Multi-wavelength Raman microscopy of nickel-based electron transport in cable bacteria.

Cable bacteria embed a network of conductive protein fibers in their cell envelope that efficiently guides electron transport over distances spanning up to several centimeters. This form of long-distance electron transport is unique in biology and is mediated by a metalloprotein with a sulfur-coordinated nickel (Ni) cofactor. However, the molecular structure of this cofactor remains presently unknown. Here, we applied multi-wavelength Raman microscopy to identify cell compounds linked to the unique cable bacterium physiology, combined with stable isotope labeling, and orientation-dependent and ultralow-frequency Raman microscopy to gain insight into the structure and organization of this novel Ni-cofactor. Raman spectra of native cable bacterium filaments reveal vibrational modes originating from cytochromes, polyphosphate granules, proteins, as well as the Ni-cofactor. After selective extraction of the conductive fiber network from the cell envelope, the Raman spectrum becomes simpler, and primarily retains vibrational modes associated with the Ni-cofactor. These Ni-cofactor modes exhibit intense Raman scattering as well as a strong orientation-dependent response. The signal intensity is particularly elevated when the polarization of incident laser light is parallel to the direction of the conductive fibers. This orientation dependence allows to selectively identify the modes that are associated with the Ni-cofactor. We identified 13 such modes, some of which display strong Raman signals across the entire range of applied wavelengths (405-1,064 nm). Assignment of vibrational modes, supported by stable isotope labeling, suggest that the structure of the Ni-cofactor shares a resemblance with that of nickel bis(1,2-dithiolene) complexes. Overall, our results indicate that cable bacteria have evolved a unique cofactor structure that does not resemble any of the known Ni-cofactors in biology.

The organo-metal-like nature of long-range conduction in cable bacteria.

Cable bacteria are filamentous, multicellular microorganisms that display an exceptional form of biological electron transport across centimeter-scale distances. Currents are guided through a network of nickel-containing protein fibers within the cell envelope. Still, the mechanism of long-range conduction remains unresolved. Here, we characterize the conductance of the fiber network under dry and wet, physiologically relevant, conditions. Our data reveal that the fiber conductivity is high (median value: 27 S cm-1; range: 2 to 564 S cm-1), does not show any redox signature, has a low thermal activation energy (Ea = 69 ± 23 meV), and is not affected by humidity or the presence of ions. These features set the nickel-based conduction mechanism in cable bacteria apart from other known forms of biological electron transport. As such, conduction resembles that of an organic semi-metal with a high charge carrier density. Our observation that biochemistry can synthesize an organo-metal-like structure opens the way for novel bio-based electronic technologies.

Efficient long-range conduction in cable bacteria through nickel protein wires.

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

Enhanced Laterally Resolved ToF-SIMS and AFM Imaging of the Electrically Conductive Structures in Cable Bacteria.

Cable bacteria are electroactive bacteria that form a long, linear chain of ridged cylindrical cells. These filamentous bacteria conduct centimeter-scale long-range electron transport through parallel, interconnected conductive pathways of which the detailed chemical and electrical properties are still unclear. Here, we combine time-of-flight secondary-ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM) to investigate the structure and composition of this naturally occurring electrical network. The enhanced lateral resolution achieved allows differentiation between the cell body and the cell-cell junctions that contain a conspicuous cartwheel structure. Three ToF-SIMS modes were compared in the study of so-called fiber sheaths (i.e., the cell material that remains after the removal of cytoplasm and membranes, and which embeds the electrical network). Among these, fast imaging delayed extraction (FI-DE) was found to balance lateral and mass resolution, thus yielding the following multiple benefits in the study of structure-composition relations in cable bacteria: (i) it enables the separate study of the cell body and cell-cell junctions; (ii) by combining FI-DE with in situ AFM, the depth of Ni-containing protein-key in the electrical transport-is determined with greater precision; and (iii) this combination prevents contamination, which is possible when using an ex situ AFM. Our results imply that the interconnects in extracted fiber sheaths are either damaged during extraction, or that their composition is different from fibers, or both. From a more general analytical perspective, the proposed methodology of ToF-SIMS in the FI-DE mode combined with in situ AFM holds great promise for studying the chemical structure of other biological systems.

