Unraveling Membrane Perturbations Caused by the Bacterial Riboregulator Hfq.

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNAs. Previously, we showed that the Hfq C-terminal region forms an amyloid-like structure and that these fibrils interact with membranes. The immediate consequence of this interaction is a disruption of the membrane, but the effect on Hfq structure was unknown. To investigate details of the mechanism of interaction, the present work uses different in vitro biophysical approaches. We show that the Hfq C-terminal region influences membrane integrity and, conversely, that the membrane specifically affects the amyloid assembly. The reported effect of this bacterial master regulator on membrane integrity is discussed in light of the possible consequence on small regulatory RNA-based regulation.

Reversible Membrane Tethering by ZipA Determines FtsZ Polymerization in Two and Three Dimensions.

In most bacteria, the early step of septum formation implies the association of soluble FtsZ polymers with the cytoplasmic membrane. ZipA, together with FtsA, provides membrane tethering to FtsZ in Escherichia coli, forming a dynamic proto-ring that serves as an assembly scaffold for the remaining elements of the divisome. Despite their importance for bacterial cell division, multivalent interactions between proto-ring elements at membrane surfaces remain poorly characterized in quantitative terms. We measured the binding of FtsZ to ZipA incorporated in supported lipid bilayers at controlled densities by using a combination of biophysical surface-sensitive techniques (quartz crystal microbalance and spectroscopic ellipsometry) and analyzed how ZipA density and FtsZ concentration control the state of assembly of FtsZ. We found that ZipA attachment enables FtsZ-GMPCPP (where GMPCPP is a GTP analogue with a reduced level of hydrolysis) to assemble in several distinct ways: (i) two-dimensional polymerization at the membrane and (ii) three-dimensional polymerization from the membrane into the solution phase where this may be associated with the formation of higher-order complexes. In these processes, ZipA is required to enrich FtsZ at the surface but the FtsZ bulk concentration defines which morphology is being formed. Moreover, we report a strong effect of the nucleotide (GDP vs GMPCPP/GTP) on the kinetics of ZipA association/dissociation of FtsZ. These results provide insights into the mode of interaction of proto-ring elements in minimal membrane systems and contribute to the completion of our understanding of the initial events of bacterial division.

Surface Orientation and Binding Strength Modulate Shape of FtsZ on Lipid Surfaces.

We have used a simple model system to test the prediction that surface attachment strength of filaments presenting a torsion would affect their shape and properties. FtsZ from E. coli containing one cysteine in position 2 was covalently attached to a lipid bilayer containing maleimide lipids either in their head group (to simulate tight attachment) or at the end of a polyethylene glycol molecule attached to the head group (to simulate loose binding). We found that filaments tightly attached grew straight, growing from both ends, until they formed a two-dimensional lattice. Further monomer additions to their sides generated a dense layer of oriented filaments that fully covered the lipid membrane. After this point the surface became unstable and the bilayer detached from the surface. Filaments with a loose binding were initially curved and later evolved into straight thicker bundles that destabilized the membrane after reaching a certain surface density. Previously described theoretical models of FtsZ filament assembly on surfaces that include lateral interactions, spontaneous curvature, torsion, anchoring to the membrane, relative geometry of the surface and the filament 'living-polymer' condition in the presence of guanosine triphosphate (GTP) can offer some clues about the driving forces inducing these filament rearrangements.

Mutations on FtsZ lateral helix H3 that disrupt cell viability hamper reorganization of polymers on lipid surfaces.

FtsZ filaments localize at the middle of the bacterial cell and participate in the formation of a contractile ring responsible for cell division. Previous studies demonstrated that the highly conserved negative charge of glutamate 83 and the positive charge of arginine 85 located in the lateral helix H3 bend of Escherichia coli FtsZ are required for in vivo cell division. In order to understand how these lateral mutations impair the formation of a contractile ring,we extend previous in vitro characterization of these mutants in solution to study their behavior on lipid modified surfaces. We study their interaction with ZipAand look at their reorganization on the surface. We found that the dynamic bundling capacity of the mutant proteins is deficient, and this impairment increases the more the composition and spatial arrangement of the reconstituted system resembles the situation inside the cell: mutant proteins completely fail to reorganize to form higher order aggregates when bound to an E.coli lipid surface through oriented ZipA.We conclude that these surface lateral point mutations affect the dynamic reorganization of FtsZ filaments into bundles on the cell membrane, suggesting that this event is relevant for generating force and completing bacterial division.

