University of Washington

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University of Washington GTL VIMSS Applied and
Environmental Microbiology Core
Analysis Updates Desulfovibrio/Methanogen
Syntrophic Co-cultures Sulfate-limited Chemostat
Growth of Desulfovibrio spp. Sulfate-reducing
Field Isolates
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Collaborators
University of Washington Sergey Stolyar Beto
Zuniga Martin Koenneke Institute for Systems
Biology Ger van den Engh ORNL Jizhong
Zhou Zhili He Qiang He Genomatica, San Diego,
CA Steve Van Dien LBNL Terry Hazen Sharon Borglin
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Syntrophic Co-culture of Desulfovibrio with
Methanococcus on lactate without sulfate
Methanococcus
Desulfovibrio
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Syntrophic co-culture of Desulfovibrio
Methanococcus sulfate depleted medium
CO2
Lactate
Interspecies Hydrogen-transfer
H2 H2 CH4
Acetate CO2
Methanococcus
Desulfovibrio
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Effect of hydrogen partial pressure on free energy
Desulfovibrio fermentation of lactate
CH3CHOHCO2- (lactate) H2O CH3COO-
(acetate) CO2 2 H2
at 10-4 atm H2
at 1 atm H2
DG0 -8.8 2 RT ln 10-4 - 54 kJ/mol
DG0 -8.8 kJ/mol
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NaCl Growth Curves on B3 media _at_ 30C
Abs _at_ 600nm
Hours
Abs _at_ 600nm
Hours
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MgSO4 Growth Curves on B3 media _at_ 30C
Abs _at_ 600nm
Hours
Abs _at_ 600nm
Hours
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Syntrophic co-culture Growth and Metabolites
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Adaptation to Syntrophic GrowthTypical growth
curves for several consecutive passages of D.
vulgaris and M. maripaludis co-cultures
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Growth curves of the six parallel batch
co-cultures of D. vulgaris and M. maripaludis C4
(8th passage)
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Initial Microarray Analyses
Batch syntrophic and Chemostat cultures harvested
for microarray analysis at ORNL Microarray
70mers for all ORFs of D. vulgaris and M.
maripaludis genomes - 3574 and 1766
oligonucleotides, respectively.
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Co-culture/24 hr Sulfate-limited Chemostat Dsv.
vulgaris H (Jul. 14, 2004) Gene ID
Co-culture/24 hr Functional annotation ORF03736
4.735 7.242 minor capsid protein C
(phage) ORF01941 4.623 3.970 hypothetical
protein ORF04118 3.087 3.516 hypothetical
protein ORF04140 2.547 3.302 Na/H antiporter
NhaC (nhaC) ORF03712 1.445 2.474 tail/DNA
circulation protein, putative (phage) ORF02762 1.
418 2.205 hypothetical protein ORF00660 1.293 2.
396 glucokinase, putative ORF02808 1.157 2.033
methyl-accepting chemotaxis protein,
putative ORF01324 0.000 0.090 Heptosyltransferas
e family ORF02917 0.000 0.090 outer membrane
protein, putative ORF02422 0.000 0.086 hypotheti
cal protein ORF05495 0.000 0.085 selenocysteine-
specific translation elongation factor
(selB) ORF03314 0.000 0.080 chemotaxis MotB
protein, putative ORF04420 0.000 0.079 cytochrom
e d ubiquinol oxidase, subunit I, POINT MUTATION
(cydA) ORF05549 0.000 0.078 hmc operon protein
4 ORF05761 0.000 0.077 hypothetical
protein ORF03449 0.000 0.076 hypothetical
protein ORF00252 0.000 0.073 histidinol
dehydrogenase (hisD) ORF01604 0.000 0.063 iron-s
ulfur cluster-binding protein ORF01996 0.000 0.06
2 hypothetical protein ORF00134 0.000 0.058 que
uine tRNA-ribosyltransferase (tgt) ORF01744 0.000
0.051 hypothetical protein ORF04935 0.000 0.048
thiosulfate reductase, putative ORF00151 0.000
0.029 cell cycle histidine kinase CckA,
putative ORF01870 0.000 0.023 periplasmic Fe
hydrogenase, small subunit (hydB) ORF04043 0.000
0.000 oxygen-independent coproporphyrinogen III
oxidase, putative ORF02953 0.000 0.000 hypotheti
cal protein ORF03023 0.000 0.000 ABC
transporter, permease protein ORF02238 0.000 0.00
0 conserved hypothetical protein ORF01057 0.000
