Unicellular Microorganisms Research Articles

Large-scale comparative metagenomics of Blastocystis, a common member of the human gut microbiome

The influence of unicellular eukaryotic microorganisms on human gut health and disease is still largely unexplored. Blastocystis spp. commonly colonize the gut, but its clinical significance and ecological role are currently unsettled. We have developed a high-sensitivity bioinformatic pipeline to detect Blastocystis subtypes (STs) from shotgun metagenomics, and applied it to 12 large data sets, comprising 1689 subjects of different geographic origin, disease status and lifestyle. We confirmed and extended previous observations on the high prevalence the microrganism in the population (14.9%), its non-random and ST-specific distribution, and its ability to cause persistent (asymptomatic) colonization. These findings, along with the higher prevalence observed in non-westernized individuals, the lack of positive association with any of the disease considered, and decreased presence in individuals with dysbiosis associated with colorectal cancer and Crohn’s disease, strongly suggest that Blastocystis is a component of the healthy gut microbiome. Further, we found an inverse association between body mass index and Blastocystis, and strong co-occurrence with archaeal organisms (Methanobrevibacter smithii) and several bacterial species. The association of specific microbial community structures with Blastocystis was confirmed by the high predictability (up to 0.91 area under the curve) of the microorganism colonization based on the species-level composition of the microbiome. Finally, we reconstructed and functionally profiled 43 new draft Blastocystis genomes and discovered a higher intra subtype variability of ST1 and ST2 compared with ST3 and ST4. Altogether, we provide an in-depth epidemiologic, ecological, and genomic analysis of Blastocystis, and show how metagenomics can be crucial to advance population genomics of human parasites.

Effects of salinity on the CO2 permeation across lipid bilayer for microalgae biofixation: a molecular dynamics study

The continuous threat of increasing CO2 concentration in the atmosphere has altered the carbon balance of our planet causing global climate change. Biological fixation of atmospheric CO2 by unicellular microorganisms such as microalgae is a promising technology pursued extensively by researchers as a means for carbon capture. The study aimed to provide an atomic level of study that will demonstrate the effect of the salinity on the mechanism of CO2 absorption across microalgae lipid bilayer. Molecular dynamics simulations were utilized to calculate the free energies of CO2 molecule as it permeates inside the microalgae cell. In thermodynamics, the transport process of a molecule can be demonstrated through its free energy gradient. Thus, calculating the free energies of CO2 molecule across microalgae lipid bilayer can elucidate the mechanisms of permeation processes. Four microalgae lipid bilayer structures were constructed that contains 128-DPPC (dipalmitoylphosphatidylcholine) lipid bilayer with 3640 water molecules with different NaCl concentrations: 0, 3, 13, and 19 NaCl molecules which correspond to a salinity level of 0, 50, 200, and 300 mM, respectively. The cavity insertion Widom method was used to calculate the free energy of CO2 molecule along the lipid bilayer. The results demonstrated that the salinity does not affect the free energies significantly, thus, it does not hamper CO2 transport across microalgae lipid membrane.

Proteins with CHADs (Conserved Histidine α-Helical Domains) Are Attached to Polyphosphate Granules In Vivo and Constitute a Novel Family of Polyphosphate-Associated Proteins (Phosins).

