TABLE 1.
Bacterial strains and plasmids used.
| Bacteria/Plasmids | Description/Features | Source |
|---|---|---|
| Strains a | ||
| E. coli Top10 | Cloning and plasmid storage host | Invitrogen, Ltd |
| E. coli Top10 (pAN2) | Top10 carrying pAN2 methylation plasmid | Heap et al. (2007) |
| C. acetobutylicum ATCC 824 | WT parental strain | Rostock, Germany |
| C. acetobutylicum ΔpyrE::dif | ΔpyrE mutant created by ACE | Ehsaan et al. (2016) |
| C. acetobutylicum ΔpheA::dif | ΔpheA auxotrophic mutant | This study |
| C. acetobutylicum ΔpurD::dif | ΔpurD auxotrophic mutant | This study |
| C. acetobutylicum ΔargH::dif | ΔargH auxotrophic mutant | This study |
| C.acetobutylicum ΔpyrE::dif, ΔargH::dif | ΔpyrE & ΔargH double auxotroph | This study |
| C. acetobutylicum ΔΔpyrE::dif, ΔpurD::dif | ΔpyrE & ΔpurD double auxotroph | This study |
| C. acetobutylicum ΔpyrE::dif, ΔpurD::dif, ΔpheA::dif | ΔpyrE, ΔpurD, ΔpheA triple auxotroph | This study |
| C. acetobutylicum 824BO1 | bgaR-P bgal -tcdR integrated at the pyrE locus | This study |
| C. acetobutylicum 824BO2 | bgaR-P bgal -tcdR at pyrE locus, P tcdB -ctfA/B-adc at the purD locus, WT pheA locus | This study |
| C. acetobutylicum 824BO3 | bgaR-P bgal -tcdR at pyrE locus, P tcdB -ctfA/B-adc at the purD locus and P tcdB -sadh at pheA | This study |
| Plasmid | ||
| pMTL82254 | Modular reporter vector for clostridia; pBP1 Gram-positive replicon, ColE1+tra Gram-negative replicon, ermB antibiotic marker with catP reporter | Heap et al. (2009) |
| pMTL85121 | Modular vector for clostridia; pIM13 Gram-positive replicon, p15a + tra Gram-negative replicon, catP antibiotic marker and multiple cloning site | Heap et al. (2009) |
| pMTL_SC7515 | Knock-out plasmid for clostridia; pIM13 Gram-positive replicon, codA in backbone; ColE1 Gram-negative replicon, catP antibiotic marker and multiple cloning site | Cartman et al. (2012) |
| pMTL-KG146 | Novel Suicide Knock-out plasmid for clostridia; codA in backbone; ColE1 replicon, marker cassette comprising catP and pyrEflanked by identical repeats of random 29 bp sequence | This Study |
| pMTL-KG147 | Novel Suicide Knock-out plasmid for clostridia; codA in backbone; ColE1 replicon, marker cassette comprising catP and pyrE flanked by identical repeats of the predicted C. acetobutylicum dif sequence. | This Study |
| pMTL-KG147_argH | pMTL-KG147 with homology arms to mediate argH knock-out | This Study |
| pMTL-KG147_pheA | pMTL-KG147 with homology arms to mediate pheA knock-out | This Study |
| pMTL-KG147_purD | pMTL-KG147 with homology arms to mediate purD knock-out | This Study |
| pMTLME6C | pyrE complementation plasmid and backbone for purD and pheA complementation/expression plasmids pIM13/ColE1 replicon; CmR/TmR | Ng et al. (2013) |
| pMTL-BO1C | purD complementation plasmid based on pMTLME6C | This Study |
| pMTL-HZ1C | pheA complementation plasmid based on pMTLME6C | This Study |
| pMTL-HZ2C | argH complementation plasmid based on pMTLME6C | This Study |
| pMTL82254::PtcdB | The reporter plasmid pMTL82254 in which the catP gene has been placed under the transcriptional control of the P tcdB promoter. | This Study |
| pMTL82254::Pfdx | The reporter plasmid pMTL82254 in which the catP gene has been placed under the transcriptional control of the P fdx promoter. | This Study |
| pMTL82254::PbgaL | The reporter plasmid pMTL82254 in which the catP gene has been placed under the transcriptional control of the bgaR-Pbgal inducible promoter system. | This Study |
| pMTL-HZ13-HZ-tcdR | ACE complementation vector for the pyrE locus carrying tcdR under the control of the bgaR-Pbgal inducible promoter system. | This Study |
| pMTL-BO16-PtcdB-ctfAB-adc | ACE complementation vector for the purD locus carrying the genes necessary for acetone production, ctfAB and adc. | This Study |
| pMTL-HZ15-PtcdB-SadH | ACE complementation vector for the pheA locus carrying the gene necessary for isopropanol production, sadh. | This Study |
a
—While auxotrophic mutants with either a dif or ran scar were made, for consistency all the in-frame deletion mutants used in the study carry the dif sequence.