TY - JOUR
AU - Sinninghe Damsté, Jaap, S.
AB - Abstract Biphytanyl membrane lipids and 16S rRNA sequences derived from marine Crenarchaeota were detected in shallow North Sea surface water in February 2002. To investigate the carbon fixation mechanism of these uncultivated archaea in situ 13C bicarbonate tracer experiments were performed with this water in the absence of light. About 70% of the detected 13C incorporation into lipids (including fatty acids and sterols) is accounted for by the crenarchaeotal biphytanyl membrane lipids. This finding indicates that marine Crenarchaeota can utilize bicarbonate or CO2 derived thereof in the absence of light and are chemoautotrophic organisms. Crenarchaeota, Chemoautotrophy, Glycerol dibiphytanyl glycerol tetraethers (GDGTs) 1 Introduction Archaea are one of the three domains of life. Until recently these were thought to be restricted to extreme environments such as high salinity or high temperature. However, by applying molecular biological techniques and lipid analyses it could be shown that archaea are far more widespread than previously thought and that they also occur in temperate environments such as the open ocean [1–5]. Recent molecular biological work showed that marine Crenarchaeota are distributed over a large depth range in both the photic and aphotic zones of the water column and make up about 20% of the picoplankton in the world's oceans [6]. Membrane lipids of archaea are unique and consist of diphytanyl glycerol diethers and glycerol dibiphytanyl glycerol tetraethers (GDGTs) [7]. Marine Crenarchaeota have a specific GDGT, containing four cyclopentane rings and one cyclohexane ring which occurs exclusively in non-thermophilic Crenarchaeota [8,9] and was called crenarchaeol. This GDGT was detected in ocean water over a large depth range [8,10,11] consistent with the 16S rRNA analyses [6]. Despite the fact that marine Crenarchaeota make up an important part of the world's oceans’ picoplankton almost nothing is known about their basic physiology. Ouverney and Fuhrmann [12] showed that marine archaea can utilize dissolved amino acids and appear to be heterotrophic organisms. However, δ13C values of GDGTs are relatively invariant, in contrast to phytoplankton lipids [13–15]. This has led several authors to suggest that marine Crenarchaeota utilize bicarbonate [13,16]. Further evidence for autotrophy in marine Crenarchaeota was provided by radiocarbon analysis of biphytanes derived from sedimentary GDGTs, which suggested that archaea are not feeding on phytoplankton biomass, but rather use ‘old’14C-depleted dissolved inorganic carbon from below the photic zone in the water column [17]. Most studies, thus, suggest that marine Crenarchaeota use bicarbonate as a carbon source but solid experimental evidence is presently lacking. Here we show by a 13C label experiment that pelagic marine Crenarchaeota are capable of light-independent bicarbonate uptake and are autotrophic organisms. This may have significant implications for our understanding of the marine carbon cycle. 2 Materials and methods 2.1 13C label experiment An in situ 13C bicarbonate label experiment was performed to study the carbon acquisition mechanism of the marine Crenarchaeota by measuring 13C incorporation into their specific lipids. Since marine Crenarchaeota are not in culture yet, this approach [18–20] is a powerful tool to study in situ substrate utilization of these microorganisms in their natural environment. The experiment was performed in the dark to eliminate photosynthetic activity and thereby limiting the competition for marine Crenarchaeota. Fully labeled H213CO3 (99%13C) was continuously added over a 7 day time period to 20 l shallow North Sea surface water, sampled in February 2002 at the NIOZ harbor. Measured average nutrient concentrations of the water in February were for nitrate 75 µmol l−1, nitrite 1 µmol l−1, ammonium 4.8 µmol l−1, phosphate 1.4 µmol l−1 and silicate 39 µmol l−1, while salinity was at 26.5‰. The water was incubated in a sealed container at 15°C in the dark, and label was added to a concentration of 100 µM, resulting in a final labeling of the natural dissolved inorganic carbon pool of about 5%. This small increase of in situ bicarbonate concentration had no significant influence on the pH or nutrient condition of the water used. A control tank was incubated under the same conditions without addition of labeled bicarbonate. 