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Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease., Competing Interests: The authors have declared that no competing interests exist.

Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination. Author summary: Antimicrobial resistance is one of the greatest threats to human health. Left unchecked, we risk a rapid escalation of untreatable infections. Plasmids are one of the most important vehicles for resistance gene carriage and transmission between bacteria, and thus an understanding of plasmid biology is crucial to controlling the spread antimicrobial resistance. Here, we combine advanced bioinformatics and a state-of-the-art genetic screen to understand the molecular mechanisms involved in the maintenance and spread of a group of plasmids strongly associated with antibiotic resistance among bacteria that cause human infection. We characterised genes involved in the replication and maintenance of these plasmids, and experimentally demonstrated that plasmid spread is dependent on a well-conserved secretion system. Our genetic screen also discovered a set of broadly conserved, uncharacterised genes that adversely impact host fitness and plasmid spread under dysregulation. Taken together, these findings describe a molecular blueprint for the biology of a group of clinically relevant antibiotic resistance plasmids found frequently in Gram-negative bacterial pathogens. [ABSTRACT FROM AUTHOR]

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. Author summary: Corynebacterium diphtheriae causes both toxin-mediated diphtheria and non-toxigenic invasive infections. Despite a vaccine to protect against diphtheria, case numbers for both invasive and diphtherial disease have increased over the last decade. Furthermore, an increase in antibiotic resistant strains are being isolated from patients. It's clear that additional treatment strategies for this organism will be needed in the future. Using high-throughput mutagenesis, this work presents the densest library of mutants for any Corynebacterium sp.. This work identifies the essential genome of C. diphtheriae; an important classification as these genes are often the target of therapeutic intervention. We identify highly conserved genes and species-specific genes unique to pathogens. This data presents an important benchmark and focus for the future development of therapeutic options. Of particular significance is the identification of uncharacterized, conserved proteins within the Diphtheria vaccine. [ABSTRACT FROM AUTHOR]

Recent and classical work has revealed biologically and medically significant subtypes in complex diseases and traits. However, relevant subtypes are often unknown, unmeasured, or actively debated, making automated statistical approaches to subtype definition valuable. We propose reverse GWAS (RGWAS) to identify and validate subtypes using genetics and multiple traits: while GWAS seeks the genetic basis of a given trait, RGWAS seeks to define trait subtypes with distinct genetic bases. Unlike existing approaches relying on off-the-shelf clustering methods, RGWAS uses a novel decomposition, MFMR, to model covariates, binary traits, and population structure. We use extensive simulations to show that modelling these features can be crucial for power and calibration. We validate RGWAS in practice by recovering a recently discovered stress subtype in major depression. We then show the utility of RGWAS by identifying three novel subtypes of metabolic traits. We biologically validate these metabolic subtypes with SNP-level tests and a novel polygenic test: the former recover known metabolic GxE SNPs; the latter suggests subtypes may explain substantial missing heritability. Crucially, statins, which are widely prescribed and theorized to increase diabetes risk, have opposing effects on blood glucose across metabolic subtypes, suggesting the subtypes have potential translational value. [ABSTRACT FROM AUTHOR]

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease. [ABSTRACT FROM AUTHOR]

Upstream open reading frames (uORFs) are major gene expression regulatory elements. In many eukaryotic mRNAs, one or more uORFs precede the initiation codon of the main coding region. Indeed, several studies have revealed that almost half of human transcripts present uORFs. Very interesting examples have shown that these uORFs can impact gene expression of the downstream main ORF by triggering mRNA decay or by regulating translation. Also, evidence from recent genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of many human diseases, including malignancies, metabolic or neurologic disorders, and inherited syndromes. In this review, we will briefly present the mechanisms through which uORFs regulate gene expression and how they can impact on the organism's response to different cell stress conditions. Then, we will emphasize the importance of these structures by illustrating, with specific examples, how disturbed uORF-mediated translational control can be involved in the etiology of human diseases, giving special importance to genotype-phenotype correlations. Identifying and studying more cases of uORF-altering mutations will help us to understand and establish genotype-phenotype associations, leading to advancements in diagnosis, prognosis, and treatment of many human disorders. [ABSTRACT FROM AUTHOR]