A Genomewide Mutagenesis Screen Identifies Multiple Genes Contributing to Vi Capsular Expression in Salmonella enterica Serovar Typhi

Pickard, Derek; Kingsley, Robert A.; Hale, Christine; Turner, A. Keith; Sivaraman, Karthikeyan; Wetter, Michael; Langridge, Gemma C.; Dougan, Gordon

doi:10.1128/jb.01632-12

Cited by 28 publications

(21 citation statements)

References 42 publications

(57 reference statements)

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“…Typhimurium , 35 bacteriophage infection of S . Typhi , 36 antibiotic resistance in Pseudomonas aeruginosa , 10 cholesterol utilization in M. tuberculosis 27 and a number of stress and nutrient conditions in S. pneumoniae 22 . Transposon-insertion sequencing of populations passed through murine models have been used to assess genes required to establish the gut commensal B. thetaiotaomicron in its niche, 7 for Hemophilus influenzae infection, 8 as well as S. pneumoniae responses to two in vivo niches, the lung and nasopharynx 22 .…”

Section: Defining Conditional Gene Requirementsmentioning

confidence: 99%

Approaches to querying bacterial genomes with transposon-insertion sequencing

2013

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In this review, we discuss transposon-insertion sequencing, variously known in the literature as TraDIS, Tn-seq, INSeq, and HITS. By monitoring a large library of single transposon-insertion mutants with high-throughput sequencing, these methods can rapidly identify genomic regions that contribute to organismal fitness under any condition assayable in the laboratory with exquisite resolution. We discuss the various protocols that have been developed and methods for analysis. We provide an overview of studies that have examined the reproducibility and accuracy of these methods, as well as studies showing the advantages offered by the high resolution and dynamic range of high-throughput sequencing over previous methods. We review a number of applications in the literature, from predicting genes essential for in vitro growth to directly assaying requirements for survival under infective conditions in vivo. We also highlight recent progress in assaying non-coding regions of the genome in addition to known coding sequences, including the combining of RNA-seq with high-throughput transposon mutagenesis.

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Section: Defining Conditional Gene Requirementsmentioning

confidence: 99%

Approaches to querying bacterial genomes with transposon-insertion sequencing

2013

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“…There have been few attempts to use genetic approaches for studying genome-wide host factors essential in phage infection. These loss-of-function (LOF) genetic screens broadly use bacterial saturation mutagenesis [48,53,[57][58][59][60] or an arrayed library of single-gene deletion strains for studying phage-host interactions [49,50,52,61,62]. Consequently, these studies have generally involved laborious experiments on relatively few phages and their hosts, and scaling the approach to characterize hundreds of phages is challenging.…”

Section: Introductionmentioning

confidence: 99%

High-throughput mapping of the phage resistance landscape inE. coli

Mutalik¹,

Adler²,

Rishi³

et al. 2020

Preprint

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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and highthroughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phagemediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.3 Introduction:

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“…This approach allows replacement of the labor-intensive process of screening for individual Tn mutants and identifying the insertion locations, while also enabling more thorough discovery of beneficial insertions. Tn-Seq has previously been used to determine genes that rendered Salmonella enterica serovar Typhi resistant to lysis following the addition of a Vi bacteriophage (78) and to determine loss-of-function mutations that were transduced using P1vir from the Keio collection (35) to E. coli K-12 MG1655 ⌬lacZ which improved growth on several amino acids as a sole carbon source (23). In this work, six different wild-type and laboratory E. coli strains [K-12 MG1655, K-12 W3110, BL21(DE3), W, C, and Crooks; Table 1] were first characterized for their growth behavior under controlled conditions by the addition of stressors during the mid-exponential phase of growth.…”

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confidence: 99%

Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

Lennen

2014

Appl Environ Microbiol

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High-cell-density fermentation for industrial production of chemicals can impose numerous stresses on cells due to high substrate, product, and by-product concentrations; high osmolarity; reactive oxygen species; and elevated temperatures. There is a need to develop platform strains of industrial microorganisms that are more tolerant toward these typical processing conditions. In this study, the growth of six industrially relevant strains of Escherichia coli was characterized under eight stress conditions representative of fed-batch fermentation, and strains W and BL21(DE3) were selected as platforms for transposon (Tn) mutagenesis due to favorable resistance characteristics. Selection experiments, followed by either targeted or genome-wide nextgeneration-sequencing-based Tn insertion site determination, were performed to identify mutants with improved growth properties under a subset of three stress conditions and two combinations of individual stresses. A subset of the identified loss-offunction mutants were selected for a combinatorial approach, where strains with combinations of two and three gene deletions were systematically constructed and tested for single and multistress resistance. These approaches allowed identification of (i) strain-background-specific stress resistance phenotypes, (ii) novel gene deletion mutants in E. coli that confer single and multistress resistance in a strain-background-dependent manner, and (iii) synergistic effects of multiple gene deletions that confer improved resistance over single deletions. The results of this study underscore the suboptimality and strain-specific variability of the genetic network regulating growth under stressful conditions and suggest that further exploration of the combinatorial gene deletion space in multiple strain backgrounds is needed for optimizing strains for microbial bioprocessing applications.

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A Genomewide Mutagenesis Screen Identifies Multiple Genes Contributing to Vi Capsular Expression in Salmonella enterica Serovar Typhi

Cited by 28 publications

References 42 publications

Approaches to querying bacterial genomes with transposon-insertion sequencing

Approaches to querying bacterial genomes with transposon-insertion sequencing

High-throughput mapping of the phage resistance landscape inE. coli

Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

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