Extracting the Shape and Size of Biomolecules Attached to a Surface as Suspended Discrete Nanoparticles.

The ability to derive information on the conformation of surface attached biomolecules by using simple techniques such as biosensors is currently considered of great importance in the fields of surface science and nanotechnology. Here we present a nanoshape sensitive biosensor where a simple mathematical expression is used to relate acoustic measurements to the geometrical features of a surface-attached biomolecule. The underlying scientific principle is that the acoustic ratio (ΔD/ΔF) is a measure of the hydrodynamic volume of the attached entity, mathematically expressed by its intrinsic viscosity [η]. A methodology is presented in order to produce surfaces with discretely bound biomolecules where their native conformation is maintained. Using DNA anchors we attached a spherical protein (streptavidin) and a rod-shaped DNA (47bp) to a quartz crystal microbalance (QCM) device in a suspended way and predicted correctly through acoustic measurements their conformation, i.e., shape and length. The methodology can be widely applied to draw conclusions on the conformation of any biomolecule or nanoentity upon specific binding on the surface of an acoustic wave device.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Dynamic and Active Proteins: Biomolecular Motors in Engineered Nanostructures.

In Nature, proteins perform functions that go well beyond controlled self-assembly at the nano scale. They are the principal components of diverse "biological machines" that can self-assemble into dynamic aggregates that achieve the cold conversion of chemical energy into motion to realize complex functions involved in cell division, cellular transport and cell motility. Nowadays, we have identified many of the proteins involved in these "molecular machines" and know much about their biochemistry, structure and biophysical behavior. Additionally, we have a rich toolbox of resources to engineer the basic dynamic working units into nanostructures to provide them with motion and the capacity to manipulate, transport, separate or sense single molecules to develop in vitro sensors and bioassays. This chapter summarizes some of the progress made in incorporating bio-molecular motors and dynamic self-organizing proteins into protein based functional nanostructures.

H2 -Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration.

ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1 F0 -ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.

Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ.

We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (T(b) ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer-monomer interactions, regardless of the nucleotide present, can adopt a curved configuration.

Induction of a proton gradient across a gold-supported biomimetic membrane by electroenzymatic H2 oxidation.

Energy-transduction mechanisms in living organisms, such as photosynthesis and respiration, store light and chemical energy in the form of an electrochemical gradient created across a lipid bilayer. Herein we show that the proton concentration at an electrode/phospholipid-bilayer interface can be controlled and monitored electrochemically by immobilizing a membrane-bound hydrogenase. Thus, the energy derived from the electroenzymatic oxidation of H2 can be used to generate a proton gradient across the supported biomimetic membrane.

Intrinsic disorder of the bacterial cell division protein ZipA: coil-to-brush conformational transition.

The full-length ZipA protein from Escherichia coli, one of the essential elements of the cell division machinery, was studied in a surface model built as adsorbed monolayers. The interplay between lateral packing and molecular conformation was probed using a combined methodology based on the scaling analysis of the surface pressure isotherms and ellipsometry measurements of the monolayer thickness. The observed behavior is compatible with the one expected for an intrinsically disordered and highly flexible protein that is preferentially structured in a random coil conformation. At low grafting densities, ZipA coils organize in a mushroom-like regime, whereas a coil-to-brush transition occurs on increasing lateral packing. The structural results suggest a functional scenario in which ZipA acts as a flexible tether anchoring bacterial proto-ring elements to the membrane during the earlier stages of division.

Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

Reconstitution of respiratory complex I on a biomimetic membrane supported on gold electrodes.

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions.

