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Decades of research have witnessed that the mechanical properties of DNA and the associated protein interactions are crucial to the structural organizations and dynamic behaviors of bacterial DNA molecules. Packaged in a restricted cellular space and respective compositions, the entropic force due to confinement is a major factor driving the basal structural formation of the bacterial chromosome and the distribution of plasmids. The dynamic responses of the DNA-protein interactions also modulate the DNA organization when the cellular functions proceed. In this review, we survey the in-vivo and in-vitro evidence and the current understandings of the cellular phenomena of DNA molecules in the sub-micron scale of the bacterial cellular environment. The physical nature and constraints of DNA in cells and the related biological factors, especially the effects induced by gene expressions, are also discussed. We anticipate that by the methodological advances in vivo and in vitro as well as their combinations, the fundamental insights of the coupling from the physical environment and DNA-protein interactions on bacterial DNA dynamics and organization may be revealed.

In E. coli, the Min-protein oscillator, together with the nucleoid occlusion (NO), destabilizes the Z-ring assembly away from the midcell to ensure faithful septation. These two inhibitory pathways are thought to be working independently for division site placement. Even though the Min-protein oscillator has been displayed by synthetic minimal systems, it is unclear the interplays of Min proteins and compartment geometry are sufficient to bolster oscillation stability in vivo. By probing if NO plays a role in the Min oscillation, we study the oscillation frequency in the anucleate and nucleoid-perturbed cells. Surprisingly, we found that the oscillation periods of the Min-protein oscillators were seriously deviated in the anucleate and nucleoid-perturbed cells, but the oscillation frequency either went up in the anucleate or down in the nucleoid-perturbed cells. Intriguingly, enhanced stability and reduced frequency were observed in the cells expressing the NO factor SlmA higher than the native level. Our results reveal an unanticipated role of the nucleoid in modulating the frequency and stability of Min-protein system. SlmA is indicated to facilitate such modulations, potentially via directly interacting with the Min-protein system. A fresh perspective is suggested that frequency modulation of Min-protein oscillator is mediated via the act of nucleoid-associated factors.

Transcription and translation of active genes play an important role in determining the global organization of the chromosome. To further elucidate this phenomenon, we examined how the expression of either the lacY or the cfp gene in the native lac operon influences adjacent chromosomal segments by fluorescently labeling loci upstream and downstream of the expressed gene. Based on the positions and motile behaviors of these loci, our results reveal that the local organization of the vicinal chromosomal segments and its position in the nucleoid are both influenced by gene expression. Furthermore, we found that the effects on local organization depend on whether the expressed gene encodes a membrane protein or a cytoplasmic protein. Our measurements showing the movement of loci toward the membrane and the correlation between the motions of the upstream and downstream loci support the conclusion that the expression of genes encoding membrane proteins greatly influences chromosome dynamics.

Bacterial high-copy-number (hcn) plasmids provide an excellent model to study the underlying physical mechanisms of DNA segment segregation in an intracellular context. Using two-color fluorescent repressor-operator systems and a synthetic repressible replication origin, we tracked the motion and segregation of single hcn plasmid molecules in individual cells. The plasmid diffusion dynamics revealed between-plasmid temporal associations (clustering) as well as entropic and elastic recoiling forces in the confined intracellular spaces outside of nucleoids. These two effects could be effectively used in models to predict the heterogeneity of segregation. Additionally, the motile behaviors of hcn plasmids provide quantitative estimates of entropic exclusion strength and dynamic associations between DNA segments. Overall, this study utilizes a, to our knowledge, novel approach to predict the polymer dynamics of DNA segments in spatially confined, crowded cellular compartments as well as during bacterial chromosome segregation.

A technique based on polarization modulation spectroscopy (PMS) has been developed to determine quantitatively the number of fluorophores in nanoparticles at the single-molecule level. The technique involves rotation of the polarization of the excitation laser on a millisecond time scale, leading to fluorescence intensity modulation. By taking account of the heterogeneous orientation among the dipoles of the fluorophores and simulating the modulation depth distribution with Monte Carlo calculations, we show that it is possible to deduce the ensemble average and number distribution of the fluorophores. We apply the technique to fluorescent nanodiamonds (FNDs) containing multiple nitrogen vacancy (NV) centers. Comparing the experimental and simulated modulation depth distributions of 11 nm FNDs, we deduce an average number of = 3, which is in good agreement with independent photon correlation measurements. The method is general, rapid, and applicable to other nanoparticles, polymers, and molecular complexes containing multiple and randomly orientated fluorophores as well.

