Transposon-directed Insertion Sequencing Research Articles

A multiomic approach to defining the essential genome of the globally important pathogen Corynebacterium diphtheriae.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.

A whole-genome assay identifies four principal gene functions that confer tolerance of meropenem stress upon Escherichia coli

We report here the identification of four gene functions of principal importance for the tolerance of meropenem stress in Escherichia coli: cell division, cell envelope synthesis and maintenance, ATP metabolism, and transcription regulation. The primary mechanism of β-lactam antibiotics such as meropenem is inhibition of penicillin binding proteins, thus interfering with peptidoglycan crosslinking, weakening the cell envelope, and promoting cell lysis. However, recent systems biology approaches have revealed numerous downstream effects that are triggered by cell envelope damage and involve diverse cell processes. Subpopulations of persister cells can also arise, which can survive elevated concentrations of meropenem despite the absence of a specific resistance factor. We used Transposon-Directed Insertion Sequencing with inducible gene expression to simultaneously assay the effects of upregulation, downregulation, and disruption of every gene in a model E. coli strain on survival of exposure to four concentrations of meropenem. Automated Gene Functional Classification and manual categorization highlighted the importance at all meropenem concentrations of genes involved in peptidoglycan remodeling during cell division, suggesting that cell division is the primary function affected by meropenem. Genes involved in cell envelope synthesis and maintenance, ATP metabolism, and transcriptional regulation were generally important at higher meropenem concentrations, suggesting that these three functions are therefore secondary or downstream targets. Our analysis revealed the importance of multiple two-component signal transduction mechanisms, suggesting an as-yet unexplored coordinated transcriptional response to meropenem stress. The inclusion of an inducible, transposon-encoded promoter allowed sensitive detection of genes involved in proton transport, ATP production and tRNA synthesis, for which modulation of expression affects survival in the presence of meropenem: a finding that would not be possible with other technologies. We were also able to suggest new targets for future antibiotic development or for synergistic effects between gene or protein inhibitors and existing antibiotics. Overall, in a single massively parallel assay we were able to recapitulate many of the findings from decades of research into β-lactam antibiotics, add to the list of genes known to be important for meropenem tolerance, and categorize the four principal gene functions involved.

A Proposed Framework to Identify Dispensable and Essential Functions in Bifidobacteria: Case Study of Bifidobacterium breve UCC2003 as a Prototype of Its Genus.

Functional genomics of bacteria commonly aims at establishing genotype-phenotype links in microorganisms of industrial, technological and biomedical relevance. In this regard, random transposon mutagenesis coupled to high-throughput next-generation sequencing approaches, termed transposon-insertion sequencing (TIS), has emerged as a robust, genome-wide alternative to perform functional genome analysis. Though these approaches have been used in a large number of studies involving pathogenic and clinically relevant bacteria, they have received little attention in the fields of commensal and potentially beneficial bacteria, including probiotic microorganisms. In this chapter, we describe the implementation of the TIS method Transposon-Directed Insertion Sequencing to describe the set of essential genes in a representative strain of a genus encompassing several commensal and potentially probiotic bacteria and discuss considerations when applying similar methodological approaches to other Bifidobacterium species/strains of interest.

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

ABSTRACTThe production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen.IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse “capsule depolymerases” which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.

Mapping the Transcriptional and Fitness Landscapes of a Pathogenic E. coli Strain: The Effects of Organic Acid Stress under Aerobic and Anaerobic Conditions.

Several methods are available to probe cellular responses to external stresses at the whole genome level. RNAseq can be used to measure changes in expression of all genes following exposure to stress, but gives no information about the contribution of these genes to an organism’s ability to survive the stress. The relative contribution of each non-essential gene in the genome to the fitness of the organism under stress can be obtained using methods that use sequencing to estimate the frequencies of members of a dense transposon library grown under different conditions, for example by transposon-directed insertion sequencing (TraDIS). These two methods thus probe different aspects of the underlying biology of the organism. We were interested to determine the extent to which the data from these two methods converge on related genes and pathways. To do this, we looked at a combination of biologically meaningful stresses. The human gut contains different organic short-chain fatty acids (SCFAs) produced by fermentation of carbon compounds, and Escherichia coli is exposed to these in its passage through the gut. Their effect is likely to depend on both the ambient pH and the level of oxygen present. We, therefore, generated RNAseq and TraDIS data on a uropathogenic E. coli strain grown at either pH 7 or pH 5.5 in the presence or absence of three SCFAs (acetic, propionic and butyric), either aerobically or anaerobically. Our analysis identifies both known and novel pathways as being likely to be important under these conditions. There is no simple correlation between gene expression and fitness, but we found a significant overlap in KEGG pathways that are predicted to be enriched following analysis of the data from the two methods, and the majority of these showed a fitness signature that would be predicted from the gene expression data, assuming expression to be adaptive. Genes which are not in the E. coli core genome were found to be particularly likely to show a positive correlation between level of expression and contribution to fitness.

A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence.

Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (Transposon-directed insertion sequencing), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella ΔmgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available 'omics' datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria.

Streptococcus pyogenes genes that promote pharyngitis in primates.

