Abstract
Cyanobacteria are prokaryotic organisms that carry out oxygenic photosynthesis. The fresh water cyanobacterium Synechococcus elongatus PCC 7942 is a model organism for the study of photosynthesis and gene regulation, and for biotechnological applications. Besides several freshwater cyanobacteria, S. elongatus 7942 also contains multiple chromosomal copies per cell at all stages of its cell cycle. Here, we describe a method for the direct visualization of multicopy chromosomes in S. elongatus 7942 by fluorescence in situ hybridization (FISH).
Keywords: Cyanobacteria, Polyploidy, Chromosome distribution, In situ hybridization, Replication origin
Background
Cyanobacteria are prokaryotic microorganisms that utilize an oxygen-producing photosynthetic system similar to that of chloroplasts in higher plants. Whereas bacteria such as Escherichia coli and Bacillus subtilis harbor a single circular chromosome, several species of freshwater cyanobacteria have multiple circular chromosomes per cell (Mann and Carr, 1974; Labarre et al., 1989; Binder and Chisholm, 1995). The freshwater cyanobacterium Synechococcus elongatus PCC 7942 (hereafter referred to as S. elongatus 7942) carries 3-8 chromosomal copies per cell (Griese et al., 2011; Watanabe et al., 2012; Watanabe et al., 2015). A fluorescence reporter-operator system has been established to acquire images of the multicopy chromosome of S. elongatus 7942 (Chen et al., 2012; Jain et al., 2012). Although this system enables live-cell imaging, it requires complex genetic constructs for labeling individual chromosomes, and, overall, this type of system is unstable over an extended period of cultivation. Thus, it is necessary to examine the chromosomal distribution in S. elongatus 7942 cells that have a simple genetic background. We recently established a fluorescence in situ hybridization (FISH) method for visualizing the oriC region and putative terC region of the multicopy chromosome in S. elongatus 7942 (Watanabe et al., 2018). A DNA probe, which covers the oriC or terC region, was synthesized and used for the FISH analysis. Labeled chromosomes in S. elongatus 7942 cells can be observed by fluorescence microscopy. Here, we describe a step-by-step protocol for the FISH method.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
This protocol was adapted from Watanabe et al. (2018). This work was supported in part by Grants-in-Aid 25850056 and 17H05451 from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.W. The authors declare no conflicts of interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.