Mono- to tetra-alkyl ether cardiolipins in a mesophilic, sulfate-reducing bacterium identified by UHPLC-HRMSn: a novel class of membrane lipids.

The composition of membrane lipids varies in a number of ways as adjustment to growth conditions. Variations in head group composition and carbon skeleton and degree of unsaturation of glycerol-bound acyl or alkyl chains results in a high structural complexity of the lipidome of bacterial cells. We studied the lipidome of the mesophilic, sulfate-reducing bacterium, Desulfatibacillum alkenivorans strain PF2803T by ultra-high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMSn). This anaerobic bacterium has been previously shown to produce high amounts of mono-and di-alkyl glycerol ethers as core membrane lipids. Our analyses revealed that these core lipids occur with phosphatidylethanomamine (PE) and phosphatidylglycerol (PG) head groups, representing each approximately one third of the phospholipids. The third class was a novel group of phospholipids, i.e., cardiolipins (CDLs) containing one (monoether/triester) to four (tetraether) ether-linked saturated straight-chain or methyl-branched alkyl chains. Tetraether CDLs have been shown to occur in archaea (with isoprenoid alkyl chains) but have not been previously reported in the bacterial Domain. Structurally related CDLs with one or two alkyl/acyl chains missing, so-called monolyso-and dilyso-CDLs, were also observed. The potential biosynthetic pathway of these novel CDLs was investigated by examining the genome of D. alkenivorans. Three CDL synthases were identified; one catalyzes the condensation of two PGs, the other two are probably involved in the condensation of a PE with a PG. A heterologous gene expression experiment showed the in vivo production of dialkylglycerols upon anaerobic expression of the glycerol ester reductase enzyme of D. alkenivorans in E. coli. Reduction of the ester bonds probably occurs first at the sn-1 and subsequently at the sn-2 position after the formation of PEs and PGs.

Nitrogen and sulfur for phosphorus: Lipidome adaptation of anaerobic sulfate-reducing bacteria in phosphorus-deprived conditions.

Understanding how microbial lipidomes adapt to environmental and nutrient stress is crucial for comprehending microbial survival and functionality. Certain anaerobic bacteria can synthesize glycerolipids with ether/ester bonds, yet the complexities of their lipidome remodeling under varying physicochemical and nutritional conditions remain largely unexplored. In this study, we thoroughly examined the lipidome adaptations of Desulfatibacillum alkenivorans strain PF2803T, a mesophilic anaerobic sulfate-reducing bacterium known for its high proportions of alkylglycerol ether lipids in its membrane, under various cultivation conditions including temperature, pH, salinity, and ammonium and phosphorous concentrations. Employing an extensive analytical and computational lipidomic methodology, we identified an assemblage of nearly 400 distinct lipids, including a range of glycerol ether/ester lipids with various polar head groups. Information theory-based analysis revealed that temperature fluctuations and phosphate scarcity profoundly influenced the lipidome's composition, leading to an enhanced diversity and specificity of novel lipids. Notably, phosphorous limitation led to the biosynthesis of novel glucuronosylglycerols and sulfur-containing aminolipids, termed butyramide cysteine glycerols, featuring various ether/ester bonds. This suggests a novel adaptive strategy for anaerobic heterotrophs to thrive under phosphorus-depleted conditions, characterized by a diverse array of nitrogen- and sulfur-containing polar head groups, moving beyond a reliance on conventional nonphospholipid types.

Distinguishing the molecular diversity, nutrient content, and energetic potential of exometabolomes produced by macroalgae and reef-building corals.

