Molecular Biogeochemistry Readings Introduction

Topics Complex lipids of extant archaea • Structural peculiarities of archaeal lipids • LCMS methods • Molecular Signatures of HT Methanogens • Low temperature Chrenarchaea • Ignicoccus sp., Nanoarchaeum equitans Common Acyclic Isoprenoids isoprene tail-to-tail head-to-tail 1 OH phytol head-to-head phytane 1 crocetane pristane 1 2,6,10,15,19-pentamethylicosane (PMI) 1 2,6,10,14,18-pentamethylicosane 1 squalane farnesane Less Common Acyclic Isoprenoids C30 HBI C25 HBI C20 HBI Diatom sources Botryococcus braunii botryococcane 17 16 1 18 2 3 4 5 19 6 7 8 2' 9 lycopene tomato carotenoid Probable algal hydrocarbon lycopane ? from lycapodiene Polar Lipid Precursors of Acyclic Isoprenoids O O OH phytane archaeol HO caldarchaeol O O O O OH biphytane O O OH O chrenarchaeol O HO Favored Mass Spectrometric Fragmentations 57 253 x50 267 71 323 85 127 183 ?? 352 Crocetane – Phytane Distinction crocetane phytane crocetane phytane GC-FID (a) 282-169; 0.6%, 1.9 Full Scan (RIC) (b) 196-127; 100%, 2.1 169 Da (RIC from FS) (c) 196-126; 63%, 2.3 169 Da (SIR) (d) 168-182; 13%, 11.7 183 Da (SIR) GC and GC-MS (SIR) (e) 182-127; 40%, 0.1 GC-MS-MS Crocetane – Phytane Distinction phytane crocetane (a) Barney Ck 169 Da (b) Barney Ck 168-126 Da (c) Barney Ck 196-127 Da nC18 Figure 4 (d) Monterey 196-127 Da Regular C25 vs PMI Distinction Ace Lake Modern Sed.PMI Wilkinson 1 C25 reg 100% C25 reg 100% (a) 266-197 Da 100% (a) (a) 76% 68% 2.4% (b) 252-197 Da (b) (b ) 26% 26% 4% (c) (c) 352-267 Da 35.48 W. Terrace 1 36.18 35.48 (c) 36.18 35.48 Ret. Time (mins) One thin peak+ one compound All fat peaks = more than one compound 36.18 Regular C25 vs PMI Distinction O R O O 2,6,10,14,14-pentamethylicosane Carbon chains of Halobacterium core lipid 2,6,10,15,19-pentamethylicosane (PMI) Found as a free hydrocarbon in some methanogens A ‘highly branched isoprenoid’ (HBI) from a diatom I25 Reg PMI Unknowns 1 2 (a) Monterey (b) Byilkaoora-3 Distinguishing C25 Isoprenoids note peak shapes (c) Monterey + Byilkaoora-3 (d) W. Terrace-1 Partial 183Figure Da (SIR) 6 chromatograms of (a) Monterey Formation showing elution position of PMI; (b) Byilkaoora-3 showing elution position of I25 reg; (c) Monterey + Byilkaoora-3 mixture showing relative elution order of PMI and I25 reg isomers (NB. only partially resolved); (d) West Terrace-1 which has a peak at the same position as the I25 reg isomer and no peak at the earlier retention time of PMI. Unknown peaks 1 (Monterey) and 2 (West Terrace-1) elute after I25 reg. Chromatogram time range = 36 sec. phytane reduction/dehydration/reduction OH E-3, 7R, 11R, 15-tetramethylhexadec-2-enol = phytol oxidation/decarboxylation/reduction = 6(R), 10(S) - pristane 6(R), 10(R) - pristane 6(S), 10(R) - pristane 6(S), 10(S) - pristane Archaeal or bacterial cell cell membrane polar lipid bi-layer one molecule O polar headgroup X O O R O R O core lipid Glycerol Stereochemistry ? sn-glycerol-1­ phosphate dehydrogenase (G1PDH) sn-glycerol-3­ phosphate dehydrogenase (G3PDH) IPL variety analyzed by HPLC-ESI-MSn bacterial O OH O P O O OH O C17H33 fatty acids ~C12 to C24 10+ different HO O C17H33 headgroups O identified… phospholipids & glycolipids, some highly specific may also be alcohols archaeal similar variety of headgroups but highly specific core lipids HO 2 basic types with some variations archaeol O O O O O O O OH caldarchaeol (GDGT: glycerol dialkyl glycerol tetraether) Data on IPL distributions in biogeochemically relevant prokaryotes are very sparse Ignicoccus islandicus & Ignicoccus pacificus Sources of samples. At the Kolbeinsey Ridge, north of Iceland, eight samples of submarine sandy sediments and venting