Characterization of Diverse Homoserine Lactone Synthases inEscherichia coli

Abstract

Quorum sensing networks have been identified in over one hundred bacterial species to date . A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux Receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach is still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum sensing synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.

Engineered orthogonal quorum sensing systems for synthetic gene regulation

“…Identifying orthogonal quorum sensing pairs is challenged by at least two factors: synthases with broad HSL profiles and regulators that show nonspecific responses to HSLs. As we have discussed in our previous report (Daer et al, 2018) , many HSL synthases generate a combination of HSL molecules when the enzymes are heterologously expressed in E. coli . Thus, Senders that express these enzymes might promiscuously induce several different Receivers.…”

“…Each constitutively expresses a regulator protein, a transcription factor that gains affinity for specific promoter sequences when the protein's binding pocket is occupied by an HSL ligand. The LuxR cassette used in our previous work (Daer et al, 2018) included a constitutively expressed Lux regulator gene upstream of an inducible GFP reporter. To avoid potential readthrough from the regulator gene into the GFP reporter in other constructs, we designed a new cassette where the inducible reporter was placed upstream (5') of the constitutively expressed regulator.…”

“…Next we measured the responses of the Receivers to HSLs produced by Sender cells that expressed one of ten different synthases. The HSLs that are expected to be produced by the synthase orthologues cover a broader range of structural diversity (described in (Daer et al, 2018) ) than the set of synthetic HSLs used in our previous induction assay (Figure 3). E. coli were transformed with a Receiver plasmid or a an HSLsynthase expressing Sender plasmid and grown overnight in liquid media.…”

“…Sender and Receiver cells were plated on solid LB agar as shown in Figure 5. Sender cells were streaked near a rectangular lawn of Receiver cells, as previously described (Daer et al, 2018) . This configuration allowed Receivers to be exposed to HSLs as the molecules diffused through the medium.…”

“…A central component of HSL signalling is the synthase enzyme, a protein that catalyzes the formation of HSLs from an acyl carrier protein or coenzyme A and Sadenosyl methionine. We previously characterized a library of synthase enzymes from various bacterial strains in an Escherichia coli host for their ability to generate HSLs that activated a Luxderived reporter (Daer et al, 2018) . This reporter vector was comprised of a constitutively expressed Lux regulator and a GFP expression cassette driven by a luxbox promoter.…”