Design of artificial cell–cell communication using gene and metabolic networks

Abstract

Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell-cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling ⌬pH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp 2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model.

Teaching bacteria a new language

“…Previous efforts achieved such coordination by taking existing quorum sensing (QS) components from a source host, Vibrio fischeri, and integrating them into a target host, Escherichia coli (5,7,8). The work of Bulter et al (11) in this issue of PNAS describes a new form of engineering cell-cell communication based on manipulating metabolic pathways. Specifically, the authors engineered the nitrogen regulation system and the acetate pathway in E. coli.…”

“…Acetyl phosphate transfers its phosphate group to NR I , a transcriptional regulator of glnAp2 promoter (12). As a result, NR I ϳP dimerizes, binds the glnAp2 promoter at two enhancer regions, and activates transcription of GFP in the engineered system (11). Through the above processes, the cytoplasmic level of NR I ϳP reflects the extracellular acetate concentration, which in turn correlates to cell density.…”

“…For example, the natural system is highly sensitive to oxygen; acetate production increases significantly under anaerobic conditions. By using a pta Ϫ strain (13), where pta is one of the main genes in the acetate pathway, the authors removed the influence of oxygen; the mutant showed similar acetate production under aerobic or anaerobic conditions (11). Another benefit to using this mutant is slower degradation of acetate (14) that increases the dynamic response range to acetate (15).…”

“…In many cases, the sender and receiver elements coexist in the same cell. For example, in the work of Bulter et al (11), amino acid synthesis functions as the sender element because it produces the acetate signal. This signal is transmitted to other cells as well as the originating cell, and is finally received by the NR I ͞glnAp2 pathway.…”

“…The work of Bulter et al (11) suggests a new paradigm for finding and exploiting preexisting nontraditional cell-cell signaling. As we improve our understanding of the crucial role cell-cell communication plays in biology and discover the rich complexities of communication protocols between cells, it becomes increasingly important to be able to engineer these protocols.…”