Summary Single-molecule spectroscopy and detection are powerful techniques for the study of single fluorescent particles and their interaction with their environment. We present a low-cost system for simultaneous real-time acquisition, storage of inter-photon arrival times and the calculation and display of the fluorescence time trace, autocorrelation function and distribution of delays histogram for single-molecule experiments. From a hardware perspective, in addition to a multi-core computer, only a standard low-cost counting board is required as processing is software-based. Software is written in a parallel programming environment with time crucial operations coded in ANSI-C. Crucial to system performance is a simple and efficient real-time autocorrelation algorithm (acf) optimized for the count rates (approximately 104 cps) encountered in single-molecule experiments. The algorithm's time complexity is independent of temporal resolution, which is maintained at all time delays. The system and algorithm's performance was validated by duplicating the signal from the photon detector and sending it to both the ordinary counter board and a commercial correlator simultaneously. The data acquisition system's robustness under typical single-molecule experimental conditions was tested by observing the diffusion of Rhodamine 6G molecules in deionized water.

Optical tweezers have become a popular manipulation and force measurement tool in cellular and molecular biology. However, there is still a lack of a sophisticated model for optical tweezers on trapping cells. In this paper, we present a novel model for optical tweezers to calculate the stiffness of trapping force upon a spherically symmetric Rayleigh sphere, which stimulates a common biological cell. A numerical simulation of this model shows that the stiffness of an optical tweezers system in trapping a cell is significantly smaller than that in trapping a polystyrene bead of the same size. Furthermore, under a small variant condition of the refractive index, the proposed model provides an approximate method which requires only the radial distribution of the trapped cell’s refractive index for calculating the stiffness.

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein-DNA interaction sites.

Bacterial plasmids play important role in gene transport between cells both vertically and horizontally, and are also versatile tools for gene engineering. Their maintenance of inherence in the host cells is believed to depend on the delicate quantity control through the replication origin and the effective management of partition. Without active partition system, high-copy number plasmids are suggested to be distributed as multiple foci but randomly partitioned during cell division. Lack of the direct evidence, the detail mechanism and dynamics of plasmid location and partition in cell is unknown. Taking the advantage of temporary tolerance on the incompatibility of replication origin, two ColE1 derivative plasmids with different antibiotic resists and repressible replication origin are co-transformed and their distributions and motions are observed at single cell level simultaneously. Based on the nature of competition between plasmids, it also provides the opportunity to actually reveal a single plasmid translocation between multiple foci, which is affected by the nucleoids of the dividing cells. Also, the variance of the inhered number of copies for both of two plasmids from parent to daughter cells provides the insight of partition mechanism.

We combine pulsed laser, supercontinuum radiation source and fast single-photon counting peripherals to obtain a multifunctional micro/nano-scope. This provides us with better spatial and temporal resolution to observe fast dynamics. Performing fluorescence correlation spectroscopy for fast dynamics (

This paper focuses on developing the platform technology ofreal-time biomolecular-interaction analysis (BIA) sensor chips.A detection scheme using the electro-optically modulated surface plasmon resonance (SPR) is suggested to advance the sensorfeatures in reducing measurement complexity and time. The SPR method of a BIA sensing system detects slight changes ofrefractive index due to the biomolecular interaction at the solid-liquid interface. The most sensitive interrogation methodamong the possible conventional schemes is to measure the SPR angle ofthe attenuated total reflection. The electro-opticalmodulation replaces the mechanism of angle measurement not only to increase the speed but also to reduce the size. Recentprogress of the multilayer SPR provides an effective mean of tailoring the microchip. Several multilayer configurations havebeen studied in this paper to realize the electro-optical SPR sensing. Especially, the long-range mode of surface plasmon wasinvestigated to achieve the high-resolution and high-sensitivity detection.Keywords: Biosensor, biomolecular-interaction analysis sensor, optical sensor, surface plasmon resonance, electro-opticalmodulation, multilayer.

