John Dutcher

Absolute Quantitation of Bacterial Biofilm Adhesion and Viscoelasticity by Microbead Force Spectroscopy

Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 +/- 15 Pa and 332 +/- 47 Pa, respectively, whereas those of PAO1 and wapR mature biofilms were 19 +/- 7 Pa and 80 +/- 22 Pa, respectively. Fitting of creep data to a Voigt Standard Linear Solid viscoelasticity model revealed that the instantaneous and delayed elastic moduli in P. aeruginosa were drastically reduced by lipopolysaccharide deficiency and biofilm maturation, whereas viscosity was decreased only for biofilm maturation. In conclusion, we have introduced a direct biophysical method for simultaneously quantifying adhesion and viscoelasticity in bacterial biofilms under native conditions. This method could prove valuable for elucidating the contribution of genetic backgrounds, growth conditions, and environmental stresses to microbial community physiology.

Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy

The cell envelope of gram-negative bacteria is responsible for many important biological functions: it plays a structural role, it accommodates the selective transfer of material across the cell wall, it undergoes changes made necessary by growth and division, and it transfers information about the environment into the cell. Thus, an accurate quantification of cell mechanical properties is required not only to understand physiological processes but also to help elucidate the relationship between cell surface structure and function. We have used a novel, atomic force microscopy (AFM)-based approach to probe the mechanical properties of single bacterial cells by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of the AFM tip due to the viscoelastic properties of the cell. For these experiments, we chose a representative gram-negative bacterium, Pseudomonas aeruginosa PAO1, and we used regular V-shaped AFM cantilevers with pyramid-shaped and colloidal tips. We find that the cell response is well described by a three-element mechanical model which describes an effective cell spring constant, k(1), and an effective time constant, tau, for the creep deformation. Adding glutaraldehyde, an agent that increases the covalent bonding of the cell surface, produced a significant increase in k(1) together with a significant decrease in tau. This work represents a new attempt toward the understanding of the nanomechanical properties of single bacteria while they are under fluid conditions, which could be of practical value for elucidating, for instance, the biomechanical effects of drugs (such as antibiotics) on pathogens.

Differential Lipopolysaccharide Core Capping Leads to Quantitative and Correlated Modifications of Mechanical and Structural Properties in Pseudomonas aeruginosa Biofilms

Bacterial biofilms are responsible for the majority of all microbial infections and have profound impact on industrial and geochemical processes. While many studies documented phenotypic differentiation and gene regulation of biofilms, the importance of their structural and mechanical properties is poorly understood. Here we investigate how changes in lipopolysaccharide (LPS) core capping in Pseudomonas aeruginosa affect biofilm structure through modification of adhesive, cohesive, and viscoelastic properties at an early stage of biofilm development. Microbead force spectroscopy and atomic force microscopy were used to characterize P. aeruginosa biofilm interactions with either glass substrata or bacterial lawns. Using isogenic migA, wapR, and rmlC mutants with defined LPS characteristics, we observed significant changes in cell mechanical properties among these strains compared to wild-type strain PAO1. Specifically, truncation of core oligosaccharides enhanced both adhesive and cohesive forces by up to 10-fold, whereas changes in instantaneous elasticity were correlated with the presence of O antigen. Using confocal laser scanning microscopy to quantify biofilm structural changes with respect to differences in LPS core capping, we observed that textural parameters varied with adhesion or the inverse of cohesion, while areal and volumetric parameters were linked to adhesion, cohesion, or the balance between them. In conclusion, this report demonstrated for the first time that changes in LPS expression resulted in quantifiable cellular mechanical changes that were correlated with structural changes in bacterial biofilms. Thus, the interplay between architectural and functional properties may be an important contributor to bacterial community survival.

PHASE SEPARATION MORPHOLOGY OF SPIN-COATED POLYMER BLEND THIN FILMS

We present the results of a study of the morphology of phase separation in thin films of two different polymer blend systems: polystyrene/polyisoprene and polystyrene/poly(methyl methacrylate). For each blend system, the two polymer components are dissolved in a common solvent. Spin coating of the ternary solutions (polymer blend/solvent) is used to confine the blends to a thin film geometry and to produce phase separation because of rapid evaporation of the solvent (solvent quench). As a quantitative measure of the phase separation morphology the average domain area of the minority component is measured as a function of the polystyrene mass fraction. For both blend systems we identify a small range of composition corresponding to a large increase in the average domain area. We show that the strong dependence of the average domain area on spin speed allows control over the quench time of the polymer blend thin films.

In Situ PM-IRRAS Studies of an Archaea Analogue Thiolipid Assembled on a Au(111) Electrode Surface

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) has been applied to determine the conformation, orientation, and hydration of a monolayer of 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-dl-alpha-lipoic acid ester (DPTL) self-assembled at a gold electrode surface. This Archaea analogue thiolipid has been recently employed to build tethered lipid bilayers. By synthesizing DPT(d16)L, a DPTL molecule with a deuterium substituted tetraethylene glycol spacer, it was possible to differentiate the C-H stretch vibrations of the phytanyl chains from the tetraethylene glycol spacer and acquire the characteristic IR spectra for the chains, spacer, and lipoic acid headgroup separately. Our results show that the structure of the monolayer displays remarkable stability in a broad range of electrode potentials and that the phytanyl chains remain in a liquid crystalline state. The tetraethylene glycol chains are coiled, and the IR spectrum for this region shows that it is in the disordered state. The most significant result of this study is the information that in contrast to expectations the spacer region is poorly hydrated. Our results have implications for the design of a tethered lipid membrane based on this thiolipid.