On the basis of bioinformatic evidence, we suspected that proteins with a CYTH (CyaB thiamine triphosphatase) domain and/or a CHAD (conserved histidine α-helical domain) motif might represent polyphosphate (polyP) granule-associated proteins. We found no evidence of polyP targeting by proteins with CYTH domains. In contrast, two CHAD motif-containing proteins from Ralstonia eutropha H16 (A0104 and B1017) that were expressed as fusions with enhanced yellow fluorescent protein (eYFP) colocalized with polyP granules. While the expression of B1017 was not detectable, the A0104 protein was specifically identified in an isolated polyP granule fraction by proteome analysis. Moreover, eYFP fusions with the CHAD motif-containing proteins MGMSRV2-1987 from Magnetospirillum gryphiswaldense and PP2307 from Pseudomonas putida also colocalized with polyP granules in a transspecies-specific manner. These data indicated that CHAD-containing proteins are generally attached to polyP granules. Together with the findings from four previously polyP-attached proteins (polyP kinases), the results of this study raised the number of polyP-associated proteins in R. eutropha to six. We suggest designating polyP granule-bound proteins with CHAD motifs as phosins (phosphate), analogous to phasins and oleosins that are specifically bound to the surface of polyhydroxyalkanoate (PHA) granules in PHA-accumulating bacteria and to oil droplets in oil seed plants, respectively.IMPORTANCE The importance of polyphosphate (polyP) for life is evident from the ubiquitous presence of polyP in all species on earth. In unicellular eukaryotic microorganisms, polyP is located in specific membrane-enclosed organelles, called acidocalcisomes. However, in most prokaryotes, polyP is present as insoluble granules that have been designated previously as volutin granules. Almost nothing is known regarding the macromolecular composition of polyP granules. Particularly, the absence or presence of cellular compounds on the surface of polyP granules has not yet been investigated. In this study, we identified a novel class of proteins that are attached to the surface of polyP granules in three model species of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria These proteins are characterized by the presence of a CHAD (conserved histidine α-helical domain) motif that functions as a polyP granule-targeting signal. We suggest designating CHAD motif-containing proteins as phosins [analogous to phasins for poly(3-hydroxybutyrate)-associated proteins and to oleosins for oil droplet-associated proteins in oil seed plants]. The expression of phosins in different species confirmed their polyP-targeting function in a transspecies-specific manner. We postulate that polyP granules in prokaryotic species generally have a complex surface structure that consists of one to several polyP kinases and phosin proteins. We suggest differentiating polyP granules from acidocalcisomes by designating them as polyphosphatosomes.

Controllable alignment of elongated microorganisms in 3D microspace using electrofluidic devices manufactured by hybrid femtosecond laser microfabrication

This paper presents a simple technique to fabricate new electrofluidic devices for the three-dimensional (3D) manipulation of microorganisms by hybrid subtractive and additive femtosecond (fs) laser microfabrication (fs laser-assisted wet etching of glass followed by water-assisted fs laser modification combined with electroless metal plating). The technique enables the formation of patterned metal electrodes in arbitrary regions in closed glass microfluidic channels, which can spatially and temporally control the direction of electric fields in 3D microfluidic environments. The fabricated electrofluidic devices were applied to nanoaquariums to demonstrate the 3D electro-orientation of Euglena gracilis (an elongated unicellular microorganism) in microfluidics with high controllability and reliability. In particular, swimming Euglena cells can be oriented along the z-direction (perpendicular to the device surface) using electrodes with square outlines formed at the top and bottom of the channel, which is quite useful for observing the motions of cells parallel to their swimming directions. Specifically, z-directional electric field control ensured efficient observation of manipulated cells on the front side (45 cells were captured in a minute in an imaging area of ~160×120 μm), resulting in a reduction of the average time required to capture the images of five Euglena cells swimming continuously along the z-direction by a factor of ~43 compared with the case of no electric field. In addition, the combination of the electrofluidic devices and dynamic imaging enabled observation of the flagella of Euglena cells, revealing that the swimming direction of each Euglena cell under the electric field application was determined by the initial body angle.

Helicobacter is preserved in yeast vacuoles! Does Koch's postulates confirm it?

The manuscript titled “Vacuoles of Candida yeast behave as a specialized niche for Helicobacter pylori (H. pylori)” not only has not been prepared in a scientific manner but the methodology used was not adequate, and therefore the conclusion reached was not correct. First of all, “yeast” is a broad terminology covering a great number of genera and species of unicellular micro-organisms. The authors should have defined the organism with its binary scientific name. This measure would allow experiment reproduction by the scientific community. Moreover, the criteria established by Robert Koch to identify a specific microorganism or pathogen was not adopted in the methodology used. Regarding the methodology applied, use of the chicken egg-yolk (IgY) antibody and PCR of the apparently tainted yeast population to prove H. pylori existence in the yeast vacuoles might be main factors for their wrong conclusions. Bacterial tropism toward yeast extract is a known phenomenon, and yeast extract is one of the main ingredients in culture media. Their internalization through phagocytosis or similar pathways does not seem possible or practical because of the thick and cellulosic yeast wall. While the small size of yeast cells does not support their ability in harboring several H. pylori, other observations such as inefficiency of anti-fungal therapy as anti-Helicobacter therapy strongly reject the conclusion reached by the above-mentioned article.

Life without dUTPase.

