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The essential process of peptidoglycan synthesis requires two enzymatic activities, transpeptidation and transglycosylation. While the PBP2 and PBP3 transpeptidases perform highly specialized functions that are widely conserved, the specific roles of different glycosyltransferases are poorly understood. For example, Caulobacter crescentus encodes six glycosyltransferase paralogs of largely unknown function. Using genetic analyses, we found that Caulobacter glycosyltransferases are primarily redundant but that PbpX is responsible for most of the essential glycosyltransferase activity. Cells containing PbpX as their sole glycosyltransferase are viable, and the loss of pbpX leads to a general defect in the integrity of the cell wall structure even in the presence of the other five glycosyltransferases. However, neither PbpX nor any of its paralogs is required for the specific processes of cell elongation or division, while the cell wall synthesis required for stalk biogenesis is only partially disrupted in several of the glycosyltransferase mutants. Despite their genetic redundancy, Caulobacter glycosyltransferases exhibit different subcellular localizations. We suggest that these enzymes have specialized roles and normally function in distinct subcomplexes but retain the ability to substitute for one another so as to ensure the robustness of the peptidoglycan synthesis process. 相似文献
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The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili. 相似文献
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In Caulobacter crescentus, the actin homologue MreB is critical for cell shape maintenance. Despite the central importance of MreB for cell morphology and viability, very little is known about MreB-interacting factors. Here, we use an overexpression approach to identify a novel MreB interactor, MbiA. MbiA interacts with MreB in both biochemical and genetic assays, colocalizes with MreB throughout the cell cycle, and relies on MreB for its localization. MbiA overexpression mimics the loss of MreB function, severely perturbing cell morphology, inhibiting growth and inducing cell lysis. Additionally, mbiA deletion shows a synthetic growth phenotype with a hypomorphic allele of the MreB interactor RodZ, suggesting that these two MreB-interacting proteins either have partially redundant functions or participate in the same functional complex. Our work thus establishes MbiA as a novel cell shape regulator that appears to function through regulating MreB, and opens avenues for discovery of more MreB-regulating factors by showing that overexpression screens are a valuable tool for uncovering potentially redundant cell shape effectors. 相似文献
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The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2-peptidoglycan biosynthesis complex. 相似文献
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Caulobacter crescentus requires RodA and MreB for stalk synthesis and prevention of ectopic pole formation 下载免费PDF全文
Caulobacter crescentus cells treated with amdinocillin, an antibiotic which specifically inhibits the cell elongation transpeptidase penicillin binding protein 2 in Escherichia coli, exhibit defects in stalk elongation and morphology, indicating that stalk synthesis may be a specialized form of cell elongation. In order to investigate this possibility further, we examined the roles of two other proteins important for cell elongation, RodA and MreB. We show that, in C. crescentus, the rodA gene is essential and that RodA depletion leads to a loss of control over stalk and cell body diameter and a stalk elongation defect. In addition, we demonstrate that MreB depletion leads to a stalk elongation defect and conclude that stalk elongation is a more constrained form of cell elongation. Our results strongly suggest that MreB by itself does not determine the diameter of the cell body or stalk. Finally, we show that cells recovering from MreB depletion exhibit a strong budding and branching cell body phenotype and possess ectopic poles, as evidenced by the presence of multiple, misplaced, and sometimes highly branched stalks at the ends of these buds and branches. This phenotype is also seen to a lesser extent in cells recovering from RodA depletion and amdinocillin treatment. We conclude that MreB, RodA, and the target(s) of amdinocillin all contribute to the maintenance of cellular polarity in C. crescentus. 相似文献
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Development in Caulobacter reflects a level of complexity once thought only to exist in eukaryotic cells. The cell cycle and development are not isolated from each other, but are interdependent processes. Checkpoints are in place to ensure that both cell cycle and developmental processes are completed accurately before the next stage is initiated. The timing of these processes is regulated by signal transduction networks that integrate signals from DNA replication, cell division and development. These signal transduction networks achieve precise timing of the cell cycle and development by regulating temporal gene expression, and protein activity by dynamic spatial localization within the cell and timed proteolysis. 相似文献
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The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of the Caulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJ results in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors. 相似文献
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Effect of Macromolecular Synthesis on the Coordinate Morphogenesis of Polar Surface Structures in Caulobacter crescentus 下载免费PDF全文
