TABLE 1.
Bacterial strains and plasmids used in this study
| Bacterial strain or plasmid | Relevant characteristic(s) and genetic marker(s)a | Source or reference |
|---|---|---|
| Strains | ||
| Escherichia coli | ||
| XL1-Blue | recA1 lac endA1 gyrA46 thi hsdR17 supE44 relA1 [F′ proAB lacIqZΔM15 Tn10 (Tetr)] | Stratagene |
| SY327λpir | Δ(lac pro) argE(Am) recA (Rifr) nalA λpir | 27 |
| SM10λpir | thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu λpir | 27 |
| Sulfur chemolithotrophs | ||
| T. kashmirensis LMG 22696 | Wild type harboring pBTK445; Apr Sox+ | 15 |
| T. kashmirensis SR | Spontaneous Smr Rifr mutant of T. kashmirensis LMG 22696 | 11 |
| T. kashmirensis C1 | pBTK445-cured strain of T. kashmirensis SR | This study |
| BD 004 | Tn5-mob insertion mutant of T. kashmirensis SR; Kmr Sox− | 11 |
| BD 004 C1 | pBTK445-cured strain of BD 004; Kmr | This study |
| Plasmids | ||
| Large plasmids, cloning and expression vectors, and sources of antibiotic markers | ||
| pBTK445 | Large (∼60-kb), low-copy-number plasmid harbored in T. kashmirensis | This study |
| pBTKCΩ6 | orf6::ΩCm (pBTK445 with orf6 inactivated) | This study |
| pBTKCΩB4-B5 | tagB4-tagB5::ΩCm (pBTK445 with tagB4 and tagB5 inactivated) | This study |
| pBluescript KS(+) | 2.96-kb cloning vector; Apr | Stratagene |
| pKAS32 | Cloning vector with dominant rpsL gene; Apr | 36 |
| pSD5B | Promoter probe vector (also used to probe the replication and transfer origins of pBTK445); Kmr | 19 |
| pQE30 | T5 promoter with His6 tag; Apr | Qiagen |
| pFH450 | 7.4-kb cloning vector containing P1 and ColE1 replicons; Cmr | 16 |
| pUC4K | pUC vector carrying the kanamycin cassette from Tn903; Apr Kmr | Amersham |
| pUC4C | pUC vector carrying the chloramphenicol cassette from pFH450; Apr Cmr | This study |
| Plasmids for shuttle vector construction, curing, mobilization, and complementation assays | ||
| pBTKE5.7 | 5.7-kb EcoRI fragment of pBTK445 cloned into pBluescript; Apr | This study |
| pBTKE5.7R | 5.7-kb EcoRI fragment of pBTK445 cloned into pBluescript in reverse direction; Apr | This study |
| pBTKS | 1.9-kb Cmr gene from pUC4C cloned into the PstI site of pBTKE5.7; Apr Cmr | This study |
| pBTKSR | 1.9-kb Cmr gene from pUC4C cloned into the PstI site of pBTKE5.7R; Apr Cmr | This study |
| pBTKAS | 5.2-kb BamHI fragment of pBTKS cloned into pKAS32; Apr Cmr | This study |
| pSUP5011 | pBR325 (Bam−)::Tn5-mob; Cmr Apr Kmr | 35 |
| pBTKSM | 3.6-kb PCR product from the moco locus of T. kashmirensis cloned into the XbaI site of pBTKS; Cmr | This study |
| Constructs for in vivo determination of oriV, oriT, and PrepA | ||
| pBTK1C | pBTKSR digested with SacII, with the larger fragment religated; Apr Cmr | This study |
| pBTKHE3.1 | pBTKE5.7 digested with HindIII and religated; Apr Cmr | This study |
| pBTK2C | 1.9-kb Cmr gene from pUC4C cloned into the BamHI site of pBTKHE3.1; Apr Cmr | This study |
| pBTK3C | pBTKS digested with ClaI and religated; Apr Cmr | This study |
| pSD5MR | 1,373-bp repA- and oriV-containing PCR product cloned into pSD5B; Kmr | This study |
| pSD5V | 264-bp oriV- and repA promoter-containing PCR product cloned into the XbaI site of pSD5B; Kmr | This study |
| pSD5T | 100-bp oriT-containing PCR product cloned into the XbaI site of pSD5B; Kmr | This study |
| Recombinant plasmids for knockout mutagenesis | ||
| pBTKHE2.5 | pBTKE5.7R digested with HindIII and religated; Apr | This study |
| pKORF6 | pKAS32 containing orf6 and orf7 as a 1.97-kb BamHI and EcoRI fragment of pBTKHE2.5; Apr | This study |
| pKORF6C | pKORF6 with a Cmr cassette inserted at the BamHI site of orf6; Apr Cmr | This study |
| pBTKH1.9 | 1.9-kb HindIII fragment containing portions of tagB4-tagB5 of pBTK445 cloned into pBluescript; Apr | This study |
| pKTAGB | pKAS32 containing partial tagB4 and tagB5 genes; Apr | This study |
| pKTAGBC | pKTAGB with tagB4 and tagB5 inactivated by a Cmr cassette; Apr Cmr | This study |
a
Sox+ strains have the ability to oxidize reduced sulfur compounds, while Sox− strains do not.