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 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.

The association of seminal plasma antioxidant levels and sperm chromatin status with genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) in infertile men with oligoasthenoteratozoospermia.

In this study we aimed to examine the effects of genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) on GST activity, seminal oxidative stress and sperm chromatin status in infertile men with oligoasthenoteratozoospermia (OAT). The study population (n=121) consisted of 95 infertile men with OAT and 26 controls with normozoospermia. Multiplex polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were utilized to detect the aforesaid genetic variants. We measured GST activity and total antioxidant capacity (TAC) of seminal plasma by spectrophotometry. Sperm chromatin integrity and maturity were assessed using toluidine blue and chromomycin A3 (CMA3-positive sperm) staining, respectively. The analysis showed that subgroups of GSTM1 null and GSTP1 C/T+T/T genotypes in comparison with GSTM1 present and GSTP1 wild type (C/C) genotypes did not have statistically significant differences in both OAT or normozoospermic men considering sperm concentration and motility, percentage of CMA3-positive sperm, seminal plasma TAC, sperm chromatin integrity and GST activity. Thus, the findings of our study suggest that there are no significant associations between GSTM1 and GSTP1 polymorphisms and sperm parameters at conventional or at molecular levels including OS status, sperm chromatin integrity or maturity in Iranian infertile men with OAT and normozoospermia. However, these polymorphisms could be related to the fertility status of the studied population but not evaluated in this study.