Transcript Slide 1

Michael Allen
November 29, 2010
UNT Health Science Center, Ft. Worth
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A Brief History of Rhodopseudomonas
palustris (Our pal Rpal)
◦ Life, liberty and the pursuit of the soluble interactome
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Global Gene Regulation
◦ Extracytoplasmic Function Sigma Factors
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Regulating the Regulators
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Getting to Know Conserved Unknowns
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Conclusions and Future Directions
You Are Here
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The dominant form of life on earth
◦ Estimated 1030 bacteria
◦ 1,000,000,000,000,000,000,000,000,000,
0
Oldest form of life
0
◦ 2.8B years before multicellular animals 0
The largest source of genetic diversity
◦ 107 distinct species in 1g of soil
Gram Negative α-Proteobacterium
Capable of Anoxygenic
photosynthesis
5.5 Mbp Genome sequenced (JGI)
and annotated
(Larimer et al. Nat. Biotech. 2004)
Recently several additional strains
have been sequenced by DOE (6
total)
High metabolic diversity
Four modes
of
metabolism
+
CO2
H
CO2
ATP
Thiosulfate H+ H+
Chemoautotrophic
1/2O
2
H2O
 
ATP
Photoautotrophic
Thiosulfate
H+
Lignin monomers
Lignin monomers
 
ATP
H+ H+
1/2
H2O
O2
Chemoheterotrophic
ATP
Photoheterotrophic
Nature Biotech. 2004
N2+16ATP+8H++8e-
2NH3+H2+16ADP+16Pi
Nitrogen Fixation
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Nitrogen fixation releases H2 as a byproduct
R. palustris has three different nitrogenases
with different metal-containing co-factors:
Molybdenum, Vanadium, Iron
N2-Fixation
Efficiency
Hydrogen
Production
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Derives Energy from Sunlight
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Needs only gaseous N2
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Grows on a wide variety of carbon sources
including CO2, lignin monomers and
xenobiotic aromatics
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For all of these reasons, chosen as a subject
for a DOE-funded Genomes To Life program
Objective: To map out the entire soluble
protein “interactome”
◦ High-throughput,
heavily automated process
2-step
affinity
isolation
tag
affinity
bead
Elution
tag
Trypsin
tag
Lysis
Cells
tag
tag
Broad host range
plasmid
expression in
R. palustris
web portal
data analysis
Distinct Affinity-tagged Gene Products:
Cloned (entry / expression)
1312
Cultured and harvested
859
Isolated and analyzed by LC-MS-MS
844
LC-MS-MS Identifications:
Proteins (distinct)
3404
Photosynthetic
complex 1
Pyruvate
dehydrogenase
Riboflavin
biosynthesis
CO2 assimilation
complex
Succinyl-CoA
synthetase
Fatty acid
biosynthetic
complex 1
Fatty acid
biosynthetic
complex 2
Photosynthetic
complex 2
Heat shock
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…Including RPA4223 (bait)
and RPA4224 (prey)
Genes adjacent on the
chromosome
RPA4223
◦ a predicted response regulator
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RPA4224
◦ “hypothetical” protein
RPA4206: beta-hydroxybutyrate dehydrogenase
RPA2552: Unknown
Response Regulator
Anti-σ
RPA4223
ECF σ Factor
RPA4225
RPA4224
Predicted Operon
HWE His Kin
/CHASE/
RPA4226
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In bacteria, sigma factors bind with RNAP to
direct transcription of large sets of genes
◦ “Global Regulators”
◦ Ex’s: σ70 Housekeeping, σ32 Heat shock
◦ E. coli has 7 total
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The genome of R. palustris encodes 19
different sigma factors
◦ 16 are Extracytoplasmic Function (ECF) Sigma
Factors (only 2 in E. coli )
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AKA Type IV σ factors
Distantly related to the σ70-type (Type I)
housekeeping σ factors
Respond to changes outside the plasma
membrane (external or periplasmic)
Often control expression of virulence factors,
biofilm formation, stress responses, etc.