Fine-tuned regulation of the cellular nucleotide pools is indispensable for faithful replication of Deoxyribonucleic Acid (DNA). The genetic information is also safeguarded by DNA damage recognition and repair processes. Uracil is one of the most frequently occurring erroneous bases in DNA; it can arise from cytosine deamination or thymine-replacing incorporation. Two enzyme activities are primarily involved in keeping DNA uracil-free: dUTPase (dUTP pyrophosphatase) activity that prevent thymine-replacing incorporation and uracil-DNA glycosylase activity that excise uracil from DNA and initiate uracil-excision repair. Both dUTPase and the most efficient uracil-DNA glycosylase (UNG) is thought to be ubiquitous in free-living organisms. In the present work, we have systematically investigated the genotype of deposited fully sequenced bacterial and Archaeal genomes. We have performed bioinformatic searches in these genomes using the already well described dUTPase and UNG gene sequences. For dUTPases, we have included the trimeric all-beta and the dimeric all-alpha families and also, the bifunctional dCTP (deoxycytidine triphosphate) deaminase-dUTPase sequences. Surprisingly, we have found that in contrast to the generally held opinion, a wide number of bacterial and Archaeal species lack all of the previously described dUTPase gene(s). The dut– genotype is present in diverse bacterial phyla indicating that loss of this (or these) gene(s) has occurred multiple times during evolution. We discuss potential survival strategies in lack of dUTPases, such as simultaneous lack or inhibition of UNG and possession of exogenous or alternate metabolic enzymes involved in uracil-DNA metabolism. The potential that genes previously not associated with dUTPase activity may still encode enzymes capable of hydrolyzing dUTP is also discussed. Our data indicate that several unicellular microorganisms may efficiently cope with a dut– genotype lacking all of the previously described dUTPase genes, and potentially leading to an unusual uracil-enrichment in their genomic DNA.

The Study and Application of Photoreceptive Membrane Protein, Rhodopsin

Abstract Microbial rhodopsins are photoreceptive seven-transmembrane proteins diversely found in unicellular microorganisms. The chromophore of microbial rhodopsin is all-trans retinal. The retinal isomerizes to 13-cis form upon light-absorption and it triggers the expression of various biological functions by light. We studied the mechanism of various types of microbial rhodopsins (H+ pump, Cl− pump and sensors) by physicochemical spectroscopic methods. On the basis of these studies, we found the importance of the residues on the third helix (helix C) for the function of microbial rhodopsin, and it led to the discovery of a new functional class of microbial rhodopsin, light-driven outward Na+ pump. The mechanism of Na+ pump rhodopsin was studied by several spectroscopic methods and X-ray crystallography. These studies provided new insights very informative for the development of novel functional artificial rhodopsins (K+ and Cs+ pumps). These new natural and artificial rhodopsins are expected to have high potential for applications such as optogenetics.

Microalgae Growth using Winery Wastewater for Energetic and Environmental Purposes

Winery wastewater (WWW), produced by winemaking activities (cleaning, transferring and storage operations), is an aqueous solution containing ethanol, organic acids, sugars, aldehydes, other microbial metabolites, soaps and detergents. Nowadays, innovative wastewater treatment processes are based on bacterial and yeast species while the role of microalgae is still unclear. Arthrospira (Spirulina) platensis and Chlorella vulgaris are unicellular prokaryotic and eukaryotic microorganisms, respectively, which can be easily grown even in non-optimal conditions. Several studies reported that the amount and quality of lipids contained in microalgal cells can differ as an outcome of changes in growth conditions or growth medium characteristics (concentration of carbon, nitrogen, phosphate, iron, etc.). In this study, we investigated the influence of different concentrations of WWW (20, 40 and 60 % v/v of the medium) on the growth and chemical composition of those photosynthetic microorganisms. Microalgae were grown into vertical glass bubblers (250 mL). The biomass concentration was quantified daily by measuring the optical density at 560 and 625 nm forplatensis and C. vulgaris, respectively. Total Carbon and total Nitrogen concentrations, both in the media (mg/L) and in microalga biomass (g/100g), were monitored by a CHNS-O analyser. In order to quantify the influence of WWW-enrich media on the lipid concentration and composition, biomass was collected at the beginning of the stationary phase and the lipid fraction was extracted. Results suggested that the two tested microalgae can growth in media enriched with WWW and the total Nitrogen concentrations decreased up to 90 and 100 % for A. platensis and C. vulgaris, respectively. In conclusion, WWW could be successfully used for the growth of the tested microalgae, leading to a reduction of the environmental impact of this wastewater.

