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Ways to cause membrane damage to microalgae and yeast?

Ways to cause membrane damage to microalgae and yeast?


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I am researching a way to monitor the membrane damage of cells. To do that I fist have to have reference points, namely, cells with damaged membranes.

I am working with Dunalliela, Hematococcus (both microalgae), and common yeast. I have been using mostly ethanol to damage the membranes so far.

What other ways are there to cause the most possible damage to microalgae (or yeast) membranes?. The idea is not total cell obliteration, but rather membrane damage (death is expected).


To get to the membrane of these species you first need to get past a formidable cell wall. The methods listed below are therefore more aimed at making cells permeable but the membranes must sustain some damage in the process.

  • At our lab we regularly use glass bead transformation for microalgae transformation. The microabrasion allows DNA to go in so I imagine the membranes must be damaged somehow.
  • I've also used hypotonic media (depending on the strain) to swell cells close to their bursting point. I imagine they become rather permeable at this point since they let several dyes in that are otherwise excluded.

Methods for Monitoring Environmental Pollution

Environmental pollution poses a big threat to the healthy existence of humankind. The Governments world over pay serious attention to continuously monitor and minimize pollution.

The public and non-governmental organizations (NGOs) are also actively involved in this venture. Broadly, there are four levels of pollution monitoring agencies or environmental protection agencies (EPAs).

This is at the district or block level. The non-governmental organizations and the rural development agencies are involved in pollution monitoring.

Existing at the state level, the monitoring is done by the respective state pollution control boards.

This is at the national/country level. Each country has its own environmental protection agency to monitor pollution.

International/inter- Governmental bodies are closely associated with monitoring of pollution which is a global phenomenon. World Health Organization and United Nation Environmental Programme are actively involved.

Biotechnological Methods for Measurement of Pollution:

In recent years, environmental pollution detection and monitoring is being done by approaches involving bio-systems. For this, purpose, several groups of plants, animals and microorganisms are utilized. The environmental protection agencies (EPAs) consider bio-monitoring of pollution as a useful device to monitor environmental pollution from the point of diagnostic, preventive and remedial measures.

Criteria for Bio-monitoring of Pollution:

The parameters or the criteria chosen for bio-monitoring of pollution are very crucial. They should be reliable, reproducible and cost-effective. Three types of criteria are mostly adopted for bio-monitoring of pollution-visual rating, genotoxicity rating and metabolic rating.

In the visual rating, the growth rate and productivity are considered. When microorganisms are used in the test assay, the growth can be measured by turbidometric analysis. In case of higher plants, growth rate of different parts, visual damage to leaves, seed viability and germination frequency are taken into account.

As regards animals (fishes are commonly used), the concept of LD50 is used i.e. the dose at which 50% of the test organism is affected. Sometimes, the presence or absence of a particular species of an organism serves as an indicator for the environmental pollution.

Genotoxicity rating:

Genotoxicity tests measure the extent of damage caused to an organism by environmental pollution at the cellular and sub-cellular levels. The genotoxic lesions may be detected on the cellular organelles (membranes commonly used), genomes, immune systems, biomolecules, etc.

Cytotoxic tests such as measurement of chromosomal damage (including breakage), sister chromatid exchange (SCE) and micronuclei counting are also useful for pollution detection. The cell viability can be measured by detecting in vitro lysosomal viability. In recent years, DNA probes are in use for the identification of disease- causing organisms in water.

Metabolic rating:

The biochemical changes with environmental pollution can be measured (qualitatively and quantitatively) in selected organisms. In fact, certain metabolic parameters can be used as biomarkers to assess the pollution stress. The biomarkers used in metabolic rating include chlorophyll, proteins, nucleic acids (DNA and RNA) and changes in enzyme activities.

The biotechnological methods adopted for pollution measurement are briefly described in the following order:

3. Molecular biological assays

Bioassays in Environmental Monitoring:

In the early years, the conventional physical and chemical methods were used for the detection of environmental pollution. Bioassays are preferred these days, since the biological responses that reflect the damages to the living organisms are very crucial for the actual assessment of pollution.

The organisms employed in the bioassays for pollution detection are expected to satisfy the following criteria:

i. It should readily take up the pollutant (absorption or adsorption).

ii. The organism should be sensitive to the pollutant.

iii. It should possess measurable features to detect pollution.

iv. The organism should have wide occurrence, and available round the year.

v. The bioassay should be simple, reproducible and cost-effective.

The most commonly used plants and animals in the bioassays are briefly described.

Plant test systems in bioassays:

Certain algae, bacteria, lichens, mosses and vascular macrophytes are commonly used in bioassays.

Among the plant systems, algal bioassays are the most commonly used. Algae are considered to be reliable indicators of pollution due to their high sensitivity and easy availability, besides simple culturing techniques. The criteria adopted for algal bioassays are the growth rate, biomass accumulation and photosynthetic efficiency.

The algae used in the test assays include Chlorella, Microcystis, Spirulina, Navicula, Scenedesmus, Anabaena, Ulva, Codium, Fucus and Laminaria. In water, organic pollution can be detected by using the blue green algae, Microcystis, while metal pollution can be measured by Navicula.

These are commonly used for the detection of fecal pollution in potable water, the most widely employed test being coliform test. Ames test that detects mutagenic pollutants is carried out by the bacterium Salmonella. Bacterial bioluminescence is a recent technique used for the measurement of gaseous pollutants and other compounds e.g. sulfur dioxide, formaldehyde, ethyl acetate. Photo bacterium phosphoreum is the organism of choice for bacterial bioluminescence.

Lichens are widely used for the detection of atmospheric gas pollution, particularly in cities. Lichens are very sensitive for the measurement of sulfur dioxide.

Environmental metal pollution can be detected by using certain forest and aquatic mosses e.g. Stereophyllum, Sphagnum, Brynus.

Vascular macrophytes in bioassays:

Water hyacinth (Eichormia crassipes) and duck weed (Lemna minor) are in use to detect aquatic metal pollution. In fact, certain biochemical parameters of macrophytes are used to serve as biomarkers of pollution e.g., peroxidase activity increases due to metal pollution inhibition of nitrate reductase activity by mercury. The other commonly used bioassay parameters are the estimation of soluble proteins, nucleic acids, chlorophyll, and assay of enzyme (e.g. catalase, peroxidase) activities.

Pollution-induced peptides in bioassays:

Very recently, some workers have identified the presence of small peptides within the plant cells which are pollution-induced. These peptides, referred to as phytochelatins, are formed as a result of metal pollution. They are reasonably reliable for the detection of metal pollution.

Animal test systems in bioassays:

Among the animals, certain fishes, protozoa and helminthes are employed in bioassays.

Toxic effects of environmental pollutants on fishes have been in use for quite some time as a measure of bioassays. In fact, the concept of LD50 (i.e. the dose of the pollutant at which 50% of the test organisms are affected) has originated from the studies on fishes.

The criteria or parameters used for assessment of fish bioassays include changes in the morphology and organs, behavioural pattern and modifications in metabolisms. The alterations in the enzyme acetylcholine esterase serve as a reliable marker for pesticide pollution. The most commonly used fishes in bioassays are Catla, Teleost, Labeo and Channa.

Protozoa in bioassays:

The ciliated protozoa serve as good bioassay systems for the detection of environmental pollution. The toxic effects of the pollutants can be measured by the changes in the behavioural patterns of protozoa, recorded on an ethogram.

Helminths in bioassays:

Rotifers are a group of helminths that grow on aquatic vegetation. They are used for the detection of organic matter in water (given by BOD). Rotifers, with round the year availability, easy cultivation, slow growth rate and easy recognition are used for bio-monitoring of water.

Pollution-induced peptides in bioassays:

As already described in case of plant bioassays (above), pollution-induced small peptides are found in animal cells also. They are collectively referred to as metallothioneins (comparable to phytochelatins in plants). Metallothioneins are useful for the detection of metal pollution.

Bio-monitoring of pollution with multiple species:

Most often, bioassays using a single organism are not adequate to detect pollution. In such a case, multiple species of organisms are used.

