An Arrayed Transposon Library of <i>Ruegeria pomeroyi</i> DSS-3

Mejia, Catalina; Rodriguez, Lidimarie Trujillo; Poudel, Ravin; Ellington, Adam J; Rivers, Adam R.; Reisch, Christopher R

doi:10.1101/2022.09.11.507510

Cited by 3 publications

(7 citation statements)

References 9 publications

(12 reference statements)

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“…Ruegeria pomeroyi DSS-3 is a representative of the Roseobacteraceae family, an important bacterial group in marine microbial communities (Newton et al 2010) with its members among the most metabolically active bacterial cells in algal blooms and coastal environments (Munson-McGee et al 2022). R. pomeroyi has been well studied in the laboratory and field (Fu et al 2020; Nowinski and Moran 2021; Schreier et al 2023); it grows well in both defined and rich media; and it is amenable to genetic alteration (Howard et al 2006; Mejia et al 2022).…”

Section: Resultsmentioning

confidence: 99%

Digital Microbe: A Genome-Informed Data Integration Framework for Collaborative Research on Emerging Model Organisms

Veseli,

DeMers,

Cooper

et al. 2024

Preprint

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The remarkable pace of genomic data generation focused on the physiology and ecology of microbes is rapidly transforming our understanding of life at the micron scale. Yet this data stream has also created challenges for finding interoperable and extensible modes of analysis. From our own experience, a single microbe often has multiple versions of its genome architecture, functional gene annotation, and gene naming system, without a straightforward mechanism for collating information and preserving crucial advances in annotation. These dispersed data sources raise barriers to collaborations, and more generally hinder community coalescence around shared datasets of model organisms. Here, we describe the “Digital Microbe” data product which provides a framework for interoperability, reproducibility, and collaborative microbial science. A Digital Microbe is an open source, community-curated data package built on a (pan)genome foundation, which is housed within an integrative software environment. Using Digital Microbes ensures real-time alignment of research efforts within collaborative teams, and, as new layers of ’omic, experimental, or modeling data are added, facilitates the generation of novel scientific insights. We describe two Digital Microbes, one for the model heterotrophic marine bacteriumRuegeria pomeroyiDSS-3 which includes >100 transcriptomic datasets from lab and field studies; and another for the pangenome of the cosmopolitan heterotrophic marine bacterial genusAlteromonasrepresented by 339 genomes. Examples are provided to demonstrate how an integrated framework that collates public (pan)genome-informed data can generate novel and reproducible findings.

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Section: Resultsmentioning

confidence: 99%

Digital Microbe: A Genome-Informed Data Integration Framework for Collaborative Research on Emerging Model Organisms

Veseli,

DeMers,

Cooper

et al. 2024

Preprint

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“…1). The gene disrupted in each arrayed mutant was determined by sequencing the transposon barcode in conjunction with indexed primers that indicated plate column and row (38), creating a library that covers 3,292 of the 4,469 protein-encoded genes in the R. pomeroyi genome (73%). From the genome annotations (52, 53) we identified 156 mutants that were predicted to encode for 104 organic compound influx transporter proteins (Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…Full methods for generating and arraying the R. pomeroyi BarSeq mutant library are provided in Mejia et al . (38). Briefly, a pool of randomly barcoded transposon mutants was constructed according to Wetmore et al .…”

Section: Methodsmentioning

confidence: 99%

“…Full methods for generating and arraying the R. pomeroyi BarSeq mutant library are provided in Mejia et al (38). Briefly, a pool of randomly barcoded transposon mutants was constructed according to Wetmore et al (26) by conjugating R. pomeroyi DSS-3 with E.coli WM3064 containing the transposome pKMW7 Tn5 library (strain APA766).…”

Section: Barseq Library Generation and Mappingmentioning

confidence: 99%

“…Recently, a modification of the BarSeq approach was used to create an inexpensive arrayed mutant library of the marine model bacterium Ruegeria pomeroyi DSS-3 (38). The method took advantage of the ease of insertion site identification in BarSeq libraries in combination with 384-well plate location barcodes to generate an arrayed library of single-gene knockout mutants for R. pomeroyi , similar to the approach used recently for the anaerobic gut microbe Bacteroides thetaiotaomicron (39).…”