Cell Cycle, Filament Growth and Synchronized Cell Division in Multicellular Cable Bacteria.

Cable bacteria are multicellular, Gram-negative filamentous bacteria that display a unique division of metabolic labor between cells. Cells in deeper sediment layers are oxidizing sulfide, while cells in the surface layers of the sediment are reducing oxygen. The electrical coupling of these two redox half reactions is ensured via long-distance electron transport through a network of conductive fibers that run in the shared cell envelope of the centimeter-long filament. Here we investigate how this unique electrogenic metabolism is linked to filament growth and cell division. Combining dual-label stable isotope probing (13C and 15N), nanoscale secondary ion mass spectrometry, fluorescence microscopy and genome analysis, we find that the cell cycle of cable bacteria cells is highly comparable to that of other, single-celled Gram-negative bacteria. However, the timing of cell growth and division appears to be tightly and uniquely controlled by long-distance electron transport, as cell division within an individual filament shows a remarkable synchronicity that extends over a millimeter length scale. To explain this, we propose the "oxygen pacemaker" model in which a filament only grows when performing long-distance transport, and the latter is only possible when a filament has access to oxygen so it can discharge electrons from its internal electrical network.

A Cross-System Comparison of Dark Carbon Fixation in Coastal Sediments.

Dark carbon fixation (DCF) by chemoautotrophic microorganisms can sustain food webs in the seafloor by local production of organic matter independent of photosynthesis. The process has received considerable attention in deep sea systems, such as hydrothermal vents, but the regulation, depth distribution, and global importance of coastal sedimentary DCF have not been systematically investigated. Here we surveyed eight coastal sediments by means of stable isotope probing (13C-DIC) combined with bacterial biomarkers (phospholipid-derived fatty acids) and compiled additional rates from literature into a global database. DCF rates in coastal sediments range from 0.07 to 36.30 mmol C m-2 day-1, and there is a linear relation between DCF and water depth. The CO2 fixation ratio (DCF/CO2 respired) also shows a trend with water depth, decreasing from 0.09 in nearshore environments to 0.04 in continental shelf sediments. Five types of depth distributions of chemoautotrophic activity are identified based on the mode of pore water transport (advective, bioturbated, and diffusive) and the dominant pathway of microbial sulfur oxidation. Extrapolated to the global coastal ocean, we estimate a DCF rate of 0.04 to 0.06 Pg C year-1, which is less than previous estimates based on indirect measurements (0.15 Pg C year-1), but remains substantially higher than the global DCF rate at deep sea hydrothermal vents (0.001-0.002 Pg C year-1).

Division of labor and growth during electrical cooperation in multicellular cable bacteria.

Multicellularity is a key evolutionary innovation, leading to coordinated activity and resource sharing among cells, which generally occurs via the physical exchange of chemical compounds. However, filamentous cable bacteria display a unique metabolism in which redox transformations in distant cells are coupled via long-distance electron transport rather than an exchange of chemicals. This challenges our understanding of organismal functioning, as the link among electron transfer, metabolism, energy conservation, and filament growth in cable bacteria remains enigmatic. Here, we show that cells within individual filaments of cable bacteria display a remarkable dichotomy in biosynthesis that coincides with redox zonation. Nanoscale secondary ion mass spectrometry combined with 13C (bicarbonate and propionate) and 15N-ammonia isotope labeling reveals that cells performing sulfide oxidation in deeper anoxic horizons have a high assimilation rate, whereas cells performing oxygen reduction in the oxic zone show very little or no label uptake. Accordingly, oxygen reduction appears to merely function as a mechanism to quickly dispense of electrons with little to no energy conservation, while biosynthesis and growth are restricted to sulfide-respiring cells. Still, cells can immediately switch roles when redox conditions change, and show no differentiation, which suggests that the "community service" performed by the cells in the oxic zone is only temporary. Overall, our data reveal a division of labor and electrical cooperation among cells that has not been seen previously in multicellular organisms.

A highly conductive fibre network enables centimetre-scale electron transport in multicellular cable bacteria.

