TABLE 2.
Oligonucleotide primers used in this study
| Targeta | Nucleotide sequence (5′ to 3′) |
|---|---|
| ΔasbA | CGCGCGGCCGCCAAGAGTATCAAGGAATGAAAATAGGAC |
| CGCCCCGGGTTGTTTCGCATGCTTCATACTTGCCCTC | |
| CGCCCCGGGGTGGCTGTTCGTTCATAAATAAGGTTAAAATTTC | |
| CGCGGATCCCTTATTACATCTTTCATATTGTGCAATCGTATC | |
| ΔasbB | CGCGCGGCCGCCAGCTAAAGGATGGTTATCCGGTC |
| CGCCCCGGGATGATACATATCCATTCTCATACCCCCT | |
| CGCCCCGGGATAGATGTGGAATCGCTAGTTCATGAAGTTCCA | |
| CGCGGATCCCCATAACAATCGGTACTTCATCTATGTCAC | |
| ΔasbC | CGCGCGGCCGCCATAAAGAAGGATTACCTGTCCGTATTGC |
| CGCCCCGGGTTCTCTATTAACAATTAGCATAATTTCCCCCT | |
| CGCCCCGGGATGGGGGAAATTGTTAAAGCGAAAGTAATC | |
| CGCGGATCCCTGTTATATCAAAGTCTCCATCCCATAGTCC | |
| ΔasbD | CGCGCGGCCGCGATGCAACAATACGGTTGTTCTGAAGC |
| CGCCCCGGGCGCTTCCCGTCTCATGTTGTAACC | |
| CGCCCCGGGCCGTTACAGGACGTAAATGTGAATAACTAAC | |
| CGCGGATCCCCCTTCCCAGCGAAAATCAGGATC | |
| ΔasbE | CGCGCGGCCGCGCAAGAGGCGATTGTATATCGAGGG |
| CGCCCCGGGCACTTTAATTGAAGTCATACTATCATCCACTTTC | |
| CGCCCCGGGGAGTCTGTCTTAGTATTTTAAAAACTTTACTG | |
| CGCGGATCCGAAGGCAACGTATCCGTTAACGTATTTG | |
| ΔasbF | CGCGCGGCCGCCAGGAGCCAACAGCAGAAATGGTAG |
| CGCCCCGGGTAGTGAATATTTCATAGGTTTGAACTCCC | |
| CGCCCCGGGGAAGTAGTAACTTCTTAATATAAAATGATGAAAGAG | |
| CGCGGATCCGGCTATTACATCTTTACTACTTCCCATTC | |
| ΔasbAB | AACTAGCGATTCCACATCCCCGGGTTGTTTCGCATGCTTCATACTTG |
| ATGAAGCATGCGAAACAACCCGGGGATGTGGAATCGCTAGTTCATG | |
| Diagnosticb | TAACATATGAGCAACGAGAAAAACAATGGG |
| CAAACTCGGAACCATTACCATATTTCTCCC | |
| TTCCCCTCGTAATTGCAGATAACGTACCAC | |
| GGGATCGCACTCGAATCACA | |
| asbA complementation | CGCGGTACCCAAGAGTATCAAGGAATGAAAATAGGACAAAAG |
| CGCGGTACCTTATGAACGAACAGCCACTTCTCTAACG | |
| asbB complementation | CGCGGTACCCAAGAGTATCAAGGAATGAAAATAGGACAAAAG |
| ATACATATCCATTCTCATACTTGCCCTCCTCTTTCTATAAACAC | |
| AAGAGGAGGGCAAGTATGAGAATGGATATGTATCATACGAAAATATTG | |
| CGCGGTACCTTAACAATTAGCATAATTTCCCCCTG |
Four oligonucleotide primers (i.e., two pairs) were used to generate each allelic exchange construct. One pair (the first two listed) generated a fragment with homology upstream to the specific gene, and the other pair (the last two listed) generated a fragment with homology downstream to the gene. The entire operon deletion (ΔasbABCDEF) was constructed using the upstream primer pair from the ΔasbA set and the downstream primer pair from the ΔasbF set. The upstream homology for the ΔasbAB mutant was generated using the first primer from the ΔasbAB set and the first primer from the ΔasbA set; the downstream homology was generated using the second primer from the ΔasbAB set and the fourth primer from the ΔasbB set.
The first and third diagnostic primers were used to identify the ΔasbA, ΔasbB, and ΔasbAB mutations; the second and fourth diagnostic primers were used to identify the ΔasbC, ΔasbD, ΔasbE, and ΔasbF mutation; the first and second diagnostic primers were used to identify the ΔasbABCDEF mutation.