TABLE 3.
Primers used in this study
| Target gene | Primer name | Sequence (5′−3′)a |
|---|---|---|
| Primers for qRT-PCR | ||
| 16S rRNA | 16S-RTF | ACAGAGGGTGCGAGCGTTAATC |
| 16S-RTR | CTGCCTTCGCCATCGGTATTCC | |
| F959_RS02655 | F959_RS02655-RTF | AGCATTCTTAGCACCGAACACAACA |
| F959_RS02655-RTR | CTCCCAATTTGCGGCACCTGAAA | |
| F959_RS06225 | F959_RS06225-RTF | GCAGTAGGTTGGCTTGCAGGTT |
| F959_RS06225-RTR | TTGTGGCGTCGTGATTGTGGTT | |
| F959_RS06795 | F959_RS06795-RTF | CATGCCATGCCACAAGGAAGTG |
| F959_RS06795-RTR | ACTGTTGCCGTGCCTGTGAG | |
| altL | altL-RTF | ACTGTGGGAGCCAACTGATGACTTT |
| altL-RTR | GCAGCAAGAATCTGACCCGTAGCA | |
| alkRa | alkRa-RTF | CCGCTATCCACAACCATCCTGA |
| alkRa-RTR | TTGGCTCGCTAAACGCATTCG | |
| alkMa | alkMa-RTF | CGATGGGTGCGATCAACGGTAT |
| alkMa-RTR | TGGTGTTGCAGCACGTTTATGG | |
| gfp | gfp-RTF | CGGTGAAGGTGAAGGTGATGC |
| gfp-RTR | CTTCTGGCATGGCAGACTTGAA | |
| Primers for gene deletion | ||
| F959_RS02655 | F959_RS02655-F1 | CGGGATCCAACTAACCCAAACGGCTTCTA |
| F959_RS02655-R1 | CACTTGTGGAATGGTCGAAAACCATGTCTCGAATCAGTTTATC | |
| F959_RS02655-F2 | GATAAACTGATTCGAGACATGGTTTTCGACCATTCCACAAGTG | |
| F959_RS02655-R2 | CCCAAGCTTATGCGGGTTAGCAGAAGTGA | |
| F959_RS06225 | F959_RS06225-F1 | CGGGATCCAGGTCTAATTTGCTTGAACCTGC |
| F959_RS06225-R1 | CAAGCCATAGAGGCATCATCCTTGTTAAACC | |
| F959_RS06225-F2 | GATGATGCCTCTATGGCTTGAAAATCGGCTAT | |
| F959_RS06225-R2 | AACTGCAGAATAACGATACGGTATAAATAGCCC | |
| F959_RS06795 | F959_RS06795-F1 | CGGGATCCTCCAACACTCTATTTGTACTTCGG |
| F959_RS06795-R1 | TCGATATCAAGATGGGGGACTCCATGTCC | |
| F959_RS06795-F2 | GTCCCCCATCTTGATATCGACCCACTGATTACA | |
| F959_RS06795-R2 | AACTGCAGTATCGCATAGTGGACCGACA | |
| altL | altL-F1 | CGGAATTCTACTGTTCAATATGGCGTCTTAG |
| altL-R1 | TCTTTTGCATCATGTCTCCTTAACGTAGTTTTTTT | |
| altL-F2 | AGGAGACATGATGCAAAAGAACAGAAATCAACG | |
| altL-R2 | GCTCTAGAGCTGATAAACGCATGTAACTTTC | |
| alkRa | alkRa-F1 | CGGGATCCCATAGAATGTTTCACCCATTTGT |
| alkRa-R1 | TGTTTAGGTGATGATTTTTTATCGACGTGTTCA | |
| alkRa-F2 | AAAAAATCATCACCTAAACAATATCGGCAGC | |
| alkRa-R2 | AACTGCAGGGTGAAGAGACGTGGTTAGAAAT | |
| almA | almA-F1 | CGGGATCCTTCTGTAAATTCAAGATAGTGCCT |
| almA-R1 | CAAGCAAAAAATACCCTGTATAAACCATGACTTT | |
| almA-F2 | TACAGGGTATTTTTTGCTTGAATAATTTGAAAC | |
| almA-R2 | AACTGCAGGTACCAATGAGATCATGAAAGAACT | |
| alkMa | alkMa-F1 | CGGGATCCTGTGGATAGCGGCTAAACTA |