Biological electron transport is classically thought to occur over nanometre distances, yet recent studies suggest that electrical currents can run along centimetre-long cable bacteria. The phenomenon remains elusive, however, as currents have not been directly measured, nor have the conductive structures been identified. Here we demonstrate that cable bacteria conduct electrons over centimetre distances via highly conductive fibres embedded in the cell envelope. Direct electrode measurements reveal nanoampere currents in intact filaments up to 10.1 mm long (>2000 adjacent cells). A network of parallel periplasmic fibres displays a high conductivity (up to 79 S cm-1), explaining currents measured through intact filaments. Conductance rapidly declines upon exposure to air, but remains stable under vacuum, demonstrating that charge transfer is electronic rather than ionic. Our finding of a biological structure that efficiently guides electrical currents over long distances greatly expands the paradigm of biological charge transport and could enable new bio-electronic applications.

Long-distance electron transport in individual, living cable bacteria.

Electron transport within living cells is essential for energy conservation in all respiring and photosynthetic organisms. While a few bacteria transport electrons over micrometer distances to their surroundings, filaments of cable bacteria are hypothesized to conduct electric currents over centimeter distances. We used resonance Raman microscopy to analyze cytochrome redox states in living cable bacteria. Cable-bacteria filaments were placed in microscope chambers with sulfide as electron source and oxygen as electron sink at opposite ends. Along individual filaments a gradient in cytochrome redox potential was detected, which immediately broke down upon removal of oxygen or laser cutting of the filaments. Without access to oxygen, a rapid shift toward more reduced cytochromes was observed, as electrons were no longer drained from the filament but accumulated in the cellular cytochromes. These results provide direct evidence for long-distance electron transport in living multicellular bacteria.

The Cell Envelope Structure of Cable Bacteria.

Cable bacteria are long, multicellular micro-organisms that are capable of transporting electrons from cell to cell along the longitudinal axis of their centimeter-long filaments. The conductive structures that mediate this long-distance electron transport are thought to be located in the cell envelope. Therefore, this study examines in detail the architecture of the cell envelope of cable bacterium filaments by combining different sample preparation methods (chemical fixation, resin-embedding, and cryo-fixation) with a portfolio of imaging techniques (scanning electron microscopy, transmission electron microscopy and tomography, focused ion beam scanning electron microscopy, and atomic force microscopy). We systematically imaged intact filaments with varying diameters. In addition, we investigated the periplasmic fiber sheath that remains after the cytoplasm and membranes were removed by chemical extraction. Based on these investigations, we present a quantitative structural model of a cable bacterium. Cable bacteria build their cell envelope by a parallel concatenation of ridge compartments that have a standard size. Larger diameter filaments simply incorporate more parallel ridge compartments. Each ridge compartment contains a ~50 nm diameter fiber in the periplasmic space. These fibers are continuous across cell-to-cell junctions, which display a conspicuous cartwheel structure that is likely made by invaginations of the outer cell membrane around the periplasmic fibers. The continuity of the periplasmic fibers across cells makes them a prime candidate for the sought-after electron conducting structure in cable bacteria.

From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton.

One of the major challenges in ecological stoichiometry is to establish how environmental changes in resource availability may affect both the biochemical composition of organisms and the species composition of communities. This is a pressing issue in many coastal waters, where anthropogenic activities have caused large changes in riverine nutrient inputs. Here we investigate variation in the biochemical composition and synthesis of amino acids, fatty acids (FA), and carbohydrates in mixed phytoplankton communities sampled from the North Sea. The communities were cultured in chemostats supplied with different concentrations of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) to establish four different types of resource limitations. Diatoms dominated under N-limited, N+P limited and P-limited conditions. Cyanobacteria became dominant in one of the N-limited chemostats and green algae dominated in the one P-limited chemostat and under light-limited conditions. Changes in nutrient availability directly affected amino acid content, which was lowest under N and N+P limitation, higher under P-limitation and highest when light was the limiting factor. Storage carbohydrate content showed the opposite trend and storage FA content seemed to be co-dependent on community composition. The synthesis of essential amino acids was affected under N and N+P limitation, as the transformation from non-essential to essential amino acids decreased at DIN:DIP ≤ 6. The simple community structure and clearly identifiable nutrient limitations confirm and clarify previous field findings in the North Sea. Our results show that different phytoplankton groups are capable of adapting their key biosynthetic rates and hence their biochemical composition to different degrees when experiencing shifts in nutrient availability. This will have implications for phytoplankton growth, community structure, and the nutritional quality of phytoplankton as food for higher trophic levels.

