TABLE 1.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristics | Reference, construction, or source |
|---|---|---|
| Strains | ||
| YMC10 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs | 8 |
| RB9060 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 | 10 |
| K | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs Δmdl-glnK::Kan | 6 |
| BK | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::Kanr | 6 |
| BKc | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::Camr | 6 |
| BKΦ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA130 [Φ(glnKp-lacZ) Kanr Cams] | 6 |
| WCH30 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnK1 (amtB+) | 1 |
| UNF3435 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnK1 (amtB+) ΔglnB2306 | 1 |
| YMC15 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 | 11 |
| BKgΦ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnK1 (amtB+) ΔglnB2306 trpDC700::putPA130 [Φ(glnKp-lacZ) Kanr Cams] | BΦ × WCH30 P1vir |
| BΦ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 trpDC700::putPA130 [Φ(glnKp-lacZ) Kanr Cams] | 6 |
| BA | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 glnA::Tn5 | 13 |
| TEΦ | recD1903::Tn10 trpDC700::putPA130 [Φ(glnKp-lacZ) Kanr Cams] | 6 |
| YMC10Φ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs trpDC700::putPA130 [Φ(glnKp-lacZ) Kanr Cams] | 6 |
| YMC15Φ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 trpDC700::putPA130 [Φ(glnKp-lacZ) Kanr Cams] | YMC15 × TEΦ P1vir |
| MAKc | recD Δmdl-glnK::Camr | 6 |
| Kc | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs Δmdl-glnK::Camr | YMC10 × MAKc P1vir |
| YMC15K | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::Camr | YMC15 × MAKc P1vir |
| YMC15B | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 ΔglnB2306 | BA × YMC15 P1vir |
| YMC15BΦ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 ΔglnB2306 trpDC700::putPA1303 [Φ(glnKp-lacZ) Kanr Cams] | YMC15B × TEΦ P1vir |
| YMC15BKΦ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA1303 [Φ(glnKp-lacZ) Kanr Cams] | YMC15BΦ × MAKc P1vir |
| YMC15KΦ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::CamrtrpDC700::putPA1303 [Φ(glnKp-lacZ) Kanr Cams] | YMC15Φ × MAKc P1vir |
| Y15KgΦ | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 ΔglnK1 (amtB+) trpDC700::putPA1303 [Φ(glnKp-lacZ) Kanr Cams] | YMC15Φ × WCH30 P1vir |
| TE2680 | recD1903::Tn10 trpDC700::putPA1303 [Kans Camrlac] | 12 |
| MABpB | recD1903::Tn10 trpDC700::putPA1303 [Kanr CamsglnB8] | TE2680 × pglnB8 DNA |
| MAKpK | recD1903::Tn10 trpDC700::putPA1303 [Kanr CamsglnK91] | TE2680 × pglnK91 DNA |
| MAKpB2 | recD1903::Tn10 trpDC700::putPA1303 [Kanr Cams Φ(glnKp-glnB)] | TE2680 × pglnKpB2 DNA |
| MABpK2 | recD1903::Tn10 trpDC700::putPA1303 [Kanr Cams Φ(glnBp-glnK)] | TE2680 × pglnBpK2 DNA |
| MAApB2 | recD1903::Tn10 trpDC700::putPA1303 [Kanr Cams Φ(glnAp-glnB)] | TE2680 × pglnApB2 DNA |
| MAApK2 | recD1903::Tn10 trpDC700::putPA1303 [Kanr Cams Φ(glnAp-glnK)] | TE2680 × pglnApK2 DNA |
| BKcBpB | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr CamsglnB8] | BKc × MABpB P1vir |
| BKcBpK2 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr Cams Φ(glnBp-glnK)] | BKc × MABpK2 P1vir |
| BKcKpK | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr CamsglnK91] | BKc × MAKpK P1vir |
| BKcKpB2 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr Cams Φ(glnKp-glnB)] | BKc × MAKpB2 P1vir |
| BKcApB2 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr Cams Φ(glnAp-glnB)] | BKc × MAApB2 P1vir |
| BKcApK2 | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs ΔglnB2306 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr Cams Φ(glnAp-glnK)] | BKc × MAApK2 P1vir |
| Y15KcBpB | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr CamsglnB8] | YMC15K × MABpB P1vir |
| Y15KcKpK | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr CamsglnK91] | YMC15K × MAKpK P1vir |
| Y15KcKpB | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::CamrtrpDC700::putPA1303 [Kanr Cams Φ(glnKp-glnB)] | YMC15K × MAKpB2 P1 vir |