2.2 Lipid analyses For lipid analyses the water was filtered through a 0.7 µm ashed glass fiber (GF) filter and subsequently through a 0.2 µm cellulose/acetate (C/A) filter. The filters were freeze-dried and for GF filters ultrasonically extracted with methanol, methanol/dichloromethane (1:1, vol/vol) and three times with dichloromethane. C/A filters were extracted three times with methanol and three times with hexane. The extracts were combined and the water was removed with a small pipette Na2SO4 column and analyzed for GDGTs by high-performance liquid chromatography/mass spectrometry (HPLC/MS) [21]. Aliquots of the extracts were derivatized with boron trifluoride methanol complex and bis-trimethylsilyltrifluoroacetamide (BSTFA)/pyridine to derivatize fatty acids and alcohols, respectively. The lipids were analyzed by gas chromatography (GC), GC/MS. The total lipid fraction was analyzed for label incorporation by isotopic-ratio-monitoring (IRM) GC/MS. To measure the 13C incorporation in archaeal membrane lipids the GDGTs were treated with HI/LiAlH4[14] to release the biphytanes and measured by IRM-GC/MS. Label incorporation into lipids is expressed in Δδ13C values (the difference between δ13C-label experiment and δ13C-control experiment). 2.3 DNA analyses For molecular biological analysis the water was filtered through a 0.2 µm polycarbonate filter and total DNA was extracted with an ultra clean soil DNA kit (Mobio, Carlsbad, CA, USA) using the conditions as described by the manufacturer. Partial archaeal 16S rRNA genes were amplified by polymerase chain reaction (PCR) using primers Parch519f (complementary reverse sequence of primer PARCH519r [22]) and GCArch915r [23] including a 40-bp long GC clamp [24] as used by Coolen et al. [25]. The amplification protocol after Coolen et al. [25] includes initial denaturation at 96°C (4 min) followed by 35 cycles including a denaturation step at 94°C (30 s), primer annealing at 57°C (40 s) and primer extension at 72°C (40 s). A final extension was performed at 72°C for 10 min. A final concentration of 0.5 µM of each primer was used. The 436-bp long PCR products was separated by denaturing gradient gel electrophoresis (DGGE) [26]. DGGE conditions were applied as described by Coolen et al. [25]. DGGE was carried out in a Bio-Rad D Gene system. PCR samples were applied directly onto 6% (wt/vol) polyamide gel (acrylamide/N, N′-methylene bisacrylamide ratio, 37:1 [wt/wt]) in 1× TAE buffer (pH 7.4). The gel contained a linear gradient of 30–60% denaturant (100% denaturant 7 M urea plus 40% [vol/vol] formamide). Electrophoresis proceeded for 5 h at 200 V and 60°C. DGGE fragments were excised from the gel and the individual 16S rRNA genes were subsequently sequenced. Comparative analysis of the various sequences was done by using the ARB program package (developed by O. Strunk and W. Ludwig; available online at http://www.biol.chemie.tu-muenchen.de/pub/ARB/). Evolutionary distances were calculated by using the Jukes–Cantor equation [27]. The phylogenetic tree was constructed by using the neighbor-joining algorithm [28]. 3 Results and discussion Several archaeal 16S rRNA gene sequences could be detected in shallow North Sea surface water sampled during the winter. The comparative analyses of the sequences showed that they all belong to the group of marine Crenarchaeota (Fig. 1). This is in good agreement with previous seasonal studies, which reported abundant marine Crenarchaeota in North Sea waters during the winter season [29]. Analysis by HPLC/MS of the archaeal lipids from shallow North Sea surface water showed abundant GDGT-0 (I) and crenarchaeol (II) (Fig. 2), the characteristic GDGT membrane lipids of marine Crenarchaeota [9,10]. 1 Open in new tabDownload slide Phylogenetic positions of four partial 16S rRNA gene sequences of Crenarchaeota recovered from shallow surface North Sea water (North Sea 1–4) as well as their closest relatives. All sequences were affiliated to the marine Crenarchaeota Group I. 1 Open in new tabDownload slide Phylogenetic positions of four partial 16S rRNA gene sequences of Crenarchaeota recovered from shallow surface North Sea water (North Sea 1–4) as well as their closest relatives. All sequences were affiliated to the marine Crenarchaeota Group I. 2 Open in new tabDownload slide HPLC/MS base peak ion chromatogram of intact archaeal tetraether membrane lipids from the 20 l 13C-label tank filled with North Sea water, showing a typical cold-water lipid pattern with the main archaeal lipids GDGT-0 (I) and crenarchaeol (II). These lipids are derived from marine pelagic Crenarchaeota [8,9]. 