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1.
Abhishekh Gupta Jason Lloyd-Price Ramakanth Neeli-Venkata Samuel?M.D. Oliveira Andre?S. Ribeiro 《Biophysical journal》2014,106(9):1928-1937
The cytoplasm of Escherichia coli is a crowded, heterogeneous environment. From single cell live imaging, we investigated the spatial kinetics and heterogeneities of synthetic RNA-protein complexes. First, although their known tendency to accumulate at the cell poles does not appear to introduce asymmetries between older and newer cell poles within a cell lifetime, these emerge with cell divisions. This suggests strong polar retention of the complexes, which we verified in their history of positions and mean escape time from the poles. Next, we show that the polar retention relies on anisotropies in the displacement distribution in the region between midcell and poles, whereas the speed is homogeneous along the major cell axis. Afterward, we establish that these regions are at the border of the nucleoid and shift outward with cell growth, due to the nucleoid’s replication. Overall, the spatiotemporal kinetics of the complexes, which is robust to suboptimal temperatures, suggests that nucleoid occlusion is a source of dynamic heterogeneities of macromolecules in E. coli that ultimately generate phenotypic differences between sister cells. 相似文献
2.
Samuel M. D. Oliveira Ramakanth Neeli‐Venkata Nadia S. M. Goncalves João A. Santinha Leonardo Martins Huy Tran Jarno Mäkelä Abhishekh Gupta Marilia Barandas Antti Häkkinen Jason Lloyd‐Price José M. Fonseca Andre S. Ribeiro 《Molecular microbiology》2016,99(4):686-699
In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub‐optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location. We present evidence that this is due to increased cytoplasm viscosity, which weakens the anisotropy in aggregate displacements at the nucleoid borders that is responsible for their preference for polar localisation. Next, we show that in plasmolysed cells, which have increased cytoplasm viscosity, aggregates are also not preferentially located at the poles. Finally, we show that the inability of cells with increased viscosity to exclude aggregates from midcell results in enhanced aggregate concentration in between the nucleoids in cells close to dividing. This weakens the asymmetries in aggregate numbers between sister cells of subsequent generations required for rejuvenating cell lineages. We conclude that the process of exclusion of protein aggregates from midcell is not immune to stress conditions affecting the cytoplasm viscosity. The findings contribute to our understanding of E. coli's internal organisation and functioning, and its fragility to stressful conditions. 相似文献
3.
Nucleoid partitioning in Escherichia coli during steady-state growth and upon recovery from chloramphenicol treatment 总被引:12,自引:7,他引:5
To distinguish between a gradual or an abrupt movement of the Escherichia coli nucleoid during partitioning we determined the distances between nucleoid borders and cell poles. Measurements were performed on fixed but hydrated cells and on living cells growing in steady state. The distance between nucleoid outer border and cell pole remained constant in cells with either one or two nucleoids. Thus the nucleoid outer borders moved gradually during the partition process. To study partitioning during recovery from protein-synthesis inhibition cells were treated with chloramphenicol. After growth resumption, cells and nucleoids first elongated before partitioning occurred. Again, no indication of a rapid displacement of the nucleoid to one-quarter and three-quarter positions in the cell was observed. 相似文献
4.
Bacterial genomic DNA is packed within the nucleoid of the cell along with various proteins and RNAs. We previously showed that the nucleoid in log phase cells consist of fibrous structures with diameters ranging from 30 to 80 nm, and that these structures, upon RNase A treatment, are converted into homogeneous thinner fibers with diameter of 10 nm. In this study, we investigated the role of major DNA-binding proteins in nucleoid organization by analyzing the nucleoid of mutant Escherichia coli strains lacking HU, IHF, H–NS, StpA, Fis, or Hfq using atomic force microscopy. Deletion of particular DNA-binding protein genes altered the nucleoid structure in different ways, but did not release the naked DNA even after the treatment with RNase A. This suggests that major DNA-binding proteins are involved in the formation of higher order structure once 10-nm fiber structure is built up from naked DNA. 相似文献
5.
