Kinetic principles of ParA2-ATP cycling guide dynamic subcellular localizations in Vibrio cholerae.

Dynamic protein gradients are exploited for the spatial organization and segregation of replicated chromosomes. However, mechanisms of protein gradient formation and how that spatially organizes chromosomes remain poorly understood. Here, we have determined the kinetic principles of subcellular localizations of ParA2 ATPase, an essential spatial regulator of chromosome 2 segregation in the multichromosome bacterium, Vibrio cholerae. We found that ParA2 gradients self-organize in V. cholerae cells into dynamic pole-to-pole oscillations. We examined the ParA2 ATPase cycle and ParA2 interactions with ParB2 and DNA. In vitro, ParA2-ATP dimers undergo a rate-limiting conformational switch, catalysed by DNA to achieve DNA-binding competence. This active ParA2 state loads onto DNA cooperatively as higher order oligomers. Our results indicate that the midcell localization of ParB2-parS2 complexes stimulate ATP hydrolysis and ParA2 release from the nucleoid, generating an asymmetric ParA2 gradient with maximal concentration toward the poles. This rapid dissociation coupled with slow nucleotide exchange and conformational switch provides for a temporal lag that allows the redistribution of ParA2 to the opposite pole for nucleoid reattachment. Based on our data, we propose a 'Tug-of-war' model that uses dynamic oscillations of ParA2 to spatially regulate symmetric segregation and positioning of bacterial chromosomes.

RNA Polymerase Pausing during Initial Transcription.

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content.

The alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important polyploid species has been largely uncharacterized. In the present study, we characterized basic cellular behavior of Z. mobilis strain Zm6 under anaerobic conditions at the single-cell level. We observed that growing Z. mobilis cells often divided at a nonmidcell position, which contributed to variant cell size at birth. However, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA content among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated with cell growth. Furthermore, slightly angled divisions might have resulted in temporary curvatures of attached Z. mobilis cells. Overall, the present study uncovers a novel bacterial cell organization in Z. mobilisIMPORTANCE With increasing environmental concerns about the use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker's yeast (Saccharomyces cerevisiae), the most-used microorganism for ethanol production. For development of a Z. mobilis culture-based biorefinery, an investigation of its uncharacterized cell biology is important, because bacterial cellular organization and metabolism are closely associated with each other in a single cell compartment. In addition, the current work demonstrates that the polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, likely reflecting its unique metabolism that does not prioritize incorporation of nutrients for cell growth. Thus, another significant result of this work is to advance our general understanding in the diversity of bacterial cell architecture.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD.

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified "burst" patterns--radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator.

ParA-mediated plasmid partition driven by protein pattern self-organization.

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.

Reconstitutions of plasmid partition systems and their mechanisms.

Bacterial plasmid and chromosome segregation systems ensure that genetic material is efficiently transmitted to progeny cells. Cell-based studies have shed light on the dynamic nature and the molecular basis of plasmid partition systems. In vitro reconstitutions, on the other hand, have proved to be an invaluable tool for studying the minimal components required to elucidate the mechanism of DNA segregation. This allows us to gain insight into the biological and biophysical processes that enable bacterial cells to move and position DNA. Here, we review the reconstitutions of plasmid partition systems in cell-free reactions, and discuss recent work that has begun to challenge long standing models of DNA segregation in bacteria.

Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism.

Increasingly diverse types of cargo are being found to be segregated and positioned by ParA-type ATPases. Several minimalistic systems described in bacteria are self-organizing and are known to affect the transport of plasmids, protein machineries, and chromosomal loci. One well-studied model is the F plasmid partition system, SopABC. In vivo, SopA ATPase forms dynamic patterns on the nucleoid in the presence of the ATPase stimulator, SopB, which binds to the sopC site on the plasmid, demarcating it as the cargo. To understand the relationship between nucleoid patterning and plasmid transport, we established a cell-free system to study plasmid partition reactions in a DNA-carpeted flowcell. We observed depletion zones of the partition ATPase on the DNA carpet surrounding partition complexes. The findings favor a diffusion-ratchet model for plasmid motion whereby partition complexes create an ATPase concentration gradient and then climb up this gradient toward higher concentrations of the ATPase. Here, we report on the dynamic properties of the Sop system on a DNA-carpet substrate, which further support the proposed diffusion-ratchet mechanism.

Sensing DNA opening in transcription using quenchable Förster resonance energy transfer.

