl sulfate-polyacrylamide gel. Proteins were separated using the Mini-Protean II protein electrophoresis system (Bio-Rad Inc., Hercules, California). Two gels were used in parallel experiments, with equal amounts of proteins from the same preparation applied on each gel. One gel was used for TrmD as a control and the other for IscS determinations. Proteins were blotted to Amersham Hybond C membranes using the wet blot transfer system Mini Trans blot cell (Bio-Rad Inc.). Blocking was performed with the ECL blocking agent (Amersham Life Sciences, GE Healthcare, Uppsala, Sweden). Primary antibodies against IscS, kindly provided by Larry E. Vickery (Irvine, California), and TrmD (27) were added, followed by incubation with the secondary antibody according to the protocol supplied with the ECL Plus kit (Amersham Life Sciences, GE Healthcare, Uppsala, Sweden). ECL detection was performed in a Fluor-S Max2 Lumi-imager (Bio-Rad Inc., Hercules, California).
RESULTS
Strategy for isolation of IscS mutants defective in either of the two tRNA thiolation pathways. Two of the thiolated nucleosides, (c)mnm5s2U34 and ms2i(o)6A37, are present in different tRNAs reading different codons and improve reading frame maintenance, since the lack of either of them induces codon-specific frameshifts (46). We have utilized this fact to select for IscS mutants deficient in either (c)mnm5s2U34 or ms2i(o)6A37. According to our working model (Fig. 2), such IscS mutants were expected to be deficient not only in (c)mnm5s2U34 or ms2i(o)6A37 but also in other thiolated nucleosides, since IscS is pivotal for the synthesis of all thiolated nucleosides (23, 34). At the sequence CCC-CAA-UAA (the sequence written in the zero frame, and a +1 frameshift must occur before the ribosome reaches the stop codon UAA), which is found in the hisC gene in a strain containing the hisC3737 mutation, the CAA codon is read by tRNAcmnm5s2UUGGln, which contains as the wobble nucleoside (c)mnm5s2U34 (6). At the sequence CCC-CAA-UAA we anticipated that a thiolation deficiency of cmnm5s2U34 would suppress the +1 frameshift mutation hisC3737, resulting in growth in the absence of histidine (a His+ phenotype) (Fig. 3A). Our strategy was to select His+ clones of the histidine-requiring hisC3737 mutant following localized mutagenesis of the iscS gene. Mutations in iscS should reduce the thiolation of the wobble nucleoside (c)mnm5s2U34 and thereby increase frameshifting.
At the sequence UUU-UAU-UAA, codons UUU and UAU are read by tRNAGAAPhe and tRNAQUATyr, respectively. A lack of the ms2 group in either of these tRNAs causes frameshifting (46) (Fig. 3B). Hence, mutants defective in the synthesis of the ms2 group of ms2i(o)6A37 in both tRNAGAAPhe and tRNAQUATyr should suppress the UUU-UAU-UAA site placed early in the hisD gene. Only mutations in iscS were characterized in this work, although iscU, iscA, hscA, or fdx mutants will also be found with this method, since these genes affect the synthesis of ms2i(o)6A (26).
To test our strategy, we introduced mutations in mnmA or miaB, which encode thiolating enzymes required for the synthesis of (c)mnm5s2U34 and ms2io6A37, respectively, into strains with one of the two different frameshifts sites hisC3737 and hisD10114. Whereas the mnmA1 mutation suppressed only the hisC3737 (CCC-CAA-UAA) mutation, the miaB1 mutation suppressed only the hisD10114 (UUU-UAU-UAA) mutation (data not shown). Thus, we anticipated that iscS mutations suppressing hisC3737 would concomitantly cause deficiency in at least (c)mnm5s2U34, whereas iscS mutations suppressing hisD10114 would cause deficiency in at least ms2i(o)6A37. Since mutations in thiI or ttcA, whose products are required for the synthesis of s4U8 and s2C32 on the tRNA, respectively, did not suppress any of these two +1 frameshift mutations (data not shown), we did not anticipate obtaining iscS mutants deficient in only either of these thiolated nucleosides. Thus, suppression of the two frameshift mutations hisC3737 and hisD10114 requires deficiency of only (c)mnm5s2U34 and only ms2i(o)6A37, respectively.
Isolation, identification, and characterization of iscS mutations found to cause defects in either of the two tRNA thiolation pathways. Strain GT6430, which has a Tn10dTc inserted just upstream of the isc operon, was mutagenized either by DinB overexpression or by treatment with nitrosoguanidine. Phage P22 was grown on such mutagenized cultures and used to infect strain GT853 (hisC3737; CCC-CAA-UAA) or strain GT6942 (hisD10114; UUU-UAU-UAA). Tcr His+ transductants were selected and backcrossed to the original parent strains to avoid mutants with compensatory mutations in the his operon. However, the frequency of such mutations ought to be low, since the his operon is located far from the isc operon. This approach should also avoid any mutations in the structural genes whose products are required for the synthesis of thiolated nucleosides, such as thiI (s4U8), mnmA [(c)mnm5s2U34], or miaB [ms2i(o)6A37], since their chromosomal locations are far from the isc operon. The mutations in the various His+ mutants characterized were all located in the iscS gene, since cotransduction frequencies between the Tcr and His+ phenotypes were such that the His+ phenotype could be caused by a mutation in iscS and since introduction of a plasmid carrying the wild-type allele of iscS complemented the mutant phenotypes (His+ and the deficiency in thiolation of tRNA) (Tables 2 and 3). The mutations were identified by sequencing of the iscS genes from the various mutants. Thus, the induced phenotype must be caused by the alteration in IscS. tRNA was isolated and degraded to nucleosides, and the distribution and quantification of nucleosides were determined by HPLC (Fig. 4; Tables 2 and 3).
Using the frameshifting site hisC3737 (CCC-CAA-UAA), we obtained, as expected, class I mutants (iscS56, -60, and -61) with reduced levels of all thiolated nucleosides (Table 2), similar to the case for the earlier characterized iscS51 mutation (34) and to that for a deletion of iscS (23). However, we also obtained iscS55 and iscS57 mutants with much reduced levels of (c)mnm5s2U34 and s4U8 and almost wild-type (iscS55) or wild-type (iscS57) levels of s2C32 and ms2i(o)6A37 (designated class II mutants). Apparently, alterations in IscS affected mainly the [Fe-S] protein-independent pathway and not at all (iscS57) or weakly (iscS55) the [Fe-S] protein-dependent pathway (Fig. 2). Moreover, we also obtained another mutant (iscS59) with a reduced level of only (c)mnm5s2U34 and normal levels of all the other thiolated nucleosides (class IIB mutant) (Table 2). In some way an alteration in IscS affected the mobilization of sulfur to only one of the enzymes required for the synthesis of the four thiolated derivatives in tRNA. A mirror image of the iscS59 mutation was the iscS64 mutation, obtained using the hisD10114 (UUU-UAU-UAA) frameshifting site, since it had a reduced synthesis of all thiolated nucleosides except (c)mnm5s2U (a class III and IIA mutant) (Table 3). Thus, in addition to class I mutants, we also isolated iscS mutants defective in the synthesis of a subset of the thiolated nucleosides.
One suppressor mutation, iscS58, of the hisD10114 (UUU-UAU-UAA) mutation was deficient in all thiolated nucleosides (Table 3). However, by using this frameshifting site we also obtained mutants defective in the synthesis of one or more of the thiolated nucleosides, such as the class IIIB mutants carrying iscS63, -65, and -66 [deficient in only ms2i(o)6A] and iscS64 [deficient in all thiolated nucleosides except (c)mnm5s2U34] (a class IIA and III mutant) (Table 2). The iscS65 and -66 mutants had a phenotype reminiscent of that of a mutant deleted for iscA, which is also deficient in only ms2i(o)6A37 (26). Note also that the iscS65 and -66 mutations, as well as deletion of iscA, reduced ms2i(o)6A to a similar level (about 50% of the wild-type level). However, the iscS65 and -66 mutants have a wild-type allele of the iscA gene as determined by DNA sequencing (data not shown), and these mutants had only a mutation in the iscS gene, because the wild-type phenotype was restored by introducing the wild-type allele of iscS+ on a plasmid (Table 3). Both mutants contained an alteration in the C-terminal part of the IscS protein. The iscS66 mutation was a frameshift mutation in codon 380 resulting in a truncated IscS. The iscS65 mutation changed His350 to Arg350.
Some amino acid substitutions may reduce the stability of the IscS protein, which would result in a lower activity in the cell and thereby lower synthesis of all thiolated nucleosides. We therefore monitored the levels of the IscS protein in the various mutants (Fig. 5). The levels of full-size IscS in the iscS56 and -58 mutants were significantly lower than that in the wild type, and, accordingly, these mutations reduced the levels of all thiolated nucleosides (Tables 2 and 3; Fig. 5). All other mutations did not reduce the level of IscS, which is why the phenotype mediated by these mutations must be caused by the activity of IscS and not by a lower level of the protein. The isc operon is controlled by IscR, which senses the [Fe-S] status of the cell (40). Thus, a nonfunctional IscS, which should decrease the [Fe-S] content of the cell, ought to derepress the isc operon, as was observed for some mutants (the iscS55, iscS57, iscS59, and iscS60 mutants) (Fig. 5).