Metabolites exuded by primary producers comprise a significant fraction of marine dissolved organic matter, a poorly characterized, heterogenous mixture that dictates microbial metabolism and biogeochemical cycling. We present a foundational untargeted molecular analysis of exudates released by coral reef primary producers using liquid chromatography-tandem mass spectrometry to examine compounds produced by two coral species and three types of algae (macroalgae, turfing microalgae, and crustose coralline algae [CCA]) from Mo'orea, French Polynesia. Of 10,568 distinct ion features recovered from reef and mesocosm waters, 1,667 were exuded by producers; the majority (86%) were organism specific, reflecting a clear divide between coral and algal exometabolomes. These data allowed us to examine two tenets of coral reef ecology at the molecular level. First, stoichiometric analyses show a significantly reduced nominal carbon oxidation state of algal exometabolites than coral exometabolites, illustrating one ecological mechanism by which algal phase shifts engender fundamental changes in the biogeochemistry of reef biomes. Second, coral and algal exometabolomes were differentially enriched in organic macronutrients, revealing a mechanism for reef nutrient-recycling. Coral exometabolomes were enriched in diverse sources of nitrogen and phosphorus, including tyrosine derivatives, oleoyl-taurines, and acyl carnitines. Exometabolites of CCA and turf algae were significantly enriched in nitrogen with distinct signals from polyketide macrolactams and alkaloids, respectively. Macroalgal exometabolomes were dominated by nonnitrogenous compounds, including diverse prenol lipids and steroids. This study provides molecular-level insights into biogeochemical cycling on coral reefs and illustrates how changing benthic cover on reefs influences reef water chemistry with implications for microbial metabolism.

Changes in the membrane lipid composition of a Sulfurimonas species depend on the electron acceptor used for sulfur oxidation.

Sulfurimonas species are among the most abundant sulfur-oxidizing bacteria in the marine environment. They are capable of using different electron acceptors, this metabolic flexibility is favorable for their niche adaptation in redoxclines. When oxygen is depleted, most Sulfurimonas spp. (e.g., Sulfurimonas gotlandica) use nitrate ([Formula: see text]) as an electron acceptor to oxidize sulfur, including sulfide (HS-), S0 and thiosulfate, for energy production. Candidatus Sulfurimonas marisnigri SoZ1 and Candidatus Sulfurimonas baltica GD2, recently isolated from the redoxclines of the Black Sea and Baltic Sea respectively, have been shown to use manganese dioxide (MnO2) rather than [Formula: see text] for sulfur oxidation. The use of different electron acceptors is also dependent on differences in the electron transport chains embedded in the cellular membrane, therefore changes in the membrane, including its lipid composition, are expected but are so far unexplored. Here, we used untargeted lipidomic analysis to reveal changes in the composition of the lipidomes of three representative Sulfurimonas species grown using either [Formula: see text] and MnO2. We found that all Sulfurimonas spp. produce a series of novel phosphatidyldiazoalkyl-diacylglycerol lipids. Ca. Sulfurimonas baltica GD2 adapts its membrane lipid composition depending on the electron acceptors it utilizes for growth and survival. When carrying out MnO2-dependent sulfur oxidation, the novel phosphatidyldiazoalkyl-diacylglycerol headgroup comprises shorter alkyl moieties than when sulfur oxidation is [Formula: see text]-dependent. This is the first report of membrane lipid adaptation when an organism is grown with different electron acceptors. We suggest novel diazoalkyl lipids have the potential to be used as a biomarker for different conditions in redox-stratified systems.

Changes in the Distribution of Membrane Lipids during Growth of Thermotoga maritima at Different Temperatures: Indications for the Potential Mechanism of Biosynthesis of Ether-Bound Diabolic Acid (Membrane-Spanning) Lipids.

