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The Bacterial Carbon-Fixing Organelle Is Formed by Shell Envelopment of Preassembled Cargo

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 3