Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides 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 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.

Genetic and Biochemical Analysis of Anaerobic Respiration in Bacteroides fragilis and Its Importance In Vivo

“…Plasmid pLGB36 was made specifically to make allelic deletions and replacements in B. fragilis 638R and other B. fragilis strains that have the same type VI secretion system (T6SS) locus as strain 638R (67). This vector was created using the backbone of plasmid pLGB13 (68), which was designed for allelic replacements in Bacteroides and Parabacteroides species using the type VI secretion effector Bfe1 of strain 638R. As pLEC13 uses the 638R effector, strains with this T6SS region are immune to this toxin.…”

Genetic Manipulation of Wild Human Gut Bacteroides

“…Given the prevalence of type VI effector-immunity pairs and orphan immunity genes across human gut Bacteroides species (25,35,36) and the observation that immunity proteins confer specific resistance to individual effectors (25), a counterselection system utilizing multiple effectors reduces the likelihood that a recipient strain would be resistant to effector-mediated selection. No orphan Bfe1 immunity genes were found by Garcia-Bayona et al (22), although another study did identify candidate orphan Bfe1 immunity genes in both human metagenomic and microbial genome sequences (36). The modular design of the plasmid ( Fig.…”

“…In this study, we introduce an approach for genetic modification of Bacteroides species utilizing an engineered cassette that provides selection through the induction of multiple toxic effectors. This system complements related techniques utilizing a single effector (Bfe1) (22). Given the prevalence of type VI effector-immunity pairs and orphan immunity genes across human gut Bacteroides species (25,35,36) and the observation that immunity proteins confer specific resistance to individual effectors (25), a counterselection system utilizing multiple effectors reduces the likelihood that a recipient strain would be resistant to effector-mediated selection.…”

“…These toxic effectors are regulated by tightly controlled inducible promoters (23), such that expression occurs only during the counterselection step. This approach complements methods for allelic exchange that rely on expression of a single antibacterial effector (22). We demonstrate functional counterselection in 21 Bacteroides isolates representing 10 species, including multiple strains that were captured directly from human donors and displayed a range of resistance to commonly used antibiotic markers.…”

“…Similarly, methods that require selection on chemically defined culture medium can be difficult to apply to fastidious strains that are readily cultured only on undefined medium. Antibacterial toxins have been employed for negative selection in Bacillus subtilis, Clostridium acetobutylicum, Enterobacteriaceae species, Proteobacteria species, and Bacteroidales species (18)(19)(20)(21)(22). These approaches, which rely on a tightly regulated inducible expression system, provide an attractive method for negative selection in wildtype bacterial strains.…”