Membrane-spanning lipids are present in a wide variety of archaea, but they are rarely in bacteria. Nevertheless, the (hyper)thermophilic members of the order Thermotogales harbor tetraester, tetraether, and mixed ether/ester membrane-spanning lipids mostly composed of core lipids derived from diabolic acids, C30, C32, and C34 dicarboxylic acids with two adjacent mid-chain methyl substituents. Lipid analysis of Thermotoga maritima across growth phases revealed a decrease of the relative abundance of fatty acids together with an increase of diabolic acids with independence of growth temperature. We also identified isomers of C30 and C32 diabolic acids, i.e., dicarboxylic acids with only one methyl group at C-15. Their distribution suggests they are products of the condensation reaction but are preferably produced when the length of the acyl chains is not optimal. Compared with growth at the optimal temperature of 80°C, an increase of glycerol ether-derived lipids was observed at 55°C. Our analysis only detected diabolic acid-containing intact polar lipids with phosphoglycerol (PG) head groups. Considering these findings, we hypothesize a biosynthetic pathway for the synthesis of membrane-spanning lipids based on PG polar lipid formation, suggesting that the protein catalyzing this process is a membrane protein. We also identified, by genomic and protein domain analyses, a gene coding for a putative plasmalogen synthase homologue in T. maritima that is also present in other bacteria producing sn-1-alkyl ether lipids but not plasmalogens, suggesting it is involved in the conversion of the ester-to-ether bond in the diabolic acids bound in membrane-spanning lipids. IMPORTANCE Membrane-spanning lipids are unique compounds found in most archaeal membranes, but they are also present in specific bacterial groups like the Thermotogales. The synthesis and physiological role of membrane-spanning lipids in bacteria represent an evolutionary and biochemical open question that points to the differentiation of the membrane lipid composition. Understanding the formation of membrane-spanning lipids is crucial to solving this question and identifying the enzymatic and biochemical mechanism performing this procedure. In the present work, we found changes at the core lipid level, and we propose that the growth phase drives the biosynthesis of these lipids rather than temperature. Our results identified physiological conditions influencing the membrane-spanning lipid biosynthetic process, which can further clarify the pathway leading to the biosynthesis of these compounds.

Lipidomics of Environmental Microbial Communities. I: Visualization of Component Distributions Using Untargeted Analysis of High-Resolution Mass Spectrometry Data.

Lipids, as one of the main building blocks of cells, can provide valuable information on microorganisms in the environment. Traditionally, gas or liquid chromatography coupled to mass spectrometry (MS) has been used to analyze environmental lipids. The resulting spectra were then processed through individual peak identification and comparison with previously published mass spectra. Here, we present an untargeted analysis of MS1 spectral data generated by ultra-high-pressure liquid chromatography coupled with high-resolution mass spectrometry of environmental microbial communities. Rather than attempting to relate each mass spectrum to a specific compound, we have treated each mass spectrum as a component, which can be clustered together with other components based on similarity in their abundance depth profiles through the water column. We present this untargeted data visualization method on lipids of suspended particles from the water column of the Black Sea, which included >14,000 components. These components form clusters that correspond with distinct microbial communities driven by the highly stratified water column. The clusters include both known and unknown compounds, predominantly lipids, demonstrating the value of this rapid approach to visualize component distributions and identify novel lipid biomarkers.

Lipidomics of Environmental Microbial Communities. II: Characterization Using Molecular Networking and Information Theory.

Structurally diverse, specialized lipids are crucial components of microbial membranes and other organelles and play essential roles in ecological functioning. The detection of such lipids in the environment can reveal not only the occurrence of specific microbes but also the physicochemical conditions to which they are adapted to. Traditionally, liquid chromatography coupled with mass spectrometry allowed for the detection of lipids based on chromatographic separation and individual peak identification, resulting in a limited data acquisition and targeting of certain lipid groups. Here, we explored a comprehensive profiling of microbial lipids throughout the water column of a marine euxinic basin (Black Sea) using ultra high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). An information theory framework combined with molecular networking based on the similarity of the mass spectra of lipids enabled us to capture lipidomic diversity and specificity in the environment, identify novel lipids, differentiate microbial sources within a lipid group, and discover potential biomarkers for biogeochemical processes. The workflow presented here allows microbial ecologists and biogeochemists to process quickly and efficiently vast amounts of lipidome data to understand microbial lipids characteristics in ecosystems.

Bridging the membrane lipid divide: bacteria of the FCB group superphylum have the potential to synthesize archaeal ether lipids.

