TABLE 1.
Bacterial strains and plasmids used in this study
| Bacterial strain or plasmida | Genotype or insert | Reference or source |
|---|---|---|
| B. subtilis strains | ||
| 1A1 (= 168 trpC2) | trpC2 | BGSCd |
| CU741 | trpC2 leuC7 | 14 |
| OA101 | Prototroph isolated from CU741 | 14 |
| BEST23 | CU741 plus rnhC151::cat | pMIB15-N1 × CU741: Cm |
| BEST138 | OA101 plus rnhB21::neo | pMIB21LNEO × OA101: Nm |
| BEST206 | 1A1 plus ypdQ44::spc | pRNHA-4 × 1A1: Sp |
| BEST218 | 1A1 plus rnhB21::neo | BEST138 × 1A1: Nm |
| BEST220 | 1A1 plus rnhC151::cat | BEST21 × 1A1: Cm |
| BEST207 | ypdQ44::spc rnhB21::neo | BEST23 × BEST206: Cm |
| BEST208 | ypdQ44::spc rnhC151::cat | BEST138 × BEST206: Nm |
| E. coli strains | ||
| JA221 | F−hsdR hsdM+ trp leu lacY recA1 | 13 |
| MIC3037b | F−rnhA339::cat recC271 hsdR hsdM+ trp leu | 15 |
| MIC2067b | F−rnhA339::cat rnhB716::kam | This study |
| MIC1021b | F−rnhA-91 recB270 | 16 |
| Plasmids | ||
| pRNHA-1 | ypdQ (0.474-kb insert) in pGEM4 | This study |
| pUC2.2 | ypdQ (2.2-kb insert) in pUC18 | N. Ohtani |
| pRNHA-4 | ypdQ44::spc in pUC18 | This study |
| pMIB21 | rnhB in pBR322 | This study |
| pMIB21LNEO | rnhB21::neo in pBR322 | This study |
| pMIB15 | rnhC in pBR322 | This study |
| pMIB15-N1 | rnhC151::cat in pBR322 | This study |
| pGEM4 | 13 | |
| pBEST6 | Chimeric plasmid pBR322 and pGEM4 | This studyc |
| pBEST517A | spc cassette | 12a |
| pBEST512 | neo cassette | 14 |
| pBEST4F | cat cassette | 20 |
a
Selection conditions for both E. coli and B. subtilis were 5-μg/ml chloramphenicol (Cm) and 50-μg/ml spectinomycin (Sp). Selection for neomycin resistance transformants was on 25-μg/ml kanamycin (Km) for E. coli and 5-μg/ml neomycin (Nm) for B. subtilis.
b
Temperature-sensitive growth phenotype. Details for construction of MIC2067 will be published elsewhere.
c
AvaI-BglI fragment of pBR322 and PvuII-BglI fragment of pGEM4 were ligated to give multiple cloning sites to the pBR322 replicon.
d
Bacillus Genetic Stock Center (Ohio State University, Columbus).