Abstract
Cyanobacteria are prokaryotic organisms that perform oxygenic photosynthesis. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, are model organisms for the study of photosynthesis, gene regulation, and biotechnological applications because they are easy to manipulate genetically. However, while studying these cyanobacteria, care has to be taken with respect to genetic heterogeneity in the establishment of gene disruptants, because these cyanobacteria contain multiple chromosomal copies per cell. Here, we describe a method for the estimation of chromosomal copy number in Synechococcus 7942. Using this method, we have recently observed that the chromosomal copy number of Synechococcus 7942 significantly changes during its growth phases. This technique is available for studying polyploidy not only in cyanobacteria, but also in other polyploid organisms.
Keywords: Cyanobacteria, Ploidy, Bacterial Cell Cycle
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. DNA content profiles of chloramphenicol-treated E. coli cells. After culturing for 2 h in M9 medium with 20 µg/ml chloramphenicol, the cells are fixed and analyzed. A. Profiles of cell size (X axis: FSC-H) and DNA content (Y axis). B. Profiles of DNA content (X axis) and cell number (Y axis). For preparing the standard peak, which corresponds to single copy chromosome in E. coli, the major peak (asterisk) was extracted. The bottom panel represents the profiles obtained after extraction by gating. Figure 2. The DNA content profiles of cells cultured in the dark (Top) and lag-phase Synechococcus 7942 cells (bottom) cultured in light conditions for 9 h. A. Profiles of cell size (X axis: FSC-H) and DNA content (Y axis). B. Profiles of DNA content (X axis) and cell number (Y axis). Figure 3. DNA content profiles of cells cultured in the dark (thin line), lag-phase Synechococcus 7942 cells cultured in light conditions for 9 h (bold line), and the chloramphenicol-treated E. coli culture (thin red line). The copy number of the Synechococcus 7942 chromosome (ca. 2.7 Mbp) was calculated based on the peak location of E. coli (ca. 4.7 Mbp).
Recipes
Acknowledgments
This protocol was adapted from Watanabe et al. (2012). This work was supported in part by a Grants-in-Aid 25850056 from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S. W.
References
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