Mistranslation Drives the Evolution of Robustness in TEM-1 Β-Lactamase
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Mistranslation drives the evolution of robustness in TEM-1 β-lactamase Sinisa Bratulica,b, Florian Gerberc, and Andreas Wagnera,b,d,1 aInstitute of Evolutionary Biology and Environmental Studies, University of Zurich, CH-8057 Zurich, Switzerland; bSwiss Institute of Bioinformatics, Quartier Sorge - Batiment Genopode, CH-1015 Lausanne, Switzerland; cInstitute of Mathematics, University of Zurich, CH-8057 Zurich, Switzerland; and dThe Santa Fe Institute, Santa Fe, NM 87501 Edited by Richard E. Lenski, Michigan State University, East Lansing, MI, and approved August 26, 2015 (received for review May 28, 2015) How biological systems such as proteins achieve robustness to A second group of mechanisms does not reduce mistranslation ubiquitous perturbations is a fundamental biological question. itself, but mitigates its deleterious consequences, and thus in- Such perturbations include errors that introduce phenotypic muta- creases the translational robustness of proteins. Some error- tions into nascent proteins during the translation of mRNA. These mitigation mechanisms are global and affect many proteins. They errors are remarkably frequent. They are also costly, because they include chaperones that can help proteins fold even if they reduce protein stability and help create toxic misfolded proteins. harbor destabilizing mutations (15). In contrast to such global Adaptive evolution might reduce these costs of protein mistrans- mechanisms, which can become overwhelmed when mistranslation lation by two principal mechanisms. The first increases the rates are high, local mechanisms are less costly. They rely on accuracy of translation via synonymous “high fidelity” codons at (genetic) mutations called suppressors, which increase the stability especially sensitive sites. The second increases the robustness of of a single protein, and thus buffer destabilizing effects of other proteins to phenotypic errors via amino acids that increase protein (phenotypic) mutations (16–18). stability. To study how these mechanisms are exploited by popu- The only experimental study of protein evolution under lations evolving in the laboratory, we evolved the antibiotic re- phenotypic mutations focused on errors in transcription (19). It Escherichia coli sistance gene TEM-1 in hosts with either normal or could not answer whether proteins evolve to reduce the mu- high rates of mistranslation. We analyzed TEM-1 populations that tational load of mistranslation by increasing their translational evolved under relaxed and stringent selection for antibiotic resis- accuracy, or by mitigating the effect of errors resulting from tance by single molecule real-time sequencing. Under relaxed selec- such accuracy. Here, we address this question by evolving genes tion, mistranslating populations reduce mistranslation costs by encoding the antibiotic resistance protein TEM-1 β-lactamase reducing TEM-1 expression. Under stringent selection, they effi- in strains of E. coli with different rates of mistranslation. We ciently purge destabilizing amino acid changes. More importantly, also ask whether relaxed and stringent selection for antibiotic they accumulate stabilizing amino acid changes rather than syn- resistance affect the adaptation to elevated mistranslation in onymous changes that increase translational accuracy. In the large populations we study, and on short evolutionary timescales, the different ways. Under relaxed selection, mistranslating pop- path of least resistance in TEM-1 evolution consists of reducing the ulations adapt by reducing TEM-1 expression through in- consequences of translation errors rather than the errors themselves. efficient initiation codons, which lowers the cost of mistranslation. Under stringent selection, where reducing gene expression molecular evolution | mutational robustness | phenotypic mutations | would be detrimental, populations increase translational ro- protein stability | antibiotic resistance bustness by accumulating stabilizing and purging destabi- lizing single nucleotide polymorphisms (SNPs). rotein synthesis or translation is a key step in genetic in- Pformation processing. Despite being fundamental to all cel- Significance lular life, translation is remarkably error prone. Mistranslation events are estimated to occur once per 102–104 codons (1–3). Translation is a fundamental biochemical process in which When mRNA is mistranslated, synthesized proteins carry phe- ribosomes use an mRNA’s nucleotide sequence as a template notypic mutations in positions where ribosomes incorrectly decoded to synthesize a protein with a specific amino acid sequence. the mRNA. A pool of proteins with such phenotypic mutations, also Errors in this