Osmolality-dependent relocation of penicillin-binding protein PBP2 to the division site in Caulobacter crescentus
15 Jun 2012-Journal of Bacteriology (American Society for Microbiology)-Vol. 194, Iss: 12, pp 3116-3127
TL;DR: Show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts, revealing an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.
Abstract: The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.
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TL;DR: Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision.
Abstract: With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills.
323 citations
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...Beyond tools for displaying cells/signals and for plotting their statistics, more advanced plots such as demographs (Hocking et al., 2012) and kymographs can be easily produced (see the Oufti website)....
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TL;DR: Bacterial cell division is facilitated by the divisome, a dynamic multiprotein assembly localizing at mid‐cell to synthesize the stress‐bearing peptidoglycan and to constrict all cell envelope layers.
Abstract: Bacterial cell division is facilitated by the divisome, a dynamic multiprotein assembly localizing at mid-cell to synthesize the stress-bearing peptidoglycan and to constrict all cell envelope layers. Divisome assembly occurs in two steps and involves multiple interactions between more than 20 essential and accessory cell division proteins. Well before constriction and while the cell is still elongating, the tubulin-like FtsZ and early cell division proteins form a ring-like structure at mid-cell. Cell division starts once certain peptidoglycan enzymes and their activators have moved to the FtsZ-ring. Gram-negative bacteria like Escherichia coli simultaneously synthesize and cleave the septum peptidoglycan during division leading to a constriction. The outer membrane constricts together with the peptidoglycan layer with the help of the transenvelope spanning Tol-Pal system.
302 citations
TL;DR: Coordination of the P BP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.
Abstract: To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.
156 citations
Cites methods from "Osmolality-dependent relocation of ..."
...For each examined protein, fluorescence profiles of 3000–5000 individual cells were sorted according to cell length to generate profile maps (Figure 4B), similar to demographs (Hocking et al., 2012)....
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TL;DR: New data is provided showing that the bifunctional P BP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions.
Abstract: Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.
153 citations
Cites background from "Osmolality-dependent relocation of ..."
...crescentus PBP2 and PBP1A—osmolarity of growth medium [129]...
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...crescentus, even small osmotic upshifts cause PBP1A and PBP2 to relocate, moving from a patchy side wall location to the position of FtsZ at mid-cell [129]....
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...coli was largely unaffected by an osmotic up-shift [129]....
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TL;DR: This review describes aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope and discusses the potential of these techniques for translational applications.
Abstract: The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology.
116 citations
Cites background from "Osmolality-dependent relocation of ..."
...For example, in the oligotrophic environmental bacterium C. crescentus, the localization of several peptidoglycan-acting proteins varies according to osmolality (Hocking et al., 2012)....
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References
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7,278 citations
TL;DR: This review is an account of the processes that mediate adaptation of bacteria to changes in their osmotic environment.
1,581 citations
"Osmolality-dependent relocation of ..." refers background in this paper
...Cells respond to an increase in external osmolality by transporting and synthesizing compatible solutes to increase their internal osmotic strength and return water to the cytoplasm (8, 38)....
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TL;DR: A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.
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1,149 citations
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Journal Article•
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TL;DR: This Review discusses how growth of the sacculus is sensitive to mechanical force and nutritional status, and describes the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.
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