Joshua W. Shaevitz

Direct observation of base-pair stepping by RNA polymerase

During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 ± 0.6 Å, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force–velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.

Backtracking by single RNA polymerase molecules observed at near-base-pair resolution

Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. Its low error rate may be achieved by means of a ‘proofreading’ mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3′ end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events (∼5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly

Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.

Probing the kinesin reaction cycle with a 2D optical force clamp.

With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADP/Pi release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.

An automated two-dimensional optical force clamp for single molecule studies.

We constructed a next-generation optical trapping instrument to study the motility of single motor proteins, such as kinesin moving along a microtubule. The instrument can be operated as a two-dimensional force clamp, applying loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, via acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Each measurement is preceded by an initialization sequence, which includes adjustment of bead height relative to the coverslip using a variant of optical force microscopy (to +/-4 nm), a two-dimensional raster scan to calibrate position detector response, and adjustment of bead lateral position relative to the microtubule substrate (to +/-3 nm). During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector. We present details of force clamp operation and preliminary data showing kinesin motor movement subject to diagonal and forward loads.

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization

Significance Across all kingdoms of life, maintaining the correct cell shape is critical for behaviors such as sensing, motility, surface attachment, and nutrient acquisition. Maintaining proper shape requires cellular-scale coordination of proteins and feedback systems that enable responses that correct local morphological perturbations. Here, we demonstrate that the MreB cytoskeleton in Escherichia coli preferentially localizes to regions of negative curvature, directing growth away from the poles and actively straightening locally curved regions of the cell. Moreover, our biophysical simulations of curvature-biased growth suggest that cell wall insertion causes surface deformations that could be responsible for the circumferential motion of MreB. Taken together, our work demonstrates that MreB’s local orchestration of persistent, bursty growth enables robust, uniform growth at the cellular scale. Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.

Massively parallel X-ray holography

Stefano Marchesini, 2 Sébastien Boutet, 4 Anne E. Sakdinawat, Michael J. Bogan, Sas̆a Bajt, Anton Barty, Henry N. Chapman, 6 Matthias Frank, Stefan P. Hau-Riege, Abraham Szöke, Congwu Cui, Malcolm R. Howells, David A. Shapiro, John C. H. Spence, Joshua W. Shaevitz, Johanna Y. Lee, Janos Hajdu, 4 and Marvin M. Seibert Lawrence Livermore National Laboratory, 7000 East Ave., Livermore, CA 94550, USA. Advanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron rd. Berkeley, CA 94720, USA∗ Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, 2575 Sand Hill Road, Menlo Park, California 94025, USA. Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3, Box 596, SE-75124 Uppsala, Sweden. Center for X-ray Optics, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. 6 Centre for Free-Electron Laser Science U. Hamburg, DESY, Notkestraße 85, Hamburg, Germany. Department of Physics and Astronomy, Arizona State University, Tempe, AZ 85287-1504, USA Department of Physics and Lewis-Sigler Institute, 150 Carl Icahn Laboratory, Princeton, New Jersey 08544, USA. Department of Plant and Microbial Biology, University of California, Berkeley, 648 Stanley Hall 3220, Berkeley, California 94720, USA. (Dated: February 9, 2008)

Motor-driven intracellular transport powers bacterial gliding motility

Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility.

Direct measurement of cell wall stress stiffening and turgor pressure in live bacterial cells.

We study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress stiffening in the E. coli cell wall, with an exponent of 1.22±0.12, such that the wall is significantly stiffer in intact cells (E=23±8  MPa and 49±20  MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29±3  kPa.

Actin-like cytoskeleton filaments contribute to cell mechanics in bacteria

A filamentous cytoskeleton largely governs the physical shape and mechanical properties of eukaryotic cells. In bacteria, proteins homologous to all three classes of eukaryotic cytoskeletal filaments have recently been discovered. These proteins are essential for the maintenance of bacterial cell shape and have been shown to guide the localization of key cell-wall-modifying enzymes. However, whether the bacterial cytoskeleton is stiff enough to affect the overall mechanical rigidity of a cell has not been probed. Here, we used an optical trap to measure the bending rigidity of live Escherichia coli cells. We find that the actin-homolog MreB contributes nearly as much to the stiffness of a cell as the peptidoglycan cell wall. By quantitatively modeling these measurements, our data indicate that the MreB is rigidly linked to the cell wall, increasing the mechanical stiffness of the overall system. These data are the first evidence that the bacterial cytoskeleton contributes to the mechanical integrity of a cell in much the same way as it does in eukaryotes.