In Situ Characterization of Differences in the Viscoelastic Response of Individual Gram-Negative and Gram-Positive Bacterial Cells

We used a novel atomic force microscopy (AFM)-based technique to compare the local viscoelastic properties of individual gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacterial cells. We found that the viscoelastic properties of the bacterial cells are well described by a three-component mechanical model that combines an instantaneous elastic response and a delayed elastic response. These experiments have allowed us to investigate the relationship between the viscoelastic properties and the structure and composition of the cell envelope. In addition, this is the first report in which the mechanical role of Lpp, the major peptidoglycan-associated lipoprotein and one of the most abundant outer membrane proteins in E. coli cells, has been quantified. We expect that our findings will be helpful in increasing the understanding of the structure-property relationships of bacterial cell envelopes.

Molecular resolution imaging of an antibiotic peptide in a lipid matrix.

In this work, we show molecular resolution scanning tunneling microscopy (STM) images of gramicidin, a model antibacterial peptide, inserted into a phospholipid matrix. The resolution of the images is superior to that obtained in previous attempts to image gramicidin in a lipid environment using atomic force microscopy (AFM). This breakthrough has allowed visualization of individual peptide molecules surrounded by lipid molecules. We have observed several important features: the peptide molecules do not aggregate, the peptide molecules adopt a single conformation corresponding to a specific ion channel form, and the lipid molecules adjacent to the peptide molecules are systematically longer than those in the lipid matrix. These results constitute a new approach to obtain structural characteristics of antibiotic peptides in lipid assemblies that is necessary for the understanding of their biological activity.

Use of Atomic Force Microscopy and Transmission Electron Microscopy for Correlative Studies of Bacterial Capsules

ABSTRACT Bacteria can possess an outermost assembly of polysaccharide molecules, a capsule, which is attached to their cell wall. We have used two complementary, high-resolution microscopy techniques, atomic force microscopy (AFM) and transmission electron microscopy (TEM), to study bacterial capsules of four different gram-negative bacterial strains: Escherichia coli K30, Pseudomonas aeruginosa FRD1, Shewanella oneidensis MR-4, and Geobacter sulfurreducens PCA. TEM analysis of bacterial cells using different preparative techniques (whole-cell mounts, conventional embeddings, and freeze-substitution) revealed capsules for some but not all of the strains. In contrast, the use of AFM allowed the unambiguous identification of the presence of capsules on all strains used in the present study, including those that were shown by TEM to be not encapsulated. In addition, the use of AFM phase imaging allowed the visualization of the bacterial cell within the capsule, with a depth sensitivity that decreased with increasing tapping frequency.

Phase separation morphology of thin films of polystyrene/polyisoprene blends

We present the results of a study of the morphology of phase separation in a thin film blend of polystyrene (PS) and polyisoprene (PI) in a common solvent of toluene. The blend is quenched by rapid solvent evaporation using a spincoating technique rather than a temperature quench. The mass fraction of polystyrene is varied to determine the effect of the substrate on thin film phase separation morphology. We compare the phase separation morphology for very thin films of the PS/PI blend cast onto three different substrates: Si(001) with a native oxide layer (Si (SINGLEBOND) SiOx), Si(001) etched in hydrofluoric acid (Si-H), and a Au/Pd alloy sputtered onto Si(001). We observe large differences between the morphologies of 1000 A thick blend films on the Si(SINGLEBOND) SiOx and Si-H substrates as the mass fraction is varied due to the difference in the wetting properties of PS on the two substrates. Smaller differences are observed between the films on the Si(SINGLEBOND) SiOx and Au/Pd substrates only for film thicknesses h < 600 A.

Phosphorylation of Thellungiella salsuginea Dehydrins TsDHN-1 and TsDHN-2 Facilitates Cation-Induced Conformational Changes and Actin Assembly

Group 2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperatures. These proteins are characterized by the presence of at least one conserved, lysine-rich K-segment and sometimes by one or more serine-rich S-segments that are phosphorylated. Dehydrins may stabilize proteins and membrane structures during environmental stress and can sequester and scavenge metal ions. Here, we investigate how the conformations of two dehydrins from Thellungiella salsuginea, denoted as TsDHN-1 (acidic) and TsDHN-2 (basic), are affected by pH, interactions with cations and membranes, and phosphorylation. Both TsDHN-1 and TsDHN-2 were expressed as SUMO fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and structural analysis by circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy. We show that the polyproline II conformation can be induced in the dehydrins by their environmental conditions, including changes in the concentration of divalent cations such as Ca(2+). The assembly of actin by these dehydrins was assessed by sedimentation assays and viewed by transmission electron and atomic force microscopy. Phosphorylation allowed both dehydrins to polymerize actin filaments. These results support the hypothesis that dehydrins stabilize the cytoskeleton under stress conditions and further that phosphorylation may be an important feature of this stabilization.