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The chloroplast is a central part of plant cells, as this is the organelle where the photosynthesis, fixation of inorganic carbon, and other key functions related to fatty acid synthesis and amino acid synthesis occur. Since this organelle should be an integral part of any genome-scale metabolic model for a microalgae or a higher plant, it is of great interest to generate a detailed and standardized chloroplast model. Additionally, we see the need for a novel type of sub-model template, or organelle model, which could be incorporated into a larger, less specific genome-scale metabolic model, while allowing for minor differences between chloroplast-containing organisms. The result of this work is the very first standardized chloroplast model, iGR774, consisting of 788 reactions, 764 metabolites, and 774 genes. The model is currently able to run in three different modes, mimicking the chloroplast metabolism of three photosynthetic microalgae-Nannochloropsis gaditana, Chlamydomonas reinhardtii and Phaeodactylum tricornutum. In addition to developing the chloroplast metabolic network reconstruction, we have developed multiple software tools for working with this novel type of sub-model in the COBRA Toolbox for MATLAB, including tools for connecting the chloroplast model to a genome-scale metabolic reconstruction in need of a chloroplast, for switching the model between running in different organism modes, and for expanding it by introducing more reactions either related to one of the current organisms included in the model, or to a new organism.

Cyanobacteria have high iron requirements due to iron-rich photosynthetic machineries. Despite the high concentrations of iron in the Earth&rsquo, s crust, iron is limiting in many marine environments due to iron&rsquo, s low solubility. Oxic conditions leave a large portion of the ocean&rsquo, s iron pool unavailable for biotic uptake, and so the physiochemical properties of iron are hugely important for iron&rsquo, s bioavailability. Our study is the first to investigate the effect of iron source on iron internalization and extracellular reduction by Synechococcus sp. PCC 7002. The results indicated that the amorphous iron hydrolysis species produced by FeCl3 better support growth in Synechococcus through more efficient iron internalization and a larger degree of extracellular reduction of iron than the crystalline FeO(OH). An analysis of dissolved iron (II) indicated that biogenic reduction took place in cultures of Synechococcus grown on both FeCl3 and FeO(OH).

The precise expression of genes is one of the foundations of biotechnology. Here we present GeneEE, a straightforward method for generating artificial gene expression systems. We demonstrate that GeneEE segments, containing a 200 nucleotide DNA with random nucleotide composition, can facilitate constitutive and inducible gene expression. To highlight the universal character of our method, we demonstrate GeneEE-mediated gene and protein expression in six bacterial species and Baker’s yeast.

Siderophores are low-molecular-weight metal chelators that function in microbial iron uptake. As iron limits primary productivity in many environments, siderophores are of great ecological importance. Additionally, their metal binding properties have attracted interest for uses in medicine and bioremediation. Here, we review the current state of knowledge concerning the siderophores produced by cyanobacteria. We give an overview of all cyanobacterial species with known siderophore production, finding siderophores produced in all but the most basal clades, and in a wide variety of environments. We explore what is known about the structure, biosynthesis, and cycling of the cyanobacterial siderophores that have been characterized: Synechobactin, schizokinen and anachelin. We also highlight alternative siderophore functionality and technological potential, finding allelopathic effects on competing phytoplankton and likely roles in limiting heavy-metal toxicity. Methodological improvements in siderophore characterization and detection are briefly described. Since most known cyanobacterial siderophores have not been structurally characterized, the application of mass spectrometry techniques will likely reveal a breadth of variation within these important molecules. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Iron is an essential, yet scarce, nutrient in marine environments. Phytoplankton, and especially cyanobacteria, have developed a wide range of mechanisms to acquire iron and maintain their iron-rich photosynthetic machinery. Iron limitation studies often utilize either oceanographic methods to understand large scale processes, or laboratory-based, molecular experiments to identify underlying molecular mechanisms on a cellular level. Here, we aim to highlight the benefits of both approaches to encourage interdisciplinary understanding of the effects of iron limitation on cyanobacteria with a focus on avoiding pitfalls in the initial phases of collaboration. In particular, we discuss the use of trace metal clean methods in combination with sterile techniques, and the challenges faced when a new collaboration is set up to combine interdisciplinary techniques. Methods necessary for producing reliable data, such as High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS), Flow Injection Analysis Chemiluminescence (FIA-CL), and 77K fluorescence emission spectroscopy are discussed and evaluated and a technical manual, including the preparation of the artificial seawater medium Aquil, cleaning procedures, and a sampling scheme for an iron limitation experiment is included. This paper provides a reference point for researchers to implement different techniques into interdisciplinary iron studies that span cyanobacteria physiology, molecular biology, and biogeochemistry. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Photosynthetic fluorescence emission spectra measurement at the temperature of 77 K (–196°C) is an often-used technique in photosynthesis research. At low temperature, biochemical and physiological processes that modulate fluorescence are mostly abolished, and the fluorescence emission of both PSI and PSII become easily distinguishable. Here we briefly review the history of low-temperature chlorophyll fluorescence methods and the characteristics of the acquired emission spectra in oxygen-producing organisms. We discuss the contribution of different photosynthetic complexes and physiological processes to fluorescence emission at 77 K in cyanobacteria, green algae, heterokont algae, and plants. Furthermore, we describe practical aspects for obtaining and presenting 77 K fluorescence spectra. © The Author(s) 2018 Open Access. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