Identification of Malaria Infection using HSV Colour Model and Dynamic Thresholding with Image Binarization

Malaria is a mosquito borne infectious disease of humans and other animals caused by unicellular parasitic micro-organism ofplasmodiumgenus underprotozoa.This disease is transmitted through the bite from an infected female Anopheles mosquito. The organism is introduced from its saliva into the host circulatory system. Within the blood stream the parasite travels to the liver where they mature and reproduce.Malaria is a serious global health problem and nearly one million deaths is reported eachyear. Malaria causes symptoms that typically include high fever and headache that under severe infection can progress to coma or death. The disease is prevalent in tropical and subtropical regions around the equator region, including much of sub-Saharan Africa, Asia, and America. Accurate diagnosis is important to control the disease. There are several diagnostic tools available but microscopic analysis is the gold standard. An image processing method to automate the diagnosis of malaria in blood smear images is proposed in this paper using image segmentation approach for detection of malaria parasite.

Identification of Malaria Infection using HSV Colour Model and Dynamic Thresholding with Image Binarization

Malaria is a mosquito borne infectious disease of humans and other animals caused by unicellular parasitic micro-organism ofplasmodiumgenus underprotozoa.This disease is transmitted through the bite from an infected female Anopheles mosquito. The organism is introduced from its saliva into the host circulatory system. Within the blood stream the parasite travels to the liver where they mature and reproduce.Malaria is a serious global health problem and nearly one million deaths is reported eachyear. Malaria causes symptoms that typically include high fever and headache that under severe infection can progress to coma or death. The disease is prevalent in tropical and subtropical regions around the equator region, including much of sub-Saharan Africa, Asia, and America. Accurate diagnosis is important to control the disease. There are several diagnostic tools available but microscopic analysis is the gold standard. An image processing method to automate the diagnosis of malaria in blood smear images is proposed in this paper using image segmentation approach for detection of malaria parasite.

Fabrication of living soft matter by symbiotic growth of unicellular microorganisms.

We report the fabrication of living soft matter made as a result of the symbiotic relationship of two unicellular microorganisms. The material is composed of bacterial cellulose produced in situ by acetobacter (Acetobacter aceti NCIMB 8132) in the presence of photosynthetic microalgae (Chlamydomonas reinhardtii cc-124), which integrates into a symbiotic consortium and gets embedded in the produced cellulose composite. The same concept of growing living materials can be applied to other symbiotic microorganism pairs similar to the combination of algae and fungi in lichens, which is widespread in Nature. We demonstrate the in situ growth and immobilisation of the C. reinhardtii cells in the bacterial cellulose matrix produced by the simultaneous growth of acetobacter. The effect of the growth media composition on the produced living materials was investigated. The microstructure and the morphology of the produced living biomaterials were dependent on the shape of the growth culture container and media stirring conditions, which control the access to oxygen. As the photosynthetic C. reinhardtii cells remain viable and produce oxygen as they spontaneously integrate into the matrix of the bacterial cellulose generated by the acetobacter, such living materials have the potential for various applications in bio-hydrogen generation from the immobilised microalgae. The proposed approach for building living soft matter can provide new ways of immobilising other commercially important microorganisms in a bacterial cellulose matrix as a result of symbiosis with acetobacter without the use of synthetic binding agents and in turn increase their production efficiency.

Facet Energy and Reactivity versus Cytotoxicity: The Surprising Behavior of CdS Nanorods

Responsible development of nanotechnology calls for improved understanding of how nanomaterial surface energy and reactivity affect potential toxicity. Here, we challenge the paradigm that cytotoxicity increases with nanoparticle reactivity. Higher-surface-energy {001}-faceted CdS nanorods (CdS-H) were less toxic to Saccharomyces cerevisiae than lower-energy ({101}-faceted) nanorods (CdS-L) of similar morphology, aggregate size, and charge. CdS-H adsorbed to the yeast's cell wall to a greater extent than CdS-L, which decreased endocytosis and cytotoxicity. Higher uptake of CdS-L decreased cell viability and increased endoplasmatic reticulum stress despite lower release of toxic Cd(2+) ions. Higher toxicity of CdS-L was confirmed with five different unicellular microorganisms. Overall, higher-energy nanocrystals may exhibit greater propensity to adsorb to or react with biological protective barriers and/or background constituents, which passivates their reactivity and reduces their bioavailability and cytotoxicity.

Cancer Cell Cannibalism: A Primeval Option to Survive.