In summary (Table 4), we obtained mutants with altered IscS that reduced the synthesis of all thiolated nucleosides (class I mutants), irrespective of which frameshifting site we used to select the suppressor mutants (iscS51, -56, -58, -60 and -61) (Table 3). For two of them (iscS56 and -58), the low level of full-size IscS (Fig. 5) could explain the deficiency of all four thiolated nucleosides. The phenotype mediated by the isc51 and -61 mutations, which did not reduce the level of IscS, may suggest a reduction of the desulfurase activity of IscS, since the synthesis of all thiolated nucleosides was reduced by these mutations. We also found mutants that were defective in the synthesis of one, two, or three of the thiolated nucleosides (the iscS55, -57, -59, -63, -64, -65, and -66 mutants) (Table 4). Class II mutants (iscS55 and iscS57), which were defective in the synthesis of both s4U and (c)mnm5s2U34, were apparently defective in sulfur mobilization from IscS to ThiI and to TusA, respectively, suggesting that a structural change in IscS influences its interaction with ThiI and TusA in a similar way. We obtained a mutant (iscS59) that was deficient in only (c)mnm5s2U (class IIB), indicating a specific defect in the interaction with TusA. No class III mutants were found, although one would think that an alteration in IscS could influence its interaction with IscU, plausibly resulting in a deficiency in s2C32 and ms2i(o)6A37 but not in s4U8 and (c)mnm5s2U34 (26). However, we obtained three mutants deficient in only ms2i(o)6A37 (iscS63, -65, and -66; class IIIB). A mutant with a more complex defect in the thiolation pattern was also obtained, and this mutant was apparently simultaneously defective in both the [Fe-S] protein-independent and -dependent pathways (iscS64; class IIB and class III) (Table 3). Apparently, different alterations in IscS affect sulfur trafficking in specific ways, suggesting that IscS does not mobilize sulfur unspecifically in the thiolation of tRNA.
Growth and suppression levels. Mutants having a low level of (c)mnm5s2U34 showed the largest reduction in growth (Table 4), whereas those deficient in only ms2i(o)6A37 showed no reduction in growth compared to the wild type. This growth behavior was expected, since the lack of only ms2i(o)6A37, only s2C32, or only s4U8 does not result in any growth defect in rich medium (11, 17, 38), whereas the lack of (c)mnm5s2U34 severely reduces the growth rate (34). Thus, the growth in rich medium is consistent with the tRNA thiolation deficiency in the various mutants (Table 4). The levels of suppression of the His– phenotype is also consistent with the reduction in the levels of the relevant thiolated nucleosides.
DISCUSSION
Using a genetic selection for iscS mutants with a defect in the synthesis of either only (c)mnm5s2U34 or only ms2i(o)6A37 in their tRNAs, we obtained, as expected, mutants that were defective in the synthesis of all thiolated nucleosides. We also isolated iscS mutants with defects in the synthesis of one or groups of two or three of the four thiolated nucleosides, suggesting that there is specificity in the way IscS transfers sulfur in the [Fe-S] protein-independent and -dependent pathways (Fig. 2).
The selection of the mutants and analysis of the thiolation level of the tRNA were done with the iscS+ gene present at its wild-type location on the chromosome. Thus, potential multicopy artifacts, which may be introduced if the IscS protein is encoded from a plasmid, or regulatory artifacts caused by misplacement of the iscS gene on the chromosome were avoided. Therefore, we believe that our results obtained from the analyses of the various mutants are true reflections of the in vivo kinetics of the synthesis of the thiolated nucleosides and thus a reflection of the efficiency of sulfur transfer and of synthesis and maintenance of [Fe-S] cluster proteins participating in the thiolation of tRNA.
Deletion of the iscS gene abolishes the synthesis of all thiolated nucleosides (23). Since iscS58 is a frameshift mutation early in the iscS gene (Table 4), we expected that this mutation also should mediate a similar phenotype. However, this was not the case, since this mutant still had significant residual levels of all thiolated nucleosides (Table 3) and, accordingly, a low level of full-size IscS was observed (Fig. 5). The explanation might be that the deficiencies of both (c)mnm5s2U34 and ms2i(o)6A37 in this mutant also mediate suppression of the iscS58 frameshift mutation per se by hypomodified tRNA(s) resulting in a small level of active IscS protein. Since the iscS mRNA might also be overexpressed because of a nonfunctional IscR (40), the level of a possible frameshift product of IscS would be elevated, explaining the observed phenotypes (Table 3; Fig. 5). There are several codons in the frameshifting window created by the iscS58 mutation that would potentially be suppressed by these hypomodified tRNAs.
Five mutations, i.e., iscS51, -56, -58, -60, and -61, reduced the synthesis of all thiolated nucleosides (Tables 2, 3, and 4). For the iscS56 and -58 mutants the low levels of all thiolated nucleosides are explained by the apparent instability of the IscS protein in these mutants (Fig. 5). The iscS51 (A327V) mutation results in an amino acid substitution next to the catalytic Cys328 present in the active-site loop region. The iscS51 mutation, which does not affect the stability of IscS (Fig. 5), abolishes the synthesis of all thiolated nucleosides (34). The phenotype caused by the A327V substitution is likely due to an activity change due to its close proximity to the catalytic Cys328. The A327V substitution might change the flexibility of the active loop (25, 45), resulting in a conformational change in the neighborhood of the catalytic Cys328 that gives a lower desulfurase activity. The iscS61 (M169V) mutation, which, like iscS51, does not affect the stability of IscS (Fig. 5), also results in a deficiency of all thiolated nucleosides, and it is located far from the catalytic Cys328 (Table 4). Therefore, we suggest that the M169V alteration influences the desulfurase activity in a way similar to that of the iscS51 mutation and not by affecting the stability of IscS. A mutation (iscS63) resulting in an alteration (V153W) rather close to the iscS61 (M169V) mutation did not reduce the levels of all thiolated nucleosides as the latter mutation did, and, accordingly, the level of IscS was not reduced (Tables 3 and 4; Fig. 5). The V153W alteration only slightly affected the [Fe-S] protein-dependent pathway [ms2i(o)6A] (Table 3), indicating that the synthesis of this thiolated nucleoside is especially sensitive to small aberrations in the desulfurase activity and/or iron mobilization.
Lauhon et al. (25) generated IscS mutants by alanine scanning mutagenesis from amino acid Ser323 to amino acid Ser336 in the active-site loop. Indeed, a C328A alteration abolishes the synthesis of all thiolated nucleosides, consistent with the suggested pivotal role of Cys328 in the function of IscS. Of the other 13 alterations, 11 did not influence the thiolation of tRNA, and the two alterations (S326A and L333A) which influence thiolation of tRNA do so in a specific way, since they reduce the synthesis of only s2C32 and ms2i6A37; i.e., they affect only the [Fe-S] protein-dependent pathway (class III mutants). Note that the Ser326 is part of the consensus sequence (S323-GS326AC328- - - - - -PS-VL339; the conserved amino acids are shown and dashes indicate nonconserved amino acids) and that it is only two amino acids away from the catalytic Cys328 and only one amino acid away from the conserved A327, which when replaced by Val reduces the synthesis of all thiolated nucleosides (34). A replacement of the nonconserved Ala by Ser (iscS64) at position 321 resulted in a complex phenotype (Table 2 and 4). Whereas the S326A and L333A alterations of IscS do not decrease the cysteine desulfurase activity (25), the A321S change does (class IIB mutant) (Table 3 and 4), as demonstrated by the reduced synthesis of s4U8, which is dependent on a desulfurase activity and not on any [Fe-S] protein (26). Thus, amino acid substitutions in the active loop and near the catalytic Cys328 induce multiple phenotypes: (i) no change in tRNA thiolation (S323, S324, G325, T329, S330, S332, E334, P335, and S336 to Ala) (25), (ii) deficiency in s2C32 and ms2i(o)6A37 (class III mutants; S326A and L333A) (25), (iii) deficiency in all thiolated nucleosides (class I mutants; A327V) (34) (Table 4), or (iv) deficiency in s4U8, s2C32, and ms2i(o)6A37(class IIB and III; A321V) (Table 4). Apparently, alterations in the vicinity of the active site have different impacts on the activity and/or the substrate specificity of IscS.
We also obtained mutants (iscS55, -57, and -59) with decreased levels of s4U8 and (c)mnm5s2U34 (iscS55 and -57) or of only (c)mnm5s2U34 (iscS59). Thus, these mutants have normal levels of s2C32 and ms2i(o)6A37, whose synthesis is dependent on [Fe-S] proteins. The alterations in the iscS55 and -57 mutants (W45R and V65F, respectively) are located far from the catalytic Cys328 and are clustered in the N-terminal -helix (Fig. 6). The [Fe-S] protein-dependent pathway seems to be intact in these mutants, implying that the activity of the altered IscS proteins is sufficient for the synthesis of s2C and ms2i(o)6A. However, the levels of IscS were increased (Fig. 5), suggesting that other [Fe-S] proteins not involved in the thiolation of tRNA are affected, giving rise to derepression of iscS. Since W45 and D65 are exposed on the surface of IscS and are far from the catalytic Cys328 (Fig. 6), they may interact with TusA and ThiI, which both receive sulfur from IscS (16, 19), although long-range conformational changes around the catalytic Cys328 cannot be excluded. The H96Y alteration, as in the iscS59 mutant, results in a decreased level of only (c)mnm5s2U34 (class IIB mutant) and is also exposed on the surface of IscS but is located rather far from the other two class II alterations (Fig. 6). The level of IscS in the iscS59 (H96Y) mutant was slightly elevated compared to that in the wild type (Fig. 5), and the mutant possesses desulfurase activity (makes s4U8). Therefore, the H96Y alteration seems to have a negative impact on the IscS-TusA interaction but not on the overall desulferase activity. We suggest that the surface composed of the N-terminal -helix of IscS, where the W45R and D65Y mutations are located, as well as the surface where H96Y is located, may make protein-protein contacts with the acceptor proteins ThiI and MnmA, which are required for the synthesis of s4U8 and (c)mnm5s2U34 ([Fe-S]-independent pathways).
