45 Cyanobacteria are the prokaryotic group of phytoplankton responsible for a significant 46 fraction of global CO 2 fixation. Like plants, cyanobacteria use the enzyme Ribulose 1,5-47 bisphosphate Carboxylase/Oxidase (RuBisCO) to fix CO 2 into organic carbon molecules via the 48 Calvin-Benson-Bassham cycle. Unlike plants, cyanobacteria evolved a carbon concentrating 49 organelle called the carboxysome -a proteinaceous compartment that encapsulates and 50 concentrates RuBisCO along with its CO 2 substrate. In the rod-shaped cyanobacterium 51 Synechococcus elongatus PCC7942, we recently identified the McdAB system responsible for 52 uniformly distributing carboxysomes along the cell length. To date, it is unclear what role 53carboxysome positioning plays with respect to cellular physiology. Here, we show that a failure 54 to distribute carboxysomes leads to a temperature-dependent decrease in cell growth rate, 55 changes in cell morphology, and a significant reduction in cellular levels of RuBisCO. 56 Unexpectedly, we also found that wild-type S. elongatus elongates and divides asymmetrically at 57 the environmentally relevant growth temperature of 20 o C. We propose that carboxysome 58 positioning by the McdAB system functions to maintain carbon-fixation efficiency of RuBisCO 59 by preventing carboxysome aggregation, which is particularly important at temperatures where 60 rod-shaped cyanobacteria adopt a filamentous morphology. 61 62 63 64 65 66 67 quantifying the subcellular organization of carboxysomes, as previously shown (MacCready et 132 al., 2018). We also performed Phase Contrast imaging to monitor for potential changes in cell 133 morphology, and Chlorophyll fluorescence imaging to verify that cells were photosynthetically 134 active and therefore viable. 135

Photosynthetic cyanobacteria are responsible for almost half of global CO 2 fixation. Due to eutrophication, rising temperatures, and increasing atmospheric CO 2 concentrations, cyanobacteria have gained notoriety for their ability to form massive blooms in both freshwater and marine ecosystems across the globe.

There has been substantial recent interest in the promise of sustainable, light-driven bioproduction using cyanobacteria, including developing efforts for microbial bioproduction using mixed autotroph/heterotroph communities, which could provide useful properties, such as division of metabolic labor. However, building stable mixed-species communities of sufficient productivity remains a challenge, partly due to the lack of strategies for synchronizing and coordinating biological activities across different species. To address this obstacle, we developed an inter-species communication system using quorum sensing (QS) modules derived from well-studied pathways in heterotrophic microbes. In the model cyanobacterium, Synechococcus elongatus PCC 7942 (S. elongatus), we designed, integrated, and characterized genetic circuits that detect acyl-homoserine lactones (AHLs), diffusible signals utilized in many QS pathways. We showed that these receiver modules sense exogenously supplied AHL molecules and activate gene expression in a dose-dependent manner. We characterized these AHL receiver circuits in parallel with Escherichia coli W (E. coli W) to dissect species-specific properties, finding broad agreement, albeit with increased basal expression in S. elongatus. Our engineered “sender” E. coli strains accumulated biologically synthesized AHLs within the supernatant and activated receiver strains similarly to exogenous AHL activation. Our results will bolster the design of sophisticated genetic circuits in cyanobacterial/heterotroph consortia and the engineering of QS-like behaviors across cyanobacterial populations.

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO 2 to HCO 3 À . The Arabidopsis genome encodes members of the a-, band c-CA families, and it has been hypothesized that bCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial bCAs, bCA1 and bCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of bCA5 induced the expression of bCA1, supporting the existence of regulatory mechanisms to control the expression of stromal bCAs. We also established that bCA1 and bCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that bCA5 had a first-order rate constant ~10-fold lower than bCA1, and that the loss of bCA5 is detrimental to growth and could be rescued by high CO 2 . Furthermore, we established that, while a bCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of bCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO 2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed bCA1 does not compensate for the loss of a less active bCA5, which in turn is involved in growth and photosynthesis at ambient CO 2 levels. These results lend support to the hypothesis that, in Arabidopsis,bCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal bCA5 and a dispensable role for bCA1.

1.AbstractThere has been substantial recent interest in the promise of sustainable, light-driven bioproduction using cyanobacteria, including developing efforts for microbial bioproduction using mixed autotroph/heterotroph communities, which could provide useful properties, such as division of metabolic labor. However, building stable mixed-species communities of sufficient productivity remains a challenge, partly due to the lack of strategies for synchronizing and coordinating biological activities across different species. To address this obstacle, we developed an inter-species communication system using quorum sensing (QS) modules derived from well-studied pathways in heterotrophic microbes. In the model cyanobacterium, Synechococcus elongatus PCC 7942 (S. elongatus), we designed, integrated, and characterized genetic circuits that detect acyl-homoserine lactones (AHLs), diffusible signals utilized in many QS pathways. We showed that these receiver modules sense exogenously supplied AHL molecules and activate gene expression in a dose-dependent manner. We characterized these AHL receiver circuits in parallel in Escherichia coli W (E. coli W) to dissect species-specific properties, finding broad agreement, albeit with increased basal expression in S. elongatus. Our engineered “sender” E. coli strains accumulated biologically synthesized AHLs within the supernatant and activated receiver strains similarly to exogenous AHL activation. Our results will bolster the design of sophisticated genetic circuits in cyanobacterial/heterotroph consortia and the engineering of QS-like behaviors across cyanobacterial populations.HighlightsDesigned, built, and tested an inter-species quorum sensing-based communication system.These genetic circuits can sense and respond to exogenous and secreted signals.Circuit function in S. elongatus was comparable to E. coli, albeit with increased basal expression and lower induction ratiosDemonstrated inter-species communication in direct co-cultivationFirst demonstration of inducible promoters and cross-species gene regulation in S. elongatus based on quorum sensing