Cancer cell cannibalism is currently defined as a phenomenon in which an ensemble of a larger cell containing a smaller one, often in a big cytoplasmic vacuole, is detected in either cultured tumor cells or a tumor sample. After almost one century of considering this phenomenon as a sort of neglected curiosity, some recent studies have first proposed tumor cell cannibalism as a sort of "aberrant phagocytosis", making malignant cells very similar to professional phagocytes. Later, further research has shown that, differently to macrophages, exclusively ingesting exogenous material, apoptotic bodies, or cell debris, tumor cells are able to engulf other cells, including lymphocytes and erythrocytes, either dead or alive, with the main purpose to feed on them. This phenomenon has been associated to the malignancy of tumors, mostly exclusive of metastatic cells, and often associated to poor prognosis. The cannibalistic behavior increased depending on the microenvironmental condition of tumor cells, such as low nutrient supply or low pH, suggesting its key survival option for malignant cancers. However, the evidence that malignant cells may cannibalize tumor-infiltrating lymphocytes that act as their killers, suggests that tumor cell cannibalism could be a very direct and efficient way to neutralize immune response, as well. Tumor cell cannibalism may represent a sign of regression to a simpler, ancestral or primeval life style, similar to that of unicellular microorganisms, such as amoebas, where the goal is to survive and propagate in an overcrowded and very hostile microenvironment. In fact, we discovered that metastatic melanoma cells share with amoebas a transmembrane protein TM9SF4, indeed related to the cannibal behavior of these cells. This review attempts to provide a comprehensive description of the current knowledge about the role of TM9SF4 in cancer, highlighting its role as a key player in the cannibal behavior of malignant cancer cells. Moreover, we discuss differences and similarities between tumor cannibalism, entosis, phagocytosis and emperipolesis.

Proteomic analyses of early response of unicellular eukaryotic microorganism Tetrahymena thermophila exposed to TiO2 particles

Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO2 nanoparticles (TiO2-NPs) and their bulk counterparts (bulk-TiO2). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO2-NPs or bulk-TiO2 particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE + nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO2 particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO2-NPs concentration differed significantly from the response to higher TiO2-NPs concentration and both bulk-TiO2 concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO2; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO2-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.

Pseudomonas spp. as models for plant-microbe interactions.

As is the case with other multicellular eukaryotes, plants are colonized by large numbers of unicellular microorganisms. They may be free-living commensals, epiphytes, symbionts (endophytes), or obligate parasites. The plant holobiont is in effect an ecosystem, and it is of interest to know how this assemblage is established and maintained, and reacts to both biotic and abiotic cues. The current view, initially elaborated in the context of coral-dwelling microbial communites, is that the multicellular organism is more inclusively described by the term “holobiont” that includes associated microbiota, and is a valid unit of natural selection (Rosenberg et al., 2007). The holobiont then, is often dependent on its microbiota for crucial functions, drastic imbalances in which, termed dysbiosis, are thought to lead to compromised or deficient functioning. The association of plants with microbes is phylogenetically ancient, going back to the macroalgae (Marshall et al., 2006). The role of the microbiota of plants, collectively termed the “phytomicrobiome,” in their overall life cycle is now under investigation, close on the heels of more extensive studies on animal, especially human, microbiota. The development of Arabidopsis thaliana (thale cress) and Brachypodium distachyon (purple false brome) as model systems for dicotyledonous and monocotyledonous plants respectively, and the availability of genome databases for Pseudomonads (Winsor et al., 2011) and plants (Duvick et al., 2008) indicate that the potential for both hypothesis-based and discovery science are indeed great. The assembly, development and maintenance of the plant holobiont is not possible without an exchange and sensing of, and responses to, biomolecular cues between its constituents. Within this overall theme, we focus on a few recently discovered, novel interand intra-species interactions of some Pseudomonas spp., indicating their utility as model systems, and highlighting some previously unforeseen mechanisms that could have a bearing on plant-phytomicrobiome interactions. Note that, for purposes of this article, we use the word “signaling” to refer generically to the sensing and response of organisms to environmental cues of both biotic and abiotic origins.

UV Sunscreens of Microbial Origin: Mycosporines and Mycosporine- like Aminoacids.