Many biological processes, such as gene transcription and replication, involve opening and closing of short regions of double-stranded DNA (dsDNA). Few techniques, however, can study these processes in real time or at the single-molecule level. Here, we present a Förster resonance energy transfer (FRET) assay that monitors the state of DNA (double- vs single-stranded) at a specific region within a DNA fragment, at both the ensemble level and the single-molecule level. The assay utilizes two closely spaced fluorophores: a FRET donor fluorophore (Cy3B) on the first DNA strand and a FRET acceptor fluorophore (ATTO647N) on the complementary strand. Because our assay is based on quenching and dequenching FRET processes, i.e., the presence or absence of contact-induced fluorescence quenching, we have named it a "quenchable FRET" assay or "quFRET". Using lac promoter DNA fragments, quFRET allowed us to sense transcription bubble expansion and compaction during abortive initiation by bacterial RNA polymerase. We also used quFRET to confirm the mode of action of gp2 (a phage-encoded protein that acts as a potent inhibitor of Escherichia coli transcription) and rifampicin (an antibiotic that blocks transcription initiation). Our results demonstrate that quFRET should find numerous applications in many processes involving DNA opening and closing, as well as in the development of new antibacterial therapies involving transcription.

Single-molecule FRET analysis of protein-DNA complexes.

We present a single-molecule method for studying protein-DNA interactions based on fluorescence resonance energy transfer (FRET) and alternating-laser excitation (ALEX) of single diffusing molecules. An application of this method to the study of a bacterial transcription initiation complex is presented.

Recent advances in fluorescence cross-correlation spectroscopy.

Fluorescence cross-correlation spectroscopy (FCCS) is a method that measures the temporal fluorescence fluctuations coming from two differently labeled molecules diffusing through a small sample volume. Cross-correlation analysis of the fluorescence signals from separate detection channels extracts information of the dynamics of the dual-labeled molecules. FCCS has become an essential tool for the characterization of diffusion coefficients, binding constants, kinetic rates of binding, and determining molecular interactions in solutions and cells. By cross-correlating between two focal spots, flow properties could also be measured. Recent developments in FCCS have been targeted at using different experimental schemes to improve on the sensitivity and address their limitations such as cross-talk and alignment issues. This review presents an overview of the different excitation and detection methodologies used in FCCS and their biological applications. This is followed by a description of the fluorescent probes currently available for the different methods. This will introduce biological readers to FCCS and its related techniques and provide a starting point to selecting which experimental scheme is suitable for their type of biological study.

The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labeling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

Red light, green light: probing single molecules using alternating-laser excitation.

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein-nucleic acid interactions.

Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation.

Fluorescence cross-correlation spectroscopy is a powerful method for the study of molecular interactions and dynamics in solution and even in living cells. Usually, in the optical setup, either two laser beams have to be superimposed in their respective confocal volumes or two-photon excitation is used for a dual-color detection system. It has been shown recently that fluorescence cross correlation can be achieved with spectrally similar fluorophores using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). In this study, we show that SW-FCCS allows the simultaneous excitation of up to three fluorophores in which the cross correlation of their fluctuation signals is detected separately in three detection channels. The experimental and theoretical model to describe triple pairwise cross correlations incorporating cross talk and possible changes in emission characteristics such as quenching upon binding are outlined. The effectiveness of SW-FCCS to detect binding of three interacting partners is experimentally verified with a standard ligand-receptor model, biotin-streptavidin, where differently labeled biotin ligands and their binding to a third-color labeled streptavidin are studied. The cross-correlation amplitudes and their changes with stoichiometric binding are analyzed and the upper limits of dissociation constants are determined. Performed with appropriate negative controls, SW-FCCS can determine interaction patterns between ligands and receptors.

Prism-based multicolor fluorescence correlation spectrometer.

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: resolution for binding studies.

It was shown recently that fluorescence cross-correlation spectroscopy (FCCS) can be performed using a single laser wavelength for excitation (SW-FCCS) [L. C. Hwang and T. Wohland, Chem. Phys. Chem 5, 549 (2004).]. This method simplifies the FCCS setup since it does not require the simultaneous alignment of two lasers to the same focal spot. But up to now the method was shown to work only with dyes possessing large Stokes' shifts, and thus was limited to the use of quantum dots and tandem dyes. In this work we show that standard organic dyes with overlapping emission spectra, for instance fluorescein and tetramethylrhodamine, can be used as fluorescent pairs in SW-FCCS. As a biological model system for ligand-receptor interactions we studied the binding of biotin to streptavidin. To investigate the applicability of SW-FCCS for binding studies we adapt the existing FCCS theory for SW-FCCS and calculate limits for the measurement of dissociation constants in dependence on sample concentration, sample purity, and spectral cross talk between the different detection channels.