Since there are amino acid sequence differences in the C terminus between different NifS and IscS homologs, this portion of IscS may participate in interactions with various acceptor proteins. A truncated IscS lacking the last 29 amino acid residues (designated IscS376-404) is still active, since it complements the reduced growth induced by a deletion of iscS (45), suggesting that it synthesizes (c)mnm5s2U34, which is vital for efficient growth (Table 4). In vitro IscS376-404 binds to IscU less well than the wild-type IscS and has a reduced [Fe-S] cluster formation activity but has a desulfurase activity similar to that of the full-length protein. Thus, apparently the C-terminally mediated binding to IscU is pivotal in [Fe-S] cluster formation. A prediction would therefore be that this mutant should be a class III mutant reducing the levels of s2C and ms2i(o)6A, i.e., influencing mainly the [Fe-S] protein-dependent pathway. Although the mechanism of how iron is delivered in the [Fe-S] cluster assembly is not known in detail, a direct role of IscA has been suggested (9). Whereas the absence of IscU abolishes the synthesis of both s2C and ms2i(o)6A ([Fe-S] protein-dependent pathways), the absence of IscA decreases the level of only ms2i(o)6A and by only 50% (26). Interestingly, our iscS65 and -66 mutants, which have IscS proteins with altered C termini, show a phenotype similar to that shown by a mutant lacking IscA (Table 3). Whereas the iscS65 mutation (H350A) is just upstream of the end of IscS376-404, the iscS66 mutation is a frameshift change at codon 380 within the part of IscS that is absent in the IscS376-404 mutant. A complete lack of IscA, an IscS with an H350S amino acid substitution, or an IscS with a completely different C terminus results in a similar twofold reduction in the level of ms2i(o)6A. Unless this similar reduction in the ability to form ms2i(o)6A is coincidental, it would suggest a role for the C-terminal part of IscS in a potential interaction with IscA. However, no stable complex between IscS and IscA has been established (44). Moreover, the iscS63 (V153W) mutation also slightly reduced the synthesis of ms2i(o)6A (Table 3), although this mutation is not located in the C-terminal end of IscS. Therefore, the reduction of the synthesis of only ms2i(o)6A by the mutations iscS63, -65, and -66 may suggest that the formation of the ms2 group of ms2i(o)6A37 is especially sensitive to minor aberrations in the formation or repair of the [Fe-S] cluster required to form this nucleoside. IscA may stimulate this reaction by optimizing the formation of mature IscU, since its presence increases by twofold the level of only ms2i(o)6A (26) (Fig. 2) and/or it may stimulate the formation of mature MiaB (Fig. 2).
These results show that IscS has a complex substrate specificity revealed by alterations in different regions distinct from the catalytic region. They further suggest that IscS-dependent sulfur delivery and IscA-mediated [Fe-S] cluster formation is target specific. One could envision that several proteins compete for surfaces on IscS and such specific protein-protein interactions result in specific sulfur and/or iron mobilization. Some of the phenotypes caused by the mutations characterized in this study are consistent with such a hypothesis. Clearly, a quite complex set of interactions are involved in the sulfur trafficking required for the formation of the thiolated nucleosides on tRNA in S. enterica.
ACKNOWLEDGMENTS
We thank Kristina Nilsson, who isolated the iscS55 mutant, and we thank Kerstin Jacobsson for the HPLC analysis. We thank Dan Andersson, Uppsala University, Sweden, for the gift of plasmid pSMP24 and Diana Downs, University of Wisconsin, Madison, for the gift of plasmid piscS. We are grateful for the generous gift of antibodies to IscS from Larry E. Vickery, University of California, Irvine. We also thank Tord Hagervall, Anders Bystrm, and Mikael Wikstrm for critical reading of the manuscript.
REFERENCES
Agar, J. N., C. Krebs, J. Frazzon, B. H. Huynh, D. R. Dean, and M. K. Johnson. 2000. IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. Biochemistry 39:7856-7862.
Auffinger, P., and E. Westhof. 1998. Location and distribution of modified nucleotides in tRNA, p. 569-576. In H. Grosjean and R. Benne (ed.), Modification and editing of RNA. ASM Press, Washington, D.C.
Barras, F., L. Loiseau, and B. Py. 2005. How Escherichia coli and Saccharomyces cerevisiae Build Fe/S proteins. Adv. Microb. Physiol. 50:41-101.
Bertani, G. 1951. Studies on lysogenesis. J. Bacteriol. 62:293-300.
Bjrk G. R., and T. G. Hagervall. 25 July 2005, posting date. Chapter 4.6.2, Transfer RNA modification. In R. Curtiss III et al. (ed.), EcoSal—Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, D.C. [Online.] http://www.ecosal.org.
Chen, P., P. F. Crain, S. J. Nsvall, S. C. Pomerantz, and G. R. Bjrk. 2005. A "gain of function" mutation in a protein mediates production of novel modified nucleosides. EMBO J. 24:1842-1851.
Cupp-Vickery, J. R., H. Urbina, and L. E. Vickery. 2003. Crystal structure of IscS, a cysteine desulfurase from Escherichia coli. J. Mol. Biol. 330:1049-1059.
Davis, W., D. Botstein, and J. R. Roth. 1980. A manual for genetic engineering: advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Ding, H., R. J. Clark, and B. Ding. 2004. IscA mediates iron delivery for assembly of iron-sulphur clusters in IscU under the limited accessible free iron conditions. J. Biol. Chem. 279:37499-37504.
Emilsson, V., and C. G. Kurland. 1990. Growth rate dependence of transfer RNA abundance in Escherichia coli. EMBO J. 9:4359-4366.
Esberg, B., and G. R. Bjrk. 1995. The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA. J. Bacteriol. 177:1967-1975.
Frazzon, J., and D. R. Dean. 2003. Formation of iron-sulfur clusters in bacteria: an emerging field in bioinorganic chemistry. Curr. Opin. Chem. Biol. 7:166-173.
Gehrke, C. W., and K. C. Kuo. 1990. Ribonucleoside analysis by reversed-phase high performance liquid chromatography, p. A3-A71. In C. W. Gehrke and K. C. T. Kuo (ed.), Chromatography and modification of nucleosides, part A. Analytical methods for major and modified nucleosides. Elsevier, Amsterdam, The Netherlands.
Gehrke, C. W., K. C. Kuo, R. A. McCune, K. O. Gerhardt, and P. F. Agris. 1982. Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides. J. Chromatogr. 230:297-308.
Hoff, K. G., J. J. Silberg, and L. E. Vickery. 2000. Interaction of the iron-sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli. Proc. Natl. Acad. Sci. USA 97:7790-7795.
Ikeuchi, Y., N. Shigi, A. Nishimura, and T. Suzuki. 2006. Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol. Cell 21:98-108.
Jger, G., R. Leipuviene, M. G. Pollard, Q. Qian, and G. R. Bjrk. 2004. The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium. J. Bacteriol. 186:750-757.
Johnson, D., D. R. Dean, A. D. Smith, and M. K. Johnson. 2005. Structure, function, and formation of iron sulphur clusters. Annu. Rev. Biochem. 74:247-281.
Kambampati, R., and C. T. Lauhon. 1999. IscS is a sulfurtransferase for the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. Biochemistry 38:16561-16568.
Kambampati, R., and C. T. Lauhon. 2000. Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. J. Biol. Chem. 275:10727-10730.
Kambampati, R., and C. T. Lauhon. 2003. MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry 42:1109-1117.
Kim, S. R., G. Maenhaut-Michel, M. Yamada, Y. Yamamoto, K. Matsui, T. Sofuni, T. Nohmi, and H. Ohmori. 1997. Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA. Proc. Natl. Acad. Sci. USA 94:13792-13797.
Lauhon, C. T. 2002. Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli. J. Bacteriol. 184:6820-6829.
Lauhon, C. T., and R. Kambampati. 2000. The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD. J. Biol. Chem. 275:20096-20103.
Lauhon, C. T., E. Skovran, H. D. Urbina, D. M. Downs, and L. E. Vickery. 2004. Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo. J. Biol. Chem. 279:19551-19558.
Leipuviene, R., Q. Qian, and G. R. Bjrk. 2004. Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways. J. Bacteriol. 186:758-766.
Li, J. N., and G. R. Bjrk. 1999. Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity. RNA 5:395-408.
Maisiner-Patin, S., J. R. Roth, . Fredriksson, T. Nystrm, O. G. Berg, and D. I. Andersson. 2005. Genomic buffering mitigates the effects of deleterious mutations in bacteria. Nat. Genet. 37:1376-1379.
Martz, E. 2002. Protein Explorer: easy yet powerful macromolecular visualization. Trends Biochem. Sci. 27:107-109.
McCloskey, J. A., D. E. Graham, S. Zhou, P. F. Crain, M. Ibba, J. Konisky, D. Soll, and G. J. Olsen. 2001. Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales. Nucleic Acids Res. 29:4699-4706.
Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Mueller, E. G., C. J. Buck, P. M. Palenchar, L. E. Barnhart, and J. L. Paulson. 1998. Identification of a gene involved in the generation of 4-thiouridine in tRNA. Nucleic Acids Res. 26:2606-2610.
