The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring, driving the motions of the peptidoglycan synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall, building increasingly smaller concentric rings of peptidoglycan to divide the cell.

We demonstrate the feasibility of a nanopore based single-molecule DNA sequencing method, which employs multi-color readout. Target DNA is converted according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state nanopores are then used to sequentially strip off the beacons, leading to a series of detectable photon bursts, at high speed. We show that signals from multiple nanopores can be detected simultaneously, allowing straightforward parallelization to large nanopore arrays.Nanopore based DNA sequencing is widely considered to be a promising next generation sequencing platform [1,2]. Two main features of the nanopore method make it exceptionally useful for single molecule-based genome analyses: First, the method's ability to electrophoretically focus and thread extremely long DNA molecules from the bulk into the pore, making it possible to analyze minute DNA samples [3]. Second, sub-5 nm pores are now routinely used to linearize long DNA coils, thus in principle, nanopores can be used to effectively 'scan' information along a long genome. These features, as well as the fact that solid-state nanopores can be fabricated in a highly dense array [4,5], allow the development of massively parallel detection, and are crucial for the realization of an amplification-free, lowcost and high-throughput sequencing [2,[6][7][8][9].A nanopore is a nanometer-sized pore in an ultra-thin membrane that separates two chambers containing an ionic solution. An external electrical field applied across the membrane creates an ionic current and a local electrical potential gradient near the pore, which draws in and threads biopolymers through the pore in a single file manner [3,10]. As a biopolymer enters the pore, it displaces a fraction of the electrolytes, giving rise to a change in the pore conductivity, which can be measured directly using an electrometer. A number of nanopore based DNA sequencing methods have recently been proposed and highlight two major challenges [1,11]: 1) The ability to discriminate among individual nucleotides (nt). The system must be capable of differentiating among the four bases on a single-molecule level.2) The method must enable parallel readout. As a single nanopore can probe only a single molecule at a time, a strategy for manufacturing an array of nanopores and simultaneously monitoring them is needed. To date, parallel readout through any nanopore-based method has not yet been demonstrated. A large number of current, and future, generation sequencing methodologies rely on the use of an enzyme (polymerase, exonuclease, etc) in the readout process. The kinetics of enzymatic activity, however, is a major bottleneck for increasing readout speed, and these + Corresponding author. Email: ameller@bu.edu. * These authors contributed equally to this work. NIH Public AccessAuthor Manuscript Nano Lett. Author manuscript; available in PMC 2011 June 9. Published in final edited form as:Nano Lett. 2010 June 9; 10(6): 2237-2244. doi:10.1021/nl1012147. NI...

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

Introductory Paragraph Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: The Rod complex moves around the cell circumference, while class A penicillin-binding proteins do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis, as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: The Rod complex reduces diameter, while aPBPs increase it. Increased Rod complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative amount of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.

We present a novel method for integrating two single-molecule measurement modalities, namely, total internal reflection microscopy and electrical detection of biomolecules using nanopores. Demonstrated here is the electrical measurement of nanopore based biosensing performed simultaneously and in-sync with optical detection of analytes. This method makes it possible, for the first time, to visualize DNA and DNA-protein complexes translocating through a nanopore with high temporal resolution ͑1000 frames/s͒ and good signal to background. This paper describes a detailed experimental design of custom optics and data acquisition hardware to achieve simultaneous high resolution electrical and optical measurements on labeled biomolecules as they traverse through a ϳ4 nm synthetic pore. In conclusion, we discuss new directions and measurements, which this technique opens up.

The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C 55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized clustered regularly interspaced short palindromic repeat (CRISPR) system with catalytically inactive ("dead") CRISPR-associated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the M -dependent cell envelope stress response, including bcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the M regulon to cell envelope homeostasis pathways. IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5′-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can significantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP- γ S. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/eIF4H can bind directly to loop structures to destabilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applicable to a myriad of DEAD-box helicases employed in RNA metabolism.

The eukaryotic translation initiation factor 4AI (eIF4AI) is the prototypical DEAD-box RNA helicase. It has a "dumbbell" structure consisting of two domains connected by a flexible linker. Previous studies demonstrated that eIF4AI, in conjunction with eIF4H, bind to loop structures and repetitively unwind RNA hairpins. Here, we probe the conformational dynamics of eIF4AI in real time using single-molecule FRET. We demonstrate that eIF4AI/eIF4H complex can repetitively unwind RNA hairpins by transitioning between an eIF4AI "open" and a "closed" conformation using the energy derived from ATP hydrolysis. Our experiments directly track the conformational changes in the catalytic cycle of eIF4AI and eIF4H, and this correlates precisely with the kinetics of RNA unwinding. Furthermore, we show that the small-molecule eIF4A inhibitor hippuristanol locks eIF4AI in the closed conformation, thus efficiently inhibiting RNA unwinding. These results indicate that the large conformational changes undertaken by eIF4A during the helicase catalytic cycle are rate limiting.