Sigma 1
Sigma 3
Sigma 2
Sigma 2
Sigma 1
Sigma 3
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Shotgun proteomics of R. palustris under
different metabolic conditions
-VerBerkmoes et al, J. Proteome Res. 2006
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1 of the 16 ECF σ dominated: RPA4225
◦ Present during stationary phase
◦ Growth on benzoate
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Cloned rpa4225 into a broad host range
vector and transformed it into WT R. palustris
CGA010
Extract total protein
Analyzed the whole proteome for changes in
protein abundance as a result of constitutive
expression of rpa4225
(15NH4)2SO4
(14NH4)2SO4
Control
Experiment
Biological
Technical
LC/MS/MS
Replicates
Mix
Replicates
Mix
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Verified expression of rpa4225 in experiment
vs. control strains containing empty vector
1
L
2
3
C1 E1
4
5
C2
6
E2
Biological Replicate 1
Locus
Name
Description
Log2
Ratio
Lower
CI
Upper
CI
Biological Replicate 2
Log2 Lower Upper
Ratio
CI
CI
RPA3310
KatE
Catalase
3.8
3.4
4.1
4
3.5
4.3
RPA3726
CDS
DUF892, YciF-like
3.7
3.2
4.2
3
2.3
3.5
RPA3568
CDS
Conserved Unknown
3
2.6
3.5
2.1
1.6
2.6
RPA3943
CDS
Conserved Hypothetical
2
1.5
2.5
2.1
1.6
2.7
RPA3309
CDS
Conserved Unknown
2.2
1.9
2.6
1.6
1.3
1.9
RPA1481
CDS
Putative CheY-like protein
1.9
1.5
2.4
1.9
1.5
2.2
RPA1500
CDS
Unknown Protein
2
1.7
2.4
1.1
0.7
1.6
RPA3510
CDS
Conserved Unknown
1.7
1.5
1.9
1.2
1
1.4
RPA3702
MetH
Methionine Synthase
1.1
0.7
1.4
0.7
0.3
1.1
RPA1274
CDS
DPS-like Protein
0.9
0.4
1.3
0.9
0.5
1.3
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Quantitative PCR performed on mRNA from
13 different genes
◦ Selected Up, Down, and Unchanged examples
◦ Included RPA4224
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Results similar for both techniques
◦ qPCR Data Indicate RPA4224 Strongly Upregulated
 Proteomics data inconclusive due to low abundance
◦ Indicates positive feedback
Including the
4224-4225
operon
1274
1481
3568
4418
3308
3726
4224
3943
3510
Consensus
-35
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
-35
-10
GGAACGCCACCGGACGCAGCGCGTTGATGAGGGGTCTAACGT-132N--GGATTCTCATCGTG
GGAACGATCGTGGGGTTGAGCCCTTGATGATCGGTCGTGTCGAC-GAGAGGATGACTGAGGTG
GGAACGCAGCGTGGAGTCCGCGGTTCTGTAGGCACCATTCAC-6N-AAGAGGGCAATCCTATG
GGAACATCCGAGGAGCCTGCCGGTTGTCTCCACGAAGCTCACAAGTGAAGGAGACATTCAATG
GGAACAGAATCGTTCGTCCCCGGTTCTCCGGTCGAGCCGCGG-15N-GGCGGAGGCAACGATG
GGAACATTCGTCGGAAGGTCGCGTTGGGCGGTTGCACTGTCA-19N-CGGAGATCACAGCATG
GGAACTTTCGCGCCGGGATCGCATT---------------------------AGGGTCCCATG
GGAACGGCGGTCGCTGTCGCTGGTTAACGACCCGAACGGCCG-46N-AAGAGGCACCCCGATG
GGAACCCAATGGCGCGCTGCGGGTTGACGTGGTGCATCTCGG-28N-TCTGGAGGGCATCATG
GGAAC
G GG G
C GGTTG G G CG
-10
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Search the genome for the sequence:
GGAAC-17N-TT
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Found in the promoter regions of ~150 genes, including
◦ DNA repair
◦ Heat shock sigma factor rpoH
◦ Superoxide dismutase sodB
Suggests RPA4225 is a major regulator of the
stress response in R. palustris
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RPA3310
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RPA3726
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RPA1481
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RPA1274
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RPA3702
◦ KatE, Catalase
2 H2O2 → 2 H2O + O2
◦ Mn Catalase Assoc (?)
