Abstract
Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization.
Highlights
The mammalian microbiota influences the biology of its host at many levels
We reconstructed the genotype of the reversible intestinal colonization prototype strain E. coli K-12 HA107 [6] in the genetic background of the less laboratory-adapted commensal E. coli strain HS [13,14] by deleting the genes alr, dadX and asd
The intestinal bacterial clearance of HS Δalr ΔdadX Δasd was delayed compared to E. coli K-12 HA107 (Fig 1A), suggesting a leaky phenotype in this strain background
Summary
The mammalian microbiota influences the biology of its host at many levels. As a consequence, a large number of human conditions are shaped by the host’s genetic predisposition, external environment and diet, and the microbiota composition. The high microbiota variability between individuals and between different experimental vivaria (often synonymously referred to as “hygiene status”) generates a growing demand for new and improved animal models that provide better experimental control over microbiota composition. Numerous studies, spanning many decades, have utilized axenic/ germ-free animals [1] and gnotobiotic animal models with simplified defined microbial compositions [2,3] to greatly advance our current understanding of host-microbial interactions. Manipulating simple microbiotas by experimentally increasing the complexity with new immigrants is generally technically easier than permanently eliminating members of an established consortia. Antibiotic treatments provide a means for the reduction of density and complexity of an already established microbiota, it is incomplete and unsustainable without continued antibiotic administration [4] and can lead to blooms of unsusceptible or resistant microbes. The recovery from the antibiotic treatment back to the original state is often incomplete and irreproducible [5], potentially causing persistent dysbiosis
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