Cell Biology in Environmental Monitoring:

Cell biology deals with the study of the structural and functional aspects of cells and the cellular organelles. It is successfully exploited for environmental pollution detection, particularly with reference to mutagens and carcinogens.

The cell biological methods primarily aim to trace the harmful effects of pollutants on different cellular components — membranes, chloroplasts, mitochondria, chromosomes. In addition, the macromolecules namely nucleic acids (particularly DNA) and proteins are also used. Further, cell biological methods help in understanding the mechanisms of toxicity of pollutants.

Some important cell biological methods used in environmental pollution monitoring are described.

Membrane damage in bioassay:

The plasma membrane, an envelope surrounding the cell, protects the cell from hostile environment. It is the first cellular component to be directly exposed to pollutants. Many toxic substances that cause damage to cell structure and its functions are known. For the purpose of bioassay, the physical damages caused by pollutants or their deposition on the membranes can be detected by light, phase contrast and electron microscopy.

This approach may not be always practicable. The alterations in the semipermeable properties of the membranes due to pollutants can be detected by leakage of enzymes (e.g., lactate dehydrogenase), efflux of electrolytes or uptake of trypan blue. Lysosomes are also useful as biomarkers for measurement of cell viability. This can be done by neutral red retention test. The damaged lysosomes cannot retain this dye.

In recent years, animal and plant tissue culture techniques are also used for pollution monitoring. This is made possible by measuring cellular damages observed in cell cycles. A good example is the use of human lymphocyte culture to monitor the persons exposed to toxic pollutants.

Cytogenetic bioassays:

The genetic damages of the cells, as reflected by changes in the chromosomes, can be effectively used in bio-monitoring of pollution. For this purpose, animals (e.g. insect Drosophila) and plants (e.g. Arabidiopsis) with shorter life cycles are preferred. Other plants such as pea, maize and soy bean are also used in cytogenetic bioassays.

The pollutants may cause several types of chromosomal damages- fragmentation, bridge formation, and disruption in cell division. The chromosomal alterations can be effectively used for pollution detection. It has been clearly established that the severity of chromosomal damage depends on the chemical nature of the pollutant.

Severe damage to chromosomes by pollutants may result in large scale fragmentation of chromosomes, followed by micronuclei formation. The degree of micronuclei development is directly related to the severity of the damage. Micronucleus test (MNT) is used for screening of mutagenic compounds.

Sister-chromatid exchange:

The damages caused by pollutants results in misexchange of chromosomal segments (chromatids) during cell division. The sister chromated exchange (SCE) can be detected by using a fluorescent dye technique.

Ames test in bioassays:

Ames test can be used for the detection of chemical mutagens and their carcinogenicity. This is very widely used bioassay for screening of various pollutants, drugs, cosmetics, food additives and metals. Ames test employs the use of a special mutant strain of bacterium namely Salmonella typhimurium (His – ). This organism cannot synthesize histidine, hence the same should be supplied in the medium for its growth.

Addition of chemical carcinogens causes mutations (reverse mutation) restoring the ability of this bacterium to synthesize histidine (His + ). By detecting the strain of Salmonella (His + ) in the colony of agar plates, the chemical mutagens can be identified. The Ames assay can detect about 90% of the chemical carcinogens.

Recently, the yeast cells (Saccharomyces cerevisae) are also used for the detection of chemical carcinogens.

Molecular Biology in Environmental Monitoring:

The use of molecular probes and immunoassays in monitoring of environmental pollution is gaining importance in recent years. Molecular biological bioassays are particularly useful for the detection of bacteria, viruses and other pathogenic organisms that cause diseases.

DNA probes and polymerase chain reaction (PCR) can be effectively used for water quality monitoring, particularly potable water. However, these techniques are expensive and not practicable at all places.

Immunological techniques are useful for the detection of pollutants (pesticides, herbicides) and identification of pathogens that exhibit immunological properties. Immunoassays are in use for the measurement of several pesticides e.g. aldrin, triazines DDT, glyphosate. Metabolic products of certain bacteria can also be detected by immunoassays. For instance, assay systems have been developed for the detection of toxins of cholera and Salmonella.

In recent years, use of monoclonal antibodies (MAbs) in the detection and bio-monitoring of environmental pollution is gaining importance. In fact, assay techniques are available for detection of pesticide and herbicide contamination in water.

Bioluminescent bioassays using Lux reporter genes:

Certain genes, referred to as Lux reporter genes, on the plasmids produce assayable signals. Whenever these genes are expressed in luminescent bacteria like Photo bacterium and Vibrio. Some bacterial strains have been developed through gene cloning (employing Lux reporter genes) for the detection of pollutants and their degradation. For instance, genetically altered Pseudomonas can be used for detecting naphthalene, xylene, toluene and salicylate.

Biosensors in Environmental Monitoring:

A biosensor is an analytical device containing an immobilized biological material (enzyme, organelle, cell) which can specifically interact with an analyte (a compound whose concentration is to be determined) and produce physical, chemical or electrical signals that can be measured. Biosensors are highly specific and accurate in their function. The details on biosensors—principles of working, types, various applications are described elsewhere Some of the important biosensors used in environmental pollution monitoring are briefly described.

Biological oxygen demand (BOD5) is a widely used test for the detection of organic pollution. This test requires five days of incubation. A BOD biosensor using the yeast Trichosporon cutaneum with oxygen probe takes just 15 minutes for detecting organic pollution.

Microbial biosensors for the detection of gases such as sulfur dioxide (SO2), methane and carbon dioxide have been developed. Thiobacillus-based biosensor can detect the pollutant SO2, while methane (CH4) can be detected by immobilized Methalomonas. For carbon dioxide monitoring, a particular strain of Pseudomonas is used.

Immunoassay biosensors:

Immunoelectrodes as biosensors are useful for the detection of low concentrations of pollutants. Pesticide specific antibodies can detect the presence of low concentrations of triazines, malathion and carbamates, by employing immunoassays.

Other biosensors:

Biosensors employing acetylcholine esterase (obtained from bovine RBC) can be used for the detection of organophosphorus compounds in water. In fact, portable pesticide monitors are commercially available in some developed countries. Biosensors for the detection of polychlorinated biphenyls (PCBs) and chlorinated hydrocarbons and certain other organic compounds have been developed.

Phenol oxidase enzyme (obtained from potatoes and mushrooms) containing biosensor is used for the detection of phenol. A graphite electrode with Cynobacterium and Synechococcus has been developed to measure the degree of electron transport inhibition during photosynthesis due to certain pollutants e.g. herbicides.

A selected list of environmental pollutants measured by employing biosensors is given in Table 54.1.


INTRODUCTION

Biological membranes are essential components of living systems. They form a boundary between the cell and its environment, mediate intracellular signaling transduction and cell-to-cell communications and establish a selective permeable boundary that only allows certain molecules to enter or leave the cell. In eukaryotic organisms, membranes divide the cell into discrete subcellular compartments that segregate vital but, in many cases, incompatible metabolic reactions. The fundamental structure of cellular membranes is the bilayer comprising two sheets of lipid molecules, into which proteins with important functions such as enzymes in energy-transducing systems, receptors and transporters are either partially or fully embedded. According to the fluid mosaic model (Singer and Nicolson, 1972 ), a critical property of biological membranes is that they are present in a fluid state in which lipids and proteins are loosely bound to one another via chemical interactions and individual molecules are generally able to rotate and move laterally. Such fluidity is important for membrane-associated functions such as transport, synthesis of biomolecules, energy transduction and cell signaling, and it is influenced by both temperature and lipid composition (Ernst et al., 2016 Los and Murata, 2004 van Meer et al., 2008 ).

In addition to their structural role, membrane lipids regulate the localization, structure and function of membrane proteins by lipid–lipid and lipid–protein interactions and by physical effects (Harayama and Riezman, 2018 van Meer et al., 2008 Nyholm, 2015 Quinn, 2012 ). Some lipids can define membrane microdomains that serve as sorting platforms and hubs for cell signal transduction machinery for a wide range of metabolic processes (Levental et al., 2020 Sezgin et al., 2017 ). Lipids also play crucial roles in membrane fusion events critical for cell division, organelle proliferation and membrane trafficking (Harayama and Riezman, 2018 van Meer et al., 2008 ). Further, some lipids are known to function directly in cell signal pathways as messengers or regulators (Sunshine and Iruela-Arispe, 2017 ).