◦ CheY-like Protein, Regulatory
◦ DPS-like Protein: Associated with DNA-protection as well as
cytoplasmic sequestration of Fe
◦ MetH, one of two methionine synthases
◦ B12-dependent, not sensitive to oxidation in E. coli, unlike MetE
All Point to Oxidative Stress Response
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“Common mechanism of cellular death
induced by bactericidal antibiotics” -Kohanski et. al
Cell ’07
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Found that 3 classes of bactericidal
antibiotics all stimulated the production of
hydroxyl radicals, causing death
End result was induction of oxidative damage
cellular death pathway
◦ Destabilization of Fe-S clusters, and Fenton
reaction
H2O2 + Fe2+  Fe3+ + OH¯+ ●OH
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Implies that:
1) Drugs targeting the bacterial response to
oxidative damage would act synergistically with
existing bactericidal antibiotics
2) There is apparently a lot we don’t know about
bacterial response to oxidative stress
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Performed qPCR on wild type R. palustris
under oxidative stress conditions
katE upregulated under H2O2
conditions as expected
rpa4224 operon also
upregulated under H2O2
conditions
rpa3568 upregulated under
H2O2 conditions but less than
predicted
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…was not particularly convincing
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H2O2 not the only kind of ROS
◦ Singlet oxygen
◦ Superoxide
◦ Alkyl peroxides
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And Rpa4225 not the only ECF σ factor
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R. palustris encodes 19 σ factors (16 ECF)
Preliminary data indicated increased
abundance of transcripts for other sigma
factors during stationary phase
Targeted rpa0550, rpa1813, rpa1819
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RpoERs associated with response to singlet
oxygen
Genes rpa0550 and chrR in R. palustris (top)
and their homologs in Rhodobacter
sphaeroides (Newman et al.
JMB ‘99)
R. pal. 5’-TGATCCAAACGATCGGCCGGCTCGTATCAGAACAAAT-3’
R. s.
5’-TGATCCAGACTGGCCCGGCCGCCGTAAGAAGGACGTT-3’
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Close proximity to each other
Also upregulated during stationary
phase/starvation
No homologs or positional clues
mRNA Fold-Change
50
40
30
pH 5.0
pH 8.0
20
10
0
rpa0550
rpa1813
rpa1819
rpa4225
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Sigma factors rpa0550 and rpa1813 had
highest response singlet oxygen
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Less known about the latter
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Back to shotgun proteomics
Locus
RPA4834
RPA4070
RPA2544
RPA4194
RPA0225
RPA1206
RPA4069
RPA1941
RPA1205
RPA1576
Log2
ratio
4.7
4.6
4.2
3.0
2.9
2.9
2.9
1.1
1.0
1.0
Description
MsrA2, pms peptide methionine sulfoxide reductase
MsrA1 possible peptide methionine sulfoxide reductase
RPA2544 conserved hypothetical protein
OsmC osmotically inducible protein OsmC
SodC putative periplasmic superoxide dismutase (Cu/Zn)
aldehyde dehydrogenase
DUF25
possible 2-nitropropane dioxygenase
putative alcohol dehydrogenase
putative glutathione S-transferase
Ezraty et al. BBA ‘05
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ECF σ4225:
◦ Controls expression of numerous genes
◦ Likely responds to multiple stresses including
oxidative and pH stress
◦ Downstream genes in turn may be controlled by
multiple regulators (e.g. OxyR)
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ECF σ1813:
◦ Controls genes related to methionine oxidation
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rpa3568 strongly associated with pH stress
Part II
Brooks and Buchanan, 2007
“Sequence homology indicates that two
component signaling activation of an ECF
sigma factor may regulate the activity of σE
from the Gram positive bacterium
Streptomyces coelicolor A3(2) [18]. However,
to date, this has not been seen in Gram
negative bacteria and thus two-component
signaling in ECF activation may be limited to
Gram positive bacteria.”
