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

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 sourc...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Extremophiles : life under extreme conditions 2019-01, Vol.23 (1), p.59-67
Hauptverfasser: Mirfeizollahi, Azadeh, Yakhchali, Bagher, Deldar, Ali Asghar, Karkhane, Ali Asghar
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung: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.
ISSN:1431-0651
1433-4909
DOI:10.1007/s00792-018-1060-5