High-throughput transposon mutagenesis in the family Enterobacteriaceae reveals core essential genes and rapid turnover of essentiality
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ABSTRACT:
PROVIDER: PRJNA891710 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21949224.fastq.gz | Fastqsanger.gz | |||
| SRR21949225.fastq.gz | Fastqsanger.gz | |||
| SRR21949226.fastq.gz | Fastqsanger.gz | |||
| SRR21949227.fastq.gz | Fastqsanger.gz | |||
| SRR21949228.fastq.gz | Fastqsanger.gz |
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Project description:The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the gut commensal and model organism Escherichia coli, as well as several major human pathogens including Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, and the E. coli Keio single-gene deletion library is often regarded as a gold standard for gene essentiality studies. However, it remains unclear how much essential genes vary between strains and species. To investigate this, we have assembled a collection of thirteen sequenced high-density transposon mutant libraries from five genera of the Enterobacteriaceae. We first benchmark a number of gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations we develop a new classifier for gene essentiality. Using gene essentiality defined by this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes, and reconstruct an ancestral essential gene set of 296 genes. Despite the presence of a large cohort of variably essential genes, we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σE extracytoplasmic stress response, which appears to have independently become essential multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts closely related to the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival, and uncovers several instances of essential stress responses masked by redundancy in free-living bacteria.
2024-08-20 | GSE216013 | GEO
Project description:Biofilms undergo a life cycle where cells attach to a surface, grow and produce a structured community before dispersing to seed biofilms in new environments. Progression through this life cycle requires controlled temporal gene expression to maximise fitness at each stage. Previous studies have focused on the essential genome for the formation of a mature biofilm, but here we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-Xpress (a massively parallel transposon mutagenesis using transposon-located promoters to assay expression of all genes in the genome) to determine how gene essentiality and expression affects the fitness of E. coli growing as a biofilm on glass beads after 12, 24 and 48 hours. An E. coli transposon mutant library of approximately 800,000 unique mutants was grown on glass beads, and a planktonic sample was taken alongside this at each time point to compare gene essentiality and expression at each time point.
2021-06-09 | E-MTAB-9873 | biostudies-arrayexpress
Project description:For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.
| S-EPMC5241402 | biostudies-literature
Project description:Transposon-insertion sequencing (TIS) methods couple high density transposon mutagenesis with next-generation sequencing and are commonly used to identify essential or important genes in bacteria. However, this approach can be work-intensive and sometimes expensive depending on the selected protocol. The difficulty to process a high number of samples in parallel using standard TIS protocols often restricts the number of replicates that can be performed and limits the deployment of this technique to large-scale projects studying gene essentiality in various strains or growth conditions. Here, we report the development of a robust and inexpensive High-Throughput Transposon Mutagenesis (HTTM) protocol and validate the method using Escherichia coli strain BW25113, the parental strain of the KEIO collection. HTTM reliably provides high insertion densities with an average of one transposon every ≤20bp along with impressive reproducibility (Spearman correlation coefficients >0.94). A detailed protocol is available at protocol.io and a graphical version is also included with this article.
| S-EPMC10089323 | biostudies-literature
Project description:Mycobacterium abscessus is an emerging opportunistic human pathogen that naturally resists most major classes of antibiotics, making infections difficult to treat. Thus far, little is known about M. abscessus physiology, pathogenesis, and drug resistance. Genome-wide analyses have comprehensively catalogued genes with essential functions in Mycobacterium tuberculosis and Mycobacterium avium subsp. hominissuis (here, M. avium) but not in M. abscessus. By optimizing transduction conditions, we achieved full saturation of TA insertion sites with Himar1 transposon mutagenesis in the M. abscessus ATCC 19977T genome, as confirmed by deep sequencing prior to essentiality analyses of annotated genes and other genomic features. The overall densities of inserted TA sites (85.7%), unoccupied TA sites (14.3%), and nonpermissive TA sites (8.1%) were similar to results in M. tuberculosis and M. avium. Of the 4,920 annotated genes, 326 were identified as essential, 269 (83%) of which have mutual homology with essential M. tuberculosis genes, while 39 (12%) are homologous to genes that are not essential in M. tuberculosis and M. avium, and 11 (3.4%) only have homologs in M. avium. Interestingly, 7 (2.1%) essential M. abscessus genes have no homologs in either M. tuberculosis or M. avium, two of which were found in phage-like elements. Most essential genes are involved in DNA replication, RNA transcription and translation, and posttranslational events to synthesize important macromolecules. Some essential genes may be involved in M. abscessus pathogenesis and antibiotics response, including certain essential tRNAs and new short open reading frames. Our findings will help to pave the way for better understanding of M. abscessus and benefit development of novel bactericidal drugs against M. abscessus. IMPORTANCE Limited knowledge regarding Mycobacterium abscessus pathogenesis and intrinsic resistance to most classes of antibiotics is a major obstacle to developing more effective strategies to prevent and mitigate disease. Using optimized procedures for Himar1 transposon mutagenesis and deep sequencing, we performed a comprehensive analysis to identify M. abscessus genetic elements essential for in vitro growth and compare them to similar data sets for M. tuberculosis and M. avium subsp. hominissuis. Most essential M. abscessus genes have mutual homology with essential M. tuberculosis genes, providing a foundation for leveraging available knowledge from M. tuberculosis to develop more effective drugs and other interventions against M. abscessus. A small number of essential genes unique to M. abscessus deserve further attention to gain insights into what makes M. abscessus different from other mycobacteria. The essential genes and other genomic features such as short open reading frames and noncoding RNA identified here will provide useful information for future study of M. abscessus pathogenicity and new drug development.
