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MOLECULAR BIOLOGY OF PATHOGENS

Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis

Krishna Kurthkoti, Umesh Varshney
Krishna Kurthkoti
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
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Umesh Varshney
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, IndiaJawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India
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  • For correspondence: varshney@mcbl.iisc.ernet.in
DOI: 10.1128/JB.00679-10
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ABSTRACT

Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.

FOOTNOTES

    • Received 13 June 2010.
    • Accepted 7 October 2010.
  • ↵▿ Published ahead of print on 22 October 2010.

  • American Society for Microbiology
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Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis
Krishna Kurthkoti, Umesh Varshney
Journal of Bacteriology Nov 2010, 192 (24) 6439-6446; DOI: 10.1128/JB.00679-10

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Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis
Krishna Kurthkoti, Umesh Varshney
Journal of Bacteriology Nov 2010, 192 (24) 6439-6446; DOI: 10.1128/JB.00679-10
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