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.