FIG 5.

Effects of other genes on xo⁵U levels. (A) HPLC analysis of iscS, yfhL, and aroD in E. coli. The iscS mutant lacks thionucleosides s²C and mnm⁵s²U and cmnm⁵s²U in this region but still retains cmo⁵U, which is decreased to a similar level as the yegQ mutant (shown in panel B). The overnight iscS mutant culture shows significant increases in s²C and some increase in cmo⁵U, similar to increases in other Fe-S-dependent modifications. The effect on cmo⁵U is not statistically significant due to variability in the log-phase data. An E. coli strain lacking the ferredoxin yfhL shows a similar HPLC spectrum as that of the yegQ mutant and a similar level of reduction in cmo⁵U (shown in panel B). In E. coli, shikimate pathway genes, such as aroD, are required for conversion of ho⁵U to cmo⁵U and for wt levels of ho⁵U, as previously reported (15, 19). Quantifying the level of ho⁵U shows the same reduction in ho⁵U as for cmo⁵U in the yegQ and yfhL mutants. (B) Quantitative analysis of HPLC data for E. coli aro, iscS, and yfhL mutants relative to those of the wt strain. *, the aroD strain lacks the ability to synthesize cmo⁵U from ho⁵U and thus the ratio reported here is for ho⁵U/Ψ. ***, P < 0.001 compared to wt (unpaired t test); **, P < 0.01 compared to wt; ns = no statistical significance among these strains or with the wt strain.