# Presentation: 236302

## 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

## Collaborators

- 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

## Methanococcus

*Methanococcus*

*Desulfovibrio*

- Syntrophic Co-culture of *Desulfovibrio*
- with *Methanococcus* on lactate without sulfate

## Syntrophic co-culture of Desulfovibrio & Methanococcus

- Syntrophic co-culture of *Desulfovibrio* & *Methanococcus*
*sulfate depleted medium*

**CO****2**

***Methanococcus***

**Lactate**

***Interspecies Hydrogen-transfer***

**H****2**** ****	****H****2**** ****	****  ****CH****4**

**Acetate + CO****2**

***Desulfovibrio***

## Effect of hydrogen partial pressure on free energy

- at 1 atm H2:

- *G**0****’****** ***=  _-8.8  kJ/mol_

- at 10-4 atm H2:

- *G**0****’******	***=  -8.8 + 2 *RT* ln [10-4]
- 	=  _- 54 kJ/mol_

*Desulfovibrio* fermentation of lactate

- CH3CHOHCO2- (lactate) + H2O              CH3COO- (acetate) + CO2 + 2 H2

## NaCl Growth Curves on B3 media @ 30 ̊C

**Hours**

**Hours**

**Abs @ 600nm**

**Abs @ 600nm**

## MgSO4 Growth Curves on B3 media @ 30 ̊C

- MgSO4 Growth Curves on B3 media @ 30 ̊C

**Hours**

**Hours**

**Abs @ 600nm**

**Abs @ 600nm**

## Syntrophic co-culture:  Growth and Metabolites

## Adaptation to Syntrophic GrowthTypical growth curves for several consecutive passages of D. vulgaris and M. maripaludis co-cultures

- Adaptation to Syntrophic GrowthTypical growth curves for several consecutive passages of *D. vulgaris* and *M. maripaludis* co-cultures

## Growth curves of the six parallel batch co-cultures of D. vulgaris and M. maripaludis C4 (8th passage)

## 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.

## 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		Heptosyltransferase family
- ORF02917	0.000	0.090		outer membrane protein, putative
- ORF02422	0.000	0.086		hypothetical 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		cytochrome 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-sulfur cluster-binding protein
- ORF01996	0.000	0.062		hypothetical protein
- ORF00134	0.000	0.058		queuine 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		hypothetical protein
- ORF03023	0.000	0.000		ABC transporter, permease protein
- ORF02238	0.000	0.000		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)

## Flux Balance Model for Analysis Desulfovibrio vulgaris Metabolism

- Flux Balance Model for Analysis *Desulfovibrio vulgaris *Metabolism

## Flux Balance Model for Analysis Methanococcus maripaludis metabolism

- Flux Balance Model for Analysis *Methanococcus maripaludis *metabolism

## Slide 15

## Slide 16

## Slide 17

## Slide 18

## 36 & 24 hr chemostat cultures of Dsv. vulgaris Hildenborough (Jul. 14, 2004)

**Elevated Expression @ 36 hr**
**   ****Gene ID****	****    36h/24****	****	****         Functional annotation**
**	**
- ORF01292	6.388991465		phosphoglucomutase, alpha-D-glucose phosphate-specific
- ORF04778	3.733083017		hypothetical protein
- ORF00693	3.33891762		cobalt ABC transporter, ATP-binding protein, putative
- ORF00130	3.216054383		sodium/alanine symporter family protein
- ORF05364	3.134338102		Ech hydrogenase, subunit EchD, putative
- ORF05417	2.854857655		hypothetical protein
- ORF04127	2.781281186		tolB protein, putative
- ORF00696	2.693513162		cobalamin biosynthesis protein CbiM (cbiM)
- ORF04738	2.432622697		hypothetical protein
- ORF05423	2.42787129		chorismate mutase/prephenate dehydratase
- ORF04347	2.394286973		flagellar basal body-associated protein, putative
- ORF00729	2.322703947		inner membrane protein, 60 kDa (yidC)
- ORF05011	2.280328617		conserved hypothetical protein
- ORF04167	2.255508735		hypothetical protein
- ORF03494	2.244561416		membrane protein, putative
- ORF01916	2.241020851		dimethyladenosine transferase (ksgA)
- ORF00937	2.179606957		ribosomal protein L32 (rpmF)
- ORF00739	2.132798695		3'- 5' exonuclease domain protein, putative
- ORF04923	2.083262494		oligopeptide/dipeptide ABC transporter, permease protein
- ORF05424	2.0594284		3-phosphoshikimate 1-carboxyvinyltransferase (aroA)
- ORF05316	2.055521362		dissimilatory sulfite reductase B
- ORF05763	2.012862065		transcriptional regulator, putative
- ORF03058	2.001378273		hypothetical protein

