Updated PubMed. Isoleucyl-tRNA synthetase (IleRS) is an essential enzyme that covalently couples isoleucine to the corresponding tRNA. Bacterial IleRSs group in two clades, ileS1 and ileS2, the latter bringing resistance to the natural antibiotic mupirocin. Generally, bacteria rely on either ileS1 or ileS2 as a standalone housekeeping gene. However, we have found an exception in Bacillus species: in family Bacillaceae ileS1 appears mandatory, but some of the Bacilli posses additional, ileS2 gene. Taking Priestia (Bacillus) megaterium as a model organism, we showed that PmIleRS1 is constitutively expressed, while PmIleRS2 is stress-induced. As part of our investigation of ileS1/ileS2 function, we explored the expression of ileS1 and ileS2 under control conditions and upon mupirocin treatment.