0.000 conserved hypothetical protein
TIGR00159 ORF01861 0.000 0.000 thiH protein
(thiH) ORF02661 0.000 0.000 single-strand
binding protein (ssb)
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Flux Balance Model for Analysis Desulfovibrio
vulgaris Metabolism
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Flux Balance Model for Analysis Methanococcus
maripaludis metabolism
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36 24 hr chemostat cultures of Dsv. vulgaris
Hildenborough (Jul. 14, 2004) Elevated Expression
_at_ 36 hr Gene ID 36h/24
Functional annotation ORF01292 6.388991465 phos
phoglucomutase, alpha-D-glucose
phosphate-specific ORF04778 3.733083017 hypotheti
cal protein ORF00693 3.33891762 cobalt ABC
transporter, ATP-binding protein,
putative ORF00130 3.216054383 sodium/alanine
symporter family protein ORF05364 3.134338102 Ec
h hydrogenase, subunit EchD, putative ORF05417 2.
854857655 hypothetical protein ORF04127 2.781281
186 tolB protein, putative ORF00696 2.693513162
cobalamin biosynthesis protein CbiM
(cbiM) ORF04738 2.432622697 hypothetical
protein ORF05423 2.42787129 chorismate
mutase/prephenate dehydratase ORF04347 2.39428697
3 flagellar basal body-associated protein,
putative ORF00729 2.322703947 inner membrane
protein, 60 kDa (yidC) ORF05011 2.280328617 cons
erved hypothetical protein ORF04167 2.255508735
hypothetical protein ORF03494 2.244561416 membra
ne protein, putative ORF01916 2.241020851 dimeth
yladenosine transferase (ksgA) ORF00937 2.1796069
57 ribosomal protein L32 (rpmF) ORF00739 2.13279
8695 3'- 5' exonuclease domain protein,
putative ORF04923 2.083262494 oligopeptide/dipep
tide ABC transporter, permease protein ORF05424 2.
0594284 3-phosphoshikimate 1-carboxyvinyltransfer
ase (aroA) ORF05316 2.055521362 dissimilatory
sulfite reductase B ORF05763 2.012862065 transcr
iptional regulator, putative ORF03058 2.001378273
hypothetical protein
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36 24 hr chemostat cultures of Dsv. vulgaris
Hildenborough (Jul. 14, 2004) Reduced Expression
_at_ 36 hr Gene ID 36h/24
Functional annotation ORF01206 0.297151263 cell
-division initiation protein, putative ORF01514 0.
295726665 molybdenum cofactor biosynthesis
family protein, putative ORF01324 0.29531482 Hept
osyltransferase family ORF02969 0.281542943 hydr
ogenase, putative ORF04191 0.280952182 hypotheti
cal protein ORF05333 0.277579617 potassium
uptake protein, TrkH family ORF04287 0.273104734
DNA-binding domain protein, excisionase
family ORF00837 0.269882625 transcriptional
regulator, Cro/CI family ORF02953 0.254067105 hy
pothetical protein ORF02808 0.251159356 methyl-a
ccepting chemotaxis protein, putative ORF05576 0.
249838232 hypothetical protein ORF02586 0.249519
915 hypothetical protein ORF02624 0.243904696 e
ndoribonuclease L-PSP, putative ORF04537 0.237363
472 proto-oncogene ski, putative ORF00568 0.2340
59704 DAK1 domain protein ORF05177 0.233716245
conserved hypothetical protein ORF00660 0.2125776
97 glucokinase, putative ORF02229 0.211697026 h
ypothetical protein ORF04140 0.204423792 Na/H
antiporter NhaC (nhaC) ORF03131 0.18762322 cell
division protein FtsZ (ftsz) ORF04091 0.184513314
integral membrane protein, putative ORF01941 0.
174340252 hypothetical protein ORF04564 0.161261
121 hypothetical protein ORF00076 0.148084514 m
embrane protein, putative ORF02691 0.144200061 R
NA methyltransferase, TrmH family ORF02130 0.1178
51129 periplasmic NiFe hydrogenase, large
subunit, isozyme 1 ORF02019 0.109922136 cold
shock domain family protein
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Sparged Chemostat Growth of Dsv. vulgaris
on Lactate minus Sulfate
Hydrogen
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Reconfiguration of Biofilm Reactor Systems
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Sample Site
FRC Enrichments Isolations
Background - FB609-01-00
Area 1 - FB060-01-00 Area 2 - FB052-01-00 Area 3
- FB056-01-17 Area 2 - FWB203-03d-04
Area 3

