Tn-seq

Staphylococcus aureus is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce highly saturated transposon mutant libraries in S. aureus and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion...

UNLABELLED: The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, Acinetobacter baumannii , is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although A. baumannii essential genes have been identified by transposon sequencing (Tn-seq), they have not been prioritized by sensitivity to knockdown or antibiotics...

UNLABELLED: Protein synthesis is catalyzed by the ribosome and a host of highly conserved elongation factors. Most elongation factors that are conserved in all domains of life are essential, such as EF-Tu (e/aEF1A) and EF-G (e/aEF2). In contrast, the universally conserved elongation factor P (EF-P/eIF5A) is essential in eukaryotes but is dispensable in most bacteria. EF-P prevents ribosome stalling at difficult-to translate sequences, especially polyprolines. Since efp deletion phenotypes range from modest to lethal in different bacterial species, we hypothesized that some bacteria encode an uncharacterized elongation factor with compensatory functions...

The possible active entry of aminoglycosides in bacterial cells has been debated since the development of this antibiotic family. Here we report the identification of their active transport mechanism in Vibrio species. We combined genome-wide transcriptional analysis and fitness screens to identify alterations driven by treatment of V. cholerae with sub-minimum inhibitory concentrations (sub-MIC) of the aminoglycoside tobramycin. RNA-seq data showed downregulation of the small non-coding RNA ncRNA586 during such treatment, while Tn-seq revealed that inactivation of this sRNA was associated with improved fitness in the presence of tobramycin...

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants...

Staphylococcus aureus is an important pathogen that leads to significant disease through multiple routes of infection. We recently published a transposon sequencing (Tn-seq) screen in a mouse acute pneumonia model and identified a hypothetical gene (SAUSA300_1902, pgl) with similarity to a lactonase of Escherichia coli involved in the pentose phosphate pathway (PPP) that was conditionally essential. Limited studies have investigated the role of the PPP in physiology and pathogenesis of S. aureus. We show here that mutation of pgl significantly impacts ATP levels and respiration...

Genetic interaction networks can help identify functional connections between genes and pathways, which can be leveraged to establish (new) gene function, drug targets, and fill pathway gaps. Since there is no optimal tool that can map genetic interactions across many different bacterial strains and species, we develop CRISPRi-TnSeq, a genome-wide tool that maps genetic interactions between essential genes and nonessential genes through the knockdown of a targeted essential gene (CRISPRi) and the simultaneous knockout of individual nonessential genes (Tn-Seq)...

Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the resistant nosocomial pathogen Acinetobacter baumannii. Our understanding of how A. baumannii defends against phage remains limited, although this information could lead to improved antimicrobial therapies. To address this problem, we identified genome-wide determinants of phage susceptibility in A. baumannii using Tn-seq. These studies focused on the lytic phage Loki, which targets Acinetobacter by unknown mechanisms...

Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid acquisition of genomic data and the (much slower) confirmation of inferred genetic function threatens to widen unless techniques for fast, high-throughput functional validation can be applied at scale. This applies equally to Mycobacterium tuberculosis, the leading infectious cause of death globally and a pathogen whose genome, despite being among the first to be sequenced two decades ago, still contains many genes of unknown function...

Dickeya and Pectobacterium species are necrotrophic pathogens that macerate stems (blackleg disease) and tubers (soft rot disease) of Solanum tuberosum . They proliferate by exploiting plant cell remains. They also colonize roots, even if no symptoms are observed. The genes involved in pre-symptomatic root colonization are poorly understood. Here, transposon-sequencing (Tn-seq) analysis of Dickeya solani living in macerated tissues revealed 126 genes important for competitive colonization of tuber lesions and 207 for stem lesions, including 96 genes common to both conditions...

