Relaxed Substrate Specificity Leads to Extensive tRNA Mischarging by Streptococcus pneumoniae Class I and Class II Aminoacyl-tRNA Synthetases Jennifer Shepherd, Michael Ibba Department of Microbiology and Center for RNA Biology, Ohio State University, Columbus, Ohio, USA ABSTRACT Aminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances by cis- and trans- editing pathways, it has been revealed that in organisms such as Streptococcus pneumoniae some aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacyla- tion profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumo- coccal IleRS mischarged tRNA Ile with both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent man- ner. IleRS substrate specificity was achieved in an editing-independent manner, indicating that tRNA mischarging would only be significant under growth conditions where Ile is depleted. Pneumococcal LysRS was found to misaminoacylate tRNA Lys with Ala and to a lesser extent Thr and Ser, with mischarging efficiency modulated by the presence of an unusual U4:G69 wobble pair in the acceptor stems of both pneumococcal tRNA Lys isoacceptors. Addition of the trans-editing factor MurM, which also functions in peptidoglycan synthesis, reduced Ala-tRNA Lys production by LysRS, providing evidence for cross talk between the protein synthesis and cell wall biogenesis pathways. Mischarging of tRNA Lys by AlaRS was also observed, and this would provide addi- tional potential MurM substrates. More broadly, the extensive mischarging activities now described for a number of Streptococ- cus pneumoniae aminoacyl-tRNA synthetases suggest that adaptive misaminoacylation may contribute significantly to the via- bility of this pathogen during amino acid starvation. IMPORTANCE Streptococcus pneumoniae is a common causative agent of several debilitating and potentially life-threatening in- fections, such as pneumonia, meningitis, and infectious endocarditis. Such infections are increasingly difficult to treat due to widespread development of penicillin resistance. High-level penicillin resistance is known to depend in part upon MurM, a pro- tein involved in both aminoacyl-tRNA-dependent synthesis of indirect amino acid cross-linkages within cell wall peptidoglycan and in translation quality control. The involvement of MurM in both protein synthesis and antibiotic resistance identify it as a potential target for the development of new and potent antibiotics for pneumococcal infections. The goals of this work were to identify and characterize S. pneumoniae pathways that can synthesize mischarged tRNAs and to relate these activities to ex- pected changes in protein and peptidoglycan biosynthesis during antibiotic and nutritional stress. Received 25 July 2014 Accepted 6 August 2014 Published 9 September 2014 Citation Shepherd J, Ibba M. 2014. Relaxed substrate specificity leads to extensive tRNA mischarging by Streptococcus pneumoniae class I and class II aminoacyl-tRNA synthetases. mBio 5(5):e01656-14. doi:10.1128/mBio.01656-14. Editor Susan Gottesman, National Cancer Institute Copyright © 2014 Shepherd and Ibba. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Michael Ibba, ibba.1@osu.edu. This article is a direct contribution from a Fellow of the American Academy of Microbiology. S treptococcus pneumoniae is a Gram-positive diplococcus that can be carried asymptomatically in the nasopharynx of healthy individuals. The bacterium is also a significant patho- gen and is the common causative agent of many community- and hospital-acquired infections, such as pneumonia and men- ingitis. In order to successfully colonize the nasopharynx in direct competition with other bacteria, including Haemophilus influenzae, S. pneumoniae routinely produces high levels of the oxidative stressor hydrogen peroxide (1–3). Increased levels of hydrogen peroxide have been directly correlated with en- hanced cellular mistranslation rates in other microorganisms (4, 5). When taken together with the finding that pneumococci lack the four typical oxidative stress regulons of other bacteria (RpoS, OxyR, SoxRS, and Mar), it is unclear how this patho- gen maintains translational fidelity during its normal life cycle (6, 7). The aminoacyl-tRNA synthetases (aaRSs) establish and main- tain the genetic code by specifically activating their cognate amino acid with ATP to form an aminoacyl-adenylate, which can then be transferred to the cognate tRNA acceptor molecule (8, 9). There are 20 aaRS enzymes in total and they correspond to the 20 stan- dard amino acids present in the cell. Each aaRS is categorized as class I or class II, based on the overall structure and function, except for lysyl-tRNA synthetase (LysRS), which has representa- tives in both classes (10–14). Common features of class I aaRSs include a HIGH/KMSKS-motif-defined Rossmann nucleotide- RESEARCH ARTICLE crossmark September/October 2014 Volume 5 Issue 5 e01656-14 ® mbio.asm.org 1