TABLE 2.
Primers used in this study
| Primer used and gene/ORF amplified | Amplicon size (bp) | Primer name | Sequence |
|---|---|---|---|
| RT-PCR | |||
| A33_1675-A33_1674 | 936 | RT toxR 2332 F | CTTGTGCTGCAGTTTGTG |
| RT 2332 toxR R | GGCATGGTGTTATTACTCAAG | ||
| A33_1674-vcsR2 | 253 | 3′VcsR2 XbaI R | TGAGTCTAGACAACTGCAACGCTTAATGG |
| RT 2332 vcsR2 NR | AGAAAGCAGCACCAACAC | ||
| vcsR2-vcsT2 | 764 | RT vcsR vcsT2 F | TACTAGAGCATGCCAAACAG |
| RT vcsT vcsR R | CCACCACGGTTTGATATAACG | ||
| vcsT2-A33_1671 | 516 | RT vcsT 2329 F | CGGTATTTAAACCGCTACTGG |
| RT 2329 vcsT R | ACGCATTTGACGAATAGACTC | ||
| A33_1671-vcsC2 | 1,061 | RT 2329 vcsC F | TGCTTCTCAAGAGGGAGATG |
| RT 2329 vcsC R | GTAAATTCCTCGGCTACCATTTAAC | ||
| vcsC2-vcsN2 | 1,002 | RT vcsC vcsN F | GATAGCGGCTGGTTAAATGG |
| RT vcsN vcsC R | GCCCTTCTCCGATAGTAAAC | ||
| vcsN2-A33_1668 | 1,012 | RT vcsN 2326 | TGCGAGCTCCAATTGAAAC |
| RT 2326 vcsN R | GCAGCCACTCAATATGATCC | ||
| A33_1668-A33_1667 | 676 | RT 2326 ORF32 F | TGAGCATGATAGCCTTAAACG |
| RT 2326 ORF32 R | AGTCTGCCGGCTAAATTG | ||
| A33_1667-vcsS2 | 458 | RT ORF32 vcsS F | CCAATTTAGCCGGCAGAC |
| RT ORF32 vcsS R | CCACCAAACTGGGTTAGTAG | ||
| vcsS2-ORF69 | 295 | 5′ ToxR2.A Del SalI | ACTAAGTCGACTTGGTGGCAGTATTTATGAG |
| RT ORF69 vcsS R | CCATAATCTTAGGTGCTTTGTAACG | ||
| ORF69-ORF65 | 317 | RT ORF69 ORF65 F | CGTTACAAAGCACCTAAGATTATGG |
| RT ORF69 ORF65 R | CATATACCAAGCCAAACTCTACC | ||
| A33_1684-A33_1683 | 1,014 | RT fw 2342 2341 | AGTTCGCAGTTTGAAGTTGG |
| RT NRv 2341 2342 | ACTTTGGGAATCGCTTTATGC | ||
| A33_1683-vcsV2 | 961 | RT Fd 2341 vcsV | GCGTCACGTTGAGTATTGAG |
| RT Rv vcsV 2341 | GATAATACCGCCAATCACTAGC | ||
| vcsV2-vcsU2 | 1,128 | RT Fd vcsV vcsU | GGCGGTATTATCATTATCTTGGG |
| RT Rv vcsV vcsU | TTGGAAATTGGCCTTTCTG | ||
| vcsU2-A33_1680 | 1,114 | RT vcsU2 Fd | AACCGAACCCAAGAAATATCC |
| RT Rv vcsU 2338 | TCTTGATCGAGCACTATGTTG | ||
| A33_1680-A33_1679 | 994 | RT 2338 2337 F | CAACATAGTGCTCGATCAAGAC |
| RT 2338 2337 R | CAACAACCTAGCAATTTCATCTC | ||
| A33_1679-A33_1678 | 1,096 | RT 2337 2336 F | GGGCATTAAAGTGACCAGTAAG |
| RT 2337 2336 R | CAGTATCGACCCAGTTCATC | ||
| A33_1678-A33_1677-vcsQ2 | 990 | RT 2336 vcsQ2 F | GATGAACTGGGTCGATACTG |
| RT vcQ 2335 R | GTCACTGCGTATTGGTCATC | ||