Membrane lipids can be grouped into four major classes: phospholipids, glycolipids, sterols and sphingolipids (Figure 1) (Harayama and Riezman, 2018 ). Phospholipids and glycolipids are glycerol-based lipids consisting of two hydrophobic fatty acids attached to the sn-1 and sn-2 positions and a phosphate group or a sugar moiety to the sn-3 position of a glycerol backbone. The phosphate group of phospholipids can be modified by a polar alcohol such as choline, ethanolamine, glycerol, inositol and serine, which gives these classes of lipids their names phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS), respectively. The fatty acids of phospholipids and glycolipids vary in chain length, the degree of saturation and double bond position. Phospholipids are the most abundant membrane lipids in both yeast and mammals. In photosynthetic tissues of plants, however, glycolipids including galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) and the sulfolipid sulfoquinovosyldiacylglycerol (SQDG) are far more abundant than phospholipids. Sterols are a subgroup of steroids with a characteristic structure consisting of four rings of carbon atoms, while sphingolipids are defined by the presence of a sphingoid base covalently linked to a fatty acid via an amide bond. In addition to the structural diversity, the different classes of membrane lipids are not distributed equally among tissues, organelles or even between two leaflets of the same membrane, but rather have specific locations, and the collective action of their bulky lipids defines the identity and function of different organelles (Harayama and Riezman, 2018 van Meer et al., 2008 ). For example, in plants, galactolipids are located exclusively in chloroplasts, while sterols and sphingolipids are enriched in lipid microdomains of the plasma membrane. Galactolipids play a key role in the biogenesis of photosynthetic membranes and are important for the optimal function of embedded photosynthetic pigment–protein complexes in higher plants (Kobayashi, 2016 ).

Schematic representation of the chemical structures of membrane lipids and lipid molecular shapes.

(a–n) Structures of membrane lipids. Membrane lipids are subdivided in four major categories: phospholipids (b–g), glycolipids (h–j), sphingolipids (a) and sterols (l). Triacylglycerol (TAG) is a storage glycerolipid (m). Classes of phospholipids are defined by the hydrophilic head groups (R) attached to the sn-3 position of the glycerol backbone. Sphingolipids constitute a large category of lipids with diverse acyl chains and head groups (X). (o) Schematic representation of lipid molecular shapes.

Lipids are the major determinants of the physicochemical properties of cellular membranes, which in turn are crucial for membrane functions (Ernst et al., 2016 Harayama and Riezman, 2018 ). Both the nature of the glycerolipid head group and the length and degree of saturation of their acyl chains influence the membrane’s physical properties such as fluidity, permeability, bilayer thickness, charge and intrinsic curvature. In this context, glycerolipids with a relatively large head group such as PC and DGDG approximate a cylindrical molecular shape and tend to form bilayer lipid phases with no curvature strain. In contrast, the shapes of PE and MGDG are more conical, due to the presence of relatively small head groups. They impose negative curvature stress on membranes and are prone to form non-bilayer lipid structures in membranes. Anionic lipids PG, SQDG, PI and PS are key determinants of membrane surface charge and hence play crucial roles in mediating lipid–protein interactions (Harayama and Riezman, 2018 Jouhet, 2013 ) (Figure 1). Sterols interact more favorably with saturated than with unsaturated acyl chains of phospholipids (Nyholm et al., 2019 Nystrom et al., 2010 ). These interactions regulate membrane fluidity, lipid bilayer stability and membrane microdomain formation. In addition to lipid class composition, membrane physical properties and function are also dependent on the fatty acid composition of lipid molecules. In general, lipids with saturated fatty acids decrease membrane fluidity due to the tight packing of straight saturated acyl tails and stronger interactions of saturated acyl chains with sterols. The packing of unsaturated lipids, on the other hand, increases membrane fluidity because cis double bonds create a rigid bend preventing tight packing of their fatty acids (Harayama and Riezman, 2018 Munro, 2003 ). In addition to the degree of fatty acid desaturation, their acyl chain length and their positional distribution on the glycerol backbone affect organization and dynamics of membranes.

Membrane lipid compositions are determined by a range of metabolic processes, including lipid biosynthesis, transport, turnover, remodeling and degradation. Glycerolipids are major structural components of cellular membranes. The enzymatic steps and pathways involved in glycerolipid biosynthesis are well defined and the mechanisms of lipid transport well studied. However, much less is known about the molecular processes underlying lipid modifications after their synthesis. This review will summarize our current knowledge about post-synthetic modifications of fatty acids and head groups, with a focus on the candidate enzymes involved in remodeling of acyl chains and head groups of glycerolipids. In addition, we will discuss the functional role of TAG metabolism in lipid remodeling. Finally, new information about the functions of membrane lipid remodeling will be summarized.


Classification of Toxins | Microbiology

Based on activ­ity, toxins are divided into three types: type I (that act the cell membrane), type II (that attack the cell membrane), and type III (that penetrate the mem­brane to act inside the cell).

1. Membrane Transducing Toxins:

These type of toxins are type I toxins which damage host cells by subtle means through inappropriate activation of cellular receptors. They send wrong message into the cell which confuses the normal routes of communication.

The examples are: the stable toxin (ST) of E. coli and emetic toxin of B. cereus. The ST binds to membrane receptors to stimulate guanyl cyclase and give rise to the intracellular message (cGMP). The cGMP activates protein kinase G and modulates several signalling pathways.

Bacterial superantigenic toxins directly stimulate immune response by acting as mitogens. They bind to the T cell receptor and MHC Class II antigen directly and activate one or several susets of about 5-20% of T cells.

Examples of superantigens are enterotoxins (causing food poisoning) and exotoxins (causing toxic sock) of S. aureus, and erythrogenic toxins of S. pyogenes.

2. Membrane-Damaging Toxins:

This group of toxins is called type II toxins which directly act on cell membrane, form holes resulting in cell death.

More than 100 toxins have been identified and categorised into several groups as given below:

i. Pore Forming Toxins:

Such toxins enter into the cell membrane as oligomers and form pores. The size of pores differ with different toxins. Some pore forming toxins have cellular activity i.e. invoke cytokine production. Be- sides, many intracellular toxins induce pores due to translocation of their catalytic domains across the membrane into cytosol.

The thio activated cholesterol-bing toxins are produced by four genera of Gram-positive bacteria, for example species of Streptococcus (e.g. streptolysin O, pneumolysin).

Listeria (listerolysin O, ivanolysin), Clostridium (tetanolysin, perfringolysin O, septicolysin O, histolyticolysin O, chauveolysin), and Bacillus (cereolysin O, alveolysin, thuringolysin O). These toxins attack cells containing cholesterol in their membrane and form pores of about 30-40 nm having 30 monomers.

Gram-negative bacteria produce RTX toxins, for example E. coli, haemolysin, leukotoxins from Pasteurella haemolytica, Proteus, Bordetella adenylate cyclase. These toxins form pores of about 1-2 nm consisting of 7 monomers and damage the normal function of host cells. The a-toxin of S. aureus form pores in host cell which results in cell death through apoptosis.

ii. Toxins that Damage Membrane Enzymatically:

There are many toxins that damage host’s cell membrane enzymatically. For example phospholipases produced by L. monocytogenes, S. aureus, P. aeruginosa, B. cereus, and Aeromonas. Phospholipase C (PLC) or a-toxin of C. perfringens has necrotic and cytolytic activity. PLC of P. aeruginosa damages lung surfactant in human. Proteases produced by Porphyromonas gingivalis is implicated in gum disease.

3. Intracellular Toxins:

These are type III toxins which act in most subtle way. They act in different stages (Fig. 27.22). They have to gain intracellular access, survive attack by proteases and protons and trick the cell into them to their target and destroy enzymatically. They are the most deadly group of toxins, for example botulinum and tetanus neurotoxins are lethal to human at a dose of about 0.1 ng and affect nerves.

