The bacterial carbon-fixing organelle is formed by shell envelopment of preassembled cargo

Abstract

Background: Cyanobacteria play a significant role in the global carbon cycle. In Synechococcuselongatus, the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is concentrated into polyhedral, proteinaceous compartments called carboxysomes.

Methodology/principal findings: Using live cell fluorescence microscopy, we show that carboxysomes are first detected as small seeds of RuBisCO that colocalize with existing carboxysomes. These seeds contain little or no shell protein, but increase in RuBisCO content over several hours, during which time they are exposed to the solvent. The maturing seed is then enclosed by shell proteins, a rapid process that seals RuBisCO from the cytosol to establish a distinct, solvent-protected microenvironment that is oxidizing relative to the cytosol. These closure events can be spatially and temporally coincident with the appearance of a nascent daughter RuBisCO seed.

Conclusions/significance: Carboxysomes assemble in a stepwise fashion, inside-to-outside, revealing that cargo is the principle organizer of this compartment's biogenesis. Our observations of the spatial relationship of seeds to previously formed carboxysomes lead us to propose a model for carboxysome replication via sequential fission, polymerization, and encapsulation of their internal cargo.

Figure 1. Carboxysomes are born one at a time at the site of preexisting carboxysomes.

(A) In pulse-chase labeling of RbcL-SNAP in live S . elongatus cells, actively assembling carboxysomes with solvent accessible RbcL-SNAP are labeled with BG dye. Red: phase contrast. Green: RbcL. Scale bar: 1µm. (B) The distribution of the number of SNAP labeled carboxysomes, indicating active assembly, in cells directly after labeling (n=442). (C) The biogenesis of carboxysomes can be monitored from long timelapses. Red: phase contrast. Green: RbcL-GFP. Scale bar: 1µm. (D) Montage showing the formation of new carboxysome at the site of a preexisting carboxysome. White arrow indicates the birth event. Panel height: 25 pixels. Time interval: 3 minutes. (E–F) RuBisCO foci elongate into bar carboxysomes that subsequently split into two carboxysomes. Scale bar: 1µm. Time interval: 75 minutes. (G–I) Kymographs of RbcL-GFP in growing and dividing cells. Carboxysome birth events are indicated by white arrows. Scale bar: 1µm. Time interval: 3 minutes. (J) Spatial distribution of 234 birth events along the long axis of the cell. Quarter cell positions are favored. (K) Relative intensity of 141 pairs of new (daughter) carboxysomes and the preexisting carboxysomes to which they initially colocalize (mothers) reveals that birth events are highly asymmetric, with mean daughter intensity being 1/4 that of the mother. Because pairs are sorted into dim (daughter) and bright (mother) pairs, no data points can fall into the shaded area. Dotted line indicates a 1:4 ratio.

Figure 2. Mapping of carboxysome lineages reveals that new organelles undergo an initial refractory period before producing daughters of their own.

(A) Example lineages of carboxysomes from three out of 25 total cells analyzed from a 522 frame movie taken at 3 minute intervals over approximately 26.1 hours. Each line represents a carboxysome tracked through time, with right-angle connectors joining daughters to mothers. Digits at the top of the panel indicate the number of times carboxysomes present at the beginning of the movie have colocalized birth events over the course of the analysis (26.1 hours), represented in the histogram in panel B. Vertical dotted lines indicate the measurable age of mothers when a daughter appears, represented in the histogram in panel C. Horizontal dotted lines indicate the time of cell division. (B) Histogram of the number of births colocalized to original carboxysome in the entire dataset (n = 65). (C) Histogram of measurable ages of mothers tabulated over the entire dataset (n = 31).

Figure 3. RuBisCO slowly forms a structured assembly prior to rapid colocalization of shell protein.

(A) RuBisCO assembly, as measured by fluorescence intensity, follows sigmoidal kinetics. Each trace represents a new carboxysome. Cell is same as that depicted in Figure 1I. Imaging interval: 3 minutes. (B) Fluorescence recovery after photobleaching of a segment of a bar carboxysome. Solid box shows bleached area. Unbleached area (dashed box) was used for photobleaching correction. Cells were imaged at regular intervals after bleaching to assay for recovery. Scale bar: 1µm. (C) Quantification of FRAP in (B). Grey bar indicates bleaching event, when fluorescence sharply decreases. No recovery was seen after 150 seconds. (D–H) Time lapse of RbcL-mOrange (green) and CcmK4-GFP (red). Arrows indicate birth events of carboxysomes. Newly born RuBisCO initially buds off without shell protein. Shell protein colocalizes to RbcL-GFP foci hours after birth. In some cases (G and H), shell protein assembly is correlated with the formation of a new RuBisCO focus. Scale bar: 1µm. Time interval: 25 minutes. (I–K) Kymograph of RbcL-mOrange (J) and ccmK4-GFP (K) assembly. Shell protein assembly (yellow arrow in K) initiates well after RuBisCO birth event (yellow arrow in J). Scale bar: 1µm. Time interval: 5 minutes. (L) Individual trace of the fluorescence intensity of a CcmK4 focus in the process of formation. Time interval: 5 minutes.

Figure 4. The carboxysome oxidizes over the course of its maturation.

(A) RbcL-roGFP1 excited with 410nm (left) and 488nm (middle) produces ratiometric (488nm/410nm) differences in emission (right). Scale bar: 1µm. (B) A histogram of this ratio measured at each carboxysome focus reveals an asymmetric distribution biased toward a relatively oxidized state. (C–F) Montages of RbcL-roGFP1 show transitions from predominantly 488nm excitation (green) to 410nm excitation (magenta) over the maturation period of carboxysomes. Carboxysomes establish an oxidizing state before the appearance of a new carboxysome, rarely reopening to the cytosol after an initial closure (G). Arrows indicate birth events. Scale bar: 1µm. Interval: 20 minutes.

Figure 5. A solvent-accessible dye pulse labels foci that subsequently divide, but do not dissipate.

(A) Montage showing one labeled RuBisCO focus partitioning into two or more daughter carboxysomes and persisting over the time interval of the experiment. Red: phase contrast. Green: RbcL-SNAP. Scale bar: 1µm. Time interval: 2 hours. (B) Intensity of the mother carboxysome (pink trace) sometimes decreases when new daughters (green and blue traces) are born. In other cases, the decrease is not detectable (grey trace). (C) Distribution of the number of new carboxysome foci formed per cell over the course of an experiment (20 hours). RuBisCO foci either persisted or divided over 20 hours (n = 220). All original foci were detectable at the end of the experiment.

Figure 6. Model of carboxysome assembly.

(A) RuBisCO seeds assemble from protomers over time. (B) Late in the assembly process, shell proteins rapidly assemble around RuBisCO. (C) Shell closure completes the carboxysome to establish an oxidizing environment, sealing RuBisCO from the cytosol. (D) A new RuBisCO nucleus forms after completion of the previous carboxysome. Colocalization may be driven by bisection of excess cargo by shell closure, or (E) by affinity of RuBisCO assemblies initiated elsewhere to the outside of the shell. (F) Rupture of a complete carboxysome would expose old RuBisCO cargo to template new assembly.