## 36 & 24 hr chemostat cultures of Dsv. vulgaris Hildenborough (Jul. 14, 2004)

**Reduced Expression @ 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		Heptosyltransferase family
- ORF02969	0.281542943		hydrogenase, putative
- ORF04191	0.280952182		hypothetical 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		hypothetical protein
- ORF02808	0.251159356		methyl-accepting chemotaxis protein, putative
- ORF05576	0.249838232		hypothetical protein
- ORF02586	0.249519915		hypothetical protein
- ORF02624	0.243904696		endoribonuclease L-PSP, putative
- ORF04537	0.237363472		proto-oncogene ski, putative
- ORF00568	0.234059704		DAK1 domain protein
- ORF05177	0.233716245		conserved hypothetical protein
- ORF00660	0.212577697		glucokinase, putative
- ORF02229	0.211697026		hypothetical 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	0.161261121		hypothetical protein
- ORF00076	0.148084514		membrane protein, putative
- ORF02691	0.144200061		RNA methyltransferase, TrmH family
- ORF02130	0.117851129		periplasmic [NiFe] hydrogenase, large subunit, isozyme 1
- ORF02019	0.109922136		cold shock domain family protein

## Sparged Chemostat Growth of Dsv. vulgaris on

- Hydrogen

- Sparged Chemostat Growth of *Dsv. vulgaris* on
- Lactate minus Sulfate

## Reconfiguration of Biofilm Reactor Systems

## Sample Site

- _[Sample Site](file:///StahlSampleSite)_

- Area 1

- Area 2

- Area 3

- S-3 Ponds

- •

- •

- •

- •

- Background - FB609-01-00

- Area 1 - FB060-01-00
- Area 2 - FB052-01-00
- Area 3 - FB056-01-17
- Area 2 - FWB203-03d-04

- FRC Enrichments & Isolations

## Sample Cores

**Sample Cores**

- Sample canister in glove bag

- Sample core in glove bag

## Enrichments - April 2002 Sample Set

- 	**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”

**Substrates:**
**Lactate (16mM)**
**Acetate (16mM)**
**Pyruvate (16mM)**
**Propionate (16mM)**
**H****2****:CO****2**

## Slide 26

## Slide 27

## Micrograph of FRC isolate growing on H2, CO2, acetate, and SO4= 

- Micrograph of FRC isolate growing on H2, CO2, acetate, and SO4=

## Total Zinc in Lake DePue, Illinois, USA

- 0

- 500

- 1000

- scale in meters

- Background concentrations

- 113 mg/kg

- 483/mg/kg

- 436 mg/kg

- Mississippi River

- Illinois River

- Backwater lakes

- Map source:	United States Geological Survey, 7.5 minute map series, DePue, Illinois Quadrangle, 1966, photorevised 1979.

- _Site 1_
- 24960 mg/kg

- _Site 2_
- 11602 mg/kg

- _Site 3_
- 6530 mg/kg

- _Site 4_
- 4475 mg/kg

- _Site 5_
- 3350 mg/kg

**Notes:**
Insert slide from ISEB with site selection criteria

## Dissolved Zn versus PLP-biomass

- Data variability represents the average of the deviation from the mean for 2 discreet depth intervals collected from 3 core samples (6 samples total).

- 0

- 50

- 100

- 150

- 200

- 250

- 300

- 0.0

- 2.0

- 4.0

- 6.0

- 8.0

**PLP (nmole P/dry g)**

**Notes:**
An apparent trend with pore water Zn

Questions:
Is correlation significant
Can trend be attributed to any other parameters?
Recall – env parameters can not explain the trend.

## Sulfate Reduction in Lake DePue compared to other sediments

- (Missing non-critical slide)

**Notes:**
Sulfate variations
 srr variations – changes with depth as expected.
 highest srr at highest metal contaminated site – result of sulfate or metal concentration?

## Lake DePue Isolates

- Enrichments on lactate fresh water sediment
- media to isolate *Desulfovibrio*-like microbes.
- Isolated using anaerobic roll tube method with FeSO4

- Samples -  Lake DePue Zinc contaminated sediment
- Sampling, Oct. 2002

- DP1 isolate

## Slide 33

## PFGE - Restriction Digestion of Lake DePue Isolates Genomic DNA

- 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

## Comparative Genomic DNA hybridization

- 				Hildenborough
**Similarity – 16S rRNA****	****   **100%		  >99%		 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.

## D. vulgaris Hildenborough chromosome map and gene clusters deleted in the Dp4 isolate genome

## GeneID	DP4/HI		Functional annotations from TIGR

**	**
**Region 1 (44 genes)****	**
- ORF00763	**0.20****	**	adenine 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****	**	hypothetical 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****	**	hypothetical protein
- ORF00827	**0.11****	**	hypothetical protein
- ORF00829	**0.09****	**	bacteriophage DNA transposition B protein, putati
- ORF00830	**0.11****	**	bacteriophage 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

## Flow Cytometry-assisted Cloning of Specific Sequence Motifs from Complex 16S ribosomal RNA Gene Libraries

- Ger van den Engh

- Nielsen, J.L., A. Schramm, A.E. Bernhard, G.J. van den Engh, and D.A. Stahl
- Appl. Environ. Microbiol. (accepted)

## 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

## 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

## 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