Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria
- Michigan State University, United States
- University of Michigan, United States
- National Heart, Lung, and Blood Institute, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
The ParA-like protein, McdA, binds nonspecifically to nucleoid DNA in the presence of ATP.
(A) Purified McdA-GFP-His slows the migration of DNA in an ATP-dependent manner in an electrophoretic mobility shift assay (EMSA). (B) In flow cell experiments containing a carpet of non-specific …
Figure 1—figure supplement 1
mNG-McdA oscillates and mNG-McdB is recruited to carboxysomes.
(A) Native McdA-mNG does not oscillate (left). MicrobeJ heatmap of McdA-mNG signal (n = 950 cells) (right). (B) Native mNG-McdA displays robust oscillations (left). MicrobeJ heatmap of McdA-mNG …
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Figure 1—figure supplement 1—source data 1
Source data for panel C.
- https://doi.org/10.7554/eLife.39723.005
Figure 2 with 1 supplement
A small, carboxysome-localized protein is required for McdA oscillatory dynamics.
(A) Candidate genes controlling McdA dynamics on either the native plasmid (left) or within the genomic neighborhood of McdA. (B) Deletion of pANL parB or (C) Synpcc7942_1835 has no effect on …
Figure 2—figure supplement 1
McdA Activity.
(A) Bacterial two-hybrid between McdA and McdB tagged at their N-termini (X-McdA, X-McdB) or C-termini (McdA-X, McdB-X) on X-gal supplemented plate. (B) Comparison of the ATPase activities of …
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Figure 2—figure supplement 1—source data 1
Source data for panel B.
- https://doi.org/10.7554/eLife.39723.011
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Figure 2—figure supplement 1—source data 2
Source data for panel D.
- https://doi.org/10.7554/eLife.39723.012
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Figure 2—figure supplement 1—source data 3
Source data for panel F.
- https://doi.org/10.7554/eLife.39723.013
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Figure 2—figure supplement 1—source data 4
Source data for panel G.
- https://doi.org/10.7554/eLife.39723.014
Figure 3 with 1 supplement
McdB interacts with the carboxysome shell.
(A) Native McdB-mNG forms diffuse and punctate-like structures, while (B) native mNG-McdB localizes to centralized foci in S. elongatus cells. (C) mNG-McdB and a carboxysome reporter - RbcS-mTQ - …
Figure 3—figure supplement 1
McdA/B bacterial two-hybrid against carboxysome shell proteins.
(A) Native McdB-mNG forms diffuse and punctate-like structures throughout cells (left). MicrobeJ heatmap of McdA-mNG signal (n = 371 cells) (right). (B) Native mNG-McdB forms central foci structures …
Figure 4
McdA and McdB are essential for distributing carboxysomes.
(A) Carboxysome (orange; rbcS-mO) and mNG-McdA (yellow) distribution in a ΔmcdB background. Cartoon schematics (top) of the genetic construct are depicted, mcdA promoter ‘P’=light grey, rbcS …
Figure 5 with 1 supplement
McdAB physically separate and regulate carboxysome ultrastructure.
(A–F) MicrobeJ average distribution heat-map of carboxysomes in the indicated genetic background strains. Quantity of cells measured in lower right corner. (G) Mean RbcS-mO foci area and standard …
Figure 5—figure supplement 1
Quantification of carboxysome size from TEM.
(A) Mean carboxysome area and standard deviation for wildtype and mutant strains. p<0.0001 denoted as (***). N = at least 16 carboxysomes measured. Additional representative TEM images for (B) …
Figure 6
Carboxysomes locally deplete McdA from the nucleoid and cause McdA oscillation at high-copy number.
(A) Cartoon schematic of the inducible carboxysome operon with the synthetic, theophylline-inducible riboswitch proceeding ccmK2. Induction of carboxysome biogenesis with either (B) 400 µM or (C) …
Figure 7 with 1 supplement
McdAB accounts for linear and hexagonal packing of carboxysomes.