Neurotoxins of C. botulinum and C. tetani act as protease. They block the function of peripheral nerves and cause a flaccid paralysis, stimulate adenylate cyclase (cAMP), the high concentration of which causes massive fluid accumulation in the lumen of the gut resulting in watery effect on immune function. Tetanus toxin attacks nerve cells in the CNS and its effects are more dramatic leading to muscle spasm and rigid paralysis.

The anthrax lethal factor (LF) is a zinc protease that cleaves the N-terminus of MAP (mitogen- activated protein) kinase to inactivate it.


Salt causes ion disequilibrium-induced programmed cell death in yeast and plants

Programmed cell death (PCD) is a fundamental cellular process conserved in metazoans, plants and yeast. Evidence is presented that salt induces PCD in yeast and plants because of an ionic, rather than osmotic, etiology. In yeast, NaCl inhibited growth and caused a time-dependent reduction in viability that was preceded by DNA fragmentation. NaCl also induced the cytological hallmarks of lysigenous-type PCD, including nuclear fragmentation, vacuolation and lysis. The human anti-apoptotic protein Bcl-2 increased salt tolerance of wild-type yeast strain and calcineurin-deficient yeast mutant (cnb1Δ) that is defective for ion homeostasis, but had no effect on the NaCl or sorbitol sensitivity of the osmotic hypersensitive hog1Δ mutant – results that further link PCD in the response to the ion disequilibrium under salt stress. Bcl-2 suppression of cnb1Δ salt sensitivity was ENA1 (P-type ATPase gene)-dependent, due in part to transcriptional activation. Salt-induced PCD (TUNEL staining and DNA laddering) in primary roots of both Arabidopsis thaliana wild type (Col-1 gl1) and sos1 (salt overly sensitive) mutant seedlings correlated positively with treatment lethality. Wild-type plants survived salt stress levels that were lethal to sos1 plants because secondary roots were produced from the shoot/root transition zone. PCD-mediated elimination of the primary root in response to salt shock appears to be an adaptive mechanism that facilitates the production of roots more able to cope with a saline environment. Both salt-sensitive mutants of yeast (cnb1Δ) and Arabidopsis (sos1) exhibit substantially more profound PCD symptoms, indicating that salt-induced PCD is mediated by ion disequilibrium.


CONCLUSIONS

Clearly, an understanding of desiccation tolerance will require a better understanding of the molecular functions of sugars and hydrophilins in desiccation. Important insights will come from identifying the critical targets of these stress effectors. Indeed, one can postulate that many proteins in the cell could aggregate without impinging on cell viability. Likewise, most aggregates can be removed and replaced with new proteins by de novo gene expression. Obviously, this replacement strategy requires that the replacement machinery, like RNA polymerase II, chaperonin proteins, or ribosomes, remain functional. These proteins may be the critical targets of trehalose and/or Hsp12. Similarly, only a subset of membranes may need to be protected. For example, loss of mitochondrial membrane integrity is irreversible and lethal, whereas transient holes in the plasma membrane can be partially tolerated if fixed (van Meer et al., 2008). Identifying the subset of proteins and membranes that must be protected will be difficult but important challenges going forward.

The study of trehalose and hydrophilins is also very likely to inform on stress biology beyond desiccation. First, in yeast and in other organisms trehalose and hydrophilins are expressed in aqueous conditions, particularly under different stresses (Singer and Lindquist, 1998 Garay-Arroyo et al., 2000 François and Parrou, 2001 Battaglia et al., 2008 De Virgilio, 2012). In addition, dividing cells lacking both trehalose and hydrophilin have an unusual phenotype, the inability to propagate a membrane-associated prion (Kim et al., 2018). These results raise the possibility that the membrane, or yet-to-be-discovered, stress effector activities of trehalose and hydrophilins may be at play even under aqueous conditions. Second, a recent study by Kurzchalia and colleagues showed that a specific metabolic pathway, the previous enigmatic glyoxylate shunt, was critical for desiccation tolerance (Erkut et al., 2016). This work inspires the search for other metabolic pathways that may be critical to other stress responses. Finally, the fact that only a subset of hydrophilins impact desiccation raises an important question. What is the function of the other hydrophilins? Localization studies of the other hydrophilins suggest they may be targeted to specific subcellular locations where they are performing yet-to-be-discovered functions (Candat et al., 2014). The study of desiccation tolerance, like previous studies of biological extremes, is opening new doors in biology.


How saturated fatty acids damage cells

In our increasingly health-conscious society, a new fad diet seems to pop up every few years. Atkins, Zone, Ketogenic, Vegetarian, Vegan, South Beach, Raw -- with so many choices and scientific evidence to back each, it's hard to know what's healthy and what's not. One message, however, has remained throughout: saturated fats are bad.

A new Columbia University study reveals why.

While doctors, nutritionists and researchers have known for a long time that saturated fats contribute to some of the leading causes of death in the United States, they haven't been able to determine how or why excess saturated fats, such as those released from lard, are toxic to cells and cause a wide variety of lipid-related diseases, while unsaturated fats, such as those from fish and olive oil, can be protective.

To find answers, Columbia researchers developed a new microscopy technique that allows for the direct tracking of fatty acids after they've been absorbed into living cells. The technique involves replacing hydrogen atoms on fatty acids with their isotope, deuterium, without changing their physicochemical properties and behavior like traditional strategies do. By making the switch, all molecules made from fatty acids can be observed inside living cells by an advanced imaging technique called stimulated Raman scattering (SRS) microscopy.

What the researchers found using this technique could have significant impact on both the understanding and treatment of obesity, diabetes and cardiovascular disease.

Published online December 1st in Proceedings of the National Academy of Sciences (PNAS), the team reports that the cellular process of building the cell membrane from saturated fatty acids results in patches of hardened membrane in which molecules are "frozen." Under healthy conditions, this membrane should be flexible and the molecules fluidic.

The researchers explained that the stiff, straight, long chains of saturated fatty acids rigidify the lipid molecules and cause them to separate from the rest of the cell's membrane. Under their microscope, the team observed that those lipid molecules then accumulate in tightly-packed "islands," or clusters, that don't move much -- a state they call "solid-like." As more saturated fatty acids enter the cell, those islands grow in size, creating increasing inelasticity of the membrane and gradually damaging the entire cell.

"For a long time, we believed that all cell membrane is liquid-like, allowing embedded proteins to change their shape and perform reactions," said Principal Investigator Wei Min, a professor of chemistry. "Solid-like membrane was hardly observed in living mammalian cells before. What we saw was quite different and surprising."

Lipid molecules made from unsaturated fatty acids on the other hand bear a kink in their chains, Min said, which makes it impossible for these lipid molecules to align closely with each other as saturated ones do. They continue to move around freely rather than forming stationary clusters. In their movement, these molecules can jostle and slide in between the tightly-packed saturated fatty acid chains.

"We found that adding unsaturated fatty acids could 'melt' the membrane islands frozen by saturated fatty acids," said First Author Yihui Shen, a graduate student in Min's lab. This new mechanism, she said, can partly explain the beneficial effect of unsaturated fatty acids and how unsaturated fats like those from fish oil can be protective in some lipid disorders.

The study represents the first time researchers were able to visualize the distribution and dynamics of fatty acids in such detail inside living cells, Shen added, and it revealed a previously unknown toxic physical state of the saturated lipid accumulation inside cellular membranes.

"The behavior of saturated fatty acids once they've entered cells contributes to major and often deadly diseases," Min said. "Visualizing how fatty acids are contributing to lipid metabolic disease gives us the direct physical information we need to begin looking for effective ways to treat them. Perhaps, for example, we can find a way to block the toxic lipid accumulation. We're excited. This finding has the potential to really impact public health, especially for lipid related diseases."


3 Major Diseases Caused by Fungi in Humans

Some agarics (mushrooms) are poisonous to living being. The most severe type of mushroom poisoning is caused by species belonging to the genus Amanita. A mistake can result in very unpleasant gastrointestinal upset or even death. Amanita phalloides (the death cap) is very poisonous and responsible for most of the mushroom poisoning deaths.