Torsion and curvature of FtsZ filaments.

FtsZ filaments participate in bacterial cell division, but it is still not clear how their dynamic polymerization and shape exert force on the underlying membrane. We present a theoretical description of individual filaments that incorporates information from molecular dynamic simulations. The structure of the crystallized Methanococcus jannaschii FtsZ dimer was used to model a FtsZ pentamer that showed a curvature and a twist. The estimated bending and torsion angles between monomers and their fluctuations were included in the theoretical description. The MD data also permitted positioning the curvature with respect to the protein coordinates and allowed us to explore the effect of the relative orientation of the preferred curvature with respect to the surface plane. We find that maximum tension is attained when filaments are firmly attached and oriented with their curvature perpendicular to the surface and that the twist serves as a valve to release or to tighten the tension exerted by the curved filaments on the membrane. The theoretical model also shows that the presence of torsion can explain the shape distribution of short filaments observed by Atomic Force Microscopy in previously published experiments. New experiments with FtsZ covalently attached to lipid membranes show that the filament on-plane curvature depends on lipid head charge, confirming the predicted monomer orientation effects. This new model underlines the fact that the combination of the three elements, filament curvature, twist and the strength and orientation of its surface attachment, can modulate the force exerted on the membrane during cell division.

FtsZ polymers bound to lipid bilayers through ZipA form dynamic two dimensional networks.

Bacteria divide by forming a contractile ring around their midcell region. FtsZ, a cytoskeletal soluble protein structurally related to tubulin, is the main component of this division machinery. It forms filaments that bundle at the inner side of the cytoplasmic membrane. These FtsZ bundles do not attach to bare lipid surfaces. In Escherichia coli they remain near the membrane surface by attaching to the membrane protein ZipA and FtsA. In order to study the structure and dynamics of the ZipA-FtsZ bundles formed on a lipid surface, we have oriented a soluble form of ZipA (sZipA), with its transmembrane domain substituted by a histidine tag, on supported lipid membranes. Atomic force microscopy has been used to visualize the polymers formed on top of this biomimetic surface. In the presence of GTP, when sZipA is present, FtsZ polymers restructure forming higher order structures. The lipid composition of the underlying membrane affects the aggregation kinetics and the shape of the structures formed. On the negatively charged E. coli lipid membranes, filaments condense from initially disperse material to form a network that is more dynamic and flexible than the one formed on phosphatidyl choline bilayers. These FtsZ-ZipA filament bundles are interconnected, retain their capacity to dynamically restructure, to fragment, to anneal and to condense laterally.

Dextran-lipase conjugates as tools for low molecular weight ligand immobilization in microarray development.

The development of effective array biosensors relies heavily on careful control of the density of surface-immobilized ligands on the transducing platform. In this paper we describe the synthesis of new dextran-lipase conjugates for use in immobilizing low molecular weight haptens onto glass planar waveguides for immunosensor development. The conjugates were synthesized by immobilizing bacterial thermoalkalophilic lipases (Geobacillus thermocatenulatus lipase 2, BTL2) on agarose macroporous beads, followed by covalent coupling to dextran networks of variable molecular weight (1500-40000). The chimeras were immobilized via nonspecific hydrophobic interactions onto glass planar waveguides modified with 1,1,1,3,3,3-hexamethyldisilazane to obtain highly ordered and homogeneous molecular architectures as confirmed by atomic force microscopy. Microcystin LR (MCLR) was covalently bound to the dextran-BTL2 conjugates. The usefulness of this approach in immunosensor development was demonstrated by determining amounts of MCLR down to a few picograms per liter with an automated array biosensor and evanescent wave excitation for fluorescence measurements of attached DyLight649-labeled secondary antibody. Modifying BTL2 with dextrans of an increased molecular weight (>6000) provided surfaces with an increased loading capacity that was ascribed to the production of three-dimensional surfaces by the effect of analyte binding deep in the volume, leading to expanded dynamic ranges (0.09-136.56 ng L(-1)), lower limits of detection (0.007 ± 0.001 ng L(-1)), and lower IC50 values (4.4 ± 0.7 ng L(-1)). These results confirm the effectiveness of our approach for the development of high-performance biosensing platforms.

