TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristics or sequence | Source, reference, or comment |
|---|---|---|
| E. coli strains | ||
| JM109 | recAl supE44 endAI hsdR17 gyrA96 relA1 thi Δ(lac-proAB) F′ (traD36 proAB lacIqlacZΔM15) | Stratagene (catalog no. 200235) |
| BL21(DE3) | hsdS gal (λcIts857 ind-l Sam7 nin-5 lacUV5-T7 gene 1) | Novagen (catalog no. 69387-3) |
| C. glutamicum strains | ||
| RES167 | Restriction-deficient mutant of ATCC 13032, Δ(cglIM-cglIR-cglIIR) | University of Bielefeld |
| RES167ΔPcaO | DNA fragment encoding amino acids 106 to 421 of pcaO was deleted | This study |
| Plasmids | ||
| pK18mobsacB | Mobilizable vector for generation of C. glutamicum mutants for deletion of pcaO | 32 |
| pK18mobsacB-ΔPcaO | This study | |
| pXMJ19-lacZ | pXMJ19 carrying lacZ | This study |
| pXMJ19-PpcaHG-lacZ | pXMJ19 carrying PpcaHG and lacZ; lacIq and Ptac were deleted | This study |
| pET28a | Expression vector | Novagen |
| pET28a-PcaO | pET28a derivative for expression of pcaO | This study |
| pEASY-T1 | Cloning vector | Transgen (Beijing, People's Republic of China) |
| Primersa | ||
| 2311F | TGCGGATCCAAAGGAGGAATGTCCCCTATACTAGGTTATTG (BamHI) | Used to generate |
| 2311R | TGATCTAGATCAGACCTTCAGTAATTCTCGAAGCTC (XbaI) | pK18mobsacB-ΔPcaO and pXMJ19-PcaO |
| 2311EF | TGCGGATCCATGTCCCCTATACTAGGTTATTG (BamHI) | Used to generate pET28a- |
| 2311ER | CTATCGCCGGCGTCAGACCTTCAGTAATTC (NotI) | PcaO |
| 2315PE | TCGTTGCTTGGGTTGCGC | Used for primer extension |
| 1516F | AGCTCCGCAGTTCACTCG | of pcaHG and DNase I footprinting |
| 2314RTF | TACAGGGAAGAACGGCGAGT | Used for RT-PCR analysis |
| 2314RTR | AATCAGAGTTGTGGATGC | of ncgl2314 |
| 2315RTF | CAACCCAAGCAACGATCTCA | Used for RT-PCR analysis |
| 2315RTR | TCCTGGAAGAACAATGGATCG | of ncgl2315 |
| rpoBF | GTTGGTGTTCGTATTGAC | Used for RT-PCR analysis |
| rpoBR | TGGTCATACGCTCACGAAC | of rpoB |
| Pro15F | CACGGATCCATGTTCACAACCAGTCTCC (PstI) | Used for generation of |
| Pro15R | TATGCGGCCGCTCAGACCTTCAGTAATTCTC (NarI) | pXMJ19-PpcaHG-lacZ |
| Intr1516Fb | GCGAGACCTTTCTGCGTC | |
| Intr1516R1b | TACTTGTCAACGGCCTCAATC | |
| Intr1516R2b | CGCGATCTGACATGTTCGC | |
| Intr1516R3b | TTGCGCTGGTGGCGAATGTTC | |
| Intr1516R4b | CTTATGCACACCCCTAGTTAACC | |
| Intr1516F5b | TATGCACACCCCTAGTTAAC | |
| Intr1516R5b | GCCACCAGCGCAAACCCC | |
| FiFc | TTATGCACACCCCTAGTTAACCCCGACCTTCGGGGTTT | |
| FaFc | TTATGCACACCCCTAGTTCCAAAAGACCTTCGGGGTTT | |
| FbFc | TTATGCACACCCCTAGTTAACCCCGACCTTTTGGTGCGCTGGTGGCGAATGTT | |
| FcFc | TTATGCACACCCCTAGTTCCAAAAGACCTTCTTGGGGTGCGCTGGTGGCGAATGTT | |
| FRc | GCGCGCGAACATTCGCCA | |
| lacZF | GCCCTGCAGATGACCATGATTACGGA (PstI) | Used for PCR of lacZ gene |
| lacZR | GGGATCCCGGGGAAATACGGGCAGACA (BamHI, SmaI) |
a
Restriction enzyme cut sites are underlined. Ribosome-binding sites are indicated by bold type.
b
Primer used for amplification of divided fragments of the intergenic sequence between pcaHG and ncgl2316 for EMSA analysis (fragment F1, bases −7 to 59; fragment F2, bases −34 to 59; fragment F3, bases −59 to 59; fragment F4, bases −95 to 59; fragment F5, bases −49 to −95).
c
Primer used for generation of mutants with mutations in the Rep4 palindromic sequence.