AFM Force Mapping Elucidates Pilus Deployment and Key Lifestyle-Dependent Surface Properties in Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is known for predation of a wide variety of Gram-negative bacteria, making it of interest as an alternative or supplement to chemical antibiotics. However, a fraction of B. bacteriovorus follows a nonpredatory, "host-independent" (HI) life cycle. In this study, live predatory and HI B. bacteriovorus were captured on a surface and examined, in buffer, by collecting force maps using atomic force microscopy (AFM). The approach curves obtained on HI cells are similar to those on other Gram-negative cells, with a short nonlinear region followed by a linear region. In contrast, the approach curves obtained on predatory cells have a large nonlinear region, reflecting the unusual flexibility of the predatory cell. As the AFM tip is retracted, it shows virtually no adhesion to predatory B. bacteriovorus but has multiple adhesion events on HI cells and the 200-500+ nm region immediately surrounding them. Measured pull-off forces, pull-off distances, and effective spring constants are consistent with the multiple stretching events of Type IV pili, both on and especially adjacent to the cells. Exposure of the HI B. bacteriovorus to a pH-neutral 10% cranberry juice solution, which contains type A proanthocyanidins that are known to interfere with the adhesion of multiple types of pili, results in a substantial reduction in adhesion. Type IV pili are required for successful predation by B. bacteriovorus, but pili used in the predation process are located at the non-flagellated pole of the cell and can retract when not in use. Such pili are rarely observed under the conditions of this study, where the predator has not encountered a prey cell. In contrast, HI cells appear to have many pili distributed on and around the whole cell, presumably ready to be utilized for a variety of HI cell activities including attachment to surfaces.

Extracellular Polymeric Substance Protects Some Cells in an Escherichia coli Biofilm from the Biomechanical Consequences of Treatment with Magainin 2.

Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully eliminate biofilms by conventional chemical or physical means. Since most bacteria are capable of forming biofilms, understanding how biofilms respond to new antibiotic compounds and components of the immune system has important ramifications. Antimicrobial peptides (AMPs) are both potential novel antibiotic compounds and part of the immune response in many different organisms. Here, we use atomic force microscopy to investigate the biomechanical changes that occur in individual cells when a biofilm is exposed to the AMP magainin 2 (MAG2), which acts by permeabilizing bacterial membranes. While MAG2 is able to prevent biofilm initiation, cells in an established biofilm can withstand exposure to high concentrations of MAG2. Treated cells in the biofilm are classified into two distinct populations after treatment: one population of cells is indistinguishable from untreated cells, maintaining cellular turgor pressure and a smooth outer surface, and the second population of cells are softer than untreated cells and have a rough outer surface after treatment. Notably, the latter population is similar to planktonic cells treated with MAG2. The EPS likely reduces the local MAG2 concentration around the stiffer cells since once the EPS was enzymatically removed, all cells became softer and had rough outer surfaces. Thus, while MAG2 appears to have the same mechanism of action in biofilm cells as in planktonic ones, MAG2 cannot eradicate a biofilm unless coupled with the removal of the EPS.

Qualitative and Quantitative Changes to Escherichia coli during Treatment with Magainin 2 Observed in Native Conditions by Atomic Force Microscopy.

The bacterial membrane has been suggested as a good target for future antibiotics, so it is important to understand how naturally occurring antibiotics like antimicrobial peptides (AMPs) disrupt those membranes. The interaction of the AMP magainin 2 (MAG2) with the bacterial cell membrane has been well characterized using supported lipid substrates, unilamellar vesicles, and spheroplasts created from bacterial cells. However, to fully understand how MAG2 kills bacteria, we must consider its effect on the outer membrane found in Gram-negative bacteria. Here, we use atomic force microscopy (AFM) to directly investigate MAG2 interaction with the outer membrane of Escherichia coli and characterize the biophysical consequences of MAG2 treatment under native conditions. While propidium iodide penetration indicates that MAG2 permeabilizes cells within seconds, a corresponding decrease in cellular turgor pressure is not observed until minutes after MAG2 application, suggesting that cellular homeostasis machinery may be responsible for helping the cell maintain turgor pressure despite a loss of membrane integrity. AFM imaging and force measurement modes applied in tandem reveal that the outer membrane becomes pitted, more flexible, and more adhesive after MAG2 treatment. MAG2 appears to have a highly disruptive effect on the outer membrane, extending the known mechanism of MAG2 to the Gram-negative outer membrane.

