Widespread Sterol Methyltransferase Participates in the Biosynthesis of Both C4α- and C4ß-Methyl Sterols.

The 4-methyl steranes serve as molecular fossils and are used for studying both eukaryotic evolution and geological history. The occurrence of 4α-methyl steranes in sediments has long been considered evidence of products of partial demethylation mediated by sterol methyl oxidases (SMOs), while 4ß-methyl steranes are attributed entirely to diagenetic generation from 4α-methyl steroids since possible biological sources of their precursor 4ß-methyl sterols are unknown. Here, we report a previously unknown C4-methyl sterol biosynthetic pathway involving a sterol methyltransferase rather than the SMOs. We show that both C4α- and C4ß-methyl sterols are end products of the sterol biosynthetic pathway in an endosymbiont of reef corals, Breviolum minutum, while this mechanism exists not only in dinoflagellates but also in eukaryotes from alveolates, haptophytes, and aschelminthes. Our discovery provides a previously untapped route for the generation of C4-methyl steranes and overturns the paradigm that all 4ß-methyl steranes are diagenetically generated from the 4α isomers. This may facilitate the interpretation of molecular fossils and understanding of the evolution of eukaryotic life in general.

Ambrein: a minor, but common constituent of mammalian faeces?

For nearly 200 years, the only natural source of the alcohol ambrein has been coproliths produced in about 1% of sperm whales and in related jetsam. However, the finding of ambrein in adipocere/faeces of human corpses, led us to hypothesise that ambrein might occur in the faeces of other mammals. Herein, we used a recently developed gas chromatography-mass spectrometry method, with suitable derivatisation of the hindered hydroxy group of ambrein, to screen a number of extracts of mammalian faeces. Minor proportions of ambrein were detected in digested human sewage sludge and in the dung of elephant, domestic cattle, giraffe and buffalo. Whether ambrein formation in the terrestrial species is associated with coprolith formation, is unknown, but solid deposits known as enteroliths and fecaliths occur in humans and some domestic animals.

Algal origin of sponge sterane biomarkers negates the oldest evidence for animals in the rock record.

The earliest fossils of animal-like organisms occur in Ediacaran rocks that are approximately 571 million years old. Yet 24-isopropylcholestanes and other C30 fossil sterol molecules have been suggested to reflect an important ecological role of demosponges as the first abundant animals by the end of the Cryogenian period (>635 million years ago). Here, we demonstrate that C30 24-isopropylcholestane is not diagnostic for sponges and probably formed in Neoproterozoic sediments through the geological methylation of C29 sterols of chlorophyte algae, the dominant eukaryotes at that time. These findings reconcile biomarker evidence with the geological record and revert the oldest evidence for animals back into the latest Ediacaran.

High-throughput analysis of ammonia oxidiser community composition via a novel, amoA-based functional gene array.

Advances in microbial ecology research are more often than not limited by the capabilities of available methodologies. Aerobic autotrophic nitrification is one of the most important and well studied microbiological processes in terrestrial and aquatic ecosystems. We have developed and validated a microbial diagnostic microarray based on the ammonia-monooxygenase subunit A (amoA) gene, enabling the in-depth analysis of the community structure of bacterial and archaeal ammonia oxidisers. The amoA microarray has been successfully applied to analyse nitrifier diversity in marine, estuarine, soil and wastewater treatment plant environments. The microarray has moderate costs for labour and consumables and enables the analysis of hundreds of environmental DNA or RNA samples per week per person. The array has been thoroughly validated with a range of individual and complex targets (amoA clones and environmental samples, respectively), combined with parallel analysis using traditional sequencing methods. The moderate cost and high throughput of the microarray makes it possible to adequately address broader questions of the ecology of microbial ammonia oxidation requiring high sample numbers and high resolution of the community composition.

Effects of estuarine sediment hypoxia on nitrogen fluxes and ammonia oxidizer gene transcription.

The effects of sediment hypoxia, resulting from increased carbon loads or decreased dissolved oxygen (DO), on nitrogen cycling in estuarine environments is poorly understood. The important role played by bacterial and archaeal ammonia oxidizers in the eventual removal of nitrogen from estuarine environments is likely to be strongly affected by hypoxic events. In this study, an analysis of the effects of different levels of sediment hypoxia (5%, 20% and 75% DO) was performed in a microcosm experiment. Changes in the nutrient fluxes related to nitrification at 5% DO were observed after 4 h. Quantification of the key nitrification gene ammonium monooxygenase (amoA) in both DNA and RNA extracts suggests that bacterial amoA transcription was reduced at both of the lower DO concentrations, while changes in DO had no significant effect on archaeal amoA transcription. There was no change in the diversity of expressed archaeal amoA, but significant change in bacterial amoA transcriptional diversity, indicative of low- and high-DO phylotypes. This study suggests that groups of ammonia oxidizers demonstrate differential responses to changes in sediment DO, which may be a significant factor in niche partitioning of different ammonia oxidizer groups.

