scholarly journals Molecular phenotyping of infection-associated small non-coding RNAs

2016 ◽  
Vol 371 (1707) ◽  
pp. 20160081 ◽  
Author(s):  
Lars Barquist ◽  
Alexander J. Westermann ◽  
Jörg Vogel
Keyword(s):  
Virulence Factors ◽  
High Throughput ◽  
Genetic Manipulation ◽  
Genetic Interaction ◽  
Forward Genetics ◽  
Model Systems ◽  
Transposon Insertion ◽  
Culture Models ◽  
Molecular Phenotypes ◽  
Transposon Insertion Sequencing

Infection is a complicated balance, with both pathogen and host struggling to tilt the result in their favour. Bacterial infection biology has relied on forward genetics for many of its advances, defining phenotype in terms of replication in model systems. However, many known virulence factors fail to produce robust phenotypes, particularly in the systems most amenable to genetic manipulation, such as cell-culture models. This has particularly been limiting for the study of the bacterial regulatory small RNAs (sRNAs) in infection. We argue that new sequencing-based technologies can work around this problem by providing a ‘molecular phenotype’, defined in terms of the specific transcriptional dysregulation in the infection system induced by gene deletion. We illustrate this using the example of our recent study of the PinT sRNA using dual RNA-seq, that is, simultaneous RNA sequencing of host and pathogen during infection. We additionally discuss how other high-throughput technologies, in particular genetic interaction mapping using transposon insertion sequencing, may be used to further dissect molecular phenotypes. We propose a strategy for how high-throughput technologies can be integrated in the study of non-coding regulators as well as bacterial virulence factors, enhancing our ability to rapidly generate hypotheses with regards to their function. This article is part of the themed issue ‘The new bacteriology’.

Transposon Insertion Sequencing, a Global Measure of Gene Function

2020 ◽  
Vol 54 (1) ◽  
pp. 337-365
Author(s):  
Tim van Opijnen ◽  
Henry L. Levin
Keyword(s):  
Dna Sequencing ◽  
High Throughput ◽  
Bacterial Infections ◽  
Single Experiment ◽  
Transposon Insertion ◽  
High Throughput Method ◽  
High Throughput Dna Sequencing ◽  
Complete Set ◽  
Living Organisms ◽  
Transposon Insertion Sequencing

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.

scholarly journals Staphylococcus aureus Virulence Factors Identified by Using a High-Throughput Caenorhabditis elegans-Killing Model

2005 ◽  
Vol 73 (2) ◽  
pp. 872-877 ◽  
Author(s):  
Jakob Begun ◽  
Costi D. Sifri ◽  
Samuel Goldman ◽  
Stephen B. Calderwood ◽  
Frederick M. Ausubel
Keyword(s):  
Staphylococcus Aureus ◽  
Caenorhabditis Elegans ◽  
Dna Replication ◽  
Virulence Factors ◽  
High Throughput ◽  
Human Pathogen ◽  
Renal Abscess ◽  
C Elegans ◽  
Transposon Insertion ◽  
Strain Nctc

ABSTRACT Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.

scholarly journals Genetic interaction mapping highlights key roles of the Tol-Pal complex

2021 ◽  
Author(s):  
Wee Boon Tan ◽  
Shu-Sin Chng
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Genetic Interaction ◽  
Strong Interactions ◽  
Growth Defects ◽  
Transposon Insertion ◽  
Complex Functions ◽  
Division Process ◽  
Transposon Insertion Sequencing ◽  
Periplasmic Glucan

AbstractThe conserved Tol-Pal trans-envelope complex is important for outer membrane (OM) stability and cell division in Gram-negative bacteria. It has been proposed to mediate OM constriction during cell division via tethering to the cell wall. Yet, recent studies suggest that the complex has additional roles in OM lipid homeostasis and septal cell wall separation. How the Tol-Pal complex functions to facilitate these many processes is unclear. To gain insights into its role(s), we applied transposon insertion sequencing, and report here a detailed network of genetic interactions with the tol-pal locus in Escherichia coli. We found one positive and >20 negative strong interactions based on fitness. Disruption of genes responsible for osmoregulated periplasmic glucan biosynthesis restores fitness and OM barrier function, but not cell division defects, in tol-pal mutants. In contrast, deletions of genes involved in OM homeostasis and cell wall remodelling give rise to synthetic growth defects in strains lacking Tol-Pal, especially exacerbating OM barrier and/or cell division defects. Notably, the ΔtolA mutant having additional defects in OM protein assembly (ΔbamB) exhibited severe division phenotypes, even under conditions where the single mutants divide normally; this highlights the possibility for OM phenotypes to indirectly influence the cell division process. Overall, our work provides insights into the intricate nature of Tol-Pal function, and reinforces the model that this complex plays crucial roles in cell wall-OM tethering, cell wall remodelling, and in particular, OM homeostasis.

scholarly journals ESSENTIALS: Software for Rapid Analysis of High Throughput Transposon Insertion Sequencing Data

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e43012 ◽  
Author(s):  
Aldert Zomer ◽  
Peter Burghout ◽  
Hester J. Bootsma ◽  
Peter W. M. Hermans ◽  
Sacha A. F. T. van Hijum
Keyword(s):  
High Throughput ◽  
Rapid Analysis ◽  
Sequencing Data ◽  
Transposon Insertion ◽  
Transposon Insertion Sequencing

scholarly journals Genome-wide identification of sexual-reproduction genes in fission yeast via transposon-insertion sequencing

