TABLE 2.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Description | Reference or source |
|---|---|---|
| C. glutamicum strains | ||
| WT | ATCC13032; biotin-auxotrophic WT strain | DSMZ |
| ΔpknG | WT derivative with an in-frame deletion of pknG (cg3046) | 6 |
| ΔglnH | WT derivative with an in-frame deletion of glnH (cg3045) | 6 |
| ΔglnX2 | WT derivative with an in-frame deletion of glnX (cg3044); 59 codons at the 5′ end and 142 codons at the 3′ end were kept; the 301 codons in between were deleted and replaced by an artificial 21-bp sequence | This work |
| ΔglnX-glnH-pknG | WT derivative with in-frame deletion of glnX-glnH-pknG (cg3044-cg3046) | This work |
| ΔodhI | WT derivative with in-frame deletion of odhI (cg1630) | 6 |
| ΔglnX2 ΔodhI | WT derivative with in-frame deletions of odhI (cg1630) and glnX (cg3044) | This work |
| E. coli strains | ||
| DH5α | F- supE44 ΔlacU169 (Φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 53 |
| BL21(DE3) | F- ompT hsdSB(rB-mB-) gal dcm (λcIts857) ind1 Sam7 nin5 lacUV5-T7 Gen 1 | 67 |
| TG1 | F- supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK-mK-)(traD36 proAB + lacIq lacZΔM15) | Lucigen Corporation |
| Plasmids | ||
| pK19mobsacB | KanR; suicide vector for allelic exchange in C. glutamicum; oriVEc oriT sacB | 68 |
| pK19mobsacB-glnX2 | KanR; pK19mobsacB derivative containing PCR products (primer ΔglnX_1–4) covering the up- and downstream regions of the glnX gene | This work |
| pK19mobsacB-glnX-glnH-pknG | KanR; pK19mobsacB derivative containing PCR products (primer ΔglnX 1+2 and ΔpknG 3+4) covering the up- and downstream regions of the glnX and pknG genes, respectively | This work |
| pK19mobsacB-odhI | KanR; pK19mobsacB derivative containing PCR products covering the up- and downstream regions of the odhI gene | 6 |
| pET-TEV | KanR; pET28b derivative for protein overproduction in E. coli, contains a His10-tag and a TEV cleavage site | 69 |
| pET-TEV-odhI | KanR; pET-TEV derivative for overproduction of OdhI with an N-terminal His10-tag and TEV cleavage site | 8 |
| pET-TEV-odhI-R87A | KanR; pET-TEV-odhI derivative, encodes OdhI-R87A with an N-terminal His10-tag and TEV cleavage site | This work |
| pET-TEV-odhI-R87P | KanR; pET-TEV-odhI derivative, encodes OdhI-R87P with an N-terminal His10-tag and TEV cleavage site | This work |
| pET-TEV-glnHΔSP | KanR; pET-TEV derivative for overproduction of GlnH lacking the signal peptide and the lipobox motif with an N-terminal His10-tag and TEV cleavage site | This work |
| pET-TEV-glnHcore | KanR; pET-TEV derivative for overproduction of GlnH lacking flexible N- and C-terminal parts (amino acid residues 48–334 of GlnH) with an N-terminal His10-tag and TEV cleavage site | This work |
| pET-TEV-glnHcore-S163T-T165S | KanR; pET-TEV-glnHcore derivative, encodes GlnHcore-S163T-T165S with an N-terminal His10-tag and TEV cleavage site | This work |
| pJC1 | KanR; E. coli-C. glutamicum shuttle vector | 70 |
| pJC1-glnXProm | KanR; pJC1 derivative carrying the glnX gene, including its native promoter region (383 bp upstream of the transcriptional start site, which is also the translational start site) | This work |
| pEKEx2 | KanR, E. coli/C. glutamicum shuttle vector; Ptac; lacIq; oriCg from pBL1; oriEc ColE1 from pUC18 | 71 |
| pEKEx2-pknG | KanR, pEKEx2 derivative encodes PknG with a C-terminal Strep-tag, contains the native RBSa of pknG | 6 |
| pAN6 | KanR; C. glutamicum/E. coli shuttle vector for regulated gene expression | 72 |
| pAN6-glnH | KanR; pAN6 derivative encodes GlnH with a C-terminal Strep-tag | This work |
| pAN6-glnH-C27A | KanR; pAN6-glnH derivative, encodes GlnH-C27A with a C-terminal Strep | This work |
| pPREx2 | KanR; pPBEx2 derivative (Ptac, lacIq, oriC.g from pBL1; oriEc ColE1 from pUC18), with a consensus RBS (AAGGAG) for C. glutamicum | 35 |
| pPREx2-glnX-(F1 to F7)-phoA | KanR; pPREx2 derivative for expression of GlnX variants of different lengths (F1–F7) fused to the alkaline phosphatase (phoA) | This work |
| pPREx2-glnX-(F1 to F7)-lacZ | KanR; pPREx2 derivative for expression of GlnX variants of different lengths (F1–F7) fused to the β-galactosidase (lacZ) | This work |
| pMA632-Ex | AmpR; E. coli plasmid for topology determination of membrane proteins via phoA-lacZ fusions (Ptac, lacIq; with RBS) used as template for phoA amplification including linker sequence | Provided by Lothar Eggeling, Forschungszentrum Jülich |
a
RBS, ribosomal binding site.