The glxR mutants grew very slowly, forming tiny colonies on the sucrose selection plate after 3 days of incubation. The deletion of the glxR gene in the mutant strain was verified by PCR (Fig. 1) and Southern blot (data not shown). To explore the growth phenotype of the glxR mutant, it was grown in MB medium containing various carbon sources, including glucose, sucrose,
acetate, pyruvate and glutamate. The glxR mutant displayed a significantly reduced growth rate GSK2126458 compared with that of the wild type, regardless of the carbon source (μ, 0.74–0.8 vs. 0.19–0.21 h−1) (Fig. 2a), which was consistent with the growth on the agar plate. The growth yields of the glxR mutant were about 75% of those of the wild type at the stationary phase when acetate or glutamate was used as the carbon source. Whereas the wild-type strain exhibited a similar growth yield and growth rate in the MB medium, independent of the carbon source, the glxR mutant entered the stationary phase earlier when the medium contained glucose or pyruvate rather than acetate or glutamate. To further verify that the phenotype observed was solely due to the deletion of the glxR gene, the mutant strain was complemented with the recombinant plasmids pCR1 and pCR2, containing the glxR and S. coelicolor crp genes, respectively. The complemented
strains displayed a growth phenotype Ibrutinib similar to that of the wild-type strain when cultivated in the MB medium (Fig. 2b). Thus, these findings indicate that the mutation in the glxR gene is responsible for the impaired growth phenotype, suggesting that GlxR is important for the growth of C. glutamicum. Previously, it has been speculated that GlxR represses the genes of the glyoxylate bypass enzymes in the presence of glucose.
In contrast, the overexpression of GlxR repressed the glyoxylate bypass enzymes, ICL and MS, on acetate, but not on a glucose medium (Kim et al., 2004); thus, the role of GlxR in the regulation of the glyoxylate bypass genes remains unclear. SDS-PAGE was performed to compare the total protein lysates from the wild type and glxR mutant grown on acetate and glucose as the carbon source. The synthesis of ICL (45 kDa) and MS (96 kDa) was found to be significantly increased in the acetate-grown Dapagliflozin glxR mutant when compared with the acetate-grown wild type (Fig. 3a) as demonstrated by an N-terminal sequence analysis of the blotted proteins. The N-terminal amino acid sequences of ICL and MS were revealed to be Met–Ser–Asn–Val–Gly–Lys –Pro–Arg–Thr–Ala and Met–Thr–Glu–Gln–Glu–Leu–Leu –Ser–Ala–Gln, respectively. The SDS-PAGE data also showed an increased production of both enzymes with the glxR mutant in the glucose medium (Fig. 3a). The specific activities of ICL and MS in the acetate-grown glxR mutant were about 1188.4 and 506.9 mU, respectively, representing a 2.