Programming microbial population dynamics by engineered cell-cell communication

Abstract

A major aim of synthetic biology is to program novel cellular behavior using engineered gene circuits. Early endeavors focused on building simple circuits that fulfill simple functions, such as logic gates, bistable toggle switches, and oscillators. These gene circuits have primarily focused on single-cell behaviors since they operate intracellularly. Thus, they are often susceptible to cell-cell variations due to stochastic gene expression. Cell-cell communication offers an efficient strategy to coordinate cellular behavior at the population level. To this end, we review recent advances in engineering cell-cell communication to achieve reliable population dynamics, spanning from communication within single species to multispecies, from one-way sender-receiver communication to two-way communication in synthetic microbial ecosystems. These engineered systems serve as well-defined model systems to better understand design principles of their naturally occurring counterparts and to facilitate novel biotechnology applications.

Figure 1

Engineering genetic circuits at the single-cell level. (A) A synthetic IMPLIES gate that is interfaced with an Inverter. As shown in the upper panel, the IMPLIES gate involves a constitutively expressed LacI repressor, which inhibits the transcription of CI from the P_lacI promoter; IPTG activates the P_lacI promoter and induces CI and ECFP expression; CI inhibits the λPRO12 promoter to repress EYFP expression. As shown in the bottom panel, the P_lacI promoter comprises an IMPLIES logic gate wherein two inputs, LacI and IPTG, induce the response of the output CI, which acts as the input to the interfaced Inverter based on the λPRO12 promoter. Arrows indicate synthesis. Blunt bars indicate repression. Figure adapted from Yokobayashi et al. [7]. (B) A synthetic riboregulated transcriptional cascade (RTC) counter circuit that can count two pulses of arabinose stimulations. The RTC two-counter includes a transcriptional cascade: promoter P_Ltet0-1 drives the transcription of T7 RNA polymerase (RNAP), whose protein binds the P_T7 promoter and mediates transcription of a green fluorescent protein (GFP). The two genes (T7 RNAP and GFP) are in turn repressed by the riboregulator (cr). Meanwhile, a short, transactivating noncoding RNA (taRNA), driven by the arabinose promoter P_BAD, can bind to cr, relieving its repression on RBS and effectively activating the translation of T7 RNAP and GFP proteins. Figure adapted from Friedland et al. [17]. (C) A tunable band-pass filter conferred by a two-arm feed-forward loop (FFL). In one arm, insufficient β-lactamase (BLA) activity causes accumulation of Amp, leading to compromised cell wall synthesis and repressed cell growth. In the second arm, cell wall (murein) breakdown of Amp results in the accumulation of aM-pentapeptide (aM-Pp), which activates the promoter P_ampC via interacting with the promoter's repressor AmpR. As a result, aM-Pp induces TetC synthesis (which confers cells' Tet resistance) and GFP expression. Alternatively, BLA can be expressed by IPTG induction of P_{tac P/O} promoter, which is repressed by LacI. Figure adapted from Sohka et al. [18].