Identification and differential production of ubiquinone-8 in the bacterial predator Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus 109J, a predatory bacterium with potential as a bacterial control agent, can exist in several lifestyles that differ both in predatory capacity and color. We determined that levels of ubiquinone-8 contribute to the distinctive but variable yellow color of different types of Bdellovibrio cells. Steady-state ubiquinone-8 concentrations did not differ markedly between conventional predatory and host-independent B. bacteriovorus despite upregulation of a suite of ubiquinone-8 synthesis genes in host-independent cells. In contrast, in spatially organized B. bacteriovorus films, the yellow inner regions contain significantly higher ubiquinone-8 concentrations than the off-white outer regions. Correspondingly, RT-PCR analysis reveals that the inner region, previously shown to consist primarily of active predators, clearly expresses two ubiquinone biosynthesis genes, while the outer region, composed mainly of quiescent or stalled bdelloplasts, expresses those genes weakly or not at all. Moreover, B. bacteriovorus cells in the inner region of week-old interfacial films, which are phenotypically attack-phase, have much higher UQ8 levels than regular attack-phase bdellovibrios, most likely because their "trapped" state prevents a high expenditure of energy to power flagellar motion.

Au nanoparticle clusters from deposition of a coalescing emulsion.

HYPOTHESIS: Nanoparticle adsorption at the oil-water interface in an unstable, coalescing emulsion leads to cluster formation. EXPERIMENTS: Stable suspensions of clusters are prepared using a facile, two-step procedure involving few reagents and neither thiolated compounds nor chlorinated solvents. First, colloidal gold nanoparticles are assembled at the aqueous-hexanol interface in an emulsion that rapidly coalesces and spontaneously deposits a film on the interior surface of the glass container. The film is dissolved in ethanol with sonication to disperse the clusters. The film and clusters are characterized by transmission electron and atomic force microscopies as well as ultraviolet-visible spectrometry. FINDINGS: Clusters are observed to contain as few as 8 to as many as 24 Au nanoparticles. The clusters are anisotropic and can also be formed from larger nanoparticles. Hydrophobic and hydrophilic interactions are implicated in the formation of these clusters within the interfacial tension gradients of a coalescing emulsion. The clusters can be re-suspended in ethanol and water, maximizing the utility of these clusters with an extinction band in the near-Infrared region of the electromagnetic spectrum.

Spatially Organized Films from Bdellovibrio bacteriovorus Prey Lysates.

Bdellovibrio bacteriovorus is a Gram-negative predator of other Gram-negative bacteria. Interestingly, Bdellovibrio bacteriovorus 109J cells grown in coculture with Escherichia coli ML-35 prey develop into a spatially organized two-dimensional film when located on a nutrient-rich surface. From deposition of 10 µl of a routine cleared coculture of B. bacteriovorus and E. coli cells, the cells multiply into a macroscopic community and segregate into an inner, yellow circular region and an outer, off-white region. Fluorescence in situ hybridization and atomic force microscopy measurements confirm that the mature film is spatially organized into two morphologically distinct Bdellovibrio populations, with primarily small, vibroid cells in the center and a complex mixture of pleomorphic cells in the outer radii. The interior region cell population exhibits the hunting phenotype while the outer region cell subpopulation does not. Crowding and high nutrient availability with limited prey appear to favor diversification of the B. bacteriovorus population into two distinct, thriving subpopulations and may be beneficial to the persistence of B. bacteriovorus in biofilms.

Characterizing pilus-mediated adhesion of biofilm-forming E. coli to chemically diverse surfaces using atomic force microscopy.

Biofilms are complex communities of microorganisms living together at an interface. Because biofilms are often associated with contamination and infection, it is critical to understand how bacterial cells adhere to surfaces in the early stages of biofilm formation. Even harmless commensal Escherichia coli naturally forms biofilms in the human digestive tract by adhering to epithelial cells, a trait that presents major concerns in the case of pathogenic E. coli strains. The laboratory strain E. coli ZK1056 provides an intriguing model system for pathogenic E. coli strains because it forms biofilms robustly on a wide range of surfaces.E. coli ZK1056 cells spontaneously form living biofilms on polylysine-coated AFM cantilevers, allowing us to measure quantitatively by AFM the adhesion between native biofilm cells and substrates of our choice. We use these biofilm-covered cantilevers to probe E. coli ZK1056 adhesion to five substrates with distinct and well-characterized surface chemistries, including fluorinated, amine-terminated, and PEG-like monolayers, as well as unmodified silicon wafer and mica. Notably, after only 0-10 s of contact time, the biofilms adhere strongly to fluorinated and amine-terminated monolayers as well as to mica and weakly to "antifouling" PEG monolayers, despite the wide variation in hydrophobicity and charge of these substrates. In each case the AFM retraction curves display distinct adhesion profiles in terms of both force and distance, highlighting the cells' ability to adapt their adhesive properties to disparate surfaces. Specific inhibition of the pilus protein FimH by a nonhydrolyzable mannose analogue leads to diminished adhesion in all cases, demonstrating the critical role of type I pili in adhesion by this strain to surfaces bearing widely different functional groups. The strong and adaptable binding of FimH to diverse surfaces has unexpected implications for the design of antifouling surfaces and antiadhesion therapies.

