, Volume 23, Issue 1, pp 59–67 | Cite as

In silico and experimental improvement of bacteriorhodopsin production in Halobacterium salinarum R1 by increasing DNA-binding affinity of Bat through Q661R/Q665R substitutions in HTH motif

  • Azadeh Mirfeizollahi
  • Bagher YakhchaliEmail author
  • Ali Asghar Deldar
  • Ali Asghar Karkhane
Original Paper


DNA-binding motif of bacterioopsin activator (Bat) protein is a Helix–Turn–Helix motif, which binds to bop promoter and induces bacterioopsin (Bop) expression under light and low oxygen tension. Bacterioopsin is linked to retinal to produce bacteriorhodopsin (BR), which in turn supplies energy source in Halobacterium salinarum. In this study, effect of Bat HTH motif–promoter DNA interaction on bacterioopsin (Bop) expression was investigated using in silico and experimental approaches. Molecular docking showed that the most stable DNA–protein complex was generated by Q661R/Q665R mutant. Based on the in silico analysis, HTH motif was mutated using site-directed mutagenesis and Hbt. salinarum recombinant strains were developed by introduction of mutant bat genes. Double positively charged amino acid substitutions (Q661R/Q665R) in second helix of HTH motif increased whereas deletion of this region decreased BR production. However, other single substitutions (Q665R and Q661H) did not change BR production. These findings represent key role of HTH motif stability for DNA binding and regulation of bacterioopsin (Bop) expression and bacteriorhodopsin (BR) production independent of environmental condition.


Amino acid substitution Bacteriorhodopsin Regulatory protein Halobacterium salinarum HTH motif Molecular docking 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Azadeh Mirfeizollahi
    • 1
  • Bagher Yakhchali
    • 1
    Email author
  • Ali Asghar Deldar
    • 2
  • Ali Asghar Karkhane
    • 1
  1. 1.Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and BiotechnologyTehranIran
  2. 2.Department of Bioscience, Institute of Bioscience and BiotechnologyMalek Ashtar University of TechnologyTehranIran

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