scholarly journals Real-Time Messenger RNA Dynamics in Bacillus subtilis

2021 ◽  
Vol 12 ◽  
Author(s):  
Laura Sattler ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Real Time ◽  
Single Molecule ◽  
Messenger Rna ◽  
Time Lapse ◽  
Single Particle Tracking ◽  
Polar Regions ◽  
Constrained Motion ◽  
Diffusion Constants

Messenger RNA molecules have been localized to different positions in cells and have been followed by time-lapse microscopy. We have used MS2-mVenus–labeled mRNA and single-particle tracking to obtain information on the dynamics of single-mRNA molecules in real time. Using single-molecule tracking, we show that several mRNA molecules visualized via two MS2-binding sites and MS2-mVenus expressed in Bacillus subtilis cells show free diffusion through the entire cell and constrained motion predominantly close to the cell membrane and at the polar regions of the cells. Because constrained motion of mRNAs likely reflects molecules complexed with ribosomes, our data support the idea that translation occurs at sites surrounding the nucleoids. Squared displacement analyses show the existence of at least two distinct populations of molecules with different diffusion constants or possibly of three populations, for example, freely mobile mRNAs, mRNAs in transition complexes, or in complex with polysomes. Diffusion constants between differently sized mRNAs did not differ dramatically and were much lower than that of cytosolic proteins. These data agree with the large size of mRNA molecules and suggest that, within the viscous cytoplasm, size variations do not translate into mobility differences. However, at observed diffusion constants, mRNA molecules would be able to reach all positions within cells in a frame of seconds. We did not observe strong differences in the location of confined motion for mRNAs encoding mostly soluble or membrane proteins, indicating that there is no strong bias for localization of membrane protein-encoding transcripts for the cell membrane.

scholarly journals Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2826 ◽  
Author(s):  
Shangguo Hou ◽  
Courtney Johnson ◽  
Kevin Welsher
Keyword(s):  
Fluorescence Spectroscopy ◽  
Real Time ◽  
Temporal Resolution ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Active Feedback

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.

scholarly journals Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

2020 ◽  
Author(s):  
Marie Burghard-Schrod ◽  
Alexandra Kilb ◽  
Kai Krämer ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Metal Binding ◽  
Outer Cell ◽  
Dna Uptake ◽  
Cell Pole ◽  
Catalytic Function ◽  
C Terminus ◽  
Capture Mechanism

AbstractIn competent gram-negative and gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility within the periplasm as ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol.ImportanceBacteria can take up DNA from the environment and incorporate it into their chromosome in case similarity to the genome exists. This process of “natural competence” can result in the uptake of novel genetic information leading to horizontal gene transfer. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells with similar dynamics as DNA receptor ComEA, and thus takes a detour to get stored before uptake across the cell membrane into the cytosol by DNA permease ComEC. The latter assembles at a single cell pole, likely by a diffusion-capture mechanism, and requires its large C-terminus, including a conserved residue thought to confer nuclease function, for proper localization, function and mobility within the membrane.

scholarly journals Complete Genome Sequence and Methylome Analysis ofBacillus globigiiATCC 49760

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Richard D. Morgan
Keyword(s):  
Bacillus Subtilis ◽  
Real Time ◽  
Genome Sequence ◽  
Single Molecule ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Restriction Enzymes ◽  
Complete Sequence ◽  
Smrt Sequencing ◽  
Methylome Analysis

Bacillus subtilis(Ehrenburg) Cohn ATCC 49760, deposited asBacillus globigii, is the source strain for the restriction enzymes BglI and BglII. Its complete sequence and full methylome were determined using single-molecule real-time (SMRT) sequencing.

scholarly journals Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Sandra Kunz ◽  
Anke Tribensky ◽  
Wieland Steinchen ◽  
Luis Oviedo-Bocanegra ◽  
Patricia Bedrunka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Live Cells ◽  
Independent Manner ◽  
Single Molecule Tracking ◽  
Content Type ◽  
Diguanylate Cyclases

ABSTRACT Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro, which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro, showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient. IMPORTANCE Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis. Thus, local-pool signal transduction operates extremely efficiently and specifically.

scholarly journals Spot-On: robust model-based analysis of single-particle tracking experiments

2017 ◽  
Author(s):  
Anders S Hansen ◽  
Maxime Woringer ◽  
Jonathan B Grimm ◽  
Luke D Lavis ◽  
Robert Tjian ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Motion Blur ◽  
Single Particle Tracking ◽  
Modeling Framework ◽  
Diffusion Constants ◽  
Diffusion Dynamics ◽  
Wide Range ◽  
And Diffusion

ABSTRACTSingle-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce “Spot-On”, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.IMPACT STATEMENTSpot-On is an easy-to-use website that makes a rigorous and bias-corrected modeling framework for analysis of single-molecule tracking experiments available to all.

scholarly journals Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking

2015 ◽  
Vol 209 (4) ◽  
pp. 609-619 ◽  
Author(s):  
Carlas S. Smith ◽  
Stephan Preibisch ◽  
Aviva Joseph ◽  
Sara Abrahamsson ◽  
Bernd Rieger ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Messenger Rna ◽  
Three Dimensional ◽  
Spatial Inhomogeneity ◽  
Live Cells ◽  
Nuclear Pore ◽  
Cell Nuclei ◽  
Entire Volume ◽  
Time Required

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

scholarly journals Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

2021 ◽  
Author(s):  
Marie Burghard-Schrod ◽  
Alexandra Kilb ◽  
Kai Krämer ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Metal Binding ◽  
Outer Cell ◽  
Dna Uptake ◽  
Dna Dynamics ◽  
Cell Pole ◽  
C Terminus ◽  
Capture Mechanism

In competent Gram-negative and Gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis , the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol. Importance Bacteria can take up DNA from the environment and incorporate it into their chromosome, termed “natural competence” that can result in the uptake of novel genetic information. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of the latter assembles at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm, and involves a high degree of free diffusion along and within the cell membrane.

