TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristicsa | Source, reference, or note | ||
|---|---|---|---|---|
| Strains | ||||
| E. coli | ||||
| 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 | ||||
| RES167 | Restriction-deficient mutant of ATCC 13032; Δ(cglIM-cglIR-cglIIR) | University of Bielefeld | ||
| RES167ΔNCgl1110 | A fragment of DNA coding for amino acids 105 to 215 of NCgl1110 was deleted | This study | ||
| RES167ΔNCgl1111 | A fragment of DNA coding for amino acids 123 to 375 of NCgl1111 was deleted | This study | ||
| RES167ΔNCgl1112 | A fragment of DNA coding for amino acids 114 to 222 of NCgl1112 was deleted | This study | ||
| RES167ΔNCgl1113 | A fragment of DNA coding for amino acids 122 to 180 of NCgl1113 was deleted | This study | ||
| RES167ΔNCgl(2950-2953) | A fragment of DNA coding for amino acids 170 to 1288 of NCgl(2950-2953) was deleted | This study | ||
| RES167ΔNCgl(2950-2953)/ΔNCgl1112 | Fragments of DNA coding for amino acids 114 to 222 of NCgl1112 and 170 to 1288 of NCgl(2950-2953) were deleted | This study | ||
| RES167ΔNCgl(2950-2953)/ΔNCgl1113 | Fragments of DNA coding for amino acids 122 to 180 of NCgl1113 and 170 to 1288 of NCgl(2950-2953) were deleted | This study | ||
| Plasmids | ||||
| pK18mobsacB | Mobilizable vector, allows for selection of double crossover in C. glutamicum | Schäfer et al. (22) | ||
| pK18mobsacB-ΔNCgl1110 | Carrying NCgl1110 deletion (see RES167ΔNCgl1110) | This study | ||
| pK18mobsacB-ΔNCgl1111 | Carrying NCgl1111 deletion (see RES167ΔNCgl1111) | This study | ||
| pK18mobsacB-ΔNCgl1112 | Carrying NCgl1112 deletion (see RES167ΔNCgl1112) | This study | ||
| pK18mobsacB-ΔNCgl1113 | Carrying NCgl1113 deletion (see RES167ΔNCgl1113) | This study | ||
| pK18mobsacB-ΔNCgl(2950-2953) | Carrying NCgl(2950-2953) deletion [see RES167ΔNCgl(2950-2953)] | This study | ||
| pXMJ19 | Shuttle vector (CamrPtac lacIqpBL1 oriVC. glutamicumpK18 oriVE. coli) | Jakoby et al. (11) | ||
| pXMJ19-NCgl1110 | Carrying NCgl1110 (to generate complementation for NCgl1110) | This study | ||
| pXMJ19-NCgl1111 | Carrying NCgl1111 (to generate complementation for NCgl1111) | This study | ||
| pXMJ19-NCgl1112 | Carrying NCgl1112 (to generate complementation for NCgl1112) | This study | ||
| pXMJ19-NCgl1113 | Carrying NCgl1113 (to generate complementation for NCgl1113) | This study | ||
| pXMJ19-NCgl2951 | Carrying NCgl2951 (to generate complementation for NCgl2951) | This study | ||
| pXMJ19-NCgl2952 | Carrying NCgl2952 (to generate complementation for NCgl2952) | This study | ||
| pET28a | Expression vector with N-terminal hexahistidine affinity tag | Novagen | ||
| pET28a-NCgl1111 | pET28a derivative for expression of NCgl1111 | |||
| pET28a-NCgl1112 | pET28a derivative for expression of NCgl1112 | This work | ||
| pGEM-T Easy | Cloning of PCR products | Promega | ||
| Primers | ||||
| 1110F(a) | GATTCTAGAAAAGGAGGACACATATGCCCACGCCTTCG (XbaI) | To generate pK18mobsacB-ΔNCgl1110 and pXMJ19-NCgl1110 | ||
| 1110R | AGCAAGCTTGAGCTCCCTGAGTCTGGTGCTT (HindIII, SacI) | |||
| 1111F(a) | GATTCTAGAAAAGGAGGACCATATGTCACCCAATAACTTCG (XbaI, NdeI) | To generate pK18mobsacB-ΔNCgl1111, pXMJ19-NCgl1111, and pET28a-NCgl1111 | ||
| 1111R | AGCAAGCTTCCCGGGCCTCACCTGAACCGAAC (HindIII, SmaI) | |||
| 1112F(a) | GATTCTAGAAAAGGAGGACAACCATGTCTTTACAGTTCGAT (XbaI) | To construct pK18mobsacB-ΔNCgl1112 and pXMJ19-NCgl1112 | ||
| 1112R | ACAGAATTCACACGAGCGTTTATACC (HindIII, SacI) | |||
| 1112EF | GTCGCTAGCATGTCTTTACAGTTCGATC (NheI) | |||
| 1112ER | AGCAAGCTTGAATTCCGTAGTTCCACTCTTCTT (HindIII, SacI) | |||
| 1113a | GATTCTAGAAAAGGAGGACAACCATGACTATTTCAGCACAA (XbaI) | To construct pK18mobsacB-ΔNCgl1113, pXMJ19-NCgl1113, and RT-PCR | ||
| 1113b | GGTATTCGCCGTTTTCTGTCATCTCCGTTTTG | |||
| 1113c | GAAAACGGCGAATACC | |||
| 1113d | AGCAAGCTTGAGCTCTCTCAAATTGCCAAAA (HindIII, SacI) | |||
| 2950-2953a | GATTCTAGAAAATGCCCCTCGCTTGAAAA (XbaI) | To generate pK18mobsacB-ΔNCgl(2950-2953) | ||
| 2950-2953b | AGCAAGCTTGTCCGCACCCCTGTCCTAAA (HindIII) | |||
| 2950-2953c | TTTAGGACAGGGGTGCGGACAAGCTTATCCTGGTCCGTAGAACTGC (HindIII) | |||
| 2950-2953d | AGCGAATTCACAGGGCTAAATGCTCCAA (EcoRI) | |||
| 1110b | AATCCGCCCCAAATCC | To perform RT-PCR on the fragments of the related genes | ||
| 1111b | AATGATGGCGGAGTTTT | |||
| 1112b | AACCATTGAGACCAGA | |||
| 2950a | GATTCTAGAAAAGGAGGACACATATGAATCTGAAAGATCTC (XbaI, NdeI) | |||
| 2950b | TCATTGAAAGCCACTC | |||
| 2951a | GATTCTAGAAAAGGAGGACACATATGACAACCACCACCG (XbaI, NdeI) | |||
| 2951b | TTCCTTCAATCCACGC | |||
| 2952a | GATTCTAGAAAAGGAGGACACATATGAACAACTCACTCGCATT (XbaI, NdeI) | |||
| 2952b | GAGCACCTTCAGATCAACA | |||
a
Restriction enzyme sites are underlined. Ribosome binding sites are boldfaced. Nucleotides that are complementary to primer 2950-2953b are italicized.