Section: Introductionmentioning

confidence: 99%

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Function and Importance of Marine Bacterial Transporters of Plankton Exometabolites

Schroer

Kepner

Uchimiya

et al. 2023

Preprint

Self Cite

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Metabolite exchange within marine microbial communities transfers carbon and other major elements through global cycles and forms the basis of microbial interactions. Yet lack of gene annotations and concern about the quality of existing ones remain major impediments to revealing the metabolite-microbial network. We employed an arrayed mutant library of the marine bacterium Ruegeria pomeroyi DSS-3 to experimentally annotate substrates of organic compound transporter systems, using mutant growth and compound drawdown analyses to link transporters to their substrates. Mutant experiments verified substrates for thirteen R. pomeroyi transporters. Four were previously hypothesized based on gene expression data (taurine, glucose/xylose, isethionate, and cadaverine/putrescine/spermidine); five were previously hypothesized based on homology to experimentally annotated transporters in other bacteria (citrate, glycerol, N-acetylglucosamine, fumarate/malate/succinate, and dimethylsulfoniopropionate); and four had no previous annotations (thymidine, carnitine, cysteate, and 3-hydroxybutyrate transporter). These bring the total number of experimentally-verified organic carbon influx transporters to 17 of 126 in the R. pomeroyi genome. In a longitudinal study of a coastal phytoplankton bloom, expression patterns of the experimentally annotated transporters linked them to different stages of the bloom, and also led to the hypothesis that citrate and 3-hydroxybutyrate were among the most highly available bacterial substrates. Improved functional knowledge of these gatekeepers of organic carbon uptake is facilitating better characterization of the surface ocean metabolite network.

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Functional annotation and importance of marine bacterial transporters of plankton exometabolites

Schroer

Kepner

Uchimiya

et al. 2023

ISME Communications

Self Cite

View full text Add to dashboard Cite

Metabolite exchange within marine microbial communities transfers carbon and other major elements through global cycles and forms the basis of microbial interactions. Yet lack of gene annotations and concern about the quality of existing ones remain major impediments to revealing currencies of carbon flux. We employed an arrayed mutant library of the marine bacterium Ruegeria pomeroyi DSS-3 to experimentally annotate substrates of organic compound transporter systems, using mutant growth and compound drawdown analyses to link transporters to their cognate substrates. Mutant experiments verified substrates for thirteen R. pomeroyi transporters. Four were previously hypothesized based on gene expression data (taurine, glucose/xylose, isethionate, and cadaverine/putrescine/spermidine); five were previously hypothesized based on homology to experimentally annotated transporters in other bacteria (citrate, glycerol, N-acetylglucosamine, fumarate/malate/succinate, and dimethylsulfoniopropionate); and four had no previous annotations (thymidine, carnitine, cysteate, and 3-hydroxybutyrate). These bring the total number of experimentally-verified organic carbon influx transporters to 18 of 126 in the R. pomeroyi genome. In a longitudinal study of a coastal phytoplankton bloom, expression patterns of the experimentally annotated transporters linked them to different stages of the bloom, and also led to the hypothesis that citrate and 3-hydroxybutyrate were among the most highly available bacterial substrates. Improved functional annotation of the gatekeepers of organic carbon uptake is critical for deciphering carbon flux and fate in microbial ecosystems.

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An Arrayed Transposon Library of Ruegeria pomeroyi DSS-3

Cited by 3 publications

References 9 publications

Digital Microbe: A Genome-Informed Data Integration Framework for Collaborative Research on Emerging Model Organisms

Digital Microbe: A Genome-Informed Data Integration Framework for Collaborative Research on Emerging Model Organisms

Function and Importance of Marine Bacterial Transporters of Plankton Exometabolites

Functional annotation and importance of marine bacterial transporters of plankton exometabolites

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