# Presentation: 256968

## Insights From Comparative Analysis of Four Sulfate Reducing Bacteria

**Eric Alm** & Adam Arkin
- [Terry Hazen & Jay Keasling]
- And a star cast of thousands
- VIMSS/LBNL

## ‘Jamboree’ Organization

## Why SRB?

- Key players in global C and S cycles
- Sulfate reduction
- Carbon mineralization
- Implicated in metal corrosion
- Problem for petroleum industry
- Souring of oil reservoirs
- Bioremediation activities
- Reduction of U(VI), Cr(VI)

## What Genome are We Annotating?

- Taxonomic classification is Desulfovibrio desulfuricans G20
- 16S sequence is >99% identical to Desulfovibrio alaskensis
- Both alaskensis and vulgaris have significantly higher GC%
- Likely not due to mobile DNA
- Alignment of single alaskensis gene sequence in GenBank
- ~5% difference between alaskensis and G20
- 7 of 8 mismatches that changed GC content had lower GC in G20
- %GC difference is real?
- Will likely change name to Desulfovibrio alaskensis G20

## ‘Genomic Signature’ of a SRB

- Considered genes present in all sequenced SRB
- Desulfovibrio vulgaris
- Desulfovibrio desulfuricans G20
- Desulfotalea psychrophila
- Archeoglobus fulgidis
- But not present in other phylogenetically related bacteria
- Geobacters
- Bdellovibrio

## Genomic Signature

- Sulfate reduction pathway
- Sat
- Aps
- Desulfoviridin
- New genes
- Ferredoxin-thioredoxin reductase
- Tri-heme cytochrome associated with Dsr locus

## Genomic Signature

**Tri-heme cytochrome**

**Ferredoxin-thioredoxin reductase**

## Unique Features of SRB and Related Genomes

**C - Energy production**
**G - Carbohydrate transport**
**N - Cell motility**
**T - Signal transduction**

## CheD: Deamidase					1

- Transmembrane Receptors				33
- CheW: Coupling Protein				4
- CheA: Histidine Kinase				2
- CheY: Response Regulator				6
- CheR: Receptor Methyltransferase			3
- CheB: Receptor Methylesterase			2
- CheZ: CheY Phosphatase				1
- CheV: CheW/CheY Fusion				4
- CheC: FliM and CheA (P2) Homologue			2
- CheD: Deamidase					1

## Energy Metabolism

**H****2**** cycling**

**?**

**?**

**?**

**?**

## Energy Metabolism

- A number of possible enzymes have been identified from the genome sequence
- Hydrogenases
- [Fe]
- [NiFe]
- [NiFeSe]
- Need periplasmic and cytoplasmic
- Transmembrane redox complexes

## Energy Metabolism

**?**

## Cytoplasmic Hydrogenases?

- Two identified in D. vulgaris
- Ech
- CooMKLXUH (CO dehydrogenase associated)
- No orthologs present in G20
- New cytoplasmic hydrogenase identified in G20
- Conserved in Desulfotalea psychrophila
- Different methods of cytoplasmic H2 generation among SRB?

## Regulon (Motif) Prediction

- Identify list of known operons for some biological process
- Search for over-represented motifs in upstream region
- Validate by looking at comparative data from D. vulgaris
- Search all upstreams to add new genes to regulon

