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When new genes evolve through modification of existing genes, there are often trade-offs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near cognate stop codons and highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds not only to alterations in the demand for RF2 activity, but also for RF1 activity.

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the T Psi C-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 angstrom resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon. Here, the authors report de novo design, optimization and characterization of tRNAs that decode UGA stop codons in E. coli. The structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon suggests that distinct A-site ligands (tRNAs versus release factors) induce distinct conformation of the stop codon within the mRNA in the decoding center.

RNase P is an essential enzyme found across all domains of life that is responsible for the 5 '-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli. In E. coli, the temperature-sensitive (ts) rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme's activity in vivo. Here, we report for the first time naturally occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the ts defect via gene amplifications of either RNase P subunit (rnpA49 or rnpβ) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.

Translation terminates by releasing the polypeptide chain in one of two chemical reactions catalyzed by the ribosome. Release is also a target for engineering, as readthrough of a stop codon enables incorporation of unnatural amino acids and treatment of genetic diseases. Hydrolysis of the ester bond of peptidyl-tRNA requires conformational changes of both a class I release factor (RF) protein and the peptidyl transferase center of a large subunit rRNA. The rate-limiting step was proposed to be hydrolysis at physiological pH and an RF conformational change at higher pH, but evidence was indirect. Here, we tested this by activating the ester electrophile at the Escherichia coli ribosomal P site using a trifluorine-substituted amino acid. Quench-flow kinetics revealed that RF1-catalyzed release could be accelerated, but only at pH 6.2-7.7 and not higher pH. This provided direct evidence for rate-limiting hydrolysis at physiological or lower pH and a different rate limitation at higher pH. Additionally, we optimized RF-free release catalyzed by unacylated tRNA or the CCA trinucleotide (in 30% acetone). We determined that these two model release reactions, although very slow, were surprisingly accelerated by the trifluorine analog but to a different extent from each other and from RF-catalyzed release. Hence, hydrolysis was rate limiting in all three reactions. Furthermore, in 20% ethanol, we found that there was significant competition between fMet-ethyl ester formation and release in all three release reactions. We thus favor proposed mechanisms for translation termination that do not require a fully-negatively-charged OH⁻ nucleophile.

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the ‘critical region’ of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg²⁺. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

Chromoproteins (CPs) are widely-used visual reporters of gene expression. We previously showed that, for coloration in Escherichia coli, CPs had to be overexpressed and that this caused large fitness costs with the most useful (darkly colored) CPs. These fitness costs were problematic because passage of plasmids encoding darkly colored CPs in liquid culture frequently resulted in loss of color due to mutations. Unexpectedly, an early variant of the monomeric red fluorescent protein 1 (mRFP1) gene that was codon-optimized for E. coli (abbreviated mRFP1E) was found here to be an ideal replacement for CP genes. When we subcloned mRFP1E in the same way as our CP genes, it produced a similarly dark color, yet affected E. coli fitness minimally. This finding facilitated testing of several hypotheses on the cause of CP cytotoxicities by gel electrophoresis and size-exclusion chromatography: toxicities correlated with the combination of amounts of expression, oligomerization and inclusion bodies, not isoelectric point. Finally, a semi-rational mutagenesis strategy created several mRFP1 protein variants with different colors without altering the fitness cost. Thus, these mutants and mRFP1E are suitable for comparative fitness costs between different strains of E. coli. We conclude that our new mRFP1E series overcomes prior limitations of CPs.

Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.

Background: The Mycobacterium genus encompasses at least 192 named species, many of which cause severe diseases such as tuberculosis. Non-tuberculosis mycobacteria (NTM) can also infect humans and animals. Some are of emerging concern because they show high resistance to commonly used antibiotics while others are used and evaluated in bioremediation or included in anticancer vaccines.

