Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

ABSTRACT Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of...

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Autores principales:	Travis J. Wiles, Elena S. Wall, Brandon H. Schlomann, Edouard A. Hay, Raghuveer Parthasarathy, Karen Guillemin
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	Tn7 allelic exchange bacterial genetics conjugation counterselection domestication Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6d927dab039044e9a29cca5aab4fbc96
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spelling	oai:doaj.org-article:6d927dab039044e9a29cca5aab4fbc962021-11-15T15:58:20ZModernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages10.1128/mBio.01877-182150-7511https://doaj.org/article/6d927dab039044e9a29cca5aab4fbc962018-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01877-18https://doaj.org/toc/2150-7511ABSTRACT Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis and in vitro assays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings. IMPORTANCE A great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.Travis J. WilesElena S. WallBrandon H. SchlomannEdouard A. HayRaghuveer ParthasarathyKaren GuilleminAmerican Society for MicrobiologyarticleTn7allelic exchangebacterial geneticsconjugationcounterselectiondomesticationMicrobiologyQR1-502ENmBio, Vol 9, Iss 5 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Tn7 allelic exchange bacterial genetics conjugation counterselection domestication Microbiology QR1-502
spellingShingle	Tn7 allelic exchange bacterial genetics conjugation counterselection domestication Microbiology QR1-502 Travis J. Wiles Elena S. Wall Brandon H. Schlomann Edouard A. Hay Raghuveer Parthasarathy Karen Guillemin Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages
description	ABSTRACT Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis and in vitro assays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings. IMPORTANCE A great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.
format	article
author	Travis J. Wiles Elena S. Wall Brandon H. Schlomann Edouard A. Hay Raghuveer Parthasarathy Karen Guillemin
author_facet	Travis J. Wiles Elena S. Wall Brandon H. Schlomann Edouard A. Hay Raghuveer Parthasarathy Karen Guillemin
author_sort	Travis J. Wiles
title	Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages
title_short	Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages
title_full	Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages
title_fullStr	Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages
title_full_unstemmed	Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages
title_sort	modernized tools for streamlined genetic manipulation and comparative study of wild and diverse proteobacterial lineages
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/6d927dab039044e9a29cca5aab4fbc96
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Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

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