The miaA tRNA modification gene was cloned and located by insertion mutagenesis and DNA sequence analysis. The miaA gene product, tRNA delta 2-isopentenylpyrophosphate (IPP) transferase, catalyzes the first step in the biosynthesis of 2-methylthio-N6-(delta 2-isopentenyl)-adenosine (ms2i6A) adjacent to the anticodon of several tRNA species. The translation start of miaA was deduced by comparison with mod5, which encodes a homologous enzyme in yeasts. Minicell experiments showed that Escherichia coli IPP transferase has a molecular mass of 33.5 kilodaltons (kDa). Transcriptional fusions, plasmid and chromosomal cassette insertion mutations, and RNase T2 mapping of in vivo miaA transcription were used to examine the relationship between miaA and mutL, which encodes a polypeptide necessary for methyl-directed mismatch repair. The combined results showed that miaA, mutL, and a gene that encodes a 47-kDa polypeptide occur very close together, are transcribed in the same direction in the order 47-kDa polypeptide gene-mutL-miaA, and likely form a complex operon containing a weak internal promoter. Three additional relationships were demonstrated between mutagenesis and the miaA gene or ms2i6A tRNA modification. First, miaA transcription was induced by 2-aminopurine. Second, chromosomal miaA insertion mutations increased the spontaneous mutation frequency with a spectrum distinct from mutL mutations. Third, limitation of miaA+ bacteria for iron, which causes tRNA undermodification from ms2i6A to i6A, also increased spontaneous mutation frequency. These results support the notion that complex operons organize metabolically related genes whose primary functions appear to be completely different. In addition, the results are consistent with the idea that mechanisms exist to increase spontaneous mutation frequency when cells need to adapt to environmental stress.