Cyanobacterial growth and morphology are influenced by carboxysome positioning and temperature

Abstract

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

Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

“…Overall, McdA mutant strains defective for ATP-binding and dimerization displayed a cell elongation phenotype, and possessed few and irregularly-spaced carboxysome aggregates. These phenotypes match what we have previously observed in the mcdA deletion strain (Rillema et al, 2020), which suggests a complete loss of function in carboxysome positioning when McdA cannot bind ATP and dimerize.…”

“…All three mutants, on the other hand, displayed increased spacing, and variability in spacing, as cell length increased (Figure 2, E-F). The average cell lengths of the ATP-binding and dimerization mutants were significantly longer compared to wild-type (Figure 2G); a change in cell morphology that mirrors the ΔmcdA phenotype (Supplementary Figure S1A) (Rillema et al, 2020).…”

“…We have previously shown that mNG-McdA is fully functional for carboxysome positioning when expressed as the only copy of McdA at its native locus (MacCready et al, 2018). Finally, we also performed Phase Contrast imaging to monitor for changes in cell morphology, as we have recently shown that carboxysome mispositioning in mcdA or mcdB deletion strains triggers cell elongation, which we proposed is a response to carbon limitation (Rillema et al, 2020). We first set out to determine the in vivo localization pattern of McdA when unbound from ATP, and its impact on carboxysome positioning.…”

Effects of CO2and RuBisCO concentration on cyanobacterial growth and carbon isotope fractionation

“…Various culture conditions including pH, temperature, CO2 concentration, and light intensity may substantially affect the growth characteristics of cyanobacteria, and thus have the potential to influence isotopic discrimination (Kuan, Duff, Posarac, & Bi, 2015;Rillema, MacCready, & Vecchiarelli, 2020;Ungerer, Lin, Chen, & Pakrasi, 2018;Yu et al, 2015). The influence of genetic factors, including the regulation of RuBisCO expression on cyanobacterial fitness, is less well known.…”

The emergence of phase separation as an organizing principle in bacteria

“…Still, the N-terminal domain of CsoS2, a required protein for α-carboxysome assembly, can form phase-separated droplets with RuBisCO(25). Since CsoS2 is an intrinsically disordered protein (IDP) that is believed to act as a scaffold for RuBisCO coalescence (103), it is possible that like CcmM, CsoS2 might use LLPS to initiate carboxysome formation.Carboxysome structure, composition, function, and organization are all highly responsive and adaptable to environmental change including changes in growth temperature, light, and CO2 concentration(94,104,105). For example, under high light intensities, the number of S.…”