Streamlined Genetic Manipulation of Diverse and Isolates from the Human Gut Microbiota.

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 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 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 and 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 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 strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools. 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 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 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 and strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut , including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.