Marangoni flow of Ag nanoparticles from the fluid-fluid interface.

Fluid flow is observed when a volume of passivated Ag nanoparticles suspended in chloroform is mixed with a water/ethanol (v/v) mixture containing acidified 11-mercaptoundecanoic acid. Following mechanical agitation, Ag nanoparticles embedded in a film are driven from the organic-aqueous interface. A reddish-brown colored film, verified by transmission electron microscopy to contain uniformly dispersed Ag nanoparticles, is observed to spontaneously climb the interior surface of an ordinary, laboratory glass vial. This phenomenon is recorded by a digital video recorder, and a measurement of the distance traveled by the film front versus time is extracted. Surface (interfacial) tension gradients due to surfactant concentration, temperature, and electrostatic potential across immiscible fluids are known to drive interface motion; this well-known phenomenon is termed Marangoni flow or the Marangoni effect. Experimental results are presented that show the observed mass transfer is dependent on an acid surfactant concentration and on the volume fraction of water in the aqueous phase, consistent with fluid flow induced by interfacial tension gradients. In addition, an effective desorption rate constant for the Marangoni flow is measured in the range of approximately 0.01 to approximately 1 s(-1) from a fit to the relative film front distance traveled versus time data. The fit is based on a time-dependent expression for the surface (interface) excess for desorption kinetics. Such flow suggests that purposeful creation of interfacial tension gradients may aid in the transfer of 2- and 3-dimensional assemblies, made with nanostructures at the liquid-liquid interface, to solid surfaces.

Quantitative changes in the elasticity and adhesive properties of Escherichia coli ZK1056 prey cells during predation by bdellovibrio bacteriovorus 109J.

Atomic force microscopy (AFM) was used to explore the changes that occur in Escherichia coli ZK1056 prey cells while they are being consumed by the bacterial predator Bdellovibrio bacteriovorus 109J. Invaded prey cells, called bdelloplasts, undergo substantial chemical and physical changes that can be directly probed by AFM. In this work, we probe the elasticity and adhesive properties of uninvaded prey cells and bdelloplasts in a completely native state in dilute aqueous buffer without chemical fixation. Under these conditions, the rounded bdelloplasts were shown to be shorter than uninvaded prey cells. More interestingly, the extension portions of force curves taken on both kinds of cells clearly demonstrate that bdelloplasts are softer than uninvaded prey cells, reflecting a decrease in bdelloplast elasticity after invasion by Bdellovibrio predators. On average, the spring constant of uninvaded E. coli cells (0.23 +/- 0.02 N/m) was 3 times stiffer than that of the bdelloplast (0.064 +/- 0.001 N/m) when measured in a HEPES-metals buffer. The retraction portions of the force curves indicate that compared to uninvaded E. coli cells bdelloplasts adhere to the AFM tip with much larger pull-off forces but over comparable retraction distances. The strength of these adhesion forces decreases with increasing ionic strength, indicating that there is an electrostatic component to the adhesion events.

Rapid isolation of host-independent Bdellovibrio bacteriovorus.

A new method of isolating host-independent Bdellovibrio bacteriovorus has been developed. Filtered suspensions of host-dependent cells are dropped in small volumes onto 0.2 microm membranes laid on rich media agar. Significant growth is observed within 1-2 days; these cells were confirmed to be B. bacteriovorus using microscopic observations and PCR.

Exploring the interaction of ruthenium(II) polypyridyl complexes with DNA using single-molecule techniques.

