Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron
Abstract Background Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-12-01
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| Series: | BMC Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12915-022-01481-2 |
| _version_ | 1798005331933003776 |
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| author | Heidi A. Arjes Jiawei Sun Hualan Liu Taylor H. Nguyen Rebecca N. Culver Arianna I. Celis Sophie Jean Walton Kimberly S. Vasquez Feiqiao Brian Yu Katherine S. Xue Daniel Newton Ricardo Zermeno Meredith Weglarz Adam Deutschbauer Kerwyn Casey Huang Anthony L. Shiver |
| author_facet | Heidi A. Arjes Jiawei Sun Hualan Liu Taylor H. Nguyen Rebecca N. Culver Arianna I. Celis Sophie Jean Walton Kimberly S. Vasquez Feiqiao Brian Yu Katherine S. Xue Daniel Newton Ricardo Zermeno Meredith Weglarz Adam Deutschbauer Kerwyn Casey Huang Anthony L. Shiver |
| author_sort | Heidi A. Arjes |
| collection | DOAJ |
| description | Abstract Background Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development. Results Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data. Conclusion We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members. |
| first_indexed | 2024-04-11T12:38:33Z |
| format | Article |
| id | doaj.art-22ce794b6e254b8ebb0ddbb93ac44327 |
| institution | Directory Open Access Journal |
| issn | 1741-7007 |
| language | English |
| last_indexed | 2024-04-11T12:38:33Z |
| publishDate | 2022-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biology |
| spelling | doaj.art-22ce794b6e254b8ebb0ddbb93ac443272022-12-22T04:23:34ZengBMCBMC Biology1741-70072022-12-0120111710.1186/s12915-022-01481-2Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicronHeidi A. Arjes0Jiawei Sun1Hualan Liu2Taylor H. Nguyen3Rebecca N. Culver4Arianna I. Celis5Sophie Jean Walton6Kimberly S. Vasquez7Feiqiao Brian Yu8Katherine S. Xue9Daniel Newton10Ricardo Zermeno11Meredith Weglarz12Adam Deutschbauer13Kerwyn Casey Huang14Anthony L. Shiver15Department of Bioengineering, Stanford UniversityDepartment of Bioengineering, Stanford UniversityEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryDepartment of Bioengineering, Stanford UniversityDepartment of Genetics, Stanford University School of MedicineDepartment of Medicine, Stanford University School of MedicineBiophysics Training Program, Stanford University School of MedicineDepartment of Microbiology and Immunology, Stanford University School of MedicineChan Zuckerberg BiohubDepartment of Bioengineering, Stanford UniversityDepartment of Bioengineering, Stanford UniversityStanford Shared FACS Facility, Center for Molecular and Genetic Medicine, Stanford UniversityStanford Shared FACS Facility, Center for Molecular and Genetic Medicine, Stanford UniversityEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryDepartment of Bioengineering, Stanford UniversityDepartment of Bioengineering, Stanford UniversityAbstract Background Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development. Results Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data. Conclusion We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.https://doi.org/10.1186/s12915-022-01481-2B. thetaTransposon mutagenesisOrdered libraryBarSeqRB-TnSeqMicrobiome |
| spellingShingle | Heidi A. Arjes Jiawei Sun Hualan Liu Taylor H. Nguyen Rebecca N. Culver Arianna I. Celis Sophie Jean Walton Kimberly S. Vasquez Feiqiao Brian Yu Katherine S. Xue Daniel Newton Ricardo Zermeno Meredith Weglarz Adam Deutschbauer Kerwyn Casey Huang Anthony L. Shiver Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron BMC Biology B. theta Transposon mutagenesis Ordered library BarSeq RB-TnSeq Microbiome |
| title | Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron |
| title_full | Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron |
| title_fullStr | Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron |
| title_full_unstemmed | Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron |
| title_short | Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron |
| title_sort | construction and characterization of a genome scale ordered mutant collection of bacteroides thetaiotaomicron |
| topic | B. theta Transposon mutagenesis Ordered library BarSeq RB-TnSeq Microbiome |
| url | https://doi.org/10.1186/s12915-022-01481-2 |
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