TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Relevant characteristicsa | Source and/or reference |
|---|---|---|
| Escherichia coli strains | ||
| DH5α | F−endA1 hsdR17(rK− mK+) supE44 thi-1 λ−gyrA96 relA1 Δ(lacZYA argF) U169 (φ80 lacZΔM15) recA | Roche Diagnostics |
| SG13009(pREP4) | F−his pyrD Δlon-100 rpsL (pREP4) | Qiagen (11) |
| BL21(DE3) | F−ompT hsdS (rB− mB−) gal dcm (DE3) | Novagen |
| Clostridium cellulolyticum strains | ||
| ATCC 35319 | Wild type | 25 |
| D3K | ATCC 35319(pD3K) | This study |
| 952K | ATCC 35319(p952K) | This study |
| 954K | ATCC 35319(p954K) | This study |
| Plasmids | ||
| pGEMT-Easy | E. coli cloning vector; Apr | Invitrogen |
| pET22b(+) | E. coli expression vector; Apr | Novagen |
| pET-K | pET22b(+) derivative carrying the 1,202-bp NdeI-XhoI fragment encoding mature Man5K | This study |
| pSOS95 | E. coli-Clostridium shuttle vector (ColEI and plM13 replicons); Apr Emr carrying the adc-ctfA-ctfB operon transcribed from the thl promoter | 29 |
| pSOSzero | pSOS95 derivative with the entire expression cassette (Pthl-adc-ctfA-ctfB) deleted | 24 |
| pSOS952 | pSOS95 derivative carrying two lac operators upstream and downstream of the thl promoter | 23 |
| pSOS954 | pSOS952 derivative carrying a thl −35 mutated box | This study |
| pD3 | pSOS95 derivative with the 377-bp fragment carrying the upstream region of the cipC open reading frame in place of the thl promoter | This study |
| p952K | pSOS952 derivative carrying the 1,313-bp BamHI-NarI cipC′-′man5K gene | This study |
| p954K | pSOS954 derivative carrying the 1,313-bp BamHI-NarI cipC′-′man5K gene | This study |
| pD3K | pD3 derivative carrying the 1,313-bp BamHI-NarI cipC′-′man5K gene | This study |
a
Abbreviations: adc, acetoacetate decarboxylase gene; ctfA, coenzyme A transferase subunit gene; ctfB, CoAT subunit B gene; thl promoter, thl promoter region for the thiolase gene (thl) of C. acetobutylicum ATCC 824; Apr, ampicillin resistant; Emr, erythromycin resistant.