Constriction and septation during cell division in caulobacters

Poindexter, Jeanne S.; Hagenzieker, J G

doi:10.1139/m81-109

Cited by 30 publications

(28 citation statements)

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“…DISTINCT CONSTRICTIVE PROCESSES 6875 form a septum (24). The high-resolution images provided here, together with the previous FLIP studies (15), show how the late stages of constriction and fission of the Caulobacter inner and outer membrane are distinct events occurring one after the other, distinctively separated in time and space.…”

Section: Vol 187 2005mentioning

confidence: 54%

Distinct Constrictive Processes, Separated in Time and Space, DivideCaulobacterInner and Outer Membranes

et al. 2005

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Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed prefission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.The early stages of bacterial cytokinesis, involving septal ring assembly and constriction, are better understood than the terminal stages. In this work we investigate the late stages of cytokinesis in the gram-negative bacterium Caulobacter crescentus. We use fluorescence loss in photobleaching (FLIP) and cryoelectron microscope tomography (cryoEM) to determine (i) the geometry of the inner and outer membranes at various stages of cell division and (ii) whether the FtsZ ring and associated cell division machinery hinder diffusion of membranebound or periplasmic molecules through the division plane in predivisional cells.Caulobacter crescentus divides asymmetrically, producing two distinct daughter cells: the swarmer cell and the stalked cell (Fig. 1). These cells differ in their transcription programs, protein composition, and behavior (3,20,28,32). The daughter cells also differ in size; the nascent swarmer-cell compartment is about two-thirds as long as the nascent stalked-cell compartment. After cell division, swarmer cells undergo an initial motile phase, and then they differentiate into stalked cells; they lose their flagellum and pili, and DNA replication is initiated. The compartmentalization of the predivisional cell is an important step in the process of creating daughter cells with different cell fates. Immediately after cytoplasmic compartmentalization, the complement of cytoplasmic proteins in the two compartments can begin to diverge, launching the two daughter cells on different developmental paths. The physical compartmentalization of the Caulobacter cytoplasm well before cell division triggers a phosphotransport-based switch mechanism that initiates the differential regulation of development in the nascent daughter cells (19,21).Many proteins involved in cytokinesis are known in Escherichia coli and Bacillus subtilis (1,17,35) and to a lesser extent in Caulobacter crescentus...

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Section: Vol 187 2005mentioning

confidence: 54%

Distinct Constrictive Processes, Separated in Time and Space, DivideCaulobacterInner and Outer Membranes

et al. 2005

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“…Sacculi from C. crescentus strain CB15N and E. coli strains MG1655 and XL-10 were prepared by using a published protocol (24). A 1-liter cell culture was grown to mid-exponential phase (OD600 Ϸ 0.2-0.4) in PYE (C. crescentus) or LB (E. coli).…”

Section: Methodsmentioning

confidence: 99%

Molecular organization of Gram-negative peptidoglycan

Gan¹,

Chen²,

Jensen

2008

Proc. Natl. Acad. Sci. U.S.A.

248

238

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The stress-bearing component of the bacterial cell wall-a multigigadalton bag-like molecule called the sacculus-is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: ''Layered'' and ''Scaffold'', in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozenhydrated sacculi through electron cryotomography. In the Gramnegative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.cell wall ͉ Cryo-EM ͉ sacculus ͉ tomography ͉ cell shape T he organization of peptidoglycan within the sacculus is a longstanding question in microbiology. Although the sacculus completely envelopes the cell, protecting it from osmotic and mechanical lysis, it is also porous, and thus sieves nutrients and small proteins. Sacculi are constantly remodeled, but they are robust enough that they can be purified from cells and still retain their original shape. Early electron microscope images of purified sacculi were difficult to interpret because the images were 2-D projections through heavy metal-stained sacculi (1, 2). Nevertheless the slender appearance of peptidoglycan in thinsectioned cells (3) and the gaps perpendicular to the polar axis produced by a peptidoglycan-specific endopeptidase (4) lead to the (Circumferential) Layered model (Fig. 1A ) in which the glycan strands lie in the plane of the cell wall and wrap around the cell. The alternative Scaffold model (5) (Fig. 1B) was proposed to fit biochemical data such as pore size and crosslinkage. Various arguments have been made in favor of (6) and against (7) the Scaffold model, but the debate intensified after a recent NMR structure of a synthetic peptidoglycan subunit dimer was interpreted to support the Scaffold model (8). To gain further insight into this issue, we imaged unstained sacculi in 3-D by electron cryotomography (9). Results and DiscussionElectron Cryotomography of Purified Sacculi. Sacculi isolated from C. crescentus strain CB15N and E. coli strains MG1655 and XL-10 were vitrified across holey carbon grids in thin (Ͻ 100 nm) ice. Whereas the gossamer sacculi were barely discernable in the low-dose, 2-D projection images, they were clearly visible in the 3-D tomograms and resembled flattened cells [ Fig. 2 and supporting information (SI) Movie S1 and Movie S2]. In stark contrast to intact cells, the sacculi were flexible and in some positions had radii of curvature less than 20 nm, highlighting how cell shape is not determined by the ''rigidity'' of the sacculus, but rather the cytosolic turgor (osmotic pressure) pushing the cytoplasmic membrane outward against the sacc...

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“…The final result of septation is the formation of two new polar caps that comprise the three layers of the gram-negative wall. The intermediate stages of septation differ from one species to another (18).…”

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confidence: 95%