Carolyn Marks

Distance Dependence of Electron Transfer Kinetics for Azurin Protein Adsorbed to Monolayer Protected Nanoparticle Film Assemblies

The distance dependence and kinetics of the heterogeneous electron transfer (ET) reaction for the redox protein azurin adsorbed to an electrode modified with a gold nanoparticle film are investigated using cyclic voltammetry. The nanoparticle films are comprised of nonaqueous nanoparticles, known as monolayer-protected clusters (MPCs), which are covalently networked with dithiol linkers. The MPC film assembly serves as an alternative adsorption platform to the traditional alkanethiolate self-assembled monolayer (SAM) modified electrodes that are commonly employed to study the ET kinetics of immobilized redox proteins, a strategy known as protein monolayer electrochemistry. Voltammetric analysis of the ET kinetics for azurin adsorbed to SAMs of increasing chain length results in quasi-reversible voltammetry with significant peak splitting. We observed rate constants (k degrees (ET)) of 12-20 s(-1) for the protein at SAMs of shorter alkanethiolates that decays exponentially (beta = 0.9/CH(2) or 0.8/A) at SAMs of longer alkanethiolates (9-11 methylene units) or an estimated distance of 1.23 nm and is representative of classical electronic tunneling behavior over increasing distance. Azurin adsorbed to the MPC film platforms of increasing thickness results in reversible voltammetry with very little voltammetric peaks splitting and nearly negligible decay of the ET rate over significant distances up to 20 nm. The apparent lack of distance dependence for heterogeneous ET reactions at MPC film assemblies is attributed to a two-step mechanism involving extremely fast electronic hopping through the MPC film architecture. These results suggest that MPC platforms may be used in protein monolayer electrochemistry to create adsorption platforms of higher architecture that can accommodate greater than monolayer protein coverage and increase the Faradaic signal, a finding with significant implications for amperometric biosensor design and development.

Proteolysis of BB0323 results in two polypeptides that impact physiologic and infectious phenotypes in Borrelia burgdorferi

Borrelia burgdorferi gene product BB0323 is required for cell fission and pathogen persistence in vivo. Here, we show that BB0323, which is conserved among globally prevalent infectious strains, supports normal spirochaete growth and morphology even at early phases of cell division. We demonstrate that native BB0323 undergoes proteolytic processing at the C‐terminus, at a site after the first 202 N‐terminal amino acids. We further identified a periplasmic BB0323 binding protein in B. burgdorferi, annotated as BB0104, having serine protease activity responsible for the primary cleavage of BB0323 to produce discrete N‐ and C‐terminal polypeptides. These two BB0323 polypeptides interact with each other, and either individually or as a complex, are associated with multiple functions in spirochaete biology and infectivity. While N‐terminal BB0323 is adequate to support cell fission, the C‐terminal LysM domain is dispensable for this process, despite its ability to bind B. burgdorferi peptidoglycan. However, the LysM domain or the precisely processed BB0323 product is essential for mammalian infection. As BB0323 is a membrane protein crucial for B. burgdorferi survival in vivo, exploring its function may suggest novel ways to interrupt infection while enhancing our understanding of the intricate spirochaete fission process.

Formation and morphology of epidermal sclerites from a deep-sea hydrothermal vent solenogaster (Helicoradomenia sp., Solenogastres, Mollusca)

The deep-sea hydrothermal vent solenogaster Helicoradomenia is covered with calcium carbonate sclerites. Light and electron microscopy reveal varying morphologies of these sclerites. Many sclerites have hollow tips and/or are pitted and etched. Bacteria are found on and in sclerites. Initial sclerite formation occurs in an extracellular crystalline chamber formed by the invagination of a cuboidal basal cell of the columnar microvillus mantle epithelium. As the sclerite grows, it fills the crystalline chamber resulting in direct contact with the microvilli of both the basal cell and neighboring secondary sclerite-forming cells. These cells shape a collar around the base of the growing sclerite. As growth continues, the sclerite-forming cells stretch around the sclerite forming a sheath in which the base of the sclerite resides. Mature sclerites grow through the cuticle into the external environment. The erosion pattern of sclerites reveals a less stable inner medullary region and a harder outer cortical region. This points to a secondary character state, where foremost hollow acicular sclerites develop into solid sclerites. This is in agreement with the systematic position of the genus Helicoradomenia within Simrothiellidae, a taxon typically with hollow sclerites.