The Caulobacter polar surface structures (flagella, pili, and the deoxyribonucleic acid phage phiCbK receptors), which are expressed at proximal sites of swarmer cells in a coordinate manner (Shapiro, Annu. Rev. Microbiol., 30:377-407, 1976) could be blocked by a single mutation. The mutant C. crescentus CB13 ple-801 did not form these surface structures when grown at 35 degrees C. Upon shift down to 25 degrees C, the mutant cells initiated the formation of the surface structures. When mitomycin C was added to the mutant culture upon shift down from 35 to 25 degrees C, phiCbK receptor formation was inhibited to a minimal level. Rifampin and chloramphenicol completely inhibited phiCbK receptor formation when added to the mutant culture upon shift down. Deoxyribonucleic acid as well as ribonucleic acid and protein synthesis seem to be required for the formation of phiCbK receptors. Penicillin V also inhibited phiCbK receptor formation, indicating the involvement of cell wall synthesis. When the mutant CB13 ple-801 cells were shifted down briefly from 35 to 25 degrees C and then shifted up to 35 degrees C, flagella and phiCbK receptors were formed even at 35 degrees C to different extents depending on how long the cells were incubated at 25 degrees C. This formation of the surface structures at 35 degrees C was inhibited by rifampin. From these results, it appears that translation, assembly, or localization processes for the formation of the surface structures are not temperature sensitive at 35 degrees C in the pleiotropic mutant CB13 ple-801. The syntheses of deoxyribonucleic acid and the cell wall do not appear to be temperature sensitive either, since the mutant grows normally at 35 degrees C. It is suggested that there exists a regulatory step that commits the cells to initiate the synthesis of requisite ribonucleic acid for the formation of the polar surface structures. 相似文献
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Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function. 相似文献
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Aleksandr Vasilyev Yan Liu Sudha Mudumana Steve Mangos Pui-Ying Lam Arindam Majumdar Jinhua Zhao Kar-Lai Poon Igor Kondrychyn Vladimir Korzh Iain A Drummond 《PLoS biology》2009,7(1)
Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis. 相似文献
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Dependence of Cell Division on the Completion of Chromosome Replication in Caulobacter crescentus 总被引:5,自引:8,他引:5 下载免费PDF全文
The relationship between chromosome replication and cell division in the stalked bacterium Caulobacter crescentus has been investigated. Two compounds, hydroxyurea and mitomycin C, were found to inhibit completely deoxyribonucleic acid (DNA) synthesis while allowing continued cell growth and elongation. When these inhibitors were added to exponentially growing cultures, cell division stopped after 38 min when hydroxyurea was used and after 33 min when mitomycin C was used. The period of continued cell division corresponds closely to the period previously determined for the postsynthetic gap (G2) in the DNA cycle of this organism. These results indicate that cell division is coupled to the completion of chromosome replication in C. crescentus. 相似文献
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Matthew T. Cabeen Michelle A. Murolo Ariane Briegel N. Khai Bui Waldemar Vollmer Nora Ausmees Grant J. Jensen Christine Jacobs-Wagner 《Journal of bacteriology》2010,192(13):3368-3378
Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.Most bacterial species display a particular cellular morphology that is generally preserved across generations. The production and maintenance of shape require coordination among multiple cellular systems positioned at different places within the cell. The peptidoglycan cell wall, located external to the cytoplasmic membrane, is an important structural element that is required for shape maintenance. The processes governing the localization and timing of cell wall growth and turnover are likewise critical (8, 9, 23). The bacterial cytoskeleton is thought to play a central role in cell morphogenesis by exerting spatiotemporal control over peptidoglycan growth (8, 9, 23). In order for it to do so, there are numerous proteins that are required to connect cytoskeletal control mechanisms to the periplasmic enzymes that directly synthesize and modify the peptidoglycan cell wall. These proteins, such as MreC, MreD, RodA, and RodZ, are essential for maintenance of cell shape and are positioned in the cytoplasmic membrane to presumably link cytoskeletal elements in the cytoplasm to the activities of peptidoglycan-modifying enzymes in the periplasm (2, 5, 9, 23, 29). Some bacterial species also contain additional components that make important contributions to cell shape, such as cell wall teichoic acids in Gram-positive bacteria (9) and periplasmic flagella in spirochetes (41).In the vibrioid bacterium Caulobacter crescentus, an intermediate filament-like protein, crescentin, is required for cell curvature (3). Crescentin forms an intracellular filamentous structure that is associated with the cell wall and is thought to mechanically govern cell wall growth to produce cell curvature (7). The crescentin structure is localized along the inner curvature of the cell under the cytoplasmic membrane (3, 7) and is highly stable, with no detectable subunit exchange (10). The association between the crescentin structure and the cell envelope appears essential for its function, since an attachment-defective crescentin mutant is unable to support cell curvature (7). The function of the actin-like protein MreB is also critical for the envelope association of the crescentin structure (10), and MreB may provide one part of the connection between the crescentin structure and the peptidoglycan cell wall.Since bacterial morphogenesis requires multiple cellular components and systems, we used a genetic screen to find other factors important for cell curvature in C. crescentus. Surprisingly, we found that an alteration in the lipopolysaccharide (LPS) biosynthesis pathway can have a catastrophic effect on the ability of the crescentin structure to associate with the cell envelope and govern cell curvature. 相似文献
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Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell. 相似文献