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Abstract The Escherichia coli chemotaxis network, by which bacteria modulate their random run/tumble swimming pattern to navigate their environment, must cope with unavoidable number fluctuations (“noise”) in its molecular constituents like other signaling networks. The probability of clockwise (CW) flagellar rotation, or CW bias, is a measure of the chemotaxis network’s output, and its temporal fluctuations provide a proxy for network noise. Here we quantify fluctuations in the chemotaxis signaling network from the switching statistics of flagella, observed using time-resolved fluorescence microscopy of individual optically trapped E. coli cells. This approach allows noise to be quantified across the dynamic range of the network. Large CW bias fluctuations are revealed at steady state, which may play a critical role in driving flagellar switching and cell tumbling. When the network is stimulated chemically to higher activity, fluctuations dramatically decrease. A stochastic theoretical model, inspired by work on gene expression noise, points to CheY activation occurring in bursts, driving CW bias fluctuations. This model also shows that an intrinsic kinetic ceiling on network activity places an upper limit on activated CheY and CW bias, which when encountered suppresses network fluctuations. This limit may also prevent cells from tumbling unproductively in steep gradients.

UvrD is a model helicase from the non-hexameric Superfamily 1. Here, the authors use optical tweezers to measure directly the stepwise translocation of UvrD along a DNA hairpin, and propose a mechanism in which UvrD moves one base pair at a time, but sequesters the nascent single strands, releasing them after a variable number of ATP hydrolysis cycles.

In viruses, multi-subunit ring-ATPases are involved in genome packaging. Here, using single-molecule techniques, the authors determine that the active bacteriophage T4 DNA packaging motor is a pentamer and show that the motor can tolerate inactive subunits, suggesting that strict coordination between the subunits is not crucial.

ABSTRACT Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar Typhimurium is motile only when nutrients are plentiful, suggesting that this bacterium uses motility for purposes other than foraging, most likely for host colonization. In this study, we investigated how nutrients affect motility in S. enterica and found that they tune the fraction of motile cells. In particular, we observed coexisting populations of motile and nonmotile cells, with the distribution being determined by the concentration of nutrients in the growth medium. Interestingly, S. enterica responds not to a single nutrient but apparently to a complex mixture of them. Using a combination of experimentation and mathematical modeling, we investigated the mechanism governing this behavior and found that it results from two antagonizing regulatory proteins, FliZ and YdiV. We also found that a positive feedback loop involving the alternate sigma factor FliA is required, although its role appears solely to amplify FliZ expression. We further demonstrate that the response is bistable: that is, genetically identical cells can exhibit different phenotypes under identical growth conditions. Together, these results uncover a new facet of the regulation of the flagellar genes in S. enterica and further demonstrate how bacteria employ phenotypic diversity as a general mechanism for adapting to change in their environment. IMPORTANCE Many bacteria employ flagella for motility. These bacteria are often not constitutively motile but become so only in response to specific environmental cues. The most common is nutrient starvation. Interestingly, in Salmonella enterica serovar Typhimurium, nutrients enhance the expression of flagella, suggesting that motility is used for purposes other than foraging. In this work, we investigated how nutrients affect motility in S. enterica and found that nutrients tune the fraction of motile cells within a population. Using both experimental and mathematical analysis, we determined the mechanism governing this tunable response. We further demonstrated that the response is bistable: that is, genetically identical cells can exhibit different phenotypes under identical growth conditions. These results reveal a new facet of motility in S. enterica and demonstrate that nutrients determine not only where these bacteria swim but also the fraction of them that do so.

Helicases utilize nucleotide triphosphate (NTP) hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins such as single-stranded DNA binding proteins. Such encounters regulate helicase function, although the underlying mechanisms remain largely unknown. Ferroplasma acidarmanus xeroderma pigmentosum group D (XPD) helicase serves as a model for understanding the molecular mechanisms of superfamily 2B helicases, and its activity is enhanced by the cognate single-stranded DNA binding protein replication protein A 2 (RPA2). Here, optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent ‘processivity switch’ in XPD. A point mutation at a regulatory DNA binding site on XPD similarly activates this switch. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.

