Summary
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.
Oufti is an interactive, open source software package built for high throughput quantification of microscopy images. Oufti performs cell detection, localization of diffraction‐limited spots as well as boundary determination of image objects beyond the diffraction limit. Due to parallel processing, it is able to perform these tasks on large (>1Gb) datasets, which can be further expanded by high performance computing centers. Oufti also provides post‐processing tools for the statistical characterization of data following image analysis.
Summary
Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and ...external cues. Quantitative, unbiased study of these spatio‐temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high‐throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini‐colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation in Caulobacter crescentus. By tracking filamentous cells, we show that the chromosomal origin at the old‐pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position‐dependent silencing is regulated by division.
Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. ...Here, we describe an image‐based quantitative screen of the single‐gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high‐dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.
Synopsis
A quantitative genomewide screen in Escherichia coli identifies genes and pathways associated with growth, cell morphology and the cell cycle, and uncovers a connection between cell morphogenesis and late cell cycle steps through the size of the nucleoid.
Measurements of growth, cell morphology and late cell cycle features are provided for each gene deletion strain of the E. coli Keio collection.
Multi‐parametric analysis identifies new phenotypic classes of mutants as well as hundreds of novel genes associated with cell morphogenesis and/or the cell cycle.
Correlation analysis across 4,000 genetic perturbations shows that growth rate is not predictive of cell size or of other scored morphological and cell cycle‐related phenotypes.
Information‐theoretic Bayesian network analysis suggests that nucleoid size connects cell size to the relative timings of nucleoid separation and cell constriction.
A quantitative genomewide screen in Escherichia coli identifies genes and pathways associated with growth, cell morphology and the cell cycle, and uncovers a connection between cell morphogenesis and late cell cycle steps through the size of the nucleoid.
What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall ...translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back‐and‐forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole‐organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self‐organizing oscillator to create motion suitable for chromosome segregation.
An analysis of the functions of polarity proteins and ParABS partitioning components in Caulobacter crescentus shows how a bacterial system for segregating multi‐copy plasmids can be adapted to faithful unidirectional chromosome movement, and at the same time provide coupling to the cell division process.
Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex ...genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
The tubulin homologue FtsZ is well known for its essential function in bacterial cell division. Here, we show that in Caulobacter crescentus, FtsZ also plays a major role in cell elongation ...by spatially regulating the location of MurG, which produces the essential lipid II peptidoglycan cell wall precursor. The early assembly of FtsZ into a highly mobile ring‐like structure during cell elongation is quickly followed by the recruitment of MurG and a major redirection of peptidoglycan precursor synthesis to the midcell region. These FtsZ‐dependent events occur well before cell constriction and contribute to cell elongation. In the absence of FtsZ, MurG fails to accumulate near midcell and cell elongation proceeds unperturbed in appearance by insertion of peptidoglycan material along the entire sidewalls. Evidence suggests that bacteria use both a FtsZ‐independent and a FtsZ‐dependent mode of peptidoglycan synthesis to elongate, the importance of each mode depending on the timing of FtsZ assembly during elongation.
Spatial organization is a hallmark of all living systems. Even bacteria, the smallest forms of cellular life, display defined shapes and complex internal organization, showcasing a highly structured ...genome, cytoskeletal filaments, localized scaffolding structures, dynamic spatial patterns, active transport, and occasionally, intracellular organelles. Spatial order is required for faithful and efficient cellular replication and offers a powerful means for the development of unique biological properties. Here, we discuss organizational features of bacterial cells and highlight how bacteria have evolved diverse spatial mechanisms to overcome challenges cells face as self-replicating entities.
In contrast to the historical view that bacteria are “simple” life forms, many studies have begun to uncover the exquisite subcellular organization that underlies many critical processes for bacterial life. This Review explores the principles of spatial organization that contribute to bacterial cell growth, division, gene expression, inheritance, motility, metabolism, and beyond.
The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament‐like protein, is required for the curved shape of Caulobacter crescentus and localizes to the ...inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.
It is now well appreciated that bacterial cells are highly organized, which is far from the initial concept that they are merely bags of randomly distributed macromolecules and chemicals. Central to ...their spatial organization is the precise positioning of certain proteins in subcellular domains of the cell. In particular, the cell poles - the ends of rod-shaped cells - constitute important platforms for cellular regulation that underlie processes as essential as cell cycle progression, cellular differentiation, virulence, chemotaxis and growth of appendages. Thus, understanding how the polar localization of specific proteins is achieved and regulated is a crucial question in bacterial cell biology. Often, polarly localized proteins are recruited to the poles through their interaction with other proteins or protein complexes that were already located there, in a so-called diffusion-and-capture mechanism. Bacteria are also starting to reveal their secrets on how the initial pole 'recognition' can occur and how this event can be regulated to generate dynamic, reproducible patterns in time (for example, during the cell cycle) and space (for example, at a specific cell pole). Here, we review the major mechanisms that have been described in the literature, with an emphasis on the self-organizing principles. We also present regulation strategies adopted by bacterial cells to obtain complex spatiotemporal patterns of protein localization.
During division of Gram-negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell ...separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast-growth conditions. Under slow-growth conditions, the ΔdipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non-canonical. DipM also carries two tandems of the PG-binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non-septal, monolayered PG.
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