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Functional Analysis of the EspR Binding Sites Upstream of espR in Mycobacterium tuberculosis

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Abstract

The ESX-1 secretion system exports substrate proteins into host cells and is crucial for the pathogenesis of Mycobacterium tuberculosis. EspR is one of the characterized transcriptional regulators that modulates the ESX-1 system by binding the conserved EspR binding sites in the promoter of espA, the encoding gene of EspA, which is also a substrate protein of the ESX-1 system and is required for the ESX-1 activity. EspR is autoregulatory and conserved EspR binding sites are present upstream of espR. In this study, we showed that these EspR sites had varying affinities for EspR, with site B being the strongest one. Point mutations of the DNA sequence at site B abolished binding of EspR to oligonucleotides containing site B alone or with other sites, further suggesting that site B is a major binding site for EspR. Complementation studies showed that constructs containing espR, and the upstream intergenic region fully restored espR expression in a ΔespR mutant strain. Although recombinant strains with mutations at more than one EspR site showed minimal differences in espR expression, reduced expression of other EspR target genes was observed, suggesting that slight changes in EspR levels can have downstream regulatory effects. These findings contribute to our understanding of the regulation of the ESX-1 system.

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Acknowledgments

The authors thank Jeffery Cox for the Erdman strain and espR mutant. The authors also thank Sarah Fortune for proving the antibodies to EspA, and BEI Resources for antibodies to Mpt32 and GroEL2. This study was supported by the Natural Science Foundation of China (NFSC81071328 and NFSC81171540 to X.P.).

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Authors and Affiliations

  1. The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China

    Guangxiang Cao, Peipei Zhang & Xiuhua Pang

  2. Department of Microbiology, University of Texas Health Science Center at Tyler, Tyler, TX, 75708, USA

    Susan T. Howard

  3. School of Medicine, Shandong University, Jinan, 250012, China

    Guihua Hou

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  1. Guangxiang Cao
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  2. Susan T. Howard
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  3. Peipei Zhang
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  4. Guihua Hou
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  5. Xiuhua Pang
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Correspondence to Xiuhua Pang.

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Cao, G., Howard, S.T., Zhang, P. et al. Functional Analysis of the EspR Binding Sites Upstream of espR in Mycobacterium tuberculosis . Curr Microbiol 67, 572–579 (2013). https://doi.org/10.1007/s00284-013-0404-8

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  • DOI: https://doi.org/10.1007/s00284-013-0404-8

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