Potential alteration of U37K' paleothermometer due to selective degradation of alkenones by marine bacteria isolated from the haptophyte Emiliania huxleyi.

The unsaturation ratio of C(37) alkenones (U(37)(K')) produced by haptophyte microalgae such as Emiliania huxleyi is often used as proxy for past sea surface temperature. In this study, 29 bacterial strains were isolated from cultures of the strain E. huxleyi TWP1. Among alkenone-degrading isolates, the strain Dietzia maris sp. S1 appeared to be able to selectively degrade alkenones leading to increases in the palaeoenvironmental proxy U(37)(K') by +0.05 to +0.10 units, which is equivalent to the change seen when the growth temperature is increased by 1.5-3.0 degrees C. This degradation was shown to involve initial epoxidation of the alkenone double bonds presumably by a monooxygenase, which showed a preference for oxidation of the omega29 double bond. Inconsistencies observed in previous studies of the aerobic microbial degradation of alkenones may simply reflect which species of bacteria were present. Our results confirm that intense aerobic bacterial degradative processes can introduce a bias in palaeotemperature reconstructions especially when there is evidence of substantial aerobic bacterial degradation of the deposited organic matter. The widespread occurrence of epoxyalkenones in the marine environment strongly suggests that selective aerobic bacterial degradation could be major source of uncertainty for palaeotemperature estimation.

Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary.

Nitrification and denitrification are key steps in nitrogen (N) cycling. The coupling of these processes, which affects the flow of N in ecosystems, requires close interaction of nitrifying and denitrifying microorganisms, both spatially and temporally. The diversity, temporal and spatial variations in the microbial communities affecting these processes was examined, in relation to N cycling, across 12 sites in the Fitzroy river estuary, which is a turbid subtropical estuary in central Queensland. The estuary is a major source of nutrients discharged to the Great Barrier Reef near-shore zone. Measurement of nitrogen fluxes showed an active denitrifying community during all sampling months. Archaeal ammonia monooxygenase (amoA of AOA, functional marker for nitrification) was significantly more abundant than Betaproteobacterial (beta-AOB) amoA. Nitrite reductase genes, functional markers for denitrification, were dominated by nirS and not nirK types at all sites during the year. AOA communities were dominated by the soil/sediment cluster of Crenarchaeota, with sequences found in estuarine sediment, marine and terrestrial environments, whereas nirS sequences were significantly more diverse (where operational taxonomic units were defined at both the threshold of 5% and 15% sequence similarity) and were closely related to sequences originating from estuarine sediments. Terminal-restriction fragment length polymorphism (T-RFLP) analysis revealed that AOA population compositions varied spatially along the estuary, whereas nirS populations changed temporally. Statistical analysis of individual T-RF dominance suggested that salinity and C:N were associated with the community succession of AOA, whereas the nirS-type denitrifier communities were related to salinity and chlorophyll-alpha in the Fitzroy river estuary.

Sources for sedimentary bacteriohopanepolyols as revealed by 16S rDNA stratigraphy.

Bacteriohopanoids are widespread lipid biomarkers in the sedimentary record. Many aerobic and anaerobic bacteria are potential sources of these lipids which sometimes complicates the use of these biomarkers as proxies for ecological and environmental changes. Therefore, we applied preserved 16S ribosomal RNA genes to identify likely Holocene biological sources of bacteriohopanepolyols (BHPs) in the sulfidic sediments of the permanently stratified postglacial Ace Lake, Antarctica. A suite of intact BHPs were identified, which revealed a variety of structural forms whose composition differed through the sediment core reflecting changes in bacterial populations induced by large changes in lake salinity. Stable isotopic compositions of the hopanols formed from periodic acid-cleaved BHPs, showed that some were substantially depleted in (13)C, indicative of their methanotrophic origin. Using sensitive molecular tools, we found that Type I and II methanotrophic bacteria (respectively Methylomonas and Methylocystis) were unique to the oldest lacustrine sediments (> 9400 years BP), but quantification of fossil DNA revealed that the Type I methanotrophs, including methanotrophs related to methanotrophic gill symbionts of deep-sea cold-seep mussels, were the main precursors of the 35-amino BHPs (i.e. aminopentol, -tetrol and -triols). After isolation of the lake approximately 3000 years ago, one Type I methanotroph of the 'methanotrophic gill symbionts cluster' remained the most obvious source of aminotetrol and -triol. We, furthermore, identified a Synechococcus phylotype related to pelagic freshwater strains in the oldest lacustrine sediments as a putative source of 2-methylbacteriohopanetetrol (2-Me BHT). This combined application of advanced geochemical and paleogenomical tools further refined our knowledge about Holocene biogeochemical processes in Ace Lake.