International audience; Molecular mechanisms of phosphorus (P) limitation are of great interest for understanding algal production in aquatic ecosystems. Previous studies point to P limitation-induced changes in lipid composition. As, in microalgae, the molecular mechanisms of this specific P stress adaptation remain unresolved, we reveal a detailed phospholipid-recycling scheme inNannochloropsis oceanicaand describe important P acquisition genes based on highly corresponding transcriptome and lipidome data. Initial responses to P limitation showed increased expression of genes involved in P uptake and an expansion of the P substrate spectrum based on purple acid phosphatases. Increase in P trafficking displayed a rearrangement between compartments by supplying P to the chloroplast and carbon to the cytosol for lipid synthesis. We propose a novel phospholipid-recycling scheme for algae that leads to the rapid reduction of phospholipids and synthesis of the P-free lipid classes. P mobilization through membrane lipid degradation is mediated mainly by two glycerophosphoryldiester phosphodiesterases and three patatin-like phospholipases A on the transcriptome level. To compensate for low phospholipids in exponential growth,N. oceanicasynthesized sulfoquinovosyldiacylglycerol and diacylglyceroltrimethylhomoserine. In this study, it was shown that anN. oceanicastrain has a unique repertoire of genes that facilitate P acquisition and the degradation of phospholipids compared with other stramenopiles. The novel phospholipid-recycling scheme opens new avenues for metabolic engineering of lipid composition in algae.

Manganese is an essential element required by cyanobacteria, as it is an essential part of the oxygen-evolving center of photosystem II. In the presence of atmospheric oxygen, manganese is present as manganese oxides, which have low solubility and consequently provide low bioavailability. It is unknown if cyanobacteria are able to utilize these manganese sources, and what mechanisms may be employed to do so. Recent evidence suggests that type IV pili in non-photosynthetic bacteria facilitate electron donation to extracellular electron acceptors, thereby enabling metal acquisition. Our present study investigates whether PilA1 (major pilin protein of type IV pili) enables the cyanobacterium Synechocystis PCC 6808 to access to Mn from manganese oxides. We present physiological and spectroscopic data, which indicate that the presence of PilA1 enhances the ability of cyanobacteria to grow on manganese oxides. These observations suggest a role of PilA1-containing pili in cyanobacterial manganese acquisition. © 2017 Lamb, Hohmann-Marriott. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background: Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrickcompatible integration modules is desirable to further simplifying chromosomal integrations. Results: We designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the Synechococcus chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for Synechococcus significantly. Conclusions: The optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in Synechococcus. We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing Synechococcus as the prominent chassis for future synthetic biology applications. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)