Archaea synthesize membranes of isoprenoid lipids that are ether-linked to glycerol-1-phosphate (G1P), while Bacteria/Eukarya produce membranes consisting of fatty acids ester-bound to glycerol-3-phosphate (G3P). This dichotomy in membrane lipid composition (i.e., the 'lipid divide') is believed to have arisen after the Last Universal Common Ancestor (LUCA). A leading hypothesis is that LUCA possessed a heterochiral 'mixed archaeal/bacterial membrane'. However, no natural microbial representatives supporting this scenario have been shown to exist today. Here, we demonstrate that bacteria of the Fibrobacteres-Chlorobi-Bacteroidetes (FCB) group superphylum encode a putative archaeal pathway for ether-bound isoprenoid membrane lipids in addition to the bacterial fatty acid membrane pathway. Key genes were expressed in the environment and their recombinant expression in Escherichia coli resulted in the formation of a 'mixed archaeal/bacterial membrane'. Genomic evidence and biochemical assays suggest that the archaeal-like lipids of members of the FCB group could possess either a G1P or G3P stereochemistry. Our results support the existence of 'mixed membranes' in natural environments and their stability over a long period in evolutionary history, thereby bridging a once-thought fundamental divide in biology.

Physiological, chemotaxonomic and genomic characterization of two novel piezotolerant bacteria of the family Marinifilaceae isolated from sulfidic waters of the Black Sea.

Diversity analyses of microbial enrichments obtained from deep sulfidic water (2000 m) collected from the Black Sea indicated the presence of eleven novel putative lineages of bacteria affiliated to the family Marinifilaceae of the phylum Bacteroidetes. Pure cultures were obtained for four strains (i.e. M1PT, M3P, A4T and 44) of this family, which could be grouped into two different clades based on their 16S rRNA gene sequences. All four strains were Gram-negative, rod-shaped and facultative anaerobic bacteria. The genomes of all strains were sequenced and physiological analyses were performed. All strains utilized a wide range of carbon sources, which was supported by the presence of the pathways involved in carbon utilization encoded by their genomes. The strains were able to grow at elevated hydrostatic pressure (up to 50 MPa), which coincided with increased production of unsaturated and branched fatty acids, and a decrease in hydroxy fatty acids. Intact polar lipid analysis of all four strains showed the production of ornithine lipids, phosphatidylethanolamines and capnine lipids as major intact polar lipids (IPLs). Genes involved in hopanoid biosynthesis were also identified. However, bacteriohopanepolyols (BHPs) were not detected in the strains. Based on distinct physiological, chemotaxonomic, genotypic and phylogenetic differences compared to other members of the genera Ancylomarina and Labilibaculum, it was concluded that strains M1PT and A4T represented two novel species for which the names Ancylomarina euxinus sp. nov. and Labilibaculum euxinus sp. nov., respectively, are proposed.

Butyrate Conversion by Sulfate-Reducing and Methanogenic Communities from Anoxic Sediments of Aarhus Bay, Denmark.

The conventional perception that the zone of sulfate reduction and methanogenesis are separated in high- and low-sulfate-containing marine sediments has recently been changed by studies demonstrating their co-occurrence in sediments. The presence of methanogens was linked to the presence of substrates that are not used by sulfate reducers. In the current study, we hypothesized that both groups can co-exist, consuming common substrates (H2 and/or acetate) in sediments. We enriched butyrate-degrading communities in sediment slurries originating from the sulfate, sulfate-methane transition, and methane zone of Aarhus Bay, Denmark. Sulfate was added at different concentrations (0, 3, 20 mM), and the slurries were incubated at 10 °C and 25 °C. During butyrate conversion, sulfate reduction and methanogenesis occurred simultaneously. The syntrophic butyrate degrader Syntrophomonas was enriched both in sulfate-amended and in sulfate-free slurries, indicating the occurrence of syntrophic conversions at both conditions. Archaeal community analysis revealed a dominance of Methanomicrobiaceae. The acetoclastic Methanosaetaceae reached high relative abundance in the absence of sulfate, while presence of acetoclastic Methanosarcinaceae was independent of the sulfate concentration, temperature, and the initial zone of the sediment. This study shows that there is no vertical separation of sulfate reducers, syntrophs, and methanogens in the sediment and that they all participate in the conversion of butyrate.

The absence of intact polar lipid-derived GDGTs in marine waters dominated by Marine Group II: Implications for lipid biosynthesis in Archaea.