water (original temperatures around 90 °C) were taken by the research submersible `Geo' at depths between 103 and 106 m (Fricke et al., 1989; Burggraf et al., 1990). Furthermore, black smoker samples were obtained during dive 3072 of the submersible `Alvin' at the East PaciÆc Rise at 9° N, 104° W at a depth of 2500 m. Arch Microbiol (2004) 182: 404-413 DOI 10.1007/s00 203-004-0725-x ORIGINAL PAPER Ulrike Jahn, Roger Summons, Helen Sturt Emmanuelle Grosjean, Harald Huber Composition of the lipids of Nanoarchaeum equitans and their origin from its host Ignicoccus sp. strain KIN4/I Received: 22 January 2004 / Revised: 30 June 2004 / Accepted: 5 August 2004 / Published online: 14 September 2004 © Springer-Verlag 2004 Archaeal or bacterial cell cell membrane polar lipid bi-layer polar lipid O polar head group X R O O O core lipid R O O RT: 0.00 - 66.00 SM: 7B P NL: 7.02E6 O O H3N O Base Peak m/z= 1560.0-1561.0+ O 1722.0-1723.0+ NH2 P 1884.0-1885.0 F: + c Full HO O O OH ms [ 500.00-2000.00] MS O Nano1pos 65 min O 33.33 100 90 Relative Abundance 80 ‘Nano’ symbiont 70 60 50 40 30 41.94 N HO 51.73 20 OH HO 2.20 10 HO 0 100 NL: 1.05E7 42.08 ‘KIN4M’ host 70 60 50 40 30 33.47 2.31 52.01 10 0 10 20 O OH X=PS O di-ester X=PG X=PC X=sugar R O R O O Archaea only O O O R O R di-ether 30 Time (min) 40 50 60 O O R R O O mixed O archaeol O O O O O tetraether Caldarchaeol glycolipid 20 0 O O O Base Peak m/z= 1560.0-1561.0+ 1722.0-1723.0+ 1884.0-1885.0 F: + c Full ms [ 500.00-2000.00] MS KIN4Mpos 65 min 90 80 HO P O OH O P O OO O X=PE O Genome Structure Nanoarchaeum has a genome with only 490 kb, which represents the smallest archaeal genome to date. Comparing ss rRNA sequences, it was noted that sequence identities were more like archaeon than bacterial species. There was no difference, however, in the sequence identity to the Crenarchaeota, Euryarchaeota, and 'Korarchaeota', indicating it represents a new archaeal phylum. Cell Structure and Metabolism These coccus cells are only 400 nm in diameter and are covered by an S-layer. They require cell-cell contact with an actively growing Ignicoccus cell in order to grow. Other nanoarchaeotal 16S rRNA genes have been obtained from the East Pacific Rise (pH 6.5), the Obsidian Pool in Yellowstone National Park (80oC, pH 6.0), and Caldera Uzon in Kamchatka, Russia (85oC, pH 5.5). RT: 0.00 - 66.00 SM: 7B 100 nano 90 Relative Abundance 3 ring (2 isomers) 2.48 80 archaeol 70 NL: 9.84E7 Base Peak m/z= 670.0-671.0+832.0-833.0+ 994.0-995.0+1156.0-1157.0+ 1318.0-1319.0+1480.0-1481.0 F: + c Full ms [ 500.00-2000.00] MS Nano1pos 65 min 36.72 43.84 4 ring 26.10 60 2 ring 50 40 30 5 ring 1 ring 20 6.30 34.39 10 0 100 NL: 1.77E8 Base Peak m/z= 670.0-671.0+832.0-833.0+ 994.0-995.0+1156.0-1157.0+ 1318.0-1319.0+1480.0-1481.0 F: + c Full ms [ 500.00-2000.00] MS KIN4Mpos 65 min 44.07 3 ring (2 isomers) 36.75 KIN4M 90 80 this is an extracted ion chromatogram showing only archaeol and the glyco caldarchaeols 4 ring 70 archaeol 60 50 2 ring 2.65 24.99 40 30 5 ring 1 ring 20 34.45 5.55 10 0 0 10 20 30 40 Time (min) 50 60 positive ion mode 65 minute run time 1.5e8 (A) Nanoarchaeum equitans (positive ion) HO OH HO O O O O HO 3GA 1156.5 m/z 3GA (isomer) 2GA 994.5 m/z 4GA 1318.6 m/z 1GPA 911.9 m/z Monoglycosylarchaeol A 670.4 m/z 5GA (trace) 1480.1 m/z 1GA (several glycosyl isomers) 832.6 m/z 1GPC (trace) 1560.7 m/z 3GPC (trace) 1884.3 m/z 2GPC 1722.5 m/z 0 8.5e6 (B) Nanoarchaeum equitans (negative ion) 3GA 1183.8 m/z M+Fo- Relative Abundance * = unidentified lipid O X O P O OH 3GA (isomer) O O 5GA (trace) 1507.6 m/z M+Fo- Monoglycosylphosphoarchaeol (1GPA) 1GPA 894.1 m/z [M-H]1GPC (trace) 1542.5 m/z [M-H]- 2GA 1021.9 m/z M+Fo- 0 1.8e8 HO 