Table 3.
Bacterial strains, plasmids and oligonucleotides.
| Strain/plasmid | Relevant genotype/features | Source/construction |
|---|---|---|
| B. subtilis | ||
| 168 | trpC2 | Laboratory stock |
| 1801 | trpC2 chr:: pJSIZDpble (Pspac‐ftsZ ble) | (Marston et al., 1998) |
| 3941 | trpC2 pbpB1–1104 b lacI aph‐A3 Pspac pbpB | pSG5601→168 |
| 3105 | trpC2 pbpC1–768 cat Pxyl gfp‐pbpC | (Scheffers et al., 2004) |
| 4000 | trpC2 pbpB1–1104lacI aph‐A3 Pspac pbpB *a | pSG5662→168 |
| 4001 | trpC2 pbpB ( S309A ) | This work |
| 4002 | trpC2 amyE Ω (spc Pxyl gfp‐pbpB) | pSG5663→168 |
| 4003 | trpC2 amyE Ω (spc Pxyl gfp‐pbpB * ) | pSG5664→168 |
| 4004 | trpC2 lacI aph‐A3 Pspac pbpB amyE Ω (spc Pxyl gfp‐pbpB * ) | This work |
| 4005 | trpC2 pbpB * pbpC1–768 cat Pxyl gfp‐pbpC | 3105→4001 |
| 4006 | trpC2 pbpB * amyE Ω (spc Pxyl gfp‐pbpB) | 4002→4001 |
| 4007 | trpC2 pbpC aph‐A3 pbpC739–2004 | pSG5665→168 |
| 4008 | trpC2 pbpC * aph‐A3 pbpC739–2004 | pSG5666→168 |
| 4009 | trpC2 pbpC * pbpB * amyE Ω (spc Pxyl gfp‐pbpB) | 4008→ 4006 |
| PS1805′ | trpC2 ΔpbpE::erm (moved into168CA) | (Popham and Setlow, 1993) |
| PS1869′ | trpC2 ΔpbpF::erm | (Popham and Setlow, 1993) |
| PS2022′ | trpC2 ΔpbpD::erm | (Popham and Setlow, 1994) |
| PS2062′ | trpC2 ΔponA::spc | (Popham and Setlow, 1995) |
| PS2352′ | trpC2 pbpC::cat | (Murray et al., 1996) |
| PS2465′ | trpC2 pbpA::(pTMM4) cat | (Murray et al., 1997) |
| HB0031′ | trpC2 ΔsigM::kan | (Luo and Helmann, 2012) |
| BSU2007′ | 168 sigMWXYZV ylaC (Δ7ECF) | (Asai et al., 2008) |
| DPVB133′ | trpC2 ΔpbpH::spc | (Wei et al., 2003) |
| 4011 | trpC2 ΔpbpE::erm | PS1805→168 |
| 4012 | trpC2 ΔpbpF::erm | PS1869→168 |
| 4013 | trpC2 ΔpbpD::erm | PS2022→168 |
| 4014 | trpC2 ΔponA::spc | PS2062→168 |
| 4015 | trpC2 pbpC::cat | PS2352→168 |
| 4016 | trpC2 pbpA::(pTMM4) cat | PS2465→168 |
| 4017 | trpC2 ΔpbpH::spc | DPVB133→168 |
| 4018 | trpC2 pbpB * ΔpbpE::erm | 4011→ 4001 |
| 4019 | trpC2 pbpB * ΔpbpF::erm | 4012→ 4001 |
| 4020 | trpC2 pbpB * ΔpbpD::erm | 4013→ 4001 |
| 4021 | trpC2 pbpB * ΔponA::spc | 4014→ 4001 |
| 4022 | trpC2 pbpB * pbpC::cat | 4015→ 4001 |
| 4023 | trpC2 pbpB * pbpA::(pTMM4) cat | 4016→ 4001 |
| 4024 | trpC2 pbpB * ΔpbpH::spc | 4017→ 4001 |
| KS50 | trpC2 amyE Ω (spc Pspank pbpC) | pKS4→168 |
| KS51 | trpC2 pbpB * amyE Ω (spc Pspank pbpC) | pKS4→4001 |
| KS52 | trpC2 amyE Ω (spc Pspank pbpC) pbpC::cat | 4015→KS50 |
| KS53 | trpC2 amyE Ω (spc Pspank pbpC * ) | pKS5→168 |
| KS54 | trpC2 pbpB * amyE Ω (spc Pspank pbpC * ) | pKS5→4001 |
| KS55 | trpC2 pbpC::cat amyE Ω (spc Pspank pbpC * ) | pKS5→4015 |
| RD300 | trpC2 ΔsigM::kan | HB0031→168 |
| RD301 | trpC2 ΔpbpC::cat ΔsigM::kan | RD300→4015 |
