Brevibacillus-Based Site-Specific Mutagenesis of S. pneumoniae

The off-the-shelf Brevibacillus cloning system (Takara Bio, Kusatsu, Shiga, Japan) was used to construct S. pneumoniae D39 rTpiA proteins with site-specific amino acid substitutions, as described in our previous study [8 (link)]. To introduce site-specific mutations into the tpiA gene, an inverse polymerase chain reaction (PCR) was performed using the primers shown in Table 1 with plasmid pBIC2-tpiA [8 (link)], where the open reading frame of the tpiA gene, except for the start codon, was inserted as the template. PCR was performed using KOD One PCR Master Mix (Toyobo, Osaka, Japan), according to the manufacturer’s instructions. To self-ligate the PCR fragments, aliquots of diluted PCR-amplified products were treated with T4 polynucleotide kinase (Takara Bio) and Ligation-High version 2 (Toyobo) and incubated at 16 °C for at least 2 h. Thereafter, B. choshinensis HPD31-SP3 was transformed with the self-ligating plasmid, and transformants were selected on MTNm plates. Colonies of the resultant transformants were inoculated into 2SYNm broth and cultured overnight at 37 °C with shaking at 120 rpm. Bacterial cells were collected, and plasmids were extracted using a QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany). The DNA inserted into the plasmid was sequenced by Eurofins Genomics (Tokyo, Japan) using the primers for sequencing (Table 1).