Mueller, E. G., P. M. Palenchar, and C. J. Buck. 2001. The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA. J. Biol. Chem. 276:33588-33595.
Nilsson, K., H. K. Lundgren, T. G. Hagervall, and G. R. Bjrk. 2002. The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium. J. Bacteriol. 184:6830-6835.
Ollagnier-de-Choudens, S., T. Mattioli, Y. Takahashi, and M. Fontecave. 2001. Iron-sulfur cluster assembly: characterization of IscA and evidence for a specific and functional complex with ferredoxin. J. Biol. Chem. 276:22604-22607.
Pierrel, F., G. R. Bjrk, M. Fontecave, and M. Atta. 2002. Enzymatic modification of tRNAs: MiaB is an iron-sulfur protein. J. Biol. Chem. 277:13367-13370.
Pierrel, F., H. L. Hernandez, M. K. Johnson, M. Fontecave, and M. Atta. 2003. MiaB protein from Thermotoga maritima. Characterization of an extremely thermophilic tRNA-methylthiotransferase. J. Biol. Chem. 278:29515-29524.
Ramabhadran, T. V., T. Fossum, and J. Jagger. 1976. Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid. J. Bacteriol. 128:671-672.
Schmieger, H. 1972. Phage P22-mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119:75-88.
Schwartz, C. J., J. L. Giel, T. Patschkowski, C. Luther, F. J. Ruzicka, H. Beinert, and P. J. Kiley. 2001. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc. Natl. Acad. Sci. USA 98:14895-14900.
Skovran, E., and D. M. Downs. 2000. Metabolic defects caused by mutations in the isc gene cluster in Salmonella enterica serovar Typhimurium: implications for thiamine synthesis. J. Bacteriol. 182:3896-3903.
Smith, A. D., J. N. Agar, K. A. Johnson, J. Frazzon, I. J. Amster, D. R. Dean, and M. K. Johnson. 2001. Sulfur transfer from IscS to IscU: the first step in iron-sulfur cluster biosynthesis. J. Am. Chem. Soc. 123:11103-11104.
Takahashi, Y., and M. Nakamura. 1999. Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli. J. Biochem. (Tokyo) 126:917-926.
Tokumoto, U., S. Nomura, Y. Minami, H. Mihara, S. Kato, T. Kurihara, N. Esaki, H. Kanazawa, H. Matsubara, and Y. Takahashi. 2002. Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli. J. Biochem. (Tokyo) 131:713-719.
Urbina, H. D., J. J. Silberg, K. G. Hoff, and L. E. Vickery. 2001. Transfer of sulfur from IscS to IscU during Fe/S cluster assembly. J. Biol. Chem. 276:44521-44526.
Urbonavicius, J., Q. Qian, J. M. Durand, T. G. Hagervall, and G. R. Bjrk. 2001. Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J. 20:4863-4873.
Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106.
Wagner, J., P. Gruz, S. R. Kim, M. Yamada, K. Matsui, R. P. Fuchs, and T. Nohmi. 1999. The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis. Mol. Cell 4:281-286.
Zheng, L., V. L. Cash, D. H. Flint, and D. R. Dean. 1998. Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. J. Biol. Chem. 273:13264-13272.(Hans K. Lundgren and Glen)
ABSTRACT
The cysteine desulfurase IscS in Salmonella enterica serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s4U) and 5-methylaminomethyl-2-thiouridine (mnm5s2U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s2C) and N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm5s2U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms2io6A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms2io6A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.
INTRODUCTION
Many nucleic acids contain, besides the four major nucleosides adenosine (A), guanosine (G), uridine (U), and cytidine (C), a variety of modified nucleosides, which are derivatives of these major nucleosides (reviewed in reference 5). One group of such modified nucleosides is the thiolated nucleosides, which are ubiquitously present in tRNAs from the three phylogenetic domains Bacteria, Eucarya, and Archaea (2, 30). tRNA from Salmonella enterica serovar Typhimurium contains six thiolated nucleosides: 2-thiocytidine (s2C), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 4-thiouridine (s4U), N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), and N6-(isopentenyl)-2-thioadenosine (ms2i6A). The locations of these thiolated nucleosides and their structures are shown in Fig. 1. All thiolated nucleosides except s4U8 are present in the anticodon loop, consistent with their importance in the fidelity and efficiency of decoding of the message (reviewed in reference 5).
The synthesis of all thiolated nucleosides in tRNA depends on IscS (23, 34). The iscS structural gene is part of the isc operon, which consists of eight phylogenetically conserved genes (iscR, iscS, iscU, iscA, hscA, hscB, fdx, and orf3) (43, 49). The IscR protein senses the status of the iron-sulfur cluster (designated the [Fe-S] cluster irrespective of which type of iron-sulfur cluster is present in a protein) in the cell and thereby regulates the expression of the isc operon (40). IscS is a pyridoxal-phosphate-dependent enzyme that mobilizes sulfur through desulfurization of cysteine to yield alanine and an IscS-bound persulfide. IscU, which is a scaffold protein in the [Fe-S] cluster assembly, accepts sulfur from IscS and in turn delivers it as an [Fe-S] cluster to the target apoprotein (1, 12, 42). IscA, which is an alternative scaffold protein in the [Fe-S] cluster assembly, may provide iron for the [Fe-S] cluster formed in IscU (9). HscA and HscB (for heat shock cognate) are chaperons apparently associated only with IscS-directed [Fe-S] cluster assembly (15). Fdx (ferredoxin), which is an iron-sulfur protein, is involved in electron transfer and complexes with IscA (35; for recent reviews of the Isc system, see references 3 and 18).
The sulfur relay in the synthesis of thiolated nucleosides is summarized in Fig. 2. ThiI transfers sulfur directly from IscS to the target nucleoside (U8) in tRNA (19, 33). A similar sulfur transfer reaction was suggested for the synthesis of the s2 group of (c)mnm5s2U34 (mnm5s2U and cmnm5s2U), in which IscS transfers the sulfur to TusA, which in turn delivers it to TusD in the TusBCD complex. TusE may accept sulfur from TusD and deliver it to MnmA, which transfers it to U8 in tRNA (16, 21). Thus, in these two cases, the IscS persulfide donates its sulfur directly to a protein [ThiI in the synthesis of s4U8 and TusA in the case of (c)mnm5s2U34], and no [Fe-S] protein is thought to participate in the synthesis of these two thiolated nucleosides (designated the [Fe-S] protein-independent pathways). TtcA is required for the synthesis of s2C32 (17), and since it contains several cysteines it may be an [Fe-S] protein, which has recently been demonstrated (Marc Fontecave and Mohamed Atta [Grenoble, France], personal communication). The MiaB protein, which belongs to the SAM-radical family of enzymes, is an [Fe-S] protein (36), and apparently it can alone catalyze the formation of the ms2 group of ms2i(o)6A37 (ms2i6A and ms2io6A), albeit at a low efficiency (37). The presence of IscU, HscA, or Fdx is required in the synthesis of s2C32 and ms2i(o)6A37 but not in the synthesis of s4U8 and (c)mnm5s2U34 (26). Therefore, it was suggested that in the synthesis of s2C32 and ms2i(o)6A37, an [Fe-S] protein(s) participates. Thus, two distinct pathways for thiolation of tRNA exist. In the [Fe-S] protein-independent pathway, which is responsible for the synthesis of s4U8 and (c)mnm5s2U34 (pathway II in Fig. 2), there is a direct transfer of sulfur from IscS to a protein which in turn transfers the sulfur to the target nucleoside in tRNA for the synthesis of s4U or to another protein in the sulfur relay for the synthesis of (c)mnm5s2U. In the [Fe-S] protein-dependent pathway, which is responsible for the synthesis of s2C32 and ms2i(o)6A37 (pathway III in Fig. 2), an [Fe-S] protein(s) participates, and its formation and maintenance are dependent on IscS (26). In the latter two cases, the sulfur is transferred from IscS to IscU, and IscA, HscA, HscB, and Fdx participate in a complex way, not so far unraveled in detail, to form [Fe-S] cluster-loaded IscU, which then transfers the cluster to the receiving apoprotein (18) (Fig. 2). A lack of any of these participating proteins except IscA almost abolishes the synthesis of s2C32 and ms2io6A37. Apparently, to optimize the formation of ms2io6A, the presence of IscA is required, since the lack of it reduces the formation of only this thiolated nucleoside (26). Since this model of two distinct pathways in the thiolation of tRNA was based on the analysis of deletions of various genes involved in thiolation of tRNA, a more elaborate pathway may exist if proteins interact in a specific manner through various protein-protein complex formations. This paper addresses this question by analyzing mutants with point mutations in iscS selected for deficiency in the synthesis of one of the end products, (c)mnm5s2U34 or ms2i(o)6A37, of the two pathways. We found that some amino acid substitutions in IscS affected the synthesis of only a subset of the thiolated nucleosides, suggesting substrate specificity of IscS in the thiolation of tRNA (Fig. 2).
MATERIALS AND METHODS
Bacteria and growth conditions. The bacterial strains used were either derivatives of S. enterica serovar Typhimurium (Table 1) or Escherichia coli. Bacteria were grown in Luria-Bertani (LB) rich liquid medium (4) or on LA plates (LB medium with 15 g/liter agar added). The minimal media were made from the basal medium E (47) supplemented with 0.2% glucose and required amino acids or vitamins at concentrations recommended previously (8). Minimal medium E agar plates were made by adding 15 g of agar per liter of culture.
