Deletion mutagenesis in the streptomycin biosynthesis regulatory gene (strR) isolated from Iranian Streptomyces griseus PTCC1127 and cloning of the new construct in E. coli

StrR is a putative pathway specific regulator of streptomycin production in Streptomyces griseus. Because of finding new spo0j domain in strR by bioinformatics methods, the purpose of this study was to suggest another role for strR gene. This domain can be seen in proteins that are involved in initiation of sporulation and normal chromosome partitioning. So, 51 bps of strR in accordance to spo0j domain was deleted to investigate effects of deletion mutation in StrR functions. A unique specific procedure, including three consecutive PCRs, known as SOEing PCR were used here for site directed mutagenesis. Application and feasibility of this PCR was studied here. Bioinformatic studies were carried out for comparison of the sequence similarities between StrR and Spo0J proteins. An exclusive procedure, including three consecutive PCRs, was designed here in order to delete a 51 bp from the native strR. Other PCRs such as Semi-Nested PCR and RFLP PCR were used for strR isolation and structural confirmation of the isolated strR and deleted strR genes. Routine genetic engineering procedures were conducted in order to clone the native and deleted strR genes into E. coli. Obtained sequence information, from Conserved Domains Database (CDD) and Clustal W program, revealed that the StrR is similar to members of ParB family. Here, the strR was initially isolated from Iranian strain of Streptomyces griseus (PTCC1127). It was then confirmed as StrR by Semi-Nested PCR and RFLP-PCR. A 51 base pair region of the strR gene was deleted by specifically designed overlapped primers. A ten nucleotide overlap region was considered for a set of these primers. The recombinant cassette pSPMstrR?17 was constructed and cloned in E. coli. The sequencing results showed that a specific deletion is produced in the desired site and region in the strR gene. Therefore the designed three steps PCRs (known as SOing PCR) is a very rapid, cheap, and precise method for introducing such a deletion in any preferred gene.