Impact of Seasonal Hypoxia on Activity and Community Structure of Chemolithoautotrophic Bacteria in a Coastal Sediment.

Seasonal hypoxia in coastal systems drastically changes the availability of electron acceptors in bottom water, which alters the sedimentary reoxidation of reduced compounds. However, the effect of seasonal hypoxia on the chemolithoautotrophic community that catalyzes these reoxidation reactions is rarely studied. Here, we examine the changes in activity and structure of the sedimentary chemolithoautotrophic bacterial community of a seasonally hypoxic saline basin under oxic (spring) and hypoxic (summer) conditions. Combined 16S rRNA gene amplicon sequencing and analysis of phospholipid-derived fatty acids indicated a major temporal shift in community structure. Aerobic sulfur-oxidizing Gammaproteobacteria (Thiotrichales) and Epsilonproteobacteria (Campylobacterales) were prevalent during spring, whereas Deltaproteobacteria (Desulfobacterales) related to sulfate-reducing bacteria prevailed during summer hypoxia. Chemolithoautotrophy rates in the surface sediment were three times higher in spring than in summer. The depth distribution of chemolithoautotrophy was linked to the distinct sulfur oxidation mechanisms identified through microsensor profiling, i.e., canonical sulfur oxidation, electrogenic sulfur oxidation by cable bacteria, and sulfide oxidation coupled to nitrate reduction by Beggiatoaceae The metabolic diversity of the sulfur-oxidizing bacterial community suggests a complex niche partitioning within the sediment, probably driven by the availability of reduced sulfur compounds (H2S, S0, and S2O32-) and electron acceptors (O2 and NO3-) regulated by seasonal hypoxia.IMPORTANCE Chemolithoautotrophic microbes in the seafloor are dependent on electron acceptors, like oxygen and nitrate, that diffuse from the overlying water. Seasonal hypoxia, however, drastically changes the availability of these electron acceptors in the bottom water; hence, one expects a strong impact of seasonal hypoxia on sedimentary chemolithoautotrophy. A multidisciplinary investigation of the sediments in a seasonally hypoxic coastal basin confirms this hypothesis. Our data show that bacterial community structure and chemolithoautotrophic activity varied with the seasonal depletion of oxygen. Unexpectedly, the dark carbon fixation was also dependent on the dominant microbial pathway of sulfur oxidation occurring in the sediment (i.e., canonical sulfur oxidation, electrogenic sulfur oxidation by cable bacteria, and sulfide oxidation coupled to nitrate reduction by Beggiatoaceae). These results suggest that a complex niche partitioning within the sulfur-oxidizing bacterial community additionally affects the chemolithoautotrophic community of seasonally hypoxic sediments.

Microbial biomass in compost during colonization of Agaricus bisporus.

Agaricus bisporus mushrooms are commercially produced on a microbe rich compost. Here, fungal and bacterial biomass was quantified in compost with and without colonization by A. bisporus. Chitin content, indicative of total fungal biomass, increased during a 26-day period from 576 to 779 nmol N-acetylglucosamine g-1 compost in the absence of A. bisporus (negative control). A similar increase was found in the presence of this mushroom forming fungus. The fungal phospholipid-derived fatty acid (PLFA) marker C18:2ω6, indicative of the living fraction of the fungal biomass, decreased from 575 to 280 nmol g-1 compost in the negative control. In contrast, it increased to 1200 nmol g-1 compost in the presence of A. bisporus. Laccase activity was absent throughout culturing in the negative control, while it correlated with the fungal PLFA marker in the presence of A. bisporus. PLFA was also used to quantify living bacterial biomass. In the negative control, the bacterial markers remained constant at 3000-3200 nmol PLFA g-1 compost. In contrast, they decreased to 850 nmol g-1 compost during vegetative growth of A. bisporus, implying that bacterial biomass decreased from 17.7 to 4.7 mg g-1 compost. The relative amount of the Gram positive associated PLFA markers a15:0 and a17:0 and the Gram negative PLFA associated markers cy17:0 and cy19:0 increased and decreased, respectively, suggesting that Gram negative bacteria are more suppressed by A. bisporus. Together, these data indicate that fungal biomass can make up 6.8% of the compost after A. bisporus colonization, 57% of which being dead. Moreover, results show that A. bisporus impacts biomass and composition of bacteria in compost.

Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates.

RATIONALE: We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of δ(13)C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence, although LC/IRMS is expected to be more accurate and precise, no direct comparison has been reported. METHODS: GC/IRMS with the aldonitrile penta-acetate (ANPA) derivatisation method was compared with LC/IRMS without derivatisation. A large number of glucose standards and a variety of natural samples were analysed for five neutral carbohydrates at natural abundance as well as at (13)C-enriched levels. Gas chromatography/chemical ionisation mass spectrometry (GC/CIMS) was applied to check for incomplete derivatisation of the carbohydrate, which would impair the accuracy of the GC/IRMS method. RESULTS: The LC/IRMS technique provided excellent precision (±0.08‰ and ±3.1‰ at natural abundance and enrichment levels, respectively) for the glucose standards and this technique proved to be superior to GC/IRMS (±0.62‰ and ±19.8‰ at natural abundance and enrichment levels, respectively). For GC/IRMS measurements the derivatisation correction and the conversion of carbohydrates into CO2 had a considerable effect on the measured δ(13)C values. However, we did not find any significant differences in the accuracy of the two techniques over the full range of natural δ(13)C abundances and (13)C-labelled glucose. The difference in the performance of GC/IRMS and LC/IRMS diminished when the δ(13)C values were measured in natural samples, because the chromatographic performance and background correction became critical factors, particularly for LC/IRMS. The derivatisation of carbohydrates for the GC/IRMS method was complete. CONCLUSIONS: Although both LC/IRMS and GC/IRMS are reliable techniques for compound-specific stable carbon isotope analysis of carbohydrates (provided that derivatisation is complete and the calibration requirements are met), LC/IRMS is the technique of choice. The reasons for this are the improved precision, simpler sample preparation, and straightforward isotopic calibration.

Microbial carbon metabolism associated with electrogenic sulphur oxidation in coastal sediments.

Recently, a novel electrogenic type of sulphur oxidation was documented in marine sediments, whereby filamentous cable bacteria (Desulfobulbaceae) are mediating electron transport over cm-scale distances. These cable bacteria are capable of developing an extensive network within days, implying a highly efficient carbon acquisition strategy. Presently, the carbon metabolism of cable bacteria is unknown, and hence we adopted a multidisciplinary approach to study the carbon substrate utilization of both cable bacteria and associated microbial community in sediment incubations. Fluorescence in situ hybridization showed rapid downward growth of cable bacteria, concomitant with high rates of electrogenic sulphur oxidation, as quantified by microelectrode profiling. We studied heterotrophy and autotrophy by following (13)C-propionate and -bicarbonate incorporation into bacterial fatty acids. This biomarker analysis showed that propionate uptake was limited to fatty acid signatures typical for the genus Desulfobulbus. The nanoscale secondary ion mass spectrometry analysis confirmed heterotrophic rather than autotrophic growth of cable bacteria. Still, high bicarbonate uptake was observed in concert with the development of cable bacteria. Clone libraries of 16S complementary DNA showed numerous sequences associated to chemoautotrophic sulphur-oxidizing Epsilon- and Gammaproteobacteria, whereas (13)C-bicarbonate biomarker labelling suggested that these sulphur-oxidizing bacteria were active far below the oxygen penetration. A targeted manipulation experiment demonstrated that chemoautotrophic carbon fixation was tightly linked to the heterotrophic activity of the cable bacteria down to cm depth. Overall, the results suggest that electrogenic sulphur oxidation is performed by a microbial consortium, consisting of chemoorganotrophic cable bacteria and chemolithoautotrophic Epsilon- and Gammaproteobacteria. The metabolic linkage between these two groups is presently unknown and needs further study.