A mixture of three toxins α-amanitine, β-amamtine and phalloidine — is the cause of poisoning. Amanita muscaria (fly agaric) and A. pantherina (panther cap) are also poisonous.

Besides Amantia, some other poisonous mushrooms are Russula, Lactarius, Boletus, Entoloma etc. Symptoms of mushroom poisoning are — nausea, vomiting, abdominal pain and visual disturbances. The affected one finally falls into a coma and may succumb.

Disease # 2. Mycotoxicosis:

Toxins produced by fungi are called mycotoxins. One of the most important mycotoxin is aflatoxin produced by some species of Aspergillus (especially A.flavus). Anatoxins can be lethal to poultry.

They may cause lever damage and are suspected to induce cancer in humans. Claviceps purpurea produces ergot alkaloids which, if mixed with rye flour, may result in severe poisoning. Fingers toes, whole arms, legs, sometimes eyes and noses become gangreneous, wither and fall off with no bleeding.

Some fungi like Stachybotrys atra, Pithomyces chartarum and some Fusarium spp. produce mycotoxins which affect large animals like norses, sheep and cattle. They develop facial eczema and liver damage while feeding on contaminated grass.

Disease # 3. Mycoses:

It is considered that around 1/5th of the global population (about 800 million) suffer or have suffered from mycoses. Mycoses can be considered of two types — superficial mycoses and deep- seated mycoses.

(i) Superficial mycoses:

Superficial mycoses are unpleasant but not lethal. Skins, hair and nails are infected. The fungi that cause superficial mycoses are called dermatophytes and the diseases they cause are called dermatophytoses.

Various species of genera Microsporon, Epidermophyton and Trichophyton are important dermatophytes. Malassezia furfur is the agent of Pityriasis versicolor (dandruff): Microsporum andouini is the agent for most cases of ring worm of scalp in children.

(ii) Deep-seated mycoses:

Deep-seated mycoses are dangerous and may become fatal if not treated. Unfortunately, the diagnosis of mycoses is often difficult because there are no specific ‘mycoses symptoms’. The isolation and identification of the pathogen is the only method to identify the disease.

About 15 deep scaled mycoses are in knowledge, e.g., Coccidioidomycosis, Blastomycosis, Candidiasis, Subcutaneous Phycomyosis, Sporotrichosis, Chromomycosis, Mucormycosis, Geotrichosis and Mycetoma.

Details of some important ones are given below:

Caused by Aspergillus fumigatus which attacks cars, lungs etc. Pulmonary aspergillosis is diagnosed as T.B.

Popular as ‘Gilchrist’s disease’. In early stages it causes cough, chest pains and weakness following the formation of subcutaneous nodules, abscesses or lesions on face and arm. Blastomyces dermitidis is the causal organism.

It is caused by Candida albicans, the mucous membrane of skin, lungs etc. are attacked. Ammons et al. (1977) have listed cutaneous candidiasis, oral candidiasis, pulmonary candidiasis, volvovaginal candidiasis and bronchocandidiasis as some of the infections.

A more or less localized and chronic infection of the skin and subcutaneous tissues by Cladosporium carrionii, Phialophora verrucosa, P. pedrosoi, etc.

Characterized by the lesions limited to the upper respiratory tract and lungs. In humans it is caused by Coccidioides immitis.

The central nervous system is affected by this disease caused by Cryptococcus neoformans, it affects the vision and causes respiratory failure.

This disease is caused by Emmonsiella capsulata. It is very widespread and serious in humans and is sometimes even fatal.

It is an oral pulmonary, bronchial or intestinal infection in humans caused by Geotrichum candidum.

However, warm blooded animals are also infected by fungi causing mycoses. Examples—Cattle (Trichophyton verrucosum), and birds (Aspergillus fumigatus, Candida albicans).


Results

N. oceanicasterol biosynthetic pathway shares features in structure and sterol profiles with those of animals and plants

Among different organisms, the core sterol biosynthetic pathway consists of a common set of enzymes that exhibit strong conservation in amino acid sequences however the pathway architecture and substrate specificity can vary significantly [1]. In silico reconstruction and comparison of sterol biosynthetic pathways among 12 selected algal species revealed intriguing structural features of the N. oceanica pathway, which include characteristics from both higher plants and animals (Figure 1 and Additional file 1).

Conservation of sterol biosynthetic genes in eukaryotic algae. The color key (top) indicates the similarity of the gene to the closest match and ranges from low similarity (black) to high similarity (red). Black areas indicate no Blastp hit below the applied e-value threshold (1e-5). Red areas indicate orthologs with Blastp e-values below 1e-100. Color in the heatmap is scaled column-wise based on the bit values of tblastn results (Additional file 1). Abbreviations: red alga Cyanidioschyzon merolae (Cm), diatom Phaeodactylum tricornutum (Pt), diatom Thalassiosira pseudonana (Tp), diatom Fragilariopsis cylindrus (Fc), eustigmatophyte N. oceanica (No), brown alga Ectocarpus siliculosus (Es), green alga Ostreococcus tauri (Ot), green alga Micromonas sp. RCC299 (Mi), green alga Chlorella variabilis NC64A (Cv), green alga Coccomyxa subellipsoidea C-169 (Cs), green alga Chlamydomonas reinhardtii (Cr), and green alga Volvox carteri (Vc). See Additional file 2: Figure S1 for the phylogenetic tree of the sampled species.

The sterol synthetic pathway of N. oceanica includes higher plant-like features. Higher plants have two sterol methyltransferase (SMT) enzymes that use different substrates to give either methylated (SMT1) or ethylated (SMT2) phytosterols. In the N. oceanica genome, two candidate genes encoding SMT were identified, which resemble those of higher plants in primary sequence. In contrast, the diatom Phaeodactylum tricornutum and several green algae including Chlamydomonas reinhardtii, Chlorella variabilis NC64A, Coccomyxa subellipsoidea C-169, and Volvox carteri have a single candidate gene encoding SMT (Figure 1, see Additional file 2: Figure S1 for the phylogenetic tree of the sampled species) that potentially catalyzes successive methylation reactions to give methylated and ethylated products.

Features that are shared with animals were also present in the sterol synthetic pathway of N. oceanica. In the sampled microalgae, the key enzyme catalyzing sterol side chain reduction is different from that of Arabidopsis and higher plants in general. In higher plants, the enzyme, namely sterol 24(28) isomerase-reductase, is encoded by the DWF1 gene in Arabidopsis and performs dual functions. It catalyzes C-24(28) double bond isomerization to form a 24(25) double bond, followed by reduction of the 24(25) double bond. In animals and yeast, the equivalent enzymes are 24-dehydrocholesterol reductase (DHCR24) and sterol C-24(28) reductase (ERG4), which only catalyze the reduction reaction. DWF1 or DHCR24 orthologs have not been found in algae except in N. oceanica and the diatom Fragilariopsis cylindrus. Amino acid sequence analysis indicates that N. oceanica sterol 24(25) reductase is clustered with that of choanoflagellates (the closest living unicellular relatives of animals [28]) and has greater similarity to animal DHCR24 than to higher plant DWF1 (Additional file 2: Figure S2). The evidence based on DWF1/DHCR24 therefore suggests features of an animal-type sterol biosynthetic pathway.

To test these predicted features of the sterol biosynthetic pathway, we characterized the chemical profile of sterols in N. oceanica IMET1, which unveiled an animal-like composition of sterols. In N. oceanica, five sterols were identified (sterol structures indicated as bold numbers in Additional file 2: Figure S3 and in the following text). Cholesterol (2) is the most abundant sterol, comprising 70% to 75% of the total (Table 1). The remaining sterols are fucosterol (11), isofucosterol (13), 24-methylcholesta-5, 25(27)-dienol (10), and 24-methylenecholesterol (7) (Additional file 2: Figure S3). In N. oceanica, sterols with a C-22 double bond are not found, supporting the absence of CYP710A (a C-22 desaturase Figure 1). Although a protein (g4528) with similarity to CYP710A was present, its primary sequence is closer to that of CYP51 than to CYP710A (Additional file 1). The lack of CYP710A is a typical feature of animals, as higher plant genomes usually encode highly conserved CYP710A. In addition, the side-chain double bond formed by SMTs is retained, supporting the presence of an animal-type DHCR24 (Additional file 2: Figure S2). This is further supported by the accumulation of a large amount of cholesterol in N. oceanica (Table 1), which is the sole sterol in animals. On the other hand, only a minor amount of the phytosterols, which are the dominant forms of sterols in higher plants, was found in N. oceanica (Table 1). Therefore, N. oceanica sterol profiles exhibit features of both animals and higher plants.

