TABLE 1.
Strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Properties | Reference(s)/source |
|---|---|---|
| E. coli | ||
| DH5α | λ− φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 | 14 |
| HB101 | F−hsdS20 (rB− mB−) supE44 ara-14 galK-2 lacY1 proA2 rpsL20 xyl-5 mtl-1 recA13 Kanr oriColE1 RK2-Mob+ RK2-Tra+ mH-1 with plasmid pRK2013 | 4, 11, 22 |
| G. oxydans | ||
| N44-1 | Cefr; derived from G. oxydans IFO 3293 | 42 |
| N44-1 mgdH::kan | Cefr Kanr; chromosomal inactivation of mgdH | DSM Nutritional Products, Kaiseraugst, Switzerland |
| N44-1 ΔmgdH | Cefr; chromosomal deletion of mgdH | This study |
| N44-1 ΔmgdH sgdH::kan | Cefr Kanr; chromosomal inactivation of mgdH and sgdH | This study |
| Plasmids | ||
| pBBR1MCS2 | KmrlacI lacZ Plac; pBBR1 replicon | 19 |
| pBBR1MCS4 | AprlacI lacZ Plac, pBBR1 replicon | 20 |
| pSUP202 | Apr Cmr Tcr; oriT from RP4; cannot replicate in G. oxydans | 41 |
| pSUP202 mgdH::kan | Apr Cmr Tcr Kmr; pSUP202 derivative containing an mgdH gene in which an internal part is replaced by a kanamycin resistance gene | DSM Nutritional Products, Kaiseraugst, Switzerland |
| pSUP202 sgdh::kan | Apr Cmr Tcr Kmr; pSUP202 derivative containing an sgdH gene in which an internal part is replaced by a kanamycin resistance gene | This study |
| pK19mobsacB | Kmr; vector for allelic exchange; cannot replicate in G. oxydans | 34 |
| pK19mobsacBΔmgdH | Kmr; pK19mobsacB derivative used for marker-free deletion of mgdH gene; contains an overlap extension PCR product covering the regions up- and downstream of mgdH | |
| Oligonucleotides | ||
| sgdh_GoxKpnIF | 5′-GGA GAG GTG AGG TAC CAT GCC TGC CCC-3′ | Amplification of sgdH; KpnI site; proof of sgdH disruption |
| sgdh_GoxSacIR | 5′-GGA TCA TCA GGA GCT CGT CTG TCC AGA CTG G-3′ | Amplification of sgdH; SacI site; proof of sgdH disruption |
| coPCR-gdh-5F | 5′-GCA CGG CCG GGC ATG CCA CTT GTG GC-3′ | Disruption of mgdH; SphI site |
| coPCR-gdh-5R | 5′-CCC ATC CAC TAA ACT TAA ACA CGT GCC GCC GAT CTG GCG GGA GAC G-3′ | Disruption of mgdH; inner primer |
| coPCR-gdh-3F | 5′-TGT TTA AGT TTA GTG GAT GGG CTC TCC ACG GCT GGC AAC CTC GGC TTC C-3′ | Disruption of mgdH; inner primer |
| coPCR-gdh-3R | 5′-CCG GTT GTG AGG CGGTCG ACT GGC AGT CGG TGG-3′ | Disruption of mgdH; SalI site |
| gdh-control-5 (for) | 5′-CGG GCA GGA ATG TTG AGC TGT AGG-3′ | Proof of mgdH disruption |
| gdh-control-3 (rev) | 5′-CCC TCG CCG TAA ACG ATT GGT CC-3′ | Proof of mgdH disruption |