Frameshift mutants used for selecting frameshift suppressors. We used two different constructs to measure the effect of hypomodification on frameshifting. Both constructs contain the hisO1242 mutation, which increases the expression of the his operon and thereby enhances the synthesis of the frameshifting product, making the system very sensitive to detect low degree of frameshifting. The first construct contains the hisC3737 mutation, which is a C insertion in the hisC gene, resulting in the sequence CCC-CAA-UAA (the inserted C underlined). The insertion results in a shift in the reading frame. To be able to make a functional HisC protein, a +1 frameshift must occur before the newly created UAA stop codon in the zero reading frame, thus enabling bypassing of the stop codon and reentrance into the zero reading frame. Such suppression of the hisC3737 mutation results in a functional HisC and growth without externally added histidine. The frameshift window in the hisC3737 mutant is fairly large (GUA-GAG-CGC-CGG-ACG-GUU-CCC-GCG-CUU-GAA-AAC-UGG-CAG- CUG-GAU-CUA-CAG-GGG-AUU-UCC-GAC-AAC-CUU-GAC-GGC-ACA-AAA-GUG-UUC-GUU-UGU-AGC-CCC-CAA-UAA; the underlined UAG is the stop codon in the +1 frame, and the underlined CCC-CAA-UAA is the potential frameshift site with the UAA stop codon in the zero reading frame). The second construct contains, in addition to the hisO1242 mutation, the hisD10114 mutation, which is a +1 frameshift mutation in the hisD gene which must be suppressed for the strain to grow without externally added histidine. The potential frameshifting site is UUU-UAU-UAA, and the frameshifting window is only eight codons (CUG-AUU-GAC-UGG-AAC-AGC-UGU-AGC-UUU-UAU-UAA; the underlined UGA is the stop codon in the +1 frame, and the underlined UUU-UAU-UAA is the potential frameshifting site constructed with the UAA stop codon in the zero frame).
Genetic procedures. Transduction with phage P22 HT105/1 (int-201) (39) was performed as described previously (8). Mutagenesis was performed using overexpression of the DinB protein (22, 48) regulated by an araC promoter harbored on a plasmid kindly supplied by Dan Andersson, Uppsala, Sweden (28).
Isolation of mutants. A crystal of nitrosoguanidine was placed on an LA rich agar plate seeded with strain GT6430 (STM2545-2525::Tn10dTc). The Tn10dTc insertion is only 2 kb away from the isc operon, and thus both the transposon and the isc operon can be present on the same DNA fragment of one P22 phage. After incubation overnight, a clear zone where no cells were growing appeared around the crystal. Cells from the area just outside the clear zone were collected, transferred to 1 ml of LB medium, and incubated for 2 h. Phage P22 was grown on such a culture and used to transduce either strain GT853 (hisC3737) or strain GT6942 (hisD10114). Five milliliters of overnight culture of the recipient cells was incubated with phage P22 for 30 min at 37°C. Cells were collected by centrifugation and washed twice with 0.9% NaCl containing 10 mM EGTA to remove unabsorbed phages before the culture was plated on selective plates. Tcr His+ transductants were selected on agar plates containing either medium E, 0.2% glucose, and 2.5 μg/ml tetracycline (Tc) or medium E, 0.2% glucose, thiamine, nicotinic acid, and 2.5 μg/ml Tc. Thiamine and nicotinic acid were included, since some mutations in iscS induce auxotrophy for these metabolites (24). Sometimes salt medium M9 (31) was used instead of medium E, with no discernible difference. The plates were incubated at 37°C for up to 7 days. The Tcr His+ transductants were purified on LA plates containing 10 mM EGTA. Phage P22 was grown on the purified transductants and backcrossed to the parent strain to obtain mutants, which did not contain mutations outside the isc region. The cross was performed on LA plates containing 15 μg/ml Tc and 10 mM EGTA, followed by replica printing on medium E plates containing 0.2% glucose with or without histidine. The cotransduction frequency between Tcr and His+ phenotypes was such that the His+ phenotype could be caused by a mutation in iscS (data not shown). One Tcr His+ transductant from each backcross was saved for further analysis. Mutational siblings were eliminated by comparing similarities in growth/suppression phenotype and/or high-pressure liquid chromatography (HPLC) analysis. Finally, mutant strains were complemented with the piscS plasmid, harboring only the wild-type allele of the iscS+ gene (41), and chromosomal sequencing of the iscS gene in the mutants was performed.
To isolate DinB-induced Tcr His+ mutants, an overnight culture of strain GT7172 (STM2545-2525::Tn10dTc/pSMP24; pSMP24 contains the dinB gene controlled by an arabinose promoter) (28) was diluted 1,000-fold in LB medium containing 0.2% arabinose and allowed to grow for 8 h, after which phage P22 was added to the culture and the phage-bacterium mixture was incubated overnight in a rotary shaker. The bacteria were removed by centrifugation, and the supernatant was treated with chloroform. Such phage stocks were used to select frameshift suppressors as described above.
DNA sequencing. Sequencing was performed on chromosomal DNA or PCR products as described in the manual for the Applied Biosystems ABI PRISM Cycle Sequencing Ready Reaction Big Dye Kit (Applied Biosystems, Foster City, California).
Analysis of modified nucleosides in tRNA. Bacterial strains were grown in LB medium (4) at 37°C to about 4 x 108 to 6 x 108 cells/ml. Cells were lysed, and total RNA was prepared (10). Total RNA was dissolved in buffer R200 (10 mM Tris-H3PO4, pH 6.3; 15% ethanol; 200 mM KCl) and applied to a Nucleobond column (AX500) equilibrated with the same buffer. tRNA was eluted with 7 ml of buffer R600 (R200 but containing 600 mM KCl), precipitated with 2.5 volumes of cold ethanol, washed twice with 80% ethanol, and dried. The dried tRNA was dissolved in water, and a portion of it was degraded to nucleosides by nuclease P1 followed by treatment with bacterial alkaline phosphatase (14). The hydrolysate was analyzed by HPLC (13) using a C18 HPLC column (250 by 4.6 mm; Phenomex Ltd., MacClesfield, Cheshire, United Kingdom).
Western blotting. Total protein was prepared from cells grown to a cell density of 5 x 108 cells/ml in LB medium by sonicating the cells using a VCX400 Vibracell sonicator (Sonics Inc). The cell debris was removed by centrifugation at 3,000 x g for 10 min. The supernatant was diluted with 1 ml of water, total protein was determined using a protein assay kit (Bio-Rad Inc., Hercules, California), and 25 μg was applied in each well on a 12% sodium dodecyl sulfate-polyacrylamide gel. Proteins were separated using the Mini-Protean II protein electrophoresis system (Bio-Rad Inc., Hercules, California). Two gels were used in parallel experiments, with equal amounts of proteins from the same preparation applied on each gel. One gel was used for TrmD as a control and the other for IscS determinations. Proteins were blotted to Amersham Hybond C membranes using the wet blot transfer system Mini Trans blot cell (Bio-Rad Inc.). Blocking was performed with the ECL blocking agent (Amersham Life Sciences, GE Healthcare, Uppsala, Sweden). Primary antibodies against IscS, kindly provided by Larry E. Vickery (Irvine, California), and TrmD (27) were added, followed by incubation with the secondary antibody according to the protocol supplied with the ECL Plus kit (Amersham Life Sciences, GE Healthcare, Uppsala, Sweden). ECL detection was performed in a Fluor-S Max2 Lumi-imager (Bio-Rad Inc., Hercules, California).
RESULTS
Strategy for isolation of IscS mutants defective in either of the two tRNA thiolation pathways. Two of the thiolated nucleosides, (c)mnm5s2U34 and ms2i(o)6A37, are present in different tRNAs reading different codons and improve reading frame maintenance, since the lack of either of them induces codon-specific frameshifts (46). We have utilized this fact to select for IscS mutants deficient in either (c)mnm5s2U34 or ms2i(o)6A37. According to our working model (Fig. 2), such IscS mutants were expected to be deficient not only in (c)mnm5s2U34 or ms2i(o)6A37 but also in other thiolated nucleosides, since IscS is pivotal for the synthesis of all thiolated nucleosides (23, 34). At the sequence CCC-CAA-UAA (the sequence written in the zero frame, and a +1 frameshift must occur before the ribosome reaches the stop codon UAA), which is found in the hisC gene in a strain containing the hisC3737 mutation, the CAA codon is read by tRNAcmnm5s2UUGGln, which contains as the wobble nucleoside (c)mnm5s2U34 (6). At the sequence CCC-CAA-UAA we anticipated that a thiolation deficiency of cmnm5s2U34 would suppress the +1 frameshift mutation hisC3737, resulting in growth in the absence of histidine (a His+ phenotype) (Fig. 3A). Our strategy was to select His+ clones of the histidine-requiring hisC3737 mutant following localized mutagenesis of the iscS gene. Mutations in iscS should reduce the thiolation of the wobble nucleoside (c)mnm5s2U34 and thereby increase frameshifting.
At the sequence UUU-UAU-UAA, codons UUU and UAU are read by tRNAGAAPhe and tRNAQUATyr, respectively. A lack of the ms2 group in either of these tRNAs causes frameshifting (46) (Fig. 3B). Hence, mutants defective in the synthesis of the ms2 group of ms2i(o)6A37 in both tRNAGAAPhe and tRNAQUATyr should suppress the UUU-UAU-UAA site placed early in the hisD gene. Only mutations in iscS were characterized in this work, although iscU, iscA, hscA, or fdx mutants will also be found with this method, since these genes affect the synthesis of ms2i(o)6A (26).
