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Journal of Bacteriology, December 2002, p. 6830-6835, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6830-6835.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Cysteine Desulfurase IscS Is Required for Synthesis of All Five Thiolated Nucleosides Present in tRNA from Salmonella enterica Serovar Typhimurium

Kristina Nilsson, Hans K. Lundgren, Tord G. Hagervall, and Glenn R. Björk*

Department of Molecular Biology, Umeå University, S-90187 Umeå University, Sweden

Received 10 June 2002/ Accepted 4 September 2002

Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations. In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene. This site may be suppressed by peptidyl-tRNAProcmo5UGG (cmo5U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNAGlnmnm5s2UUG (mnm5s2U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site. We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS. Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm5s2U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S. enterica were reduced in the iscS51 mutant. In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level. After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm5s2U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNAGlnmnm5s2UUG due to mnm5s2U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mnmA) lacking only mnm5s2U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm5s2U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant.

* Corresponding author. Mailing address: Department of Molecular Biology, Umeå University, S-90187 Umeå University, Sweden. Phone: 46 90 7856756. Fax: 46 90 772630. E-mail: glenn.bjork{at}molbiol.umu.se.

Journal of Bacteriology, December 2002, p. 6830-6835, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6830-6835.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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