S-3 Ponds



Area 1
Area 2
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Sample Cores
Sample core in glove bag
Sample canister in glove bag
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Enrichments - April 2002 Sample Set

• Substrates
• Lactate (16mM)
• Acetate (16mM)
• Pyruvate (16mM)
• Propionate (16mM)
• H2CO2

Site Sample depth Area 1 FB060-01-00
204 223 Area 2 FB052-01-00 290 295
Area 3 FB056-01-17 175 193
Background FB609-01-00 220 230
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Micrograph of FRC isolate growing on H2, CO2,
acetate, and SO4
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Total Zinc in Lake DePue, Illinois, USA
Background concentrations
Mississippi River
Illinois River
Backwater lakes
Site 1 24960 mg/kg
113 mg/kg
483/mg/kg
436 mg/kg
Site 4 4475 mg/kg
Site 2 11602 mg/kg
Site 5 3350 mg/kg
Site 3 6530 mg/kg
0
500
1000
scale in meters
Map source United States Geological Survey, 7.5
minute map series, DePue, Illinois Quadrangle,
1966, photorevised 1979.
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Dissolved Zn versus PLP-biomass
300
250
200
PLP (nmole P/dry g)
150
100
50
0
0.0
2.0
4.0
6.0
8.0
Data variability represents the average of the
deviation from the mean for 2 discreet depth
intervals collected from 3 core samples (6
samples total).
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Sulfate Reduction in Lake DePue compared to other
sediments
(Missing non-critical slide)
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Lake DePue Isolates
Samples - Lake DePue Zinc contaminated sediment
Sampling, Oct. 2002
DP1 isolate
Enrichments on lactate fresh water sediment media
to isolate Desulfovibrio-like microbes. Isolated
using anaerobic roll tube method with FeSO4
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PFGE - Restriction Digestion of Lake DePue
Isolates Genomic DNA
1) Size markers (Saccharomyces cerevisiae) 2,10)
D. vulgaris Hildenborough (DvH) (undigested
genomic DNA) 3, 11) L. DePue isolates DP4 and
DP8 undigested genomic DNA 4) DvH DNA digested
with I-CeuI 5,6- DP4 and DP8 DNA digested with
I-CeuI 7) DvH gDNA digested with NotI 8,9) DP4
and DP8 DNA digested with NotI Arrows indicate
extrachromosomal (plasmid) DNA red diamonds
indicate I CeuI fragments of DvH
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Comparative Genomic DNA hybridization
D. vulgaris Desulfovibrio DP4
Desulfovibrio PT2 Hildenborough Similarity
16S rRNA 100 gt99 95 Similarity (
hybridization trends) 100 90
60 Potential deletion regions
No Regions 1-7 Regions 5
6 Hybridization signals of most plasmid
genes in both Desulfovibrio DP4 and PT2 were
stronger than those in D. vulgaris Hidenborough,
suggesting possible loss of plasmid from D.
vulgaris hildenborough laboratory strain.
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D. vulgaris Hildenborough chromosome map and gene
clusters deleted in the Dp4 isolate genome
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GeneID DP4/HI Functional annotations from
TIGR Region 1 (44 genes) ORF00763 0.20 aden
ine specific DNA methyltransferase,
putative ORF00765 0.38 tail fiber assembly
protein, putative ORF00766 0.29 tail fiber
protein, putative ORF00771 0.23 hypothetical
protein ORF00774 0.15 baseplate assembly
protein, putative ORF00777 0.13 baseplate
assembly protein, putative ORF00778 0.12 hypothe
tical protein ORF00779 0.18 hypothetical
protein ORF00781 0.15 tail tape measure
protein, FRAMESHIFT ORF00784 0.13 hypothetical
protein ORF00786 0.35 ATPase domain
protein ORF00788 0.14 hypothetical
protein ORF00790 0.16 hypothetical
protein ORF00794 0.21 hypothetical
protein ORF00795 0.23 virion morphogenesis
protein ORF00796 0.15 conserved hypothetical
protein ORF00797 0.12 hypothetical
protein ORF00798 0.16 hypothetical
protein ORF00800 0.17 conserved hypothetical
protein ORF00801 1.14 virion morphogenesis
protein ORF00802 0.09 conserved hypothetical
protein ORF00803 0.26 hypothetical
protein ORF00805 0.33 portal protein,
putative ORF00807 0.10 conserved domain
protein ORF00810 0.12 hypothetical
protein ORF00811 0.46 hypothetical
protein ORF00812 0.24 lipoprotein,
putative ORF00815 0.11 lysozyme,
putative ORF00816 0.10 conserved hypothetical
protein ORF00817 0.09 DNA-binding domain
protein ORF00818 0.08 hypothetical
protein ORF00819 0.13 conserved hypothetical
protein ORF00820 0.25 hypothetical
protein ORF00822 0.14 DNA-binding protein HU,
beta subunit (hupB) ORF00823 0.10 conserved
domain protein ORF00824 0.12 host-nuclease
inhibitor protein Gam, putative ORF00826 0.12 hy
pothetical protein ORF00827 0.11 hypothetical
protein ORF00829 0.09 bacteriophage DNA
transposition B protein, putati ORF00830 0.11 bac
teriophage transposase A protein,
putative ORF00832 0.08 hypothetical
protein ORF00833 0.14 transcriptional
regulator, putative ORF00836 0.11 hypothetical
protein ORF00837 0.28 transcriptional
regulator, Cro/CI family
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Institute for Systems Biology Ger van den Engh
Flow Cytometry-assisted Cloning of Specific
Sequence Motifs from Complex 16S ribosomal RNA
Gene Libraries Nielsen, J.L., A. Schramm, A.E.
Bernhard, G.J. van den Engh, and D.A. Stahl Appl.
Environ. Microbiol. (accepted)
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Critical path forward and group interaction