3GPC (trace) 1867.4 m/z [M-H]- 2GPC 1704.3 m/z [M-H]- * 4GA (C) N. equitans/Ignicoccus sp. KIN4/I co-culture (positive ion) * = unidentified lipids 4GA 1345.7 m/z M+Fo- 3GA O O O O O O P O X Monoglycosylphosphocaldarchaeol (1GPC) OH A 2GA 3GA (isomer) 5GA (trace) 1GPA 3GPC (trace) 2GPC 1GPC (trace) 1GA * 0 0 10 * 20 30 * 40 Time (min) Fig. 2 Base peak chromatograms of full-scan (650-2,000 m/z) mass spectra of * 50 N. equitans (a positive ion; b negative ion) and a N. equitans/ignicoccus sp. strain KIN4/I co-culture (c positive ion). Identifications were made on the basis of MSN spectra in both positive and negative ion modes from LC-MS and from direct infusion of total lipid extracts. Peaks are labeled as follows: I-5 number of glycosyl units, G glycolipid, GP phospholipid, A archaeol lipid core, C caldarchaeol lipid core and * unidentified lipid. The dominant lipids are glycolipids with an archaeol core, with smaller amounts of phospholipids which consist of a phosphate group with glycosyl headgroup and either an archaeol or caldarchaeol lipid core. 241.1 Relative Abundance 241 O X O P O OH 894.7 [M-1] O O 731.7 731 200 Relative Abundance (A) 1GPA 33.0 min -H2O 300 400 595.1 500 Da 600 700 800 1543.2 [M-1]- (B) 1GPC 32.6 min HO O O O O 900 O O P O X OH 1381.2 1381 400 600 800 1000 1200 1400 Relative Abundance Da 1705.3 [M-1] (C) 2GPC 41.1 min HO HO HO OH O O O O O O 1543 (alternate) O O P O X OH 1543.0 1543 400 600 800 1000 Da 1200 1400 1600 Fig. 4 Negative ion mode MS/MS spectra of three glycosylbearing phospholipid species observed in the total lipid extracts of both N. equitans (shown above) and Ignicoccus sp. strain KIN4/I. Unlike glycolipids, these phospholipids do not form formate adducts and are directly observed as [M-H]-ions. Spectrum a is identified as a glycosylphosphoarchaeol (at 33 min in Fig. 2), since the 241-Da ion is diagnostic for phosphoinositol lipids and corresponds to the dehydrated glycophosphate headgroup. The 731-Da ion corresponds to an archaeol phosphate ion and is observed in phosphoarchaeol standards with different headgroups. The exact structure of the glycosyl unit attached to the phosphate is unknown since these MSN analyses do not give detailed structural information on the nature of the glycosyl units. Spectra b, c are identified as phosphoglycosyl caldarchaeols with one phosphate and one (at 32.6 min in Fig. 2) or two (at 41.1 min in Fig. 2) glycosyl groups, respectively, attached either via the phosphate group or the glycerol moiety. Co-culture Isotopes phytane biphytane nano -16.1185 -16.0045 igni -16.023 -16.297 igni 77C -15.219 -15.249 igni 90C -42.087 -43.603 igni 95C -18.623 -19.767 Yellowstone National Park ‘Ojo Caliente’ hot spring sample of biomass from a ‘streamer’ community membrane lipids complex distribution of lipids shows diverse population bacterial membrane lipids lipids common to cyanobacteria Data; Fredricks, Summons, Hinrichs TIME lipids common to Aquificales MOLECULAR MASS archaeal APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 0099-2240/97/$04.0010 Jan. 1997, p. 50–56 Vol. 63, No. 1 Copyright q 1997, American Society for Microbiology Vertical Distribution and Phylogenetic Characterization of Marine Planktonic Archaea in the Santa Barbara Channel RAMON MASSANA,1 ALISON E. MURRAY,2 CHRISTINA M. PRESTON,2 AND EDWARD F. DELONG2* Marine Science Institute1 and Department of Ecology, Evolution and Marine Biology,2University of California, Santa Barbara, Santa Barbara, California 93106 Received 1 August 1996/Accepted 11 October 1996 Newly described phylogenetic lineages within the domain Archaea have recently been found to be significant components of marine picoplankton assemblages. To