Free radical oxidation (autoxidation) of alkenones and other lipids in cells of Emiliania huxleyi.

Cells of the coccolithophorid Emiliania huxleyi strain CS-57 grown under an atmosphere of air+0.5% CO(2) showed oxidative damage after 10 days growth with concomitant and major changes to the lipid composition. The fatty acid profile was strongly altered and lacked appreciable amounts of the polyunsaturated fatty acids (PUFA: C(18:5), C(18:3) and C(22:6)) typical of healthy cells. Oxidation products of these PUFA could not be detected, but monounsaturated fatty acids proved to be good indicators of oxidative processes. The presence (after NaBH(4)-reduction) of a high proportion of 11-hydroxyoctadec-cis-9-enoic and 8-hydroxyoctadec-cis-9-enoic acids showed that the degradation of oleic acid involved mainly free radical oxidation processes (70-75% autoxidation and 20-25% photooxidation). We also detected large amounts of degradation products of the oxidation product 9,10-epoxyoctadecanoic acid including diols, methoxyhydrins and chlorohydrins. These oxidative effects were found in all the lipid classes examined. Products included significant amounts of chlorophyll side-chain autooxidation products Z- and E-3,7,11,15-tetramethylhexadec-3-ene-1,2-diols and Z-and E-3,7,11,15-tetramethylhexadec-2-ene-1,4-diols, while phytyldiol was present in relatively low proportions. Delta(5)-3beta,7-epimeric unsaturated steroidal diols arising from the autooxidation of the Delta(5) double bond of epi-brassicasterol and minor amounts of Delta(4)-3beta,6-diols were also detected. Long-chain unsaturated ketone (alkenone) content per cell was much higher in the presence of 0.5% CO(2) likely due to carbon storage under these conditions. The proportions of di- and tri-unsaturated alkenones was relatively stable throughout the growth cycle in the absence of additional CO(2), but not when grown with 0.5% CO(2). The detection of characteristic alkenone autoxidation products in cells grown under these latter conditions allowed us to attribute the significant increase in index observed to the involvement of free radical oxidation processes.

Cryptophyceae and rhodophyceae; chemotaxonomy, phylogeny, and application.

The biochemical compositions of seven strains of marine cryptomonad and a rhodophyte were determined in logarithmic phase batch (1.4 L flask) and semi-continuous (10 L carboy) culture. Lipid ranged from 13% to 28%, protein ranged from 53% to 68%, and carbohydrate ranged from 9% to 24% of the organic weight. The major lipid classes in the species examined were polar lipids (78-88% of total lipid). The major sterol in the Cryptophyceae and the Rhodophyceae was 24-methylcholesta-5,22E-dien-3beta-ol (62-99% of total sterols); which is also the major sterol in some diatoms and haptophytes. Smaller proportions of cholest-5-en-3beta-ol (1-17.7%) were also found in the Cryptophyceae. Most cryptomonads contained high proportions of the n-3 polyunsaturated fatty acids (PUFA), 18:3n-3 (20.7-29.9% of the total fatty acids), 18:4n-3 (12.5-30.2%), 20:5n-3 (7.6-13.2%) and 22:6n-3 (6.4-10.8%). However, the blue-green cryptomonad Chroomonas placoidea was characterized by a low proportion of 22:6n-3 (0.2% of total fatty acids), and a significant proportion of 22:5n-6 (4.5%), and the presence of 24-ethylcholesta-5,22E-dien-3beta-ol (35.5% of total sterols). The fatty acid composition of the rhodophyte Rhodosorus sp. was similar to those of the Cryptophyceae except for lower proportions of 18:4n-3 and lack of C21 and C22 PUFA. It is postulated that the primary endosymbiosis of a photosynthetic n-3 C18 PUFA-producing prokaryote and a eukaryotic host capable of chain elongation and desaturation of exogenous PUFA, resulted in the Rhodophyceae capable of producing n-3 C20 PUFA. The secondary endosymbiosis of a photosynthetic n-3 C20 PUFA-producing eukaryote (such as a Rhodosorus sp. like-rhodophyte) and a eukaryotic host capable of further chain elongation and desaturation, resulted in the Cryptophyceae being capable of producing n-3 C20 and C22 PUFA de novo. Selected isolates were examined further in feeding trials with juvenile Pacific oysters (Crassostrea gigas). Rhodomonas salina CS-24(containing elevated 22:6n-3) produced high growth rates in oysters; equivalent to the microalga commonly used in aquaculture, Isochrysis sp. (T.ISO).

Unsaturated diether lipids in the psychrotrophic archaeon Halorubrum lacusprofundi.

