Multiplexed Competition in a Synthetic Squid Light Organ Microbiome Using Barcode-Tagged Gene Deletions
Autor: | Emanuel F. Burgos, Garret Suen, Mark J. Mandel, Andrew J. Steinberger, Hector L. Burgos |
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Rok vydání: | 2020 |
Předmět: |
Transposable element
Candidate gene Euprymna scolopes sequence-tagged gene deletions Physiology Vibrio fischeri Computational biology Bacterial genome size Biochemistry Microbiology DNA sequencing Host-Microbe Biology barcode sequencing 03 medical and health sciences Genetics Bioluminescence Aliivibrio fischeri Overlap extension polymerase chain reaction Microbiome Molecular Biology Ecology Evolution Behavior and Systematics 030304 developmental biology 0303 health sciences amplicon sequencing biology 030306 microbiology biology.organism_classification QR1-502 Vibrio Computer Science Applications Transformation (genetics) Modeling and Simulation BarSeq Research Article |
Zdroj: | mSystems mSystems, Vol 5, Iss 6 (2020) |
ISSN: | 2379-5077 |
Popis: | Beneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Beneficial symbioses between microbes and their eukaryotic hosts are ubiquitous and have widespread impacts on host health and development. The binary symbiosis between the bioluminescent bacterium Vibrio fischeri and its squid host Euprymna scolopes serves as a model system to study molecular mechanisms at the microbe-animal interface. To identify colonization factors in this system, our lab previously conducted a global transposon insertion sequencing (INSeq) screen and identified over 300 putative novel squid colonization factors in V. fischeri. To pursue mechanistic studies on these candidate genes, we present an approach to quickly generate barcode-tagged gene deletions and perform high-throughput squid competition experiments with detection of the proportion of each strain in the mixture by barcode sequencing (BarSeq). Our deletion approach improves on previous techniques based on splicing by overlap extension PCR (SOE-PCR) and tfoX-based natural transformation by incorporating a randomized barcode that results in unique DNA sequences within each deletion scar. Amplicon sequencing of the pool of barcoded strains before and after colonization faithfully reports on known colonization factors and provides increased sensitivity over colony counting methods. BarSeq enables rapid and sensitive characterization of the molecular factors involved in establishing the Vibrio-squid symbiosis and provides a valuable tool to interrogate the molecular dialogue at microbe-animal host interfaces. IMPORTANCE Beneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Model symbioses are therefore invaluable for studying the mechanisms of beneficial microbe-host interactions. Here, we present a combined barcode-tagged deletion and BarSeq approach to interrogate the molecular dialogue that ensures specific and reproducible colonization of the Hawaiian bobtail squid by Vibrio fischeri. The ability to precisely manipulate the bacterial genome, combined with multiplex colonization assays, will accelerate the use of this valuable model system for mechanistic studies of how environmental microbes—both beneficial and pathogenic—colonize specific animal hosts. |
Databáze: | OpenAIRE |
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