2021 ◽  
Author(s):  
R. Blake Billmyre ◽  
Michael T. Eickbush ◽  
Caroline J. Craig ◽  
Jeffrey J. Lange ◽  
Christopher Wood ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Fission Yeast ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Spore Viability ◽  
Transposon Insertion ◽  
Uncharacterized Gene ◽  
Genome Wide ◽  
Cell Densities ◽  
Transposon Insertion Sequencing

AbstractMany genes required for sexual reproduction remain to be identified. Moreover, many of the genes that are known have been characterized in distinct experiments using different conditions, which complicates understanding the relative contributions of genes to sex. To address these challenges, we developed an assay in Schizosaccharomyces pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wildtype. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

Conditionally immortalized cell lines as model systems for high-throughput biology in drug discovery

2002 ◽  
Vol 30 (4) ◽  
pp. 800-802 ◽  
Author(s):  
N. Daniele ◽  
R. Halse ◽  
E. Grinyo ◽  
S.J. Yeaman ◽  
P. R. Shepherd
Keyword(s):  
Cell Culture ◽  
Drug Discovery ◽  
Cell Lines ◽  
High Throughput ◽  
Biological Data ◽  
Model Systems ◽  
Drug Discovery Process ◽  
Culture Models ◽  
Immortalized Cell ◽  
Cell Culture Models

There is an increasing emphasis on the need for high-quality biological data much earlier in the drug-discovery process. This has led to the development of high-throughput approaches to biology, many of which rely on the use of cell-culture models. Unfortunately, available cell-culture models often reflect poorly the characteristics of the tissue they are supposed to represent. However, the conditional-immortalization approach as applied by Xcellsyz offers the possibility of producing human cell lines on demand, which are truly representative of the tissue from which they derive.

scholarly journals NetR and AttR, Two New Bioinformatic Tools to Integrate Diverse Datasets into Cytoscape Network and Attribute Files

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 423
Author(s):  
Armen Halajyan ◽  
Natalie Weingart ◽  
Mirza Yeahia ◽  
Mariano Loza-Coll
Keyword(s):  
High Throughput ◽  
Genetic Interaction ◽  
Model Systems ◽  
Published Data ◽  
Data Generation ◽  
Bioinformatic Tools ◽  
Genome Wide ◽  
Genome Wide Data ◽  
Experimental Model Systems ◽  
Genetic Interaction Data

High-throughput technologies have allowed researchers to obtain genome-wide data from a wide array of experimental model systems. Unfortunately, however, new data generation tends to significantly outpace data re-utilization, and most high throughput datasets are only rarely used in subsequent studies or to generate new hypotheses to be tested experimentally. The reasons behind such data underutilization include a widespread lack of programming expertise among experimentalist biologists to carry out the necessary file reformatting that is often necessary to integrate published data from disparate sources. We have developed two programs (NetR and AttR), which allow experimental biologists with little to no programming background to integrate publicly available datasets into files that can be later visualized with Cytoscape to display hypothetical networks that result from combining individual datasets, as well as a series of published attributes related to the genes or proteins in the network. NetR also allows users to import protein and genetic interaction data from InterMine, which can further enrich a network model based on curated information. We expect that NetR/AttR will allow experimental biologists to mine a largely unexploited wealth of data in their fields and facilitate their integration into hypothetical models to be tested experimentally.

scholarly journals High-throughput discovery of phage receptors using transposon insertion sequencing of bacteria

2020 ◽  
Vol 117 (31) ◽  
pp. 18670-18679 ◽  
Author(s):  
Kaitlyn E. Kortright ◽  
Benjamin K. Chan ◽  
Paul E. Turner
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Environmental Samples ◽  
Proof Of Concept ◽  
Cellular Receptors ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Phage Receptor ◽  
Bacterial Genes ◽  
Transposon Insertion Sequencing

As the most abundant microbes on Earth, novel bacteriophages (phages; bacteria-specific viruses) are readily isolated from environmental samples. However, it remains challenging to characterize phage–bacteria interactions, such as the host receptor(s) phages bind to initiate infection. Here, we tested whether transposon insertion sequencing (INSeq) could be used to identify bacterial genes involved in phage binding. As proof of concept, results showed that INSeq screens successfully identified genes encoding known receptors for previously characterized viruses ofEscherichia coli(phages T6, T2, T4, and T7). INSeq screens were then used to identify genes involved during infection of six newly isolated coliphages. Results showed that candidate receptors could be successfully identified for the majority (five of six) of the phages; furthermore, genes encoding the phage receptor(s) were the top hit(s) in the analyses of the successful screens. INSeq screens provide a generally useful method for high-throughput discovery of phage receptors. We discuss limitations of our approach when examining uncharacterized phages, as well as usefulness of the method for exploring the evolution of broad versus narrow use of cellular receptors among phages in the biosphere.

scholarly journals A method for increasing electroporation competency of Gram-negative clinical isolates by Polymyxin B nonapeptide

2022 ◽  
Author(s):  
Jilong Qin ◽  
Yaoqin Hong ◽  
Karthik Pullela ◽  
Renato Morona ◽  
Ian R. Henderson ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Protein Complexes ◽  
Fold Increase ◽  
Polymyxin B ◽  
Transformation Frequency ◽  
Clinical Isolates ◽  
Transposon Insertion ◽  
Efficient Delivery ◽  
Natural Isolates ◽  
Transposon Insertion Sequencing

Abstract The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a 5-fold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by 8-fold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA-protein complexes into clinically important bacteria.

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