process are deleterious because they can alter a ’ called statistical proteins (4), can differ from error-free proteins in protein s structure. Yet such errors are surprisingly frequent. sequence, structure, and function. Here we ask whether and how evolution can affect the ability Comparative genomics studies show that mistranslation can of proteins to cope with these errors. In principle, evolution help explain why highly expressed proteins evolve slowly (5–8). could reduce the rate of such errors, or it could leave this rate All else being equal, highly expressed genes experience more unchanged but reduce the damaging effects of errors. We find that populations of proteins evolving in the laboratory translation events, and thus give rise to a higher number of mis- pursue the second route, increasing their robustness to translated proteins (6, 7). Mistranslation is costly because it can translation errors. Evolution may preferentially mitigate damage destabilize proteins, and increases proteotoxic stress by promoting to a biological system than reduce the source of this damage. protein misfolding and aggregation (2, 9, 10). Natural selection can reduce mistranslation costs via two non- Author contributions: S.B. and A.W. designed research; S.B. performed research; S.B., F.G., mutually-exclusive groups of mechanisms. The first increases and A.W. analyzed data; and S.B., F.G., and A.W. wrote the paper. translational accuracy, i.e., it reduces the rate at which trans- The authors declare no conflict of interest. lational errors occur. A global increase in accuracy, for example This article is a PNAS Direct Submission. through hyper-accurate ribosomes, affects all proteins but comes Data deposition: Sequences reported in this paper have been deposited in the GenBank with high energetic and kinetic costs (11). Alternatively, trans- database (accession codes KT391064–KT423096). lational accuracy can increase locally at amino acid sites where See Commentary on page 12553. (phenotypic) mutations would cause the largest fitness defects 1To whom correspondence should be addressed. Email: [email protected]. (12). This increase is possible through synonymous mutations to- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ward codons with a low propensity for mistranslation (1, 13, 14). 1073/pnas.1510071112/-/DCSupplemental. 12758–12763 | PNAS | October 13, 2015 | vol. 112 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1510071112 Downloaded by guest on October 1, 2021 Results per variant, implying a mutation rate of ≈ 8 × 10−4 per nucleo- To study how proteins adapt to mistranslation in evolving labo- tide. Consistent with reports from previous studies (21, 22), our SEE COMMENTARY ratory populations, we experimentally evolved TEM-1 β-lacta- mutagenesis protocol is A →G and T →Cbiased(SI Appendix, mase independently in two E. coli strains, the wild type, and the Table S2). mistranslating, or error-prone rpsD12 strain (Fig. 1). The error- prone strain carries a mutation in the ribosomal protein S4, Mistranslation Slows down TEM-1 Evolution. The TEM-1 protein which results in increased missense, readthrough, and frameshift has two parts: the N-terminal signal peptide (the first 25 residues (phenotypic) mutations during protein synthesis (1). Specifically, in Ambler numbering; ref. 23), and the mature enzyme. The we evolved TEM-1 in populations of 108–109 individuals for signal peptide guides the translocation of TEM-1 to the peri- eight cycles of PCR-based mutagenesis and selection (Fig. 1), plasmic space. Once translocation is complete, the signal se- with four replicate populations for each of the two host strains quence is cleaved and the mature TEM-1 folds into its active − and each of the two selection conditions (relaxed: 25 μgmL 1 conformation. The signal peptide controls the expression, and − ampicillin; stringent: 250 μgmL 1 ampicillin). We also evolved the localization of TEM-1. We observe that SNPs in the signal two replicate control populations per strain. These populations peptide have frequencies up to ≈ 26% (SI Appendix, Fig. S1 and were mutagenized in the same way as evolved populations, but Table S7). In contrast, SNPs in the mature part of TEM-1 all experienced no selection for β-lactamase activity. We sequenced have frequencies below 5% (SI Appendix, Fig. S1 and Tables S5 more than 500 evolved molecules per population using single and S6). molecule real-time (SMRT) sequencing (20) (SI Appendix, Next, we compared the average number of SNPs per TEM-1 Table S1). variant among the two strains. Wild-type populations have a From the sequenced library of ancestral TEM-1 (395 se- higher relative frequency of variants with more SNPs than error- quences), we estimated the compound sequencing and variant prone populations (Fig. 2A), and selection makes