To test our strategy, we introduced mutations in mnmA or miaB, which encode thiolating enzymes required for the synthesis of (c)mnm5s2U34 and ms2io6A37, respectively, into strains with one of the two different frameshifts sites hisC3737 and hisD10114. Whereas the mnmA1 mutation suppressed only the hisC3737 (CCC-CAA-UAA) mutation, the miaB1 mutation suppressed only the hisD10114 (UUU-UAU-UAA) mutation (data not shown). Thus, we anticipated that iscS mutations suppressing hisC3737 would concomitantly cause deficiency in at least (c)mnm5s2U34, whereas iscS mutations suppressing hisD10114 would cause deficiency in at least ms2i(o)6A37. Since mutations in thiI or ttcA, whose products are required for the synthesis of s4U8 and s2C32 on the tRNA, respectively, did not suppress any of these two +1 frameshift mutations (data not shown), we did not anticipate obtaining iscS mutants deficient in only either of these thiolated nucleosides. Thus, suppression of the two frameshift mutations hisC3737 and hisD10114 requires deficiency of only (c)mnm5s2U34 and only ms2i(o)6A37, respectively.
Isolation, identification, and characterization of iscS mutations found to cause defects in either of the two tRNA thiolation pathways. Strain GT6430, which has a Tn10dTc inserted just upstream of the isc operon, was mutagenized either by DinB overexpression or by treatment with nitrosoguanidine. Phage P22 was grown on such mutagenized cultures and used to infect strain GT853 (hisC3737; CCC-CAA-UAA) or strain GT6942 (hisD10114; UUU-UAU-UAA). Tcr His+ transductants were selected and backcrossed to the original parent strains to avoid mutants with compensatory mutations in the his operon. However, the frequency of such mutations ought to be low, since the his operon is located far from the isc operon. This approach should also avoid any mutations in the structural genes whose products are required for the synthesis of thiolated nucleosides, such as thiI (s4U8), mnmA [(c)mnm5s2U34], or miaB [ms2i(o)6A37], since their chromosomal locations are far from the isc operon. The mutations in the various His+ mutants characterized were all located in the iscS gene, since cotransduction frequencies between the Tcr and His+ phenotypes were such that the His+ phenotype could be caused by a mutation in iscS and since introduction of a plasmid carrying the wild-type allele of iscS complemented the mutant phenotypes (His+ and the deficiency in thiolation of tRNA) (Tables 2 and 3). The mutations were identified by sequencing of the iscS genes from the various mutants. Thus, the induced phenotype must be caused by the alteration in IscS. tRNA was isolated and degraded to nucleosides, and the distribution and quantification of nucleosides were determined by HPLC (Fig. 4; Tables 2 and 3).
Using the frameshifting site hisC3737 (CCC-CAA-UAA), we obtained, as expected, class I mutants (iscS56, -60, and -61) with reduced levels of all thiolated nucleosides (Table 2), similar to the case for the earlier characterized iscS51 mutation (34) and to that for a deletion of iscS (23). However, we also obtained iscS55 and iscS57 mutants with much reduced levels of (c)mnm5s2U34 and s4U8 and almost wild-type (iscS55) or wild-type (iscS57) levels of s2C32 and ms2i(o)6A37 (designated class II mutants). Apparently, alterations in IscS affected mainly the [Fe-S] protein-independent pathway and not at all (iscS57) or weakly (iscS55) the [Fe-S] protein-dependent pathway (Fig. 2). Moreover, we also obtained another mutant (iscS59) with a reduced level of only (c)mnm5s2U34 and normal levels of all the other thiolated nucleosides (class IIB mutant) (Table 2). In some way an alteration in IscS affected the mobilization of sulfur to only one of the enzymes required for the synthesis of the four thiolated derivatives in tRNA. A mirror image of the iscS59 mutation was the iscS64 mutation, obtained using the hisD10114 (UUU-UAU-UAA) frameshifting site, since it had a reduced synthesis of all thiolated nucleosides except (c)mnm5s2U (a class III and IIA mutant) (Table 3). Thus, in addition to class I mutants, we also isolated iscS mutants defective in the synthesis of a subset of the thiolated nucleosides.
One suppressor mutation, iscS58, of the hisD10114 (UUU-UAU-UAA) mutation was deficient in all thiolated nucleosides (Table 3). However, by using this frameshifting site we also obtained mutants defective in the synthesis of one or more of the thiolated nucleosides, such as the class IIIB mutants carrying iscS63, -65, and -66 [deficient in only ms2i(o)6A] and iscS64 [deficient in all thiolated nucleosides except (c)mnm5s2U34] (a class IIA and III mutant) (Table 2). The iscS65 and -66 mutants had a phenotype reminiscent of that of a mutant deleted for iscA, which is also deficient in only ms2i(o)6A37 (26). Note also that the iscS65 and -66 mutations, as well as deletion of iscA, reduced ms2i(o)6A to a similar level (about 50% of the wild-type level). However, the iscS65 and -66 mutants have a wild-type allele of the iscA gene as determined by DNA sequencing (data not shown), and these mutants had only a mutation in the iscS gene, because the wild-type phenotype was restored by introducing the wild-type allele of iscS+ on a plasmid (Table 3). Both mutants contained an alteration in the C-terminal part of the IscS protein. The iscS66 mutation was a frameshift mutation in codon 380 resulting in a truncated IscS. The iscS65 mutation changed His350 to Arg350.
Some amino acid substitutions may reduce the stability of the IscS protein, which would result in a lower activity in the cell and thereby lower synthesis of all thiolated nucleosides. We therefore monitored the levels of the IscS protein in the various mutants (Fig. 5). The levels of full-size IscS in the iscS56 and -58 mutants were significantly lower than that in the wild type, and, accordingly, these mutations reduced the levels of all thiolated nucleosides (Tables 2 and 3; Fig. 5). All other mutations did not reduce the level of IscS, which is why the phenotype mediated by these mutations must be caused by the activity of IscS and not by a lower level of the protein. The isc operon is controlled by IscR, which senses the [Fe-S] status of the cell (40). Thus, a nonfunctional IscS, which should decrease the [Fe-S] content of the cell, ought to derepress the isc operon, as was observed for some mutants (the iscS55, iscS57, iscS59, and iscS60 mutants) (Fig. 5).
In summary (Table 4), we obtained mutants with altered IscS that reduced the synthesis of all thiolated nucleosides (class I mutants), irrespective of which frameshifting site we used to select the suppressor mutants (iscS51, -56, -58, -60 and -61) (Table 3). For two of them (iscS56 and -58), the low level of full-size IscS (Fig. 5) could explain the deficiency of all four thiolated nucleosides. The phenotype mediated by the isc51 and -61 mutations, which did not reduce the level of IscS, may suggest a reduction of the desulfurase activity of IscS, since the synthesis of all thiolated nucleosides was reduced by these mutations. We also found mutants that were defective in the synthesis of one, two, or three of the thiolated nucleosides (the iscS55, -57, -59, -63, -64, -65, and -66 mutants) (Table 4). Class II mutants (iscS55 and iscS57), which were defective in the synthesis of both s4U and (c)mnm5s2U34, were apparently defective in sulfur mobilization from IscS to ThiI and to TusA, respectively, suggesting that a structural change in IscS influences its interaction with ThiI and TusA in a similar way. We obtained a mutant (iscS59) that was deficient in only (c)mnm5s2U (class IIB), indicating a specific defect in the interaction with TusA. No class III mutants were found, although one would think that an alteration in IscS could influence its interaction with IscU, plausibly resulting in a deficiency in s2C32 and ms2i(o)6A37 but not in s4U8 and (c)mnm5s2U34 (26). However, we obtained three mutants deficient in only ms2i(o)6A37 (iscS63, -65, and -66; class IIIB). A mutant with a more complex defect in the thiolation pattern was also obtained, and this mutant was apparently simultaneously defective in both the [Fe-S] protein-independent and -dependent pathways (iscS64; class IIB and class III) (Table 3). Apparently, different alterations in IscS affect sulfur trafficking in specific ways, suggesting that IscS does not mobilize sulfur unspecifically in the thiolation of tRNA.
Growth and suppression levels. Mutants having a low level of (c)mnm5s2U34 showed the largest reduction in growth (Table 4), whereas those deficient in only ms2i(o)6A37 showed no reduction in growth compared to the wild type. This growth behavior was expected, since the lack of only ms2i(o)6A37, only s2C32, or only s4U8 does not result in any growth defect in rich medium (11, 17, 38), whereas the lack of (c)mnm5s2U34 severely reduces the growth rate (34). Thus, the growth in rich medium is consistent with the tRNA thiolation deficiency in the various mutants (Table 4). The levels of suppression of the His– phenotype is also consistent with the reduction in the levels of the relevant thiolated nucleosides.
DISCUSSION
Using a genetic selection for iscS mutants with a defect in the synthesis of either only (c)mnm5s2U34 or only ms2i(o)6A37 in their tRNAs, we obtained, as expected, mutants that were defective in the synthesis of all thiolated nucleosides. We also isolated iscS mutants with defects in the synthesis of one or groups of two or three of the four thiolated nucleosides, suggesting that there is specificity in the way IscS transfers sulfur in the [Fe-S] protein-independent and -dependent pathways (Fig. 2).
The selection of the mutants and analysis of the thiolation level of the tRNA were done with the iscS+ gene present at its wild-type location on the chromosome. Thus, potential multicopy artifacts, which may be introduced if the IscS protein is encoded from a plasmid, or regulatory artifacts caused by misplacement of the iscS gene on the chromosome were avoided. Therefore, we believe that our results obtained from the analyses of the various mutants are true reflections of the in vivo kinetics of the synthesis of the thiolated nucleosides and thus a reflection of the efficiency of sulfur transfer and of synthesis and maintenance of [Fe-S] cluster proteins participating in the thiolation of tRNA.