| Y15BBpB | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 ΔglnB2306 trpDC700::putPA1303 [Kanr CamsglnB8] | YMC15B × MABpB P1vir |
| Y15BKpK | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 ΔglnB2306 trpDC700::putPA1303 [Kanr CamsglnK91] | YMC15B × MAKpK P1vir |
| Y15BKpB | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 ΔglnB2306 trpDC700::putPA1303 [Kanr Cams Φ(glnKp-glnB)] | YMC15B × MAKpB2 P1vir |
| Y15BKcBpB | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::Camr ΔglnB2306 trpDC700::putPA1303 [Kanr CamsglnB8] | Y15BBpB × MAKc P1vir |
| Y15BKcKpK | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::Camr ΔglnB2306 trpDC700::putPA1303 [Kanr CamsglnK91] | Y15BKpK × MAKc P1vir/PICK> |
| Y15BKcKpB | endA1 thi-1 hsdR17 supE44 ΔlacU169 hutCklebs glnL2302 Δmdl-glnK::Camr ΔglnB2306 trpDC700::putPA1303 [Kamr Cams Φ(glnKp-glnB)] | Y15BKpB × MAKc P1vir |
| Plasmids | ||
| pRS551 | lac fusion vector | 36 |
| pAlter-1 | Amps mutagenesis vector | Promega Corporation |
| pUC18N | pUC18, with NdeI site destroyed | 6 |
| pUC18NS | pUC18, with NdeI and SalI sites destroyed | |
| pglnB5 | glnB PCR product cloned as BamHI fragment into pAlter-1 | |
| pglnB6 | NdeI site introduced at glnB start codon of pglnB5 | |
| pglnB8 | BamHI fragment containing glnB cloned into pRS551 from pglnB6 | |
| pglnB10 | glnB cloned into pUCN as EcoRI/HindIII fragment from pglnB6 | |
| pglnK51 | glnK PCR product cloned as BamHI fragment into pAlter-1 | |
| pglnK71 | NdeI site introduced at glnK start codon of pglnK5 | |
| pglnK81 | BamHI fragment containing glnK cloned into pUCNS from pglnK71 | |
| pglnK91 | BamHI fragment containing glnK cloned into pRS551 from pglnK71 | |
| pglnKpB1 | glnK coding region of pglnK81 exchanged for the glnB coding region from pglnB10 as NdeI/SalI fragment | |
| pglnKpB2 | BamHI fragment containing glnKp-glnB fusion gene cloned into pRS551 | |
| pglnBpK1 | glnB coding region of pglnB10 exchanged for the glnK coding region of pglnK71 | |
| pglnBpK2 | BamHI fragment containing glnBp-glnK fusion gene cloned into pRS551 | |
| pglnApB1 | glnB promoter of pglnB10 exchanged for glnA promoter generated by PCR as a KpnI/NdeI fragment | |
| pglnApB2 | BamHI fragment containing glnAp-glnB fusion gene cloned into pRS551 | |
| pglnApK1 | glnB coding region of glnAp-glnB fusion gene exchanged for the glnK coding region of glnK71 as NdeI/HindIII fragment | |
| pglnApK2 | BamHI fragment containing glnAp-glnK fusion gene cloned into pRS551 | |
| Primers | ||
| 113D | GGGGATCCGCGGCCCGGTTGGCAACATGC, primer downstream of glnB for cloning (BamHI) | |
| 7158I | GCTGCAGGGATCCCTCAACTATTTGCGTAAGCTGCTGC, cloning primer located upstream of glnB promoter (PstI/BamHI) | |
| 7159I | GCTGCAGGGATCCCATTGAGCGCCTGAATAGCGC, cloning primer located upstream of glnk promoter (PstI/BamHI) | |
| 5048F | CGGATCCGTCGACTTCCTGTTGCTGTGTGCCAGAG, primer downstream of glnK for cloning and sequencing (SalI/BamHI) | |
| 4158J | CGGTACCGGATCCCCTCCGCAAACAAGTATTGCAGAGTCCC, cloning primer located upstream of glnA promoter (KpnI/BamHI) | |
| 4157J | CCATATGTTAACTCTCCTGGATTGGTCATGGTCGTCGTG downstream primer introduces NdeI site at glnA ATG start codon for cloning the glnA promoter region (NdeI) | |
| 9829I | GACCGGAGGGGACATATGAAGCTGGTGACC, internal primer for site-specific mutagenesis introducing an NdeI | |
| site at the glnK ATG start codon | ||
| 9251I | TTTTCAAGGAATCATATGAAAAAGATTGAT, internal primer for site-specific mutagenesis introducing an NdeI site at the glnB ATG start codon | |
| 4588E | GGTCATCAGCAATCGCCAC, internal glnK sequencing primer | |
| 5551E | CGTCAGTAAGGCGGCTTACAC, internal glnK sequencing primer | |
| 4929F | GATTATCACGGTCACCAGCTTC, internal glnK sequencing primer | |
| 2820 | ACGGTGACCGAAGTGAAAGGC, internal glnB sequencing primer | |
| 2866 | CAGCTCGGTATGGCCTTTC, internal glnB sequencing primer | |
| 3098 | CGATCCGGCAACCCTTGACGCGG, internal glnB sequencing primer |