Fig 6.

B. subtilis mnm⁵s²U pathway involves YtqA and MnmM. (A) The inactivation of ytqA and mnnM genes affects the relative accumulation of mnm⁵s²U and pathway intermediates. Modified nucleosides were extracted from B. subtilis wild type, DytqA, and DmnmM strains were cultured in LB medium until OD of 1. (B) B. subtilis YtqA-MnmM complements defects associated with E. coli DmnmC inactivation. E. coli wild-type MG1655 and DmnmC strain transformed cells with empty plasmid (pBAD), pDS358 (pBAD-ytqA-mnmM), pDS359 (pBAD-ytqA), or pDS365 (pBAD-mnmM) were cultured in LB ampicillin and arabinose until OD of 1. tRNA samples were isolated using the PDS method and analyzed using LC-MS method 1. Relative levels of each modification were determined by normalizing the mass abundance associated with each modification to the mass abundance of dihydrouridine in the same sample. The reported averages were calculated based on data acquired from three independent growth experiments. The star mark denotes the absence of modification or levels below the detection limit.