Nic Mullin

Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale

Bacteria of the Bacillus cereus family form highly resistant spores, which in the case of the pathogen B. anthracis act as the agents of infection. The outermost layer, the exosporium, enveloping spores of the B. cereus family as well as a number of Clostridia, plays roles in spore adhesion, dissemination, targeting, and germination control. We have analyzed two naturally crystalline layers associated with the exosporium, one representing the “basal” layer to which the outermost spore layer (“hairy nap”) is attached, and the other likely representing a subsurface (“parasporal”) layer. We have used electron cryomicroscopy at a resolution of 0.8–0.6 nm and circular dichroism spectroscopic measurements to reveal a highly α-helical structure for both layers. The helices are assembled into 2D arrays of “cups” or “crowns.” High-resolution atomic force microscopy of the outermost layer showed that the open ends of these cups face the external environment and the highly immunogenic collagen-like fibrils of the hairy nap (BclA) are attached to this surface. Based on our findings, we present a molecular model for the spore surface and propose how this surface can act as a semipermeable barrier and a matrix for binding of molecules involved in defense, germination control, and other interactions of the spore with the environment.

Torsional resonance atomic force microscopy in water

An atomic force microscope for use in liquids including water, in which a torsionally resonating microcantilever controls the tip-sample interaction, is presented. Magnetostrictive actuators made from a particulate composite of Terfenol-D are used to excite the torsional oscillations of the cantilever. The high quality factor (Q) of torsional oscillation gives high sensitivity to tip-sample forces, and the ability to work using the true resonance peak of the cantilever allows “phase” images corresponding to real mechanical contrast to be collected under liquid. High quality topographic and phase images of a delicate crystallizing polymer sample submerged in water are presented.

Torsional tapping atomic force microscopy using T-shaped cantilevers

Torsional oscillation of atomic force microscope cantilevers has been shown to offer increased optical lever sensitivity, quality factor, resonant frequency, and stiffness as compared to flexural oscillation. In this letter, T-shaped cantilevers are oscillated torsionally to give a tapping motion at the tip. This gives many of the advantages of small cantilevers, without the requirement for specialized detection optics. In order to demonstrate the capability of this technique, high resolution images of LH2 membrane protein crystal structures are presented. Reduced settle time and tip-sample force under error signal are also demonstrated.

Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy

The function of bioenergetic membranes is strongly influenced by the spatial arrangement of their constituent membrane proteins. Atomic force microscopy (AFM) can be used to probe protein organization at high resolution, allowing individual proteins to be identified. However, previous AFM studies of biological membranes have typically required that curved membranes are ruptured and flattened during sample preparation, with the possibility of disruption of the native protein arrangement or loss of proteins. Imaging native, curved membranes requires minimal tip–sample interaction in both lateral and vertical directions. Here, long-range tip–sample interactions are reduced by optimizing the imaging buffer. Tapping mode AFM with high-resonance-frequency small and soft cantilevers, in combination with a high-speed AFM, reduces the forces due to feedback error and enables application of an average imaging force of tens of piconewtons. Using this approach, we have imaged the membrane organization of intact vesicular bacterial photosynthetic “organelles”, chromatophores. Despite the highly curved nature of the chromatophore membrane and lack of direct support, the resolution was sufficient to identify the photosystem complexes and quantify their arrangement in the native state. Successive imaging showed the proteins remain surprisingly static, with minimal rotation or translation over several-minute time scales. High-order assemblies of RC-LH1-PufX complexes are observed, and intact ATPases are successfully imaged. The methods developed here are likely to be applicable to a broad range of protein-rich vesicles or curved membrane systems, which are an almost ubiquitous feature of native organelles.

