Figure 1.
Interaction of GlxR with SprA in the two-hybrid system.
The interaction was assayed by monitoring the growth of cells harboring bait and target proteins. Positive control cells harbored well-established interacting proteins such as LGF2 as bait and Gal11p as the target. Negative control cells harbored empty pBT (bait) and pTRG (target) vectors. NCgl0550p, NCgl0550-encoded protein; and CFU, colony-forming units.
Table 1.
Screened proteins and peptide sequences that interact with GlxR.
Figure 2.
Expression of glxR and sprA during growth.
Cells were grown in glucose MCGC media, and mRNA levels were measured by RT-qPCR as described in the Materials and Methods. The data represent three independent experiments. Filled circles indicate the growth of C. glutamicum. Bars indicate mRNA levels of glxR (grey) and sprA (black), respectively. OD, optical density.
Figure 3.
Growth characteristics of C. glutamicum wild type and mutants.
Cells were grown on glucose (A) or acetate (B) MCGC minimal media. The data represent three independent experiments. Symbols: •, wild-type C. glutamicum; ○, C. glutamicum ΔsprA; ▾, C. glutamicum harboring pSL509 (P180-sprA); ▵, C. glutamicum harboring pSL535 (pMT1-sprA).
Figure 4.
Purification and self-proteolysis of SprA.
Purified His6-SprA (A) was incubated at 4°C or 30°C for 14 h as described in the Materials and Methods (B). The arrows indicate purified His6-SprA. The self-proteolysis of His6-SprA is shown in brackets 1 and 2 of B. M, molecular weight marker.
Figure 5.
In vitro digestion of MBP-GlxR by His6-SprA.
Protein purification and proteolytic assays were performed as described in the Materials and Methods. Proteins were incubated at 4°C or 30°C for 24 h, analyzed on a 15% SDS-PAGE gel, and then visualized by staining with Coomassie brilliant blue G250. Molecular weights are shown in kDa. MBP, maltose-binding protein; M, molecular weight markers; and OD, optical density.
Figure 6.
Proteolytic specificity of His6-SprA.
Proteolytic assays were performed as described in the Materials and Methods. Proteins were incubated at 4°C or 30°C for 24 h, analyzed on a 15% SDS-PAGE gel, and then visualized by staining with Coomassie brilliant blue G250. Lanes 1 and 2 (A) show digested His6-SprA (identical to lane 6 in Figure 5) and purified MBP, respectively. (B) Digestion of bovine serum albumin (BSA) with His6-SprA. Molecular weights are shown in kDa, and M denotes the protein marker.
Figure 7.
Effects of cAMP on in vitro digestion of GlxR by His6-SprA.
Protein purification and proteolytic assays were performed as described in the Materials and Methods. Proteins were incubated at 4°C or 30°C for 20 h, analyzed on a 15% SDS-PAGE gel, and then visualized by staining with Coomassie brilliant blue G250. Molecular weights are shown in kDa. M, molecular weight markers; and MBP, maltose-binding protein.
Table 2.
Enzyme activities of isocitrate lyase (ICL), malate synthase (MS), and isocitrate dehydrogenase (ICDH) in cell extracts of C. glutamicum cellsa.
Figure 8.
Identification of protein affected in sprA-overexpressing C. glutamicum (P180-sprA).
2D-PAGE was performed as described in the Materials and Methods. A total of 150 μg of protein was loaded onto each gel. The identity of each protein spot was determined by electrospray ionization mass spectroscopy.
Figure 9.
Transcription of genes in C. glutamicum strains.
Cells were grown in glucose MCGC media, and mRNA levels were measured by RT-qPCR as described in the Materials and Methods. The data represent three independent experiments. Bars indicate mRNA levels in wild-type (empty bars), ΔsprA (light grey bars), and P180-sprA (dark grey bars) strains.