Figure 2

Programming single-species' populations via engineered cell-cell communication. (A) A population-control circuit which modulates population density of a single species. The circuit uses the LuxR/LuxI QS circuit, wherein both LuxR and LuxI genes are under the control of the P_lac promoter, inducible by IPTG. LuxI produces 3OC6HSL, which can bind and activate LuxR at a sufficiently high concentration. Activated LuxR-3OC6HSL complex (R*) in turn binds to the P_luxI promoter to induce expression of a killer gene, ccdB. High expression of ccdB leads to cell killing, thus reducing population density. Figure adapted from You et al. [41]. (B) Synthetic QS circuit to control bacterial invasion of malignant cancer cells. Employing a similar network structure as (A), the killer gene (LacZα-ccdB) in (A) is replaced by an invasion gene inv. Thereby, the invasion of mammalian cells by the programmed bacteria is under the control of QS. Figure adapted from Anderson et al. [42]. (C) Three network architectures for QS gene circuits synthesized by circuit reshuffling. Architecture A: LuxI and LuxR are constitutively expressed, GFP(lva) is under the control of the P_luxI promoter, which can be activated by the LuxR-autoinducer complex (LuxR*). Architecture B: LuxR is constitutively expressed; LuxI synthesizes 3OC6HSL and autoactivates expression of itself upon binding with LuxR, forming a positive-feedback loop. Architecture C: Expression of LuxR and LuxI are all under the control of the positive-feedback loop consisting of LuxR-autoinducer and the P_luxI promoter. Figure adapted from Haseltine and Arnold [43]. (D) The genetic diagram of a synchronized genetic oscillator. The P_lux promoter induces the production of three genes (LuxI, aiiA and yemGFP). The activated LuxR-AHL complex (R*) binds the P_lux promoter and activates gene expression. AiiA is an effective protease that catalyzes the cleavage of AHL. The overall circuit functions as coupled negative and positive feedback loops, wherein AHL activates synthesis of itself (positive feedback), and induces AiiA expression, which in turn represses AHL's accumulation by catalyzing AHL's degradation (negative feedback). Figure adapted from Danino et al. [44]. (E) The genetic circuit dictating an edge detection algorithm. The genetic cascade includes three sequential parts: First, the dark sensor part includes a light-sensitive kinase protein Cph8. In the presence of red light, the Cph8 kinase activity is inhibited, precluding the transfer of a phosphoryl group to the response regulator OmpR and subsequent transcriptional activation of the OmpC promoter (P_ompC). The dark sensor therefore functions as a NOT logic gate. Second, in the X and (NOT Y) logic gate, the P_ompC promoter drives the synthesis of LuxI and CI. LuxI produces a quorum-sensing signal AHL. CI inhibits the P_{lux- λ} promoter. LuxR-AHL complex (R*) activates the P_{lux- λ} promoter. Therefore, P_{lux- λ} functions as an X and (NOT Y) gate. Lastly, the P_{lux- λ} promoter drives the synthesis of LacZ, which produces β-galactosidase, an enzyme that subsequently cleaves a substrate in the media to produce black pigment. Figure adapted from Tabor et al. [45]. (F) Artificial cell-cell communication using a metabolite signal. Amino acid metabolism results in a metabolite, acetate, as a byproduct. Acetic acid (HOAc), diffuses freely across the cell membrane, playing the role of an artificial cell-cell communication signal. In the cytoplasm, ^-OAc can be phosphorylated to acetyl phosphate (Ac∼P), which can transfer its phosphate group to NR1. Upon phosphorylation, NR1∼P becomes a transcriptional regulator of the glnAp2 promoter which activates expression of GFP. Therefore, the GFP level reflects the acetate concentration, which in turn is correlated with cell density. Figure adapted from Butler et al. [38]. (G) Metabolator, a synthetic gene-metabolic oscillator that integrates transcriptional regulation with metabolism. The left panel shows the conceptual diagram of the circuit design in which the two metabolite pools (M1 and M2) are controlled by two enzymes (E1 and E2). The right panel illustrates the biological realization of the conceptual design. Acetyl-CoA is converted to acetyl phosphate (Ac∼P) by phosphate acetyltransferase (Pta), which converts to acetate (^-OAc) by acetate kinase (Ack). Meanwhile, the enzyme acetyl-CoA synthetase (Acs) is induced in the presence of acetate (^-OAc). When Ac∼P reaches a critical concentration, it represses the expression of Pta. Figure adapted from Fung et al. [46].