The marine pelagic archaeal community is dominated by three major groups, the marine group I (MGI) Thaumarchaeota, and the marine groups II and III (MGII and MGIII) Euryarchaeota. Studies of both MGI cultures and the environment have shown that the MGI core membrane lipids are predominantly composed of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids and the diether lipid archaeol. However, there are no cultured representatives of MGII and III archaea and, therefore, both their membrane lipid composition and potential contribution to the marine archaeal lipid pool remain unknown. Here, we show that GDGTs present in suspended particulate matter of the (sub)surface waters of the North Atlantic Ocean and the coastal North Sea are derived from MGI archaea, and that MGII archaea do not significantly contribute to the pool of GDGTs and archaeol. This implies, in contrast to previous suggestions, that their lipids do not affect the widely used sea surface temperature proxy TEX86. These findings also indicate that MGII archaea are not able to produce any known archaeal lipids, implying that our understanding of the evolution of membrane lipid biosynthesis in Archaea is far from complete.

Human-induced fire regime shifts during 19th century industrialization: A robust fire regime reconstruction using northern Polish lake sediments.

Fire regime shifts are driven by climate and natural vegetation changes, but can be strongly affected by human land management. Yet, it is poorly known how humans have influenced fire regimes prior to active wildfire suppression. Among the last 250 years, the human contribution to the global increase in fire occurrence during the mid-19th century is especially unclear, as data sources are limited. Here, we test the extent to which forest management has driven fire regime shifts in a temperate forest landscape. We combine multiple fire proxies (macroscopic charcoal and fire-related biomarkers) derived from highly resolved lake sediments (i.e., 3-5 years per sample), and apply a new statistical approach to classify source area- and temperature-specific fire regimes (biomass burnt, fire episodes). We compare these records with independent climate and vegetation reconstructions. We find two prominent fire regime shifts during the 19th and 20th centuries, driven by an adaptive socio-ecological cycle in human forest management. Although individual fire episodes were triggered mainly by arson (as described in historical documents) during dry summers, the biomass burnt increased unintentionally during the mid-19th century due to the plantation of flammable, fast-growing pine tree monocultures needed for industrialization. State forest management reacted with active fire management and suppression during the 20th century. However, pine cover has been increasing since the 1990s and climate projections predict increasingly dry conditions, suggesting a renewed need for adaptations to reduce the increasing fire risk.

New Insights Into the Polar Lipid Composition of Extremely Halo(alkali)philic Euryarchaea From Hypersaline Lakes.

We analyzed the polar membrane lipids of 13 strains of halo(alkali)philic euryarchaea from hypersaline lakes. Nine belong to the class Halobacteria, representing two functional groups: aerobic polysaccharide utilizers and sulfur-respiring anaerobes. The other four strains represent halo(alkali)philic methanogens from the class Methanomicrobia and a recently discovered class Methanonatronarchaeia. A wide range of polar lipids were detected across the 13 strains including dialkyl glycerol diethers (archaeols), membrane-spanning glycerol tetraethers and diether-based cardiolipins. The archaeols contained a range of core lipid structures, including combinations of C20 and C25 isoprenoidal alkyl chains, unsaturations, and hydroxy moieties. Several diether lipids were novel, including: (a) a phosphatidylglycerolhexose (PG-Gly) headgroup, (b) a N,N,N-trimethyl aminopentanetetrol (APT)-like lipid with a methoxy group in place of a hydroxy group on the pentanetetrol, (c) a series of polar lipids with a headgroup with elemental composition of either C12H25NO13S or C12H25NO16S2, and (d) novel cardiolipins containing a putative phosphatidylglycerolphosphate glycerophosphate (PGPGP) polar moiety. We found that the lipid distribution of the 13 strains could be generally separated into two groups, the methanogens (group) and the Halobacteria (class) based on the presence of specific core lipids. Within the methanogens, adaption to a high or more moderate salt concentration resulted in different ratios of glycerol dialkyl glycerol tetraethers (GDGTs) to archaeol. The methanogen Methanosalsum natronophilum AME2T had the most complex diether lipid composition of any of the 13 strains, including hydroxy archaeol and macrocyclic archaeol which we surmise is an order-specific membrane adaption. The zwitterionic headgroups APT and APT-Me were detected only in the Methanomicrobiales member Methanocalculus alkaliphilus AMF2T which also contained the highest level of unsaturated lipids. Only alkaliphilic members of the Natrialbales order contained PGPGP cardiolipins and the PG-Gly headgroup. The four analyzed neutrophilic members of the Halobacteria were characterized by the presence of sulfur-containing headgroups and glycolipids. The presence of cardiolipins with one or more i-C25 alkyl chains, generally termed extended archaeol (EXT-AR), in one of the Methanonatronarchaeia strains was unexpected as only one other order of methanogenic archaea has been reported to produce EXT-AR. We examined this further by looking into the genomic potential of various archaea to produce EXT-AR.