Seasonal variation of Fatty acids and stable carbon isotopes in sponges as indicators for nutrition: biomarkers in sponges identified.

To get a better understanding of sponge feeding biology and efficiencies, the fatty acid (FA) composition and (13)C natural abundance of sponges and of suspended particulate matter (SPM) from surrounding seawater was studied in different seasons at three locations. Haliclona oculata and Haliclona xena from the Oosterschelde, the Netherlands, Halichondria panicea and H. xena from Lake Veere, the Netherlands, and Aplysina aerophoba and Dysidea avara from the Mediterranean, Spain, were studied. Several FA biomarkers for different algal groups, bacteria and sponge biomass were identified in all sponges. The FA concentration variation in sponges was related to changes in fatty acid concentration in SPM. Stable carbon isotopic ratios (δ(13)C) in sponge specific FAs showed very limited seasonal variation at all sites. Algal FAs in sponges were mainly acquired from the SPM through active filtration in all seasons. At the two sites in the Netherlands only in May (spring), the sponge specific FAs had similar δ(13)C ratios as algal FAs, suggesting that sponges were mainly growing during spring and probably summer. During autumn and winter, they were still actively filtering, but the food collected during this period had little effect on sponge δ(13)C values suggesting limited incorporation of filtered material into the sponge body. The sponge A. aerophoba relied mostly on the symbiotic bacteria. In conclusion, fatty acid composition in combination with stable carbon isotope analysis can be used to analyze the food source of sponges.

Chemoautotrophic carbon fixation rates and active bacterial communities in intertidal marine sediments.

Chemoautotrophy has been little studied in typical coastal marine sediments, but may be an important component of carbon recycling as intense anaerobic mineralization processes in these sediments lead to accumulation of high amounts of reduced compounds, such as sulfides and ammonium. We studied chemoautotrophy by measuring dark-fixation of 13C-bicarbonate into phospholipid derived fatty acid (PLFA) biomarkers at two coastal sediment sites with contrasting sulfur chemistry in the Eastern Scheldt estuary, The Netherlands. At one site where free sulfide accumulated in the pore water right to the top of the sediment, PLFA labeling was restricted to compounds typically found in sulfur and ammonium oxidizing bacteria. At the other site, with no detectable free sulfide in the pore water, a very different PLFA labeling pattern was found with high amounts of label in branched i- and a-PLFA besides the typical compounds for sulfur and ammonium oxidizing bacteria. This suggests that other types of chemoautotrophic bacteria were also active, most likely Deltaproteobacteria related to sulfate reducers. Maximum rates of chemoautotrophy were detected in first 1 to 2 centimeters of both sediments and chemosynthetic biomass production was high ranging from 3 to 36 mmol C m(-2) d(-1). Average dark carbon fixation to sediment oxygen uptake ratios were 0.22±0.07 mol C (mol O2)(-1), which is in the range of the maximum growth yields reported for sulfur oxidizing bacteria indicating highly efficient growth. Chemoautotrophic biomass production was similar to carbon mineralization rates in the top of the free sulfide site, suggesting that chemoautotrophic bacteria could play a crucial role in the microbial food web and labeling in eukaryotic poly-unsaturated PLFA was indeed detectable. Our study shows that dark carbon fixation by chemoautotrophic bacteria is a major process in the carbon cycle of coastal sediments, and should therefore receive more attention in future studies on sediment biogeochemistry and microbial ecology.

A versatile method for simultaneous stable carbon isotope analysis of DNA and RNA nucleotides by liquid chromatography/isotope ratio mass spectrometry.

