TABLE 3.
Primers used in this study
| Primer name | Sequencea | Use(s)b |
|---|---|---|
| oAA01 | TTTTTCACCTTAATGCTTTGCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGG | Amplification of pET28a(+) |
| oAA02 | ACCTCGGTGCGAAAAGATTCCACCACCACCACCACCACT | Amplification of pET28a(+) |
| oAA03 | TAAGAAGGAGATATACCATGCAAAGCATTAAGGTGAAAAAAGGCGAC | Amplification of lytE from bases 76–1002 |
| oAA04 | CAGTGGTGGTGGTGGTGGTGGAATCTTTTCGCACCGAGGTAACG | Amplification of lytE from bases 76–1002 |
| oAA05 | ACGCCGAAACAAGCGCTC | Check the pET28a-lytE-his plasmid |
| oAA06 | GCTAGTTATTGCTCAGCG | Check the pET28a-lytE-his plasmid |
| oAG18 | GTTGACTTTATCTACAAGGT | Check the insert cloned into pLOSS-Pspac-lytE |
| oAG19 | GGTACCAGTAGTTCACCAC | Check the insert cloned into pLOSS-Pspac-lytE |
| oAG81 | GAATCGCCTGGCTCACATCATC | Check B. subtilis mprF::TnYLB-1 |
| oAG82 | CGGCAAAGAACAGTCCCAAG | Check B. subtilis mprF::TnYLB-1 |
| oAG103 | CCTGTATGTCTGGCACTCAC | Amplification upstream of mprF |
| oAG104 | GCTCTAGATGGTCTCTCCAATCATATTC | Amplification upstream of mprF (EcoRI) |
| oAG105 | CGGAATTCTAAGACGGAGTCTTTTTTTATTTCG | Amplification downstream of mprF (XbaI) |
| oAG106 | CGGAGATGCGAGAGGGTTTC | Amplification downstream of mprF |
| oAG107 | TGCGGCATGCTCATATCCAG | Check B. subtilis ΔmprF |
| oAG108 | TGGGCCAATCAGTGGACAAG | Check B. subtilis ΔmprF |
| oAG122 | GCGTCTGCCTGAAATTAACC | Check B. subtilis Δmbl |
| oAG123 | GCAATCATTGCGGATGTTGC | Check B. subtilis Δmbl |
| oAG124 | CCATCATCTGGTGCGAAAGG | Check B. subtilis ΔponAc |
| oAG125 | CCGCAAAGCCGATTAATTGG | |
| oAG126 | TCTATTGGCGAGTGCTTC | Check B. subtilis ΔpbpFc |
| oAG127 | AGCATCGACTCCGTATTG | |
| oAG128 | TTATTCGGAATGGCGATGGG | Check B. subtilis ΔpbpDc |
| oAG129 | CCTTAATGGCTGCAGTTGAC | |
| oAG165 | CTAACCTGGCTGACATTCAC | Check B. subtilis gtaB::TnYLB-1 |
| oAG166 | TGATCAGGTCTTCGCAGTTG | Check B. subtilis gtaB::TnYLB-1 |
| oAG197 | GCTCTAGAGCACCCATTAGTTCAACAAACG | Amplification of cat (XbaI) |
| oAG198 | GCGGATCCAGTACAGTCGGCATTATCTC | Amplification of cat (BamHI) |
| oAG261 | GCCAGTGAATCGCTGGAAAG | Check B. subtilis ΔugtP |
| oAG262 | ATCTGGGAGCACCCGTCAAG | Check B. subtilis ΔugtP |
| oAG271 | GGGGTCGACAAAGGAGATTCCTAGGATGCTGATTAAAAAGAATGCT | Construction of pDR111-Physpank-mprF |
| oAG272 | GGGGCATGCTTGATGATATTGAAACCT | Construction of pDR111-Physpank-mprF |
| oAG300 | CAATCACGAAACAATAATTGG | Check the insert cloned into pDR111 |
| oAG301 | GTTGACTTTATCTACAAGGTG | Check the insert cloned into pDR111 |
| oAG308 | GGGGCTAGCAAAGGAGATTCCTAGGATGAAAACATTTATAAAAGAAAGAGG | Construction of pDR111-Physpank-ltaS (NheI) |
| oAG309 | CCGGCATGCCCGAATGTGGAATTTGC | Construction of pDR111-Physpank-ltaS (SphI) |
| oAG310 | GGGGCTAGCAAAGGAGATTCCTAGGATGAAGAAACTTTTTTCTTACAAAC | Construction of pDR111-Physpank-yfnI (NheI) |
| oAG311 | CCGGCATGCTGTAATGATATGAGAGAAAGC | Construction of pDR111-Physpank-yfnI (SphI) |
