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Determining the Roles of a Conserved α-Helix in a Global Virulence Regulator from Staphylococcus aureus

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Abstract

SarA, a pleiotropic transcription regulator, is encoded by Staphylococcus aureus, a pathogenic bacterium. The expression of many virulence and non-virulence genes in S. aureus is modulated by this regulator. Structural studies have shown it to be a winged-helix DNA-binding protein carrying two monomers. Each SarA monomer is composed of five α-helices (α1–α5), three β-strands (β1–β3) and multiple loops. The putative DNA binding region of SarA is constituted with α3, α4, β2, and β3, whereas, its dimerization seems to occur using α1, α2, and α5. Interestingly, many SarA-like proteins are dimeric and use three or more helices for their dimerization. To clearly understand the roles of helix α1 in the dimerization, we have constructed and purified a SarA mutant (Δα1) that lacks helix α1. Our in-depth studies with Δα1 indicate that the helix α1 is critical for preserving the structure, DNA binding activity and thermodynamic stability of SarA. However, the helix has little affected its dimerization ability. Possible reasons for such anomaly have been discussed at length.

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Abbreviations

rSarA:

Recombinant SarA

Δα1:

rSarA lacking helix α1

PCR:

Polymerase chain reaction

IPTG:

Isopropyl-β-D-1-thiogalactopyranoside

PMSF:

Phenylmethane sulfonylfluoride

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

CD:

Circular dichroism

hla:

Promoter region of S. aureus α-toxin-encoding gene

Ni-NTA:

Nickel nitrilotriacetic acid

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Acknowledgements

The authors thank Mr. J. Guin, Mr. A. Banerjee, Mr. M. Das and Mr. J. Chatterjee for their excellent technical support. Mr. A. Mahapa and Mr. S. Mandal are the recipients of Senior Research Fellowship from the Council of Scientific and Industrial Research (Government of India, New Delhi). The work was supported by the grants from CSIR and BRNS/DAE (Government of India, Mumbai) to KS and SS, respectively.

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Authors and Affiliations

  1. Department of Biotechnology, Haldia Institute of Technology, PO-HIT, Dist-Purba, Medinipur, 721657, West Bengal, India

    Avisek Mahapa & Keya Sau

  2. Department of Biochemistry, Bose Institute, Kolkata, West Bengal, India

    Sukhendu Mandal, Debabrata Sinha & Subrata Sau

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  1. Avisek Mahapa
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  2. Sukhendu Mandal
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  3. Debabrata Sinha
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  4. Subrata Sau
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  5. Keya Sau
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Correspondence to Subrata Sau or Keya Sau.

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The present studies have not been performed with any human or animal.

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Mahapa, A., Mandal, S., Sinha, D. et al. Determining the Roles of a Conserved α-Helix in a Global Virulence Regulator from Staphylococcus aureus. Protein J 37, 103–112 (2018). https://doi.org/10.1007/s10930-018-9762-1

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