Site-directed mutagenesis:
The role of the conserved amino acids present in the NPDKV motif and DGGGR motif of RpoE10was validated by PCR-based site-directed mutagenesis. The native rpoE10 gene was PCR-amplified with gene-specific primers (RpoE10F/RpoE10R, Supplemental Table S1) containing Bam HI and Hind III restriction enzyme sites in the forward and reverse primers, respectively. After purification, the amplicon of rpoE10 was cloned in pGEMT easy vector (Promega) by TA cloning method, and the recombinant plasmid (pAKVSS1, Supplemental Table S2) harboringrpoE10 gene was directly used for PCR-based mutagenesis. Restriction sites (Bam HI and Hind III) were required to generate mutated versions of rpoE10 after PCR-based mutagenesis and in-frame cloning in the expression vector, pMMB206. In the C-terminal, NPDKV motif was replaced by NAAAV motif in RpoE10(Mut1), and the DGGGR motif was replaced by AAAGR motif in RpoE10(Mut-2) using sets of complementary primers (RpoE10MOT1F/ RpoE10MOT1R for NPDKV motif and RpoE10MOT2F/ RpoE10MOT2R for DGGGR motif listed in Supplemental Table S2) having mutations flanked by unmodified nucleotides for the described amino acids. Site-directed mutagenesis was carried out by using QuikChange II site-directed mutagenesis kit (Agilent), and the PCR cycling conditions consisted of initial denaturation step, at 950C for 3 min followed by amplification step of 18 cycles, which was further composed of three sub-steps including denaturation at 940C for 1min, annealing at 460C for 1 min and extension at 680C for 12 min. The final extension step was at 680C for 30 min. Mutagenesis-PCR was performed in a 50 µl reaction mixture containing 1X Pfu Ultra HF DNA polymerase buffer, 1 mMdNTPs, 0.5 µM forward and reverse primers, 1% DMSO, and 2-unit Pfu Ultra HF DNA polymerase. After amplification 1 µl of the Dpn I restriction enzyme (10 U/µl) was directly added to each amplification reaction and kept at 370C for 3 h to digest the parental DNA. After that, 5-10 µl of each Dpn I-digested DNA sample was directly transformed in XL1-Blue super-competent cells. Recombinant plasmids having mutations in the NPDKV motif (pAKVSS2) and DGGGR motif (pAKVSS3) were confirmed by DNA sequencing using gene-specific primers (RpoE10F/RpoE10R, Supplemental Table S2). After mutations, modified inserts of rpoE10 were generated by restriction digestion withBam HI and Hind III and cloned in the similarly digested and eluted vector backbone of pMMB206.