TABLE 1.
Strains, plasmids, and synthetic oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristicsa or sequence | Source or reference |
|---|---|---|
| Strains | ||
| A. eutrophus | ||
| H16 | MBH+ SH+ | DSM428, ATCC 17699 |
| HF18 | H16 hoxA18, MBH− SH− | 17 |
| HF09 | H16 rpoN09, MBH− SH− | 37 |
| HF457 | H16 hoxTΩR-B2 | This study |
| HF491 | H16 hoxKΔ171-R4 | This study |
| E. coli | ||
| S17-1 | recA pro thi hsdR chr::RP4-2 Tra+ | 41 |
| JM109 | recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 Δ(lac-proAB) e14−(F′ lacIqlacZΔM15 proAB traD36) | 48 |
| Plasmids: | ||
| pEDY305 | Tcr RK2 ori Mob+, promoterless lacZ gene | 39 |
| pPHU234 | Tcr RK2 ori Mob+, ′lacZ translational fusion vector | 20 |
| pPHU278 | Φ(aph-lacZ), Paph deleted | 20 |
| pGMΩ1 | Derivative of pUC18 carrying a polar Ω cassette | 40 |
| pLO2 | sacB oriTRP4 ColE1 ori, Kmr Mob+, counterselectable suicide vector | 25 |
| pRZ10 | Φ(acoR-lacZ) | 23 |
| pCH103 | Derivative of pSUP202 carrying the hyp locus | 11 |
| pCH104 | Derivative of pSUP202 carrying the hyp locus | 11 |
| pCH300 | 2.2-kb SalI-BamHI fragment of pGE43 in pBluescript KS+ | This study |
| pCH301 | 2.2-kb PstI-KpnI fragment of pGE43 in pBluescript KS+ | This study |
| pCH303 | 2.6-kb XhoI-SalI fragment (Klenow enzyme treated) of pGE43 in pBluescript SK+ | This study |
| pCH304 | 2.7-kb SalI-XhoI fragment of pGE43 in pBluescript KS+ | This study |
| pCH427 | hoxT subcloned in pLO1, KmrsacB oriTRP4 ColE1 ori | 4 |
| pCH679 | Ω cassette of pGMΩ1 inserted as a SmaI fragment into the EcoRV site of pCH427 | This study |
| pCH680 | Deletion derivative lacking 171 bp of the hoxK upstream region introduced into pLO2 as 686-bp PstI-XhoI fragment | This study |
| pGE43 | Derivative of pVK101 carrying the hyp region | 10 |
| pGE281 | 8.5-kb EcoRI (Klenow enzyme-treated)-HpaI fragment of pCH104 in pPHU234, Φ(hoxA-lacZ) | This study |
| pGE282 | 6.1-kb BstEII (Klenow enzyme-treated)-HpaI fragment of pCH104 in pPHU234, Φ(hoxA-lacZ) | This study |
| pGE283 | 1.9-kb BspEI (Klenow enzyme-treated)-HpaI fragment of pCH104 in pPHU234, Φ(hoxA-lacZ) | This study |
| pGE319 | Derivative of pEDY305 with PMBH upstream of lacZ | 39 |
| pGE320 | Derivative of pEDY305 with PSH upstream of lacZ | 39 |
| pGE322 | 667-bp XhoI-PvuII fragment of pCH104 in pEDY305 | O. Lenz and B. Friedrich |
| pGE323 | 516-bp PvuII fragment of pCH303 in pEDY305 | O. Lenz and B. Friedrich |
| pGE324 | 711-bp BamHI-NheI fragment pCH104 in pEDY305 | This study |
| pGE325 | 1739-bp NheI-HpaI fragment of pCH104 in pEDY305 | O. Lenz and B. Friedrich |
| pGE326 | 463-bp SnaBI fragment of pCH301 in pEDY305 | This study |
| pGE327 | 721-bp FspI-Eco47III fragment of pCH303 in pEDY305 | This study |
| pGE328 | 233-bp segment of pRZ10 subcloned as a PCR product in pEDY305 | This study |
| pGE413 | 2.1-kb EcoRI-PvuII fragment of pCH103 in pPHU234, Φ(hypB1-lacZ) | This study |
| pGE417 | 1-kb AscI-NheI fragment of pCH301 in pEDY305 | This study |
| pGE418 | 350-bp segment of pCH301 subcloned as PCR product in pEDY305 | This study |
| pGE420 | 393-bp segment of pCH103 subcloned as a PCR product in pEDY305 | This study |
| Oligonucleotides | ||
| BF213 | 5′-gcgGATCCATGACCACCAGG-3′ (M90471, 150–166) | |
| BF214 | 5′-tggccCGGGCGCGTGCTGCGG-3′ (M90471, 383–368) | |
| BF250 | 5′-ccagCTAGCGATTCCCGCACG-3′ (X70183, 2466–2482) | |
| BF251 | 5′-ccgCGCGCCTCAGCGCGAACG-3′ (X70183, 2802–2785) | |
| BF360 | 5′-acagagatcTCGCGGAGTTGGTGTTGC-3′ (M96433, 8579–8596) | |
| BF361 | 5′-gcaacaGCTGAGCTCATGCATGGACAG-3′ (M96433, 8971–8951) |
a
MBH, membrane-bound hydrogenase activity; SH, NAD-reducing hydrogenase activity; Tra, transfer of mobilizable plasmids; ori, origin of replication; chr::RP4-2, chromosomally integrated copy of RP4-2; Mob, mobilizability. For oligonucleotides, foreign or altered nucleotides are shown in lowercase, and accession numbers of DDBJ/EMBL/GenBank entries and coordinates are given in parentheses.