Michael AllenNovember 29, 2010

UNT Health Science Center, Ft. Worth

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

You Are Here

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

N2+16ATP+8H++8e- 2NH3+H2+16ADP+16Pi

Nature Biotech. 2004

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.

Sigma 1

Sigma 1

Sigma 2Sigma 2

Sigma 3Sigma 3

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

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

…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

mR

NA

Fol

d-C

hang

e

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

Ezraty et al. BBA ‘05

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

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-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

Methylobacterium extorquens Francez-Charlot et al. PNAS ‘09

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

RPA4223-RPA4224 shown to interact by pull-down assay and LC/MS-MS

What about RPA4223-RPA4226?

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

2μm2μm

RPA4226-GFP + RPA4223-DivIVARPA4226-GFP

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

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?

UNT◦ Leslie Perry◦ Sarah Martinez◦ Stephanie Simon◦ David Visi

ORNL◦ Dale Pelletier◦ Greg Hurst◦ Jenny Morrell-Falvey◦ and many others…

Funding Provided by:

                              

Office of Research

University of North Texas Health Science Center