Signal Amplification in Synthetic Bacterial Communication
Chapter 1 Introduction
Research in synthetic biology is an exercise in constant humility. Every breakthrough in this field demands a comparison to the astounding brilliance of the natural work it is imitating. It is like designing the first moon lander while seeing aliens pass through our solar system on intergalactic spacecraft without saying so much as “hello”.
Such is the relationship of a synthetic biologist to all living things. The majority of research and commercial applications in this field rely on domesticated cell lines to express synthetic gene circuits without significantly altering the genomic programming for the cell line’s key biological features, such as self-replication and metabolic homeostasis. In a 2020 comment paper published in Nature Communications, Voigt highlighted six commercial products that were manufactured using genetically modified oranganisms. Three were products produced by engineered cells and the other three were themselves engineered cells (Voigt (2020)). The commercial success of these commodities derived from engienered cells illustrates that bioengineered cellular factories represent a significant industrial source of valuable chemicals and materials, and the living commodities show that the future is bright for applications of engineered organisms.
While these indicate synthetic biology’s growing relevance in the commercial sector, the article notes that the next phase of industrial synthetic biology would need to develop on engineering cells as embedded controllers for biosynthesis and biochemical environments of interest. Consider for example one of the living commodities highlighted in Voigt’s article: a rhizosphere bacteria strain engineered to overexpress nitrogen-fixing genes marketed under the name PROVEN (Willits (2020), Temme (2019)). The modification alters a conditionally-expressed gene cluster to instead be persistently active, thereby increasing soil nitrogen and crop yields while reducing the need for chemical fertilizers. Other known benefits of bacteria-plant symbiosis, however, cannot be achieved by unconditional overexpression of the relevant biosynthesis pathways (Sarma et al. (2015)). Species of the genus Rhizobium associate with legume roots and secrete biocontrol agents that suppress pathogens, but the same mechanisms of action that target pathogenic bacteria also antagonize other Rhizobia (Avis et al. (2008)). Improper balancing of strain demographics in the root microbiome or adjustments to the control circuits of antagonistic genes could spoil the potential benefit provided to the plant (Jain et al. (2012)). Action by the beneficial microbial consortia associated with the root system must also properly respond to chemical signals emitted by the plant itself in order to be effective (Avis et al. (2008)). The next generation of engineered microbial interventions for complex deployment scenarios must include the capability to sense environmental signals and respond accordingly.
One avenue for expanding the functional capabilities of synthetic bacterial devices is through engineering bacterial communities. As opposed to populations of synthetic bacteria made up of a single genotype, synthetic bacterial consortia include multiple genetically distinct strains that cooperate in completing a shared task. Strains that compose a synthetic microbial consortium can be specialized in different subroutines of the overall task. Dividing the requisite labor between component strains and optimizing each strain individually reduces the overall difficulty of the engineering task in comparison to optimizing a single strain to perform all functions. Division of labor has been successfully applied in various bioproduction applications to increase yield and titer of biochemicals (Tsoi et al. (2018), Saini et al. (2016), Zhang and Stephanopoulos (2016)). Furthermore a consortium may include multiple species to broaden the natural mechanisms in the consortium (Kim, Du, and Ismagilov (2011)). A unique challenge in consortia engineering, however, is making the intended function robust to variations in the population balance and spatial patterning of the component strains (Johns et al. (2016), Zomorrodi and Segrè (2016)).
Coordination between members of a synthetic bacterial consortium is most often achieved by modified quorum sensing systems. Quorum sensing refers to a positive-feedback gene regulatory motif, commonly found in bacteria, that is composed of a signal synthase, a signal receptor protein, and a receptor-controlled promoter. Communities of wild microbes use this motif to coordinate group behaviors in a manner that is conventionally understood to be density-dependent. The cell-cell signaling chemicals of quorum sensing systems are acyl-homoserine lactones (AHLs); the composition of the acyl chain varies between instances of this motif. AHL signaling molecules are created by the signal synthase protein and can diffuse freely through cell membranes. The receptor proteins bind to AHL molecules, forming a complex that activates transcription at associated promoters containing the receptor’s binding sequence. These components are a popular choice in the design of synthetic bacterial consortia due to their simplicity and portability. Because AHL molecules undergo passive transport through cell membranes, AHL-mediated communication can be implemented using only the two protein components and one AHL-induced promoter.
