Table 1.
Bacterial strains, plasmids, and oligonucleotide primers used in this study
| Strain or plasmid | Relevant characteristic(s) or sequencea | Source or reference |
|---|---|---|
| R. leguminosarum bv. trifolii strains | ||
| 24.2 | Wild type, Rifr Nxr | 18 |
| 2472 | 24.2 with Tn5 insertion at bp 151/152 of rosR | 5 |
| 5819 | 24.2 with Tn5 insertion at bp 363/364 of pssA | 5 |
| 1012 | 24.2 with Tn5 insertion at bp −253/−254 of pssA | This work |
| 4440 | 24.2 with Tn5 insertion at bp −77/−78 of pssA | This work |
| R. leguminosarum bv. viciae strains | ||
| J251 | Wild type, 8401(pRL1JI), Smr | 54 |
| J386 | J251 with Tn5 lac insertion in irrA, Smr Gmr Kmr | 55 |
| J397 | J251 with Tn5 insertion in rirA, Smr Kmr | 31 |
| S. meliloti strains | ||
| Rm1021 | SU47 str-21 | 56 |
| RmH406 | ΩphoB3::Tn5-132 | T. M. Finan, McMaster University, Hamilton, Ontario, Canada |
| E. coli strains | ||
| DH5α | λ− ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 | 23 |
| VH1000 | MG1655 derivative, lacZ lacI pyrE+ | 57 |
| VH1000 cyaA | ΔcyaA::Kmr derivative of VH1000 | 57 |
| M182 Δcrp | Δcrp39 Δlac derivative of M182 | 57 |
| S17-1 | 294 derivative; thi RP4-2 (Tc::Mu-Km::Tn7) | 58 |
| Plasmids | ||
| pUC19 | Cloning and sequencing vector, Apr | 23 |
| pMP220 | IncP, mob, promoterless lacZ, Tcr | 59 |
| pBBR1MCS-2 | mob, lacZα, Kmr cloning vector | 60 |
| pSUP202 | pBR325 derivative, mob, Cmr Tcr Apr | 58 |
| pM18 | pUC19 with a 1.7-kb HindIII fragment containing 24.2 pssA | This work |
| pBR12 | pBBR1MCS-2 with a 1.2-kb EcoRI fragment containing 24.2 rosR | This work |
| pM34 | pUC19 containing a 3.4-kb EcoRI fragment with pssA | 5 |
| pMJ440 | pUC19 with a 0.4-kb EcoRI-PstI fragment of the pssA upstream region | This work |
| pMSUP4 | pSUP202 containing a 4.6-kb EcoRI fragment with Tn5 inserted at bp 77/78 upstream of pssA ORF | This work |
| pMSUP10 | pSUP202 containing a 4.6-kb EcoRI fragment with Tn5 inserted at bp 253/254 upstream of the pssA ORF | This work |
| pAX1 | pBBR1MCS-2 with a 0.75-kb EcoRI-XbaI fragment containing the pssA upstream region (bp −750 to bp −2) | This work |
| pAX2 | pBBR1MCS-2 with a 0.53-kb EcoRI-XbaI fragment containing the pssA upstream region (bp −538 to bp −2) | This work |
| pAX3 | pBBR1MCS-2 with a 0.37-kb EcoRI-XbaI fragment containing the pssA upstream region (bp −374 to bp −2) | This work |
| pAX4 | pBBR1MCS-2 with a 0.28-kb EcoRI-XbaI fragment containing the pssA upstream region (bp −284 to bp −2) | This work |
| pAX5 | pBBR1MCS-2 with a 0.13-kb EcoRI-XbaI fragment containing the pssA upstream region (bp −134 to bp −2) | This work |
| pAX6 | pBBR1MCS-2 containing a 1.55-kb EcoRI-XbaI fragment with pssA | This work |
| pAX7 | pBBR1MCS-2 containing a 1-kb EcoRI-XbaI fragment with pssA | This work |
| pPA1 | pMP220 carrying the −750-bp to +152-bp fragment of the pssA regulatory region | This work |
| pPA2 | pMP220 carrying the −538-bp to +152-bp fragment of the pssA regulatory region | This work |
| pPA3 | pMP220 carrying the −374-bp to +152-bp fragment of the pssA regulatory region | This work |
| pPA4 | pMP220 carrying the −284-bp to +152-bp fragment of the pssA regulatory region | This work |
| pPA4-A | pMP220 carrying the −265-bp to +152-bp fragment of the pssA regulatory region | This work |
| pPA4-B | pMP220 carrying the −261-bp to +152-bp fragment of the pssA regulatory region | This work |
| pPA4-C | pMP220 carrying the −284-bp to +152-bp fragment of the pssA regulatory region with a deletion of Pho box 3 (−38 bp to −14 bp sequence) | This work |
| pPA5 | pMP220 carrying the −134-bp to +152-bp fragment of the pssA regulatory region | This work |
| Primers (5′–3′)b | ||
| PA1 | AGATCGTTGAATTCCGGCAAGCG | This work |
| PA2 | TCTTTTGAATTCGCTGCGCGCCAT | This work |
| PA3 | CCCTGTGAATTCCTGCCAAAGGC | This work |
| PA4 | GCGAAAGAATTCCTGCGATGCGA | This work |
| PA4-A | TGCGATGAATTCTTTCTCTGGACAC | This work |
| PA4-B | GCGATAGAATTCTGGACACCATTAC | This work |
| PAC-F | GATGCGGATCCGGCGCCGTTGGT | This work |
| PAC-R | TAACAGGGATCCGGATCTTCAGG | This work |
| PA5 | CAACAGAATTCGATCCTTGAGCATC | This work |
| PAR1 | ATGGCCCTGCAGTTCTTGCCCCA | This work |
| PAR2 | ACCAGATCTAGAAACGGCGCCTG | This work |
| PAR3 | CAGGTTATCTAGAGAGCCCGATG | This work |
| EMSA13 | TGGGTCACGTGTCGAAACGGCACGT | This work |
| EMSA14 | CGCATCGCAGCAAATCTTTCGCATG | This work |
a
Nxr, nalidixic acid resistance; Rifr, rifampin resistance; Tcr, tetracycline resistance; Apr, ampicillin resistance; Kmr, kanamycin resistance; Smr, streptomycin resistance; Gmr, gentamicin resistance. The sequences for the EcoRI, BamHI, XbaI, and PstI restriction sites are underlined.
b
Amplification of the pssA promoter region for construction of pPA fusions and EMSAs.