## Periplasmic Hydrogenases

**[Fe] only**

**Nickel Regulon**

**Hydrogenases**

## Sulfate Reduction Regulon

** ****TGTCGGCnnGCCGACA**

**TTGTgAnnnnnnTcACAA**

**Predicted cytoplasmic hydrogenase**

## Phylogenetic Tree of the CRP-FNR-like proteins

**Phylogenetic Tree of the CRP-FNR-like proteins**

## Binding Mode of Fnr Family Regulators

## New Regulators and Old Motifs

**DD COOA TELRLALTTEQLSLHMGAT****R****Q****TVS****T****LLNNLVR**
**DV COOA PDIHLELTMEQLAGLVGTT****R****Q****TAS****T****LLNDMIR**
**EC CRP  DGMQIKITRQEIGQIVGCS****R****E****TVG****R****ILKMLED**
**YP CRP  DGMQIKXTRQEIGQIVGCS****R****E****TVG****R****ILKMLED**
**VC CRP  DGMQIKITRQEIGQIVGCS****R****E****TVG****R****ILKMLEE**
**EC FNR  REFRLTMTRGDIGNYLGLT****V****E****TIS****R****LLGRFQK**
**YP FNR  REFRLTMTRGDIGNYLGLT****V****E****TIS****R****LLGRFQK**
**VC FNR  REFRLTMTRGDIGNYLGLT****V****E****TIS****R****LLGRFQK**
**DD new  DVIRLDVSKSLLAGVLGTA****R****E****TLS****R****ALAKLVE**
**DV new  DTFTLDVTKGLLAGLLGTA****R****E****TLS****R****CLSRMVE**

** ****TGTCGGCnnGCCGACA**

**TTGTgAnnnnnnTcACAA**

**TTGTGAnnnnnnTCACAA**

** ****TTGATnnnnATCAA**

-   CooA in *Desulfovibrio *sp.
-   CRP in Gamma-proteobacteria
-   FNR in Gamma-proteobacteria
-   A new CRP-FNR-family factor in  *Desulfovibrio *sp.

## Phylogenetic Tree of the CRP-FNR-like proteins

**Phylogenetic Tree of the CRP-FNR-like proteins**

## Phylogenetic Tree of the CRP-FNR-like proteins

**Phylogenetic Tree of the CRP-FNR-like proteins**

**FNR**
**Bacillus**
**TTGTGAnnnnnnTCACAA**

## Horizontal Gene Transfer

## Analysis of HTGs Using GO/COG

## HGT of Phosphonate-related Genes

- tRNAs

- Most genes have closest homolog in Alphaproteobacteria
- Likely transfer of operon

## Phosphonate Metabolism?

- Phosphonates are a minor component of phosphorous content in soil
- Operon not necessary for growth in laboratory conditions (growth on phosphate)
- Isolated from transposon library as being essential for growth in natural environment
- Possible case of HTG from niche-adapted bacteria becoming essential for adaptation of new bacteria to same niche
- Proposed experiment
- Compare growth of wt and mutant strains in laboratory using phosphonate as only P source

## Phylogeny of Proteobacteria

- Why study phylogeny?
- Important to understanding comparative data
- Key to detecting HGT
- Proteobacterial phylogeny
- One of the most ancient bacterial clades
- Origin of eukarya likely occurred after divergence of alpha lineage
- Many model organisms
- E. coli
- Agrobacterium
- Helicobacter
- Caulobacter
- Desulfovibrio

## epsilon

**epsilon**

**beta**

**gamma**

**alpha**

**Out**

**delta**

## epsilon

**epsilon**

**beta**

**gamma**

**alpha**

**Out**

**delta**

## Ongoing Questions

- Incomplete Methionine synthesis regulon
- Incomplete pathway for assimilatory sulfate reduction in G20
- GC content discrepancy
- Rapid evolution and duplication of histidine sensor kinase domains
- Pseudogene remnants support rapid evolution hypothesis
- New genes of unknown function common to SRB
- Photo-lyase genes identified
- Phage derived operon conserved between G20 and Desulfotalea

## Acknowledgments

**Eric Alm**
- Chris Rao
- Ralf Rabus
- Toby Richardson
- Gerrit Voordouw
- Shelley Haveman
- Judy Wall
- Larry Barton
- Mikhael Gelfand
- Dmitry Rodionov

- VIMSS Computational Group
- Katherine Huang
- Morgan Price
- Richard Koche
- Keith Keller
- JGI
- Inna Dubchak
- Paramvir Dehal
- Pilar Francino
- The SRB Annotation Team