1. Towards Systematic Identification of cdiGMP Binding Proteins
Kevin Roelofs
Laboratory of Dr. Vincent Lee
University of Maryland, College Park

2. What is the total regulatory potential of a second messenger?
Nucletoide Second Messengers:
cAMP
ppGpp(p)
cdiGMP
cdiAMP
c-AMP/GMP
Receptors:
Membrane Proteins
Cytosolic Enzymes
Transcription Factors
Phenotypes

3. CdiGMP in Vibrio cholerae
In V. cholerae cdiGMP regulates:
Biofilm Formation
Host Colonization
Virulence
Environmental dissemination
51 proteins with domains for synthesis and degradation of cdiGMP
4 Currently Identified Receptors
Tamayo Ann Rev Microbiol 2007

4. Current approaches in second messenger receptor identification
1) Genetics
2) Biochemistry
3) Bioinformatics
Identify target genes required for phenotype regulated by second messenger
Systematically purify and test target proteins for binding to second messenger
Pull down proteins with second messenger as bait
Identify second messenger in crystal structures of purified proteins
Identify receptors with conserved binding domains

5. Problems with current approaches
Genetics – Receptors not always associated with observable phenotype
Biochemistry – Receptor must be expressed
Bioinformatics – Limited to identification of previously described binding motifs
None of these approaches systematically identify all genomically encoded receptors

6. Differential Radial Capillary Action of Ligand Assay (DRaCALA)
Protein and bound ligand are immobilized on nitrocellulose membrane while free ligand is distributed uniformly by capillary action

7. DRaCALA detects specific binding interactions
Proof of Principle:
Purify 10 µM protein
Mix with nM amounts of radiolabeled nucleotide

8. Quantitative Detection of Binding Interactions in E
Quantitative Detection of Binding Interactions in E. coli Whole Cell Lysate
Mix with 32P cdiGMP:
Purified Alg44
Purified Alg44 added to BL21 E. coli cell lysate
BL21 E. coli cell lysate with endogenously expressed Alg44
Plot Fraction Bound vs Protein Concentration

9. Design of DRaCALA ORFeome Screen
Obtain Open Reading Frame (ORF) library
Contains all annotated ORFs from individual organism
V. cholerae ORF library contains 3866 unique clones
Express individual V. cholerae ORFs in E. coli T7IQ
2 libraries: N-terminal His- or HisMBP-
Screen whole cell lysates in 96-well format for increased cdiGMP binding

10. Results of Partial (938 ORFs) Screen
Buffer control
Other
PilZ domain
EAL domain
Positive Clone
Positive Control
3 standard deviation positive cutoff
Average Fraction Bound

11. Biochemical Classification of Positive Hits
Vector
control
ORF expressed
1 mM Competitor NC NC
cdiGMP
cGMP
cAMP
GMP
GDP
GTP
CTP
ATP
UTP
His-PlzE
His-VC1641
2x Serial Dilutions of E. coli WCL into binding buffer
His-PlzE
His-VC1641

12. DRaCALA ORFeome Screens
A screen of Staph aureus ORFeome has identifed 4 novel cdiAMP receptors
Systematically identify genomically encoded receptors of metabolites
Identify processes regulated by second messenger systems
Systems-level understanding of regulatory potential of second messenger

13. Acknowledgements Lee Lab Members
Funding University of Maryland NIH R21 NIH T32 Training Grant DOD DTRA Cystic Fibrosis Foundation
Lee Lab Members

14. DRaCALA detects specific binding of soluble and insoluble proteins in WCL
PAGE of Whole Cell Lysate (W) and Soluble Fraction (S)

15. Nucleotide Second Messengers Regulate Bacterial Metabolism
Nucletoides:
cAMP
cdiGMP
pGpG
cdiAMP
c-AMP/GMP
ppGpp(p)
Receptors:
Protein
RNA
What is the total regulatory potential of a second messenger?

16. What is the biological function of any small molecule?
cAMP has one known protein receptor in E. coli: CRP
Mona Wu
“Intracellular cAMP levels affect bacterial chemotaxis responses by inhibiting CheA”
Poster # _______________
Glucose
cAMP
Alternative Carbohydrate Utilization
CRP ?
E. coli whole cell lysates from strains lacking CRP still bind cAMP

17. SM – less story (use other slide)
Detect – put this in comparisons
DRaCALA – no animation
Example -
ORF
Staph (use figure that was made remove names thank grundling lab) - Vibrio

18. Differentiating ORFs that directly and indirectly increase cdiGMP binding
CdiGMP Binding Proteins
Receptors, Diguanylate Cyclases, Phosphodiesterases
2. Conserved non-binding regulators of cdiGMP signaling
Transcription factors
ORF
ORF
Mills et al Cell Micro