• I. Co-culture (in association with ORNL and
  LBNL)
• Priority
• Continued expression analysis of Dsv. vulgaris
  H./M. maripaludis co-cultures
• Expression analysis of sulfate-minus purged
  chemostat Dsv. vulgaris H.
• EM characterization of co-culture ultrastructure
  (Yuri Gorby)
• Secondary
• Characterize expression changes associated with
  adaptation
• Pair Dsv. vulgaris with hydrogenotrophic
  methanogen of higher Ks for H2
• Pair M. maripaludis with related Desulfovibrio
  species (strain PT2, Lake Depue Isolates)
• Characterize response of Co-cultures to stress
  (O2, NaCl,)
• II. Biofilm Growth Characterization (in
  association with ORNL and LBNL)
• Priority
• Establish criteria to monitor biofilm growth for
  harvesting under controlled conditions
• Harvest Biofilm for microarray analysis

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Critical path forward and group interaction

• III. Isolation and Characterization of Sulfate
  Reducing Microorganisms from FRC and
  Metals-impacted Field Sites (in association with
  ORNL and LBNL)
• Priority
• Comparative stress response of Dsv. vulgaris H.
  and closely related phage-less Lake Depue
  desulfovibrio isolates
• Complete sequence analysis of 16S rRNA and
  dissimilatory sulfite reductase genes of FRC
  isolates.
• IV. FACS-cloning Recovery of Conserved Gene
  Families by Hybridization (in possible
  association with Diversa?)
• Priority
• Demonstrate recovery of dissimilatory reductase
  genes from environmental shotgun libraries

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Critical path forward and group interaction

• V. Initiate Discussion
• Stress Experiments
• Co-cultures
• Sulfate-limited/minus Chemostat cultures
• Proteome/Metabolome analysis
• Co-cultures
• Mutant constructs based on Co-culture expression
  data
• VI. Personnel Changes
• Ongoing Search for Vice-Beto position
• New postdoctoral hire
• Kristina Stredwick (Lenski laboratory) to join
  laboratory in fall