Ruchirej Yongsunthon

Specific Bonds between an Iron Oxide Surface and Outer Membrane Cytochromes MtrC and OmcA from Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is purported to express outer membrane cytochromes (e.g., MtrC and OmcA) that transfer electrons directly to Fe(III) in a mineral during anaerobic respiration. A prerequisite for this type of reaction would be the formation of a stable bond between a cytochrome and an iron oxide surface. Atomic force microscopy (AFM) was used to detect whether a specific bond forms between a hematite (Fe(2)O(3)) thin film, created with oxygen plasma-assisted molecular beam epitaxy, and recombinant MtrC or OmcA molecules coupled to gold substrates. Force spectra displayed a unique force signature indicative of a specific bond between each cytochrome and the hematite surface. The strength of the OmcA-hematite bond was approximately twice that of the MtrC-hematite bond, but direct binding to hematite was twice as favorable for MtrC. Reversible folding/unfolding reactions were observed for mechanically denatured MtrC molecules bound to hematite. The force measurements for the hematite-cytochrome pairs were compared to spectra collected for an iron oxide and S. oneidensis under anaerobic conditions. There is a strong correlation between the whole-cell and pure-protein force spectra, suggesting that the unique binding attributes of each cytochrome complement one another and allow both MtrC and OmcA to play a prominent role in the transfer of electrons to Fe(III) in minerals. Finally, by comparing the magnitudes of binding force for the whole-cell versus pure-protein data, we were able to estimate that a single bacterium of S. oneidensis (2 by 0.5 microm) expresses approximately 10(4) cytochromes on its outer surface.

Antibody recognition force microscopy shows that outer membrane cytochromes OmcA and MtrC are expressed on the exterior surface of Shewanella oneidensis MR-1

ABSTRACT Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe2O3), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.

Polymorphisms in fibronectin binding protein A of Staphylococcus aureus are associated with infection of cardiovascular devices

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

A Tactile Response in Staphylococcus aureus

It is well established that bacteria are able to respond to temporal gradients (e.g., by chemotaxis). However, it is widely held that prokaryotes are too small to sense spatial gradients. This contradicts the common observation that the vast majority of bacteria live on the surface of a solid substrate (e.g., as a biofilm). Herein we report direct experimental evidence that the nonmotile bacterium Staphylococcus aureus possesses a tactile response, or primitive sense of touch, that allows it to respond to spatial gradients. Attached cells recognize their substrate interface and localize adhesins toward that region. Braille-like avidity maps reflect a cells biochemical sensory response and reveal ultrastructural regions defined by the actual binding activity of specific proteins.

Force Measurements Between a Bacterium and Another Surface In Situ.

Publisher Summary This chapter discusses that the Atomic force microscope (AFM) measurements can investigate aspects of bacteria‐surface interactions inaccessible to more traditional microbiology techniques, such as optical microscopy or batch culture experiments.. AFM is a scanning probe microscopy technique that provides information about a localized portion of a sample by sensing the behavior of a small tip‐cantilever system, which interacts with the sample. The chapter covers AFM force measurement by functionalizing the AFM probe that is done by colloid bead probe and bacteria probe or biologically active force probe method. Force-sensing cantilevers can be used to measure forces between a bacterium on a glass cover slip and silicon or silicon nitride tip. A bead coated with living cells is attached to the end of a cantilever to measure forces between two cells or between a bacterium and a specific face of a mineral. This chapter concludes by presenting two examples of AFM force measurements for a bacterium and material substrate in aqueous solution: forces detected as a bacterium approaches a material surface and forces detected as a bacterium is pulled away from a material surface. A great deal of care and perseverance are required to extract valid and meaningful conclusions from the force measurements. In spite of the difficulties involved, there is no doubt that AFM experiments will continue to enhance the understanding of the nature of bacteria‐surface interactions.

Surface Electromigration and Current Crowding

Steps on macroscopic surfaces provide a useful model system for quantifying electron scattering at defects in nanostructures, where the large surface/volume ratio will cause surface effects to dominate. Here, the effects of electron scattering at surface steps are quantified using thin silver films with (111) surface orientation. Using real-time scanning tunneling microscopy (STM) measurements while large current densities are applied to the films, changes in step fluctuations and island motion are observed and quantified. Applying the tools of the continuum step model, the observations are analyzed in terms of step free energies and kinetics, yielding quantitative values of the electromigration force driving the observed mass displacements. The derived magnitudes are surprisingly large in comparison with classical calculations of the force due to electron scattering at the internal surface of a conductor. This result indicates that the specific atomistic characteristics of the scattering sites, in this case kinks at the step edge, may greatly enhance the electromigration force. Within the classical ballistic picture of ballistic momentum transfer, specific mechanisms for such enhancement include enhanced geometric “blocking” at the kinked step edges, changes in carrier density near kinks, and current crowding. Quantum transmission effects at atomic-scale defect sites may also be responsible for the observed enhancement. The nature of classical current crowding as a function of the shape and size of defect was characterized using magnetic force microscopy (MFM) of fabricated micron-scale model structures. Techniques were developed to remove the effects of instrumental broadening using deconvolution, so that full three-dimensional maps of the magnetic fields above the current line are determined. A Green function inversion technique is then used to invert the field distribution to determine the spatial variations in the current density in the sample. Current enhancement is highly localized near defects and is maximized by sharp variations in geometry that require strong deflections of the current path. Current enhancements up to a factor of 4 are found at the most strongly deflecting defects, while small notches of various shapes typically cause local enhancements of tens of percent to a factor of 2. The perpendicular component of the current flow around defects forms a dipole pattern with length scale determined by the length of the defect along the direction of the current flow. The shape and localization of the dipole pattern vary with the sharpness and symmetry of the defect. The current crowding affect alone is not sufficient to explain the greatly enhanced electromigration force observed for scattering at kink sites at steps.

Binding Forces Associated withStaphylococcus aureusBiofilms on Medical Implants

Staphylococcus aureus is one of the most frequently isolated bacteria from infected medical implants. S. aureus has the capacity to adhere to the surface of an implant where it forms a biofilm. We used atomic force microscopy to probe binding forces between a fibronectin-coated tip and isolates of S. aureus, which were obtained from either patients with infected prostheses or healthy humans. A unique force-signature was observed for binding events between the tip and the cells. There is a statistically significant difference in the binding force-signature observed for S. aureus isolated from the infected vs. healthy populations. This observation suggests a fundamental correlation between nanometer scale binding forces and the clinical outcome of patients with implanted medical devices.