Exposure to ultraviolet radiation (UVR) is harmful to living organisms, causing damage to macromolecules such as DNA, RNA, proteins and lipids. Depending on the wavelength, the injury could be direct or indirect through reactive oxygen intermediates, so it is desirable to find compounds that can reduce both. Many organic chemicals used in commercial sunscreen possess estrogenic activity in vivo. In this report we analyzed recent patents related to UV sunscreens of microbial origin, in particular mycosporines (MYC) and mycosporine-like aminoacids (MAA). Both are promising natural alternatives for both direct (UV-absorption) and indirect (antioxidant) protection, given they show strong photostability and absence of cytotoxicity. It becomes clear that although the search for natural photoprotective molecules is relatively recent, efforts have been invested mainly in marine environments, remaining still many potential photoprotective molecules to find in other type of habitats. Furthermore, unicellular microorganisms have several advantages for the production of metabolites of interest, since they improve the production costs due to its simplicity of culture and easy genetic manipulation. The knowledge of the biosynthesis pathway of MYC and MAA is essential to improve rationally their expression levels. Currently, only the MAA pathway in bacteria has been reported, remaining the MYC pathway unclear. Future perspectives include the heterologous expression of MYC and/or MAA in industrially friendly microorganisms (bacteria and yeast) in order to co-produce different UV-protective molecules and thus cover a broader UV spectrum and simplify the production process.

Eukaryotic cell encystation and cancer cell dormancy: is a greater devil veiled in the details of a lesser evil?

Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. These dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expression profiles, immunocytochemistry tools, and siRNAs during the process of encystation, understanding the covert features of cancer cell dormancy as proposed could be possible. This knowledge can be extended to dormant cancer cells to uncover the mechanisms that underlie this ghost, yet dangerous state of human cancers. We propose a strategy to induce dormancy and exit this state by application of knowledge gained from the encystation induction and retrieval processes in pathogenic eukaryotic microorganisms. Given that early detection and characterization of dormant malignant tumor cells is important as a general strategy to monitor and prevent the development of overt metastatic disease, this homology may enable the design of therapies that could either awake the dormant cell from dormancy to make it available for therapies or prolong such a phase to make cancer appear as a chronic disease.

Osmosensing and osmoregulation in unicellular eukaryotes.

Eukaryotic microorganisms possess mechanisms to detect osmotic variations in their surroundings, from specialized receptors and membrane transporters, to sophisticated systems such as two-component histidine kinases. Osmotic stimuli are transduced through conserved phosphorylation cascades that result in a rapid response to mitigate stress. This response allows for the maintenance of an optimal biochemical environment for cell functioning, as well as a suitable recovery in suboptimal environments that would otherwise endanger cell survival. The molecular basis of these responses has been largely studied in yeasts and bacteria. However, fewer studies have been published concerning the molecular basis of osmoregulation in other eukaryotic microorganisms such as protozoans and microalgae. Here, we review the main osmosensors reported in unicellular eukaryotic microorganisms (yeasts, microalgae and protozoa) and the pathways that maintain homeostasis in cells encountering hyperosmotic challenges.

Plant membrane assays with cytokinin receptors underpin the unique role of free cytokinin bases as biologically active ligands.

Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N (9)-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents β6-β7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.

Isolation and characterization of yeasts capable of efficient utilization of hemicellulosic hydrolyzate as the carbon source.

Few yeasts have shown the potential to efficiently utilize hemicellulosic hydrolyzate as the carbon source. In this study, microorganisms isolated from the Manaus region in Amazonas, Brazil, were characterized based on their utilization of the pentoses, xylose, and arabinose. The yeasts that showed a potential to assimilate these sugars were selected for the better utilization of lignocellulosic biomass. Two hundred and thirty seven colonies of unicellular microorganisms grown on hemicellulosic hydrolyzate, xylose, arabinose, and yeast nitrogen base selective medium were analyzed. Of these, 231 colonies were subjected to sugar assimilation tests. One hundred and twenty five of these were shown to utilize hydrolyzed hemicellulose, xylose, or arabinose as the carbon source for growth. The colonies that showed the best growth (N = 57) were selected, and their internal transcribed spacer-5.8S rDNA was sequenced. The sequenced strains formed four distinct groups in the phylogenetic tree, and showed a high percentage of similarity with Meyerozyma caribbica, Meyerozyma guilliermondii, Trichosporon mycotoxinivorans, Trichosporon loubieri, Pichia kudriavzevii, Candida lignohabitans, and Candida ethanolica. The discovery of these xylose-fermenting yeasts could attract widespread interest, as these can be used in the cost-effective production of liquid fuel from lignocellulosic materials.