Furthermore, we adopted a chemical biology approach to probe the architecture of the sterol biosynthetic pathway, in which N. oceanica was treated with a series of isoprenoid and sterol biosynthetic inhibitors (SBIs). (See Figure 2 for the target enzymes and Additional file 2: Figure S4 for the inhibition ratios.) The phenotypes of sterol biosynthetic mutants can be mimicked by the application of specific chemical inhibitors, which are powerful tools for elucidating the biosynthesis and functions of sterols [29–33], particularly when a targeted gene knockdown system is not yet available for N. oceanica IMET1. The chemical inhibitors we employed here have been well studied and the specificities to their corresponding enzymes established [34–38].

Deduced pathway of sterol biosynthesis in N. oceanica , comparison among plants, humans and yeasts, and sites of action of inhibitors. Enzyme nomenclature is that commonly used in Arabidopsis, because although many human and yeast enzymes have different names and abbreviations, they catalyze equivalent reactions to those of the Arabidopsis enzymes. An exception is Arabidopsis DWF1 (a dual isomerase-reductase), which is absent in Nannochloropsis instead, this alga has a human-type DHCR24 (the yeast equivalent is ERG4). Full names and functions of enzymes are provided in Additional file 3: Table S1. Lanosterol synthase (LAS) is found in Arabidopsis but has a minor role. Enzyme abbreviations: DXS, 1-deoxy-D-xylulose 5-phosphate synthase HMGR, hydroxy-methyl-glutaryl-CoA reductase HMGS, hydroxy-methyl-glutaryl-CoA synthase SQE, squalene epoxidase CAS, cycloartenol synthase LAS, lanosterol synthase SMT, sterol methytransferase SMO, sterol 4-methyl oxidase CPI, cycloeucalenol cycloisomerase CYP51, sterol 14-alpha demethylase FK, sterol C-14 reductase HYD, sterol C-8 isomerase DWF7, delta7 sterol C-5 desaturase DWF5, sterol C-7 reductase DWF1, sterol C-24(28) isomerase-reductase CYP710A, sterol C-22 desaturase DHCR24, dihydrocholesterol reductase ERG4, sterol delta 24(28) reductase. Abbreviations for metabolites: MEP, methylerythritol phosphate IPP, isopentenyl pyrophosphate MVA, mevalonic acid. Abbreviations for inhibitors: CLO, clomazone TBF, terbinafine 25-AZA, 25-azalanosterol TDM, tridemorph TEB, tebuconazole.

The inhibitor clomazone (CLO) acts on 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a key regulatory enzyme for chloroplast IPP biosynthesis in higher plants [36]. DXS plays an equivalent role to animal HMGR, which is a key regulatory enzyme for cytosolic IPP biosynthesis via the MVA pathway. CLO had a small effect on sterol profiles (Table 1), but it reduced to 70% of the total sterol amount of the control, presumably by limiting IPP supplies to the sterol pathway (Figure 2). Terbinafine (TBF) specifically targets squalene epoxidase (SQE) [34], which is the second committed enzyme of the sterol biosynthetic pathway (Figure 2). It is a critical point for inhibition of sterol biosynthesis, as it reduces sterol content but has no direct effect on the biosynthesis of other isoprenoids [34]. TBF-treated cells accumulated a significant amount of squalene (1), supporting the presence and function of SQE (Table 1) and the total amount of sterol was reduced by about 20%. Cells inhibited by tridemorph (TDM), the cycloeucalenol cycloisomerasesterol isomerase (CPI) and sterol C-8 isomerase inhibitor [38], accumulated 9,19-cyclopropyl sterol (pollinastanol, 5) and delta-8 sterols [obtusifoliol, (12), cholest-8-enol (3), and stigmasta-8,24(28)-dienols (14, 16)] (Table 1). Thus, CPI is required for sterol biosynthesis, implying that N. oceanica uses cycloartenol as a precursor in the biosynthesis of other sterols, consistent with the presence of a cycloartenol synthase (CAS) gene (Figure 1). Cycloartenol has been shown to be the signature sterol in almost all photosynthetic organisms [39]. Treatment with tebuconazole (TEB), an inhibitor of the cytochrome P450 CYP51 [37], resulted in accumulation of 4,4,14-trimethyl (8, 15, 17) and 4,14-dimethyl sterols (6). The relatively large quantities of cycloartanol (17) and cycloartenol (15) (Table 1) further indicate that cycloartenol is the major precursor for the N. oceanica sterol biosynthetic pathway (Figure 2). Moreover, the predominance of sterols without side-chain methylation implies that the 14α-demethylation step occurs before C-24 methylation and C-4 demethylation (Figure 2). This architecture of the biosynthetic pathway differs from that of land plants and is more similar to the cholesterol biosynthetic pathway in animals and the ergosterol biosynthetic pathway in fungi [40]. 25-azalanosterol (25-AZA) is a specific inhibitor of SMT, which determines sterol compositions [35]. Application of 25-AZA resulted in desmosterol (4) accumulation (Table 1), revealing a similarity to yeast SMT which exhibits a preference for 4,4-desmethylsterols, unlike other algal or plant SMTs [7].

In summary, the collective findings allowed us to propose a sterol biosynthetic pathway in N. oceanica that exhibits both common and distinct features from those in fungi, animals, and green plants (including green algae and land plants) (Figure 2).

The role of sterols in the growth of N. oceanica

To probe the functional roles of sterols in N. oceanica, we next investigated the dynamics of sterol profiles and the expression of their biosynthetic genes during proliferation or cessation of cell division (Figure 3A). Sterol levels exhibited modest increases during the early growth stages of N. oceanica cultures, and then increased rapidly at later stages of the culture cycle between 4 × 10 7 and 10 8 cells ml -1 (Figure 3B). The increase of cholesterol accounts for a significant proportion of the increase in total sterols (Figure 3B). Meanwhile, sterol biosynthetic genes showed a coordinated adaptation in the late culture cycle (Figure 3C). All studied genes except DXS and SMT1 were transcriptionally elevated, with maximum levels at the late log phase. Thus, sterol biosynthesis and accumulation appears to be a feature of late cell growth as the culture approaches stationary phase.

Chemical analysis and transcripts of sterol biosynthesis during N. oceanica growth. (A) Growth curve of N. oceanica. (B) Changes in total sterol and sterol composition at different growth stages. (C) Transcript levels of sterol biosynthetic genes at different growth stages. The values are the means of three replicates. Asterisks (*) indicate P values < 0.05.

Nitrogen depletion is reported to inhibit cell division in N. oceanica[41]. To investigate its effects on sterol biosynthesis, we transferred cells to N-replete and N-depleted media for six days. The sterol level in N-depleted cells was about 33% that of N-replete cells (Additional file 2: Figure S5a). This difference is largely explained by differences in the amount of cholesterol, but surprisingly isofucosterol was disproportionately low, especially compared to fucosterol. This could be explained by differential responses of SMTs to nitrogen depletion. Studies of gene expression during the first 24 h of nitrogen depletion showed that genes of the MEP pathway were appreciably down-regulated (Additional file 2: Figure S5b), which may explain the subsequent decline in sterol levels. While some genes of sterol biosynthesis were initially up-regulated (for example, FK and SMT1), others (for example, SMT2 and DWF5) were rapidly down-regulated (Additional file 2: Figure S5b). These results suggest a complex response to nitrogen depletion but point to a central role for phytosterols, reflecting the importance of SMTs and potentially the methyl donor S-adenosyl methionine.

