engleski

Two IleRSs in Bacillus megaterium: is there a difference?

Isoleucyl-tRNA synthetase (IleRS) catalyses covalent coupling of tRNAIle with cognate isoleucine, thus providing aminoacylated tRNA for protein biosynthesis. During aminoacylation, isoleucine is firstly activated to form isoleucyl- adenylate intermediate. The second step then comprises the transfer of Ile to the cognate tRNAIle. Based on phylogenetic analysis, bacterial IleRSs group in two distinct clades: bIleRS1 (bacteria-like) and bIleRS2 (so-called eukaryote- like) [1]. The two IleRS types differ in mupirocin resistance. Mupirocin is an antibiotic that binds with a high affinity to the synthetic site of bIleRS1, thus inhibiting formation of Ile-tRNAIle and blocking protein biosynthesis. At the same time, bIleRS2 is less susceptible to mupirocin inhibition. Generally, either bIleRS1 or bIleRS2 is present in the cell. However, several Bacilli species, like Bacillus megaterium, are known to have both bacterial IleRS types in their genome. To gather insight into a difference between two IleRSs in B. megaterium, BmIleRS1 and BmIleRS2, both ileS genes were cloned, overexpressed in E. coli, and purified using Ni-NTA affinity chromatography. Native total tRNA was isolated from B. megaterium and specific tRNAIleGAT isoacceptor was purified using complementary biotinylated oligonucleotide. In parallel, the gene for tRNAIleGAT was cloned and overexpressed in E. coli. Kinetic analysis revealed that BmIleRS2 shows an unusually high Km for isoleucine in the activation step. The Km (Ile) was 30-fold higher compared to the Km (Ile) for BmIleRS1. When aminoacylation was followed, a substantially smaller difference in the Km values for isoleucine between BmIleRS1 and BmIleRS2 was observed. Thus, our results indicate that BmIleRS1 and BmIleRS2 exhibit some differences in the aminoacylation mechanisms. Based on our preliminary data, BmIleRS2 displays lower affinity than BmIleRS1 towards mupirocin. However, detailed kinetic analyses are undergoing to address that issue. In silico analysis revealed that the gene for BmIleRS1 is under a constitutive promoter, while transcription of BmIleRS2 seems to be induced under stress conditions. This is in agreement with expected lower mupirocin sensitivity of BmIleRS2. We hypothesised that stress conditions could also be related to a temperature stress, so we measured protein’s thermal stability. Interestingly, BmIleRS2 possessed a significantly higher Tm (65 °C) compared to BmIleRS1 (55 °C). This is surprising as usually antibiotic resistance evolves at the expense of protein stability. In conclusion, BmIleRS1 and BmIleRS2 differ in some aspects of the aminoacylation mechanism, transcriptional regulation and likely mupirocin susceptibility. Why BmIleRS2 didn’t prevail, and both IleRS types exist in B. megaterium, remains unanswered. Further in vivo and in vitro analysis will show whether the requirement for antibiotic resistance in B. megaterium compromised BmIleRS2 catalytic efficiency/accuracy, to a point where presence of BmIleRS1 is essential. However, a possibility that it is an evolution’s dead-end could not be excluded at this point.

isoleucyl-tRNA synthetase, Bacillus megaterium, mupirocin resistance

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