2 Open in new tabDownload slide HPLC/MS base peak ion chromatogram of intact archaeal tetraether membrane lipids from the 20 l 13C-label tank filled with North Sea water, showing a typical cold-water lipid pattern with the main archaeal lipids GDGT-0 (I) and crenarchaeol (II). These lipids are derived from marine pelagic Crenarchaeota [8,9]. The lipids analyses of the label incorporation study revealed that hardly any 13C label incorporation could be detected in the C32 hopanoic acids, C24–26 fatty acids and C26 alcohol (Fig. 3). Since these compounds are likely derived from dead bacterial and higher plant material, respectively, this is not surprising. Very low 13C incorporation with Δδ13C values of 7‰ could be measured for eukaryotic C27–C29 sterols (Fig. 3). This is probably due to the lack of light, which inhibits photosynthesis by eukaryotes in the North Sea water and thereby uptake of 13C-labeled bicarbonate, or CO2. Minor 13C enrichment could be detected in the anteiso-C15 fatty acid, a compound which occurs exclusively in bacteria, with Δδ13C values of 10‰ (Fig. 3). Compounds derived from both bacteria and eukaryotes such as the C14–C20 fatty acids and C18–C20 alcohols showed higher label incorporation. The Δδ13C values for fatty acids were up to 80‰ for the C14 fatty acid and for the alcohols up to 35‰ for the C18 alcohol (Fig. 3). In contrast to the eukaryotic and bacterial lipids, the crenarchaeotal membrane lipids were heavily labeled in 13C with Δδ13C values between 400 and 440‰ (Fig. 3) for the crenarchaeotal biphytanes derived from GDGT-0 (I) and crenarchaeol (II). 3 Open in new tabDownload slide Results of 13C-label experiment with 20 l shallow North Sea water incubated in the dark. The figure shows Δδ13C values from different lipids, where Δδ13C is defined as δ13C of the label tank −δ13C of the control tank. There is minor 13C incorporation into short-chain bacterial and eukaryotic fatty acids (FA), C18–C20 alkanols (AL) and eukaryotic C27–C29 sterols. The main 13C enrichment is found in the acyclic, bicyclic and tricyclic archaeal biphytanes (BIP) derived from GDGT-0 (I) and crenarchaeol (II). 3 Open in new tabDownload slide Results of 13C-label experiment with 20 l shallow North Sea water incubated in the dark. The figure shows Δδ13C values from different lipids, where Δδ13C is defined as δ13C of the label tank −δ13C of the control tank. There is minor 13C incorporation into short-chain bacterial and eukaryotic fatty acids (FA), C18–C20 alkanols (AL) and eukaryotic C27–C29 sterols. The main 13C enrichment is found in the acyclic, bicyclic and tricyclic archaeal biphytanes (BIP) derived from GDGT-0 (I) and crenarchaeol (II). Quantification of the 13C uptake in lipids revealed that about 70% of the detected 13C incorporation was accounted for by the crenarchaeotal biphytanes. The 13C enrichment in the crenarchaeotal membrane lipids clearly shows that Crenarchaeota actively incorporate the inorganic carbon label into their biomass. Transfer of algal fixed carbon to bacteria or Crenarchaeota through a DOC pool is not likely for several reasons: Firstly, the algal lipids show hardly any 13C incorporation, suggesting no substantial uptake by algae. Secondly, lipids of heterotrophic bacteria, specifically the anteiso-C15 fatty acids [30], also incorporated only minor amounts of label, suggesting that no 13C-labeled organic matter was released. Our experiment thus indicates that a cosmopolitan group of marine picoplankton, the marine Crenarchaeota, are autotrophic organisms. Experiments are now underway to estimate the dark 13C uptake rates of marine Crenarchaeota. Our 13C tracer experiment does not provide any information about the energy source of marine Crenarchaeota. Therefore, we have to rely on available ecological studies. Marine Crenarchaeota dwell in surface as well as in deep waters (e.g. [3,6]) and grow under dark conditions as our experiment shows and, thus, are not phototrophic organisms. Studies from the Arabian Sea [9] and Black Sea [11] showed that marine Crenarchaeota do not seem to require oxygen for their metabolism and can thrive at very low levels of oxygen or even anaerobically, suggesting they are aerotolerant anaerobes. A correlation of increasing abundance of marine Crenarchaeota and a distinct nitrate minimum and nitrite maximum in the oxygen minimum zone of the Arabian Sea led Sinninghe Damsté et al. [9] to suggest that marine Crenarchaeota may reduce nitrate to nitrite. This metabolism is known in the kingdom Crenarchaeota from the hyperthermophilic autotrophic archaeon Pyrobaculum aerophilum, which reduces nitrate to nitrite using hydrogen. Nitrate is generally abundant in the water column and is only limited during the phytoplankton blooms, which may explain the reported negative correlation between chlorophyll a concentration and archaeal abundance [4] in Antarctic surface waters. 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TI - Bicarbonate uptake by marine Crenarchaeota
JF - FEMS Microbiology Letters
DO - 10.1016/S0378-1097(03)00060-0
DA - 2003-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/bicarbonate-uptake-by-marine-crenarchaeota-3jWnpvmz32
SP - 203
EP - 207
VL - 219
IS - 2
DP - DeepDyve
ER -