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.

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.

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.

TiO2-photocatalyzed As(III) oxidation in a fixed-bed, flow-through reactor.

Compliance with the U.S. drinking water standard for arsenic (As) of 10 microg L(-1) is required in January 2006. This will necessitate implementation of treatment technologies for As removal by thousands of water suppliers. Although a variety of such technologies is available, most require preoxidation of As(III) to As(V) for efficient performance. Previous batch studies with illuminated TiO2 slurries have demonstrated that TiO2-photocatalyzed AS(III) oxidation occurs rapidly. This study examined reaction efficiency in a flow-through, fixed-bed reactor that provides a better model for treatment in practice. Glass beads were coated with mixed P25/sol gel TiO2 and employed in an upflow reactor irradiated from above. The reactor residence time, influent As(III) concentration, number of TiO2 coatings on the beads, solution matrix, and light source were varied to characterize this reaction and determine its feasibility for water treatment. Repeated usage of the same beads in multiple experiments or extended use was found to affect effluent As(V) concentrations but not the steady-state effluent As(III) concentration, which suggests that As(III) oxidation at the TiO2 surface undergoes dynamic sorption equilibration. Catalyst poisoning was not observed either from As(V) or from competitively adsorbing anions, although the higher steady-state effluent As(III) concentrations in synthetic groundwater compared to 5 mM NaNO3 indicated that competitive sorbates in the matrix partially hinder the reaction. A reactive transport model with rate constants proportional to incident light at each bead layer fit the experimental data well despite simplifying assumptions. TiO2-photocatalyzed oxidation of As(III) was also effective under natural sunlight. Limitations to the efficiency of As(III) oxidation in the fixed-bed reactor were attributable to constraints of the reactor geometry, which could be overcome by improved design. The fixed-bed TiO2 reactor offers an environmentally benign method for As(III) oxidation.

TiO2-photocatalyzed As(II) oxidation in aqueous suspensions: reaction kinetics and effects of adsorption.

Oxidation of arsenite, As(III), to arsenate, As(V), is required for the efficient removal of arsenic by many water treatment technologies. The photocatalyzed oxidation of As(III) on titanium dioxide, TiO2, offers an environmentally benign method for this unit operation. In this study, we explore the efficacy and mechanism of TiO2-photocatalyzed As(III) oxidation at circumneutral pH and over a range of As(III) concentrations approaching those typically encountered in water treatment systems. We focus on the effect of As adsorption on observed rates of photooxidation. Adsorption (in the dark) of both As(III) and As(V) on Degussa P25 TiO2 was examined at pH 6.3 over a range in dissolved arsenic concentrations, [As]diss, of 0.10-89 microM and 0.2 or 0.05 g L(-1) TiO2 for As(III) and As(V), respectively. Adsorption isotherms generally followed the Langmuir-Hinshelwood model with As(III) exhibiting an adsorption maxima of 32 micromol g(-1). As(V) adsorption did not reach a plateau under the experimental conditions examined; the maximum adsorbed concentration observed was 130 micromol g(-1). The extent of As(III) and As(V) adsorption observed at the beginning and end of the kinetic studies was consistent with that observed in the adsorption isotherms. Kinetic studies were performed in batch systems at pH 6.3 with 0.8-42 microM As(III) and 0.05 g L(-1) TiO2; complete oxidation of As(III) was observed within 10-60 min of irradiation at 365 nm. The observed effect of As(III) concentration on reaction kinetics was consistent with surface saturation at higher concentrations. Addition of phosphate at 0.5-10 microM had little effect on either As(III) sorption or its photooxidation rate but did inhibit adsorption of the product As(V). The selective use of hydroxyl radical quenchers and superoxide dismutase demonstrated that superoxide, O2-, plays a major role in the oxidation of As(III) to As(V).