The Search for Hypermutability:
Determining Frequency of
Mutation in Mycobacterium
smegmatis mc2155
___________
Susan Puckett
Mentors: Dr. Digby Warner, Dr. Valerie Mizrahi
MRC/NHLS/WITS Molecular Mycobacteriology Research Unit,
School of Pathology, University of the Witwatersrand and National Health
Laboratory Service,
Johannesburg, South Africa
Funded by the Howard Hughes Medical Institute
Background: Tuberculosis
(TB)
• Contagious, airborne disease caused by Mycobacterium
tuberculosis (MTB)
– Coughing, weakness, weight loss, fever, death
• Over 1/3 of the world’s population infected, with 5-10%
projected to become infectious at some point
Estimated Death Toll in 2005
USA
1,345 people
South Africa
101,989 people
World
1,600,000 people
Source: World Health Organization (WHO) website
http://www.who.int/mediacentre/factsheets/fs104/en/index.html
http://www.who.int/globalatlas/dataQuery/default.asp
TB Treatment and Drug
Resistance
• BCG Vaccine, Antibiotics
• MDR-TB
– Isoniazid, rifampicin resistant
• XDR-TB
– Isoniazid, rifampicin resistant
– Fluoroquinolone resistant
– Resistant to at least one of three second-line drugs
• capreomycin, kanamycin, and amikacin
• All drug resistance is chromosomally encoded
Understanding Mutation
• Problem of drug resistance caused by
mutations in genes required for antibiotic
effectiveness
• Link between mutagenesis and drug
resistance:
Boshoff, H., Reed, M., Barry, C., V. Mizrahi. 2003. DnaE2
Polymerase Contributes to In Vivo Survival and the Emergence of
Drug Resistance in Mycobacterium tuberculosis. Cell 113: 183-193.
Questions to Answer
• Do mutations occur more frequently under
certain conditions?
• Is there a hypermutable state in
Mycobacterium?
• Mycobacterium smegmatis (MSM)
– Not pathogenic, genetically similar, faster grower
• Hypothesis: Stress-induced hypermutability
• Targeting mutation during a hypermutable
state could be an effective way of combating
TB
Karunakaran, P., and J. Davies. 2000. Genetic
Antagonism and Hypermutability in Mycobacterium
smegmatis. J. Bacteriol. 182: 3331-3335.
FIG. 4. Growth phase-dependent hypermutability in M. smegmatis, shown
as frequencies of the appearance of resistant mutants
Experiment
• Test MSM strains mc2155 and mc26
using method outlined in the paper,
with different types of media
– Incubate cultures and plate on
rifampicin plates during log phase and
stationary phase over a period of
several days
Expectations:
-- More RifR mutants in stationary
phase than in log phase
-- mc2155 and mc26 generate
similar numbers of RifR mutants
-- Process is growth medium
independent
Results with 7H9, 7H10
• First tested with 7H9 and 7H10, media commonly used to grow
MSM and MTB. With and without TW80.
• Only a few RifR colonies per plate after plating 0.4 ml undiluted
culture
• NO increase in frequencies of mutation in stationary phase vs.
log phase (mutation frequency ~10-8)
Results with TSB and TSA
• Later tested with tryptic soy broth (TSB) and tryptic soy agar
(TSA), the media used in the paper. No TW80.
• Log phase: a few mutants and a haze of smaller colonies on
rifampicin plates
• Stationary phase: a lawn of growth on rifampicin plates
• Seems to correlate with paper
Final TSB experiment
• mc2155 and mc26 grown in TSB at 30ºC
with TW80
• 7H10 and TSA used for plating
• Simultaneous experiment: sample from
starting culture diluted with new media
every day and incubated at same
conditions as main culture
Results
• Contamination issues, plates reflect
trends as seen in last experiment with
TSB
• Simultaneous experiment: contaminated
• However, certain plates show
interesting results
mc2155 grows differently on
TSA
Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80
mc2155 on TSA
mc2155 on 7H10
mc26 on TSA
mc26 on 7H10
• Colonies are smaller, grow more slowly
• Consistently different than mc26 results on
TSA
mc2155 grows differently on
TSA
Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80
mc2155 on TSA
mc2155 on 7H10
mc26 on TSA
mc26 on 7H10
• Sometimes, colonies do not appear at all.
Perhaps longer incubation is needed.
mc2155 RIFR mutants grow on
TSA
Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80
mc2155 on TSA (RIF)
mc2155 on 7H10 (RIF)
• Stationary phase cultures
• Contamination problems with mc26
TW80-less TSB encourages
growth on TSA RIF plates
Before plating, these were incubated in TSB at 30ºC, 100RPM, without TW80
CONTAMINATED
mc2155 on TSA (RIF)
mc2155 on 7H10 (RIF)
mc26 on TSA (RIF)
mc26 on 7H10 (RIF)
• Cultures under similar log phase conditions
with TW80 had 0-5 mutants per TSA plate
and 0-1 mutants per 7H10 plate
Conclusion
• MSM behaves differently under different
conditions
– Media: TSB vs. 7H9, TSA vs. 7H10
– Presence or lack of TW80
• Greater number of RIFR mutants with TSB, TSA
• mc2155 behaves differently than mc26 on TSA but
not on 7H10
• Frequency of mutation: more testing needed
• Comparable results to Karunakaran and Davies
paper
Analysis
• If stationary phase hypermutability occurs, it is only
under certain growth conditions
• Other explanations: competitive advantage
Perkins, A., W. Nicholson. 2007. Uncovering New Metabolic
Capabilities of Bacillus sublitis Using Phenotype Profiling of RifampicinResistant rpoB Mutants. J. Bact. 190: 807-814.
Wrande, M., Roth, J., D. Hughes. 2008. Accumulation of Mutants in
“Aging” Bacterial Colonies is Due to Growth Under Selection, Not
Stress-Induced Mutagenesis. Proc. Natl. Acad. Sci. USA . 105: 1186311868
• Clustering of cells in TW80-less media could act like
biofilm, allowing RIF susceptible cells to survive
Significance
• Hypermutability is questionable due to error
catastrophe
• Significance: cannot assume hypermutability
in MTB from in vitro studies, since there is so
much variability
– Emphasizes need for better understanding
of in vivo conditions in order to mimic them
in vitro
Acknowledgements
•
•
•
•
•
Dr. Digby Warner
Dr. Valerie Mizrahi
Dr. Kevin Ahern
Mizrahi lab
University of the
Witwatersrand
• Howard Hughes
Medical Institute
(HHMI)