The importance of n-3 long chain polyunsaturated fatty acids (LC-PUFAs) for human health has received more focus the last decades, and the global consumption of n-3 LC-PUFA has increased. Seafood, the natural n-3 LC-PUFA source, is harvested beyond a sustainable capacity, and it is therefore imperative to develop alternative n-3 LC-PUFA sources for both eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Genera of algae such as Nannochloropsis, Schizochytrium, Isochrysis and Phaedactylum within the kingdom Chromista have received attention due to their ability to produce n-3 LC-PUFAs. Knowledge of LC-PUFA synthesis and its regulation in algae at the molecular level is fragmentary and represents a bottleneck for attempts to enhance the n-3 LC-PUFA levels for industrial production. In the present review, Phaeodactylum tricornutum has been used to exemplify the synthesis and compartmentalization of n-3 LC-PUFAs. Based on recent transcriptome data a co-expression network of 106 genes involved in lipid metabolism has been created. Together with recent molecular biological and metabolic studies, a model pathway for n-3 LC-PUFA synthesis in P. tricornutum has been proposed, and is compared to industrialized species of Chromista. Limitations of the n-3 LC-PUFA synthesis by enzymes such as thioesterases, elongases, acyl-CoA synthetases and acyltransferases are discussed and metabolic bottlenecks are hypothesized such as the supply of the acetyl-CoA and NADPH. A future industrialization will depend on optimization of chemical compositions and increased biomass production, which can be achieved by exploitation of the physiological potential, by selective breeding and by genetic engineering. © 2013 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/3.0/).

The chlorophyll content is an important experimental parameter in agronomy and plant biology research. In this report, we explore the feasibility of determining total concentration of extracts containing chlorophyll a and chlorophyll b by chlorophyll fluorescence. We found that an excitation at 457 nm results in the same integrated fluorescence emission for a molecule of chlorophyll a and a molecule of chlorophyll b. The fluorescence yield induced by 457 nm is therefore proportional to total molar chlorophyll concentration. Based on this observation, we designed an instrument to determine total chlorophyll concentrations. A single light emitting diode (LED) is used to excite chlorophyll extracts. After passing through a long-pass filter, the fluorescence emission is assessed by a photodiode. We demonstrate that this instrument facilitates the determination of total chlorophyll concentrations. We further extended the functionality of the instrument by including LEDs emitting at 435 and 470 nm wavelengths, thereby preferentially exciting chlorophyll a and chlorophyll b. This instrument can be used to determine chlorophyll a and chlorophyll b concentrations in a variety of organisms containing different ratios of chlorophylls. Monte-Carlo simulations are in agreement with experimental data such that a precise determination of chlorophyll concentrations in carotenoid-containing biological samples containing a concentration of less than 5 nmol/mL total chlorophyll can be achieved.

Energy conversion of sunlight by photosynthetic organisms has changed Earth and life on it. Photosynthesis arose early in Earth's history, and the earliest forms of photosynthetic life were almost certainly anoxygenic (non-oxygen evolving). The invention of oxygenic photosynthesis and the subsequent rise of atmospheric oxygen approximately 2.4 billion years ago revolutionized the energetic and enzymatic fundamentals of life. The repercussions of this revolution are manifested in novel biosynthetic pathways of photosynthetic cofactors and the modification of electron carriers, pigments, and existing and alternative modes of photosynthetic carbon fixation. The evolutionary history of photosynthetic organisms is further complicated by lateral gene transfer that involved photosynthetic components as well as by endosymbiotic events. An expanding wealth of genetic information, together with biochemical, biophysical, and physiological data, reveals a mosaic of photosynthetic features. In combination, these data provide an increasingly robust framework to formulate and evaluate hypotheses concerning the origin and evolution of photosynthesis.

It is shown that dark-field images collected in the scanning transmission electron microscope (STEM) at two different camera lengths yield quantitative distributions of both the heavy and light atoms in a stained biological specimen. Quantitative analysis of the paired STEM images requires knowledge of the elastic scattering cross sections, which are calculated from the NIST elastic scattering cross section database. The results reveal quantitative information about the distribution of fixative and stain within the biological matrix, and provide a basis for assessing detection limits for heavy-metal clusters used to label intracellular proteins. In sectioned cells that have been stained only with osmium tetroxide, we find an average of 1.2 ± 0.1 Os atom per nm 3 , corresponding to an atomic ratio of Os:C atoms of approximately 0.02, which indicates that small heavy atom clusters of Undecagold and Nanogold can be detected in lightly stained specimens.