(A) In a field of S. elongatus cells, carboxysomes are found in either a linear (yellow arrow) or hexagonal (blue arrow) arrangement. Scale bar = 5 µm. (B) Linear or hexagonal arrangement of …
Figure 7—figure supplement 1
Carboxysomes fall between nucleoids in elongated cells lacking McdA or McdB.
(A) Carboxysomes are confined to nucleoid ‘islands’. (B) Carboxysomes are routinely localized away from nucleoids (orange arrow) in the absence of McdB. (C) Carboxysomes are routinely localized away …
Figure 8
Evolutionary conservation of McdAB.
(A) Cartoon illustration of McdAB operon structures in S. elongatus and Gloeobacter kilaueensis JS1. (B) Gloeobacter kilaueensis JS1 McdB colocalizes with S. elongatus carboxysomes (RbcS-mO). Scale …
Author response image 1
Author response image 2
Videos
Video 1
McdA binds DNA only in the presence of ATP.
After 3 min, a wash buffer is flowed in.
Video 2
Oscillation of mNG-McdA is observed across a population of cells.
https://doi.org/10.7554/eLife.39723.007
Video 3
Oscillation of mNG-McdA (magenta) occurs while carboxysomes (blue) are fluorescently labeled.
https://doi.org/10.7554/eLife.39723.008
Video 4
Carboxysomes (blue; RbsS-mTQ) move towards increased concentrations of mNG-McdA (magenta); notable examples of this phenomenon are denoted by white arrows. Each frame is 30 seconds.
https://doi.org/10.7554/eLife.39723.021
Video 5
Zoomed-in single cell representation of carboxysomes (blue; RbsS-mTQ) moving towards increased concentrations of mNG-McdA (red). Each frame is 15 seconds.
https://doi.org/10.7554/eLife.39723.022Tables
Table 1
Cyanobacterial strains used in this study.
https://doi.org/10.7554/eLife.39723.026| Strain Name | Description/Genotype |
|---|---|
| JSM-201 | mNG-McdA |
| JSM-202 | McdA-mNG |
| JSM-203 | mNG-McdB |
| JSM-204 | McdB-mNG |
| JSM-205 | RbcS-mO |
| JSM-206 | mNG-McdA + RbcS-mTQ |
| JSM-207 | mNG-McdB + RbcS-mTQ |
| JSM-208 | mNG-McdA∆parB |
| JSM-209 | mNG-McdA∆ Synpcc7942_1834 |
| JSM-210 | mNG-McdA∆ Synpcc7942_1835 |
| JSM-211 | mNG-McdA + ∆mcdB + RbcS-mO |
| JSM-212 | ∆mcdA + mTQ McdB+RbcS-mO |
| JSM-213 | ∆mcdA + ∆mcdB + RbcS-mO |
| JSM-214 | mNG-McdA + ∆mcdB + RbcS mO+RS::mTQ-McdB |
| JSM-215 | ∆mcdA + mTQ McdB+RbcS mO+RS::mNG-McdA |
| JSM-216 | mNG-McdA + RbcS mTQ + ∆ccmK2-ccmO |
| JSM-217 | mNG-McdB + RbcS mTQ + ∆ccmK2-ccmO |
| JSM-218 | mNG-McdA + RbcS mTQ+RS:: CcmK2 |
| JSM-219 | mNG-McdA + RbcS mTQ+Ptrc::CcmK2 |
| JSM-220 | RbcS-mTQ + RS::Cdv3 |
| JSM-221 | mNG-McdA + ∆mcdB + RbcS mO+RS::Cdv3 |
| JSM-222 | ∆mcdA + mTQ McdB+RbcS mO+RS::Cdv3 |
| JSM-223 | mNG-McdA + ∆mcdB + RbcS mO+RS::mTQ-McdB(Gk) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.39723.027