better understand the ecology of these microorganisms, we investigated the relative abundance, distribution, and phylogenetic composition of Archaea in the Santa Barbara Channel. Significant amounts of archaeal rRNA and rDNA (genes coding for rRNA) were detected in all samples analyzed. A Few Cosmopolitan Phylotypes Dominate Planktonic Archaeal Assemblages in Widely Different Oceanic Provinces RAMON MASSANA,1* EDWARD F. DELONG,2 AND CARLOS PEDRO´ S-ALIO´ 1 Institut de Cie`ncies del Mar, CSIC, 08039 Barcelona, Catalunya, Spain,1 and Monterey Bay Aquarium Research Institute, Moss Landing, California 950392 Received 4 October 1999/Accepted 2 February 2000 We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces………… letters to nature Nature 371, 695 - 697 (2002); doi:10.1038/371695a0 High abundance of Archaea in Antarctic marine picoplankton Edward F. DeLong, Ke Ying Wu, Barbara B. Prézelin & Raffael V. M. Jovine ARCHAEA (archaebacteria) constitute one of the three major evolu-tionary lineages of life on Earth13. Previously these prokaryotes were thought to predominate in only a few unusual and disparate niches, characterized by hypersaline, extremely hot, or strictly anoxic conditions47. Recently, novel (uncultivated) phylotypes of Archaea have been detected in coastal8 and subsurface9,10 marine waters, but their abundance, distribution, physiology and ecology remain largely undescribed. Here we report exceptionally high archaeal abundance in frigid marine surface waters of Antarctica. Pelagic Archaea constituted up to 34% of the prokaryotic biomass in coastal Antarctic surface waters, and they were also abundant in a variety of other cold, pelagic marine environments. Because they can make up a significant fraction of picoplankton biomass in the vast habitats encompassed by cold and deep marine waters, these pelagic Archaea represent an unexpectedly abundant component of the Earth's biota. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 0099-2240/98/$04.0010 Mar. 1998, p. 1133–1138 Vol. 64, No. 3 Copyright © 1998, American Society for Microbiology Dibiphytanyl Ether Lipids in Nonthermophilic Crenarchaeotes EDWARD F. DELONG,1* LINDA L. KING,2 RAMON MASSANA,1 HENRY CITTONE,1 ALISON MURRAY,1 CHRISTA SCHLEPER,1 AND STUART G. WAKEHAM2 The Marine Science Institute, University of California, Santa Barbara, California 93106,1 andSkidaway Institute of Oceanography, Savannah, Georgia 314112 Received 30 September 1997/Accepted 23 December 1997 The kingdom Crenarchaeota is now known to include archaea which inhabit a wide variety of low-temperature environments. We report here lipid analyses of nonthermophilic crenarchaeotes, which revealed the presence of cyclic and acyclic dibiphytanylglycerol tetraether lipids. Nonthermophilic crenarchaeotes appear to be a major biological source of tetraether lipids in marine planktonic environments. Parallel Molecular Signatures EUCARYA HO micros poridia plants ciliates slime molds man animals HO fungi BACTERIA green sulfur bacteria OH OH OH OH grampositives protcobacteria R cyanobacteria avobacteria Thermocrinis Aquifex CH2O CHOR CH2OR COOH HO flagellates HO diplomonads ARCHAEA Sulfolobus Desulfurococcus Thermofilum Thermoproteus Pyrobaculum Thermotoga Pyrodictium Pyrococcus Methano- Methanobacterium thermus Archaeoglobus >2.7 Ga Halococcus 1 2 Halobacterium 3 Methanoplanus Methanopyrus 4 Methanococcus Methanospirillum 1 jannaschii Methanosarcina O O 2 igneus OH 3 thermolithotrophicus 4 vanniellii Acid-labile cyclopropyl lipids from Aquificales CH2O CHO CH2OPO3H2 acid-labile cyclopropyl CH2O CHOR CH2OPO3H2 COOH Alkaline methanolysis OK for FAME but Mild Acid Hydrolysis needed to liberate ether lipids