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Intact Polar Lipids in the Environment
MOG April 21, 2011 Kim Popendorf
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Intact Polar Lipids:
• Structure • Diversity • Biogeochemical significance • Lipid analytical methods • Studying microbial lipid sources • Other applications of membrane
lipids
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Basic structure of Intact Polar Lipids (IPLs)
intact polar lipid
lipid bi‐layer cell membrane
cell
‘head group’
‘faBy acids’ glycerol
IP‐DAG Made by
prokaryotes & eukaryotes
IP‐AEG IP‐DEG Made by archaea
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Structure: FaBy acids
• Major defining features: – carbon chain length – unsaturations
• Define the fluidity of membrane – Length and unsaturation varies with
• Microbial source • Temperature • Pressure (ie depth)
• Common range of FAs: – C14 to C24 – Most abundant in ocean are C16 & C18 – Even c#’s dominate (acetogenic) – Odd carbon chains usually from
bacteria • Analysis of FAs by GC-FID, -MS, -IRMS • Specific FAs used as biomarkers for
microbes
Examples of fatty acids:
C18:3
C14:0
Popendorf unpublished data
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Structure: FaBy acids
Van Mooy & Fredricks, GCA 2010
Diversity of IP-DAGs in the South Pacific euphotic zone
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Structure: Headgroups
• Intact polar lipids = headgroup+fatty acid • Headgroup bond to glycerol is labile => IPLs represent live cells • Analysis of intact polar lipids (headgroup+fatty acids) by HPLC-MS
Major headgroups for marine microbes:
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Importance for biogeochemical cycles:
• Membrane lipids compose 11-23% of planktonic carbon (Wakeham et al. DSR 1997)
• Phospholipids can be 1-28% of the cellular phosphate needs (Van Mooy et al. PNAS 2006)
• Substantial and variable cellular nutrient requirement
Contains P Contains C Contains N, no P
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Idea of membrane lipid substitution: • A lot of cellular demands for nutrients:
• As an adaptation to phosphate stress, microbes can substitute non-phospholipids for phospholipids in their membranes
• Proposed in 1990’s by Christoph Benning, followed up by studies in the environment & cultures by Ben Van Mooy 2000’s
DIP
DIN
Cellular P
Cellular N
Lipids DNA Proteins
DON PON
DOP POP
Phosphate replete: Phosphate deplete:
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Van Mooy et al. Nature 2009
Across different environments, P‐lipid synthesis is variable
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SubsZtuZon occurs rapidly in culture Thalassiosira pseudonana
MarZn, Van Mooy, Heithoff, Dyhrman ISME Journal 2010 (modified with colored lines)
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Van Mooy et al. Nature 2009
When faced with P‐stress, adjust membrane composiZon
Ability (may be) limited to specific groups of microbes! 11
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What can lipids tell us about what microbes are present?
PG: associated with prokaryotes, chloroplast memb. and heterotrophic bacteria
PE: Associated with heterotrophic bacteria
PC: Associated with eukaryotic phytoplankton & with het bac
Glycolipids: Associated with prok., mostly phytoplankton
Betaine lipids: Associated with eukaryotes
SQDG: Associated with chloroplast membrane
Phospholipids: Associated with prokaryotes & eukaryotes
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Lipid extracZon one method: Bligh and Dyer solvent extracZon
Bligh & Dyer, Canadian Journal of Biochemistry and Physiology 1959
One phase: Aqueous (eg water based buffer) + organic solvents (eg methanol & dichloromethane)
RaZo: Water:MeOH:DCM 0.8:2:1
Filter with parZculate maBer, or sediment sample, Zssue sample, etc
Sonicate, vortex, or otherwise break up cellular material
Separate phases:
Aqueous phase (water + methanol)
Organic phase (dichloromethane) ‐> Lipids<‐
RaZo: Water:MeOH:DCM 1.8:2:2
Extract lipids in dichloromethane or chloroform Use a change in raZo of solvents to go from one phase to two
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Lipid analysis:
HPLC- ESI – MS High pressure Electrospray Mass Liquid chromatography Ionization Spectrometry