Archaeal Sources of Intact Membrane Lipid Biomarkers in the Oxygen Deficient Zone of the Eastern Tropical South Pacific.

Archaea are ubiquitous in the modern ocean where they are involved in the carbon and nitrogen biogeochemical cycles. However, the majority of Archaea remain uncultured. Archaeal specific membrane intact polar lipids (IPLs) are biomarkers of the presence and abundance of living cells. They comprise archaeol and glycerol dibiphytanyl glycerol tetraethers (GDGTs) attached to various polar headgroups. However, little is known of the IPLs of uncultured marine Archaea, complicating their use as biomarkers. Here, we analyzed suspended particulate matter (SPM) obtained in high depth resolution from a coastal and open ocean site in the eastern tropical South Pacific (ETSP) oxygen deficient zone (ODZ) with the aim of determining possible biological sources of archaeal IPL by comparing their composition by Ultra High Pressure Liquid Chromatography coupled to high resolution mass spectrometry with the archaeal diversity by 16S rRNA gene amplicon sequencing and their abundance by quantitative PCR. Thaumarchaeotal Marine Group I (MGI) closely related to Ca. Nitrosopelagicus and Nitrosopumilus dominated the oxic surface and upper ODZ water together with Marine Euryarchaeota Group II (MGII). High relative abundance of hexose phosphohexose- (HPH) crenarchaeol, the specific biomarker for living Thaumarchaeota, and HPH-GDGT-0, dihexose- (DH) GDGT-3 and -4 were detected in these water masses. Within the ODZ, DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaea) of the Woesearchaeota DHVE-6 group and Marine Euryarchaeota Group III (MGIII) were present together with a higher proportion of archaeol-based IPLs, which were likely made by MGIII, since DPANN archaea are supposedly unable to synthesize their own IPLs and possibly have a symbiotic or parasitic partnership with MGIII. Finally, in deep suboxic/oxic waters a different MGI population occurred with HPH-GDGT-1, -2 and DH-GDGT-0 and -crenarchaeol, indicating that here MGI synthesize membranes with IPLs in a different relative abundance which could be attributed to the different detected population or to an environmental adaptation. Our study sheds light on the complex archaeal community of one of the most prominent ODZs and on the IPL biomarkers they potentially synthesize.

A combined lipidomic and 16S rRNA gene amplicon sequencing approach reveals archaeal sources of intact polar lipids in the stratified Black Sea water column.

Archaea are important players in marine biogeochemical cycles, and their membrane lipids are useful biomarkers in environmental and geobiological studies. However, many archaeal groups remain uncultured and their lipid composition unknown. Here, we aim to expand the knowledge on archaeal lipid biomarkers and determine the potential sources of those lipids in the water column of the euxinic Black Sea. The archaeal community was evaluated by 16S rRNA gene amplicon sequencing and by quantitative PCR. The archaeal intact polar lipids (IPLs) were investigated by ultra-high-pressure liquid chromatography coupled to high-resolution mass spectrometry. Our study revealed both a complex archaeal community and large changes with water depth in the IPL assemblages. In the oxic/upper suboxic waters (<105 m), the archaeal community was dominated by marine group (MG) I Thaumarchaeota, coinciding with a higher relative abundance of hexose phosphohexose crenarchaeol, a known marker for Thaumarchaeota. In the suboxic waters (80-110 m), MGI Nitrosopumilus sp. dominated and produced predominantly monohexose glycerol dibiphytanyl glycerol tetraethers (GDGTs) and hydroxy-GDGTs. Two clades of MGII Euryarchaeota were present in the oxic and upper suboxic zones in much lower abundances, preventing the detection of their specific IPLs. In the deep sulfidic waters (>110 m), archaea belonging to the DPANN Woesearchaeota, Bathyarchaeota, and ANME-1b clades dominated. Correlation analyses suggest that the IPLs GDGT-0, GDGT-1, and GDGT-2 with two phosphatidylglycerol (PG) head groups and archaeol with a PG, phosphatidylethanolamine, and phosphatidylserine head groups were produced by ANME-1b archaea. Bathyarchaeota represented 55% of the archaea in the deeper part of the euxinic zone and likely produces archaeol with phospho-dihexose and hexose-glucuronic acid head groups.