Effects of light on sterol biosynthesis in N. oceanica

To probe the relationship between sterol biosynthesis and light, we next examined the responses of sterol profiles and their biosynthetic genes to changes in light intensity. Cultures of N. oceanica are typically grown at or below the light saturation point of about 100 μmol photons m -2 s -1 , whereas 300 μmol photons m -2 s -1 poses a high light stress. High light is known to cause biochemical damage to the photosynthetic system in higher plants, reducing the efficiency of light utilization.

First, cultures with the same cell density were transferred to constant light intensities of 100 and 300 μmol photons m -2 s -1 for 96 h. RNA and sterols were isolated at the end of the light treatments. The sterol content was lower in cells under 300 μmol photons m -2 s -1 compared with those under 100 μmol photons m -2 s -1 (Figure 4A). This is the result of a significantly reduced cholesterol level, whereas phytosterols (fucosterol and isofucosterol) were significantly increased under 300 μmol photons m -2 s -1 (Figure 4A). This indicates a relationship between light stress and sterol metabolism. All sterol biosynthetic genes examined were expressed at a significantly lower level under 300 μmol photons m -2 s -1 (Figure 4B). Therefore, in response to high light stress, sterol biosynthesis as a whole is repressed, while some specific phytosterols increase in concentration. This observation is consistent with observations that sterol biosynthesis is modified in the high light response of the green alga Dunaliella bardawil[42] and higher plants [43].

Transcript levels of sterol metabolic genes of N. oceanica in response to changes in light intensity. (A) Sterol contents under different light intensities. (B) Expression of sterol metabolic genes of N. oceanica under different light intensities. The transcript levels under 300 μmol photons m -2 s -1 are normalized to 1.0. Cells at mid-log phase were transferred to constant light intensities of 100 and 300 μmol photons m -2 s -1 . Cells were taken for transcript and sterol analysis after 96 h. (C) Maximum photosynthetic efficiency of photosystem II (PSII) of N. oceanica in response to the transfer from dark to light. Cells at mid-log phase were transferred to dark for 12 h and then transferred to 300 or 100 μmol photons m -2 s -1 light. (D) Relative mRNA abundance of sterol metabolic genes in N. oceanica in response to the transfer from dark to light. (E) Relative mRNA abundance of sterol metabolic genes in N. oceanica in response to a light intensity change from 300 to 100 μmol photons m -2 s -1 . Cells at mid-log phase were transferred to 300 μmol photons m -2 s -1 for 12 h, then to 100 μmol photons m -2 s -1 . Samples were collected after 12 and 24 h. (F) Relative mRNA abundance of sterol metabolic genes in N. oceanica in response to a light intensity change from 100 to 50 μmol photons m -2 s -1 . Cells at mid-log phase were transferred to 100 μmol photons m -2 s -1 for 12 h, then to 50 μmol photons m -2 s -1. Samples were collected after 12 and 24 h. Values are the means of three replicates. Asterisks (*) indicate P values < 0.05.

To study the short-term effects of high light, cells were first grown under 50 μmol photons m -2 s -1 to mid-log phase, were then darkness-adapted for 12 h to simulate night, and finally transferred to 100 or 300 μmol photons m -2 s -1 for 12 h. After this time, the photosynthetic efficiency was measured. Samples were collected for analysis of gene expression at the beginning and the end of the 12-h light treatment. The photosynthetic efficiency was significantly higher at 100 μmol photons m -2 s -1 than at 300 μmol photons m -2 s -1 (Figure 4C). The transcript levels of all studied genes were also higher at 100 μmol photons m -2 s -1 than at 300 μmol photons m -2 s -1 (Figure 4D). This further indicates that high light treatment rapidly represses sterol biosynthesis genes and impairs photosynthesis.

To determine if such a repression of gene expression is reversible, cells were treated for 12 h at 300 μmol photons m -2 s -1 and then transferred to 100 μmol photons m -2 s -1 for 24 h. RNA was isolated at the end of the high light treatment and after 12 and 24 h adaptation to 100 μmol photons m -2 s -1 , for analysis of gene expression. The results showed that expression of sterol biosynthesis genes increased significantly after 24 h at 100 μmol photons m -2 s -1 (Figure 4E). Thus, sterol biosynthesis gene expression is highly responsive to such light treatments, implying a key role for sterols in adaptation to high light. For comparison, transfer of cells from 100 to 50 μmol photons m -2 s -1 resulted in only minor changes in expression of sterol biosynthesis genes after 12 and 24 h (Figure 4F), implying that sterol biosynthesis does not play a major role in adaptation to light levels below the saturation point.

Inhibition of sterol biosynthesis in N. oceanicaleads to depressed photosynthetic efficiency

The preceding observations provide evidence of a regulatory role of light in microalgal sterol biosynthesis and the involvement of sterol biosynthesis in photodamage at both the metabolic and gene expression levels. However, whether changes of sterol biosynthesis modulate photosynthesis is still unknown. Thus, we further probed the cellular changes that occurred in response to pharmacological perturbation of sterol biosynthesis. Cells were grown for 96 h under 50 μmol photons m -2 s -1 in the presence of 20 mg l -1 CLO, which inhibits DXS activity. This led to a decrease in photosynthetic efficiency (Figure 5A). RBCL is the large subunit of RuBisCO, which incorporates inorganic CO2 into organic forms during photosynthesis [44]. RBCL was transcriptionally depressed following CLO administration (Figure 5B). DXS inhibition led to reduced accumulation of sterols in particular, and of carotenoids and chlorophylls to a much lesser extent (Figure 5C). CLO-treated cells revealed less dense cytoplasm and less distinct organelles. Plastids showed a reduced number of thylakoid membrane structures and a deficiency of normal thylakoid stacking compared with the wild-type cells (Figure 5D,E). This likely explains the decreased photosynthetic efficiency of these cells. Although we showed that sterols were significantly reduced by CLO treatment, determining the contribution of sterols to photosynthetic function required the use of a more specific inhibitor. Inhibition of the post-squalene sterol biosynthetic pathway by TBF led to a 24% drop in photosynthetic efficiency relative to the control within 96 h (Figure 5A). Meanwhile, RBCL was transcriptionally decreased (Figure 5B). TBF-treated cells also contained fewer sterols and more carotenoids and had slightly reduced chlorophyll content (Figure 5C). The cells displayed an aberrant membrane structure with severely affected chloroplasts (Figure 5F). In addition, TBF-treated cells were misshapen compared to untreated cells (Figure 5G,H), implying a defect in membrane structure and function. Therefore, sterols are apparently required for membrane structure, including those of the chloroplast, and this is manifested in reduced photosynthetic function when sterol biosynthesis is inhibited.

The consequence of inhibition of sterol biosynthesis on the photosynthetic activity and apparatus of N. oceanica. (A) Effect of CLO (20 mg l -1 ), and TBF (2.5 mg l -1 ) on the maximum photosynthetic efficiency of PSII. (B) Transcript levels of RBCL gene of N. oceanica induced by CLO (20 mg l -1 ) or TBF (2.5 mg l -1 ). (C) Levels of sterols, chlorophylls, and carotenoids in the presence of CLO and TBF. Values were normalized (100% for mock control). (D-F) Transmission electron microscopy analysis of algal cells cultured with DMSO control (D), CLO (E), and TBF (F). (G-H) Scanning electron microscopy analysis of algal cells treated with DMSO control (G) or TBF (H). Algal cells were grown in medium containing CLO, TBF, or an equivalent amount of DMSO (control) for 96 h under 50 μmol photons m -2 s -1 light. Scale bars represent 0.5 μm. Asterisks (*) indicate P values < 0.05.

