TABLE 1.
Bacterial strains and plasmids used in this study
| Strains and plasmids | Relevant characteristics | Source or reference |
|---|---|---|
| Strains | ||
| E. coli DH5α | F− f80lacZDM15 D(lacZYA-argF) U169 endA1 recA1 hsdR17 (rk−, mk+) supE44 thi-1 gyrA96 relA1 phoA, host for cloning purposes | Invitrogen |
| E. coli BL21(DE3) with pLysS | F−ompT hsdSB (rB−, mB−) gal dcm (DE3); host for overproduction of RosR | Ref. 60 |
| C. glutamicum ATCC 13032 | Biotin-auxotrophic wild-type strain | American Type Culture Collection |
| C. glutamicum ΔrosR | Strain with an in-frame deletion of the rosR (cg1324) gene | This work |
| C. glutamicum Δcg1322 | Strain with an in-frame deletion of the cg1322 gene encoding a putative polyisoprenoid-binding protein | This work |
| Plasmids | ||
| pK19mobsacB | KanR; vector for allelic exchange in C. glutamicum (pK18 oriVE.c., sacB, lacZa) | Ref. 61 |
| pK19mobsacB-ΔrosR | KanR; pK19mobsacB derivative containing an overlap extension PCR product composed of the up- and downstream regions of rosR | This work |
| pK19mobsacB Δcg1322 | KanR; pK19mobsacB derivative containing an overlap extension PCR product composed of the up- and downstream regions of cg1322 | This work |
| pET16b | AmpR; plasmid for overexpression of genes in E. coli, adding an N-terminal decahistidine tag and a factor Xa cleavage site to the target protein (pBR322 oriVE.c., PT7, lacI) | Novagen |
| pET16b-rosR | KanR; pET16b derivative for overproduction of RosR with an N-terminal decahistidine tag | This work |
| pET16b-rosRC64S | pET16b-rosR derivative coding for a RosR protein with a Cys-64 → Ser exchange | This work |
| pET16b-rosRC92S | pET16b-rosR derivative coding for a RosR protein with a Cys-92 → Ser exchange | This work |
| pET16b-rosRC151S | pET16b-rosR derivative coding for a RosR protein with a Cys-151 → Ser exchange | This work |
| pET16b-rosRC64S,C92S | pET16b-rosR derivative coding for a RosR protein with Cys-64,Cys-92 → Ser exchanges | This work |
| pET16b-rosRC64S,C151S | pET16b-rosR derivative coding for a RosR protein with Cys-64,Cys-151 → Ser exchanges | This work |
| pET16b-rosRC92S,C151S | pET16b-rosR derivative coding for a RosR protein with Cys-92,Cys-151 → Ser exchanges | This work |
| pET16b-rosRC64S,C92S,C151S | pET16b-rosR derivative coding for a RosR protein with Cys-64,Cys-92,Cys-151 → Ser exchanges | This work |
| pET28b | KanR; plasmid for overexpression of genes in E. coli, adding an N-terminal hexahistdine tag and a thrombin cleavage site to the target protein (pBR322 oriVE.c., PT7, lacI) | Novagen |
| pET-TEV | KanR; pET28b derivative for overexpression of genes in E. coli, adding an N-terminal decahistdine tag and a TEV protease cleavage site to the target protein (pBR322 oriVE.c., PT7, lacI) | This work |
| pET-TEV-rosR | KanR; pET-TEV derivative for overproduction of RosR with an N-terminal decahistidine tag, which can be cleaved off using TEV protease | This work |
| pAN6 | KanR; C. glutamicum/E. coli shuttle vector for regulated gene expression; derivative of pEKEx2 (Ptac, lacIq, pBL1 oriVC.g., pUC18 oriVE.c.) | Ref. 14 |
| pAN6-rosR | KanR; pAN6 derivative carrying the rosR gene under the control of an IPTGa-inducible tac promoter | This work |
| pET2 | KanR; promoter probe plasmid for C. glutamicum | Ref. 26 |
| pET2-rosR | KanR; pET2-derivative with a 284-bp fragment covering the C. glutamicum rosR promoter | This work |
a Isopropyl 1-thio-β-d-galactopyranoside.