TABLE 2.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Genotype or description | Source or reference |
|---|---|---|
| E. coli strains | ||
| DH5α | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 deoR Δ(lacZYA-argF)U169 λ (φ80d lacZΔM15) | Laboratory collection (CGSC#12384) |
| S17-1 | recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 | Laboratory collection |
| BL21(DE3) | Host strain for recombinant protein expression | Laboratory collection |
| R. sphaeroides strains | ||
| 2.4.1 | Wild type | ATCC 17023 |
| Δcls | 2.4.1 containing a kanamycin resistance cassette in place of the genomic cls | 11 |
| TyL1 | 2.4.1 murG::gfp-murG | This study |
| TyL2 | Δcls murG::gfp-murG | This study |
| Plasmids | ||
| pIND5 | A derivative of pIND4 in which the NcoI site is replaced with an NdeI site; pIND4 is an IPTG-inducible expression vector for R. sphaeroides; kanamycin resistance |
8 |
| pIND5TetR | A variant of pIND5 with a tetracycline resistance cassette in place of the kanamycin resistance cassette |
This study |
| gfp-pIND5 | pIND5 containing gfp | This study |
| gfp-murG-pIND5 | pIND5 containing gfp-murG | This study |
| gfp-pIND5TetR | pIND5TetR containing gfp | This study |
| gfp-murG-pIND5TetR | pIND5TetR containing gfp-murG | This study |
| gfp-murGR77E-pIND5 | pIND5 containing gfp-murGR77E | This study |
| gfp-murGL76E-pIND5 | pIND5 containing gfp-murGL76E | This study |
| pK18mobsacB | Suicide plasmid for allele exchange; kanamycin resistance | Laboratory collection |
| pK18mobsacBTetR | A variant of pK18mobsacB with a tetracycline resistance cassette in place of the kanamycin resistance cassette |
This study |
| 1k-gfp-murG-pK18 mobsacBTetR |
pK18mobsacBTetR containing murG and its 5′ upstream DNA sequence, with a gfp inserted between the two |
This study |
| pET28b(+) | A vector for expression of proteins with an N-terminal His tag/thrombin/T7 tag in the E. coli pET system |
Novagen |
| murG-pET28b(+) | pET28b(+) containing murG | This study |
| murGR77E-pET28b(+) | pET28b(+) containing murGR77E | This study |
| cls-pIND5sp | pIND5sp containing cls; pIND5sp is a variant of pIND5 with a spectinomycin resistance cassette in place of the kanamycin resistance cassette |
8 |