Synthetic bacterial consortia can make use of modified quorum sensing circuits to autonomously balance their strain composition and activity. In gene circuits engineered to limit or balance strain populations, a positive-feedback loop similar to natural quorum sensing circuits is connected to genes that lead to autolysis or expression of antibiotic compounds. Cells expressing these circuits limit their own population by implementing self-killing measures in a density-dependent fashion (Scott et al. (2017)). Consortia may also make use of mutualistic or antagonistic relationships between strains. These interactions are effected by metabolic relationships or targeted antibiotic interactions to achieve programmatic strain balancing (Balagaddé et al. (2008), Kong et al. (2018), Taillefumier et al. (2017)). The emergent behavior can also be made more robust without population-limiting circuits by encoding density-dependence through cell-cell signaling circuits. Chen et al. (2015) show in simulation that a two-strain relaxation oscillator is made more robust to demographic variation by the addition of a negative feedback loop used by one strain to attenuate its own activity in a density-dependent fashion. These examples demonstrate several approaches to engineering consortia that autonomously balance their strain demographics in well-mixed media.
Cell-cell communication circuits that apply spatiotemporal control over strain composition or behavior in media that is not well mixed are called pattern forming circuits. In diffusive environments, the absence of convection or turbulence allows for the formation of chemical gradients and non-homogeneous spatial patterning of the cells making up a consortium. As a result, cells in a consortium may sense different chemical signals depending on their position in these gradients. Pattern forming systems exploit spatial gradients in signaling molecules, cell density, and nutrients to generate complex spatial patterns out of growing consortia.
Ring-forming systems have been described several times in the literature (Liu et al. (2011), Schaerli et al. (2014), Xue, Xue, and Tang (2018), Basu et al. (2005), Cao et al. (2016), Potvin-Trottier et al. (2016)). Given the radial symmetry of bacterial colonies that grow from a small number of close founding cells, ring-forming systems are the first choice for demonstrating that a cell-cell communication system can be used to program spatially heterogeneous behaviors. Cao et al. (2016) and Liu et al. (2011) describe ring-forming bacteria that coordinate using quorum sensing components. On the other hand, Potvin-Trottier et al. (2016) describe a system that relies on a transcriptional oscillator circuit with the remarkable feature that, over many generations of cell division, the oscillations in daughter cells remain synchronized. As a colony of these oscillators grows, then, their synchronized oscillations give rise to uniform rings.
While these examples achieve similar spatial patterns through drastically different mechanisms, they all rely on the spatial heterogeneity of nutrient availability as a key component of pattern formation. Liu et al. (2011) employ a combination of AHL-mediated cell-cell signaling and synthetic chemotaxis to program a consortium to form stripes of high and low cell density. However, monotonically decreasing nutrient availability at each position is key to this mechanism: falling resource concentrations eventually fix cells in place. Indeed, the fact that cells struggle to express transgenic circuits when nutrients are low is a common feature in these ring-forming systems. Cao et al. (2016) identify that the gradient in gene expression capacity along the radius of a colony is critical to achieving the scale-free ring patterns observed in their experiments. Gene programs that create synchronized oscillations in time at the colony edge will produce oscillations in space along the colony radius as a result of the difference in gene expression capacity between the colony’s interior and exterior.
These pattern formation systems underscore the fact that spatially heterogeneous gene expression is inherent to consortia growing in diffusive environments. While nutrient-dependent growth and gene expression can be leveraged in pattern-forming systems, these factors can be an enormous obstacle to coordination within multi-component consortia. The distance separating two consortium components, components that together form a signaling or metabolic relationship, dramatically impacts their emergent behavior (Gupta et al. (2020), Langebrake et al. (2014), Macia et al. (2016)). A study investigating the capability of a sender-receiver pair composed of engineered Pseudomonas putida strains to communicate within the rhizosphere found that the introduced cells grow in sparsely-distributed groups of dozens of cells that can communicate reliably only over tens of microns (Gantner et al. (2006)). The limited signaling distances and the impact of nutrient availability highlight the need for communication networks in engineered bacterial consortia that can overcome not only spatial heterogeneity in strain demographics, but in cellular resources and relevant environmental events as well.
Agents in nature utilize traveling waves of signaling activity to share localized information over longer distances than can be achieved by diffusion from an isolated source. Slime molds such as Dictyostelium emit waves of cAMP, a nucleotide derivative, as they approach starvation. As neighboring amoebae join in the signaling activity, the emergent behavior appears to be an election of a community leader that serves as the target for chemotaxis by local members (Goldbeter (2006), Noorbakhsh et al. (2015)). Bacillus subtilis propagate waves of potassium signaling to coordinate resource sharing between the interior and exterior of growing colonies (Larkin et al. (2018), Prindle et al. (2015)). Cheng and Ferrell (2018) describes a self-regenerating front of apoptotic activity that travels through cell-free extracts of Xenopus eggs in response to localized initiation. The authors of this study demonstrate a nearly 5-fold range in wave speeds under a variety of perturbations to the feedback loops supporting the traveling wave of apoptosis-related activity. In each of these articles, the researchers discuss a well-known result in traveling wave phenomena: positive feedback and local tethering are key to long-distance chemical signaling (Gelens, Anderson, and Ferrell (2014), Oleinik, Kolmogorov, and Piskunov (2019)).
