Diether and Tetraether Phospholipids and ... - ACS Publications

Chapter 3

Diether and Tetraether Phospholipids and Glycolipids as Molecular Markers for Archaebacteria (Archaea)

Downloaded by STANFORD UNIV GREEN LIBR on October 5, 2012 | http://pubs.acs.org Publication Date: July 1, 1997 | doi: 10.1021/bk-1997-0671.ch003

Morris Kates Department of Biochemistry, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada

Archaebacteria (Archaea) inhabit extreme environments globally: extreme halophiles in nearly saturated salty locales; methanogens in anoxic environments; and extreme thermophiles or thermoacidophiles at high temperatures or at both high temperatures and low pH. A new group of archaebacteria has recently been found to be highly concentrated and widely distributed in marine bacterioplankton. Archaea are clearly delineatedfromall other organisms by their unique ribosomal RNA (rRNA) sequences, cell wall structures and membrane lipids. The latter are derivedfromdiphytanylglycerol diether (archaeol) and its dimer, dibiphytanyldiglycerol tetraether (caldarchaeol), as lipid cores (hydrophobic moieties) instead of from diacylglycerol diesters. Archaeal phospholipids, glycolipids and archaeol or caldarchaeol lipid cores should be useful molecular markers as aids for identification of archaeal subgroups and in some cases of individual genera, and for determination of their biomass in mixed microbial communities. These molecular markers should be useful as an adjunct to rRNA sequencing for detecting and characterizing new archaeal genera in the environment, such as those recently discovered in oceanic bacterioplankton. Archaea (Archaebacteria) are prokaryotic (non-nucleated) microorganisms that have been assigned separate Domain status, distinct from the two previous Domains, Bacteria and Eukarya (nucleated), but more closely related to the Eukarya than to the Bacteria (7). Archaea, which presumably arose in the Archaean era, are generally known to inhabit extreme environments globally: a) extreme halophiles in saturated or nearly-saturated salty locales (salt flats, salt lakes such as Great Salt Lake, alkaline lakes, and inland seas, such as the Dead Sea, etc.); b) methanogens in anaerobic environments (marshes, rice paddies, wetlands, rumen of herbivores, sewage, marine sediments, deep-ocean hydrothermal vents, etc.); and c) extreme thermophiles and thermoacidophiles in high temperature locales and at both high temperature and low pH, respectively (hot springs such as in Yellowstone National Park, volcanic soils and solfatara, * etc.)(2). Recently, however, Archaea have been detected, in high concentration, in marine bacterioplankton samplesfromthe Pacific Ocean, Antarctica © 1997 American Chemical Society

In Molecular Markers in Environmental Geochemistry; Eganhouse, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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MOLECULAR MARKERS IN ENVIRONMENTAL GEOCHEMISTRY

and Alaska (3,4), showing (hat Archaea are not confined to extreme environmental niches, but are widespread and abundant in the global biota (3-5). Archaea are clearly delineated from all other organisms by their ribosomal RNA (rRNA) sequences, their glycosylated polypeptide and other non-murein (nonBacterial) cell wall structures, and their unique glycerol ether-type membrane lipids (6). The latter (7,8) are derived from the lipid core diphytanylglycerol diether (l)(termed C C -archaeol (9)) and variants such as C^C^- (1A), C^C^- (IB), C^macrocyclic (1C), and hydroxylated (1D,1E) archaeols (Figure 1) and/or its dimer, dibiphytanyldiglycerol tetraether (2) (caldarchaeol (9)) and variants such as calchtoglycerocaldarchaeol (2A)(9,10) with or without cyclopentane rings (2B2E)(Figure 2), instead offromdiacylglycerol diesters as in all other organisms. Note that the term "lipid core" refers to the hydrophobic or lipid portion of membrane lipids. Within the Archaea, the compositions (profiles) of lipid cores (Figures 1 & 2) and phospholipids and glycolipids (Figures 3 to 6) can clearly distinguish between the three subgroups of Archaea: extreme halophiles, methanogens and extreme thermophiles or thermoacidophiles, and also to some extent between genera within each subgroup (Tables I and II) (see below). The present paper, apart from presenting the specific structures of these archaeal polar lipid molecular markers (Figures 1 to 6) and their distribution and taxonomic correlations among the Archaea (Tables I & II) (see below), includes a review of procedures for extraction, isolation and structure determination of polar lipids from halophiles, methanogens and thermophiles and for preparation and analysis of their lipid cores and other derivatives. Methods for extraction of lipids from marine sediments and soil samples, for analysis of both fatty acids and glycerol diethers/tetraethers and for biomass estimation in such samples of mixed microbial communities will also be reviewed. The significance of these archaeal lipid markers to environmental geochemistry and to ongoing environmental research will be discussed, in particular, the recent reports of the identification of archaea in microbial communities in marine bacterioplankton (3,4).


