N 7-methylguanine at position 46 (m 7G46) in tRNA is produced by tRNA (m 7G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant ( ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m 7G46 modification in tRNA Phe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m 7G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m 1G37, suggesting that the m 7G46 positively affects their formations. Although the lack of the m 7G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA Phe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNA Phe and tRNA Ile. 35S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m 7G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m 7G46 modification supports introduction of other modifications.