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A key regulatory decision for many bacteria is the switch between biofilm formation and motile dispersal, and this dynamic is well illustrated in the light‐organ symbiosis between the bioluminescent bacteriumVibrio fischeriand the Hawaiian bobtail squid. Biofilm formation mediated by thesypgene cluster helpsV. fischeritransition from a dispersed planktonic lifestyle to a robust aggregate on the surface of the nascent symbiotic organ. However, the bacteria must then swim to pores and down into the deeper crypt tissues that they ultimately colonize. A number of positive and negative regulators controlsypexpression and biofilm formation, but until recently the environmental inputs controlling this clash between opposing regulatory mechanisms have been unclear. Thompsonet al. have now shown that Syp‐mediated biofilms can be repressed by a well‐known host‐derived molecule: nitric oxide. This regulation is accomplished by the NO sensor HnoX exerting control over the biofilm regulator HahK. The discoveries reported here by Thompsonet al. cast new light on a critical early stage of symbiotic initiation in theV. fischeri‐squid model symbiosis, and more broadly it adds to a growing understanding of the role(s) that NO and HnoX play in biofilm regulation by many bacteria.

 

ABSTRACT Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal “on” expression when PhoB is active, minimal background in the “off” state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at 19 additional intervening nucleotide positions for which we did not predict sequence-specific effects, the bases were randomized. Eighty-three such constructs were screened in Vibrio fischeri , enabling us to identify bases at particular randomized positions that significantly correlated with high-level “on” or low-level “off” expression. A second round of promoter design rationally constrained 13 additional positions, leading to a reporter with high-level PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized variants of green fluorescent protein (GFP), the latter of which has a half-life of 81 min in V. fischeri . In culture, PhoB induced the reporter when phosphate was depleted to a concentration below 10 μM. During symbiotic colonization of its host squid, Euprymna scolopes , the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other members of the Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and reengineering. IMPORTANCE Transcriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between the “on” and “off” states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over 34 billion sequence variants, we identified bases correlated with favorable performance by screening only 83 candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of V. fischeri . This promoter design strategy will facilitate the engineering of other regulator-specific reporters. 

ABSTRACT Pheromone signaling (PS) underlies many important bacterial behaviors, yet its ecological functions remain unresolved. Because pheromone-mediated behaviors require high cell density, the term “quorum sensing” is widely used to describe and make sense of PS. However, while this term has unified and popularized the field, bacterial PS clearly has roles beyond census taking, and the complexities of PS circuits indicate broader functional capacities. Two common features of bacterial PS are its regulation in response to environmental conditions and positive-feedback loops. Combined, these could enable PS to coordinate quorum-dependent group behaviors in response to heterogeneous environmental cues. Particularly in PS systems where positive feedback is strong, cells that are relatively far from a stimulatory environment could be recruited to a group response. Testing this model will benefit from in situ examination of relevant environmental cues and PS outputs in cells across populations, with and without positive feedback, in heterogeneous environments.