Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota

ABSTRACT Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Leonor García-Bayona, Laurie E. Comstock
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Bacteroides microbiota mutant construction vectors Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1fc06d832ea54e9aa87c5317f5763514
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:1fc06d832ea54e9aa87c5317f5763514
record_format	dspace
spelling	oai:doaj.org-article:1fc06d832ea54e9aa87c5317f57635142021-11-15T16:22:10ZStreamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota10.1128/mBio.01762-192150-7511https://doaj.org/article/1fc06d832ea54e9aa87c5317f57635142019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01762-19https://doaj.org/toc/2150-7511ABSTRACT Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis. Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools. IMPORTANCE We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.Leonor García-BayonaLaurie E. ComstockAmerican Society for MicrobiologyarticleBacteroidesmicrobiotamutant constructionvectorsMicrobiologyQR1-502ENmBio, Vol 10, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Bacteroides microbiota mutant construction vectors Microbiology QR1-502
spellingShingle	Bacteroides microbiota mutant construction vectors Microbiology QR1-502 Leonor García-Bayona Laurie E. Comstock Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota
description	ABSTRACT Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis. Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools. IMPORTANCE We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.
format	article
author	Leonor García-Bayona Laurie E. Comstock
author_facet	Leonor García-Bayona Laurie E. Comstock
author_sort	Leonor García-Bayona
title	Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota
title_short	Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota
title_full	Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota
title_fullStr	Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota
title_full_unstemmed	Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota
title_sort	streamlined genetic manipulation of diverse <italic toggle="yes">bacteroides</italic> and <italic toggle="yes">parabacteroides</italic> isolates from the human gut microbiota
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/1fc06d832ea54e9aa87c5317f5763514
work_keys_str_mv	AT leonorgarciabayona streamlinedgeneticmanipulationofdiverseitalictoggleyesbacteroidesitalicanditalictoggleyesparabacteroidesitalicisolatesfromthehumangutmicrobiota AT laurieecomstock streamlinedgeneticmanipulationofdiverseitalictoggleyesbacteroidesitalicanditalictoggleyesparabacteroidesitalicisolatesfromthehumangutmicrobiota
_version_	1718426891240603648

Streamlined Genetic Manipulation of Diverse <italic toggle="yes">Bacteroides</italic> and <italic toggle="yes">Parabacteroides</italic> Isolates from the Human Gut Microbiota

Ejemplares similares