Figure 2

Programming single-species' populations via engineered cell-cell communication. (A) A population-control circuit which modulates population density of a single species. The circuit uses the LuxR/LuxI QS circuit, wherein both LuxR and LuxI genes are under the control of the P_lac promoter, inducible by IPTG. LuxI produces 3OC6HSL, which can bind and activate LuxR at a sufficiently high concentration. Activated LuxR-3OC6HSL complex (R*) in turn binds to the P_luxI promoter to induce expression of a killer gene, ccdB. High expression of ccdB leads to cell killing, thus reducing population density. Figure adapted from You et al. [41]. (B) Synthetic QS circuit to control bacterial invasion of malignant cancer cells. Employing a similar network structure as (A), the killer gene (LacZα-ccdB) in (A) is replaced by an invasion gene inv. Thereby, the invasion of mammalian cells by the programmed bacteria is under the control of QS. Figure adapted from Anderson et al. [42]. (C) Three network architectures for QS gene circuits synthesized by circuit reshuffling. Architecture A: LuxI and LuxR are constitutively expressed, GFP(lva) is under the control of the P_luxI promoter, which can be activated by the LuxR-autoinducer complex (LuxR*). Architecture B: LuxR is constitutively expressed; LuxI synthesizes 3OC6HSL and autoactivates expression of itself upon binding with LuxR, forming a positive-feedback loop. Architecture C: Expression of LuxR and LuxI are all under the control of the positive-feedback loop consisting of LuxR-autoinducer and the P_luxI promoter. Figure adapted from Haseltine and Arnold [43]. (D) The genetic diagram of a synchronized genetic oscillator. The P_lux promoter induces the production of three genes (LuxI, aiiA and yemGFP). The activated LuxR-AHL complex (R*) binds the P_lux promoter and activates gene expression. AiiA is an effective protease that catalyzes the cleavage of AHL. The overall circuit functions as coupled negative and positive feedback loops, wherein AHL activates synthesis of itself (positive feedback), and induces AiiA expression, which in turn represses AHL's accumulation by catalyzing AHL's degradation (negative feedback). Figure adapted from Danino et al. [44]. (E) The genetic circuit dictating an edge detection algorithm. The genetic cascade includes three sequential parts: First, the dark sensor part includes a light-sensitive kinase protein Cph8. In the presence of red light, the Cph8 kinase activity is inhibited, precluding the transfer of a phosphoryl group to the response regulator OmpR and subsequent transcriptional activation of the OmpC promoter (P_ompC). The dark sensor therefore functions as a NOT logic gate. Second, in the X and (NOT Y) logic gate, the P_ompC promoter drives the synthesis of LuxI and CI. LuxI produces a quorum-sensing signal AHL. CI inhibits the P_{lux- λ} promoter. LuxR-AHL complex (R*) activates the P_{lux- λ} promoter. Therefore, P_{lux- λ} functions as an X and (NOT Y) gate. Lastly, the P_{lux- λ} promoter drives the synthesis of LacZ, which produces β-galactosidase, an enzyme that subsequently cleaves a substrate in the media to produce black pigment. Figure adapted from Tabor et al. [45]. (F) Artificial cell-cell communication using a metabolite signal. Amino acid metabolism results in a metabolite, acetate, as a byproduct. Acetic acid (HOAc), diffuses freely across the cell membrane, playing the role of an artificial cell-cell communication signal. In the cytoplasm, ^-OAc can be phosphorylated to acetyl phosphate (Ac∼P), which can transfer its phosphate group to NR1. Upon phosphorylation, NR1∼P becomes a transcriptional regulator of the glnAp2 promoter which activates expression of GFP. Therefore, the GFP level reflects the acetate concentration, which in turn is correlated with cell density. Figure adapted from Butler et al. [38]. (G) Metabolator, a synthetic gene-metabolic oscillator that integrates transcriptional regulation with metabolism. The left panel shows the conceptual diagram of the circuit design in which the two metabolite pools (M1 and M2) are controlled by two enzymes (E1 and E2). The right panel illustrates the biological realization of the conceptual design. Acetyl-CoA is converted to acetyl phosphate (Ac∼P) by phosphate acetyltransferase (Pta), which converts to acetate (^-OAc) by acetate kinase (Ack). Meanwhile, the enzyme acetyl-CoA synthetase (Acs) is induced in the presence of acetate (^-OAc). When Ac∼P reaches a critical concentration, it represses the expression of Pta. Figure adapted from Fung et al. [46].

