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      Identification of genes required for the fitness of Streptococcus equi subsp. equi in whole equine blood and hydrogen peroxide

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          The availability of next-generation sequencing techniques provides an unprecedented opportunity for the assignment of gene function. Streptococcus equi subspecies equi is the causative agent of strangles in horses, one of the most prevalent and important diseases of equids worldwide. However, the live attenuated vaccines that are utilized to control this disease cause adverse reactions in some animals. Here, we employ transposon-directed insertion-site sequencing (TraDIS) to identify genes that are required for the fitness of S. equi in whole equine blood or in the presence of H 2O 2 to model selective pressures exerted by the equine immune response during infection. We report the fitness values of 1503 and 1471 genes, representing 94.5 and 92.5 % of non-essential genes in S. equi , following incubation in whole blood and in the presence of H 2O 2, respectively. Of these genes, 36 and 15 were identified as being important to the fitness of S. equi in whole blood or H 2O 2, respectively, with 14 genes being important in both conditions. Allelic replacement mutants were generated to validate the fitness results. Our data identify genes that are important for S. equi to resist aspects of the immune response in vitro, which can be exploited for the development of safer live attenuated vaccines to prevent strangles.

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          Tn-seq; high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms

          Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing of the flanking regions en masse. These changes are used to calculate each mutant’s fitness. Fitness was determined for each gene of the gram-positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions identified both alleviating and aggravating interactions that could be further divided into seven distinct categories. Due to the wide activity of the Mariner transposon, Tn-seq has the potential to contribute to the exploration of complex pathways across many different species.
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            Identifying genetic determinants needed to establish a human gut symbiont in its habitat.

            The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B(12)). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems.
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              Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.

              Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.

                Author and article information

                Microb Genom
                Microb Genom
                Microbial Genomics
                Microbiology Society
                April 2020
                31 March 2020
                31 March 2020
                : 6
                : 4
                [ 1] Animal Health Trust, Lanwades Park , Newmarket, Suffolk, UK
                [ 2] departmentDepartment of Veterinary Medicine , University of Cambridge , Cambridge, UK
                [ 3] departmentSchool of Veterinary Medicine , University of Surrey , Guildford, UK
                [ 4] departmentDepartment of Molecular Sciences , Macquarie University , Sydney, Australia
                [ 5] School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington , Leicestershire, UK
                [ 6] University of Melbourne , Victoria, Australia
                Author notes
                *Correspondence: Amelia R. L. Charbonneau, Amy@
                © 2020 The Authors

                This is an open-access article distributed under the terms of the Creative Commons Attribution License.

                Funded by: Australian Research Council Discovery Early Career Research Award
                Award ID: DE180100929
                Award Recipient : Amy K. Cain
                Funded by: Biotechnology and Biological Sciences Research Counci
                Award ID: 1503883
                Award Recipient : Duncan J. Maskell
                Funded by: Horse Trust
                Award ID: G4104
                Award Recipient : Andrew S. Waller
                Research Article
                Genomic Methodologies: Genome-phenotype association
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