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Transcriptional programming in a Bacteroides consortium.

ABSTRACT: Bacteroides species are prominent members of the human gut microbiota. The prevalence and stability of Bacteroides in humans make them ideal candidates to engineer as programmable living therapeutics. Here we report a biotic decision-making technology in a community of Bacteroides (consortium transcriptional programming) with genetic circuit compression. Circuit compression requires systematic pairing of engineered transcription factors with cognate regulatable promoters. In turn, we demonstrate the compression workflow by designing, building, and testing all fundamental two-input logic gates dependent on the inputs isopropyl-β-D-1-thiogalactopyranoside and D-ribose. We then deploy complete sets of logical operations in five human donor Bacteroides, with which we demonstrate sequential gain-of-function control in co-culture. Finally, we couple transcriptional programs with CRISPR interference to achieve loss-of-function regulation of endogenous genes-demonstrating complex control over community composition in co-culture. This work provides a powerful toolkit to program gene expression in Bacteroides for the development of bespoke therapeutic bacteria.

SUBMITTER: Huang BD

PROVIDER: S-EPMC9259675 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Whole-transcriptome analysis was used to investigate the molecular interplay between three bacterial species that are members of the human gut microbiota. Bacteroides ovatus, Subdoligranulum variabile, and Hungatella hathewayi formed associations in cocultures fed barley β-glucan, a constituent of dietary fiber. B. ovatus depolymerized β-glucan and released, but did not utilize, 3-O-β-cellobiosyl-d-glucose (DP3) and 3-O-β-cellotriosyl-d-glucose (DP4). These oligosaccharides provided growth substrates for S. variabile and H. hathewayi with a preference for DP4 in the case of the latter species. There was increased transcription of a B. ovatus mixed-linkage-β-glucan utilization locus, as well as carbohydrate transporters in S. variabile and H. hathewayi when in batch coculture. Increased transcription of the β-glucan utilization locus did not occur in continuous culture. Evidence for interactions relating to provision of cobalamin, alterations to signaling, and modulation of the "stringent response" (an adaptation to nutrient deprivation) were detected. Overall, we established a bacterial consortium based on barley β-glucan in vitro, which can be used to investigate aspects of the functional blueprint of the human gut microbiota.IMPORTANCE The microbial community, mostly composed of bacterial species, residing in the human gut degrades and ferments polysaccharides derived from plants (dietary fiber) that would not otherwise be digested. In this way, the collective metabolic actions of community members extract additional energy from the human diet. While the variety of bacteria present in the microbial community is well known, the formation of bacterial consortia, and the consequent interactions that result in the digestion of dietary polysaccharides, has not been studied extensively. The importance of our work was the establishment, under laboratory conditions, of a consortium of gut bacteria that formed around a dietary constituent commonly present in cereals. This enabled the metabolic interplay between the bacterial species to be studied. This kind of knowledge is required to construct an interactive, metabolic blueprint of the microbial community that inhabits the human gut.

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Transcriptional programming in a Bacteroides consortium.

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