Chaperonin 60 (Cpn60) is a member of the heat shock protein family Hsp60.

1. Geneticanalysis of Mycobacterium tuberculosisgroEL homologues cpn60.1 and cpn60.2C. M. Santosh Kumar, Yashaswini Kannan, C. V. Srikanth, Rohini Qamra, Abhijit A. Sardesai and Shekhar C. MandeCentre for DNA Fingerprinting and Diagnostics (CDFD) Hyderabad

2. ABSTRACT • Chaperonin 60 (Cpn60) is a member of the heat shock protein family Hsp60. • Cpn60s are ATP dependent molecular chaperones. • Essential at all temperatures in E. coli.

3. GroEL-GroES Chaperonin Complex • Best studied molecular chaperones • Two proteins – GroEL & GroES • GroEL • Tetradecamer of 58kDa subunits • Two isologus heptameric rings • Hydrophobic cavities bind protein substates • GroESHeptamer of 10kDa, acts as a lid GroES GroEL

4. Chaperonins and Substrates • Assist protein folding • Broad range of substrates • Non specific interactions • Oligomeric ring is essential

5. Unusual Chaperonin 60s of M. tuberculosis Two copies cpn60s: M.groEL1 (cpn60.1) and M.groEL2 (cpn60.2) Unusual properties: • Share 70% homology with E. coli counterpart GroEL • Dimeric form • ATP independent • Cannot help refolding • Prevent aggregation Qamra, Srinivas and Mande (2004) J. Mol. Biol. 342, 605-617. Qamra and Mande (2004) J. Bacteriol. 186, 8105-8113. • Slow growing M. tuberculosis independent of GroEL?

6. Complementation Studies Strain Back Ground Cloning • M.groEL1 and M.groEL2 cloned along with cohort M.groES • Genes are co-expressed in E. coli E. coli groEL44 (SV2) • Derivative of E. coli TG1 strain • GroEL TS mutant • Grows well at 300C not at 420C • Does not allow phage λ plaquing

7. Growth at Elevated Temperature Experiment Results E. coli SV2 pBAD Constructs LB amp Arabinose LB amp Glucose LB agar Amp Glucose 300C 300C Single Colonies LB amp Arabinose plates & LB amp Glucose plates 420C Incubated at 300C and 420C • M.groEL1 and M.groEL2 do not support growth at elevated temperature

8. Spotting Assay for Quantitation Experiment Results 420C 300C E. coli SV2/pBAD constructs 1 2 3 4 1 2 3 4 LB amp Glucose 300C Serial Dilutions Spotted on LB amp Arabinose Incubated at 300C and 420C 1: M.groEL1 2: M.groEL2 3: E.groEL 4: pBAD • Negligible complementation

9. Other functions-Plaque assay Experiment Results E. coli SV2/pBAD constructs E.groEL LB amp 0.4% Maltose 300C M.groEL1 Pellet, wash & resuspend in LB MgSO4 M.groEL2 Overlay in Soft Agar on LB amp arabinose + 10mM MgSO4 pBAD λCIB2 phage serially diluted and spotted 1 X 10-5 1 X 10-1 Incubated at 300C 1 X 10-3 1 X 10-7 • M.groELs are not capable of allowing phage plaquing

10. Are the Genes Expressed? Experiment Results Western blotting Primary probing Anti-M.GroEL1: IT56 Anti-M.GroEL2: mAb67-2 Secondary probing M.GroEL2 Pure protein E.GroEL M.GroEL1 IT56 : anti-rabbit mAb67-2 : anti-mouse Detection ECL plus Kit • M.groEL genes are expressed

11. Which featuresof E. coli groEL are absent inM.tb groELs ?

12. M.groEL1 M.groEL2 NH2OH DNaseI digest C HAC A Primerless PCR GC AT PCR with Specific primers pBAD24 Cloning Selection and Characterization Random Mutagenesis Gene Shuffling Random Chemical Mutagenesis Mutator strain XL1-RED • mutS • (Error-prone mismatch repair) • mutD • (DNA Pol III 3´- to 5´-exonuclease) • mutT • (Hydrolyze 8-oxodGTP)

13. Clones to Come Primerless PCR PCR DNaseI Digestion PCR with Specific Primers Experiments Gene Shuffling Experiments with Hydroxylamine and XL1-RED are in progress

14. Summary M.groELs • Expressed in E. coli SV2. • Not able to support growth at elevated temperatures. • Not able to allow lambda plaquing. • Mutant pool of groEL is generated by gene shuffling. • M.groELs are not able to complement the loss of E. coligroEL

15. Acknowledgements • CDFD, Hyderabad • Department of Biotechnology, New Delhi • Council of Scientific and Industrial Research • Wellcome Trust, UK