Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli

The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is defic ient in the methylthio group of the modified nucleoside N-6-(4-hydroxyisope ntenyl)-2-methylthioadenosine (ms(2)io(6)A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into the f474 (yleA) open rea ding frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508:: Tn10dCm mutation with a minimal subcloned f474 fragment, we showed that f474 could be ident ified as the miaB gene, which is transcribed in the counterclockwise direct ion on the bacterial chromosome. Transcriptional studies revealed two promo ters upstream of miaB in E. coli and S. typhimurium. A Rho-independent term inator was identified downstream of the miaB gene, at which the majority (9 6%) of the miaB transcripts terminate in E. coli, showing that the miaB gen e is part of a monocistronic operon. A highly conserved motif with three cy steine residues was present in MiaB. This motif resembles iron-binding site s in other proteins. Only a weak similarity to an AdoMet-binding site was f ound, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms(2)i(o)(6)A37 formation.