Deletion of the iscS gene abolishes the synthesis of all thiolated nucleosides (23). Since iscS58 is a frameshift mutation early in the iscS gene (Table 4), we expected that this mutation also should mediate a similar phenotype. However, this was not the case, since this mutant still had significant residual levels of all thiolated nucleosides (Table 3) and, accordingly, a low level of full-size IscS was observed (Fig. 5). The explanation might be that the deficiencies of both (c)mnm5s2U34 and ms2i(o)6A37 in this mutant also mediate suppression of the iscS58 frameshift mutation per se by hypomodified tRNA(s) resulting in a small level of active IscS protein. Since the iscS mRNA might also be overexpressed because of a nonfunctional IscR (40), the level of a possible frameshift product of IscS would be elevated, explaining the observed phenotypes (Table 3; Fig. 5). There are several codons in the frameshifting window created by the iscS58 mutation that would potentially be suppressed by these hypomodified tRNAs.
Five mutations, i.e., iscS51, -56, -58, -60, and -61, reduced the synthesis of all thiolated nucleosides (Tables 2, 3, and 4). For the iscS56 and -58 mutants the low levels of all thiolated nucleosides are explained by the apparent instability of the IscS protein in these mutants (Fig. 5). The iscS51 (A327V) mutation results in an amino acid substitution next to the catalytic Cys328 present in the active-site loop region. The iscS51 mutation, which does not affect the stability of IscS (Fig. 5), abolishes the synthesis of all thiolated nucleosides (34). The phenotype caused by the A327V substitution is likely due to an activity change due to its close proximity to the catalytic Cys328. The A327V substitution might change the flexibility of the active loop (25, 45), resulting in a conformational change in the neighborhood of the catalytic Cys328 that gives a lower desulfurase activity. The iscS61 (M169V) mutation, which, like iscS51, does not affect the stability of IscS (Fig. 5), also results in a deficiency of all thiolated nucleosides, and it is located far from the catalytic Cys328 (Table 4). Therefore, we suggest that the M169V alteration influences the desulfurase activity in a way similar to that of the iscS51 mutation and not by affecting the stability of IscS. A mutation (iscS63) resulting in an alteration (V153W) rather close to the iscS61 (M169V) mutation did not reduce the levels of all thiolated nucleosides as the latter mutation did, and, accordingly, the level of IscS was not reduced (Tables 3 and 4; Fig. 5). The V153W alteration only slightly affected the [Fe-S] protein-dependent pathway [ms2i(o)6A] (Table 3), indicating that the synthesis of this thiolated nucleoside is especially sensitive to small aberrations in the desulfurase activity and/or iron mobilization.
Lauhon et al. (25) generated IscS mutants by alanine scanning mutagenesis from amino acid Ser323 to amino acid Ser336 in the active-site loop. Indeed, a C328A alteration abolishes the synthesis of all thiolated nucleosides, consistent with the suggested pivotal role of Cys328 in the function of IscS. Of the other 13 alterations, 11 did not influence the thiolation of tRNA, and the two alterations (S326A and L333A) which influence thiolation of tRNA do so in a specific way, since they reduce the synthesis of only s2C32 and ms2i6A37; i.e., they affect only the [Fe-S] protein-dependent pathway (class III mutants). Note that the Ser326 is part of the consensus sequence (S323-GS326AC328- - - - - -PS-VL339; the conserved amino acids are shown and dashes indicate nonconserved amino acids) and that it is only two amino acids away from the catalytic Cys328 and only one amino acid away from the conserved A327, which when replaced by Val reduces the synthesis of all thiolated nucleosides (34). A replacement of the nonconserved Ala by Ser (iscS64) at position 321 resulted in a complex phenotype (Table 2 and 4). Whereas the S326A and L333A alterations of IscS do not decrease the cysteine desulfurase activity (25), the A321S change does (class IIB mutant) (Table 3 and 4), as demonstrated by the reduced synthesis of s4U8, which is dependent on a desulfurase activity and not on any [Fe-S] protein (26). Thus, amino acid substitutions in the active loop and near the catalytic Cys328 induce multiple phenotypes: (i) no change in tRNA thiolation (S323, S324, G325, T329, S330, S332, E334, P335, and S336 to Ala) (25), (ii) deficiency in s2C32 and ms2i(o)6A37 (class III mutants; S326A and L333A) (25), (iii) deficiency in all thiolated nucleosides (class I mutants; A327V) (34) (Table 4), or (iv) deficiency in s4U8, s2C32, and ms2i(o)6A37(class IIB and III; A321V) (Table 4). Apparently, alterations in the vicinity of the active site have different impacts on the activity and/or the substrate specificity of IscS.
We also obtained mutants (iscS55, -57, and -59) with decreased levels of s4U8 and (c)mnm5s2U34 (iscS55 and -57) or of only (c)mnm5s2U34 (iscS59). Thus, these mutants have normal levels of s2C32 and ms2i(o)6A37, whose synthesis is dependent on [Fe-S] proteins. The alterations in the iscS55 and -57 mutants (W45R and V65F, respectively) are located far from the catalytic Cys328 and are clustered in the N-terminal -helix (Fig. 6). The [Fe-S] protein-dependent pathway seems to be intact in these mutants, implying that the activity of the altered IscS proteins is sufficient for the synthesis of s2C and ms2i(o)6A. However, the levels of IscS were increased (Fig. 5), suggesting that other [Fe-S] proteins not involved in the thiolation of tRNA are affected, giving rise to derepression of iscS. Since W45 and D65 are exposed on the surface of IscS and are far from the catalytic Cys328 (Fig. 6), they may interact with TusA and ThiI, which both receive sulfur from IscS (16, 19), although long-range conformational changes around the catalytic Cys328 cannot be excluded. The H96Y alteration, as in the iscS59 mutant, results in a decreased level of only (c)mnm5s2U34 (class IIB mutant) and is also exposed on the surface of IscS but is located rather far from the other two class II alterations (Fig. 6). The level of IscS in the iscS59 (H96Y) mutant was slightly elevated compared to that in the wild type (Fig. 5), and the mutant possesses desulfurase activity (makes s4U8). Therefore, the H96Y alteration seems to have a negative impact on the IscS-TusA interaction but not on the overall desulferase activity. We suggest that the surface composed of the N-terminal -helix of IscS, where the W45R and D65Y mutations are located, as well as the surface where H96Y is located, may make protein-protein contacts with the acceptor proteins ThiI and MnmA, which are required for the synthesis of s4U8 and (c)mnm5s2U34 ([Fe-S]-independent pathways).
Since there are amino acid sequence differences in the C terminus between different NifS and IscS homologs, this portion of IscS may participate in interactions with various acceptor proteins. A truncated IscS lacking the last 29 amino acid residues (designated IscS376-404) is still active, since it complements the reduced growth induced by a deletion of iscS (45), suggesting that it synthesizes (c)mnm5s2U34, which is vital for efficient growth (Table 4). In vitro IscS376-404 binds to IscU less well than the wild-type IscS and has a reduced [Fe-S] cluster formation activity but has a desulfurase activity similar to that of the full-length protein. Thus, apparently the C-terminally mediated binding to IscU is pivotal in [Fe-S] cluster formation. A prediction would therefore be that this mutant should be a class III mutant reducing the levels of s2C and ms2i(o)6A, i.e., influencing mainly the [Fe-S] protein-dependent pathway. Although the mechanism of how iron is delivered in the [Fe-S] cluster assembly is not known in detail, a direct role of IscA has been suggested (9). Whereas the absence of IscU abolishes the synthesis of both s2C and ms2i(o)6A ([Fe-S] protein-dependent pathways), the absence of IscA decreases the level of only ms2i(o)6A and by only 50% (26). Interestingly, our iscS65 and -66 mutants, which have IscS proteins with altered C termini, show a phenotype similar to that shown by a mutant lacking IscA (Table 3). Whereas the iscS65 mutation (H350A) is just upstream of the end of IscS376-404, the iscS66 mutation is a frameshift change at codon 380 within the part of IscS that is absent in the IscS376-404 mutant. A complete lack of IscA, an IscS with an H350S amino acid substitution, or an IscS with a completely different C terminus results in a similar twofold reduction in the level of ms2i(o)6A. Unless this similar reduction in the ability to form ms2i(o)6A is coincidental, it would suggest a role for the C-terminal part of IscS in a potential interaction with IscA. However, no stable complex between IscS and IscA has been established (44). Moreover, the iscS63 (V153W) mutation also slightly reduced the synthesis of ms2i(o)6A (Table 3), although this mutation is not located in the C-terminal end of IscS. Therefore, the reduction of the synthesis of only ms2i(o)6A by the mutations iscS63, -65, and -66 may suggest that the formation of the ms2 group of ms2i(o)6A37 is especially sensitive to minor aberrations in the formation or repair of the [Fe-S] cluster required to form this nucleoside. IscA may stimulate this reaction by optimizing the formation of mature IscU, since its presence increases by twofold the level of only ms2i(o)6A (26) (Fig. 2) and/or it may stimulate the formation of mature MiaB (Fig. 2).