| E. coli | ||
| DH 5α | F‐endA1 hsdR17 supE44 thi‐1 λ‐recA1 gyrA96 relA1 Δ(lacZYA‐argF) U169 Φ80 δlacZ ΔM15 | Laboratory stock |
| NM554 | recA13 araD139 Δ(ara‐leu)7696 Δ(lac)l7A galU galK hsdR rpsL (Strr) mcrA mcrB | Laboratory stock |
| Plasmids | ||
| pSG441 | bla aph‐A3 lacI Pspac | (Illing and Errington, 1991) |
| pSG5601 | bla aph‐A3 lacI Pspac pbpB1–1104 | This work |
| pSG5662 | bla aph‐A3 lacI Pspac pbpB1–1104 * | This work |
| pQE31 | ori (ColE1) bla T5 promotor/lac operator | Qiagen |
| pSG5670 | pQE31 pbpB | This work |
| pSG5671 | pQE31 pbpB * | This work |
| pSG1729 | bla amyE3′ spc Pxyl gfp amyE5′ | (Lewis and Marston, 1999) |
| pSG5663 | bla amyE3′ spc Pxyl gfp‐pbpB amyE5′ | This work |
| pSG5664 | bla amyE3′ spc Pxyl gfp‐pbpB * amyE5′ | This work |
| pSG5045 | bla cat Pxyl gfp‐pbpC1–768 | (Scheffers et al., 2004) |
| pUK19 | aph‐A3 bla | Gift from W. G. Haldenwang |
| pSG5665 | aph‐A3 bla pbpC739–2004 | This work |
| pSG5666 | aph‐A3 bla pbpC739–2004 * | This work |
| pDR111 | bla amyE3′ spc Pspank lacI amyE5′ | (Vavrová et al., 2010) |
| pKS4 | bla amyE3′spc Pspank pbpC lacI amyE5′ | This work |
| pKS5 | bla amyE3′ spc Pspank Ω pbpC * lacI amyE5′ | This work |
| Oligonucleotides d | ||
| PBPB‐F | GATggatCCAAAAAAGAATAAATTTATGAATAGAGGAGC | |
| PBPB‐R | ACTggtaccTTAATCAGGATTTTTAAACTTAACCTTG | |
| SDM B‐F | GAACCCGGGGCCACGATGAAGATCTTTACACTCGC | |
| SDM B‐R | CGTGGCCCCGGGTTCATACGCATATGAAATCAAATC | |
| pbpB‐1 | GCAtctagaAGCGCATTATGGACATTG | |
| pbpB‐2 | CAGTTTgcatgcAGCGACATTCGACGACCTTAG | |
| pbpB‐3 | ATCggatccCCAAAAAAGAATAAATTTATGAATAGAGGAGCAGC | |
| pbpB‐4 | ATCgtcgacTTTAATCAGGATTTTTAAACTTAACCTTGATTACGG | |
| yllA‐F | AGAATTCAAAATAGCATTAAGCCGCTTCTTGCG | |
| ftsL‐R | GCATTTGAATCATTCCTGTATGTTTTTCACTTTTTTATCTTTTAAATTCAAGCCG | |
| pbpC‐1 | ATCggatccTCAGCTTCAAGAATACTGCTGTGCTG | |
| pbpC‐2 | ATGctgcagTTTAATTCGATTGAAATTGCTTTTTCGCTTTCTC | |
| SDM C‐F | GACATACGCGCCAGGTGCTACCATTAAACCGATTGCGGC | |
| SDM C‐R | GGTTTAATGGTAGCACCTGGCGCGTATGTCTTATTGAAT | |
| pbpC‐3 | GATgcatgcGAGGGGAAAGTCATGTTAAAAAAGTGTATTCTACTAG | |
| pbpC‐4 | ATCgcatgcGCCCCCTTACTAGTTCATTCGGCCTCAGATCC |
a. Numbers indicate the base coordinates of the gene cloned from http://genolist.pasteur.fr/SubtiList/.
b. Numerical values indicate the region of coding sequence present (starting from the initiation codon).
a *indicates that the active site of the PBP was mutated, in the case of pbpB this corresponds to S309A, where the codon corresponding to residue S 309 of protein was mutated to A. For pbpC, serine 410 was mutated to alanine.
d. Lower case letters show where restriction sites were introduced into the oligonucleotide sequence.