Table 2.
Plasmids and oligonucleotides used in this study
| Plasmid or primer name (description) | Primer used, 5′-3′ sequence; reference or source and/or other detailsa |
|---|---|
| pAM505 | Conjugal shuttle plasmid vector (32) |
| pAM1951 (PpatS-gfp) | 32 |
| pAM1956 (promoterless gfp) | 32 |
| pAM2178 | Conjugal suicide plasmid vector (16) |
| pAM3264 (PhetR-gfp) | 30 |
| pAM3826 (pAM1956-all2334) | AMO-1561, GTCGTCGACTCGTACATTCTAGAGCTTG |
| AMO-1562, GGTGGTACCTTGACGACACAGAGATTA | |
| pAM3828 (pAM1956-pknE) | AMO-1563, GTCGTCGACTATCCTCGCTACAGAGGAA |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM3830 (pAM1956-all4668) | AMO-1565, GTCGTCGACTTGCCCACCCTAAAGATA |
| AMO-1566, GGTGGTACCGTCTTCCGCTAGATAGGT | |
| pAM3832 (pAM1956-all4838) | AMO-1567, GTCGTCGACGAAGATAGGAGATTGGGGAA |
| AMO-1568, TTCACGGTACCTATTGATGC | |
| pAM3851 (pK18-PpknE) | AMO-1563, GTCGTCGACTATCCTCGCTACAGAGGAA |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM3853 (pAM1956-pknE-P) | AMO-1668, GTCGTCGACGCCTATGATAAAGACAGC |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM3855 (pAM1956-pknE-P1) | AMO-1669, GTCGTCGACAATAGTCTTACTTTTTCA |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM3857 (pAM1956-pknE-P2) | AMO-1670, GTCGTCGACAAGTTCTTAATTTTGGAT |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM3859 (pAM1956-pknE-P3) | AMO-1671, GTCGTCGACAGCACGCAATCAAGAG |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM3882 (pQE30-alr2339) | AMO-1675, GGAGGATCCAGTAACGACATCGATCTG |
| AMO-1490, GAGGAGCTCTTAATCTTCTTTTCTACCA | |
| pAM3918 (pK18-SDM-PpknE) | AMO-1684, TTAGCACGCAATCAAGAGAAT |
| AMO-1685, TTCCGGAAAGAGAAATTACTCTAAG | |
| pAM3919 (pAM1956-PpknE-PmutntcA) | AMO-1668, GTCGTCGACGCCTATGATAAAGACAGC |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM3920 (pAM1956-PpknE-P2mutntcA) | AMO-1670, GTCGTCGACAAGTTCTTAATTTTGGAT |
| AMO-1564, GGTGGTACCTTCAGTTTCTGTCAGAAAA | |
| pAM4004 (pAM2178-pknE) | AMO-1686, GGAGGATCCATCGGTAAAGTACTGCAA |
| AMO-1767, AAGAAGCTTACGTCTGATTAAATTTTCTGG | |
| pAM4012 (pAM1956-pknE-P with Ω Spr/Smr) | Ω Spr/Smr XbaI cassette from pDW9 cloned into pAM3853 |
| pAM4146 (pAM505-pknE-OE) | AMO-1768, GAGGAGCTCTAGAAAGTGAACCCG |
| AMO-1824, GTCGTCGACTACTAGCGAATATTTACTTCT | |
| pAM4171 (pAM505-pknE-OE with Ω Spr/Smr) | Ω Spr/Smr XbaI cassette from pDW9 cloned into pAM4146 |
| pAM4375 (pAM505-hetR-6His) | AMO-1763, CCCCCCGGGACTTATAATCAAAACAAATG |
| AMO-1764, AGGAGCTCTTAACGATGGTGATGGTGATGGTGATCTTCTTTTCTACCAAACACC | |
| pAM4378 (pAM2770-pknE-OE) | AMO-1762, CATCATATGATCGGTAAAGTACTGCAA |
| AMO-1672, GGTGGTACCTAGCGAATATTTACTTCTG | |
| pGEX-4T1 | IPTG-inducible GST protein overexpression; GE Healthcare |
| pGEX-4T1-hetR | IPTG-inducible GST-HetR protein overexpression (27) |
| Primers for PpknE | AMO-1926, TAGCGTGCTTGCAGTACTTT |
| AMO-1670, GTCGTCGACAAGTTCTTAATTTTGGAT | |
| Primers for PhetR | AMO-1135, TTCTCGAGCAGATAAGTTCCGGATAATAG |
| AMO-1136, TCAGAGCTCCGTAATTTATGGCATATAAC | |
| Primers for noncoding fragment from pAM505 for EMSAb | AMO-367, ACCCGTCGAACTGCGCGC |
| AMO-369, CGCTCTGCTGAAGCCAG | |
| pQE30 | IPTG-inducible overexpression of His-tagged proteins; Qiagen |
a
Nucleotides in italics indicate a restriction site, and underlined nucleotides indicate a site-directed mutation.
b
EMSA, electrophoretic mobility shift assay.