In optical tweezers applications, tracking a trapped particle is essential for force measurement. One of the most popular techniques for single-particle tracking is achieved by analyzing the forward and backward light pattern, scattered by the target particle trapped by a trap laser beam, of an additional probe-laser beam with different wavelength whose focus is slightly apart from the trapping center. However, the optimized focal offset has never been discussed. In this paper, we investigate the tracking range and sensitivity as a function of the focal offset between the trapping and the probe-laser beams. As a result, the optimized focal offsets are a 3.3-fold radius ahead and a 2.0-fold radius behind the trapping laser focus in the forward tracking and the backward tracking, respectively. The experimental result agrees well with a theoretical prediction using the Mie scattering theory.

Faithful cell division of bacterium E. coli relies on remarkable oscillations of the MinCDE system to perform accurate and symmetric septation. Perturbing this bio-oscillator by disrupting minB locus or altering MinDE expressions is known to cause mini-celling and otherwise filamentous phenotypes. Recent reports suggested MinE plays a pivotal role in spatiotemporal pattern formation of MinDE cohorts; yet it remains elusive if their pattern transitions led to changes in cell morphology, and vice versa.In the present study, the coupling dynamics of MinDE interplays is investigated by time-lapse microscopy. We used multicolor fluorescence imaging to detail the mode transitions in distinct MinDE pattern formations, and photoactivated localization microscopy (PALM) to probe the nanoscale fine structures of the MinDE filaments/patterns in living cells. Despite E-ring capping has been viewed as a drive of pole-to-pole MinD oscillations, we found peculiar expression signatures in MinD and MinE spatiotemporal distributions, corresponding to MinDE patterns other than the E-ring type, correlate to their relative expression levels. Alternatively, bacteria were cultured and confined in micro/nanofluidic devices, to mimic various curvature changes of cell peripherals. Interestingly, under strong nanoslit confinement of 400 nm depth, bacteria are able to proliferate, but show irregular pancake-like morphology and intermittent oscillation episodes were observed in MinDE dynamics. The transitions between intermittent episodes display period-doubling signature of bifurcation by analyzing image series via spatial time-frequency method.Our results indicate MinDE pattern fluctuations / transitions correspond to aberrancy in septation and morphology. The study synergizes the join merits of in vivo imaging, single cell analysis, and nanofluidics to grasp the insight of noisedriven pattern transitions and phenotypic changes in bacteria.

The number of negatively charged nitrogen-vacancy centers (N-V)− in fluorescent nanodiamond (FND) has been determined by photon correlation spectroscopy and Monte Carlo simulations at the single particle level. By taking account of the random dipole orientation of the multiple (N-V)− fluorophores and simulating the probability distribution of their effective numbers (Ne), we found that the actual number (Na) of the fluorophores is in linear correlation with Ne, with correction factors of 1.8 and 1.2 in measurements using linearly and circularly polarized lights, respectively. We determined Na=8±1 for 28 nm FND particles prepared by 3 MeV proton irradiation.

Since their invention in 1986, optical tweezers have become a popular manipulation and force measurement tool in cellular and molecular biology. However, until recently there has not been a sophisticated model for optical tweezers on trapping cells in the ray-optics regime. We present a model for optical tweezers to calculate the optical force upon a spherically symmetric multilayer sphere representing a common biological cell. A numerical simulation of this model shows that not only is the magnitude of the optical force upon a Chinese hamster ovary cell significantly three times smaller than that upon a polystyrene bead of the same size, but the distribution of the optical force upon a cell is also much different from that upon a uniform particle, and there is a 30% difference in the optical trapping stiffness of these two cases. Furthermore, under a small variant condition for the refractive indices of any adjacent layers of the sphere, this model provides a simple approximation to calculate the optical force and the stiffness of an optical tweezers system.

Using AFM and magnetic tweezers, we performed single molecule rupture force measurements on the binding of Topoisomerase (TOPO) II to double-stranded DNA. With a few hundred pulling events, using DNA and disulfide bond cross-linked mutant TOPOII as controls, we found the rupture force between wild type TOPOII-DNA complex is ∼45 pN at a 50% accumulative probability.