TABLE 2.
Oligonucleotides used in this study
| Primers | Sequencea | Use |
|---|---|---|
| 5′HisaccR | 5′-CGGGATCCTTACCGCACCGTCCGCCGTA (BamHI) | Amplification of the 5′-end of accR; used to construct His-tagged accR and accRD60E genes |
| 3′HisaccR | 5′-AACTGCAGTCAGCACGTTCTGCACGAGTTCG (PstI) | Amplification of the 3′-end of accR gene; used to construct the accR, CaccR, and accRD60E genes |
| 5′6HisCAccR | 5′-CGGGATCCTTCGGCAGCTCGAGGCGGAGA (BamHI) | Amplification of the 5′-end of CaccR; used to construct His-tagged CaccR |
| 5′AccRD60E | 5′-CTCGTGCTCGAAGTGCGCATG | Internal accR primers used to introduce the accRD60E substitution |
| 3′AccRD60E | 5′-CATGCGCACTTCGAGCACGAG | |
| 5′AccSR | 5′-ACGCGTCGACTGACCTAAGGAGGTAAATAATGTCCGACTCTGCCGAATCCG (SalI) | Used to amplify and clone the accSR genes into plasmid pIZAccSR |
| 3′AccSR | 5′-GGACTAGTTCAGCGGCCGCCACCTCC (SpeI) | |
| 5′AccRext | 5′-CGGGATCCTAGTTAACTAGGAAGGGGGTACCATCTTCTC (BamHI) | Amplification of the accR gene into plasmid pBBR5AccR |
| 3′AccRext | 5′-GCTCTAGACAGGCCGGCACATCCGTCAGCG (XbaI) | |
| 5′AccRmut(BamHI) | 5′-CGGGATCCGCGGAGGTCTCGCGCCAGC (BamHI) | Amplification of a 762-bp BamHI/XbaI fragment, spanning the upstream region of accR, for constructing the ΔaccR allele in pKNG101ΔaccR |
| 3′AccRmut(XbaI) | 5′-GCTCTAGACATTGTTCAGGTACGGCGGACG (XbaI) | |
| 5′AccRmut(XbaI) | 5′-GCTCTAGAGGCGGCCGCTGACGGATG (XbaI) | Amplification of a 642-bp XbaI/SpeI fragment, spanning the downstream region of accR, for constructing the ΔaccR allele in pKNG101ΔaccR |
| 3′AccRmut(SpeI) | 5′-CCACTAGTCCATTACGCTGAGGAACCTTGCG (SpeI) | |
| 5′pboxdQ | 5′-CAACTCCAGTGCCTGTGCG | RT-PCR amplification of cDNA, a 153-bp product that reports PD promoter activity |
| 3′pboxdQ | 5′-GAGAGGAGACAATCAGGTGAAGC | |
| 5′RTpN1 | 5′-GCAACACATCAGAGGAGATAG | RT-PCR amplification of cDNA, a 141-bp product that reports PN promoter activity |
| 3′RTpN2 | 5′-GTGTAGGTACACATCGTTGC | |
| 5′POLIIIHK | 5′-CGAAACGTCGGATGCACGC | RT-PCR amplification of cDNA, a 166-bp product of dnaE used as internal control in RT-PCR assays |
| 3′POLIIIHK | 5′-GCGCAGGCCTAGGAAGTCGAAC | |
| FPAdh 5′ | CGGAATTCGCACCTTCGATCCATTGCCC (EcoRI) | Amplification of a 234-bp boxDR intergenic fragment, PD probe |
| FPAdh 3′ bis | AAAAGTACTCGGTTGCTGTGATGCTGTGTC (ScaI) | |
| 5IVTPN | CGGAATTCCGTGCATCAATGATCCGGCAAG(EcoRI) | 5′-end of PN promoter, used with 3IVTPN to amplify the 376-bp PN probe |
| 3IVTPN | CGGAATTCCATCGAACTATCTCCTCTGATG(EcoRI) | 3′-end of PN promoter, used to amplify the PN, PN2, and PN2mut probes |
| 5′PN3 | TTTCCGCTCGCGTTCGCTCTC | Amplification of the 93-bp PN3 probe |
| 3′PN3 | AATCTTTCTTGCCGCAACGC | |
| 5′PN2 | AATGCAATCAAGTGCATGCAAACG | Used with 3IVTPN to amplify the 162-bp PN2 probe |
| 5′PN mut3 | AAACCCCTGATCAAGTGCATGCA | Used with 3IVTPN to amplify the 165-bp PN2mut probe |
| 5′ScaI PB1 | AAAAGTACTGGTATTACGGTAAGTGCTCCA (ScaI) | Amplification of the 251-bp PB1 probe |
| 3′EcoRI PB1 | CCGGAATTCCCTGCGCGCGGCACTATG (EcoRI) |
a Engineered restriction sites are underlined, and the corresponding restriction enzyme is shown in parentheses.