Green sulfur bacteria possess a complex photosynthetic machinery. The dominant light harvesting systems are chlorosomes, which consist of bacteriochlorophyll c, d or e oligomers with small amounts of protein. The chlorosomes are energetically coupled to the membrane-embedded iron sulfur-type reaction center via a bacteriochlorophyll a-containing baseplate protein and the Fenna–Matthews–Olson (FMO) antenna protein. The fluorescence yield and spectral properties of these photosynthetic complexes were investigated in intact cells of several species of green sulfur bacteria under physiological, anaerobic conditions. Surprisingly, green sulfur bacteria show a complex modulation of fluorescence yield upon illumination that is very similar to that observed in oxygenic phototrophs. Within a few seconds of illumination, the fluorescence reaches a maximum, which decreases within a minute of illumination to a lower steady state. Fluorescence spectroscopy reveals that the fluorescence yield during both processes is primarily modulated on the FMO-protein level, while the emission from chlorosomes remains mostly unchanged. The two most likely candidates that modulate bacteriochlorophyll fluorescence are (1) direct excitation quenching at the FMO-protein level and (2) indirect modulation of FMO-protein fluorescence by the reduction state of electron carriers that are part of the reaction center.

The fluorescence emission spectrum of photosynthetic microorganisms at liquid nitrogen temperature (77 K) provides important insights into the organization of the photosynthetic machinery of bacteria and eukaryotes, which cannot be observed at room temperature. Conventionally, to obtain such spectra, a large and costly table-top fluorometer is required. Recently portable, reliable, and largely maintenance-free instruments have become available that can be utilized to accomplish a wide variety of spectroscopy-based measurements in photosynthesis research. In this report, we show how to build such an instrument in order to record 77K fluorescence spectra. This instrument consists of a low power monochromatic light-emitting diode (LED), and a portable CCD array based spectrometer. The optical components are coupled together using a fiber optic cable, and a custom made housing that also supports a dewar flask. We demonstrate that this instrument facilitates the reliable determination of chlorophyll fluorescence emission spectra for the cyanobacterium Synechocystis sp. PCC 6803, and the green alga Chlamydomonas reinhardtii. © 2015 Lamb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.

The precise determination of object positions within a specimen grid is important for many applications in electron microscopy. For example, real-time position determination is necessary for current statistical approaches and the efficient mapping and relocation of objects. Unfortunately, precise real-time position determination is not available on many older electron microscopes with manual stage controls. This report demonstrates the cost-effective and flexible implementation of a digital position determination system that can be adapted to many hand-operated electron microscopes. A customized solution that includes the hardware and software to accomplish position determination is presented. Lists of required parts, instructions for building the hardware, and descriptions of the developed programs are included. Two LED-photodiode assemblies detect x and y movements via an optical wheel that is in physical contact with the mechanical x and y stage control elements. These detector assemblies are interfaced with an integrated circuit that converts movement information into serial port-compatible signals, which are interpreted by a computer with specialized software. Two electron microscopes, a Philips CM12 (S)TEM and a Philips 201 TEM, were equipped with the described digital position determination system. The position fidelity and position fidelity after reloading of grids were determined for both microscopes. The determined position deviation was 1.06 microm in the x axis and 0.565 microm in the y axis for the Philips CM12 (S)TEM, and 0.303 microm in the x axis and 0.545 microm in the y axis for the Philips 201 TEM. After reloading and computational realigning, the determined average position variation was 2.66 microm in the x axis and 2.61 microm in the y axis for the Philips CM12 (S)TEM, and 1.13 microm in the x axis and 1.27 microm in the y axis for the Philips 201 TEM.

