TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Genotype, phenotype, or sequence of primera (5′ to 3′) | Reference, origin, or description |
|---|---|---|
| R. palustris strains | ||
| CGA009 | hupV mutant; spontaneous frameshift (4-bp deletion) | 25 |
| CGA010 | hupV repaired derivative of CGA009; 4 bp inserted in hupV | This study |
| CGA553 | ΔhoxJ mutant of CGA010; 1,095 bp deleted | This study |
| CGA554 | ΔRPA0981 mutant of CGA010; 2,217 bp deleted | This study |
| CGA550 | hupS mutant of CGA010; lacZ-Km cassette inserted in hupS | This study |
| CGA555 | rpoN mutant of CGA010; rpoN::Tn5 | 17 |
| CGA2023 | ΔregSR mutant of CGA010 | F. R. Tabita |
| RCH100 | Environmental isolate of R. palustris | Iowa City, IA |
| RCH200 | Environmental isolate of R. palustris | Iowa City, IA |
| RCH350 | Environmental isolate of R. palustris | Iowa City, IA |
| RCH500 | Environmental isolate of R. palustris | Woods Hole, MA |
| RCH550 | Environmental isolate of R. palustris | Woods Hole, MA |
| E. coli strains | ||
| DH5α | F− λ−recA1 Δ(lacZYA-argF)U169 hsdR17 thi-1 gyrA96 supE44 endA1 relA1 φ80lacZΔM15 | GIBCO-BRL |
| S17-1 | thi pro hdsR hdsM+recA; chromosomal insertion of RP4-2 (Tc::Mu Km::Tn7) | 37 |
| Plasmids | ||
| pJQ200KS | Gmr, sacB; mobilizable suicide vector | 33 |
| pUC19 | Apr; high-copy-number cloning vector | 44 |
| pUTmini-Tn5lacZ1 | Apr, Kmr; delivery plasmid for mini-Tn5 lacZ1 | 7 |
| pHRP309 | Gmr, IncQ; lacZ transcriptional fusion vector | 31 |
| pHRP314 | Apr, Smr, Spr; cohort cloning vector containing a 5.3-kb lacZ-Km cassette | 31 |
| pHRP316 | Apr, Smr, Spr; cohort cloning vector for use with pHRP309 | 31 |
| pHRP316-PhupS | Apr, Smr, Spr; hupS promoter region cloned into BamHI and SmaI sites of pHRP316 | This study |
| pHRP309-PhupS | Gmr, Smr, Spr; hupS-lacZ transcriptional fusion cloned into SmaI and XbaI sites of pHRP309 | This study |
| pUC-ΔhoxJ | Apr; 2.2-kb fragment containing a 1,095 bp in-frame deletion of hoxJ constructed by PCR and cloned into XbaI sites of pUCI9 | This study |
| pJQ-ΔhoxJ | Gmr; 2.2-kb fragment containing a 1,095 bp in-frame deletion of hoxJ constructed by PCR and cloned into XbaI sites of pJQ200KS | This study |
| pUC-hupV | Apr; 1.4-kb fragment containing repaired hupV gene cloned into BamHI sites of pUC19 | This study |
| pJQ-hupV | Gmr; 1.4-kb fragment containing repaired hupV gene cloned into BamHI sites of pJQ200KS | This study |
| pUC-ΔRPA0981 | Apr; 2.1-kb fragment containing a 2,217 bp in-frame deletion of RPA0981 constructed by PCR and cloned into XbaI site of pUC19 | This study |
| pJQ-ΔRPA981 | Gmr; 2.1-kb fragment containing a 1,095 bp in-frame deletion of hoxJ constructed by PCR and cloned into the XbaI site of pJQ200KS | This study |
| pUC-hupS | Apr; 1.3-kb fragment containing hupS and contiguous DNA cloned into the XbaI site of pUC19 | This study |
| pUC-hupS::lacZKm | Apr, Kmr; pUC-hupS containing lacZ-Km cassette from pHRP314 into the BsmI site of hupS | This study |
| pJQ-hupS::lacZKm | Gmr, Kmr; hupS::lacZ-Km fragment cloned into the XbaI site of pJQ200KS | This study |
| Primers | ||
| hupVS1 | AGGATGCACCGCTGTTTGCGG | Sequencing hupV forward primer |
| hupVS2 | ACAATCGCGGATCAGCGGATC | Sequencing hupV reverse primer |
| hupVP1-BamHI | CGGGATCCCGATGACGCGACGGATCACGGTCGG | hupV upstream primer |
| hupVP2-BamHI | CGGGATCCCGTTAGTGCGCGGTGCACACCATGCA | hupV downstream primer |
| hupV-I1 | GCCAGTGCGGCGCGGACGTCGTTCGATCCTGCCGCGATCACCGAGGATGTGG | hupV 4-bp insertion forward primer |
| hupV-I2 | CCACATCCTCGGTGATCGCGGCAGGATCGAACGACGTCCGCGCCGCACTGGC | hupV 4-bp insertion reverse primer |
| hupS-XbaI-1 | GCTCTAGACAGCGCCGCAACCGGCTTATCA | hupS upstream primer |
| hupS-XbaI-2 | GCTCTAGATTAAGCCGACTTGCCGTTGGAG | hupS downstream primer |
| hupS1-BamHI | CGGGATCCCGCTCGAACAAGCGCTGGTTGGCACC | hupS promoter forward primer |
| hupS2-SmaI | TCCCCCGGGGGAGGTGCATTCCAGCCCGTGCATCCA | hupS promoter reverse primer |
| hoxJP1-XbaI | GCTCTAGAGCAAGCGTGCTGCCGAATT | hoxJ upstream primer |
| hoxJP2-XbaI | GCTCTAGAGCCAGGATCGCGCGGAGCT | hoxJ downstream primer |
| hoxJD1 | GTTTACGCCGACCTGCTGCGCACCGGGCTCGGCTTGTGGATC | hoxJ in-frame deletion forward primer |
| hoxJD2 | GATCCACAAGCCGAGCCCGGTGCGCAGCAGGTCGGCGTAAAC | hoxJ in-frame deletion reverse primer |
| RPA0981P1-XbaI | GCTCTAGAGCTGGCGCCACCCGCTGCGA | RPA0981 upstream primer |
| RPA0981P2-XbaI | GCTCTAGAGCAACTGCCTCTAAAGAGCA | RPA0981 downstream primer |
| RPA0981D1 | GAAGTTCAGCGCATCGCCAAATACAGCAGCGTGCTCGCCGAG | RPA0981 in-frame deletion forward primer |
| RPA0981D2 | CTCGGCGAGCACGCTGCTGTATTTGGCGATGCGCTGAACTTC | RPA0981 in-frame deletion reverse primer |
a
Restriction sites are underlined; the four extra bases that were added to correct the frameshift in CGA009 hupV are in bold.