TABLE 1.
Strains, plasmids, and primers
| Strain, plasmid, or primer | Relevant features or sequence | Source or reference |
|---|---|---|
| Strains | ||
| DH5α pir+ | E. coli cloning strain | Lab collection |
| DK1217 | A− S+ aglB1 | 13 |
| DK8601 | A− S− aglB1 ΔpilA::Tc Tcr | 12 |
| DK8606 | A− S− aglB1 ΔpilA PpilA-gfp Kmr | 12 |
| DK8615 | A+ S− ΔpilQ | 13 |
| DK10410 | A+ S− ΔpilA | 12 |
| DW709 | DK10410 ΔpilA PpilA-gfp Kmr | 28 |
| DW1056 | DK8606 PpilA-SSOM-mCherry(pXW6) PpilA-gfp Smr Tcr Kmr | 2 |
| DW1415 | DK8615 traA::pDP2 Kmr | 3 |
| DW1466 | A− S− ΔcglC Δtgl::Tc Tcr | 3 |
| DW1467 | DK8601 aglB1 ΔtraA ΔpilA::Tc Tcr | 11 |
| DW1478 | DK8615 PpilA-SSOM-mCherry(pXW6) Smr | This study |
| DW1479 | DW1478 traA::pDP2 Kmr Smr | This study |
| DW1601 | DK8615 omrA::EZ-Tn5 Kmr | This study |
| DW1602 | DK8615 omrA::mini-Himar-lacZ Kmr | This study |
| DW1603 | DK8615 omrA::mini-Tn5 Kmr | This study |
| DW1604 | DK8601 aglB1 ΔpilA::Tc omrA::mini-Tn5 Tcr Kmr | This study |
| DW1605 | DK1217 omrA::mini-Tn5 Kmr | This study |
| DW1606 | DK8615 ΔphpA (markerless) | This study |
| DW1607 | DK1217 ΔphpA (markerless) | This study |
| DW1608 | DK8615 ΔMXAN_4428 (markerless) | This study |
| DW1609 | DK8615 omrB::pAD3 Kmr Zmr | This study |
| DW1610 | DK8601 omrB::pAD3 Kmr Zmr | This study |
| DW1611 | DW1603 PpilA-SSOM-mCherry(pXW6) Smr Kmr | This study |
| DW1612 | DW1466 omrA::mini-Tn5 Kmr Tcr | This study |
| Plasmids | ||
| pMini-Himar-lacZ | mariner-derived transposon with lacZ oriR6K, Kmr | Heidi Kaplan |
| pRL27 | Mini-Tn5 oriR6K hyperactive transposase, Kmr | 14 |
| pCR2.1 TOPO XL | Cloning vector, Kmr Zmr | Life Technologies |
| pCR2.1 TOPO TA | Cloning vector, Kmr | Life Technologies |
| pDP2 | traA (insertion cassette) in pCR2.1 TOPO TA, Kmr | 3 |
| pDP21 | PpilA-RBSsyn-traAB cloned in pSWU1, Kmr | 3 |
| pXW6 | PpilA-SSOM-mCherry cassette cloned in pKSAT, Smr | 2 |
| pAD1 | ΔphpA cassette in pBJ114, Kmr | This study |
| pAD2 | ΔMXAN_4428 cassette in pBJ114, Kmr | This study |
| pAD3 | omrB (insertion cassette) in pCR2.1 TOPO XL, Kmr Zmr | This study |
| Primers | ||
| Mariner primer | TGTGTTTTTCTTTGTTAGACCG | |
| EZ-Tn5 KAN-2 Fwd | ACCTACAACAAAGCTCTCATCAACC | |
| EZ-Tn5 KAN-2 Rev | GCAATGTAACATCAGAGATTTTGAG | |
| Tpn RL13 (mini-Tn5) | CAGCAACACCTTCTTCACGA | |
| Tpn RL17 (mini-Tn5) | AACAAGCCAGGGATGTAACG | |
| MXAN_4428delupstrfwd | TATGACCATGATTACGCCAAGCTTAGGTTCACCTCGGTGGC | |
| MXAN_4428delupstrrev | ATCCCAGCGCTGTGAAGCTGTGTGGATGGGACACGTCAGGGT | |
| MXAN_4428deldwnstrfwd | CACACAGCTTCACAGCGCTGGGATGCACGCCAGCACGGCGAC | |
| MXAN_4428deldwnstrrev | CGTTGTAAAACGACGGCCAGTGAATTCCCTCATGAGACGGGCGAAGC | |
| MXAN_4427delupstrfwd | TATGACCATGATTACGCCAAGCTTCTCTCGCTCGTGGGTGAC | |
| MXAN_4427delupstrrev | CCAGCGCTGTGAAGCTGTGTGGCAATGCAGGTCGACAAAG | |
| MXAN_4427deldwnstrfwd | CACACAGCTTCACAGCGCTGGAATCCCGCGACTTTGTTG | |
| MXAN_4427deldwnstrrev | CGTTGTAAAACGACGGCCAGTGAATTCAGTACAGCGCCGTGTAGAGG | |
| MXAN_6818 KO FWD | GGACAGACGGTGTTCTCGTT | |
| MXAN_6818 KO REV | ACAGGAAGCCCACCACCT |