| vcsQ2-A33_1675 | 280 | 3′ ToxR2PRMkd R XbaI | GATTTCTAGAGATGACCAATACGCAGTGAC |
| ToxR2B R SB | CTTAACATCGTTGAACCCTTTCAC | ||
| Transcriptional reporter fusions (plasmid and single copy) | |||
| Upstream region of A33_1674 (vcsRTCNS2) | 969 | 3′ PM 2332 XbaI | TTTTTCTAGATGCGCTGATTTCTTCATTTGC |
| 5′ PM 2332 PstI | GCTGCTGCAGGTGCGCCAGCAATATCACG | ||
| Upstream region of A33_1683 (vcsVUQ2) | 390 | 3′ PM 2341 XbaI | CAACTCTAGATTTAACAACTCAGGGAATGG |
| 5′ PM 2341 PstI | AATCCTGCAGTGCGACGCATCTAATTCG | ||
| Upstream region of A33_1689 (vspD) | 460 | 3′ VspD R XbaI | AAATTCTAGATTCAAATTATGCGTGACGAACG |
| 5′ VspD F PstI | CACACTGCAGCTTGGTTCTCTGCGATATTCAC | ||
| Upstream region of A33_1693(vcsJ2) | 527 | 3′ VcsJ2 R XbaI | CAATTCTAGAGGAGCATAAGGAGAGTAAGC |
| 5′ VcsJ2 F PstIN | CCGGCTGCAGAACCCGAGACAATCAGAGC | ||
| Upstream region of A33_1696 (vopF) | 421 | 5′ WH2 PM F XbaI | TTGTTCTAGACAGCCCGACATTACTATGC |
| 3′ WH2 PM R PstI | AGAGCTGCAGGTTGTGCCGTGTCACTGG | ||
| Upstream region of A33_1675 (vttRB) | 310 | 3′ ToxR2PRMkd R XbaI | GATTTCTAGAGATGACCAATACGCAGTGAC |
| 5′ ToxR2PMRkd F PstI | AAAACTGCAGGGTTGCCTAGTAAGTAGTTTC | ||
| Upstream region of A33_1664 (vttRA) | 412 | ToxR2A F XbaI | GGGATCTAGACTGCTCCTTGCTTAACACTC |
| ToxR2A R PstI | AATCCTGCAGAAAGGGTGAGTCAGATGAGAG | ||
| Deletion constructions | |||
| Deletion of toxR (VC0984 and A33_0921) | |||
| 3′ toxRdel | TTAGCGGTCCGGATGTTTTGGTGCATGG | ||
| 5′ toxRdel OL | CTGGGACATTAGATGTTCATCAAAGTGTGTGAGTAGG | ||
| 5′ toxRdel | TATATTAATTAACCATGCTTATGTTCACGATTG | ||
| 3′ toxR OL | CCTACTCACACACTTTGATGAACATCTAATGTCCCAG | ||
| Deletion of vttRB (A33_1675) | |||
| 3′ ToxR2Rkd SacI | GACGGAGCTCTTACGATGAACTGGGTCGATACTG | ||
| 5′ ToxR2Fkd SalI | AACCCGTCGACAGGCATTGGCAGGACTTAATAATTC | ||
| 3′ SOEa toxR2del | GTGAAAGGGTTCAACGATGTTAAGATCTGCGCTGATTTCTTCATTTGC | ||
| 5′ SOEToxR2Del | GCAAATGAAGAAATCAGCGCAGATCTTAACATCGTTGAACCCTTTCAC | ||
| Deletion of vttRA (A33_1664) | |||
| 3′ SOE N ToxR2A | GAATTGATCTCAAAAATGTATAAACGTGAGCATATAATAAATGAAATC | ||
| 5′ ToxR2.A SOE F | GATTTCATTTATTATATGCTCACGTTTATACATTTTTGAGATCAATTC | ||
| 3′ ToxR2.A del SacI | TATACGAGCTCCTTTCTACAGAACGACTTGAG | ||
| 5′ ToxR2.A Del SalI | ACTAAGTCGACTTGGTGGCAGTATTTATGAG |
a
SOE, splicing by overlapping extension.