TABLE 1.
B. subtilis strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Genotype or characteristicsa | Reference, source, or derivationb |
|---|---|---|
| Strains | ||
| CU1065 | W168 trpC2 attSPβ | Lab stock |
| JH642 | W168 trpC2 pheA1 | Lab stock |
| ZB307A | W168 SPβc2Δ2::Tn917::pSK10Δ6; MLSr | 22 |
| HB4247 | JH642 sigW::kan | pKF90→JH642 |
| HB0020 | CU1065 sigW::kan | HB4247→CU1065 |
| HB0010 | CU1065 rsiW::kan | pXH51→CU1065 |
| HB0008 | CU1065 fosB::pAG4041 (Cmr) | pAG4041→CU1065 |
| HB8083 | ZB307A SPβ PfosB-cat-lacZ (MLSr Neor) | pAG3839→ZB307A |
| HB0052 | CU1065 SPβPfosB-cat-lacZ (MLSr Neor) | Transduction |
| HB0023 | HB0020 SPβ PfosB-cat-lacZ (MLSr Kanr) | Transduction |
| HB0012 | HB0010 SPβ PfosB-cat-lacZ (MLSr Kanr) | Transduction |
| HB0050 | CU1065 SPβ Pw-cat-lacZ (MLSr Neor) | 9 |
| HB0080 | CU1065::pMC82 (PxylA-fosB) | This work |
| HB0081 | HB0020::pMC82; sigW::kan (PxylA-fosB) | This work |
| HB0082 | HB0008::pMC82; fosB::cat(PxylA-fosB) | This work |
| Plasmids | ||
| pET17b | T7 RNAP driven overexpression plasmid | Novagen |
| pXT | Derivative of pDG1731; allows fusion of genes to xylose-inducible xylA promoter; integrates at thrC locus | T. Msadek |
| pJPM122 | Vector for integration of reporter fusions into SPβ (Apr Neor) | 18 |
| pJM114 | Kanamycin resistance cassette vector | 16 |
| pGEM-cat-3Zf(+) | Cloning vector | 20 |
| pDG783 | Kanamycin resistance cassette vector | 6 |
| pKF84 | Contains sigW | Lab stock |
| pKF90 | Contains sigW::kan | Construction analogous to sigW::MLS (reference 9) |
| pXH50 | pGEM-cat-3Zf(+) carrying rsiW | Lab stock |
| pXH51 | rsiW::kan in pGEM-cat-3Zf(+) | Lab stock |
| pAG4041 | pGEM-cat-3Zf(+) carrying internal fragment (330 bp) of fosB (PCR primers 275, 276) | This work |
| pAG3839 | pJPM122 carrying PfosB (PCR from 273, 274) | This work |
| pMC82 | pXT carrying fosB (PCR from 470, 309) | This work |
| pMC50 | pET17b with fosB (PCR from 308, 309) | This work |
| Oligonucleotidesc | ||
| 275 | CGGAATTCAGTTTCGCATTTGGATACA | fosB (internal; F) |
| 276 | CGTCTAGAAGCCTGTCTTGAAGGGTT | fosB (internal; R) |
| 273 | GCGAAGCTTCCGTTTTTGTTTACACTGGTA | fosB promoter F |
| 274 | GCGGATCCTAGCAAGTGATTGATTCCTTTTA | fosB promoter R |
| 470 | CGCGGATCCATTCATAATGGTCATGTT | fosB F |
| 309 | CCGGAATTCTGGTTGTGCTATCAAA | fosB R |
| 370 | CCCTTTACCAAAAGCTTTGCACC | fosB (for primer extension; R) |
| 308 | CTAGTCTAGACAGTCCGTTTTTGTT | fosB (for overproduction; F) |
a
MLS, macrolides-lincosamides-streptogramin B; Neo, neomycin; Kan, Kanamycin, Ap, ampicillin; Cm, chloramphenicol.
b
Arrows indicate transformation with either plasmid or chromosomal DNA as indicated.
c
Introduced restriction sites used for cloning are underlined. F, forward; R, reverse (relative to fosB).