The major phospholipids of Halorubrum lacusprofundi grown at 25 degrees C were archaeol phosphatidylglycerol, archaeol phosphatidylglycerylsulphate and archaeol phosphatidylglycerylphosphate methyl ester. Glycolipids included a monoglycosyl archaeol and the sulphate ester of a diglycosyl archaeol. Cultures grown at 12 degrees C contained the same suite of phospho- and glycolipids, with the addition of a series of unsaturated analogues with up to six double bonds. The patterns of unsaturation were similar for all the phospholipid series, but a different pattern occurred in the glycolipids. The analytical techniques used in this study allow facile detection of unsaturated archaeal cell membrane lipids that are degraded by commonly used chemical derivatization procedures.

Regiospecific oxygenation of alkenones in the benthic haptophyte Chrysotila lamellosa Anand HAP 17.

Two groups of previously unidentified C37-C39 epoxyalkenones and alkenediones were detected in late stationary phase cultures of the haptophyte microalga Chrysotila lamellosa. The formation of these compounds is attributed to the involvement of enzymatic processes acting specifically on the C-21 or C-22 allylic carbon and the omega15 double bond of methyl and ethyl alkenones respectively. Thus, the epoxyalkenones appear to be derivatives of alkenones where the omega15 double bond is oxidized to the epoxide. These epoxyalkenones disappear as the cells age to be replaced by a series of alkenediones. The structures of these compounds indicate that they are derivatives of methyl and ethyl alkenones with an additional carbonyl group on the C-21 or C-22 carbon respectively and without the omega15 double bond. We propose that these compounds are formed by an initial regiospecific lipoxygenase-catalyzed peroxidation of methyl and ethyl alkenones on their C-21 or C-22 allylic carbon, respectively. Lipohydroperoxidase-catalyzed homolytic cleavage of the O-O bond could then result in the formation of conjugated ketones which may then undergo a saturation reaction to form the diketones identified. This work demonstrates that alkenones can be degraded by enzymatic reactions in senescent cells, and by implication this could also occur in the natural environment.

Long-chain alkenones and related compounds in the benthic haptophyte Chrysotila lamellosa Anand HAP 17.

The neutral lipid compositions of the coastal haptophyte Chrysotila lamellosa HAP 17 grown in batch culture at 10 and 20 degrees C have been determined. A comparison was also made between the lipid compositions of cells harvested in early and late stationary phase. This species contains a suite of very long-chain C(37)-C(40) alkenones and alkenoates as found in a few microalgae from the Haptophyta. The distributions of these compounds show some differences to earlier reports of different strains of this alga, which are only in part attributable to culture conditions. A suite of long-chain alkenols, the reduced form of the alkenones, was characterized for the first time. The abundance of these compounds was only 1.5% of that of the corresponding alkenones, and the relative proportion of C(37)-C(38) constituents depended on growth temperature. These data show that haptophyte algae are a possible source of the alkenols found in some marine sediments, but the small amounts found suggest that other sources such as bacterial reduction of alkenones are more likely in highly reducing sediments. A mixture of C(29)-C(33) n-alkenes, dominated by the C(31:1) monoene, was found in marked contrast to previous analyses of other strains which reported only the presence of a C(31:2) diene. The sterol distribution included the common haptophyte sterol 24alpha-methylcholesta-5,22E-dien-3beta-ol (epi-brassicasterol) as well as significant amounts of Delta(5)- and Delta(5,22)-C(29) sterols.

The effect of growth phase on the lipid class, fatty acid and sterol composition in the marine dinoflagellate, Gymnodinium sp. in batch culture.

We have studied the effects of growth phase on the lipid composition in batch cultures of Gymnodinium sp. CS-380/3 over 43 days of culturing. The lipid content increased two fold, from late logarithmic (day 6) to linear growth phase (day 22) then decreased at stationary phase (day 43) while the lipid yield (mg l(-1)) increased 30-fold from day 6 to 30 mg l(-1) at day 43. Changes in fatty acid content mirrored those observed for the total lipid, while the sterol content continued to increase with culture age through to stationary phase. The largest changes occurred in the lipid classes, especially the polar lipids and triacylglycerols (oil). The proportion of triacylglycerols increased from 8% (of total lipids) at day 6 to 30% at day 43, with a concomitant decrease in the polar lipid fraction. The proportions of 16:0 and DHA [22:6(n-3)] increased while those of 18:5(n-3) and EPA [20:5(n-3)] decreased with increasing culture age. The proportion of the major sterol, dinosterol, decreased from 41% (day 6) to 29% (day 43), while the major dinostanol epimer (23R,24R) increased from 33% (day 6) to 38% (day 22). Despite small changes in the proportion of the main sterols, the same sterols were present at all stages of growth, indicating their value as a chemotaxonomic tool for distinguishing between strains within the same genus. Growth phase could be a useful variable for optimising the oil and DHA content with potential for aquaculture feeds and a source of DHA-rich oils for nutraceuticals.