Results: We provide the genome sequences for 114 mycobacterial type strains and together with 130 available mycobacterial genomes we generated a phylogenetic tree based on 387 core genes and supported by average nucleotide identity (ANI) data. The 244 genome sequences cover most of the species constituting the Mycobacterium genus. The genome sizes ranged from 3.2 to 8.1 Mb with an average of 5.7 Mb, and we identified 14 new plasmids. Moreover, mycobacterial genomes consisted of phage-like sequences ranging between 0 and 4.64% dependent on mycobacteria while the number of IS elements varied between 1 and 290. Our data also revealed that, depending on the mycobacteria, the number of tRNA and non-coding (nc) RNA genes differ and that their positions on the chromosome varied. We identified a conserved core set of 12 ncRNAs, 43 tRNAs and 18 aminoacyl-tRNA synthetases among mycobacteria.

Conclusions; Phages, IS elements, tRNA and ncRNAs appear to have contributed to the evolution of the Mycobacterium genus where several tRNA and ncRNA genes have been horizontally transferred. On the basis of our phylogenetic analysis, we identified several isolates of unnamed species as new mycobacterial species or strains of known mycobacteria. The predicted number of coding sequences correlates with genome size while the number of tRNA, rRNA and ncRNA genes does not. Together these findings expand our insight into the evolution of the Mycobacterium genus and as such they establish a platform to understand mycobacterial pathogenicity, their evolution, antibiotic resistance/tolerance as well as the function and evolution of ncRNA among mycobacteria.

Background: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members.

Results: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete Mmuc(T) (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNA(Ile)TAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members.

Conclusions: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.

Nontuberculous mycobacteria, NTM, are of growing concern and among these members of the Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clades can cause infections in humans and they are resistant to first-line anti-tuberculosis drugs. They can be isolated from different ecological niches such as soil, tap water and ground water. Mycobacteria, such as Mmuc and Mneo, are classified as rapid growing mycobacteria, RGM, while the most familiar, Mycobacterium tuberculosis, belongs to the slow growing mycobacteria, SGM. Modern "omics" approaches have provided new insights into our understanding of the biology and evolution of this group of bacteria. Here we present comparative genomics data for seventeen NTM of which sixteen belong to the Mmuc- and Mneo-clades. Focusing on virulence genes, including genes encoding sigma/anti-sigma factors, serine threonine protein kinases (STPK), type VII (ESX genes) secretion systems and mammalian cell entry (Mce) factors we provide insight into their presence as well as phylogenetic relationship in the case of the sigma/anti-sigma factors and STPKs. Our data further suggest that these NTM lack ESX-5 and Mce2 genes, which are known to affect virulence. In this context, Mmuc- and Mneo-clade members lack several of the genes in the glycopeptidolipid (GLP) locus, which have roles in colony morphotype appearance and virulence. For the M. mucogenicum type strain, Mmuc(T), we provide RNASeq data focusing on mRNA levels for sigma factors, STPK, ESX proteins and Mce proteins. These data are discussed and compared to in particular the SGM and fish pathogen Mycobacterium marinum. Finally, we provide insight into as to why members of the Mmuc- and Mneo-clades show resistance to rifampin and isoniazid, and why Mmuc(T) forms a rough colony morphotype.

The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.

Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids transformed into Escherichia coli cells. From in vivo measurements, we found that these promoters in tandem formation can have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters’ positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which is known to hamper RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation in these constructs emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to falloff. Finally, the broad spectrum of possible, externally regulated, attenuation strengths observed in our synthetic tandem promoters suggests that they could become useful as externally controllable regulators of future synthetic circuits.

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2 '-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2 '-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2 '-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Background

The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic β-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance.

Methods

CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing.

Results

The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2.

Conclusion

These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.

Several RNA-binding proteins undergo reversible liquid-liquid phase transitions, which, in pathological conditions, might evolve into transitions to solid-state phases, giving rise to amyloid structures. Amyloidogenic and prion-like proteins, such as the tumor suppressor protein p53 and the mammalian prion protein (PrP), bind RNAs specifically or nonspecifically, resulting in changes in their propensity to undergo aggregation. Mutant p53 aggregation seems to play a crucial role in cancer through loss of function, negative dominance and gain of function. PrP conversion modulated by RNA results in highly toxic aggregates. Here, we review data on the modulatory action of RNAs on the aggregation of both proteins.