Proteomic analysis of the endophytic fungus Undifilum oxytropis

The filamentous Ascomycete fungus Undifilum oxytropis is an endophyte of locoweed plants of the genera Oxytropis that produces a toxic alkaloid swainsonine. Swainsonine, an alpha-mannosidase inhibitor causes a general toxicosis and neurological problems (locoism) when consumed by grazing animals. Swainsonine is also being assessed for its anti-cancer properties. While the ecology of U. oxytropis has been studied, little is known about the genetics and proteomics of any swainsonine-producing fungus. To help understand the proteins in U. oxytropis, the proteome of U. oxytropis was analyzed using 2-dimensional electrophoresis (2-DE). Proteins from U. oxytropis mycelia were extracted and separated by in-gel isoelectric focusing (IEF). The entire immobilized pH gradient (IPG) strip was cut into a set of gel sections and each gel section was digested with trypsin and then identified using liquid chromatography tandem mass spectrometry (LC-MS/MS). 2-DE maps were also developed for U. oxytropis to define its proteome. In the isoelectric point (pI) range of 3-11 and 10-250 kDa ranges, more than 450 spots were detected in 2-DE silver-stained gels, and 52 proteins were identified by LC-MS/MS. Most of the identified proteins were involved in energy production, oxidoreductase activity, carbohydrate metabolic process, amino acid and cellular ketone metabolic process. A large group of identified proteins were related to stress proteins and heat shock proteins. This work presents the first two-dimensional reference map of this alkaloid-producing fungus. Details of the proteome serve as a baseline for further study of this swainsonine-producing fungus and are essential for a reverse genetic analysis of the fungus.Keywords: Undifilum oxytropis fungus, two-dimensional gel electrophoresis, proteome reference map, liquid chromatography tandem mass spectrometry, swainsonin

Molecular-level evidence of early lipid transformations throughout oceanic depths

Our understanding of lipid biogeochemistry of the ocean’s interior is still in its infancy. Here we focus on early lipid transformation and the formation of lipid degradation products in the NE Atlantic Ocean (49.0°N, 16.5°W). We employed high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), a method that allows observation and elemental composition assignment of thousands of lipids in a single sample. Using these data, we infer molecular-level changes that occur during lipid transformation in the oceanic water column to shed new light on early lipid transformation processes and the formation of lipid decomposition intermediates, here termed CHO compounds (i.e., lipid-derived species that contain carbon, hydrogen and oxygen in their molecular formula). We considered the distribution of molecular rings and/or double bonds (DBE), H/C and O/C ratios, molecular diversity based on the number of mass spectral signals for monoisotopic species, and carbon number in CHO molecules. Data are elaborated for the four ocean zones, the epipelagic, mesopelagic, bathypelagic and abyssal. The highest molecular diversity characterizes CHO compounds associated with the epipelagic zone, which is explained by numerous and diverse planktonic communities inhabiting the epipelagic and by the effects of both biotic and abiotic processes on lipid transformation. Lipid transformations include crosslinking (condensation), partial degradation or fragmentation, double bond reduction, oxidation, hydrogenation, dehydrogenation and cyclization. Crosslinking likely results in a unimodal distribution of carbon number of CHO compounds, in contrast to cell lipids (referred to as Reported lipids based on the Lipid Maps Database), which have a bimodal distribution of carbon number. CHO compounds that appear to be formed by fragmentation (decrease of the number of C atoms) and ring/double bond reduction were more stable to further transformation and remained longer in the water column, i.e., these compounds were transferred deeper into the water column. Low unsaturation and fast transport to depth promotes CHO compound preservation in the water column. Dehydrogenation leads to increased unsaturation (average DBE up to 21.3), condensation, and cyclization, resulting in high molecular weight compounds with a high degree of unsaturation. Our data demonstrate that lipids with cyclic structures are more refractory than those with acyclic structures. The formation of aromatic structures is not a significant process during early lipid transformation in the oceanic water column

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and 13C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700+ reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic 13CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates

New Mexico State University Nannochloropsis Negative Collection - Mass Spectral Peaklist/Nannochloropsis Negative Ion Mode - Test No. 1 - 200 degrees C

We illustrate a detailed compositional characterization of hydrothermal liquefaction (HTL) oils derived from two biochemically distinct microalgae, Nannochloropsis gaditana and Chlorella sp. (DOE 1412), for a range of reaction temperature as observed by high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The unique capability to unequivocally derive molecular formulae directly from FT-ICR MS-measured mass-to- charge ratio (for several thousand compounds in each oil) shows that lipids are completely reacted/converted for any reaction temperature above 200 degrees C and reveals the formation of non-lipid reaction products with increasing temperature. Specifically, lipid-rich oil is obtained at low reaction temperature (less than 225 degrees C) for both microalgal strains. For positive ion mode, the major lipid components in Chlorella sp. and N. gaditana HTL oils are betaine lipids and acylglycerols, respectively. Acidic species in the HTL oils (observed by negative ion mode) are dominated by free fatty acids (FFA) regardless of reaction temperature. HTL oils obtained at higher-temperatures (ge 225 degrees C) are comprised of a variety of basic nitrogen- and oxygen-containing compounds that originate from protein and carbohydrate degradation at elevated temperature. Similar structural features are observed for the abundant nitrogen heterocyclics between the two strains with slightly lower carbon number for Chlorella sp., overall

New Mexico State University Chlorella Negative Collection - Mass Spectral Peaklist/Chlorella Negative Class Graph

We illustrate a detailed compositional characterization of hydrothermal liquefaction (HTL) oils derived from two biochemically distinct microalgae, Nannochloropsis gaditana and Chlorella sp. (DOE 1412), for a range of reaction temperature as observed by high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The unique capability to unequivocally derive molecular formulae directly from FT-ICR MS-measured mass-to- charge ratio (for several thousand compounds in each oil) shows that lipids are completely reacted/converted for any reaction temperature above 200 degrees C and reveals the formation of non-lipid reaction products with increasing temperature. Specifically, lipid-rich oil is obtained at low reaction temperature (less than 225 degrees C) for both microalgal strains. For positive ion mode, the major lipid components in Chlorella sp. and N. gaditana HTL oils are betaine lipids and acylglycerols, respectively. Acidic species in the HTL oils (observed by negative ion mode) are dominated by free fatty acids (FFA) regardless of reaction temperature. HTL oils obtained at higher-temperatures (ge 225 degrees C) are comprised of a variety of basic nitrogen- and oxygen-containing compounds that originate from protein and carbohydrate degradation at elevated temperature. Similar structural features are observed for the abundant nitrogen heterocyclics between the two strains with slightly lower carbon number for Chlorella sp., overall

New Mexico State University Nannochloropsis Negative Collection - Mass Spectral Peaklist/Nannochloropsis Negative Ion Mode - Test No. 1 - 250 degrees C

We illustrate a detailed compositional characterization of hydrothermal liquefaction (HTL) oils derived from two biochemically distinct microalgae, Nannochloropsis gaditana and Chlorella sp. (DOE 1412), for a range of reaction temperature as observed by high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The unique capability to unequivocally derive molecular formulae directly from FT-ICR MS-measured mass-to- charge ratio (for several thousand compounds in each oil) shows that lipids are completely reacted/converted for any reaction temperature above 200 degrees C and reveals the formation of non-lipid reaction products with increasing temperature. Specifically, lipid-rich oil is obtained at low reaction temperature (less than 225 degrees C) for both microalgal strains. For positive ion mode, the major lipid components in Chlorella sp. and N. gaditana HTL oils are betaine lipids and acylglycerols, respectively. Acidic species in the HTL oils (observed by negative ion mode) are dominated by free fatty acids (FFA) regardless of reaction temperature. HTL oils obtained at higher-temperatures (ge 225 degrees C) are comprised of a variety of basic nitrogen- and oxygen-containing compounds that originate from protein and carbohydrate degradation at elevated temperature. Similar structural features are observed for the abundant nitrogen heterocyclics between the two strains with slightly lower carbon number for Chlorella sp., overall