Transcript Aim 2 - CLU-IN

Metabolic Interactions
Supporting Effective TCE
Bioremediation under Various
Biogeochemical Conditions
Grant 1R01ES024255-01
Lisa Alvarez-Cohen
UC Berkeley
Technical Background
Anaerobic microbial reductive dechlorination
PCE
TCE
cis-DCE
VC
ETH
Dehalococcoides
mccartyi
Clostridium, Dehalobacter,
Dehalospirillum, Desulfitobacterium,
Desulfomonile, Desulfuromonas,
Sulfurospirillum, Geobacter, etc
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•
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Electron acceptors: chlorinated ethenes
Electron donor: H2
Carbon source: acetate
Some important RDase genes:
pceA, tceA, bvcA and vcrA
(all require corrinoids)
Material exchanges in dechlorinating
communities
• D. mccartyi do not live alone in nature. Important to determine
how environmental changes affect material exchanges in
communities.
Organic Substrate
(lactate/whey/molasses)
CO
2
Hydrogenotrophic
Acetogens
Vitamin B12
Acetate
TCE
Fermenters
H2
Acetate
Microbial consortia ferment
organics to hydrogen, providing
electron donor required for
Dehalococcoides to respire TCE
Methanogens
Vitamin B12
Dehalococcoides mccartyi
???
DMB,
thiamine,
biotin
Ethene
“Microorganisms do not exist in isolation but
form complex ecological interaction webs”
Karoline Faust & Jeroen Raes
Nature Reviews Microbiology 2012 10, 538-550
Stable Isotope Probing of Enrichment growth of
dechlorinating community without external cobalamin
RNA-SIP Fractionation
heavy
light
PF
DvH
cDNA, PCR, 35 cycles
Desulfovibrio vulgaris
Hildenbrough (DvH)
Pelosinus fermentens
(PF)
5
Bin-genomes Recovered from Metagenomic Binning
Veillonellaceae
Dehalococcoides
Coverage (HiTCE)
Desulfovibrio
Sedimentibacter
Spirochaetaceae
Bacteroides
Clostridium
With nearly complete corrinoid biosynthesis pathway
Coverage (HiTCEB12)
6
Pathway compilation of
Selected Genomes in
Groundwater
Enrichment
KEGG mapped Porphyrin and
chlorophyll pathway (B12
generation)
from Dehalococcoides,
Veillonellaceae and Desulfovibrio
genomes derived from
metagenome
Sequence similarity
Tri-Culture of D. mccartyi 195 by corrinoid salvaging
and remodeling in defined tri-culture
Men et al., 2014 Environ. Microbiol. DOI: 10.1111/1462-2920.12500
Aim 3:
Isotopomer Metabolomics
CO, an obligate byproduct from an
imcomplete WoodLjungdahl
Carbon Fixation
Pathway of
D. mccartyi
Zhuang et al., 2014 PNAS 111: 6419–6424
CO accumulation in Dhc195 and
DvH/Dhc195 co-culture
Men et al., (2012) ISME J.
Zhuang et al., 2014 PNAS 111: 6419–6424
CO serves as a potential energy source
for Syntrophomonas wolfei growth
Syntrophomonas Wolfei/Dhc co-cultures
1011
0.10 B
A
20 C
pure S. wolfei
co-culture
control
0.6 µmol
1.4 µmol
109
3.2 µmol
6.4 µmol
8.0 µmol
108
0
5
10
Time (day)
15
20
neg
CO (µmol/bottle)
1010
CO (µmol/bottle)
cell number/bottle
0.08
0.06
0.04
abiotic
live
15
10
5
0.02
0.00
0
0
4
8
12
16
day 0
day 24
time (day)
a) CO effect on S. wolfei growth, b) CO production from S. wolfei growth, c) CO consumption by S. wolfei
day 42
Technical Objectives
Aim 1: Construct TCE-dechlorinating consortia of
fully sequenced organisms and maintain in
chemostats
Aim 2: Identify changes in microbial community
that occur in response to geochemical
perturbations
Aim 3: Elucidate networked interactions in the
consortia that occur in response to geochemical
perturbations
Technical Approach
1) Construct defined consortia
(and inoculate chemostats)
2) Perturb chemostats
(Identify changes in
microbial community)
Defined
consortia
Cell activity &
metabolite
exchange
TCE
Expression array
RMT analysis
Quantitative
correlation
qPCR
3) Apply random matrix theory (RMT) and metabolomics
(Elucidate networked interactions)
Ethene
Aim 1:
Construct TCE-dechlorinating consortia
• Begin with a lactate fermenter and two D. mccartyi strains (with
different reductive dehalogenases)
• Sequentially add
microorganisms that
D. vulgaris Hildenborough
represent
lactate fermentation
homoacetogenic,
Lactate
hydrogenotrophic
methanogenesis
methanogenic and
acetoclastic
methanogenic functions
acetogenesis
CO2 H2 Acetate
D. mccartyi
strains
dechlorination
PCE
VC,
ETH
Aim 1:
Inoculate and Optimize Chemostats
• Inoculate chemostats with defined consortia
D. vulgaris Hildenborough
lactate fermentation
acetogenesis
Lactate
CO2 H2 Acetate
methanogenesis
D. mccartyi
strains
dechlorination
PCE
VC,
ETH
• Then optimize chemostats to
retain all desired functions
Aim 2:
Perturb Chemostats with Geochemical
Stresses
• Changes in pH, salinity, acetate, sulfate, sulfide, iron
species
• Amendments with alternative terminal electron acceptors
qPCR
Steady state reactor
Apply environmental stress
Monitor TCE reduction,
cell growth, changes in
metabolite pool, etc.
Aim 2:
Microarray-based genome and transtricptome
analysis
Cell lysis
Reverse
transcription
RNA isolation
DNA removal
Labeling and
hybridization
ss-cDNA
Cell lysis
DNA isolation
Cells
Purified
DNA/RNA
Labeling and
hybridization
Scanning
BAV1 PCE vs TCE
TCE Expression
10000
1000
Data
Analysis
100
10
10
100
1000
PCE Expression
10000
Aim 2:
Identify Changes in Intercellular Metabolites
Phelan et al., Nature Chemical Biology 2012 8, 26-35
Aim 3:
Map Gene Network and Interactions
Zhou et al., mBio, Sept/Oct 2010, 4.
Aim 3:
Validate Interrelationships
• Use quantitative analysis on targeted
metabolites
– qPCR
– 13C stable isotope labeling
– Targeted metabolomics + GC/MS
qPCR
GC/MS
Overall Project Plan
Aim 1: Construct consortia
● D. mccartyi strains and fermenters
● Methanogens and homoacetogens
● Inoculate chemostats
Aim 2: Identify changes due to stress
● Apply environmental stresses
● Genomic and transcriptomic analysis
● metabolomic analysis
Aim 3: Define networked interactions
● Map gene networks
● Validate identified relationships
● Investigate engineered solutions
YEAR 1
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YEAR 4