Figure 2

Programming single-species' populations via engineered cell-cell communication. (A) A population-control circuit which modulates population density of a single species. The circuit uses the LuxR/LuxI QS circuit, wherein both LuxR and LuxI genes are under the control of the P_lac promoter, inducible by IPTG. LuxI produces 3OC6HSL, which can bind and activate LuxR at a sufficiently high concentration. Activated LuxR-3OC6HSL complex (R*) in turn binds to the P_luxI promoter to induce expression of a killer gene, ccdB. High expression of ccdB leads to cell killing, thus reducing population density. Figure adapted from You et al. [41]. (B) Synthetic QS circuit to control bacterial invasion of malignant cancer cells. Employing a similar network structure as (A), the killer gene (LacZα-ccdB) in (A) is replaced by an invasion gene inv. Thereby, the invasion of mammalian cells by the programmed bacteria is under the control of QS. Figure adapted from Anderson et al. [42]. (C) Three network architectures for QS gene circuits synthesized by circuit reshuffling. Architecture A: LuxI and LuxR are constitutively expressed, GFP(lva) is under the control of the P_luxI promoter, which can be activated by the LuxR-autoinducer complex (LuxR*). Architecture B: LuxR is constitutively expressed; LuxI synthesizes 3OC6HSL and autoactivates expression of itself upon binding with LuxR, forming a positive-feedback loop. Architecture C: Expression of LuxR and LuxI are all under the control of the positive-feedback loop consisting of LuxR-autoinducer and the P_luxI promoter. Figure adapted from Haseltine and Arnold [43]. (D) The genetic diagram of a synchronized genetic oscillator. The P_lux promoter induces the production of three genes (LuxI, aiiA and yemGFP). The activated LuxR-AHL complex (R*) binds the P_lux promoter and activates gene expression. AiiA is an effective protease that catalyzes the cleavage of AHL. The overall circuit functions as coupled negative and positive feedback loops, wherein AHL activates synthesis of itself (positive feedback), and induces AiiA expression, which in turn represses AHL's accumulation by catalyzing AHL's degradation (negative feedback). Figure adapted from Danino et al. [44]. (E) The genetic circuit dictating an edge detection algorithm. The genetic cascade includes three sequential parts: First, the dark sensor part includes a light-sensitive kinase protein Cph8. In the presence of red light, the Cph8 kinase activity is inhibited, precluding the transfer of a phosphoryl group to the response regulator OmpR and subsequent transcriptional activation of the OmpC promoter (P_ompC). The dark sensor therefore functions as a NOT logic gate. Second, in the X and (NOT Y) logic gate, the P_ompC promoter drives the synthesis of LuxI and CI. LuxI produces a quorum-sensing signal AHL. CI inhibits the P_{lux- λ} promoter. LuxR-AHL complex (R*) activates the P_{lux- λ} promoter. Therefore, P_{lux- λ} functions as an X and (NOT Y) gate. Lastly, the P_{lux- λ} promoter drives the synthesis of LacZ, which produces β-galactosidase, an enzyme that subsequently cleaves a substrate in the media to produce black pigment. Figure adapted from Tabor et al. [45]. (F) Artificial cell-cell communication using a metabolite signal. Amino acid metabolism results in a metabolite, acetate, as a byproduct. Acetic acid (HOAc), diffuses freely across the cell membrane, playing the role of an artificial cell-cell communication signal. In the cytoplasm, ^-OAc can be phosphorylated to acetyl phosphate (Ac∼P), which can transfer its phosphate group to NR1. Upon phosphorylation, NR1∼P becomes a transcriptional regulator of the glnAp2 promoter which activates expression of GFP. Therefore, the GFP level reflects the acetate concentration, which in turn is correlated with cell density. Figure adapted from Butler et al. [38]. (G) Metabolator, a synthetic gene-metabolic oscillator that integrates transcriptional regulation with metabolism. The left panel shows the conceptual diagram of the circuit design in which the two metabolite pools (M1 and M2) are controlled by two enzymes (E1 and E2). The right panel illustrates the biological realization of the conceptual design. Acetyl-CoA is converted to acetyl phosphate (Ac∼P) by phosphate acetyltransferase (Pta), which converts to acetate (^-OAc) by acetate kinase (Ack). Meanwhile, the enzyme acetyl-CoA synthetase (Acs) is induced in the presence of acetate (^-OAc). When Ac∼P reaches a critical concentration, it represses the expression of Pta. Figure adapted from Fung et al. [46].