| oAG312 | GGGGCTAGCAAAGGAGATTCCTAGGATGCGAAAAACGTTTTTTTCGAAG | Construction of pDR111-Physpank-yqgS (NheI) |
| oAG313 | CCGGCATGCGAAAGCCTCCCGCTCACTTC | Construction of pDR111-Physpank-yqgS (SphI) |
| oAG314 | TCGTCCAGTGATTGGTTTCC | Check B. subtilis ΔyfnI |
| oAG315 | GACCGCTTTCATCTCTACCC | Check B. subtilis ΔyfnI |
| oAG316 | CGCCACTTTCTCCCTCATAC | Check B. subtilis ΔltaS |
| oAG317 | GTCAAATCGGGCGGGCAATC | Check B. subtilis ΔltaS |
| oAG318 | CGTATCGAGAGCCGGAGAAC | Check B. subtilis ΔyqgS |
| oAG319 | GAAGCTCTTTGCCGCTATGC | Check B. subtilis ΔyqgS |
| oAG340 | GTCACATGAAGTCAAGACTATT | Check B. subtilis ΔlytE |
| oAG341 | ACGGTTTATCAAGGAAGGACTC | Check B. subtilis ΔlytE |
| oAG348 | GTGGCGGCCGCATGAAAAAGCAAATCATTACAG | Construction of pLOSS-Pspac-lytE (NotI) |
| oAG349 | CCGGGATCCGATTGCCCTTTATGAAAATAAG | Construction of pLOSS-Pspac-lytE (BamHI) |
| oAG350 | GTCTGTGCTTGAGGATAAGG | Amplification upstream of lytE for construction of ΔlytE |
| oAG351 | GGGTCTAGACTGCTGTCGTAGCTGTAATG | Amplification upstream of lytE (XbaI) for construction of ΔlytE |
| oAG352 | CCGGGATCCGCGAAAAGATTCTAATTTTTAG | Amplification downstream of lytE (BamHI) for construction of ΔlytE |
| oAG353 | GATCCGTTTGCGTGTTTC | Amplification downstream of lytE for construction of ΔlytE |
| oAG354 | GGGGAATTCGGCGGAACGACAACTTCAG | Construction of pMUTin-′lytE-his, amplification of the 3′ terminus of ′lytE (no stop codon) |
| oAG355 | GGGCTCGAGGAATCTTTTCGCACCGAGG | Construction of pMUTin-′lytE-his, amplification of the 3′ terminus of ′lytE (no stop codon) |
| oAG360 | GGCGAATTCGATTTATTCTCTAAAGACCACG | Construction of pMUTin-′yfnI-his, amplification of the 3′ terminus of ′yfnI (no stop codon) |
| oAG361 | GGGCTCGAGTTTGATTTCTTTCTCCTTGCCG | Construction of pMUTin-′yfnI-his, amplification of the 3′ terminus of ′yfnI (no stop codon) |
| oAG366 | GGCGAATTCCGAAACGGAGACTTTATTTCAC | Construction of pMUTin-′ltaS-his, amplification of the 3′ terminus of ′ltaS (no stop codon) |
| oAG367 | GGGCTCGAGTTTATCTTCGTTATCCTTTGAC | Construction of pMUTin-′ltaS-his, amplification of the 3′ terminus of ′ltaS (no stop codon) |
| oAG368 | CAAGGTGTGGCATAATGT | Check integration into pMUTinHis |
| oAG369 | GACATCCAGAGGCACTTC | Check integration in pMUTinHis |
| oAG412 | CACCTGGTTTTCTGCTATAGT | Check B. subtilis ΔgtaB |
| oAG413 | GTTTAACTCTACAATCAGTG | Check B. subtilis ΔgtaB |
| PhleoXbaI-fw | GCTCTAGATCTTCCTTCAGGTTATGAC | Amplification of phleo from pIC22 (XbaI) |
| PhleoEcoRI-rev | CGAATTCCGCGCCCGATTGCTGAAC | Amplification of phleo from pIC22 (EcoRI) |
a
Restriction enzyme sites are in boldface type.
b
cat, chloramphenicol resistance; phleo, phleomycin resistance cassette. Pspac and Physpank are promoters inducible with IPTG, the latter of which is known to be stronger. TnYLB-1 is a transposon associated with kanamycin resistance.
c
See reference 12.