The lipid-producing model alga Nannochloropsis oceanica has a distinct photosynthetic machinery. This organism possesses chlorophyll a as its only chlorophyll species, and has a high ratio of PSI to PSII. This high ratio of PSI to PSII may affect the redox state of the plastoquinone pool during exposure to light, and consequently may play a role in activating photoprotection mechanisms. We utilized pulse-amplitude modulated fluorometry to investigate the redox state of the plastoquinone pool during and after bright light pulses. Our data indicate that even very intense (5910 μmol photons s-1m-2 of blue light having a wavelength of 440 nm) light pulses of 0.8 second duration are not sufficient to completely reduce the plastoquinone pool in Nannochloropsis. In order to achieve extensive reduction of the plastoquinone pool by bright light pulses, anaerobic conditions or an inhibitor of the photosynthetic electron transport chain has to be utilized. The implication of this finding for the application of the widely used saturating pulse method in algae is discussed. © 2017 Røkke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cyanobacteria require large quantities of iron to maintain their photosynthetic machinery; however, in most environments iron is present in the form of insoluble iron oxides. Whether cyanobacteria can utilize these sources of iron, and the potential molecular mechanisms involved remains to be defined. There is increasing evidence that pili can facilitate electron donation to extracellular electron acceptors, like iron oxides in non-photosynthetic bacteria. In these organisms, the donation of electrons to iron oxides is thought to be crucial for maintaining respiration in the absence of oxygen. Our study investigates if PilA1 (major pilin protein) may also provide a mechanism to convert insoluble ferric iron into soluble ferrous iron. Growth experiments supported by spectroscopic data of a strain deficient in pilA1 indicate that the presence of the pilA1 gene enhances the ability to grow on iron oxides. These observations suggest a novel function of PilA1 in cyanobacterial iron acquisition. © 2014 Lamb et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Using an axial detector, scanning transmission electron microscopy allows three-dimensional tomographic reconstruction of micrometer-thick sections of biological samples, at a resolution comparable to that obtained on thin sections. Electron tomography provides three-dimensional structural information about supramolecular assemblies and organelles in a cellular context, but image degradation, caused by scattering of transmitted electrons, limits applicability in specimens thicker than 300 nm. We found that scanning transmission electron tomography of 1,000-nm-thick samples using axial detection provided resolution comparable to that of conventional electron tomography. We demonstrated the method by reconstructing a human erythrocyte infected with the malaria parasite Plasmodium falciparum.

The concentration of microorganisms in growth medium is an important parameter in microbiological research. One of the approaches to determine this parameter is based on the physical interaction of small particles with light that results in light scattering. Table-top spectrophotometers can be used to determine the scattering properties of a sample as a change in light transmission. However, a portable, reliable, and maintenance-free instrument that can be built from inexpensive parts could provide new research opportunities. In this report, we show how to build such an instrument. This instrument consists of a low power monochromatic light-emitting diode, a monolithic photodiode, and a microcontroller. We demonstrate that this instrument facilitates the precise determination of cell concentrations for the bacteria Escherichia coli and Pseudomonas aeruginosa as well as the cyanobacterium Synechocystis sp. PCC 6803 and the green alga Chlamydomonas reinhardtii.

Photosynthesis, the conversion of sunlight into energy that is available to sustain cellular metabolism, is accomplished by a diverse group of organisms. The present photosynthetic diversity has been shaped over billions of years through the interactions of the genetic makeup and metabolic capabilities of each organism with its environment. Some photosynthetic bacteria found today can live in anaerobic conditions as must have been the case with the first photosynthetic organisms found on the primordial Earth. The oxygen of our present atmosphere was generated by ancient cyanobacteria. Cyanobacteria were incorporated into non-photosynthetic organisms in a process called endosymbiosis, giving rise to all the photosynthetic eukaryotes. Competition for light led to the development of a multitude of pigments that together span the entire solar spectrum. These pigments are arranged in special light-harvesting antenna systems that have evolved to efficiently channel excited-state energy to the reaction centers. In reaction centers, electrons are stripped away from donor molecules. This charge separation event, and successive electron transfer reactions, are catalyzed by multisubunit membrane-embedded protein complexes that are connected together via mobile electron carriers. The electron transfer reactions are ultimately used to convert inorganic carbon into organic carbon compounds. Most ecosystems rely on consuming photosynthesis-derived organic molecules and photosynthesis-derived oxygen to sustain life.

The effect of the plastoquionone (PQ) pool oxidation state on minimum chlorophyll fluorescence was studied in the green alga Chlamydomonas reinhardtii. In wild type and a mutant strain that lacks both photosystems but retains light harvesting complexes, oxygen depletion induced a rise in minimum chlorophyll fluorescence. An increase in minimum fluorescence yield is also observed when the PQ pool becomes reduced in the presence of oxygen and after application of an ionophore that collapses the transmembrane proton gradient. Together these results indicate that minimum chlorophyll fluorescence is modulated by the PQ oxidation state.

