Regulation of expression of general components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) by the global regulator SugR in Corynebacterium glutamicum

The phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) catalyzes transport of carbohydrates by coupling carbohydrate translocation and phosphorylation. Enzyme I and HPr, encoded in ptsI and ptsH, respectively, are cytoplasmic proteins commonly used for transport of variety of PTS sugars. In this study, we investigated the role of SugR on the expression of the ptsI and ptsH which increases in the presence of PTS sugars in Corynebacterium glutamicum. Disruption of sugR resulted in the increased expression of ptsI and ptsH in the absence of PTS sugar. Introduction of a plasmid containing sugR gene complemented the effect of sugR disruption. SugR was purified and binding to the promoter regions of ptsI and ptsH was indicated by EMSA. DNase I footprinting analysis indicated the binding sites of SugR on the promoter region of divergently transcribed ptsI gene and fructose-pts operon. The binding sites contain a possible SugR binding motif which is conserved in the promoter regions of general and sugar-specific pts genes. Mutations in this motif resulted in the decrease of SugR binding to the ptsI promoter. These results suggest that SugR represses ptsI and ptsH in the absence of PTS sugar and derepression is the mechanism for the induction of the general components of PTS.     

Characterization of glk, a gene coding for glucose kinase of Corynebacterium glutamicum

The glk gene from Corynebacterium glutamicum was isolated by complementation using Escherichia coli ZSC113 (ptsG ptsM glk). We sequenced a total of 3072 bp containing the 969-bp open reading frame encoding glucose kinase (Glk). The glk gene has a deduced molecular mass of 34.2 kDa and contains a typical ATP binding site. Comparison with protein sequences revealed homologies to Glk from Streptomyces coelicolor (43%) and Bacillus megaterium (35%). The glk gene in C. glutamicum was inactivated on the chromosome via single crossover homologous recombination and the resulting glk mutant was characterized. Interestingly, the C. glutamicum glk mutant showed poor growth on rich medium such as LB medium or brain heart infusion medium in the presence or absence of glucose, fructose, maltose or sucrose as the sole carbon source. Growth yield was reduced significantly when maltose was used as the sole carbon source using minimal medium. The growth defect of glk mutant on rich medium was complemented by a plasmid-encoded glk gene. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression, and it was found that glk was expressed constitutively under all tested conditions with different carbon sources.     

The phosphotransferase system of Corynebacterium glutamicum: features of sugar transport and carbon regulation

In this review, we describe the phosphotransferase system (PTS) of Corynebacterium glutamicum and discuss genes for putative global carbon regulation associated with the PTS. C. glutamicum ATCC 13032 has PTS genes encoding the general phosphotransferases enzyme I, HPr and four enzyme II permeases, specific for glucose, fructose, sucrose and one yet unknown substrate. C. gluamicum has a peculiar sugar transport system involving fructose efflux after hydrolyzing sucrose transported via sucrose EII. Also, in addition to their primary PTS, fructose and glucose are each transported by a second transporter, glucose EII and a non-PTS permease, respectively. Interestingly, C. glutamicum does not show any preference for glucose, and thus co-metabolizes glucose with other sugars or organic acids. Studies on PTS-mediated sugar uptake and its related regulation in C. glutamicum are important because the production yield of lysine and cell growth are dependent on the PTS sugars used as substrates for fermentation. In many bacteria, the PTS is also involved in several regulatory processes. However, the detailed molecular mechanism of global carbon regulation associated with the PTS in this organism has not yet been revealed.     

Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering

The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates.     

Analysis of mutations affecting the expression of catabolite-sensitive operons in Escherichia coli K12 mutants defective in the HPr-component of the carbohydrate transport system

Expression of catabolite-sensitive operons in mutants devoid of HPr (a component of the glucose transport system) is severely repressed. ptsH mutants do not utilize substrates of the phosphoenolpyruvate: carbohydrate system (PTS) and many other sugars. Analysis of mutations suppressing the effect of the delta ptsH mutation revealed a new class of reversions which restore the growth of bacteria on different substrates. This mutation (named ptsS) intensifies the growth rate of ptsH mutants and increases the differential rate of beta-galactosidase production. ptsS mutation was mapped in the region of ptsF gene (coding for the fructose specific enzyme II of the PTS) on the 46th min. of the E. coli chromosome map. The effect of the ptsS mutation on the expression of catabolite-sensitive operons manifests only in the presence of the intact enzyme I of the PTS.     

Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.