Programmable bacteria detect and record an environmental signal in the mammalian gut

Abstract

The mammalian gut is a dynamic community of symbiotic microbes that interact with the host to impact health, disease, and metabolism. We constructed engineered bacteria that survive in the mammalian gut and sense, remember, and report on their experiences. Based on previous genetic memory systems, we constructed a two-part system with a "trigger element" in which the lambda Cro gene is transcribed from a tetracycline-inducible promoter, and a "memory element" derived from the cI/Cro region of phage lambda. The memory element has an extremely stable cI state and a Cro state that is stable for many cell divisions. When Escherichia coli bearing the memory system are administered to mice treated with anhydrotetracycline, the recovered bacteria all have switched to the Cro state, whereas those administered to untreated mice remain in the cI state. The trigger and memory elements were transferred from E. coli K12 to a newly isolated murine E. coli strain; the stability and switching properties of the memory element were essentially identical in vitro and during passage through mice, but the engineered murine E. coli was more stably established in the mouse gut. This work lays a foundation for the use of synthetic genetic circuits as monitoring systems in complex, ill-defined environments, and may lead to the development of living diagnostics and therapeutics.

Keywords: genetic switch; synthetic biology.

Fig. 1.

Schematic of the memory circuit. (A) An abstraction of the genetic circuit used in this study combining the elements of a trigger/memory system and a toggle switch (–, –, –23) (TF-1 and TF-2 symbolize generic antagonistic transcription factors). (B) The lambda cI/Cro-based transcriptional memory element used in this work. (C) The tetP-Cro trigger element used in this study. Construction of both elements is detailed in Methods.

Fig. 2.

Engineered bacteria sense and remember ATC exposure in vitro. (A) Schematic of experimental design, as described in Methods. (B) In vitro triggering of PAS132. (C) In vitro memory of ATC exposure. (D) PAS132 was grown in liquid culture in the presence of ATC at the indicated concentration for 4 h, then plated on M9 glucose X-gal plates to determine the minimum dose of ATC required to switch the cells from the cI state to the Cro state. For all panels, means ± SD of three or more independent samples are shown. (E) Competitive growth assay to compare fitness of parental and engineered E. coli strains (Results, Methods). Mixed cultures of MG1655 rpsL and PAS132 were titered before (gray bars) and after (black bars) subculturing with a 10¹⁵-fold net serial dilution.

Fig. 3.

Engineered bacteria record, remember and report ATC exposure from the mammalian gut. (A) Schematic of experimental design, as described in Methods. (B) Mice were given ATC (0.1 mg/mL) and streptomycin (0.5 mg/mL) in drinking water on day 8. PAS132 cells were administered to the mice via oral gavage on day 9. Streptomycin and ATC were removed from the cage on days 10 and 11, respectively (means ± SD from four mice). (C) Total PAS132 cells in the cI and Cro states. Bars represent the average colony counts from four +ATC mice, and four −ATC mice. (D) The CFU of PAS132 and culturable bacteria (including PAS132) throughout the experiment (means ± SD from eight mice). (E) PAS132 cells were administered on day 9. ATC (0.1 mg/mL) was added to the drinking water on day 10 after streptomycin was removed. ATC was removed from the drinking water on day 11. All PAS132 cells triggered into the cro state within 1 d of ATC exposure, and remembered ATC exposure for more than 7 d (means ± SD from four mice).

Fig. 4.

Memory behavior of an endogenous murine E. coli strain engineered to contain the memory circuit. (A) The 16S ribosomal subunits of PAS132 and PAS133 were sequenced and compared against known gut microbes (46). (B) PAS133 was grown in M9 glucose + casamino acids liquid medium +ATC (100 ng/mL), or −ATC for 0–6 h. PAS133 was unable to grow in M9 glucose media without casamino acids. Aliquots were titered hourly on MacConkey Lactose plates to evaluate switching from the cI state to the cro state in response to ATC (means ± SD of three independent samples). (C) PAS133 was administered by oral gavage to mice exposed to antibiotics, and gut flora were characterized following the same protocol as in Fig. 3. On day 11 the ATC was removed from the cage (means ± SD from four mice). (D) Comparison of survival of PAS133 and PAS132, engineered E. coli K12 in the mouse gut. Shown are PAS132, PAS133, and anaerobic gut flora CFU per milligram of fecal sample on corresponding days (means ± SD from eight mice).