Spatial and temporal organization of chromosome duplication and segregation in the cyanobacterium Synechococcus elongatus PCC 7942

Abstract

The spatial and temporal control of chromosome duplication and segregation is crucial for proper cell division. While this process is well studied in eukaryotic and some prokaryotic organisms, relatively little is known about it in prokaryotic polyploids such as Synechococcus elongatus PCC 7942, which is known to possess one to eight copies of its single chromosome. Using a fluorescent repressor-operator system, S. elongatus chromosomes and chromosome replication forks were tagged and visualized. We found that chromosomal duplication is asynchronous and that the total number of chromosomes is correlated with cell length. Thus, replication is independent of cell cycle and coupled to cell growth. Replication events occur in a spatially random fashion. However, once assembled, replisomes move in a constrained manner. On the other hand, we found that segregation displays a striking spatial organization in some cells. Chromosomes transiently align along the major axis of the cell and timing of alignment was correlated to cell division. This mechanism likely contributes to the non-random segregation of chromosome copies to daughter cells.

Figure 1. Chromosomes in the polyploid bacterium S. elongatus can be visualized using a fluorescent repressor-operator system.

(A) Bacteria contain different genomic arrangements. Here, each color represents a different chromosome. They can possess a single copy of one chromosome (I), or have multipartite genomes (II-III) with one large chromosome (red) and one or more smaller chromosomes (green and blue). Some species of bacteria, such as cyanobacteria Synechococcus elongatus PCC 7942, are polyploid. That is, they have multiple complete copies of one chromosome (IV). (B) Chromosomes can be tagged and observed in vivo using a fluorescent repressor-operator system. lacO arrays were integrated either near the origin of replication (NS1) or the predicted terminus in the S. elongatus chromosome. 10 bp spacers with random sequences were inserted between the operator sites to avoid recombination (black). The protein fusion Lacl-GFP (blue and green) bound to multiple repeats of its cognate lacO operator site (pink). (C) The fluorescent repressor-operator system from (B) was transformed into S. elongatus. The origins of replication of each chromosome appear as foci (green) in cells (red) when imaged using wide field fluorescence microscopy. Origins of replication are seen throughout the cell. (D) Cells with lacO arrays integrated near the putative terminus region at 1.59 Mb in the genome were visualized. Foci appear throughout the cell, similar to (C).

Figure 2. Chromosome duplication is correlated to cell length.

(A) Distribution of chromosome number per cell is not significantly different from a log-normal distribution (n = 681, χ² goodness of fit test, h = 0, p = 0.2621). Most cells contain 4 chromosomes with values ranging from 1 to 10. (B) Chromosome copy number is correlated to cell length (n = 660, r = 0.7519, p<0.001), suggesting chromosome duplication is coupled to cell growth.

Figure 3. Chromosome duplication is asynchronous and is not coupled to cell division.

(A) Single stranded binding (SSB) protein was tagged with mOrange. These were co-expressed in cells with LacI-GFP (NSl-lacO). Replisome localization appeared as foci and co-localized with tagged chromosomes (merge), indicating that tagging did not interfere with functioning of SSB. A cell with two chromosomes (left, green arrows) only contains one actively replicating chromosome (left, blue arrows). A cell with three chromosomes (right, green arrows) also only has one replisome (right, blue arrows). (B) Most cells contain one actively duplicating chromosome (85%), while the remaining contain two or three replisomes. Since most cells contain multiple chromosomes but only one replisome, this shows that chromosome duplication is asynchronous.

Figure 4. Replication occurs in a spatially random manner but replisomes undergo constrained movement.

(A) Chromosome duplication events have no spatial preference within the cell. Cells (n = 297) were sub-segmented along the major axis into 20 smaller regions and SSB foci localization was binned. The distribution of SSB localization was not found to be significantly different from a uniform distribution (KS test, h = 0, p = 0.4867, k = 0.0456), suggesting spatially random replication events. Cell poles were excluded in this analysis because of boundary effects due to reduced cell volume and the volume of the nucleoid decreasing the likelihood of SSB foci found at the edges of rod shaped cells. (B) To confirm the random localization of chromosome duplication, newly synthesized chromosomes were visualized using fluorescent nucleotide incorporation. Fluorescent foci also show a uniform distribution (KS test, h = 0, p = 0.1027, k = 0.0579), confirming the results shown in (A). (C) Two replisomes (red and green) were tracked over the time scale of a complete replication cycle with images taken every two minutes. Replisomes show restricted occupation of domains in the cytoplasm, remaining close to their initial position, suggesting that chromosomes are spooled through the replisomes.

Figure 5. Chromosomes align transiently before non-random segregation occurs.

(A) Chromosomes in cells visualized through Lacl-GFP in an NSl-lacO background showed two different spatial arrangements. The chromosomes are either aligned along the major axis of the cell (I) or randomly localized (II). (B) Time-lapse imaging of single cells revealed a transient alignment of the chromosomes (4 hours, yellow arrows) approximately three hours before the cell entered cytokinesis. (C) Almost all sibling cells descended from mother cells containing eight chromosomes inherit four chromosomes, thus chromosome segregation is highly non-random.

Figure 6. Model of chromosome replication and segregation in the polyploid bacterium S. elongatus PCC 7942.

S. elongatus possess multiple copies of a single chromosome, shown in red (A). Chromosomes are duplicated asynchronously and coupled to cell growth (B, D, E). Newly synthesized chromosomes (orange) are synthesized in a spatially random manner (D,E,F). Replisomes (blue) assemble on a spatially random chromosome (B,D,E), but once initiated, their motion remains confined within the same region of the cell (grey box, B to C). Chromosomes transiently align (F) before non-random segregation and cytokinesis (F to G &A).