Rapid Lipid Biomarker Analysis for Quantitative Assessment of Microbial Community Composition and Activity David C. White, Cory Lytle, Aaron Peacock, Yun-Juan Chang, Jonas S. Almeida, Ying Dong Gan, Institute for Applied Microbiology, 10515 Research Drive, Suite 300, Knoxville, TN,37932-2575, University of Tennessee
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David C. White, Cory Lytle, Aaron Peacock, Yun-Juan Chang, Jonas S. Almeida, Ying Dong Gan,
Rapid Lipid Biomarker Analysis for Quantitative Assessment of Microbial Community Composition and Activity. David C. White, Cory Lytle, Aaron Peacock, Yun-Juan Chang, Jonas S. Almeida, Ying Dong Gan, Institute for Applied Microbiology, 10515 Research Drive, Suite 300, - PowerPoint PPT Presentation
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Rapid Lipid Biomarker Analysis for Quantitative Assessment of Microbial Community Composition and Activity
David C. White, Cory Lytle, Aaron Peacock, Yun-Juan Chang, Jonas S. Almeida, Ying Dong Gan,
Institute for Applied Microbiology, 10515 Research Drive, Suite 300,Knoxville, TN,37932-2575,
University of Tennessee
In-situ Microbial Community Assessment
What do you want to know? Characterization of the microbial community: 1. Viable and Total biomass ( < 0.1% culturable &
VBNC ) 2. Community Composition
General + proportions of clades Specific organisms (? Pathogens)
3. Physiological/Nutritional Status ~ Evidence for
4 Metabolic Activities (Genes +Enzymes + Action)
5.Community Interactions & Communications
In-situ Microbial Community Assessment
Classical Plate Count < 1.0 to 0.1% of community, takes days, lose community interactions & Physiology
Two Biomarker Methods: DNA: Recover from surface, Amplify with PCR
using rDNA primers , Separate with denaturing gradient gel electrophoresis (DGGE), sequence for identification and phylogenetic relationship. Great specificity
Lipids: Extract, concentrate, structural analysisQuantitative, Insight into: viable biomass, community composition,Nutritional-physiological status, evidence for metabolic activity
Cathedral from a Brick Predict impact of Cr contamination (from 50-200,000 ppm) on soil microbial community by artificial neural network (ANN) analysis
DNA is “non compressible” ~ perfect code not so influencedBy microniche conditions as cell membranes PLFA is compressible as contains physiological status input Contains “holistic’ information & responds to perturbations Predict it is a Cathedral or a Prison : DNA a perfect brick PLFA a non-linear mixture of bricks and a window
Signature Lipid Biomarker Analysis
Detection of Specific genes or rDNA
1. Recover DNA from samples (often aqueous of lipid extract is best)
2. Amplify with PCR using rDNA eubacterial primers
3. Separate Amplicons with Denaturating Gel Gradient Electrophoresis (DGGE)
4. Isolate Bands, 5. Sequence and match with rDNA database 6. Phylogenetic analysis
DGGE analysis of bacterial communities in sediment samples. Amplified product was separated on a gradient of 20%-65% denaturant
*& Na+,Mg+
+,Cl-,SO4
--,K+,
Bands Phylogenetic group
Metal Metabolism / Metal Transport Properties Associated with Group
A Arthrobacter Plasmid-borne heavy metal resistance
B Bacillus Known resistance to metals incl. Hg, Cd, Pb, Zn, etc.
C Vibrio Heavy metal resistant (via membrane permeability and transport)
D Shewanella Facultative anaerobes (anaerobic respiration utilizes Fe3+, Mn4+, U6+)
E Pseudomonas Metal efflux genes on both plasmid and chromosome (commonly detected at metal contaminated sites); also ability to reduce metal
F Marinomonas As above (Pseudomonas)
G Pedomicrobium Metal (Fe3+, Mn4+) oxidizers
Table: Identification of sequences derived from DGGE bands
Umtra DSR group A
Umtra DSR group B
Umtra DSR group C
Umtra DSR group D
Umtra DSR group E
Umtra DSR group F
Umtra DSR group G
Umtra DSR group H
10071
90 79
71
50
50
91
75
55
75
84
92
94100
100
77
100
8897
100
100 99
96 81
67100
100
96
100
97
100
100
94
94
99 100
100
100
100
81
81
100
75
LIPID Biomarker Analysis
1. Intact Membranes essential for Earth-based life
2. Membranes contain Phospholipids
3. Phospholipids have a rapid turnover from endogenous phospholipases .
4. Sufficiently complex to provide biomarkers for viable biomass, community composition, nutritional/physiological status
5. Analysis with extraction provides concentration & purification
6. Structure identifiable by Electrospray Ionization Mass Spectrometry at attomoles/uL (near single bacterial cell)7. Surface localization, high concentration ideal for organic
1. Aerobic microniche/high redox potential.~ high respiratory benzoquinone/PLFA ratio, high proportions of Actinomycetes, and low levels of i15:0/a15:0 (< 0.1) characteristic of Gram-positive Micrococci type bacteria, Sphinganine from Sphingomonas 2. Anaerobic microniches ~high plasmalogen/PLFA ratios (plasmalogens are characteristic Clostridia), the isoprenoid ether lipids of the methanogenic Archae.
