TABLE 1.
Bacterial strains, plasmids, and oligonucleotide used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristics or sequencea | Reference or source |
|---|---|---|
| Strains | ||
| Agrobacterium tumefaciens C58 | Wild type, Kms | |
| A. tumefaciens ΔphaC mutant | Deletion of PHB synthase gene phaC (Atu_1604) | This study |
| A. tumefaciens ΔhppA mutant | Deletion of H+-pyrophosphatase hppA (Atu_1174) | This study |
| A. tumefaciens Δppk1 mutant | Deletion of polyP kinase PPK1 gene ppk1 (Atu_1144) | This study |
| A. tumefaciens Δppk2 mutant | Deletion of polyP kinase PPK2 gene ppk2 (Atu_0418) | This study |
| A. tumefaciens Δppk1 Δppk2 mutant | Deletion of ppk1 and ppk2 | This study |
| A. tumefaciens eyfp-hppA | Chromosomal replacement of hppA by eyfp-hppA | This study |
| Ralstonia eutropha H16 | Wild type, forms PHB and polyP granules but no acidocalcisomes (no hppA gene) | DSMZ428 |
| Plasmids | ||
| pBBR1MCS2-PphaC-eyfp-c1 | Broad-host-range vector for construction of gene fusions with eyfp, confers Kmr, constitutive expression from PphaC | 34 |
| pBBR1MCS2-PphaC-dsred2EC-c1 | Broad-host-range vector for construction of gene fusions with Dsred2EC constitutive expression from PphaC | 15 |
| pBBR1MCS2-PphaC-eyfp-hppA | Constitutive expression of eyfp-hppA | This study |
| pBBR1MCS2-PphaC-eyfp-ppk2 | Constitutive expression of eyfp-ppk2 | This study |
| pBBR1MCS2-PphaC-ppk1-mcherry | Constitutive expression of ppk1-mcherry | This study |
| pBBR1MCS2-PphaC-dsred2EC-lactC2 | Constitutive expression of dsred2EC-lactC2 | 15 |
| pBBR1MCS2-PphaC-eyfp-pptA | Constitutive expression of eyfp-pptA | 23 |
| pHis_EcPPX1 | Source of PPX | A. Saiardi, UC London |
| Oligonucleotides | ||
| Sacl_upF_atu1144 | CGATCAGAGCTCGACCGGCGGGGTTTTGACGATACG | |
| upR_atu1144 | GCCGTTAATTAAGCCGGCCCTGTTCCGTCCCCTGAC | |
| downF_atu1144 | CGGCTTAATTAACGGCATGACTCGATCAGAAGCACAG | |
| SpeI_downR_atu1144 | GCACTAGTCGAAGGATTTGACGTGTTTTCTGGC | |
| Sacl_upF_atu0418 | CGATCAGAGCTCCCTTACGCCGGAAATGACGTT | |
| upR_atu0418 | GCCGTTAATTAAGCCGGGTTTCCTCCGGATGTTGT | |
| downF_atu0418 | CGGCTTAATTAACGGCGACCGGGAGACG | |
| SpeI_downR_atu0418 | GCACTAGTTGACTGGGTCGAGGTCTTTAATGC | |
| Sacl_upF_atu1604 | CGATCAGAGCTCCTTATATTCGGCTTCCGAA | |
| upR_atu1604 | GCCGTTAATTAAGCCGGCACGATCCTCCA | |
| downF_atu16004 | CGGCTTAATTAACGGCTCTCATCCGCCACTCTC | |
| SpeI_downR_atu1604 | GCACTAGTCGCCACGTTTGGTGGCTGTA | |
| 500bp_up_1174_fwd | tcgagctcggtacccgggggcgcgccTCTGGGCAGCGCTCATCC | |
| 500bp_up_1174_rev | actatgggtattacggtcatGCGCACTTACCTCCCAAAAG | |
| eYFP_atu1174_fwd | cttttgggaggtaagtgcgcATGACCGTAATACCCATAGTAATTTTATGCG | |
| eYFP_atu1174_rev | cagtctagctatcgccatgactagtTCAGTGGGCAAGAACTGC | |
| PPK1_mcherry_fwd | tcgagctcggtacccgggggcgcgccATGGACGCTATCGCGCAG | |
| PPK1_mcherry_rev | cccctgtgcttctgatcgagTTACTTGTACAGCTCGTCCATG | |
| 500bp_down_ppk1_fwd | tggacgagctgtacaagtaaCTCGATCAGAAGCACAGGGG | |
| 500bp_down_ppk1_rev | cagtctagctatcgccatgactagtCGGCAGGGTGATGCCCTC |
a
Uppercase letters indicate bases complementary to the respective gene sequence. Kms, kanamycin sensitive; Kmr, kanamycin resistant.