Combinatorial Mapping of Substrate Step Edge Effects on Diblock Copolymer Thin Film Morphology and Orientation

We have used a combinatorial gradient technique to map precisely how the terrace structure and microdomain lattice alignment in a thin film of a sphere-forming diblock copolymer are affected by both the thickness of the copolymer film and the height of a series of parallel step edges fabricated on the substrate. We find that for film thicknesses slightly incommensurate with integer numbers of sphere layers, the step edges act as nucleation sites for regions with one more or one fewer layers of spheres. We also find that for our system, the hexagonal lattice formed by a single layer of spheres on the low side of a step edge is aligned along the direction of the step edge only where the film on the high side is sufficiently thin to support only a wetting layer of copolymer material. This work will guide the tuning of film thickness and step height in future studies and applications of graphoepitaxy in block copolymer films.

Incorporating Advanced Microscopy Techniques into the Undergraduate Curriculum

One topic I think we can all agree on as microscopist, is the need to train and educate today’s students in the area of advanced microscopy so that we have microscopy experts in the future. However, Microscopy is not for everyone. So, how do you reach the subset of students who may have an interest in microscopy? How do you recruit them into training? You take them as undergraduates, and give them hands-on exposure, the earlier in their studies, the better.

Microbial Symbionts and Sponge Heterotrophy; Morphological Aspects of Sponge: Symbiont Integration via SEM Analysis

The ecological and evolutionary relationship between sponges and their symbiotic microflora remains poorly understood, which limits our ability to understand broad scale patterns in benthicpelagic coupling on coral reefs. Sponges can process > 40 ml of water per kg of tissue per hour, removing microbes and dissolved organic matter during the process. Some sponge species are known to harbor extraordinarily dense populations of microbes (high microbial abundance (HMA) sponges), while other species maintain bacterial populations at very low levels (low microbial abundance sponges (LMA)). HMA and LMA sponges have significantly different pumping rates and trophic states. The role these different feeding strategies play in benthic-pelagic coupling on coral reefs may be significant. Previous work in this area examined the importance of sponge pumping rates. It has been suggested that the dense tissue in the HMA sponges would result in smaller water canals, which would contribute to a decrease in water flow [1]. Previous work has confirmed these predictions given that HMA sponges displayed a slower pumping rate than LMA sponges [2]. The differences in microbial abundance suggested that there is a morphological explanation for the differences in pumping rate, which also provides evidence of significant differences between HMA and LMA sponge morphology and physiology.

Investigating Bacterial Endosymbiosis in Leptogorgia virgulata Using a Molecular and SEM Approach

The common sea-whip, Leptogorgia virgulata is reported to be asymbiotic in terms of endosymbiotic dinoflagellates. Though we tend to believe that L. virgulata uses possible bacterial symbionts as a mechanism to reduce acidic environments within the gorgonian tissue to form calcium carbonate spicules. This study investigates urease production by bacteria within L. virgulata to reduce acidity. The goal of the study is to characterize possible bacterial symbionts and using SEM determine locality within L. virgulata tissue. Possible microbe symbionts were isolated from L. virgulata tissue through a series of dilutions where bacteria colonies unique to surface bacteria were cultured and tested for urease production. Bacteria that were positive for urease production were then prepared for SEM analysis and a series of magnification SEM references for cultured bacteria were compared to freeze fractured L. virgulata SEM prepared samples to determine possible microbe symbiont location in the gorgonian tissue. Possible bacterial endosymbionts were characterized using univ518r primers typical for identifying universal bacteria. Preliminary molecular data suggests there are unique microbes inhabiting L. virgulata tissue, though their location is still being investigated. Microsc Microanal 15(Suppl 2), 2009 Copyright 2009 Microscopy Society of America doi: 10.1017/S1431927609098675 836