The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation - PubMed (original) (raw)

The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation

R B Jensen et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The highly conserved SMC (Structural Maintenance of Chromosomes) proteins function in chromosome condensation, segregation, and other aspects of chromosome dynamics in both eukaryotes and prokaryotes. A null mutation in the Caulobacter crescentus smc gene is conditionally lethal and causes a cell cycle arrest at the predivisional cell stage. Chromosome segregation in wild-type and smc null mutant cells was examined by monitoring the intracellular localization of the replication origin and terminus by using fluorescence in situ hybridization. In wild-type cells, the origin is located at the flagellated pole of swarmer cells and, immediately after the initiation of DNA replication in stalked cells, one of the origins moves to the opposite pole, giving a bipolar localization of the origins. The terminus moves from the end of the swarmer cell opposite the origin to midcell. A subpopulation of the smc null mutant cells had mislocalized origins or termini, showing that the smc null mutation gives DNA segregation defects. Nucleoid morphology was also abnormal. Thus, we propose that the Caulobacter chromosomal origins have specific cellular addresses and that the SMC protein plays important roles in maintaining chromosome structure and in partitioning. The specific cell cycle arrest in the smc null mutant indicates the presence of a cell cycle checkpoint that senses perturbations in chromosome organization or segregation.

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Figures

Figure 1

Figure 1

Organization of the Caulobacter chromosomal region containing the smc gene and SMC protein domain structure. (A) Predicted genetic organization of the smc chromosomal locus. Bars above the line represent genes transcribed from left to right; bars below the line represent genes transcribed in the opposite direction. The orfA and orfB genes are putative ORFs that show homology to genes of unknown function in other bacteria. (B) Predicted domain structure of the Caulobacter SMC protein. The domains were assigned by homology to other SMC proteins (24) and the locations of the coiled-coil regions were predicted as described (40).

Figure 2

Figure 2

Nucleoid morphology in wild-type and smc null mutant cells. (A) The parent strain CB15N grown at 20°C. (B) CB15N grown at 20°C and shifted to 30°C for 4 hr. (C) The smc null mutant strain CB15NΔsmc grown at 20°C. (D) CB15NΔsmc grown at 20°C and shifted to 30°C for 4 hr. Nomarski differential interference contrast microscopy images of the cells are shown in the first column, DAPI-stained nucleoids are shown in the second column, and the third column is an overlay of the images. Arrows indicate cells with abnormal nucleoid morphology, and the white scale bar represents 2 μm.

Figure 3

Figure 3

Characterization of the cell cycle block in the smc mutant strain. Swarmer cells from the parent strain CB15N (A) and the smc null mutant strain CB15NΔsmc (B) grown at 20°C were isolated and shifted to 32°C. Progression through the cell cycle was examined by measuring the DNA content of chromomycin A3-stained cells by using flow cytometry. Chromosome equivalents are indicated on the _x_-axis and relative number of cells on the _y_-axis. The times (in minutes) when the samples were removed and fixed are indicated. Cell cycle progression is shown schematically. In the wild-type strain, swarmer cells are present at 0 min, stalked cells at 60 min, early predivisional cells at 90 min, and late predivisional cells at 120 min. Cell division occurs at 140 min.

Figure 4

Figure 4

Presence of the McpA chemoreceptor, the CtrA cell cycle regulator, and flagellin during the cell cycle in wild-type and Δ_smc_ cells. The McpA, CtrA, and 25-kDa flagellin proteins were detected by immunoblotting samples from synchronous cultures of CB15N (A) or CB15NΔsmc (B) grown at the restrictive temperature of 32°C. Samples were taken every 20 min, and equal amounts of cells were applied to each lane. The cell cycle progression of each strain is shown schematically. Gray shading indicates the presence of CtrA in the different cell types as determined previously (29).

Figure 5

Figure 5

Localization of the origin and terminus proximal regions of the Caulobacter chromosome by using FISH. Wild-type (A and B) and smc null mutant (C) swarmer cells were isolated and allowed to progress synchronously through the cell cycle at 32°C. At the indicated times (in minutes), the cells were fixed and hybridized simultaneously with a Cy3-labeled origin probe (red) and a FluorX-labeled terminus probe (green) (A and C). In B, the origin was localized by using a Cy3-labeled origin probe (red), and the McpA chemoreceptor was visualized by immunofluorescence staining with FITC-labeled secondary antibodies (green). The McpA protein localizes at the flagellated pole in swarmer cells (30). The nucleoids were visualized by DAPI staining (blue). Because no nucleoid-free regions are present in wild-type Caulobacter, the DAPI staining outlines the cells. Arrows indicate cells with abnormal localization of the origin or the terminus, and the white scale bar represents 2 μm. The cell cycle of each strain is diagrammed. Blue ovals and theta structures represent nonreplicating and replicating chromosomes, respectively. The intracellular localization of the origin (red dot), the terminus (green dot), and the McpA chemoreceptor (purple dot) are shown. The presence of CtrA (29) is indicated with shading of the cell.

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