Conventional bright-field electron tomography (ET) provides valuable information about three-dimensional cellular architecture at 5–10 nm spatial resolution [1–3]. Reconstructed volumes can be obtained from heavy-atom stained plastic sections imaged with amplitude contrast, or from frozen-hydrated specimens imaged with phase contrast. Spatial resolution in conventional ET, as limited by stain artifacts and by radiation damage, is adequate for visualizing certain large macromolecular complexes, but smaller molecular assemblies often remain difficult to identify. We have investigated the use of alternative contrast mechanisms to help localize molecular structures of interest.

A Monte Carlo electron-trajectory calculation has been implemented to assess the optimal detector configuration for scanning transmission electron microscopy (STEM) tomography of thick biological sections. By modeling specimens containing 2 and 3 atomic % osmium in a carbon matrix, it was found that for 1-μm thick samples the bright-field (BF) and annular dark-field (ADF) signals give similar contrast and signal-to-noise ratio provided the ADF inner angle and BF outer angle are chosen optimally. Spatial resolution in STEM imaging of thick sections is compromised by multiple elastic scattering which results in a spread of scattering angles and thus a spread in lateral distances of the electrons leaving the bottom surface. However, the simulations reveal that a large fraction of these multiply scattered electrons are excluded from the BF detector, which results in higher spatial resolution in BF than in high-angle ADF images for objects situated towards the bottom of the sample. The calculations imply that STEM electron tomography of thick sections should be performed using a BF rather than an ADF detector. This advantage was verified by recording simultaneous BF and high-angle ADF-STEM tomographic tilt series from a stained 600-nm thick section of C. elegans. It was found that loss of spatial resolution occurred markedly at the bottom surface of the specimen in the ADF-STEM but significantly less in the BF-STEM tomographic reconstruction. Our results indicate that it might be feasible to use BF-STEM tomography to determine the 3D structure of whole eukaryotic microorganisms prepared by freeze-substitution, embedding, and sectioning.

By studying the inactivation of malaria parasite culture by cysteine protease inhibition using confocal microscopy of living cells, and electron microscopy of high-pressure frozen and freeze-substituted cells, we report the precise step in the release of malaria parasites from erythrocytes that is likely regulated by cysteine proteases: the opening of the erythrocyte membrane, liberating parasites for the next round of infection. Inhibition of cysteine proteases within the last few minutes of cycle does not affect rupture of the parasitophorus vacuole but irreversibly blocks the subsequent rupture of the host cell membrane, locking in resident parasites, which die within a few hours of captivity. This irreversible inactivation of mature parasites inside host cells makes plasmodial cysteine proteases attractive targets for anti-malarials, as parasite-specific cysteine protease inhibitors may significantly augment multi-target drug cocktails.

Chlorosomes are specialized compartments that constitute the main light harvesting system of green sulfur bacteria (GSB) and some filamentous anoxygenic phototrophs (FAP). Chlorosome biogenesis promises to be a complex process requiring the generation of a unilayer membrane and the targeting of bacteriochlorophyll, carotenoids, quinones, and proteins to the chlorosome. The biogenesis of chlorosomes as well as their presence in two distinct bacterial groups, GSB and FAP, remains enigmatic. The photosynthetic machinery and overall metabolic characteristics of these two bacterial groups are very different, and horizontal gene transfer has been proposed to explain chlorosome distribution. Chlorosomes have been considered to be unique structures that require a specific assembly machinery. We propose that no special machinery is required for chlorosome assembly. Instead, it is suggested that chlorosomes are a special form of lipid body. We present a model for chlorosome biogenesis that combines aspects of lipid body biogenesis with established chlorosome characteristics and may help explain the presence of chlorosomes in two metabolically diverse organism groups.

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.