| alkMa-R1 | CTACTTGAGTCATCTTTTCCATGTCTTTGTATA | |
| alkMa-F2 | GGAAAAGATGACTCAAGTAGAAGTAGTCGCTGA | |
| alkMa-R2 | AACTGCAGTTCTATATCAGGTAATGAGGGCT | |
| altLPa | altLPa-F1 | GGAATTCGCGGCGCTGATGCTGTCT |
| altLPa-R1 | CAGCCCTCAGAAGGTCGCGGGTTACTGGGCCTTC | |
| altLPa-F2 | GCCCAGTAACCCGCGACCTTCTGAGGGCTGGAGC | |
| altLPa-R2 | CAAGCTTCGATGAAAGCGTAGCGATACC | |
| Sequencing primers for gene deletion mutants | ||
| F959_RS02655 | seq_F | GCACCACCAATGAAGGGG |
| seq_R | GCACCATCAACAACTAAAGCC | |
| F959_RS06225 | seq_F | TCAGCATTGCAGCTAAAGTAA |
| seq_R | CGCTATTCATCTATGGTGGC | |
| F959_RS06795 | seq_F | GATACAATCTGTCGCAACCA |
| seq_R | CCAATCAATAAGCGGAAAA | |
| altL | seq_F | CGGTTAAGGGTTGAAGAGG |
| seq_R | TCAAGAAAGCAACGGTCAT | |
| alkRa | seq_F | GCAACAATTCATTGTCTGTAGACC |
| seq_R | ATTACGTTCGCATGTTGTAGTTG | |
| almA | seq_F | AAGTTTAGGCGTTGATGCAG |
| seq_R | CCTGTTACGGCTGTAGATGC | |
| alkMa | seq_F | GCTAAACGCATTCGATGTT |
| seq_R | CAAGCCTACCTGAGACCCT | |
| Primers for gene knock-in | ||
| gfp | alkMa-up-F | ATTCGAGCTCGGTACCCGGGTGTGGATAGCGGCTAAACTA |
| alkMa-up-R | CTTTAGACATCATCTTTTCCATGTCTTTGTATA | |
| gfp-F | GGAAAAGATGATGTCTAAAGGTGAAGAATTATTCA | |
| gfp-R | TGTCTTTAAATTATTTGTACAATTCATCCATACCA | |
| alkMa-dn-F | GTACAAATAATTTAAAGACATGATCAAATGAAAGC | |
| alkMa-dn-R | GCCAAGCTTGCATGCCTGCAAACAAGCCTACCTGAGACCCT | |
| Primers for gene overexpression | ||
| alkRa-gfp | pMK-F | GTACCCGGGGATCCTCTAGAGTC |
| pMK-R | AGCCTTTTCTGAGCATGGTATTT | |
| alkRa-gfp-F | TACCATGCTCAGAAAAGGCTTTATGTTTCATTTTGAATATATTGC | |
| alkRa-gfp-R | TCTAGAGGATCCCCGGGTACTTATTTGTACAATTCATCCATACCA | |
| Primers for gene complementation | ||
| altL | altL-F | ATTCGAGCTCGGTACCCGGGTTGGAAAAAAACTACGTTAAGG |
| altL-R | CCTGCAGGTCGACTCTAGAGTTGTATAGAAACCATCTGCATATAG | |
| alkRa | alkRa-F | CAATTTCACACAGGAAACAGATGGATGCTTTAAGTAAAATATTTG |
| alkRa-R | TCTAGAGGATCCCCGGGTACTTATGTTTCATTTTGAATATATTGC | |
| altLPa | altLPa-F | CAATTTCACACAGGAAACAGAATTCAGTAACCCGCGAGCGAGGC |
| altLPa-R | TCTAGAGGATCCCCGGGTACCTCAGAAGGTCGTACGGTAGGTCAT | |
| Sequencing primers for plasmids | ||
| pMMB67EH | pMM-F | AGCGGATAACAATTTCACACAG |
| pMM-R | CGCTTCTGCGTTCTGATTTAA |
a
Nucleotides in bold represent restriction enzyme sites added to the 5′ region of the primers. Underlines nucleotides represent overlap sequences.