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HPLC separaZon by headgroup Solvent gradient + column selected to separate IPLs by headgroup, roughly less polar to more polar
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Electrospray IonizaZon
Ion Transfer Tube to MS ESI Needle +/‐ 3 kV
Solvent evapora,on and Ion desolva,on
Inlet from HPLC
“soj ionizaZon” does not break labile headgroup bond *Does not change ionizaBon of molecules!* For IP‐DAGs not naturally ions (MGDG, DGDG, SQDG, PG) use ion adducts in HPLC eluent 16
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QOO QO Q1 Hyperquad
Q3 Hyperquad
Q2 Collision Cell
DetecZon System
Ion Source Interface
Ion Transfer Tube Mass Analyzer
Mass Spectrometry example of triple quadropole system
Quadropole 1: detect masses Quadropole 2: fragment ions Quadropole 3: detect fragment masses
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HPLC‐MS lipid analysis
In environmental samples, a large diversity of molecules exist for each headgroup class
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Lipid idenZficaZon: relies on characterisZc fragmentaZon of the headgroup
MGDG Loss of headgroup gives constant neutral loss=mass of headgroup (for MGDG cnl=197)
MS MS2
PC Loss of headgroup gives product ion= mass of headgroup (for PC product ion=184)
MS MS2
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718
732
690
716
746
285
C14:0
311
C16:1
285
C14:0
309
C16:2
285
C14:0
309
C16:2
285
C14:0
339
C18:1
313
C16:0
Base peak molecular ions for MGDG
ms2 ms2
325
C17:1
257
C12:0
285
C14:0
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Examples of studies with lipids: In the environment, who makes which lipid?
• Stable isotope tracing
• Cell sorting flow cytometry
• Targeted incubations Filter seawater to remove grazers, incubate in the dark to select for heterotrophs
13C‐labeled bicarbonate
13C‐labeled lipids made by autotrophs
13C‐labeled glucose
13C‐labeled lipids made by heterotrophs
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10 15 20 25 30 35 RetenZon Time (min)
0
20 30 40 50 60 70 80 90 100
RelaZve Abu
ndance
10
MGDG
Int. Std.
DGTS PG
DGTA PE
PC
DGCC
SQDG
Methods: IncubaBon condiBon: Light Dark
Substrate addiBon:
13C‐bicarbonate
13C‐glucose
BL photoautotrophs
GL
BD control
GD osmotrophic heterotrophs
Separate IPLs by prep‐HPLC
Transesterify to faBy acids
Measure with GC‐IRMS
FaHy acid methyl esters
Abundance
Enrichment
0
10
20
30
40
50
C14 C15 C16 unid'ed
"C17"
C18 C20 C22 C24
% a
bu
nd
an
ce o
f fa
tty a
cid
-50
0
50
100
150
200
250
300
C14 C15 C16 unid'ed
"C17"
C18 C20 C22 C24
fatty acid chain length
!1
3C
(‰
) r
ela
tive t
o c
on
tro
l
Examples of studies with lipids: In the environment, who makes which lipid?
Stable Isotope tracing
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In the Sargasso Sea: SQDG and DGTS made by photoautotrophs PG made by heterotrophs
Examples of studies with lipids: In the environment, who makes which lipid?
Stable Isotope tracing
Popendorf et al. Org. Geochem. submiBed
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Examples of studies with lipids: In the environment, who makes which lipid? Cell sorZng flow cytometry in the Sargasso Sea
Popendorf et al. Org. Geochem. submiBed
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Examples of studies with lipids: In the environment, who makes which lipid?
Targeted incubaZons 90% filtered seawater, 10% whole seawater, in the dark
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ApplicaZons of membrane lipid studies: Phospholipid production as a measure of biomass production • Membrane production is obligate with cell growth
• Use addition of radioactive phosphate (33PO4) to trace production of phospholipids in a timed incubation
References: White L&O 1977 Fuhrman & Azam 1982 Van Mooy BGS 2008
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ApplicaZons of membrane lipid studies: Phospholipid producZon rate as a measure of biomass producZon
Van Mooy & Rappé ASLO presentaZon 2008
Van Mooy et al. Biogeoscienses 2008
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ApplicaZons of membrane lipid studies: Membrane lipids as chemical signals
Vardi et al. Science 2009 A glycosphingolipid can induce programmed cell death in diatoms (E.hux)
Glycosphingolipid producZon is induced by viruses ‐> may play a key role in ending diatom blooms ‐> thus a role in carbon flux
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