These results show that IscS has a complex substrate specificity revealed by alterations in different regions distinct from the catalytic region. They further suggest that IscS-dependent sulfur delivery and IscA-mediated [Fe-S] cluster formation is target specific. One could envision that several proteins compete for surfaces on IscS and such specific protein-protein interactions result in specific sulfur and/or iron mobilization. Some of the phenotypes caused by the mutations characterized in this study are consistent with such a hypothesis. Clearly, a quite complex set of interactions are involved in the sulfur trafficking required for the formation of the thiolated nucleosides on tRNA in S. enterica.
ACKNOWLEDGMENTS
We thank Kristina Nilsson, who isolated the iscS55 mutant, and we thank Kerstin Jacobsson for the HPLC analysis. We thank Dan Andersson, Uppsala University, Sweden, for the gift of plasmid pSMP24 and Diana Downs, University of Wisconsin, Madison, for the gift of plasmid piscS. We are grateful for the generous gift of antibodies to IscS from Larry E. Vickery, University of California, Irvine. We also thank Tord Hagervall, Anders Bystrm, and Mikael Wikstrm for critical reading of the manuscript.
REFERENCES
Agar, J. N., C. Krebs, J. Frazzon, B. H. Huynh, D. R. Dean, and M. K. Johnson. 2000. IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. Biochemistry 39:7856-7862.
Auffinger, P., and E. Westhof. 1998. Location and distribution of modified nucleotides in tRNA, p. 569-576. In H. Grosjean and R. Benne (ed.), Modification and editing of RNA. ASM Press, Washington, D.C.
Barras, F., L. Loiseau, and B. Py. 2005. How Escherichia coli and Saccharomyces cerevisiae Build Fe/S proteins. Adv. Microb. Physiol. 50:41-101.
Bertani, G. 1951. Studies on lysogenesis. J. Bacteriol. 62:293-300.
Bjrk G. R., and T. G. Hagervall. 25 July 2005, posting date. Chapter 4.6.2, Transfer RNA modification. In R. Curtiss III et al. (ed.), EcoSal—Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, D.C. [Online.] http://www.ecosal.org.
Chen, P., P. F. Crain, S. J. Nsvall, S. C. Pomerantz, and G. R. Bjrk. 2005. A "gain of function" mutation in a protein mediates production of novel modified nucleosides. EMBO J. 24:1842-1851.
Cupp-Vickery, J. R., H. Urbina, and L. E. Vickery. 2003. Crystal structure of IscS, a cysteine desulfurase from Escherichia coli. J. Mol. Biol. 330:1049-1059.
Davis, W., D. Botstein, and J. R. Roth. 1980. A manual for genetic engineering: advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Ding, H., R. J. Clark, and B. Ding. 2004. IscA mediates iron delivery for assembly of iron-sulphur clusters in IscU under the limited accessible free iron conditions. J. Biol. Chem. 279:37499-37504.
Emilsson, V., and C. G. Kurland. 1990. Growth rate dependence of transfer RNA abundance in Escherichia coli. EMBO J. 9:4359-4366.
Esberg, B., and G. R. Bjrk. 1995. The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA. J. Bacteriol. 177:1967-1975.
Frazzon, J., and D. R. Dean. 2003. Formation of iron-sulfur clusters in bacteria: an emerging field in bioinorganic chemistry. Curr. Opin. Chem. Biol. 7:166-173.
Gehrke, C. W., and K. C. Kuo. 1990. Ribonucleoside analysis by reversed-phase high performance liquid chromatography, p. A3-A71. In C. W. Gehrke and K. C. T. Kuo (ed.), Chromatography and modification of nucleosides, part A. Analytical methods for major and modified nucleosides. Elsevier, Amsterdam, The Netherlands.
Gehrke, C. W., K. C. Kuo, R. A. McCune, K. O. Gerhardt, and P. F. Agris. 1982. Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides. J. Chromatogr. 230:297-308.
Hoff, K. G., J. J. Silberg, and L. E. Vickery. 2000. Interaction of the iron-sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli. Proc. Natl. Acad. Sci. USA 97:7790-7795.
Ikeuchi, Y., N. Shigi, A. Nishimura, and T. Suzuki. 2006. Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol. Cell 21:98-108.
Jger, G., R. Leipuviene, M. G. Pollard, Q. Qian, and G. R. Bjrk. 2004. The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium. J. Bacteriol. 186:750-757.
Johnson, D., D. R. Dean, A. D. Smith, and M. K. Johnson. 2005. Structure, function, and formation of iron sulphur clusters. Annu. Rev. Biochem. 74:247-281.
Kambampati, R., and C. T. Lauhon. 1999. IscS is a sulfurtransferase for the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. Biochemistry 38:16561-16568.
Kambampati, R., and C. T. Lauhon. 2000. Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. J. Biol. Chem. 275:10727-10730.
Kambampati, R., and C. T. Lauhon. 2003. MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry 42:1109-1117.
Kim, S. R., G. Maenhaut-Michel, M. Yamada, Y. Yamamoto, K. Matsui, T. Sofuni, T. Nohmi, and H. Ohmori. 1997. Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA. Proc. Natl. Acad. Sci. USA 94:13792-13797.
Lauhon, C. T. 2002. Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli. J. Bacteriol. 184:6820-6829.
Lauhon, C. T., and R. Kambampati. 2000. The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD. J. Biol. Chem. 275:20096-20103.
Lauhon, C. T., E. Skovran, H. D. Urbina, D. M. Downs, and L. E. Vickery. 2004. Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo. J. Biol. Chem. 279:19551-19558.
Leipuviene, R., Q. Qian, and G. R. Bjrk. 2004. Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways. J. Bacteriol. 186:758-766.
Li, J. N., and G. R. Bjrk. 1999. Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity. RNA 5:395-408.
Maisiner-Patin, S., J. R. Roth, . Fredriksson, T. Nystrm, O. G. Berg, and D. I. Andersson. 2005. Genomic buffering mitigates the effects of deleterious mutations in bacteria. Nat. Genet. 37:1376-1379.
Martz, E. 2002. Protein Explorer: easy yet powerful macromolecular visualization. Trends Biochem. Sci. 27:107-109.
McCloskey, J. A., D. E. Graham, S. Zhou, P. F. Crain, M. Ibba, J. Konisky, D. Soll, and G. J. Olsen. 2001. Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales. Nucleic Acids Res. 29:4699-4706.
Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Mueller, E. G., C. J. Buck, P. M. Palenchar, L. E. Barnhart, and J. L. Paulson. 1998. Identification of a gene involved in the generation of 4-thiouridine in tRNA. Nucleic Acids Res. 26:2606-2610.
Mueller, E. G., P. M. Palenchar, and C. J. Buck. 2001. The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA. J. Biol. Chem. 276:33588-33595.
Nilsson, K., H. K. Lundgren, T. G. Hagervall, and G. R. Bjrk. 2002. The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium. J. Bacteriol. 184:6830-6835.
Ollagnier-de-Choudens, S., T. Mattioli, Y. Takahashi, and M. Fontecave. 2001. Iron-sulfur cluster assembly: characterization of IscA and evidence for a specific and functional complex with ferredoxin. J. Biol. Chem. 276:22604-22607.
Pierrel, F., G. R. Bjrk, M. Fontecave, and M. Atta. 2002. Enzymatic modification of tRNAs: MiaB is an iron-sulfur protein. J. Biol. Chem. 277:13367-13370.
Pierrel, F., H. L. Hernandez, M. K. Johnson, M. Fontecave, and M. Atta. 2003. MiaB protein from Thermotoga maritima. Characterization of an extremely thermophilic tRNA-methylthiotransferase. J. Biol. Chem. 278:29515-29524.
Ramabhadran, T. V., T. Fossum, and J. Jagger. 1976. Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid. J. Bacteriol. 128:671-672.
Schmieger, H. 1972. Phage P22-mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119:75-88.
Schwartz, C. J., J. L. Giel, T. Patschkowski, C. Luther, F. J. Ruzicka, H. Beinert, and P. J. Kiley. 2001. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc. Natl. Acad. Sci. USA 98:14895-14900.
Skovran, E., and D. M. Downs. 2000. Metabolic defects caused by mutations in the isc gene cluster in Salmonella enterica serovar Typhimurium: implications for thiamine synthesis. J. Bacteriol. 182:3896-3903.
Smith, A. D., J. N. Agar, K. A. Johnson, J. Frazzon, I. J. Amster, D. R. Dean, and M. K. Johnson. 2001. Sulfur transfer from IscS to IscU: the first step in iron-sulfur cluster biosynthesis. J. Am. Chem. Soc. 123:11103-11104.
Takahashi, Y., and M. Nakamura. 1999. Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli. J. Biochem. (Tokyo) 126:917-926.
Tokumoto, U., S. Nomura, Y. Minami, H. Mihara, S. Kato, T. Kurihara, N. Esaki, H. Kanazawa, H. Matsubara, and Y. Takahashi. 2002. Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli. J. Biochem. (Tokyo) 131:713-719.
Urbina, H. D., J. J. Silberg, K. G. Hoff, and L. E. Vickery. 2001. Transfer of sulfur from IscS to IscU during Fe/S cluster assembly. J. Biol. Chem. 276:44521-44526.
Urbonavicius, J., Q. Qian, J. M. Durand, T. G. Hagervall, and G. R. Bjrk. 2001. Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J. 20:4863-4873.
Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106.
Wagner, J., P. Gruz, S. R. Kim, M. Yamada, K. Matsui, R. P. Fuchs, and T. Nohmi. 1999. The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis. Mol. Cell 4:281-286.
Zheng, L., V. L. Cash, D. H. Flint, and D. R. Dean. 1998. Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. J. Biol. Chem. 273:13264-13272.(Hans K. Lundgren and Glen)