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Extraction and Fractionation of Polar Lipids Procedures for extraction andfractionationof lipidsfromArchaea cells, soil samples, etc., and preparation of lipid core and other lipid markers are summarized in Figure 7. a) From Halophilic Archaea. Total lipids of extreme halophiles with easily broken cell walls (e.g., Halobacterium, Haloferax, Haloarcula species) can be extracted quantitatively by a modified neutral Bligh-Dyer extraction procedure (11,12). For halophile cells with rigid cell walls (e.g. Halococcus species), cell suspensions are subjected to sonication before extraction by the neutral Bligh/Dyer procedure (extraction with cWoroform/methanol/water, 1:2:0.8, v/v; then formation of a biphasic system, chloroform/methano/ water, 1:1:0.9, v/v) (11). The total lipids in the lower chloroform layer are then separated into polar and non-polar lipids by acetone precipitation (11,12). The precipitated polar lipids are analyzed for phospholipids and glycolipids by quantitation of lipid phosphate (lipid-P) and sugars (12), respectively, and are separated into individual components by preparative silicic acid thin-layer chromatography (TLC)(11,12). Alternatively, the total lipids may be fractionated by silicic acid column chromatography into a neutral lipid fraction (elution with chloroform), a glycolipid fraction (elution with chloroform/acetone, 1:1, v/v, then acetone) and a phospholipid fraction (elution with methanol)(72). The glycolipid and phospholipidfractionscan then be separated into individual lipids by preparative TLC (11,12).


3.

Molecular Markers for Archaebacteria (Archaea)

KATES

H

2

3

37

C-0

H-C—O (1)

H 'C0H 2


H,C—O

I I

C20-C2S.

(1AI

H COH 2

C25-C25

(IE)

Figure 1. Structures of archaeol and variant lipid cores (7,8,35-37): (1) diphytanylglycerol (C2o-C o-archaeol) in extreme halophiles and methanogens; (1A) C2o-C25-archaeol and (IB) C 5-C 5-archaeol in haloalkaliphiles and some halococci; (1C) C o-macrocyclic diether in M. jannaschii; (ID & IE) 3-hydroxyarchaeols m Methanosaeta (Methanothrix) and Methanosarcina species. 2

2

2

4


38


CHOR


2

(2A)

R = C6H11O5

(2B) - (2E) R=H or R = C H n 0 6

5

Figure 2. Structures of caldarchaeol and some variant lipid cores (7,8,10,35+37): (2) dibiphytanyldiglycerol tetraether (caldarchaeol) in methanogens and thermophiles; (2A) dibiphytanyl glycerol calditol tetraether (calditoglycerocaldarchaeol) in thermoacidophiles; (2B-2E) cyclized caldarchaeols and calditoglycerocaldarchaeols (1 to 4 cyclopentane rings per chain) in extreme thermophiles and thermoacidophiles, respectively.


KATES

Molecular Markers for Archaebacteria (Archaea)

CH -0-PO-(OH) 2

^H -0-JO-0-CH

2

R-O-C-H

I

R-O-CH2

2

R-O-C-H

H-C-OH

R-O-CH2

H2C-OH

Phosphatidylglycerol (PG)

Phosphatide acid (PA)


2

;H

j:H -O-^O-O-(j:H2

2

2

R-O-C-H

H-C-OH

R-O-CH2

H2C-O-SO3

R-O^C-H

H-C-OH

R-O-CH2

H2C-0-l£0-OCH3

T

Phosphatidylglycerolmethylphosphate (PGP-Me)

Phosphatidylglycerosulfate (PGS)

CH CH R = Phytanyl group = CH [CH(CH )3]3CH(CH ) 3

3

2

2

2

2

Figure 3. Structures of archaeol phospholipids in extreme halophiles (7,8). R —0—CH 2

H0-CH

2

2

|R — 0 — C — H R —O—CH

2

GENUS DGA, R =H, TGA-1, R =P-galp, TGA-2, R =p-glcp S-DGA, R =-S0 -OH S-TGA-1 R =3-S0 '-P-galp S-TeGA, R =3-S0 "-p-galp 2

2

2

2

2

2

3

2

3

R =H R =H R =H R =H R =H R =cc-gay 3

3

3

3

3

3

Haloferax Halobacterium Haloarcula Haloferax Halobacterium Halobacterium

R=phytanyl group:

Figure 4. Structures of some archaeol glycolipids in extreme halophiles (7,8).


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CH2-0-R2 H2C-O-(C40H80) -0-

Diether and Tetraether Phospholipids and ... - ACS Publications

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