The interaction between an antibiotic and bacterium is not merely restricted to the drug and its direct target, rather antibiotic induced stress seems to resonate through the bacterium, creating ...selective pressures that drive the emergence of adaptive mutations not only in the direct target, but in genes involved in many different fundamental processes as well. Surprisingly, it has been shown that adaptive mutations do not necessarily have the same effect in all species, indicating that the genetic background influences how phenotypes are manifested. However, to what extent the genetic background affects the manner in which a bacterium experiences antibiotic stress, and how this stress is processed is unclear. Here we employ the genome-wide tool Tn-Seq to construct daptomycin-sensitivity profiles for two strains of the bacterial pathogen Streptococcus pneumoniae. Remarkably, over half of the genes that are important for dealing with antibiotic-induced stress in one strain are dispensable in another. By confirming over 100 genotype-phenotype relationships, probing potassium-loss, employing genetic interaction mapping as well as temporal gene-expression experiments we reveal genome-wide conditionally important/essential genes, we discover roles for genes with unknown function, and uncover parts of the antibiotic's mode-of-action. Moreover, by mapping the underlying genomic network for two query genes we encounter little conservation in network connectivity between strains as well as profound differences in regulatory relationships. Our approach uniquely enables genome-wide fitness comparisons across strains, facilitating the discovery that antibiotic responses are complex events that can vary widely between strains, which suggests that in some cases the emergence of resistance could be strain specific and at least for species with a large pan-genome less predictable.
It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the ...fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.
A large fraction of the genes from sequenced organisms are of unknown function. This limits biological insight, and for pathogenic microorganisms hampers the development of new approaches to battle ...infections. There is thus a great need for novel strategies that link genotypes to phenotypes for microorganisms. We describe a high-throughput strategy based on the method Tn-seq that can be applied to any genetically manipulatable microorganism. By screening 17 in vitro and two in vivo (carriage and infection) conditions for the pathogen Streptococcus pneumoniae, we create a resource consisting of >1800 interactions that is rich in new genotype-phenotype relationships. We describe genes that are involved in differential carbon source utilization in the host, as well as genes that are involved both in virulence and in resistance against specific in vitro stresses, thereby revealing selection pressures that the pathogen experiences in vivo. We reveal the secondary response to an antibiotic, including a dual role efflux pump also involved in resistance to pH stress. Through genetic-interaction mapping and gene-expression analysis we define the mechanism of attenuation and the regulatory relationship between a two-component system and a core biosynthetic pathway specific to microorganisms. Thus, we have generated a resource that provides detailed insight into the biology and virulence of S. pneumoniae and provided a road map for similar discovery in other microorganisms.
The goal of genomics and systems biology is to understand how complex systems of factors assemble into pathways and structures that combine to form living organisms. Great advances in understanding ...biological processes result from determining the function of individual genes, a process that has classically relied on characterizing single mutations. Advances in DNA sequencing has made available the complete set of genetic instructions for an astonishing and growing number of species. To understand the function of this ever-increasing number of genes, a high-throughput method was developed that in a single experiment can measure the function of genes across the genome of an organism. This occurred approximately 10 years ago, when high-throughput DNA sequencing was combined with advances in transposon-mediated mutagenesis in a method termed transposon insertion sequencing (TIS). In the subsequent years, TIS succeeded in addressing fundamental questions regarding the genes of bacteria, many of which have been shown to play central roles in bacterial infections that result in major human diseases. The field of TIS has matured and resulted in studies of hundreds of species that include significant innovations with a number of transposons. Here, we summarize a number of TIS experiments to provide an understanding of the method and explanation of approaches that are instructive when designing a study. Importantly, we emphasize critical aspects of a TIS experiment and highlight the extension and applicability of TIS into nonbacterial species such as yeast.
Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) ...that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.
Using an optimized virus‐assisted directed evolution Scheme in mammalian cells, an enhanced variant of the M. mazei pyrrolysyl tRNA, PyOtR, was developed. PyOtR enabled improved incorporation of many noncanonical amino acids (ncAAs) into proteins expressed in mammalian cells, and enabled facile generation of cell lines stably expressing the ncAA incorporation machinery.
Small noncoding RNAs (sRNAs) play important roles in gene regulation in both prokaryotes and eukaryotes. Thus far, no sRNA has been assigned a definitive role in virulence in the major human pathogen ...Streptococcus pneumoniae. Based on the potential coding capacity of intergenic regions, we hypothesized that the pneumococcus produces many sRNAs and that they would play an important role in pathogenesis. We describe the application of whole-genome transcriptional sequencing to systematically identify the sRNAs of Streptococcus pneumoniae. Using this approach, we have identified 89 putative sRNAs, 56 of which are newly identified. Furthermore, using targeted genetic approaches and Tn-seq transposon screening, we demonstrate that many of the identified sRNAs have important global and niche-specific roles in virulence. These data constitute the most comprehensive analysis of pneumococcal sRNAs and provide the first evidence of the extensive roles of sRNAs in pneumococcal pathogenesis.
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.
Current approaches explore bacterial genes that change transcriptionally upon stress exposure as diagnostics to predict antibiotic sensitivity. However, transcriptional changes are often specific to ...a species or antibiotic, limiting implementation to known settings only. While a generalizable approach, predicting bacterial fitness independent of strain, species or type of stress, would eliminate such limitations, it is unclear whether a stress-response can be universally captured. By generating a multi-stress and species RNA-Seq and experimental evolution dataset, we highlight the strengths and limitations of existing gene-panel based methods. Subsequently, we build a generalizable method around the observation that global transcriptional disorder seems to be a common, low-fitness, stress response. We quantify this disorder using entropy, which is a specific measure of randomness, and find that in low fitness cases increasing entropy and transcriptional disorder results from a loss of regulatory gene-dependencies. Using entropy as a single feature, we show that fitness and quantitative antibiotic sensitivity predictions can be made that generalize well beyond training data. Furthermore, we validate entropy-based predictions in 7 species under antibiotic and non-antibiotic conditions. By demonstrating the feasibility of universal predictions of bacterial fitness, this work establishes the fundamentals for potentially new approaches in infectious disease diagnostics.
Omics-profiling is a collection of increasingly prominent approaches that result in large-scale biological datasets, for instance capturing an organism's behavior and response in an environment. It ...can be daunting to manually analyze and interpret such large datasets without some programming experience. Additionally, with increasing amounts of data; management, storage and sharing challenges arise.
Here, we present ShinyOmics, a web-based application that allows rapid collaborative exploration of omics-data. By using Tn-Seq, RNA-Seq, microarray and proteomics datasets from two human pathogens, we exemplify several conclusions that can be drawn from a rich dataset. We identify a protease and several chaperone proteins upregulated under aminoglycoside stress, show that antibiotics with the same mechanism of action trigger similar transcriptomic responses, point out the dissimilarity in different omics-profiles, and overlay the transcriptional response on a metabolic network.
ShinyOmics is easy to set up and customize, and can utilize user supplied metadata. It offers several visualization and comparison options that are designed to assist in novel hypothesis generation, as well as data management, online sharing and exploration. Moreover, ShinyOmics can be used as an interactive supplement accompanying research articles or presentations.
Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are ...mediated in part by
uter
embrane
esicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in
(
). We identified a family of
ual
embrane-spanning
nti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of
. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.
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