Imaging the behavior of molecules in biological systems: breaking the 3D speed barrier with 3D multi-resolution microscopy

2015 ◽  
Vol 184 ◽  
pp. 359-379 ◽  
Author(s):  
Kevin Welsher ◽  
Haw Yang
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Pid Control ◽  
Single Particle Tracking ◽  
Three Dimensions ◽  
Temporal Precision ◽  
Optical Sensitivity ◽  
Position Sensitive

The overwhelming effort in the development of new microscopy methods has been focused on increasing the spatial and temporal resolution in all three dimensions to enable the measurement of the molecular scale phenomena at the heart of biological processes. However, there exists a significant speed barrier to existing 3D imaging methods, which is associated with the overhead required to image large volumes. This overhead can be overcome to provide nearly unlimited temporal precision by simply focusing on a single molecule or particle via real-time 3D single-particle tracking and the newly developed 3D Multi-resolution Microscopy (3D-MM). Here, we investigate the optical and mechanical limits of real-time 3D single-particle tracking in the context of other methods. In particular, we investigate the use of an optical cantilever for position sensitive detection, finding that this method yields system magnifications of over 3000×. We also investigate the ideal PID control parameters and their effect on the power spectrum of simulated trajectories. Taken together, these data suggest that the speed limit in real-time 3D single particle-tracking is a result of slow piezoelectric stage response as opposed to optical sensitivity or PID control.

scholarly journals Real-time assembly of an artificial virus elucidated at the single-particle level

2019 ◽  
Author(s):  
Margherita Marchetti ◽  
Douwe Kamsma ◽  
Ernesto Cazares Vargas ◽  
Armando Hernandez García ◽  
Paul van der Schoot ◽  
...  
Keyword(s):  
Real Time ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Single Particle ◽  
Self Assembly ◽  
De Novo ◽  
Particle Growth ◽  
Single Particle Tracking ◽  
Contour Length ◽  
Assembly Pathways

AbstractWhile the structure of a variety of viruses has been resolved at atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues in assembly are the nature of the critical nucleus starting particle growth, the subsequent self-assembly reaction and the manner in which the viral genome is compacted. These issues are difficult to address in bulk approaches and are effectively only accessible by tracking the dynamics of assembly of individual particles in real time, as we show here. With a combination of single-molecule techniques we study the assembly into rod-shaped virus-like particles (VLPs) of artificial capsid polypeptides, de-novo designed previously. Using fluorescence optical tweezers we establish that oligomers that have pre-assembled in solution bind to our DNA template. If the oligomer is smaller than a pentamer, it performs one-dimensional diffusion along the DNA, but pentamers and larger oligomers are essentially immobile and nucleate VLP growth. Next, using real-time multiplexed acoustic force spectroscopy, we show that DNA is compacted in regular steps during VLP growth. These steps, of ∼30 nm of DNA contour length, fit with a DNA packaging mechanism based on helical wrapping of the DNA around the central protein core of the VLP. By revealing how real-time, single particle tracking of VLP assembly lays bare nucleation and growth principles, our work opens the doors to a new fundamental understanding of the complex assembly pathways of natural virus particles.

scholarly journals A bacterial dynamin-like protein confers a novel phage resistance strategy on the population level in Bacillus subtilis

2021 ◽  
Author(s):  
Lijun Guo ◽  
Laura Sattler ◽  
Peter Graumann ◽  
Marc Bramkamp
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Host Cell ◽  
Single Molecule ◽  
Membrane Integrity ◽  
Resistance Mechanism ◽  
Cell Lysis ◽  
Phage Infection ◽  
Host Cells ◽  
Protective Effects

Bacillus subtilis DynA is a member of the dynamin superfamily, involved in membrane remodeling processes. DynA was shown to catalyze full membrane fusion and it plays a role in membrane surveillance against antibiotics. We show here that DynA also provides a novel resistance mechanism against phage infection. Cells lacking DynA are efficiently lysed after phage infection and virus replication. DynA does not prevent phage infection and replication in individual cells, but significantly delays host cell lysis, thereby slowing down the release of phage progeny from the host cells. During the process, DynA forms large, almost immobile clusters on the cell membrane that seem to support membrane integrity. Single molecule tracking revealed a shift of freely diffusive molecules within the cytosol towards extended, confined motion at the cell membrane following phage induction. Thus, the bacterial dynamins are the first anti-phage system reported to delay host cell lysis and the last line of defense of a multilayered antiviral defense. DynA is therefore providing protective effects on the population, but not on single cell level.

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