A pair of isoleucyl-tRNA synthetases in Bacilli fulfill complementary roles to keep fast translation and provide antibiotic resistance

Abstract

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.

Antibiotic hyper-resistance in a class I aminoacyl-tRNA synthetase with altered active site signature motif

“…Mupirocin strongly inhibits IleRS1 and modestly IleRS2, with the difference in K i ranging from 10 3 -10 5 -fold. Accordingly, the presence of IleRS2 as a sole 16 or a second 19 gene in the genome or on a plasmid 20 confers the resistance. Antibiotic resistance is a major public health problem worldwide 3 and resolving the underlying mechanisms by which it evolves and operates broadens our capacity to tackle it.…”

“…However, did the same selective forces hold for both IleRS types? IleRS1 is found preferentially in faster-while IleRS2 in slower-growing bacteria 19 , raising the question whether a negative selection against catalytic trade-offs more prominently shaped IleRS1. That said, clinical isolates of S. aureus, including MRSA strains 33 , could evolve only a low resistance by IleRS1 mutations, while bacterial isolates with a higher IleRS1 resistance can be obtained exclusively in the laboratory because of their compromised fitness.…”

“…In some cases, they even reach millimolar levels 16 , which we term here "hyper-resistance". IleRS1 and IleRS2 generally occur individually in bacteria, but there is a small group of Bacillaceae that carry both genes in the genome 19 . IleRS2 plays an important role in providing mupirocin resistance to bacteria in hospital environments, since mupirocin-resistant Staphylococcus aureus isolates acquired the ileS2 gene on a plasmid 20 .…”

Diversification of aminoacyl-tRNA synthetase activities via genomic duplication

“…These strains have acquired a plasmid encoded IleRS that is insensitive to mupirocin (Hodgson et al, 1994). Barring the low activity of IleRS2, its retained editing capacity and amino acid specificity compensates for the sensitivity of IleRS1 to mupirocin (Brown et al, 2003;Zanki et al, 2022).…”

“…IleRS2 is resistant to mupirocin(Zanki et al, 2022) (a) SbzA transfers Ile onto altemicidin(Hu et al, 2019) LeuRS (a) LeuRS-I produces leucyl-adenylates(Weitzel et al, 2020) -(b)LeuRS2 produces low levels of Leu-tRNA Leu(Fang et al, 2014) LysRS -(a) LysU produces Lys-tRNA Lys under stress(Brevet et al, 1995) (b) PoxA (GenX, YjeA) transfers β-lysine onto EF-P(Yanagisawa et al, 2010;Roy et al, 2011) (c) LysX transfers Lys to peptidoglycan(Maloney et al, 2009) ProRS -(a) ProRSx inserts Pro at Ala codons (Vargas-Rodriguez et al, 2021) PylRS -(a) PylRS2 aminoacylates cognate tRNA Pyl (Zhang et al, 2022) SerRS (a) SerRS2 is resistant to albomycin (Zhou et al, 2019) (a) SLIMP regulates cell-cycle progression (Picchioni et al, 2019) (b) aa:CP ligases add Ser to Ppant which is linked to a carrier protein (Mocibob et al, 2010) ThrRS (a) T2 produces Ser-tRNA Thr in low zinc conditions (Rubio et al, 2015) (a) ThrRS-L plays a role in the MSC and recycles tRNA Thr for ThrRS under stress (Zhou et al, 2019) (b) ThrRS-L produces Thr-tRNA Thr but with poor editing activity (Zhou et al, 2013) TrpRS (a) TrpRSII nitrates Trp on Trp-tRNA Trp in toxic environments (Buddha et al, 2004a; Buddha et al, 2004b) -(b) TrpRS1 is resistant to indolmycin (Kitabatake et al, 2002; Vecchione and Sello, 2009) TyrRS (a) TyrZ produces Tyr-tRNA Tyr with high selectivity for l-Tyr under stress (Williams-Wagner et al, 2015) -(b) Two fused TyrRSs produce 1 or 2 Tyr-tRNA Tyr(Duchêne et al, 2005;Larson et al, 2011) …”