Here we explore DNA binding by a family of ruthenium(II) polypyridyl complexes using an atomic force microscope (AFM) and optical tweezers. We demonstrate using AFM that Ru(bpy)2dppz2+ intercalates into DNA (K(b) = 1.5 x 10(5) M(-1)), as does its close relative Ru(bpy)2dppx2+ (K(b) = 1.5 x 10(5) M(-1)). However, intercalation by Ru(phen)3(2+) and other Ru(II) complexes with K(b) values lower than that of Ru(bpy)2dppz2+ is difficult to determine using AFM because of competing aggregation and surface-binding phenomena. At the high Ru(II) concentrations required to evaluate intercalation, most of the DNA strands acquire a twisted, curled conformation that is impossible to measure accurately. The condensation of DNA on mica in the presence of polycations is well known, but it clearly precludes the accurate assessment by AFM of DNA intercalation by most Ru(II) complexes, though not by ethidium bromide and other monovalent intercalators. When stretching individual DNA molecules using optical tweezers, the same limitation on high metal concentration does not exist. Using optical tweezers, we show that Ru(phen)2dppz2+ intercalates avidly (K(b) = 3.2 x 10(6) M(-1)) whereas Ru(bpy)3(2+) does not intercalate, even at micromolar ruthenium concentrations. Ru(phen)3(2+) is shown to intercalate weakly (i.e., at micromolar concentrations (K(b) = 8.8 x 10(3) M(-1))). The distinct differences in DNA stretching behavior between Ru(phen)3(2+) and Ru(bpy)3(2+) clearly illustrate that intercalation can be distinguished from groove binding by pulling the DNA with optical tweezers. Our results demonstrate both the benefits and challenges of two single-molecule methods of exploring DNA binding and help to elucidate the mode of binding of Ru(phen)3(2+).

Atomic force microscopy of bacterial communities.

This chapter discusses atomic force microscopy (AFM) for the benefit of microbiologists who are interested in using this technique to examine the structures and dynamics of bacteria. AFM is a powerful technique for imaging biological samples at the nanometer to micrometer scale under nondestructive conditions. In order to be imaged with AFM, bacteria must be supported by a surface, which presents challenges because many laboratory strains of bacteria are planktonic. Still, in nature many bacteria live at surfaces and interfaces. This chapter discusses the benefits and difficulties of different methods that have been used to support bacteria on surfaces for AFM imaging and presents two methods in detail used to successfully grow and image bacteria at solid-liquid and solid-air interfaces. Using these methods it is possible to study bacterial morphology and interactions in a native state. These explorations by AFM have important applications to the study of different kinds of bacteria, interfacial bacterial communities, and biofilms.

Predation, death, and survival in a biofilm: Bdellovibrio investigated by atomic force microscopy.

Biofilms are complex microbial communities that are resistant to attack by bacteriophages and to removal by drugs and chemicals. Here we use atomic force microscopy (AFM) to image the attack on Escherichia coli biofilms by Bdellovibrio bacteriovorus 109J. Bdellovibrio is a small, predatory bacterium that invades and devours other Gram-negative bacteria. We demonstrate that under dilute nutrient conditions, bdellovibrios can prevent the formation of simple bacterial biofilms and destroy established biofilms; under richer conditions the prey bacteria persist and are not eradicated, but may be shifted toward solution populations. Using AFM we explore these bacterial interactions with more detail and accuracy than available by more traditional staining assays or optical microscopy. AFM also allows us to investigate the nanoscale morphological changes of the predator, especially those related to motility. This demonstration of Bdellovibrio's successful predation in a biofilm inspires us to consider ways that it might be used productively for industrial, medical, agricultural, and biodefensive purposes.

Film formation of Ag nanoparticles at the organic-aqueous liquid interface.

We report a wet-chemical method to make films by spontaneous assembly of passivated Ag nanoparticles at the organic-aqueous liquid interface. The interfacial films exhibit a blue opalescence and are characterized with transmission electron microscopy and UV-vis spectrophotometry. Measurements indicate that nanoparticles in the interfacial film can form superlattices and in some cases nanostructures.

Investigations into the life cycle of the bacterial predator Bdellovibrio bacteriovorus 109J at an interface by atomic force microscopy.

Atomic force microscopy was used to image Bdellovibrio bacteriovorus 109J, a gram-negative bacterial predator that consumes a variety of other gram-negative bacteria. In predator-prey communities grown on filters at hydrated air-solid interfaces, repeated cycles of hunting, invasion, growth, and lysis occurred readily even though the cells were limited to near two-dimensional movement. This system allowed us to image the bacteria directly without extensive preparation or modification, and many of the cells remained alive during imaging. Presented are images of the life cycle in two species of prey organisms, both Escherichia coli (a small prey bacterium that grows two-dimensionally on a surface) and Aquaspirillum serpens (a large prey bacterium that grows three-dimensionally on a surface), including high-resolution images of invaded prey cells called bdelloplasts. We obtained evidence for multiple invasions per prey cell, as well as significant heterogeneity in morphology of bdellovibrios. Mutant host-independent bdellovibrios were observed to have flagella and to excrete a coating that causes the predators to clump together on a surface. Most interestingly, changes in the texture of the cell surface membranes were measured during the course of the invasion cycle. Thus, coupled with our preparation method, atomic force microscopy allowed new observations to be made about Bdellovibrio at an interface. These studies raise important questions about the ways in which bacterial predation at interfaces (air-solid or liquid-solid) may be similar to or different from predation in solution.