Physicochemical characterization of Acidiphilium sp. biofilms.

The biofilm formation of a strain of the extremophile bacterium Acidiphilium sp., capable of donating electrons directly to electrodes, was studied by different surface characterization techniques. We develop a method that allows the simultaneous study of bacterial biofilms by means of fluorescence microscopy and atomic force microscopy (AFM), in which transparent graphitic flakes deposited on a glass substrate are used as a support for the biofilm. The majority of the cells present on the surface were viable, and the growth of the biofilms over time showed a critical increase of the extracellular polymeric substances (EPS) as well as the formation of nanosized particles inside the biofilm. Also, the presence of Fe in Acidiphilium biofilms was determined by X-ray photoelectron spectroscopy (XPS), whereas surface-enhanced infrared absorption spectroscopy indicated the presence of redox-active proteins.

Polymorphism of FtsZ filaments on lipid surfaces: role of monomer orientation.

FtsZ is a bacterial cytoskeletal protein involved in cell division. It forms a ringlike structure that attaches to the membrane to complete bacterial division. It binds and hydrolyzes GTP, assembling into polymers in a GTP-dependent manner. To test how the orientation of the monomers affects the curvature of the filaments on a surface, we performed site-directed mutagenesis on the E. coli FtsZ protein to insert cysteine residues at lateral locations to orient FtsZ on planar lipid bilayers. The E93C and S255C mutants were overproduced, purified, and found to be functionally active in solution, as well as being capable of sustaining cell division in vivo in complementation assays. Atomic force microscopy was used to observe the shape of the filament fibers formed on the surface. The FtsZ mutants were covalently linked to the lipids and could be polymerized on the bilayer surface in the presence of GTP. Unexpectedly, both mutants assembled into straight structures. E93C formed a well-defined lattice with monomers interacting at 60° and 120° angles, whereas S255C formed a more open array of straight thicker filament aggregates. These results indicate that filament curvature and bending are not fixed and that they can be modulated by the orientation of the monomers with respect to the membrane surface. As filament curvature has been associated with the force generation mechanism, these results point to a possible role of filament membrane attachment in lateral association and curvature, elements currently identified as relevant for force generation.

Electro-enzymatic ATP regeneration coupled to biocatalytic phosphorylation reactions.

Adenosine-5-triphosphate (ATP) is the main energy vector in biological systems, thus its regeneration is an important issue for the application of many enzymes of interest in biocatalysis and synthetic biology. We have developed an electroenzymatic ATP regeneration system consisting in a gold electrode modified with a floating phospholipid bilayer that allows coupling the catalytic activity of two membrane-bound enzymes: NiFeSe hydrogenase from Desulfovibrio vulgaris and F1Fo-ATP synthase from Escherichia coli. Thus, H2 is used as a fuel for producing ATP. This electro-enzymatic assembly is studied as ATP regeneration system of phosphorylation reactions catalysed by kinases, such as hexokinase and NAD+-kinase for respectively producing glucose-6-phosphate and NADP+.

Active membrane viscoelasticity by the bacterial FtsZ-division protein.

At the early stages of the division process in Escherichia coli, the protein FtsZ forms a septal ring at the midcell. This Z-ring causes membrane constriction during bacterial division. The Z-ring associates to the lipid membrane through several membrane proteins, ZipA among them. Here, a simplified FtsZ-ZipA model was reconstituted onto Langmuir monolayers based in E. coli polar lipid extract. Brewster angle and atomic force microscopy have revealed membrane FtsZ-polymerization upon GTP hydrolysis. The compression viscoelasticity of these monolayers has been also investigated. The presence of protein induced softening and fluidization with respect to the bare lipid membrane. An active mechanism, based on the internal forces stressed by FtsZ filaments and transduced to the lipid membrane by ZipA, was suggested to underlie the observed behavior.