1. Genetic analysis of Mycobacterium tuberculosis groEL homologues cpn60
Genetic analysis of Mycobacterium tuberculosis groEL homologues cpn60.1 and cpn60.2 C. M. Santosh Kumar, Yashaswini Kannan, C. V. Srikanth, Rohini Qamra, Abhijit A. Sardesai and Shekhar C. Mande Centre 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
GroES Heptamer 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,
Qamra and Mande (2004) J. Bacteriol. 186,
Slow growing M. tuberculosis independent of GroEL?

6. Complementation Studies
Cloning
M.groEL1 and M.groEL2 cloned along with cohort M.groES
Genes are co-expressed in E. coli
Strain Back Ground
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. LB amp Arabinose plates
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
E. coli SV2/pBAD constructs
300C
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 10-7
1 X 10-3
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 features of E. coli groEL are absent in M.tb groELs ?

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

13. Experiments Gene Shuffling
PCR
DNaseI
Digestion
Primerless
PCR with
Specific Primers
Clones to
Come
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. coli groEL

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