Cyanobacteria Maintain Constant Protein Concentration despite Genome Copy-Number Variation  Xiao-yu Zheng, Erin K. O’Shea  Cell Reports  Volume 19, Issue 3, Pages 497-504 (April 2017) DOI: 10.1016/j.celrep.2017.03.067 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 497-504DOI: (10.1016/j.celrep.2017.03.067) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Protein Concentration Is Constant in Cells with Different Chromosome Copy Numbers (A) Each chromosome was labeled with mTurquoise2-tagged tetracycline repressor protein (TetR-mTurq2) and tetracycline operator arrays (tetO). The YFP marker protein was integrated into each chromosome. (B) Using a fluorescence microscope, chromosomes can be visualized and YFP fluorescence can be measured. A single z section of representative images in each channel is shown. (C) Computational analysis enabled automatic cell segmentation and chromosome foci identification. Green lines outline the cells segmented by the Oufti software, overlaid with a single z section of the image from the YFP channel (top). Red dots show chromosomes identified by custom-written scripts, overlaid with the maximal projection from the z stack image from the CFP channel (bottom). (D) YFP fluorescence intensity concentration in arbitrary units per cubic micrometer (A.U./μm3) for each cell is plotted against chromosome copy number. Data shown are from the strain with BbbJ23119::YFP. Each blue point represents one single-cell measurement. Violin plots represent distributions of single-cell data. Yellow diamonds represent the sample mean. Error bars represent SD. Top panel bar graph shows the coefficient of variation from cells with the same chromosome copy number. Bottom histogram shows the distribution of chromosome copy numbers. N shows the total sample size. Data from one biological replicate is shown, but similar results were obtained in three biological replicates. (E) Mean YFP concentrations from groups of cells with the same chromosome copy number for the pkaiBC::YFP strain are shown. Blue and red colors represent data obtained at the peak phase and trough phase of the circadian cycle, respectively. The corresponding distributions of chromosome copy numbers are presented in the top and bottom histograms. Error bars represent SD. Data from one biological replicate is shown, but similar results were obtained in two biological replicates. See also Figure S1. Cell Reports 2017 19, 497-504DOI: (10.1016/j.celrep.2017.03.067) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Cell Volume and Total YFP Amount, Respectively, Are Positively, Linearly Correlated with Chromosome Copy Number (A and B) Data of cell volume (A) and total YFP amount (B) from the S. elongatus strain with BbbJ23119::YFP is presented. Each blue point represents one single-cell measurement. Violin plots represent distributions of single-cell data. Yellow diamonds represent the sample mean. Error bars represent SD. The histogram shows the distribution of chromosome copy numbers. N shows the total sample size. The red line shows the best linear fit, the parameters of which are listed in Table S1. (C) Total YFP fluorescence intensity was plotted against cell volume. Each point represents a single-cell measurement. Green, blue, and red represent cells with two, four, and six chromosome copies, respectively. Histograms show the corresponding volume distributions. For chromosome copy number 2 and 6, data presented are from all 185 cells and 184 cells measured, respectively. For chromosome copy number 4, a random 180-cell subset of 876 cells is shown. The black line shows the best linear fit of total YFP intensity versus cell volume based on data from all 2,575 cells. For all subfigures, data are from the same biological replicate as that shown in Figure 1D. Similar results were obtained from three biological replicates. See also Figure S2 and Table S1. Cell Reports 2017 19, 497-504DOI: (10.1016/j.celrep.2017.03.067) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Cells Grown in Low Light Intensity or Darkness Maintain the Linear Correlation between Cell Volume and Genome Copy Number and Maintain Homogeneous Protein Concentration Despite Genome Copy-Number Variation (A) Cells (the BbbJ23119::YFP strain) were grown under constant illumination at low light intensity (9 μE m−2s−1). (B) Cells (the BbbJ23119::YFP strain) were first grown in constant light (30 μE m−2s−1) until reaching exponential growth, then shifted to darkness for 24 hr. The histogram shows the distribution of chromosome copy numbers. Data from cells with a chromosome copy number frequency less than 20 are not shown. N shows the total sample size presented. Each blue point represents one single-cell measurement. Violin plots represent distributions of single-cell data. Yellow diamonds represent the sample mean. Error bars represent SD. The red line shows the best linear fit, the parameters of which are listed in Table S1. Data shown are from one biological replicate. Similar results were obtained in two biological replicates for the low light intensity growth condition. See also Figure S3 and Table S1. Cell Reports 2017 19, 497-504DOI: (10.1016/j.celrep.2017.03.067) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 The ΔminD Mutant that Is Defective in the Correct Placement of the Division Septum Still Maintains Homogeneous Protein Concentration in Cells with Different Chromosome Copy Numbers (A) In wild-type cells, the division septum is placed in the middle of the cell and chromosome copies are evenly distributed along the long axis of the cell; thus, daughter cells inherit approximately equal numbers of chromosome copies from the mother cell. In the ΔminD mutant, the division septum is randomly placed but the chromosome spatial ordering maintains; thus, daughter cells inherit unequal numbers of chromosome copies. (B–D) For the ΔminD mutant strain with BbbJ23119::YFP, YFP fluorescence concentration (B), cell volume (C), and total YFP intensity (D) were plotted against chromosome copy number, respectively. The histogram shows the distribution of chromosome copy numbers. N shows the total sample size presented. Each blue point represents one single-cell measurement. Violin plots represent distributions of single-cell data. Yellow diamonds represent the sample mean. Error bars represent SD. The red line shows the best linear fit, the parameters of which are listed in Table S1. Similar results were obtained in three biological replicates. See also Figure S4 and Table S1. Cell Reports 2017 19, 497-504DOI: (10.1016/j.celrep.2017.03.067) Copyright © 2017 The Author(s) Terms and Conditions