Matthew W. Bailey Paola Bisicchia Boyd T. Warren David J. Sherratt Jaan M?nnik 《PLoS genetics》2014,10(8)
Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. coli than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins. 相似文献
6.
Although many proteins are known to localize in bacterial cells, for the most part our understanding of how such localization takes place is limited. Recent evidence that the phospholipid cardiolipin localizes to the poles of rod-shaped bacteria suggests that targeting of some proteins may rely on the heterogeneous distribution of membrane lipids. Membrane curvature has been proposed as a factor in the polar localization of high-intrinsic-curvature lipids, but the small size of lipids compared to the dimensions of the cell means that single molecules cannot stably localize. At the other extreme, phase separation of the membrane energetically favors a single domain of such lipids at one pole. We have proposed a physical mechanism in which osmotic pinning of the membrane to the cell wall naturally produces microphase separation, i.e., lipid domains of finite size, whose aggregate sensitivity to cell curvature can support spontaneous and stable localization to both poles. Here, we demonstrate that variations in the strength of pinning of the membrane to the cell wall can also act as a strong localization mechanism, in agreement with observations of cardiolipin relocalization from the poles to the septum during sporulation in the bacterium Bacillus subtilis. In addition, we rigorously determine the relationship between localization and the domain-size distribution including the effects of entropy, and quantify the strength of domain-domain interactions. Our model predicts a critical concentration of cardiolipin below which domains will not form and hence polar localization will not take place. This observation is consistent with recent experiments showing that in Escherichia coli cells with reduced cardiolipin concentrations, cardiolipin and the osmoregulatory protein ProP fail to localize to the poles. 相似文献
7.
Harry EJ 《Molecular microbiology》2001,40(4):795-803
The earliest stage of cell division in bacteria is the formation of a Z ring, composed of a polymer of the FtsZ protein, at the division site. Z rings appear to be synthesized in a bi‐directional manner from a nucleation site (NS) located on the inside of the cytoplasmic membrane. It is the utilization of a NS specifically at the site of septum formation that determines where and when division will occur. However, a Z ring can be made to form at positions other than at the division site. How does a cell regulate utilization of a NS at the correct location and at the right time? In rod‐shaped bacteria such as Escherichia coli and Bacillus subtilis, two factors involved in this regulation are the Min system and nucleoid occlusion. It is suggested that in B. subtilis, the main role of the Min proteins is to inhibit division at the nucleoid‐free cell poles. In E. coli it is currently not clear whether the Min system can direct a Z ring to the division site at mid‐cell or whether its main role is to ensure that division inhibition occurs away from mid‐cell, a role analogous to that in B. subtilis. While the nucleoid negatively influences Z‐ring formation in its vicinity in these rod‐shaped organisms, the exact relationship between nucleoid occlusion and the ability to form a mid‐cell Z ring is unresolved. Recent evidence suggests that in B. subtilis and Caulobacter crescentus, utilization of the NS at the division site is intimately linked to the progress of a round of chromosome replication and this may form the basis of achieving co‐ordination between chromosome replication and cell division. 相似文献
8.
Clément Carel Kanjana Nukdee Sylvain Cantaloube Mélanie Bonne Cheikh T. Diagne Fran?oise Laval Mamadou Daffé Didier Zerbib 《PloS one》2014,9(5)
Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles'' heel of the bacillus at all its growing stages. 相似文献
9.
Andrew N. Gray Zaoping Li Josephine Henderson-Frost Marcia B. Goldberg 《Journal of bacteriology》2014,196(3):624-632
Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion. 相似文献
10.