Development and comparison of chromatographic methods for the analysis of long chain diols and alkenones in biological materials and sediment.

We have compared and assessed the suitability of several chromatographic methods for the analysis of long chain alkenones and long chain diols and the associated paleotemperature proxies (UK'37 and LDI). We evaluated the traditional methods for the analysis of the UK'37 and the LDI, gas chromatography (GC) - flame ionization detection (FID) and GC mass spectrometry (MS) using selected ion monitoring (SIM), respectively, and developed a new method using GC-MS/MS in multiple reaction monitoring mode (MRM) for the analysis of long chain diols as well as a method for automatic silylation of diols using a robot autosampler. Finally, we evaluated liquid chromatography (LC) methods to simultaneously measure the UK'37 and the LDI, using ultra high performance LC (UHPLC) with low (nominal mass) resolution MS in SIM mode, and UHPLC with high resolution MS (HRMS). Detection and quantification limits and reproducibility were assessed by means of serial dilutions of culture extracts. Automated silylation by a robot autosampler showed similar reproducibility as off-line silylation while substantially decreasing sample preparation time. The novel MRM method had a slightly lower limit of quantification (LOQ; i.e. 0.3pgC28 1,13-diol injected on-column) than the traditional method (0.5pg) and improved reproducibility while allowing more unambiguous identification of LCDs in complex matrices. For diols, UHPLC-MS using SIM had the highest LOQ (i.e. 15pg) and a comparable reproducibility as GC-MS. UHPLC-HRMS had a LOQ of ca. 1.5pg, and an improved reproducibility for diol analysis. For alkenone analysis, both UHPLC-HRMS and UHPLC-MS using SIM were 2-3 orders of magnitude more sensitive (LOQ ca. 20 and 2pgC37:2 alkenone injected on-column, respectively) than GC-FID (LOD ca. 3ng), with a similar reproducibility of the UK'37 index. Hence, UHPLC-HRMS allows simultaneous analysis of the UK'37 and LDI at an increased sensitivity. In addition, it allows simultaneous measurement of TEX86, a temperature proxy based on the isoprenoid glycerol dialkyl glycerol tetraethers. This reduces the preparation time by excluding the need of derivatization and separation of the ketone (containing the long chain alkenones) and polar fractions (containing the long chain diols and GDGTs). However, synthetic standards are required to fully assess the accuracy of the new methods for determination of the LDI and UK'37.

Abundance and Diversity of Denitrifying and Anammox Bacteria in Seasonally Hypoxic and Sulfidic Sediments of the Saline Lake Grevelingen.

Denitrifying and anammox bacteria are involved in the nitrogen cycling in marine sediments but the environmental factors that regulate the relative importance of these processes are not well constrained. Here, we evaluated the abundance, diversity, and potential activity of denitrifying, anammox, and sulfide-dependent denitrifying bacteria in the sediments of the seasonally hypoxic saline Lake Grevelingen, known to harbor an active microbial community involved in sulfur oxidation pathways. Depth distributions of 16S rRNA gene, nirS gene of denitrifying and anammox bacteria, aprA gene of sulfur-oxidizing and sulfate-reducing bacteria, and ladderane lipids of anammox bacteria were studied in sediments impacted by seasonally hypoxic bottom waters. Samples were collected down to 5 cm depth (1 cm resolution) at three different locations before (March) and during summer hypoxia (August). The abundance of denitrifying bacteria did not vary despite of differences in oxygen and sulfide availability in the sediments, whereas anammox bacteria were more abundant in the summer hypoxia but in those sediments with lower sulfide concentrations. The potential activity of denitrifying and anammox bacteria as well as of sulfur-oxidizing, including sulfide-dependent denitrifiers and sulfate-reducing bacteria, was potentially inhibited by the competition for nitrate and nitrite with cable and/or Beggiatoa-like bacteria in March and by the accumulation of sulfide in the summer hypoxia. The simultaneous presence and activity of organoheterotrophic denitrifying bacteria, sulfide-dependent denitrifiers, and anammox bacteria suggests a tight network of bacteria coupling carbon-, nitrogen-, and sulfur cycling in Lake Grevelingen sediments.