RATIONALE: Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is currently the most accurate and precise technique for the measurement of compound-specific stable carbon isotope ratios ((13)C/(12)C) in biological metabolites, at their natural abundance. However, until now this technique could not be applied for the analysis of nucleic acids, the building blocks of the carriers of genetic information in living cells and viruses, DNA and RNA. METHODS: Mixed-mode chromatography (MMC) was applied to obtain the complete separation of nine nucleotides (eight originating from DNA/RNA and one nucleotide (inosine monophosphate) that may serve as an internal standard) in a single run using LC/IRMS. We also developed and validated a method for DNA and RNA extraction and an enzymatic hydrolysis protocol for natural samples, which is compatible with LC/IRMS analysis as it minimizes the carbon blank. The method was used to measure the concentration and stable carbon isotope ratio of DNA and RNA nucleotides in marine sediment and in the common marine macro alga (Ulva sp.) at natural abundance levels as well as for (13)C-enriched samples. RESULTS: The detection limit of the LC/IRMS method varied between 1.0 nmol for most nucleotides and 2.0 nmol for late-eluting compounds. The intraday and interday reproducibility of nucleotide concentration measurements was better than, respectively, 4.1% and 8.9% and for δ(13)C measurements better than, respectively, 0.3‰ and 0.5‰. The obtained nucleic acid concentrations and nucleic acid synthesis rates were in good agreement with values reported in the literature. CONCLUSIONS: This new method gives reproducible results for the concentration and δ(13)C values of nine nucleotides. This solvent-free chromatographic method may also be used for other purposes, such as for instance to determine nucleotide concentrations using spectrophotometric detection. This sensitive method offers a new avenue for the study of DNA and RNA biosynthesis that can be applied in various fields of research.

Phospholipid-derived fatty acids and quinones as markers for bacterial biomass and community structure in marine sediments.

Phospholipid-derived fatty acids (PLFA) and respiratory quinones (RQ) are microbial compounds that have been utilized as biomarkers to quantify bacterial biomass and to characterize microbial community structure in sediments, waters, and soils. While PLFAs have been widely used as quantitative bacterial biomarkers in marine sediments, applications of quinone analysis in marine sediments are very limited. In this study, we investigated the relation between both groups of bacterial biomarkers in a broad range of marine sediments from the intertidal zone to the deep sea. We found a good log-log correlation between concentrations of bacterial PLFA and RQ over several orders of magnitude. This relationship is probably due to metabolic variation in quinone concentrations in bacterial cells in different environments, whereas PLFA concentrations are relatively stable under different conditions. We also found a good agreement in the community structure classifications based on the bacterial PLFAs and RQs. These results strengthen the application of both compounds as quantitative bacterial biomarkers. Moreover, the bacterial PLFA- and RQ profiles revealed a comparable dissimilarity pattern of the sampled sediments, but with a higher level of dissimilarity for the RQs. This means that the quinone method has a higher resolution for resolving differences in bacterial community composition. Combining PLFA and quinone analysis as a complementary method is a good strategy to yield higher resolving power in bacterial community structure.

Natural occurrence of microbial sulphur oxidation by long-range electron transport in the seafloor.

Recently, a novel mode of sulphur oxidation was described in marine sediments, in which sulphide oxidation in deeper anoxic layers was electrically coupled to oxygen reduction at the sediment surface. Subsequent experimental evidence identified that long filamentous bacteria belonging to the family Desulfobulbaceae likely mediated the electron transport across the centimetre-scale distances. Such long-range electron transfer challenges some long-held views in microbial ecology and could have profound implications for sulphur cycling in marine sediments. But, so far, this process of electrogenic sulphur oxidation has been documented only in laboratory experiments and so its imprint on the seafloor remains unknown. Here we show that the geochemical signature of electrogenic sulphur oxidation occurs in a variety of coastal sediment environments, including a salt marsh, a seasonally hypoxic basin, and a subtidal coastal mud plain. In all cases, electrogenic sulphur oxidation was detected together with an abundance of Desulfobulbaceae filaments. Complementary laboratory experiments in intertidal sands demonstrated that mechanical disturbance by bioturbating fauna destroys the electrogenic sulphur oxidation signal. A survey of published geochemical data and 16S rRNA gene sequences identified that electrogenic sulphide oxidation is likely present in a variety of marine sediments with high sulphide generation and restricted bioturbation, such as mangrove swamps, aquaculture areas, seasonally hypoxic basins, cold sulphide seeps and possibly hydrothermal vent environments. This study shows for the first time that electrogenic sulphur oxidation occurs in a wide range of marine sediments and that bioturbation may exert a dominant control on its natural distribution.