In the wobble position of tRNAs specific for Gln, Lys, and Glu a universally conserved 5-methylene- 2-thiouridine derivative (xm(5) s(2) U34, x denotes any of several chemical substituents and 34 denotes the wobble position) is present, which is 5-(carboxy) methylaminomethyl- 2-thiouridine ((c) mnm(5) s(2) U34) in Bacteria and 5-methylcarboxymethyl-2-thiouridine (mcm(5) s(2) U34) in Eukarya. Here we show that mutants of the bacterium Salmonella enterica Serovar Typhimurium LT2 lacking either the s(2) - or the (c) mnm(5) -group of (c) mnm(5) s(2) U34 grow poorly especially at low temperature and do not grow at all at 15 degrees C in both rich and glucose minimal media. A double mutant of S. enterica lacking both the s(2)- and the (c) mnm(5)-groups, and that thus has an unmodified uridine as wobble nucleoside, is nonviable at different temperatures. Overexpression of tRN(cmnm5s2UUG)(AGln) lacking either the s(2) - or the (c) mnm(5)-group and of tRNA(mnm5s2UUU)(Lys) lacking the s(2) -group exaggerated the reduced growth induced by the modification deficiency, whereas overexpression of tRNA(mnm5s2UUU)(Lys) lacking the mnm(5)-group did not. From these results we suggest that the primary function of cmnm(5) s(2) U34 in bacterial tRNA(cmnm5s2UUG)(Gln) and mnm(5) s(2) U34 in tRNA(Lys) (mnm5s2UUU) is to prevent missense errors, but the mnm(5) -group of tRNA(Lys) (mnm5s2UUU) does not. However, other translational errors causing the growth defect cannot be excluded. These results are in contrast to what is found in yeast, since overexpression of the corresponding hypomodified yeast tRNAs instead counteracts the modification deficient induced phenotypes. Accordingly, it was suggested that the primary function of mcm(5) s(2) U34 in these yeast tRNAs is to improve cognate codon reading rather than prevents missense errors. Thus, although the xm(5) s(2) U34 derivatives are universally conserved, their major functional impact on bacterial and eukaryotic tRNAs may be different.