Arabidopsis stromal carbonic anhydrases exhibit non-overlapping roles in photosynthetic efficiency and development.

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO2 to HCO3 - . The Arabidopsis genome encodes members of the α-, ß- and γ-CA families, and it has been hypothesized that ßCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial ßCAs, ßCA1 and ßCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of ßCA5 induced the expression of ßCA1, supporting the existence of regulatory mechanisms to control the expression of stromal ßCAs. We also established that ßCA1 and ßCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that ßCA5 had a first-order rate constant ~10-fold lower than ßCA1, and that the loss of ßCA5 is detrimental to growth and could be rescued by high CO2 . Furthermore, we established that, while a ßCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of ßCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed ßCA1 does not compensate for the loss of a less active ßCA5, which in turn is involved in growth and photosynthesis at ambient CO2 levels. These results lend support to the hypothesis that, in Arabidopsis,ßCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal ßCA5 and a dispensable role for ßCA1.

Developing Cyanobacterial Quorum Sensing Toolkits: Toward Interspecies Coordination in Mixed Autotroph/Heterotroph Communities.

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.

Carboxysome Mispositioning Alters Growth, Morphology, and Rubisco Level of the Cyanobacterium Synechococcus elongatus PCC 7942.

Cyanobacteria are the prokaryotic group of phytoplankton responsible for a significant fraction of global CO2 fixation. Like plants, cyanobacteria use the enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco) to fix CO2 into organic carbon molecules via the Calvin-Benson-Bassham cycle. Unlike plants, cyanobacteria evolved a carbon-concentrating organelle called the carboxysome-a proteinaceous compartment that encapsulates and concentrates Rubisco along with its CO2 substrate. In the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942, we recently identified the McdAB system responsible for uniformly distributing carboxysomes along the cell length. It remains unknown what role carboxysome positioning plays with respect to cellular physiology. Here, we show that a failure to distribute carboxysomes leads to slower cell growth, cell elongation, asymmetric cell division, and elevated levels of cellular Rubisco. Unexpectedly, we also report that even wild-type S. elongatus undergoes cell elongation and asymmetric cell division when grown at the cool, but environmentally relevant, growth temperature of 20°C or when switched from a high- to ambient-CO2 environment. The findings suggest that carboxysome positioning by the McdAB system functions to maintain the carbon fixation efficiency of Rubisco by preventing carboxysome aggregation, which is particularly important under growth conditions where rod-shaped cyanobacteria adopt a filamentous morphology. IMPORTANCE Photosynthetic cyanobacteria are responsible for almost half of global CO2 fixation. Due to eutrophication, rising temperatures, and increasing atmospheric CO2 concentrations, cyanobacteria have gained notoriety for their ability to form massive blooms in both freshwater and marine ecosystems across the globe. Like plants, cyanobacteria use the most abundant enzyme on Earth, Rubisco, to provide the sole source of organic carbon required for its photosynthetic growth. Unlike plants, cyanobacteria have evolved a carbon-concentrating organelle called the carboxysome that encapsulates and concentrates Rubisco with its CO2 substrate to significantly increase carbon fixation efficiency and cell growth. We recently identified the positioning system that distributes carboxysomes in cyanobacteria. However, the physiological consequence of carboxysome mispositioning in the absence of this distribution system remains unknown. Here, we find that carboxysome mispositioning triggers changes in cell growth and morphology as well as elevated levels of cellular Rubisco.