Post on 03-Jan-2016
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A Brief History of Rhodopseudomonas palustris (Our pal Rpal)◦ Life, liberty and the pursuit of the soluble interactome
Global Gene Regulation◦ Extracytoplasmic Function Sigma Factors
Regulating the Regulators
Getting to Know Conserved Unknowns
Conclusions and Future Directions
The dominant form of life on earth◦Estimated 1030 bacteria◦1,000,000,000,000,000,000,000,000,000,
0 0 0
Oldest form of life
◦ 2.8B years before multicellular animals
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
H+
CO2
H+ H+ H2O1/2O2
H+
Lignin monomers
H+ H+ H2O1/2 O2
CO2
Thiosulfate
ATP
Lignin monomers
Chemoautotrophic
Chemoheterotrophic
Photoautotrophic
Photoheterotrophic
Thiosulfate
ATP
ATP
ATP
Four modes of metabolismFour modes of metabolism
Nitrogen fixation releases H2 as a byproduct R. palustris has three different nitrogenases
with different metal-containing co-factors: Molybdenum, Vanadium, Iron
HydrogenProduction
N2-Fixation Efficiency
Mo
V Fe
Derives Energy from Sunlight
Needs only gaseous N2
Grows on a wide variety of carbon sources including CO2, lignin monomers and xenobiotic aromatics
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
tag
Broad host range plasmid
expression in R. palustris
tag
Lysis
tag
2-step affinity isolation
tag Elution
affinitybead
Cells
web portal data analysis
Trypsintag
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:LC-MS-MS Identifications:
Proteins (distinct) 3404
Pyruvate dehydrogena
se
Succinyl-CoA
synthetase
Fatty acid biosynthetic
complex 1
Photosynthetic complex 1
Riboflavin biosynthesis
Heat shockPhotosynthetic complex 2
CO2
assimilation complex
Fatty acid biosynthetic complex 2
…Including RPA4223 (bait) and RPA4224 (prey)
Genes adjacent on the chromosome
RPA4223◦ a predicted response regulator
RPA4224◦ “hypothetical” protein
RPA4206: beta-hydroxybutyrate dehydrogenaseRPA2552: Unknown
HWE His Kin
RPA4223 RPA4226RPA4225RPA4224
Predicted Operon
/CHASE/
Response Regulator Anti-σ ECF σ Factor
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
The genome of R. palustris encodes 19 different sigma factors◦ 16 are Extracytoplasmic Function (ECF) Sigma
Factors (only 2 in E. coli )
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.
Shotgun proteomics of R. palustris under different metabolic conditions
1 of the 16 ECF σ dominated: RPA4225
◦ Present during stationary phase
◦ Growth on benzoate
-VerBerkmoes et al, J. Proteome Res. 2006
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
Mix Mix
LC/MS/MS
Biological T
echnical
Replicates R
eplicates
Verified expression of rpa4225 in experiment vs. control strains containing empty vector
1 2 3 4 5 6
L C1 E1 C2 E2
Biological Replicate 1 Biological Replicate 2
Locus Name DescriptionLog2
RatioLower
CIUpper
CILog2
RatioLower
CIUpper
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
Quantitative PCR performed on mRNA from 13 different genes◦ Selected Up, Down, and Unchanged examples◦ Included RPA4224
Results similar for both techniques◦ qPCR Data Indicate RPA4224 Strongly
Upregulated Proteomics data inconclusive due to low abundance
◦ Indicates positive feedback
-35 -10 1274 (1) GGAACGCCACCGGACGCAGCGCGTTGATGAGGGGTCTAACGT-132N--GGATTCTCATCGTG 1481 (1) GGAACGATCGTGGGGTTGAGCCCTTGATGATCGGTCGTGTCGAC-GAGAGGATGACTGAGGTG 3568 (1) GGAACGCAGCGTGGAGTCCGCGGTTCTGTAGGCACCATTCAC-6N-AAGAGGGCAATCCTATG 4418 (1) GGAACATCCGAGGAGCCTGCCGGTTGTCTCCACGAAGCTCACAAGTGAAGGAGACATTCAATG 3308 (1) GGAACAGAATCGTTCGTCCCCGGTTCTCCGGTCGAGCCGCGG-15N-GGCGGAGGCAACGATG 3726 (1) GGAACATTCGTCGGAAGGTCGCGTTGGGCGGTTGCACTGTCA-19N-CGGAGATCACAGCATG 4224 (1) GGAACTTTCGCGCCGGGATCGCATT---------------------------AGGGTCCCATG 3943 (1) GGAACGGCGGTCGCTGTCGCTGGTTAACGACCCGAACGGCCG-46N-AAGAGGCACCCCGATG 3510 (1) GGAACCCAATGGCGCGCTGCGGGTTGACGTGGTGCATCTCGG-28N-TCTGGAGGGCATCATG Consensus (1) GGAAC G GG G C GGTTG G G CG
Including the 4224-4225
operon
-10-35
Search the genome for the sequence:
GGAAC-17N-TT
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
RPA3310◦ KatE, Catalase
RPA3726◦ Mn Catalase Assoc (?)
