Insights From Comparative Analysis of Four Sulfate Reducing Bacteria

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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
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Jamboree Organization
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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)

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What Genome are We Annotating?

• Taxonomic classification is Desulfovibrio
  desulfuricans G20
• 16S sequence is gt99 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

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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

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Genomic Signature

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

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Genomic Signature
Tri-heme cytochrome
Ferredoxin-thioredoxin reductase
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Unique Features of SRB and Related Genomes
C - Energy production G - Carbohydrate
transport N - Cell motility T - Signal
transduction
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Chemotaxis Proteins D. desulfuricans Transmembra
ne 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 Che
V CheW/CheY Fusion 4 CheC FliM and CheA (P2)
Homologue 2 CheD Deamidase 1
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Energy Metabolism
H2 cycling
?
?
?
?
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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

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Energy Metabolism
?
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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?

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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

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Periplasmic Hydrogenases
Fe only
Nickel Regulon
Hydrogenases
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Sulfate Reduction Regulon
Predicted cytoplasmic hydrogenase
TGTCGGCnnGCCGACA
TTGTgAnnnnnnTcACAA
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Phylogenetic Tree of the CRP-FNR-like proteins
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Binding Mode of Fnr Family Regulators
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New Regulators and Old Motifs

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

DD COOA TELRLALTTEQLSLHMGATRQTVSTLLNNLVR DV COOA
PDIHLELTMEQLAGLVGTTRQTASTLLNDMIR EC CRP
DGMQIKITRQEIGQIVGCSRETVGRILKMLED YP CRP
DGMQIKXTRQEIGQIVGCSRETVGRILKMLED VC CRP
DGMQIKITRQEIGQIVGCSRETVGRILKMLEE EC FNR
REFRLTMTRGDIGNYLGLTVETISRLLGRFQK YP FNR
REFRLTMTRGDIGNYLGLTVETISRLLGRFQK VC FNR
REFRLTMTRGDIGNYLGLTVETISRLLGRFQK DD new
DVIRLDVSKSLLAGVLGTARETLSRALAKLVE DV new
DTFTLDVTKGLLAGLLGTARETLSRCLSRMVE
TGTCGGCnnGCCGACA
TTGTGAnnnnnnTCACAA
TTGATnnnnATCAA
TTGTgAnnnnnnTcACAA
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Phylogenetic Tree of the CRP-FNR-like proteins
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Phylogenetic Tree of the CRP-FNR-like proteins
FNR Bacillus TTGTGAnnnnnnTCACAA
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Horizontal Gene Transfer
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Analysis of HTGs Using GO/COG
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HGT of Phosphonate-related Genes
tRNAs

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

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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

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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

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Out
epsilon
beta
gamma
alpha
delta
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Out
epsilon
beta
gamma
alpha
delta
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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

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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