Asymmetric cell division in bacteria is achieved through cell polarization, where regulatory proteins are directed to specific cell poles. In Caulobacter crescentus, both poles contain a membraneless microdomain, established by the polar assembly hub PopZ, through most of the cell cycle, yet many PopZ clients are unipolar and transiently localized. We find that PopZ's interaction with the response regulator CpdR is controlled by phosphorylation, via the histidine kinase CckA.
View Article and Find Full Text PDFAsymmetric cell division in bacteria is achieved through cell polarization, where regulatory proteins are directed to specific cell poles. Curiously, both poles contain a membraneless microdomain, established by the polar assembly hub PopZ, through most of the cell cycle, yet many PopZ clients are unipolar and transiently localized. We find that PopZ's interaction with the response regulator CpdR is controlled by phosphorylation, via the histidine kinase CckA.
View Article and Find Full Text PDFVirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer.
View Article and Find Full Text PDFIndustrial bioreactors use microbial organisms as living factories to produce a wide range of commercial products. For most applications, yields eventually become limited by the proliferation of "escape mutants" that acquire a growth advantage by losing the ability to make product. The goal of this work is to use mathematical models to determine whether this problem could be addressed in continuous flow bioreactors that include a "stem cell" population that multiplies rapidly and could be used to compete against the emergence of cheater mutants.
View Article and Find Full Text PDFThe polar organizing protein Z (PopZ) is necessary for the formation of three-dimensional microdomains at the cell poles in Caulobacter crescentus, where it functions as a hub protein that recruits multiple regulatory proteins from the cytoplasm. Although a large portion of the protein is predicted to be natively unstructured, in reconstituted systems PopZ can self-assemble into a macromolecular scaffold that directly binds to at least ten different proteins. Here we report the solution NMR structure of PopZ, a truncated form of PopZ that does not self-assemble but retains the ability to interact with heterologous proteins.
View Article and Find Full Text PDFMulticellular organisms achieve greater complexity through cell divisions that generate different cell types. We engineered a simple genetic circuit that induces asymmetric cell division and subsequent cell differentiation in Escherichia coli. The circuit involves a scaffolding protein, PopZ, that is stably maintained at a single cell pole over multiple asymmetric cell divisions.
View Article and Find Full Text PDFIn most rod-shaped bacteria, DNA replication is quickly followed by chromosome segregation, when one of the newly duplicated centromeres moves across the cell to the opposite (or 'new') pole. Two proteins in Caulobacter crescentus, PopZ and TipN, provide directional cues at the new pole that guide the translocating chromosome to its destination. We show that centromere translocation can be inhibited by an evolutionarily conserved pole-localized protein that we have named SpbR.
View Article and Find Full Text PDFIn the endocytic pathway of animals, two related complexes, called CORVET (class C core vacuole/endosome transport) and HOPS (homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease, including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes, but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent, while CORVET-specific subunits have proliferated.
View Article and Find Full Text PDFDespite being perceived as relatively simple organisms, many bacteria exhibit an impressive degree of subcellular organization. In , the evolutionarily conserved polar organizing protein PopZ facilitates cytoplasmic organization by recruiting chromosome centromeres and regulatory proteins to the cell poles. Here, we characterize the localization and function of PopZ in , a genetically related species with distinct anatomy.
View Article and Find Full Text PDFis a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The homolog of the polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor.
View Article and Find Full Text PDFDespite their relative simplicity, bacteria have complex anatomy at the subcellular level. At the cell poles of Caulobacter crescentus, a 177-amino acid (aa) protein called PopZ self-assembles into 3D polymeric superstructures. Remarkably, we find that this assemblage interacts directly with at least eight different proteins, which are involved in cell cycle regulation and chromosome segregation.
View Article and Find Full Text PDFUnlabelled: The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance.
View Article and Find Full Text PDFBacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system.
View Article and Find Full Text PDFThe ubiquitous bacterium Caulobacter crescentus holds promise to be used in bioremediation applications due to its ability to mineralize U(VI) under aerobic conditions. Here, cell free extracts of C. crescentus grown in the presence of uranyl nitrate [U(VI)], potassium chromate [Cr(VI)], or cadmium sulfate [Cd(II)] were used for label-free proteomic analysis.
View Article and Find Full Text PDFIn Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub-cellular localization and recruitment activity. We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation.
View Article and Find Full Text PDFMany recent studies have revealed exquisite subcellular localization of proteins, DNA, and other molecules within bacterial cells, giving credence to the concept of prokaryotic anatomy. Common sites for localized components are the poles of rod-shaped cells, which are dynamically modified in composition and function in order to control cellular physiology. An impressively diverse array of mechanisms underlies bacterial polarity, including oscillatory systems, phospho-signaling pathways, the sensing of membrane curvature, and the integration of cell cycle regulators with polar maturation.
View Article and Find Full Text PDFSuperresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
March 2010
Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries.
View Article and Find Full Text PDFThe bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. The parS/ParB chromosomal centromere is tethered to PopZ at one pole prior to the initiation of DNA replication.
View Article and Find Full Text PDFBacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin.
View Article and Find Full Text PDFThe commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging.
View Article and Find Full Text PDFIn addition to a family of structurally related proteins encoded by the Granule lattice (GRL) genes, the dense core granules in Tetrahymena thermophila contain a second, more heterogeneous family of proteins that can be defined by the presence of a domain homologous to beta/gamma-crystallins. The founding members of the family, Induced during Granule Regeneration 1 (IGR1) and Granule Tip 1 (GRT1), were identified in previous screens for granule components. Analysis of the recently sequenced T.
View Article and Find Full Text PDFIn some cells, the polypeptides stored in dense core secretory granules condense as ordered arrays. In ciliates such as Tetrahymena thermophila, the resulting crystals function as projectiles, expanding upon exocytosis. Isolation of granule contents previously defined five Granule lattice (Grl) proteins as abundant core constituents, whereas a functional screen identified a sixth family member.
View Article and Find Full Text PDFThe formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible.
View Article and Find Full Text PDFRespiratory and arousal state control are heritable traits in mice. B6.V-Lep(ob) (ob) mice are leptin deficient and differ from C57BL/6J (B6) mice by a variation in the gene coding for leptin.
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