Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells.

Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-transfer RNA synthetase-tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often have poor intrinsic activity, presumably due to suboptimal interaction with a non-native translation system. This limitation can be addressed in Escherichia coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which uses a double-sieve selection scheme to facilitate single-step enrichment of active yet orthogonal tRNA mutants from naive libraries. Using VADER we developed improved mutants of Methanosarcina mazei pyrrolysyl-tRNA, as well as a bacterial tyrosyl-tRNA. We also show that the higher activity of the most efficient mutant pyrrolysyl-tRNA is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation apparatus.

A bacterial pan-genome makes gene essentiality strain-dependent and evolvable.

Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips that of any single bacterial genome. Here we investigate how a bacterial pan-genome might influence gene essentiality and whether essential genes that are initially critical for the survival of an organism can evolve to become non-essential. By using Transposon insertion sequencing (Tn-seq), whole-genome sequencing and RNA-seq on a set of 36 clinical Streptococcus pneumoniae strains representative of >68% of the species' pan-genome, we identify a species-wide 'essentialome' that can be subdivided into universal, core strain-specific and accessory essential genes. By employing 'forced-evolution experiments', we show that specific genetic changes allow bacteria to bypass essentiality. Moreover, by untangling several genetic mechanisms, we show that gene essentiality can be highly influenced by and/or be dependent on: (1) the composition of the accessory genome, (2) the accumulation of toxic intermediates, (3) functional redundancy, (4) efficient recycling of critical metabolites and (5) pathway rewiring. While this functional characterization underscores the evolvability potential of many essential genes, we also show that genes with differential essentiality remain important antimicrobial drug target candidates, as their inactivation almost always has a severe fitness cost in vivo.

Yersinia pseudotuberculosis doxycycline tolerance strategies include modulating expression of genes involved in cell permeability and tRNA modifications.

Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, promotes survival during treatment with an antibiotic classically viewed as bacteriostatic. We believe this may be the first observation of bactericidal activity of doxycycline under physiological conditions, which was revealed by reversing tusB downregulation.

Author Correction: Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication.

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis.IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.

Sampled ensemble neutrality as a feature to classify potential structured RNAs.

BACKGROUND: Structured RNAs have many biological functions ranging from catalysis of chemical reactions to gene regulation. Yet, many homologous structured RNAs display most of their conservation at the secondary or tertiary structure level. As a result, strategies for structured RNA discovery rely heavily on identification of sequences sharing a common stable secondary structure. However, correctly distinguishing structured RNAs from surrounding genomic sequence remains challenging, especially during de novo discovery. RNA also has a long history as a computational model for evolution due to the direct link between genotype (sequence) and phenotype (structure). From these studies it is clear that evolved RNA structures, like protein structures, can be considered robust to point mutations. In this context, an RNA sequence is considered robust if its neutrality (extent to which single mutant neighbors maintain the same secondary structure) is greater than that expected for an artificial sequence with the same minimum free energy structure. RESULTS: In this work, we bring concepts from evolutionary biology to bear on the structured RNA de novo discovery process. We hypothesize that alignments corresponding to structured RNAs should consist of neutral sequences. We evaluate several measures of neutrality for their ability to distinguish between alignments of structured RNA sequences drawn from Rfam and various decoy alignments. We also introduce a new measure of RNA structural neutrality, the structure ensemble neutrality (SEN). SEN seeks to increase the biological relevance of existing neutrality measures in two ways. First, it uses information from an alignment of homologous sequences to identify a conserved biologically relevant structure for comparison. Second, it only counts base-pairs of the original structure that are absent in the comparison structure and does not penalize the formation of additional base-pairs. CONCLUSION: We find that several measures of neutrality are effective at separating structured RNAs from decoy sequences, including both shuffled alignments and flanking genomic sequence. Furthermore, as an independent feature classifier to identify structured RNAs, SEN yields comparable performance to current approaches that consider a variety of features including stability and sequence identity. Finally, SEN outperforms other measures of neutrality at detecting mutational robustness in bacterial regulatory RNA structures.

Discovery and validation of novel and distinct RNA regulators for ribosomal protein S15 in diverse bacterial phyla.

BACKGROUND: Autogenous cis-regulators of ribosomal protein synthesis play a critical role in maintaining the stoichiometry of ribosome components. Structured portions within an mRNA transcript typically interact with specific ribosomal proteins to prevent expression of the entire operon, thus balancing levels of ribosomal proteins across transcriptional units. Three distinct RNA structures from different bacterial phyla have demonstrated interactions with S15 to regulate gene expression; however, these RNAs are distributed across a small fraction of bacterial diversity. RESULTS: We used comparative genomics in combination with analysis of existing transcriptomic data to identify three novel putative RNA structures associated with the S15 coding region in microbial genomes. These structures are completely distinct from those previously published and encompass potential regulatory regions including ribosome-binding sites. To validate the biological relevance of our findings, we demonstrate that an example of the Alphaproteobacterial RNA from Rhizobium radiobacter specifically interacts with S15 in vitro, and allows in vivo regulation of gene expression in an E. coli reporter system. In addition, structural probing and nuclease protection assays confirm the predicted secondary structure and indicate nucleotides required for protein interaction. CONCLUSIONS: This work illustrates the importance of integrating comparative genomic and transcriptomic approaches during de novo ncRNA identification and reveals a diversity of distinct natural RNA regulators that support analogous biological functions. Furthermore, this work indicates that many additional uncharacterized RNA regulators likely exist within bacterial genomes and that the plasticity of RNA structure allows unique, and likely independently derived, solutions to the same biological problem.

RNA structures regulating ribosomal protein biosynthesis in bacilli.

In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species.

Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria.

In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria.

Scribl: an HTML5 Canvas-based graphics library for visualizing genomic data over the web.

MOTIVATION: High-throughput biological research requires simultaneous visualization as well as analysis of genomic data, e.g. read alignments, variant calls and genomic annotations. Traditionally, such integrative analysis required desktop applications operating on locally stored data. Many current terabyte-size datasets generated by large public consortia projects, however, are already only feasibly stored at specialist genome analysis centers. As even small laboratories can afford very large datasets, local storage and analysis are becoming increasingly limiting, and it is likely that most such datasets will soon be stored remotely, e.g. in the cloud. These developments will require web-based tools that enable users to access, analyze and view vast remotely stored data with a level of sophistication and interactivity that approximates desktop applications. As rapidly dropping cost enables researchers to collect data intended to answer questions in very specialized contexts, developers must also provide software libraries that empower users to implement customized data analyses and data views for their particular application. Such specialized, yet lightweight, applications would empower scientists to better answer specific biological questions than possible with general-purpose genome browsers currently available. RESULTS: Using recent advances in core web technologies (HTML5), we developed Scribl, a flexible genomic visualization library specifically targeting coordinate-based data such as genomic features, DNA sequence and genetic variants. Scribl simplifies the development of sophisticated web-based graphical tools that approach the dynamism and interactivity of desktop applications. AVAILABILITY AND IMPLEMENTATION: Software is freely available online at http://chmille4.github.com/Scribl/ and is implemented in JavaScript with all modern browsers supported.

Lateral subtalar joint dislocation: a case with calcaneal fracture.

Subtalar joint dislocation is a relatively rare injury, with lateral dislocation occurring less frequently than medial dislocation. Associated fractures alter the treatment plan and the prognosis, but they are often missed on plain film radiographs. A brief review of the anatomy, pathomechanics, treatment, prognosis, and complications of subtalar joint dislocation is presented. An interesting case of lateral subtalar joint dislocation with an associated calcaneal fracture not evident on plain film radiographs but delineated with computed tomography is presented.