RPA1481 ◦ CheY-like Protein, Regulatory
RPA1274◦ DPS-like Protein: Associated with DNA-protection as well as
cytoplasmic sequestration of Fe
RPA3702◦ MetH, one of two methionine synthases◦ B12-dependent, not sensitive to oxidation in E. coli, unlike MetE
2 H2O2 → 2 H2O + O2
All Point to Oxidative Stress Response
“Common mechanism of cellular death induced by bactericidal antibiotics” -Kohanski et. al Cell ’07
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
reactionH2O2 + Fe2+ Fe3+ + OH¯+ ●OH
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
katE upregulated under H2O2 conditions as expected
rpa4224 operon also upregulated under H2O2 conditions
rpa3568 upregulated under H2O2 conditions but less than predicted
…was not particularly convincing
H2O2 not the only kind of ROS◦ Singlet oxygen◦ Superoxide◦ Alkyl peroxides
And Rpa4225 not the only ECF σ factor
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
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’R. pal. 5’-TGATCCAAACGATCGGCCGGCTCGTATCAGAACAAAT-3’R. s. 5’-TGATCCAGACTGGCCCGGCCGCCGTAAGAAGGACGTT-3’
Close proximity to each other
Also upregulated during stationary phase/starvation
No homologs or positional clues
Sigma factors rpa0550 and rpa1813 had highest response singlet oxygen
Less known about the latter
Back to shotgun proteomics
LocusLog2ratio Description
RPA4834 4.7 MsrA2, pms peptide methionine sulfoxide reductaseRPA4070 4.6 MsrA1 possible peptide methionine sulfoxide reductaseRPA2544 4.2 RPA2544 conserved hypothetical proteinRPA4194 3.0 OsmC osmotically inducible protein OsmCRPA0225 2.9 SodC putative periplasmic superoxide dismutase (Cu/Zn)RPA1206 2.9 aldehyde dehydrogenaseRPA4069 2.9 DUF25RPA1941 1.1 possible 2-nitropropane dioxygenaseRPA1205 1.0 putative alcohol dehydrogenaseRPA1576 1.0 putative glutathione S-transferase
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)
ECF σ1813:◦ Controls genes related to methionine oxidation
rpa3568 strongly associated with pH stress
“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-componentsignaling in ECF activation may be limited to Gram positive bacteria.”
Brooks and Buchanan, 2007
HWE His Kin
RPA4223 RPA4226RPA4225RPA4224
/CHASE/
Response Regulator Anti-σ ECF σ Factor
Smc01504 Smc01507rpoE2
R. palustris
Sinorhizobium meliloti
7001 70047003Bradyrhizobium sp.
1644 16461645Brucella abortus
This ORF has not been annotated but two-way translated BLAST analysis reveals
protein homology with rpa4224
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
RPA4225 (181 aa)
CHASE HWE_HK
σ70_r4_2
σ70_r2 σ70_r4_2
RR
RPA4226 (588 aa)
RPA4223 (268 aa)
RPA4224 (70 aa)No Recognizable
Domains
ECFRPA4225
Anti-
RPA4224
’
Histidine kinase sensor
RNA polymerase
Resp RegRPA4223
RPA4226
ECF-RPA4225
Anti-RPA4224
1. Environmental Signal
3. RR Activation
4. Sequestration/Degradation of anti-sigma factor
5. Active Complex
ATP
ADPPO4
2. Autophosphorylation
PO4
Resp RegRPA4223PO4
Green Fluorescent Protein (GFP) cytoplasmic distribution
DivIVA E. coli division protein
localizes to poles
E. coli
GFP fused to protein X
DivIVA fused to protein Y
X-
Y-
-X
-X
-X
-X
X/ -X
-X X-
-X
-X -X
-X Y Y Y Y
Y Y Y Y
Y Y Y Y
Y Y Y Y
XXXX
XXXX
X-Y Interaction
No Interaction
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
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
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
RPA3568 strongly linked to pH stress
CHASE domain linked to stress response
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
Still attempting deletion of RPA4223-4226◦ Screen for stress sensitivity phenotype◦ Mutational analysis of HK and RR
In vitro phosphotransfer
Reconstitution in E. coli
Investigate 4226 CHASE-domain binding affinities◦ Domain found in bacteria, plants◦ No solid information on target (plants cytokinin)◦ Direct oxidation? Lipid peroxides?