Advances in bacterial cell biology have demonstrated the importance of protein localization for protein function. In general, proteins are thought to localize to the sites where they are active. Here we demonstrate that in Escherichia coli, MurG, the enzyme that mediates the last step in peptidoglycan subunit biosynthesis, becomes polarly localized when expressed at high cellular concentrations. MurG only becomes polarly localized at levels that saturate MurG''s cellular requirement for growth, and E. coli cells do not insert peptidoglycan at the cell poles, indicating that the polar MurG is not active. Fluorescence recovery after photobleaching (FRAP) and single-cell biochemistry experiments demonstrate that polar MurG is dynamic. Polar MurG foci are distinct from inclusion body aggregates, and polar MurG can be remobilized when MurG levels drop. These results suggest that polar MurG represents a temporary storage mechanism for excess protein that can later be remobilized into the active pool. We investigated and ruled out several candidate pathways for polar MurG localization, including peptidoglycan biosynthesis, the MreB cytoskeleton, and polar cardiolipin, as well as MurG enzymatic activity and lipid binding, suggesting that polar MurG is localized by a novel mechanism. Together, our results imply that inactive MurG is dynamically sequestered at the cell poles and that prokaryotes can thus utilize subcellular localization as a mechanism for negatively regulating enzymatic activity.Cells need ways to deal with having more of a specific protein than they need. Left unchecked, excess protein can be toxic to the cell and interfere with essential processes. In prokaryotes, a common mechanism for dealing with excess protein is degradation (30). Bacterial proteases can break down proteins, salvaging amino acids to produce new protein. This process costs time and energy, especially if the protein being degraded is essential and will need to be resynthesized later. Excess protein can also aggregate into insoluble inclusion bodies. In inclusion bodies, proteins are generally misfolded, and though in some cases these proteins can be refolded (24, 35), inclusion body proteins are not readily accessible for use by the cell (11). A potential alternative strategy for dealing with excess protein is to temporarily store the protein in an inactive form that can later be dynamically remobilized when needed. Here we propose that Escherichia coli uses subcellular localization of MurG to accomplish such dynamic storage.MurG is an essential, membrane-associated N-acetylglucosaminyl transferase involved in catalyzing the final step of peptidoglycan subunit biosynthesis (4, 21). In E. coli, the peptidoglycan cell wall determines both cell shape and growth rate (17). During growth and division, E. coli cells add new peptidoglycan both along the lateral cylindrical portion of the cell and at the division plane, but no new peptidoglycan is added at the cell poles (10). Previous efforts to study the localization of MurG have found that MurG localizes to the cell periphery and division plane in E. coli (25). In this study, we demonstrate that E. coli MurG also localizes to the cell poles in a concentration-dependent manner. We find that the polar MurG represents a dynamic pool of excess protein, suggesting that polar accumulation represents an accessible form of temporary storage. 相似文献
11.
Protein misfolding and aggregation are inevitable but detrimental cellular processes. Cells therefore possess protein quality control mechanisms based on chaperones and proteases that (re)fold or hydrolyze unfolded, misfolded, and aggregated proteins. Besides these conserved quality control mechanisms, the spatial organization of protein aggregates (PAs) inside the cell has been proposed as an important additional strategy to deal with their cytotoxicity. In the bacterium Escherichia coli, however, it remained unclear how this spatial organization is established and how this process of assembling PAs in the cell poles affects cellular physiology. In this report, high hydrostatic pressure was used to transiently reverse protein aggregation in living E. coli cells, allowing the subsequent (re)assembly of PAs to be studied in detail. This approach revealed PA assembly to be dependent on intracellular energy and metabolic activity, with the resulting PA structure being confined to the cell pole by nucleoid occlusion. Moreover, a correlation could be observed between the time needed for PA reassembly and the individual lag time of the cells, which might prevent symmetric segregation of cytotoxic PAs among siblings to occur and ensure rapid spatial clearance of molecular damage throughout the emerging population. 相似文献
12.
It has been proposed that forces resulting from the physical exclusion of macromolecules from the bacterial nucleoid play a central role in organizing the bacterial cell, yet this proposal has not been quantitatively tested. To investigate this hypothesis, we mapped the generic motion of large protein complexes in the bacterial cytoplasm through quantitative analysis of thousands of complete cell-cycle trajectories of fluorescently tagged ectopic MS2-mRNA complexes. We find the motion of these complexes in the cytoplasm is strongly dependent on their spatial position along the long axis of the cell, and that their dynamics are consistent with a quantitative model that requires only nucleoid exclusion and membrane confinement. This analysis also reveals that the nucleoid increases the mobility of MS2-mRNA complexes, resulting in a fourfold increase in diffusion coefficients between regions of the lowest and highest nucleoid density. These data provide strong quantitative support for two modes of nucleoid action: the widely accepted mechanism of nucleoid exclusion in organizing the cell and a newly proposed mode, in which the nucleoid facilitates rapid motion throughout the cytoplasm. 相似文献
13.