Figure 3

Engineering one-way cell-cell communication. (A) A synthetic pulse-generator. It consists of one-way communication between the sender and the receiver by a QS mechanism. In the sender cells, upon aTc induction, LuxI synthesizes the autoinducer 3OC6HSL. The receiver cells include a feed-forward network comprised by two arms of regulation: in the first arm, the LuxR-3OC6HSL complex (LuxR*) activates expression of repressor CI, which in turn represses GFP expression; in the second arm, LuxR* induces GFP expression. Figure adapted from Basu et al. [47]. (B) Synthetic self-organized pattern formation. The sender cells produce 3OC6HSL upon induction by aTc. The receiver contains a feed-forward network with two arms: in the first, LuxR* activates expression of a mutated LacI (LacI_M1), which then inhibits the output of the circuit, GFP; in the second arm, LuxR* activates expression of the repressor CI, which inhibits expression of LacI; LacI further inhibits GFP expression. The receiver cells are thus conferred with the function of concentration-band detection. Figure adapted from Basu et al. [48]. (C) Simpson's paradox demonstrated by a synthetic microbial ecosystem consisting of QS signal (C4HSL) producers (P) and non-producers (NP). In P, promoter P_R drives the synthesis of autoinducer synthase (RhlI), which produces C4HSL. C4HSL diffuses across cell membranes and binds the constitutively expressed RhlR to form an activated complex RhlR-C4HSL (R*). This complex in turn drives expression of catLVA, which confers cells with chloramphenicol resistance. The sdiA null background (sdiAΔ) reduces other Rhl-independent gene expression. In the NP strain, the rhlI gene is absent. The P (with GFP) and NP (no GFP) strains can be differentiated by GFP. Figure adapted from Chuang et al. [51]. (D) An XOR logic gate utilizing cell-cell communication between colonies on a plate. It includes four colonies spatially distributed on an agar plate, in which three colonies contain NOR gates and one contains a buffer gate. The four colonies are spatially arranged such that the computation progresses from left to right; that is, the later layer receives QS signal from the previous layer, thus realizing the function of a XOR logic gate via cell-cell communication. Ara and aTc are inputs. The output of the XOR gate is a yellow fluorescent protein (YFP). Figure adapted from Tamsir et al. [52]. (E) An engineered yeast microbial consortium which utilizes cell-cell communication to achieve the OR logic gate. The microbial consortium includes three cell strains. Two cells receive input stimuli of NaCl and galactose, respectively, and consequently produce the yeast pheromone alpha-factor as a signaling molecule for cell-cell communication in the microbial consortium. The alpha-factor in turn induces GFP expression in the output cell. Figure adapted from Regot et al. [53].

