Genomic and proteomic characterization of two novel siphovirus infecting the sedentary facultative epibiont cyanobacterium Acaryochloris marina.

Acaryochloris marina is a symbiotic species of cyanobacteria that is capable of utilizing far-red light. We report the characterization of the phages A-HIS1 and A-HIS2, capable of infecting Acaryochloris. Morphological characterization of these phages places them in the family Siphoviridae. However, molecular characterization reveals that they do not show genetic similarity with any known siphoviruses. While the phages do show synteny between each other, the nucleotide identity between the phages is low at 45-67%, suggesting they diverged from each other some time ago. The greatest number of genes shared with another phage (a myovirus infecting marine Synechococcus) was four. Unlike most other cyanophages and in common with the Siphoviridae infecting Synechococcus, no photosynthesis-related genes were found in the genome. CRISPR (clustered regularly interspaced short palindromic repeats) spacers from the host Acaryochloris had partial matches to sequences found within the phages, which is the first time CRISPRs have been reported in a cyanobacterial/cyanophage system. The phages also encode a homologue of the proteobacterial RNase T. The potential function of RNase T in the mark-up or digestion of crRNA hints at a novel mechanism for evading the host CRISPR system.

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

Quantitative analysis and application of tip position modulation-scanning electrochemical microscopy.

Tip position modulation (TPM) involves moving the ultramicroelectrode (UME) tip of a scanning electrochemical microscope (SECM) perpendicular to the substrate in a sinusoidal fashion with a small amplitude compared to the tip/sample separation. The UME, which serves as the working electrode in a conventional voltammetric setup, is held at a potential to detect a species in solution at a transport-limited rate and the resulting current (ac and dc) is measured. This paper shows that tip-induced convection is an important factor in TPM. A model has been developed that describes the TPM response for the most challenging case of an inert substrate, where tip-induced convective effects compared to diffusion are greatest. The model provides an improved description of the ac response compared to existing treatment, as evidenced by the analysis of TPM-SECM approach curves (current-distance characteristics). The extension of the model to SECM-induced transfer is considered and it is shown that one can extract highly precise information on the permeability of a sample from such measurements, for which experiments and theory are compared. The prospects for using the technique more widely are highlighted and routes to improving the theoretical analysis further are briefly discussed.

Scanning electrochemical microscopy (SECM) studies of catalytic EC' processes: theory and experiment for feedback, generation/collection and imaging measurements.

This paper describes the use of scanning electrochemical microscopy (SECM) in the tip generation/substrate collection (TG/SC), or feedback, mode and substrate generation/tip collection (SG/TC) mode to measure homogeneous kinetics in the catalytic EC' process. Theoretical analyses of both configurations have been developed numerically to allow the optimal conditions for sensitive kinetic measurements to be determined. This is shown to involve collection efficiency measurements as a function of tip-substrate electrode distance in the case of TG/SC measurements and tip (collector current) images in a plane normal to the substrate electrode for the SG/TC mode. An important consideration for the SECM configuration (particularly for TG/SC and feedback measurements) is that the electroinactive co-reactant may be depleted more significantly than with other electrode geometries, because of cycling of the redox couple in the tip/substrate electrode gap, while the co-reactant can only enter this gap by hindered diffusion. The approaches described are examined through studies of the oxidation of amidopyrine by electrogenerated Fe(CN) in 0.5 mol dm(-3) aqueous KOH solution. A second-order rate constant of 390 ± 80 dm(3) mol(-1) s(-1) is obtained from TG/SC measurements, consistent with SG/TC quantitative imaging measurements. The consistency of the kinetic measurements confirms the validity of the approaches described. The kinetic constant is lower than expected based on previous ultramicroelectrode (UME) studies, and this is attributed to the fact that background currents for the direct heterogeneous oxidation of amidopyrine are more significant with conventional UME measurements, which will tend to enhance the current measured and may therefore lead to an overestimation of kinetic constants. The TG/SC approach, on the other hand, provides a means of making dual-electrode collection efficiency measurements with diffusional feedback of the redox couple, leading to superior voltammetric responses and enabling more accurate kinetic determination.