INTRODUCTION: DNA damage repair (DDR) is an essential process for living organisms and contributes to genome maintenance and evolution. DDR involves different pathways including Homologous recombination (HR), Nucleotide Excision Repair (NER) and Base excision repair (BER) for example. The activity of each pathway is revealed with particular drug inducing lesions, but the repair of most DNA lesions depends on concomitant or subsequent action of the multiple pathways. METHODS: In the present study, we used two genotoxic antibiotics, mitomycin C (MMC) and Bleomycin (BLM), to decipher the interplays between these different pathways in E...

Antibiotic resistance in bacterial pathogens is an ongoing public health concern. The arylomycins are a class of natural product antibiotics that target the type I signal peptidase, which carries out the terminal step in protein secretion. Here, we used transposon sequencing (Tn-Seq) to profile the effects of the optimized arylomycin derivative G0775 in Staphylococcus aureus. Our transposon libraries include both upregulation and inactivation mutants, allowing us to identify resistance mechanisms and targets for synergism...

Salmonella enterica in agricultural environments has become an important concern, due to its potential transmission to humans and the associated public health risks. To identify genes contributing to Salmonella adaptation to such environments, transposon sequencing has been used in recent years. However, isolating Salmonella from atypical hosts, such as plant leaves, can pose technical challenges due to low bacterial content and the difficulty to separate an adequate number of bacteria from host tissues. In this study, we describe a modified methodology using a combination of sonication and filtration to recover S...

Pathogenic microbial ecosystems are often polymicrobial, and interbacterial interactions drive emergent properties of these communities. In the oral cavity, Streptococcus gordonii is a foundational species in the development of plaque biofilms, which can contribute to periodontal disease and, after gaining access to the bloodstream, target remote sites such as heart valves. Here, we used a transposon sequencing (Tn-Seq) library of S. gordonii to identify genes that influence fitness in a murine abscess model, both as a monoinfection and as a coinfection with an oral partner species, Porphyromonas gingivalis...

Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for facilitating epidemic outbreaks. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42°C. The mutants with the most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress...

Mycobacterium abscessus ( Mabs ) is an emerging nontuberculosis mycobacterial (NTM) pathogen responsible for a wide variety of respiratory and cutaneous infections that are difficult to treat with standard antibacterial therapy. Mabs has a high degree of both innate and acquired antibiotic resistance to most clinically relevant drugs, including standard anti-mycobacterial agents. Ethionamide (ETH), an inhibitor of mycolic acid biosynthesis, is currently utilized as a second-line agent for treating multidrug-resistant tuberculosis infections...

In bacteria, natural transposon mobilization can drive adaptive genomic rearrangements. Here, we build on this capability and develop an inducible, self-propagating transposon platform for continuous genome-wide mutagenesis and the dynamic rewiring of gene networks in bacteria. We first use the platform to study the impact of transposon functionalization on the evolution of parallel Escherichia coli populations toward diverse carbon source utilization and antibiotic resistance phenotypes. We then develop a modular, combinatorial assembly pipeline for the functionalization of transposons with synthetic or endogenous gene regulatory elements (e...

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites...

Transposon mutagenesis has been the method of choice for genetic screens and selections in bacteria by virtue of the transposon being linked to the disrupted gene, simplifying its identification. Transposon sequencing (Tn-seq) is a high-throughput version of transposon mutant screening, in which massively parallel sequencing is used to simultaneously follow the fitness of all mutants in a complex library. In a single experiment, one can use Tn-seq to interrogate the contribution of all genes of a bacterium to fitness under a condition of interest...

Ralstonia solanacearum, the causal agent of the devastating bacterial wilt disease, is of particular interest to the scientific community. The repertoire of type III effectors plays an important role in the evasion of plant immunity, but tolerance to plant immunity is also crucial for the survival and virulence of R. solanacearum. Nevertheless, a systematic study of R. solanacearum tolerance to plant immunity is lacking. In this study, we used exogenous salicylic acid (SA) to improve the immunity of tomato plants, followed by transposon insertion sequencing (Tn-seq) analysis and the identification of R...