| S-EPMC8262987 | biostudies-literature
Project description:In vivo transposon mutagenesis, coupled with deep sequencing, enables large-scale genome-wide mutant screens for genes essential in different growth conditions. We analyzed six large-scale studies performed on haploid strains of three yeast species (Saccharomyces cerevisiae, Schizosaccaromyces pombe, and Candida albicans), each mutagenized with two of three different heterologous transposons (AcDs, Hermes, and PiggyBac). Using a machine-learning approach, we evaluated the ability of the data to predict gene essentiality. Important data features included sufficient numbers and distribution of independent insertion events. All transposons showed some bias in insertion site preference because of jackpot events, and preferences for specific insertion sequences and short-distance vs long-distance insertions. For PiggyBac, a stringent target sequence limited the ability to predict essentiality in genes with few or no target sequences. The machine learning approach also robustly predicted gene function in less well-studied species by leveraging cross-species orthologs. Finally, comparisons of isogenic diploid versus haploid S. cerevisiae isolates identified several genes that are haplo-insufficient, while most essential genes, as expected, were recessive. We provide recommendations for the choice of transposons and the inference of gene essentiality in genome-wide studies of eukaryotic haploid microbes such as yeasts, including species that have been less amenable to classical genetic studies.
| S-EPMC7599172 | biostudies-literature
Project description:Mycobacterium kansasii (Mk) is an opportunistic pathogen that is frequently isolated from urban water systems, posing a health risk to susceptible individuals. Despite its ability to cause tuberculosis-like pulmonary disease, very few studies have probed the genetics of this opportunistic pathogen. Here, we report a comprehensive essentiality analysis of the Mk genome. Deep sequencing of a high-density library of Mk Himar1 transposon mutants revealed that 86.8% of the chromosomal thymine-adenine (TA) dinucleotide target sites were permissive to insertion, leaving 13.2% TA sites unoccupied. Our analysis identified 394 of the 5,350 annotated open reading frames (ORFs) as essential. The majority of these essential ORFs (84.8%) share essential mutual orthologs with Mycobacterium tuberculosis (Mtb). A comparative genomics analysis identified 139 Mk essential ORFs that share essential orthologs in four other species of mycobacteria. Thirteen Mk essential ORFs share orthologs in all four species that were identified as being not essential, while only two Mk essential ORFs are absent in all species compared. We used the essentiality data and a comparative genomics analysis reported here to highlight differences in essentiality between candidate Mtb drug targets and the corresponding Mk orthologs. Our findings suggest that the Mk genome encodes redundant or additional pathways that may confound validation of potential Mtb drugs and drug target candidates against the opportunistic pathogen. Additionally, we identified 57 intergenic regions containing four or more consecutive unoccupied TA sites. A disproportionally large number of these regions were located upstream of pe/ppe genes. Finally, we present an essentiality and orthology analysis of the Mk pRAW-like plasmid, pMK1248. IMPORTANCE Mk is one of the most common nontuberculous mycobacterial pathogens associated with tuberculosis-like pulmonary disease. Drug resistance emergence is a threat to the control of Mk infections, which already requires long-term, multidrug courses. A comprehensive understanding of Mk biology is critical to facilitate the development of new and more efficacious therapeutics against Mk. We combined transposon-based mutagenesis with analysis of insertion site identification data to uncover genes and other genomic regions required for Mk growth. We also compared the gene essentiality data set of Mk to those available for several other mycobacteria. This analysis highlighted key similarities and differences in the biology of Mk compared to these other species. Altogether, the genome-wide essentiality information generated and the results of the cross-species comparative genomics analysis represent valuable resources to assist the process of identifying and prioritizing potential Mk drug target candidates and to guide future studies on Mk biology.
| S-EPMC10470612 | biostudies-literature