Scanning ion conductance microscopy: a model for experimentally realistic conditions and image interpretation.

Scanning ion conductance microscopy (SICM) is a scanned probe microscopy technique in which the probe is a fine glass pipet filled with a contact (reference) electrode and an electrolyte solution. The current flow between the reference electrode and a second reference electrode positioned in bulk solution when the two electrodes are biased externally can be used as a feedback signal to maintain a constant separation between the tip and a surface during imaging. In usual practice the tip position is modulated over a small amplitude perpendicular to the surface, and the resulting alternating current (AC) is used as the feedback signal, although the direct current can also be used. A comprehensive model for the current response is reported. Laplace's equation has been solved for the electrolyte solution for a range of tip geometries, enabling the factors controlling the tip current to be identified. The approach developed is shown to represent an improvement over earlier semiempirical treatments. To explore the influence of surface topography on the (AC) current response, various surfaces have been considered, including a tip moved toward a planar surface (in the normal direction) and tips scanned over a pit and a step in the surface. The results have allowed a critical assessment of the SICM response as a means of probing surface topography. Features identified through simulation have been found in experiments through studies of two model substrates for which imaging results are reported. In typical experimental practice, the response of the SICM tip to surface features occurs over much greater lateral distances than the size of the pipet aperture.

Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina.

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 microE m(-2) s(-1) without shaking and 100 microE m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410.

Scanning electrochemical microscopy: principles and applications to biophysical systems.

This review highlights numerous and wide ranging biophysical and biochemical applications of scanning electrochemical microscopy (SECM). SECM instrumentation and theoretical modelling, necessary for experimental interpretation, are outlined, followed by a detailed discussion of the diverse applications of this technique. These include the measurement of flow through membranes, the determination of kinetic parameters of reactions, the investigation of the permeability of small molecules in tissues and monitoring biological processes, such as the production of oxygen or nitric oxide by cells. The significant impact of micro-electrochemical techniques on our understanding of basic physicochemical processes at biologically relevant interfaces is also considered. Studies reviewed include transport across and within bilayers and monolayers. Recent advances in SECM include the combination of SECM with other techniques, such as atomic force microscopy and optical microscopy. These developments are highlighted, along with prospects for the future.

Polyaniline Langmuir-Blodgett films: formation and properties.

The deposition and characterisation of Langmuir-Blodgett (LB) layers of polyaniline (PAN) on solid supports is described. Langmuir films were spread as a mixture of PAN and dodecylbenzenesulfonic acid (DBSA) at the water/air interface and deposited on either glass or indium tin oxide (ITO). Mono- and multi-layer films of PAN/DBSA were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), absorption spectroscopy and cyclic voltammetry (CV). The ultrathin films produced were found to be highly uniform and very stable. Further characterisation of the films was accomplished by scanning electrochemical microscopy (SECM) in the feedback mode. It was found that the conductivity depended strongly on the pH of the solution and the number of layers deposited. Values for the pH-dependent lateral conductivity of PAN LB films are reported.

Fluorescence confocal laser scanning microscopy as a probe of pH gradients in electrode reactions and surface activity.

The application of fluorescence confocal laser scanning microscopy (CLSM) to quantify three-dimensional pH gradients near electrode surfaces is described. The methodology utilizes a trace quantity of a fluorescent dye, fluorescein, in solution, which fluoresces strongly above pH 6.5, to map the pH adjacent to various ultramicroelectrodes undergoing electrochemical processes that lead to pH changes. The experimental fluorescence profiles, determined by CLSM, have been compared to models by solving the underlying mass transport equations, including the effect of natural convection, using the finite element method. The methodology has been validated through studies of the galvanostatic reduction of water at both disk and ring ultramicroelectrodes. The fluorescence profiles were found to be highly sensitive to both the initial bulk solution pH and applied current in a predictable fashion. The potentiostatic reduction of oxygen has been investigated at 25- and 10-microm-diameter platinum electrodes to confirm the effective number of electrons transferred in the reaction. Finally, the application of this methodology to observe defects in microelectrode arrays, particularly those that cannot be seen by optical microscopy, is described.