Streptococcus pyogenes (group A streptococcus; GAS) causes 600 million cases of pharyngitis annually worldwide. There is no licensed human GAS vaccine despite a century of research. Although the human oropharynx is the primary site of GAS infection, the pathogenic genes and molecular processes used to colonize, cause disease, and persist in the upper respiratory tract are poorly understood. Using dense transposon mutant libraries made with serotype M1 and M28 GAS strains and transposon-directed insertion sequencing, we performed genome-wide screens in the nonhuman primate (NHP) oropharynx. We identified many potentially novel GAS fitness genes, including a common set of 115 genes that contribute to fitness in both genetically distinct GAS strains during experimental NHP pharyngitis. Targeted deletion of 4 identified fitness genes/operons confirmed that our newly identified targets are critical for GAS virulence during experimental pharyngitis. Our screens discovered many surface-exposed or secreted proteins - substrates for vaccine research - that potentially contribute to GAS pharyngitis, including lipoprotein HitA. Pooled human immune globulin reacted with purified HitA, suggesting that humans produce antibodies against this lipoprotein. Our findings provide new information about GAS fitness in the upper respiratory tract that may assist in translational research, including developing novel vaccines.

Use of cellular and high throughput genetic approaches to unravel the antibacterial mechanism of honey

The medical importance of honey has been extensively demonstrated. Although high osmolarity, acidity and hydrogen peroxide (H2O2) proved to be the most prevalent factors in honey’s activity, the underlying antimicrobial mechanism remains obscure. Our aim is to provide insight into the physiological changes and genetic responses in honey-treated bacteria, thus improving our understanding of this natural product as a potential novel antimicrobial. A model honey composed of sugars, gluconic acid, and H2O2as they are accumulated in honey after enzymatic reaction happens, was used the investigation of honey’s activity. The bactericidal action of the model was tested on E. coli K-12 strain MG1655. Flow cytometry (FC) and Atomic Force Microscopy (AFM) identified physiological changes such as membrane potential, blebbing, and cell lysis. Reactive Oxygen Species (ROS) accumulation was observed in individual cells by FC. Transposon Directed Insertion Sequencing (TraDIS) identified mutants’ fitness over a time course of E. coli treatment by model honey. The loss of selenocysteine (selAB) and formate dehydrogenase (fdhDE) mutants, proved the redox- balancing activity as essential for the repression of ROS in stressed cells. High susceptibility of energy metabolism (atpABD) and peptidoglycan synthesis (prc) mutants, indicated the strain unable to maintain the reductive cell environment necessary for cellular activities, post honey exposure. Our findings identified some of the honey’s targets when acting as an antimicrobial. The synergies observed support the use of honey as an antimicrobial; however, the identification of mutations that led to enhanced resistance to honey is an important finding that needs further study.

Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model.

A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.

The essential genomic landscape of the commensal Bifidobacterium breve UCC2003

Bifidobacteria are common gut commensals with purported health-promoting effects. This has encouraged scientific research into bifidobacteria, though recalcitrance to genetic manipulation and scarcity of molecular tools has hampered our knowledge on the precise molecular determinants of their health-promoting attributes and gut adaptation. To overcome this problem and facilitate functional genomic analyses in bifidobacteria, we created a large Tn5 transposon mutant library of the commensal Bifidobacterium breve UCC2003 that was further characterized by means of a Transposon Directed Insertion Sequencing (TraDIS) approach. Statistical analysis of transposon insertion distribution revealed a set of 453 genes that are essential for or markedly contribute to growth of this strain under laboratory conditions. These essential genes encode functions involved in the so-called bifid-shunt, most enzymes related to nucleotide biosynthesis and a range of housekeeping functions. Comparison to the Bifidobacterium and B. breve core genomes highlights a high degree of conservation of essential genes at the species and genus level, while comparison to essential gene datasets from other gut bacteria identified essential genes that appear specific to bifidobacteria. This work establishes a useful molecular tool for scientific discovery of bifidobacteria and identifies targets for further studies aimed at characterizing essential functions not previously examined in bifidobacteria.

Physical enrichment of transposon mutants from saturation mutant libraries using the TraDISort approach.

Transposon-insertion sequencing methods are finding their way into the molecular toolbox of many fields of microbiology. These methods can identify the genomic locations and density of transposon insertions in saturated transposon mutant libraries and can be used to make inferences on gene function. For example, where no insertions or very few insertions are identified within a gene in a mutant library grown under permissive conditions, the gene may be essential. Furthermore, where mutations are enriched or lost in a gene after passaging the library through a selective process, the gene is likely to be involved in phenotypes linked to the process. Typically, a fitness based selection such as a stress condition is used in these experiments and the processed sequencing data are used to identify genes required for fitness under the selection. Our research team recently expanded the utility of the transposon directed insertion sequencing (TraDIS) method by applying a physical separation of a transposon mutant library mediated by fluorescence activated cell sorting, rather than a fitness-based selection. This approach, which we have named "TraDISort" is significant because it allows the study of phenotypes that are not linked to cell survival. The TraDISort approach has a broad range of future applications, in drug development, metabolic engineering and in studies of basic bacterial cell physiology.

Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii.

ABSTRACTMultidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii.

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

Summary: Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology.Availability and implementation: The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at https://github.com/sanger-pathogens/Bio-TradisContact: parkhill@sanger.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

Escherichia coli genes and pathways involved in surviving extreme exposure to ionizing radiation.

To further an improved understanding of the mechanisms used by bacterial cells to survive extreme exposure to ionizing radiation (IR), we broadly screened nonessential Escherichia coli genes for those involved in IR resistance by using transposon-directed insertion sequencing (TraDIS). Forty-six genes were identified, most of which become essential upon heavy IR exposure. Most of these were subjected to direct validation. The results reinforced the notion that survival after high doses of ionizing radiation does not depend on a single mechanism or process, but instead is multifaceted. Many identified genes affect either DNA repair or the cellular response to oxidative damage. However, contributions by genes involved in cell wall structure/function, cell division, and intermediary metabolism were also evident. About half of the identified genes have not previously been associated with IR resistance or recovery from IR exposure, including eight genes of unknown function.