3. Microeukaryote predation ~ high proportions of phospholipid polyenoic fatty acids in phosphatidylcholine (PC) and cardiolipin (CL). Decrease Viable biomass (total PLFA) 4. Cell lysis ~ high diglyceride/PLFA ratio.
Signature Lipid Biomarker Analysis
Microniche Properties from Lipids
5. Microniches with carbon & terminal electron acceptors with limiting N or Trace growth factors ~ high ( > 0.2) poly β-hydroxyalkonate (PHA)/PLFA ratios
6. Microniches with suboptimal growth conditions (low water activity, nutrients or trace components) ~ high ( > 1) cyclopropane to monoenoic fatty acid ratios in the PG and PE, as well as greater ratios of cardiolipin (CL) to PG ratios.
7. Inadequate bioavailable phosphate ~ high lipid ornithine levels
8. Low pH ~ high lysyl esters of phosphatidyl glycerol (PG) in Gram-positive Micrococci.
9. Toxic exposure ~ high Trans/Cis monoenoic PLFA
Signature Lipid Biomarker Analysis
Phospholipid Fatty Acid [PLFA] Biomarker Analysis = Single most quantitative, comprehensive insight into in-situ microbial community
*Macnaughton, S. J., T. L. Jenkins, M. H. Wimpee, M. R. Cormier, and D. C. White. 1997. Rapid extraction of lipid biomarkers from pure culture and environmental samples using pressurized accelerated hot solvent extraction. J. Microbial Methods 31: 19-27(1997)
Feasibility of “Flash” Extraction
ASE vs B&D solvent extraction*
Bacteria = B&D, no distortionFungal Spores = 2 x B&D Bacterial Spores = 3 x B&D Eukaryotic = 3 x polyenoic FA
PCA 2 Analysis of Forest Community Soil total PLFAP
CA
1
PCA Analysis Sugar Maple-Basswood Black Oak- White Oak Sugar Maple- Red Oak
Figure 1. DGGE analysis bacterial community in water and shrimp gut samples. Amplified 16S rDNAs were separated on a gradient of 20% to 65% denaturant.
Wat
er 8
31
Wat
er 8
17
Sta
ndar
d
For
e gu
t
Hin
d gu
t
Water changed composition between Aug 17 & 31st, much > diversity than shrimp gut, Fore gut less diverse than Hind gut.
Major bands have been RecoveredFor sequencing& Phylogenetic analysis
Microbial Community in Water (W), Fore Gut (F), Hind Gut (H)
W F H W F H W F H W F H W F H
0%
20%
40%
60%
80%
100%
8020
1
8020
1F
8020
1H
8030
1
8030
1F
8030
1H
8100
1
8100
1F
8100
1H
8230
1
8230
1F
8230
1H
8310
1
8310
1F
8310
1H
Monos
Bmonos
TBSats
MBSats
NSats
Microbial Viable Biomass: Water (W), Fore Gut (F), Hind Gut (H)
W F H W F H W F H W F H W F H
Biomass PLFA
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
80201
80201F
80201H
80301
80301F
80301H
81001
81001F
81001H
82301
82301F
82301H
83101
83101F
83101H
pm
ol/g
Note Log scale
Shrimp In Mariculture Water & Gut Microbial Community
Shifts Gut & Water Microbiota in 52 days of growth [pathogen-controlled shrimp outgrowth in a closed system, can be solar heated] • Water microbial biomass~same, Algal and Microeukaryotes decrease • Desulfobacter increase Desulfovibrio slight decrease • Gram-negative bacteria increase then decrease • Gut Community very different from water
• DGGE shows Hepatopancreas Mycobacteria, Propionobacteria, SRB and algae (chloroplast > BIOMASS THAN WATER
• DGGE shows Hind Gut Vibrio exclusively less diverse community• Gut 2-order of magnitude > viable microbial biomass than water
• Gut and Water different PLFA from Shrimp food
Problem: Rapid Non-invasive Detection of Infection or Metabolic stress for Emergency room Triage