Figure 3

Engineering one-way cell-cell communication. (A) A synthetic pulse-generator. It consists of one-way communication between the sender and the receiver by a QS mechanism. In the sender cells, upon aTc induction, LuxI synthesizes the autoinducer 3OC6HSL. The receiver cells include a feed-forward network comprised by two arms of regulation: in the first arm, the LuxR-3OC6HSL complex (LuxR*) activates expression of repressor CI, which in turn represses GFP expression; in the second arm, LuxR* induces GFP expression. Figure adapted from Basu et al. [47]. (B) Synthetic self-organized pattern formation. The sender cells produce 3OC6HSL upon induction by aTc. The receiver contains a feed-forward network with two arms: in the first, LuxR* activates expression of a mutated LacI (LacI_M1), which then inhibits the output of the circuit, GFP; in the second arm, LuxR* activates expression of the repressor CI, which inhibits expression of LacI; LacI further inhibits GFP expression. The receiver cells are thus conferred with the function of concentration-band detection. Figure adapted from Basu et al. [48]. (C) Simpson's paradox demonstrated by a synthetic microbial ecosystem consisting of QS signal (C4HSL) producers (P) and non-producers (NP). In P, promoter P_R drives the synthesis of autoinducer synthase (RhlI), which produces C4HSL. C4HSL diffuses across cell membranes and binds the constitutively expressed RhlR to form an activated complex RhlR-C4HSL (R*). This complex in turn drives expression of catLVA, which confers cells with chloramphenicol resistance. The sdiA null background (sdiAΔ) reduces other Rhl-independent gene expression. In the NP strain, the rhlI gene is absent. The P (with GFP) and NP (no GFP) strains can be differentiated by GFP. Figure adapted from Chuang et al. [51]. (D) An XOR logic gate utilizing cell-cell communication between colonies on a plate. It includes four colonies spatially distributed on an agar plate, in which three colonies contain NOR gates and one contains a buffer gate. The four colonies are spatially arranged such that the computation progresses from left to right; that is, the later layer receives QS signal from the previous layer, thus realizing the function of a XOR logic gate via cell-cell communication. Ara and aTc are inputs. The output of the XOR gate is a yellow fluorescent protein (YFP). Figure adapted from Tamsir et al. [52]. (E) An engineered yeast microbial consortium which utilizes cell-cell communication to achieve the OR logic gate. The microbial consortium includes three cell strains. Two cells receive input stimuli of NaCl and galactose, respectively, and consequently produce the yeast pheromone alpha-factor as a signaling molecule for cell-cell communication in the microbial consortium. The alpha-factor in turn induces GFP expression in the output cell. Figure adapted from Regot et al. [53].

Figure 4

Engineering two-way cell-cell communication. (A) A microbial consensus consortium (MCC) circuit. It implements two-way cell-cell communication using two QS modules, LasR/LasI and RhlR/RhlI, to achieve a consensus response. In Circuit A, LasI produces 3OC12HSL, which diffuses into cells containing Circuit B and binds LasR, thereby inducing expression of RhlI. RhlI in Circuit B synthesizes C4HSL, which diffuses into cells containing Circuit A and binds RhlR to activate LasI expression, closing the positive feedback loop. Figure adapted from Brenner et al. [31]. (B) A synthetic obligatory cooperative system achieved by exchanging metabolites in a two-way communication system. This system consists of two auxotrophic yeast strains that depend on each other for survival. One strain, ${\mathbf{\text{R}}}_{\to \text{lys}}^{\leftarrow \text{ade}},$ is unable to synthesize adenine but overproduces lysine. In contrast, another strain, ${\mathbf{\text{Y}}}_{\to \text{ade}}^{\leftarrow \text{lys}},$ is unable to synthesize lysine but overproduces adenine. Figure adapted from Shou et al. [40]. (C) An engineered cooperative microbial consortium, in which only the mixture of the two bacteria can chemotax toward the combined gradient of asparagine and phenylacetyl glycine (PAG). One strain synthesizes penicillin acylase (Pac), which enzymatically hydrolyzes phenylacetyl glycine (PAG) to produce phenylacetic acid (PAA), a chemoattractant to E. coli cells with Tar chemoreceptors. The other strain expresses AnsB gene, which encodes Asparaginase. Asparaginase enzymatically converts Asparagine to Aspartate, functioning as a ligand (or chemoattractant) to E. coli cells with TarPA chemoreceptors. This engineered mutualistic consortium is not able to chemotax toward a gradient consisting of only one of the two compounds (Asparagine and PAA). Figure adapted from Goldberg et al. [55]. (D) Schematic of the gene circuit in a synthetic predator-prey ecosystem. The system consists of two types of engineered E. coli. Each controls the other's survival and death via two QS circuits, LuxR/LuxI from V. fisheri and LasR/LasI from P. aeruginosa. The predator synthesizes 3OC12HSL, which diffuses into the prey and binds to LasR to induce expression of the ccdB killer protein, thereby killing the prey. On the other hand, the prey synthesizes 3OC6HSL, which diffuses into the predator and binds to LuxR to induce expression of the ccdA antidote protein. This protein neutralizes the toxicity of the ccdB killer protein, thereby rescuing the predator. Figures adapted from Balagadde et al. [32].