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 by noticing that Bacillus species with genomic ileS2 consistently also keep ileS1, which appears mandatory in the family Bacillaceae. Taking Priestia (Bacillus) megaterium as a model organism, we showed that PmIleRS1 is constitutively expressed, while PmIleRS2 is stress‐induced. Both enzymes share the same level of the aminoacylation accuracy. Yet, PmIleRS1 exhibited a two‐fold faster aminoacylation turnover (kcat) than PmIleRS2 and permitted a notably faster cell‐free translation. At the same time, PmIleRS2 displayed a 104‐fold increase in its Ki for mupirocin, arguing that the aminoacylation turnover in IleRS2 could have been traded‐off for antibiotic resistance. As expected, a P. megaterium strain deleted for ileS2 was mupirocin‐sensitive. Interestingly, an attempt to construct a mupirocin‐resistant strain lacking ileS1, a solution not found among species of the family Bacillaceae in nature, led to a viable but compromised strain. Our data suggest that PmIleRS1 is kept to promote fast translation, whereas PmIleRS2 is maintained to provide antibiotic resistance when needed. This is consistent with an emerging picture in which fast‐growing organisms predominantly use IleRS1 for competitive survival.

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 by noticing that Bacillus species with genomic ileS2 consistently also keep ileS1, which appears mandatory in the genus Bacillus. Taking Bacillus megaterium as a model organism, we showed that BmIleRS1 is constitutively expressed, while BmIleRS2 is stress-induced. Both enzymes share the same level of the aminoacylation accuracy. Yet, BmIleRS1 exhibited a two-fold faster aminoacylation turnover (kcat) than BmIleRS2 and permitted a notably faster cell-free translation. At the same time, BmIleRS2 displayed a 104-fold increase in its Ki for mupirocin, arguing that the aminoacylation turnover in IleRS2 could have been traded-off for antibiotic resistance. As expected, a B. megaterium strain deleted for ileS2 was mupirocin-sensitive. We also succeed in the construction of a mupirocin-resistant strain lacking ileS1, a solution not found among Bacilli in nature. However, B. megaterium ΔileS1 displayed a severe fitness loss and was seriously compromised in biofilm formation. Neither of these phenotypes were rescued by BmIleRS2 expressed from the constitutive promotor of ileS1. Thus, BmIleRS1 safeguards B. megaterium fitness, likely by promoting faster translation than BmIleRS2, whereas the later enzyme is maintained to provide antibiotic resistance when needed. Our data are consistent with an emerging picture in which fast-growing organisms predominantly use IleRS1 for competitive survival.