Table 3.
List of primers, plasmids, and strains
| Category | ID | Sequence (5’ to 3’) or Construct | Purpose | Source |
|---|---|---|---|---|
| Primer | oJW902 | AAAGAGGCGCTTTTGACGTG | Verify relA::cat genotype | |
| Primer | oJW903 | TTGTTGACCCGGGACATGGA | Verify relA::cat genotype | |
| Primer | oJW2298 | ACTCAGTGGCACGAACTTGT | Amplify xpt::kan | |
| Primer | oJW2303 | GCGGATGGAATAATAGTCGTGAAAG | Amplify xpt::kan | |
| Primer | oJW492 | GCTTTGTTAGCAGCCGGATCAG | Amplify pLIC-trPC-HA insert | |
| Primer | oJW1124 | CCGCACCTGTGGCGCCGGTG | Amplify pLIC-trPC-HA insert | |
| Primer | oJW1974 | TACTTCCAATCCAATGCAATGGAAGCACTGAAACGGAA | Amplify B. subtilis xpt for LIC cloning | |
| Primer | oJW1975 | TTATCCACTTCCAATGTTATTATGAATGAACCTCCTGTACGA | Amplify B. subtilis xpt for LIC cloning | |
| Primer | oJW3364 | CTGTCCATTTGCCAATAAATCATCGATAATCAGCACATGATCCT | QuikChange mutagenesis for XPRT F126L | |
| Primer | oJW3365 | AGGATCATGTGCTGATTATCGATGATTTATTGGCAAATGGACAG | QuikChange mutagenesis for XPRT F126L | |
| Primer | oJW3366 | GCCTGTCCATTTGCCAAAATATCATCGATAATCAGCACA | QuikChange mutagenesis for XPRT F126I | |
| Primer | oJW3367 | TGTGCTGATTATCGATGATATTTTGGCAAATGGACAGGC | QuikChange mutagenesis for XPRT F126I | |
| Primer | oJW3368 | GCGCTGCCTGTCCAGCTGCCAAAAAATCATCGATAATCAGCA | QuikChange mutagenesis for XPRT N129A | |
| Primer | oJW3369 | TGCTGATTATCGATGATTTTTTGGCAGCTGGACAGGCAGCGC | QuikChange mutagenesis for XPRT N129A | |
| Primer | oJW3372 | TCCCGGCTGAAATGACTCTTCAATAACAATGCCGATTC | QuikChange mutagenesis for XPRT K156E | |
| Primer | oJW3373 | GAATCGGCATTGTTATTGAAGAGTCATTTCAGCCGGGA | QuikChange mutagenesis for XPRT K156E | |
| Primer | oJW3926 | GATTTATGCTTTGCCGCGAAGAC | Megaprimer mutagenesis for XPRT R80A | |
| Primer | oJW3927 | GATTTATGCTTTTCCGCGAAGAC | Megaprimer mutagenesis for XPRT R80E | |
| Plasmid | pMCSG7 | pLIC-trPC-HA | Expression vector | [43] |
| Plasmid | pJW536 | pLIC-trPC-HA B. subtilis xprT | Expression of B. subtilis XPRT | This work |
| Plasmid | pJW725 | pLIC-trPC-HA B. subtilis xprT (F126I) | Expression of B. subtilis XPRT F126L | This work |
| Plasmid | pJW726 | pLIC-trPC-HA B. subtilis xprT (F126L) | Expression of B. subtilis XPRT F126I | This work |
| Plasmid | pJW727 | pLIC-trPC-HA B. subtilis xprT (N129A) | Expression of B. subtilis XPRT N129A | This work |
| Plasmid | pJW728 | pLIC-trPC-HA B. subtilis xprT (K156E) | Expression of B. subtilis XPRT K156E | This work |
| Plasmid | pJW729 | pLIC-trPC-HA B. subtilis xprT (R80A) | Expression of B. subtilis XPRT R80A | This work |
| Plasmid | pJW730 | pLIC-trPC-HA B. subtilis xprT (R80E) | Expression of B. subtilis XPRT R80E | This work |
| Strain | E. coli BL21(DE3) | Protein expression strain | NEB | |
| Strain | JDW2144 | Bacillus subtilis NCIB 3610 lacking pBS32 | Wild type B. subtilis | [55] |
| Strain | JDW2231 | Bacillus subtilis NCIB 3610 lacking pBS32 ΔyjbM ΔywaC relA::mls | (p)ppGpp0 B. subtilis | [56] |
| Strain | JDW4008 | JDW2144 xprT::kan | Δxpt | This work |
| Strain | JDW4022 | Bacillus subtilis NCIB 3610 lacking pBS32 ΔyjbM ΔywaC relA::cat xprT::kan | (p)ppGpp0 Δxpt | This work |
| Strain | CF5766 | BL21(DE3)/pHM504 (pT7 GppA) Ap | Express GppA for ppGpp synthesis | [40] |
| Strain | CF7955 | BL21(DE3)/pHM138 1 (pUM99) (pT7 RelSeq1–385H) Ap | Express RelSeq for (p)ppGpp synthesis | [40] |