Elucidation and identification of amino acid containing membrane lipids using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: Intact polar lipids (IPLs) are the building blocks of cell membranes, and amino acid containing IPLs have been observed to be involved in response to changing environmental conditions in various species of bacteria. High-performance liquid chromatography/mass spectrometry (HPLC/MS) has become the primary method for analysis of IPLs. Many glycerol-free amino acid containing membrane lipids (AA-IPLs), which are structurally different than abundant aminophospholipids, have not been characterized using HPLC/MS. This results in many lipids remaining unrecognized in IPL analysis of microbial cultures and environmental samples, hampering the study of their occurrence and functionality. METHODS: We analyzed the amino acid containing IPLs of a number of bacteria (i.e. Gluconobacter cerinus, Cyclobacterium marinus, Rhodobacter sphaeroides, and Pedobacter heparinus) in order to decipher fragmentation pathways, and explore potential novel lipid structures using HPLC/electrospray ionization ion trap MS (HPLC/ESI-IT-MS) and HPLC/high-resolution MS (HPLC/HRMS). RESULTS: We report differentiation between glutamine and lysine lipids with the same nominal masses, novel MS fragmentation pathways of cytolipin, the lipopeptides cerilipin and flavolipin, head group hydroxylated ornithine lipids, and the novel identification of cerilipin with a hydroxylated fatty acid. CONCLUSIONS: Non-glycerol AA lipids can be readily recognized as their fragmentation follows a clear pattern with initial dehydration or other loss from the head group, followed by fatty acid losses resulting in a diagnostic fragment ion. Higher level MSn and HRMS are valuable tools in characterizing AA lipid head group structural components.

Factors Controlling the Stable Nitrogen Isotopic Composition (δ15N) of Lipids in Marine Animals.

Lipid extraction of biomass prior to stable isotope analysis is known to cause variable changes in the stable nitrogen isotopic composition (δ15N) of residual biomass. However, the underlying factors causing these changes are not yet clear. Here we address this issue by comparing the δ15N of bulk and residual biomass of several marine animal tissues (fish, crab, cockle, oyster, and polychaete), as well as the δ15N of the extracted lipids. As observed previously, lipid extraction led to a variable offset in δ15N of biomass (differences ranging from -2.3 to +1.8 ‰). Importantly, the total lipid extract (TLE) was highly depleted in 15N compared to bulk biomass, and also highly variable (differences ranging from -14 to +0.7 ‰). The TLE consisted mainly of phosphatidylcholines, a group of lipids with one nitrogen atom in the headgroup. To elucidate the cause for the 15N-depletion in the TLE, the δ15N of amino acids was determined, including serine because it is one of the main sources of nitrogen to N-containing lipids. Serine δ15N values differed by -7 to +2 ‰ from bulk biomass δ15N, and correlated well with the 15N depletion in TLEs. On average, serine was less depleted (-3‰) than the TLE (-7 ‰), possibly due to fractionation during biosynthesis of N-containing headgroups, or that other nitrogen-containing compounds, such as urea and choline, or recycled nitrogen contribute to the nitrogen isotopic composition of the TLE. The depletion in 15N of the TLE relative to biomass increased with the trophic level of the organisms.

Lysine and novel hydroxylysine lipids in soil bacteria: amino acid membrane lipid response to temperature and pH in Pseudopedobacter saltans.

Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress response of soil bacteria.