Body-centered cubic phase in 3-arm star mesogens: a torsional tapping AFM and GISAXS study

The mode of liquid crystal (LC) self-assembly of asymmetric three-arm star oligobenzoate mesogens has been investigated by torsional tapping mode AFM imaging, and by bulk and grazing-incidence X-ray diffraction. It was confirmed that the cubic liquid crystal phase has Imm symmetry and established that it consists of spherical aggregates arranged on a body-centered lattice, rather than having a bicontinuous structure. Molecular simulation suggests that, in spite of their perceived rigidity, the oligobenzoate mesogens are folded, often sharply, within the supramolecular spheres, and that they act effectively as conical objects. The recently introduced torsional tapping AFM technique has allowed high resolution and contrast to be obtained from the soft mobile surface of the samples, showing the (110) plane of highly ordered supramolecular spheres. Notably the fine structure of the observed steps reveals that nearly isolated micelles still preserve their integrity. Finally, the equilibrium habits of the cubic LC droplets were shown by optical microscopy to be polyhedral with crystallographic facets, rather than spherical. This suggests that the air–LC interface is below the roughening transition temperature, which is attributed to the whole micelles rather than the isolated molecules acting as the interacting units.

The Interplay between Cell Wall Mechanical Properties and the Cell Cycle in Staphylococcus aureus

The nanoscale mechanical properties of live Staphylococcus aureus cells during different phases of growth were studied by atomic force microscopy. Indentation to different depths provided access to both local cell wall mechanical properties and whole-cell properties, including a component related to cell turgor pressure. Local cell wall properties were found to change in a characteristic manner throughout the division cycle. Splitting of the cell into two daughter cells followed a local softening of the cell wall along the division circumference, with the cell wall on either side of the division circumference becoming stiffer. Once exposed, the newly formed septum was found to be stiffer than the surrounding, older cell wall. Deeper indentations, which were affected by cell turgor pressure, did not show a change in stiffness throughout the division cycle, implying that enzymatic cell wall remodeling and local variations in wall properties are responsible for the evolution of cell shape through division.

A non-contact, thermal noise based method for the calibration of lateral deflection sensitivity in atomic force microscopy

Calibration of lateral forces and displacements has been a long standing problem in lateral force microscopies. Recently, it was shown by Wagner et al. that the thermal noise spectrum of the first torsional mode may be used to calibrate the deflection sensitivity of the detector. This method is quick, non-destructive and may be performed in situ in air or liquid. Here we make a full quantitative comparison of the lateral inverse optical lever sensitivity obtained by the lateral thermal noise method and the shape independent method developed by Anderson et al. We find that the thermal method provides accurate results for a wide variety of rectangular cantilevers, provided that the geometry of the cantilever is suitable for torsional stiffness calibration by the torsional Sader method, in-plane bending of the cantilever may be eliminated or accounted for and that any scaling of the lateral deflection signal between the measurement of the lateral thermal noise and the measurement of the lateral deflection is eliminated or corrected for. We also demonstrate that the thermal method may be used to characterize the linearity of the detector signal as a function of position, and find a deviation of less than 8% for the instrument used.

‘Watching’ processes in soft matter with SPM

Scanning probe microscopy (SPM) techniques can obtain nanoscale images of soft materials in almost any environment and over a wide range of temperatures. Being non-destructive, processes such as crystallization can be followed in-situ, and the effect of changes in temperature on structures can be monitored at the nanometre scale. The application of these techniques over recent years has lead to a real change in our understanding of many fundamental processes. The capabilities of scanning probe microscopes are continuously being enhanced, with recent developments in high speed scanning and material property mapping promising to significantly broaden soft matter applications. Here a personal overview of progress over the last decade in the development and application of SPM to following processes in soft matter will be provided, and a look forward to future developments in the field.

X-shaped plasmonic antenna on a quantum cascade laser

We report an x-shaped plasmonic antenna design patterned onto the gold coated facet of a mid-infrared quantum cascade laser. Using apertureless scanning near-field optical microscopy we measure a single enhanced region in the optical near-field at the center of the x-antenna, with a full-width-at-half-maximum of ∼100 nm for the operating wavelength of ∼8.8 μm. This design provides complete suppression of near-field signal away from the center, with concomitant improvements in imaging contrast expected. Our experimental results are also in good agreement with finite difference time domain simulations, which show a full-width-at-half-maximum of ∼80 nm.

Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75–100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.