The theoretical conditions necessary for traveling wave phenomena in cell-cell signaling circuits can be derived from mathematical models. For their 2020 eLife article, authors Dieterle et al. (2020) constructed reaction-diffusion models describing signaling molecule behavior in various wave-generating microbial consortia wherein a small “initiating colony” elicits a wave of signaling activity through a semi-infinite region of “relay cells”. In each scenario considered, the authors derived relationships between the model parameters, such as cell density and signal emission rate, and characteristics of the traveling wave propagated by the relay cells such as velocity and signal concentration profile. These relationships were derived for consortia employing switch-like, pulsatile, or Hill-like activation functions defining their signal emission rates as a function of the local signal concentration. Just as in the natural examples described above, the authors found each of the activation functions to be capable of producing traveling waves. Furthermore, the signaling fronts propagated by relay cells yielded higher signaling concentrations and traveled faster than fronts produced from initiating colony alone. The benefit of relay cells was more pronounced in scenarios where the dimensionality of the diffusive medium was greater than that of the consortium (e.g., signal molecules diffusing in three dimensions while the consortium cells exist in a two-dimensional plane. Other studies also suggest that local amplification in cell-cell signaling circuits could enable long-distance signaling in synthetic microbial consortia and may overcome environmental obstacles to group consensus (Langebrake et al. (2014), Holzer, Doelman, and Kaper (2013)).
Self-propagating signaling fronts that include a motif of positive-feedback and local tethering have been demonstrated experimentally in both synthetic bacteria and active chemical media.(^The earliest described active chemical media being the inorganic Belousov-Zhabotinsky reaction ( Belousov 1959).) Cell-free approaches have also demonstrated that positive feedback circuits elicit traveling waves in response to localized initiation. By exploiting the precise control over the chemical composition of cell-free active media, these studies provide further validation and context for the theoretical results relating wave characteristics to reaction dynamics (Gines et al. (2017), Tayar et al. (2015)). Synthetic bacteria expressing synchronized oscillator circuits generate traveling waves when grown in a low-turbulence microfluidic device (Danino et al. (2010)). Much like the leader selection performed by Dictyostelium, synchronized oscillators tend towards a out-of-equilibrium state in which a minority initiates oscillations that trigger wave propagation through neighboring cells (Garcia-Ojalvo, Elowitz, and Strogatz (2004), Dalchau et al. (2018), Watts and Strogatz (1998)). These results suggest that a similar mechanism could enable sender cells to generate non-oscillatory traveling pulses through nearby propagator cells. Traveling pulse circuits could be used by consortium members to share local information, thereby enabling well-informed group decision-making from spatially heterogeneous environmental conditions.
The research presented in this thesis introduces an approach to cell-cell communication networks that supports sender-receiver relationships when the spatial patterning of deployed consortium members cannot be pre-determined. In a consortium, a sender-receiver pairing implies three subpopulations: sender, receiver, and bystander. Augmenting the bystander strains with a signal amplifier circuit enables them to generate traveling waves in response to initiating signals released by a sender population. These traveling waves would enable a consortium to share local information over longer distances than by the action of the sender population alone. Amplifier activity could also compensate for variations in the amount of sender cells by increasing the overall signal molecule concentration to counteract attenuated emission from a diminished sender population. The amplifier gene circuit in this approach is pulsatile, rather than bistable or oscillatory, which allows for repeated amplifier activity in response to periodic initiation from sender cells. Together, these features improve signaling from senders to receivers over variations in the spatial patterning of consortia and geometry of the diffusive environment.
This approach is investigated in the context of pulsatile amplifier cells and sender cells growing together on the surface of agarose hydrogels. We demonstrate that the amplifier strain generates a traveling wave of signaling activity that propagates messages from a sender population more quickly than by passive transport alone. This principle is then investigated in consortia founded by a small number of sender cells and many amplifier cells. We demonstrate that, without active signal propagation from the amplifier cells, the sender population could not marshal a response from its neighboring receiver cells. On the other hand, consortia with pulsatile activation responded quickly to their sparse sender populations. A mathematical model was developed to match the observed behavior of these sender-amplifier consortia and was used to investigate the behavior of hypothetical consortia in silico applied to a two-dimensional diffusive environment.
Chapter 2 describes the composition of the pulsatile amplifier circuit, its characterization through liquid-culture experiments, and applications of the amplifier strain to extend communication distances within a spatially-structured sender-receiver consortium growing in semi-solid media. In Chapter 3, the same consortium is investigated at a smaller length scale and with random spatial organization. Chapter 4 introduces a finite-differences approach to simulating the amplifier strain in various diffusive environments and ends with an in silico investigation of how amplifier strains could benefit computation within hypothetical engineered consortia.
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