Chemical genetic analysis reveals feedback regulation of sterol biosynthesis in N. oceanica

In the N. oceanica IMET1 genome, genes for a full MEP pathway for the production of IPP as the building block for isoprenoids were identified (Figure 1). For the MVA pathway, a gene encoding hydroxy-methyl-glutaryl-CoA synthase (HMGS, g249), the first committed enzyme in the MVA pathway, was identified however, genes encoding enzymes of the remaining steps (including the key regulatory enzyme hydroxy-methyl-glutaryl-CoA reductase HMGR) were apparently absent (Figure 1). Since the MEP pathway is the sole source of isoprenoids, including sterols, in N. oceanica, the control of sterol biosynthesis in the context of IPP biosynthesis is of physiological importance. In animals, cholesterol biosynthesis is highly regulated. Cholesterol over-accumulation leads to decreased biosynthesis of cholesterol and FA, whereas low cholesterol stimulates their synthesis. HMGR, the committed enzyme in isoprenoid and sterol biosynthesis, serves as the primary feedback regulation site to ensure maintenance of lipid homeostasis [45]. Although animal and fungal HMGR is the key target for such regulation, and all such organisms employ an HMGR-binding protein called ‘Insig’, the signals and molecular mechanisms vary across these species [46]. Higher plants use both MEP and MVA pathways for isoprenoid biosynthesis. Within plant cells, IPP can be exchanged between the cytosol and plastids. Depletion of endogenous sterols by TBF triggers a significant increase in HMGR enzyme activity [34], implying a feedback mechanism in plants similar to that of mammals or fungi. However, the possible regulation of DXS in such a positive feedback regulation is still poorly understood. In higher plants the contribution of IPP from the MVA pathway might attenuate any feedback regulation caused by sterol depletion because of crosstalk between the MEP and MVA pathways. As a result, N. oceanica, which only possesses the MEP pathway, can be an ideal research model for the regulatory role of sterols with respect to DXS. Therefore, we investigated the transcriptional changes of DXS in N. oceanica cells in response to sterol depletion caused by CLO and TBF treatments, as well as the cellular response to adding cholesterol to the growth medium.

DXS transcript abundance increased markedly within 48 h following CLO administration and then decreased, which is a typical inhibition-adaptation response (Figure 6A). The total sterol level was reduced to about 76% relative to the control after 96 h of CLO treatment, which can be explained by the reduced supply of IPP due to inhibition of DXS activity (Figure 5C). Moreover, levels of carotenoids and chlorophylls also decreased (Figure 5C), further confirming the low activity of isoprenoid biosynthesis and the committed role of DXS in isoprenoid biosynthesis.

Feedback regulation of sterol biosynthesis at the point of DXS gene. (A) Transcript levels of DXS and PSY genes of N. oceanica induced by CLO (20 mg l -1 ). (B) Transcript levels of DXS and PSY genes in N. oceanica induced by TBF (2.5 mg l -1 ). (C) Transcript levels of DXS and PSY genes relative to actin gene (g3056) in response to cholesterol treatment (1.1 mg l -1 ). Samples cultured with DMSO are set as mock controls. Values are normalized to 1. Asterisks (*) indicate P values < 0.05.

Following TBF application, the total sterol content declined about 20% (Figure 5C), whereas DXS was transcriptionally induced after 48 h of treatment (Figure 6B). The induction cannot be explained by the depletion of IPP, since a high level of squalene accumulated in TBF-treated cells (Table 1), suggesting a sufficient supply of IPP for triterpene synthesis. Furthermore, SQS mRNA was reduced to 30% after 12 h of treatment (Figure 7A), a result that could potentially be caused by product inhibition by squalene accumulation. On the other hand, carotenoid levels were elevated by 40% (Figure 5B), and the phytoene synthase gene (PSY) encoding the key committed enzyme step for carotenoid biosynthesis was also transcriptionally up-regulated (Figure 6B). These observations point to stimulated isoprenoid biosynthesis that may be caused by sterol depletion. In addition, the entire MEP pathway and committed cholesterogenic genes were transcriptionally induced by TBF (Figure 7A). Together, the results suggest that TBF-induced sterol starvation stimulates the transcription of DXS and sterol biosynthetic genes.

Changes in transcripts and lipids of N. oceanica in response to sterol starvation induced by TBF. (A) Transcript profiles of cholesterogenic genes and fatty acid (FA) biosynthetic genes in response to TBF (2.5 mg l -1 ). Red and green indicate up- and down-regulated genes, respectively. (B) Transcript levels of FAS-g927 gene in N. oceanica induced by TBF (2.5 mg l -1 ) and nitrogen depletion (N - ). (C) Changes in total FA content induced by TBF. (D) Changes of total TAG content induced by TBF. (E) Changes of glycerol lipid classes (other than TAG) induced by TBF. For all experiments, cultures in exponential phase were treated with TBF (2.5 mg l -1 ) or an equivalent amount of DMSO (control). FA content was analyzed at two, four and six days, and glycerolipid content was analyzed at six days. Asterisks (*) indicate P values < 0.05.

In the case of sterol accumulation achieved by exogenously added cholesterol, the DXS transcript level was markedly reduced (Figure 6C). Although the carotenoid content did not change, PSY was transcriptionally reduced, supporting the proposal that sterol can act as a feedback regulator to the overall isoprenoid biosynthesis pathway. In summary, the transcription of DXS may be controlled by sterol levels, and sterol biosynthesis potentially exerts feedback regulation on isoprenoid biosynthesis, including cholesterogenesis.

Homeostasis between sterols and lipids in N. oceanica

Sterol and FA biosynthesis are two major lipid synthetic pathways in eukaryotes, but it is not clear whether they are co-regulated in microalgae. In animals, type I fatty acid synthase (FAS) and other biosynthetic genes are regulated by cellular sterol levels, as are genes that encode important proteins of cholesterol metabolism [13]. To test the link between sterol and FA biosynthesis in N. oceanica, the transcript abundances of lipid metabolism genes were investigated following TBF-induced sterol depletion. TBF triggered an increase in type I FAS (g927) transcription after 12 and 24 h treatment (Figure 7A). This gene was also transcriptionally elevated in cells treated with nitrogen depletion, which is a critical trigger for lipid accumulation (Figure 7B). Consistent with this, the FA level increased over a period of several days following TBF administration (Figure 7C). Next, we analyzed the TAG content after six days of TBF treatment and observed that the TAG concentration was greatly reduced compared to that of the untreated controls (Figure 7D). Therefore, inhibition of sterol biosynthesis by TBF results in an increase in FAs, but these are not accumulated in TAG. This result is supported by thin layer chromatography of FAs (Figure 7D, inset). Simultaneously, the membrane glycerolipids, particularly the photosynthetic membrane lipids such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and phosphatidylglycerol (PG), decreased significantly following TBF treatment (Figure 7E), which can explain the deformed chloroplast and the decreased photosynthetic efficiency of TBF-treated algal cells. Moreover, a remarkable reduction of phosphatidylinositol (PI) and diacylglyceroltrimethylhomoserine (DGTS) was observed (Figure 7E). Taken together, the evidence suggested that the elevated level of total FA in TBF inhibited cells was derived from the increase in de novo FA biosynthesis other than that of storage lipids or membrane lipids. Moreover, free FA should represent a large proportion of the increased amount of total FA.

We therefore propose a feedback system in Nannochloropsis for the regulation of both sterol and FA homeostasis, which is characterized by (i) the induction of sterol and FA biosynthesis by sterol depletion or the inhibition by sterol accumulation and (ii) transcriptional feedback regulation of DXS ensuring maintenance of lipid homeostasis. Interestingly, in animals, HMGR of the MVA pathway is the committed enzyme in isoprenoid and sterol biosynthesis, and serves as the primary site of the feedback regulation [45], suggesting similarity between the algal system and the animal model [17].


The authors acknowledge Mr. Anand, DST project fellow, Biotechnology Division, CSIR-NIIST, Trivandrum, for his help in preparing this manuscript. We also thank Mr. T. Balaji Prasad, Scientific Publishing Services Pvt. Ltd., Chennai, for his timely help in improving the language of the manuscript.

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Keywords: microalgae, biomass energy, lipid extraction methods, algae biofuels, biodiesel, energy efficiency

Citation: Ranjith Kumar R, Hanumantha Rao P and Arumugam M (2015) Lipid extraction methods from microalgae: a comprehensive review. Front. Energy Res. 2:61. doi: 10.3389/fenrg.2014.00061

Received: 12 November 2014 Accepted: 10 December 2014
Published online: 08 January 2015.

Junye Wang, Athabasca University, Canada

Reeta Rani Singhania, Blaise Pascal University, France
Lijuan Long, Chinese Academy of Sciences, China

Copyright: © 2015 Ranjith Kumar, Hanumantha Rao and Arumugam. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


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