Eva Garmendia

Synonymous mutations are changes to DNA sequence, which occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation elongation factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels as well as global polysome abundance on RNA transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes. IMPORTANCE: This study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein elongation factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

Synonymous mutations are changes to DNA sequence that occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation Elongation Factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels, as well as the polysome abundance on global transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes. Importance: This study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein Elongation Factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

Bacterial chromosomes frequently carry multiple copies of genes at separate chromosomal locations. In Salmonella , these include the 7 rrn operons and the duplicate tuf genes.

A feature of bacterial chromosomes is that highly expressed essential genes are usually located close to the origin of replication. Because bacteria have overlapping cycles of replication, genes located close to the origin will often be present in multiple copies, and this is thought to be of selective benefit where high levels of expression support high growth rate. However, the magnitude of this selective effect and whether other forces could be at play are poorly understood. To study this, we translocated a highly expressed essential operon, tufB , to different locations and measured growth fitness. We found that transcriptional regulation buffered the effects of translocation and that even under conditions where growth rate was reduced, genetic changes that increased the expression of tufB were easily and rapidly selected. We conclude, at least for tufB , that forces other than gene dosage may be significant in selecting for chromosomal location.

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenoustufAgene in theEscherichia coligenome with its extant and ancestral homologs. The extant homologs representedtufvariants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrectedtufsequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreigntufgenes are transferable to theE. coligenome, provided that an additional copy of the EF-Tu gene,tufB, remains present in theE. coligenome. However, when thetufBgene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability ofE. coli tufwith its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extanttufhomologs inserted into theE. coligenome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression oftufpartially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCEHorizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacingtufwith ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrectedtufsequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability ofE. coli tufwith its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Summary: RNase E is an essential bacterial endoribonuclease with a central role in processing tRNAs and rRNA, and turning over mRNAs. Previous studies in strains carrying mutations in the rne structural gene have shown that tRNA processing is likely to be an essential function of RNase E but have not determined whether mRNA turnover is also an essential function. To address this we selected extragenic suppressors of temperature‐sensitive mutations in rne that cause a large increase in mRNA half‐life at the non‐permissive temperature. Fifteen suppressors were mapped to three different loci: relBE (toxin‐antitoxin system); vacB (RNase R); and rpsA (ribosomal protein S1). Each suppressor class has the potential to interact with mRNA and each restores wild‐type levels of mRNA turnover but does not reverse the minor defects in tRNA and rRNA processing. RelE toxin is especially interesting because its only known activity is to cleave mRNAs in the ribosomal A‐site. The relBE suppressor mutations increase transcription of relE, and controlled overexpression of RelE alone was sufficient to suppress the rne ts phenotype. Suppression increased turnover of some major mRNAs (tufA, ompA) but not all mRNAs. We propose that turnover of some mRNAs is one of the essential functions of RNase E.

Hepatitis C virus (HCV) replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE), located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80%) of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.