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Isoleucyl-tRNA synthetase (IleRS) catalyzes covalent coupling of isoleucine and tRNA1le for protein synthesis. Several Bacilli species have two distinct types of ileS gene. While ileS1 seems to be following species phylogeny tree, ileS2 distribution among species is best explained by horizontal gene transfer. To understand requirements for having both ileS genes, we used B. megaterium as a model organism and performed kinetic analyses on the isolated enzymes and in vivo analyses on the constructed knockout strains. We found that BmIleRS2 exhibits 25-fold higher KM for isoleucine than BmIleRS1 and at the same time a 1000-fold higher Ki for antibiotic mupirocin. Interestingly, while mupirocin acts as a fast- on/fast-off competitive inhibitor of BmIleRS2, it displays a slow-tight binding type of inhibition with BmIleRS1. Our data further indicate that interaction of BmIleRS1 and BmIleRS2 with isoleucine is modulated by tRNA1le but in a different way. Whether and how distinct mupirocin interaction with BmIleRS1 and BmIleRS2 is related to the observed different KM values of these enzymes toward isoleucine is still not clear, however, it may suggest that aminoacylation mechanisms could have distinctly evolved in these enzymes to account for the trade-off between mupirocin resistance and amino acid affinity. Knockout strains lacking either bmileS1(ΔileS1) or bmileS2 (ΔileS2) were created to address cellular demands for both ileS genes. Although both knockout strains were viable, ΔileS1 exhibited slower growth, inefficient sporulation and deviation in biofilm formation compared to the wild-type strain. In a minimal medium, ΔileS1 strain is outcompeted by ΔileS2 strain, suggesting bmileS1 in under constant selective pressure to remain in the genome. These results show that bmileS2, carrying antibiotic resistance, can complement bmileS1 as a sole housekeeping gene, however, BmIleRS1 is essential for overall fitness of B. megaterium. |