Brooks and Buchanan, 2007
Response Regulator
Anti-σ
ECF σ Factor
HWE His Kin
RPA4224
RPA4223
RPA4225
/CHASE/
RPA4226
R. palustris
Smc01504
Sinorhizobium meliloti
rpoE2
Smc01507
7001
Bradyrhizobium sp.
7003
7004
1644
Brucella abortus
1645
1646
This ORF has not been annotated but twoway translated BLAST analysis reveals
protein homology with rpa4224
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Organization of four genes conserved among
multiple α-Proteobacteria
The homolog of RPA4224 in S. meliloti,
Smc01505, was shown to be a negative
regulator of ECF σ factor RpoE2
Presence of Response Regulator and HWE
Histidine Kinase flanking σ/anti-σ pair strong
evidence of their involvement in regulation
CHASE
HWE_HK
σ70_r4_2
RR
σ70_r2
RPA4226 (588 aa)
RPA4223 (268 aa)
σ70_r4_2
RPA4225 (181 aa)
RPA4224 (70 aa)
No Recognizable
Domains
Methylobacterium extorquens Francez-Charlot et al. PNAS ‘09
Histidine kinase sensor
1. Environmental Signal
RPA4226
ATP
2. Autophosphorylation
PO4
ADP
3. RR Activation
PO4
Resp Reg
RPA4223
b
Anti-s
RPA4224
PO4
Resp Reg
RPA4223
Anti-s
RPA4224
4. Sequestration/Degradation of
anti-sigma factor
s-ECF
RPA4225
ECF-s
RPA4225
b’
a
a
RNA polymerase
5. Active Complex
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RPA4223-RPA4224 shown to interact by pulldown assay and LC/MS-MS
What about RPA4223-RPA4226?
E. coli
Green Fluorescent
Protein (GFP)
cytoplasmic distribution
DivIVA E. coli
division protein
localizes to poles
No Interaction
X-
Y-
GFP fused to
protein X
-X X
Y -X
Y
-X
Y
-XY
/
-X
-X
Y
Y
-X
Y X- -X
-X
-X Y
DivIVA fused to
protein Y
X-Y Interaction
YX
YX
YX
YX
XY
XY
XY
XY
RPA4226-GFP +
RPA4223-DivIVA
RPA4226-GFP
2μm
2μm
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PhyR-NepR system conserved in R. palustris
RPA4226 Histidine Kinase appears to be
signal transducer for PhyR (RPA4223)
Response of system oxidative, pH stress
suggests role for CHASE domain in bacteria
Part III
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Unknowns – proteins without known function
Conserved unknowns – broadly distributed,
evolutionarily conserved of the above
Hypothetical proteins – Predicted based on
bioinformatics but no data on transcript or
protein
◦ Ghosts in the machine
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In E. coli (and bacteria in general)
◦ We know the function of about 1/3 of the genes
◦ We think we know functions of about another 1/3
◦ The last third we are almost entirely clueless about
katE upregulated under pH
stress
rpa4224 operon upregulated
under pH stress
rpa3568 dramatically
upregulated by pH shift
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RPA3568 strongly linked to pH stress
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CHASE domain linked to stress response
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Analysis of conserved unknowns to be the
subject of an HHMI-sponsored undergrad
research-based class
◦ Generate and screen KO’s for stress sensitivity
◦ Investigate auxotrophies under stress conditions
◦ High risk/high reward
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Still attempting deletion of RPA4223-4226
◦ Screen for stress sensitivity phenotype
◦ Mutational analysis of HK and RR
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In vitro phosphotransfer
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Reconstitution in E. coli
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Investigate 4226 CHASE-domain binding
affinities
◦ Domain found in bacteria, plants
◦ No solid information on target (plants cytokinin)
◦ Direct oxidation? Lipid peroxides?
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UNT
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Leslie Perry
Sarah Martinez
Stephanie Simon
David Visi
Funding Provided by:
ORNL
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Dale Pelletier
Greg Hurst
Jenny Morrell-Falvey
and many others…
Office of
Research
University of North
Texas Health
Science Center