The cyanobacterial genus Acaryochloris is the only known group of oxygenic phototrophs that contain chlorophyll d rather than chlorophyll a as the major photosynthetic pigment. Studies on this organism are still in their earliest stages, and biochemical analysis has rapidly outpaced growth optimization. We have investigated culture growth of the major strains of Acaryochloris marina (MBIC11017 and MBIC10697) by using several published and some newly developed growth media. It was determined that heavy addition of iron significantly enhanced culture longevity. These high-iron cultures showed an ultrastructure with thylakoid stacks that resemble traditional cyanobacteria (unlike previous studies). These cultures also show a novel reversal in the pigment ratios of the photosystem II signature components chlorophyll a and pheophytin a , as opposed to those in previous studies.

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

BackgroundBathycoccus prasinos is an extremely small cosmopolitan marine green alga whose cells are covered with intricate spider's web patterned scales that develop within the Golgi cisternae before their transport to the cell surface. The objective of this work is to sequence and analyze its genome, and to present a comparative analysis with other known genomes of the green lineage.ResearchIts small genome of 15 Mb consists of 19 chromosomes and lacks transposons. Although 70% of all B. prasinos genes share similarities with other Viridiplantae genes, up to 428 genes were probably acquired by horizontal gene transfer, mainly from other eukaryotes. Two chromosomes, one big and one small, are atypical, an unusual synapomorphic feature within the Mamiellales. Genes on these atypical outlier chromosomes show lower GC content and a significant fraction of putative horizontal gene transfer genes. Whereas the small outlier chromosome lacks colinearity with other Mamiellales and contains many unknown genes without homologs in other species, the big outlier shows a higher intron content, increased expression levels and a unique clustering pattern of housekeeping functionalities. Four gene families are highly expanded in B. prasinos, including sialyltransferases, sialidases, ankyrin repeats and zinc ion-binding genes, and we hypothesize that these genes are associated with the process of scale biogenesis.ConclusionThe minimal genomes of the Mamiellophyceae provide a baseline for evolutionary and functional analyses of metabolic processes in green plants.

Background Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. Results In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42 %, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. Conclusions Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven plasmids. However, there were differences at the pathway level as genes associated with degradation of phenolic compounds, nicotine, phenylalanine, styrene, ethylbenzene, and ethanolamine were detected only in the Psychrobacter strains reported in this study while they were absent among the other publicly available Psychrobacter genomes. In addition, six gene candidates were identified by genome mining and shown to possess T1, T2 and T3 copper binding sites as the main signature of the three-domain laccases. P11F6-LMCO and P11G5-LMCO2 were recombinantly expressed and shown to be active when ABTS and guaiacol were used as substrates. © 2016 Moghadam et al.Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

The metabolic capacity of the eukaryotic cell to convert free energy contained in nutrients into ATP is a process accomplished by a multi-step system: the mitochondrial respiratory chain. This chain involves a series of electron-transferring enzymes and redox co-factors, whose biochemical characterization is the collective result of more than 50 years of scientists’ endeavors. The current knowledge describes in detail the structure and function of the individual proton-translocating “core” complexes of the respiratory chain (Complex I, III, IV). However, a holistic approach to the study of electrons transport from NAD-dependent substrates to oxygen has recently directed our attention to the existence of specific albeit dynamic interactions between the respiratory complexes. In this context, the respiratory complexes are envisaged to be either in form of highly ordered assemblies (i.e. supercomplexes) or as individual enzymes randomly distributed in the mitochondrial membrane. Either model of organization has functional consequences, which can be discussed in terms of the structural stability of the protein complexes and the kinetic efficiency of inter-complex electron transfer. Available experimental evidence suggests that Complex I and Complex III behave as assembled supercomplexes (ubiquinone-channeling) or as individual enzymes (ubiquinone-pool), depending on the lipid environment of the membrane. On the contrary, a strict association of Complexes III and Complex IV is not required for electron transfer via cytochrome c, although there are supercomplexes in bovine heart mitochondria, known as the respirasomes, that also include some molecules of Complex IV. Our recent experimental results demonstrate that the disruption of the supercomplex I1–III2 enhances the propensity of Complex I to generate the superoxide anion; we propose that any primary source of oxidative stress in mitochondria may perpetuate generation of reactive oxygen species by a vicious cycle involving supercomplex dissociation as a major determinant.