14.
Thiago M. A. Santos Ti‐Yu Lin Madhusudan Rajendran Samantha M. Anderson Douglas B. Weibel 《Molecular microbiology》2014,92(5):985-1004
Subcellular biomolecular localization is critical for the metabolic and structural properties of the cell. The functional implications of the spatiotemporal distribution of protein complexes during the bacterial cell cycle have long been acknowledged; however, the molecular mechanisms for generating and maintaining their dynamic localization in bacteria are not completely understood. Here we demonstrate that the trans‐envelope Tol–Pal complex, a widely conserved component of the cell envelope of Gram‐negative bacteria, is required to maintain the polar positioning of chemoreceptor clusters in Escherichia coli. Localization of the chemoreceptors was independent of phospholipid composition of the membrane and the curvature of the cell wall. Instead, our data indicate that chemoreceptors interact with components of the Tol–Pal complex and that this interaction is required to polarly localize chemoreceptor clusters. We found that disruption of the Tol–Pal complex perturbs the polar localization of chemoreceptors, alters cell motility, and affects chemotaxis. We propose that the E. coli Tol–Pal complex restricts mobility of the chemoreceptor clusters at the cell poles and may be involved in regulatory mechanisms that co‐ordinate cell division and segregation of the chemosensory machinery. 相似文献
15.
Fundamental structural units of the Escherichia coli nucleoid revealed by atomic force microscopy 总被引:5,自引:0,他引:5
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Kim J Yoshimura SH Hizume K Ohniwa RL Ishihama A Takeyasu K 《Nucleic acids research》2004,32(6):1982-1992
A small container of several to a few hundred µm3 (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of ~80 nm was found. In addition to this ‘80 nm fiber’, a thinner ‘40 nm fiber’ and a higher order ‘loop’ structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a ‘coral reef structure’ that also possessed the 80 nm fiber units, and, finally, into a ‘tightly compacted nucleoid’ that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid. 相似文献
16.
A method is described for gently dissociating large DNA-protein complexes and for visualizing and quantitating the substructures by autoradiography. Using this technique, it is shown that nucleoids isolated from exponentially growing Escherichia coli (mean generation time = 35 min) contain on average 2.8 genome equivalents of DNA and that this nucleoid can be dissociated by deproteinization into two substructures having on average 1.4 genome equivalents. This result is correlated with previous sedimentation studies on the unfolded nucleoid DNA to explain prior inconsistencies. Scanning electron microscopy studies demonstrate that the shape and size of the isolated nucleoid is consistent with the proposed subunit structure of the in vivo nucleoid. 相似文献
17.
Alex Boehm Markus Arnoldini Tobias Bergmiller Thomas R??sli Colette Bigosch Martin Ackermann 《PLoS genetics》2016,12(4)
In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported. 相似文献
18.
Patrick D. Scheu Philipp A. Steinmetz Felix Dempwolff Peter L. Graumann Gottfried Unden 《PloS one》2014,9(12)
The C4-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of E. coli is membrane-bound and reveals a polar localization. DcuS uses the C4-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when expressed without DcuS, but co-localize with DcuS when co-expressed at appropriate levels. Thus, DcuS is required for location of DctA and DcuR at the poles and formation of tripartite DctA/DcuS/DcuR sensor/regulator complexes. Vice versa, DctA, DcuR and the alternative succinate transporter DauA were not essential for polar localization of DcuS, suggesting that the polar trapping occurs by DcuS. Cardiolipin, the high curvature at the cell poles, and the cytoskeletal protein MreB were not required for polar localization. In contrast, polar localization of DcuS required the presence of the cytoplasmic PASC and the kinase domains of DcuS. 相似文献
19.
20.
Morphological and Physiological Changes Induced by High Hydrostatic Pressure in Exponential- and Stationary-Phase Cells of Escherichia coli: Relationship with Cell Death 总被引:1,自引:0,他引:1
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The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells. 相似文献