How cargoes move within a crowded cell—over long distances and at speeds nearly the same as when moving on unimpeded pathway—has long been mysterious. Through an in vitro force-gliding assay, which involves measuring nanometer displacement and piconewtons of force, we show that multiple mammalian kinesin-1 (from 2 to 8) communicate in a team by inducing tension (up to 4 pN) on the cargo. Kinesins adopt two distinct states, with one-third slowing down the microtubule and two-thirds speeding it up. Resisting kinesins tend to come off more rapidly than, and speed up when pulled by driving kinesins, implying an asymmetric tug-of-war. Furthermore, kinesins dynamically interact to overcome roadblocks, occasionally combining their forces. Consequently, multiple kinesins acting as a team may play a significant role in facilitating smooth cargo motion in a dense environment. This is one of few cases in which single molecule behavior can be connected to ensemble behavior of multiple motors.

Helicases play key roles in genome maintenance, yet it remains elusive how these enzymes change conformations and how transitions between different conformational states regulate nucleic acid reshaping. Here, we developed a computational technique combining structural bioinformatics approaches and atomic-level free-energy simulations to characterize how the Escherichia coli DNA repair enzyme UvrD changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The lowest free-energy path shows that UvrD opens the interface between two domains, allowing the bound ssDNA to escape. The simulation results predict a key metastable 'tilted' state during ssDNA strand switching. By simulating FRET distributions with fluorophores attached to UvrD, we show that the new state is supported quantitatively by single-molecule measurements. The present study deciphers key elements for the 'hyper-helicase' behavior of a mutant and provides an effective framework to characterize directly structure-function relationships in molecular machines.

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

How simple is the underlying control mechanism for the complex locomotion of vertebrates? We explore this question for the swimming behavior of zebrafish larvae. A parameter-independent method, similar to that used in studies of worms and flies, is applied to analyze swimming movies of fish. The motion itself yields a natural set of fish "eigenshapes" as coordinates, rather than the experimenter imposing a choice of coordinates. Three eigenshape coordinates are sufficient to construct a quantitative "postural space" that captures >96% of the observed zebrafish locomotion. Viewed in postural space, swim bouts are manifested as trajectories consisting of cycles of shapes repeated in succession. To classify behavioral patterns quantitatively and to understand behavioral variations among an ensemble of fish, we construct a "behavioral space" using multi-dimensional scaling (MDS). This method turns each cycle of a trajectory into a single point in behavioral space, and clusters points based on behavioral similarity. Clustering analysis reveals three known behavioral patterns-scoots, turns, rests-but shows that these do not represent discrete states, but rather extremes of a continuum. The behavioral space not only classifies fish by their behavior but also distinguishes fish by age. With the insight into fish behavior from postural space and behavioral space, we construct a two-channel neural network model for fish locomotion, which produces strikingly similar postural space and behavioral space dynamics compared to real zebrafish.

Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell’s flagella cooperatively determine whether the cell ‘runs’ or ‘tumbles’ remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell’s run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an ‘effective number’ of flagella—smaller than the actual number—enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella.

Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones.

Dynamic biological processes such as enzyme catalysis, molecular motor translocation, and protein and nucleic acid conformational dynamics are inherently stochastic processes. However, when such processes are studied on a nonsynchronized ensemble, the inherent fluctuations are lost, and only the average rate of the process can be measured. With the recent development of methods of single-molecule manipulation and detection, it is now possible to follow the progress of an individual molecule, measuring not just the average rate but the fluctuations in this rate as well. These fluctuations can provide a great deal of detail about the underlying kinetic cycle that governs the dynamical behavior of the system. However, extracting this information from experiments requires the ability to calculate the general properties of arbitrarily complex theoretical kinetic schemes. We present here a general technique that determines the exact analytical solution for the mean velocity and for measures of the fluctuations. We adopt a formalism based on the master equation and show how the probability density for the position of a molecular motor at a given time can be solved exactly in Fourier−Laplace space. With this analytic solution, we can then calculate the mean velocity and fluctuation-related parameters, such as the randomness parameter (a dimensionless ratio of the diffusion constant and the velocity) and the dwell time distributions, which fully characterize the fluctuations of the system, both commonly used kinetic parameters in single-molecule measurements. Furthermore, we show that this formalism allows calculation of these parameters for a much wider class of general kinetic models than demonstrated with previous methods. [ABSTRACT FROM AUTHOR]

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.