TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Genotype, antibiotic resistance, or relevant feature(s)a | Reference or source |
|---|---|---|
| Strains | ||
| B. subtilis | ||
| 1012 | leuA8 metB5 | 35 |
| 1012MM002 | 1012 PvegI ribF ermAM @ sacB | This study |
| 1012MM003 | 1012 PvegI FADI ermAM @ sacB | This study |
| 1012MM004 | 1012 ΔribCΩneo PvegI ribF ermAM @ sacB | This study |
| 1012MM006 | 1012 ΔribCΩneo PvegI FADI ermAM @ sacB | This study |
| 1012mro87 | Riboflavin-secreting ribO mutant (G37 GG→A37AA) | 23 |
| RB52 | Dcr-15 ribC820 guaC3 metC7 trpC2 | 34 |
| RB52MM100 | RB52 PvegI ribC ermAM @ sacB | This study |
| RB52MM110 | RB52 PvegI ribC820 ermAM @ sacB | This study |
| RB52p210 | RB52 ribC locus @ sacB | 12 |
| RB52p211 | RB52 ribC820 locus @ sacB | 12 |
| E. coli | ||
| BL21 | Used for gene cloning and expression | 40 |
| BL21MM01 | E. coli BL21 containing pMM01 | This study |
| BL21MM02 | E. coli BL21 containing pMM02 | This study |
| XL1-Blue | Used for gene cloning | 10 |
| Plasmids | ||
| pBluescriptII SK− | High-copy-number cloning vector, amp | Stratagene |
| pJF119HE | Low-copy-number expression vector, amp Ptac lacIq | 15 |
| pMM01 | pJF119HE containing a 1.143-kbp BamHI/EcoRI fragment carrying B. subtilis ribC | This study |
| pMM02 | pJF119HE containing a 1.143-kbp BamHI/EcoRI fragment carrying B. subtilis ribC820 | This study |
| pMM10 | pIXI12 containing a 1.073-kbp NdeI/EcoRI fragment carrying B. subtilis ribC | This study |
| pMM11 | pIXI12 containing a 1.073-kbp NdeI/EcoRI fragment carrying B. subtilis ribC820 | This study |
| pMM20 | pIXI12 containing a 0.960-kbp NdeI/EcoRI fragment carrying E. coli ribF | This study |
| pMM30 | pIXI12 containing a 0.955-kbp NdeI/EcoRI fragment carrying S. cerevisiae FADI | This study |
| pMM60 | pBluescriptSK− containing a 2.609-kbp XhoI/SpeI fragment carrying the B. subtilis ribC locus; ribC of this locus was replaced by a neomycin resistance cassette (21) | This study |
| pXI12 | pBR322 sacB5′ ermAM cryT RBS PvegI sacB3′ amp; B. subtilis integration/expression vector | 20 |
a
Abbreviations: amp, ampicillin resistance cassette; @ sacB, integration of genes by homologous double-crossover recombination at B. subtilis sacB (296°); Dcr-15, decoyinine resistance; ermAM, erythromycin resistance cassette; FADI, FAD-synthetase gene from S. cerevisiae; guaC3, 8-azaguanine resistance; leuA8, leucine auxotroph; metB5, methionine auxotroph; metC7, methionine auxotroph; neo, neomycin resistance cassette; PvegI, medium-strength vegI promoter; ribC, B. subtilis flavokinase/FAD-synthetase gene; ribC820, ribC gene with mutation G820A; ribF, E. coli flavokinase/FAD-synthetase gene; trpC2, tryptophan auxotroph.