Brita Weil

Corynebacterial protein kinase G controls 2-oxoglutarate dehydrogenase activity via the phosphorylation status of the OdhI protein.

A novel regulatory mechanism for control of the ubiquitous 2-oxoglutarate dehydrogenase complex (ODH), a key enzyme of the tricarboxylic acid cycle, was discovered in the actinomycete Corynebacterium glutamicum, a close relative of important human pathogens like Corynebacterium diphtheriae and Mycobacterium tuberculosis. Based on the finding that a C. glutamicum mutant lacking serine/threonine protein kinase G (PknG) was impaired in glutamine utilization, proteome comparisons led to the identification of OdhI as a putative substrate of PknG. OdhI is a 15-kDa protein with a forkhead-associated domain and a homolog of mycobacterial GarA. By using purified proteins, PknG was shown to phosphorylate OdhI at threonine 14. The glutamine utilization defect of the ΔpknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a ΔodhI mutant with a plasmid encoding OdhI-T14A caused a defect in glutamine utilization. Affinity purification of OdhI-T14A led to the specific copurification of OdhA, the E1 subunit of ODH. Because ODH is essential for glutamine utilization, we assumed that unphosphorylated OdhI inhibits ODH activity. In fact, OdhI was shown to strongly inhibit ODH activity with a Ki value of 2.4 nm. The regulatory mechanism described offers a molecular clue for the reduced ODH activity that is essential for the industrial production of 1.5 million tons/year of glutamate with C. glutamicum. Moreover, because this signaling cascade is likely to operate also in mycobacteria, our results suggest that the attenuated pathogenicity of mycobacteria lacking PknG might be caused by a disturbed tricarboxylic acid cycle.

A high-resolution reference map for cytoplasmic and membrane-associated proteins of Corynebacterium glutamicum

We present a high‐resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pIrange from 4–6 and the molecular mass range from 5–100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second‐dimensional separation on SDS‐polyacrylamide gels and colloidal Coomassie‐staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty‐five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pI were detected. The N‐termini of eight proteins were determined by post‐source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane‐bound proteins within the pI range 4–7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.

Functional and Genetic Characterization of the (Methyl)ammonium Uptake Carrier of Corynebacterium glutamicum

Under nitrogen starvation conditions, Corynebacterium glutamicum was found to take up methylammonium at a rate of 20 ± 5 nmol•min•(mg dry weight). The specific activity of this uptake was 10-fold lower when growing the cells under sufficient nitrogen supply, indicating a tight regulation on the expression level. The methylammonium uptake showed Michaelis-Menten kinetics with an K of 44 ± 7 μM and was completely inhibited by the addition of 10 μM ammonium. This finding and the fact that methylammonium was not metabolized by C. glutamicum strongly suggests that the uptake carrier actually represents an ammonium uptake system. Methylammonium uptake was strictly dependent on the membrane potential. From the pH optimum and the accumulation of methylammonium in equilibrium, it could be deduced that only one net charge is transported and, thus, that methylammonium is taken up in its protonated form via an uniport mechanism. The amt gene encoding the (methyl)ammonium uptake system was isolated and characterized. The predicted gene product of amt consists of 452 amino acids (M = 47,699) and shows 26-33% identity to ammonium transporter proteins from Saccharomyces cerevisiae and Arabidopsis thaliana. According to the hydrophobicity profile, it is an integral membrane protein containing 10 or 11 membrane-spanning segments.

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (Km[Ile]=5.4 μM, Km[Leu]=9.0 μM, Km[Val]=9.5 μM). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na+ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na+-coupled symport mechanism.

Isoleucine excretion in Corynebacterium glutamicum: evidence for a specific efflux carrier system

SummaryCorynebacterium glutamicum effectively secretes isoleucine when the precursor 2-ketobutyrate is added to the medium. Isoleucine secretion was studied under different conditions with respect to various parameters, i.e. rate of isoleucine excretion and uptake, concentration gradients of isoleucine, other amino acids and ions, and membrane potential. By comparing these parameters in the presence and absence of the amino acid precursor it has been shown that the efflux of isoleucine in C. glutamicum can neither be explained by a passive diffusion mechanism nor by a process involving functional inversion of the isoleucine uptake carrier. Based on our results concerning the distribution of metabolites and the kinetics of excretion we conclude that isoleucine is excreted in C. glutamicum by a separate, presumably active efflux carrier system.

Urea uptake and urease activity in Corynebacterium glutamicum.

Abstract When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 × 10–7 cm s–1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a Km for urea of 9 μM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (Km approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2–3.5 nmol min–1 (mg dry wt.)–1].

Lysine secretion by wild-type Corynebacterium glutamicum triggered by dipeptide uptake

SUMMARY: In Corynebacterium glutamicum peptide uptake increases the internal concentration of amino acids and thus triggers amino acid secretion. The peptide uptake system is stimulated by a factor of two in cells grown on pure peptone medium in comparison to peptone media with additional carbon sources. Uptake depends on the proton-motive force and shows a broad substrate spectrum. Peptide uptake is characterized by a K m of about 230 μM and a V max of 12 nmol min-1 (mg dry wt)-1 for the peptide lysyl-alanine (Lys-Ala). Lysine secretion in the wild-type of C. glutamicum does not show Michaelis-Menten-type kinetics as reported for the producing strains DG 52-5 and MH 20-22B. The secretion of lysine depends on the composition of the medium in which the cells were grown prior to the initiation of secretion by peptide uptake. The lack of secretion activity when the cells are shifted to peptone medium in the presence of chloramphenicol indicates that protein synthesis is necessary for this regulatory process.

Carrier-mediated acetate uptake in Corynebacterium glutamicum

Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The Km of acetate uptake was 50 μM and the Vmax 25–35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by propionate with a Ki of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane potential, but independent of the presence of Na+-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism.

Glutamate excretion in Escherichia coli : dependency on the relA and spoT genotype

Glutamate excretion due to amino acid starvation was investigated in “stringent” and “relaxed” strains ofEscherichia coli. The observed excretion process isrelA-dependent, carrier-mediated, and glutamate-specific. After induction, excretion was detected within less than 2 min and continued for more than 5h with a rate of 7–10 nmol (mg dry weight)−1 min−1. Using carbonyl cyanidem-chlorophenylhydrazone or polymyxin B nonapeptide, together with valinomycin, it was shown that glutamate excretion is driven by the membrane potential.

Glutamine uptake by a sodium-dependent secondary transport system inCorynebacterium glutamicum

Corynebacterium glutamicum took up glutamine by a sodium-dependent secondary transport system. Both the membrane potential and the sodium gradient were driving forces. Glutamine uptake showed Michaelis-Menten kinetics, with aKm of 36 μM and aVmax of 12.5 nmol min−1 (mg dry weight)−1 at pH 7. Despite a pH optimum in the alkaline range around pH 9, it was shown that uncharged glutamine is the transported species. The affinity for the cotransported sodium was relatively low; an apparentKm of 1.4 mM was determined. Among various substrates tested, only asparagine, when added in 50-fold excess, led to an inhibition of glutamine transport. It was concluded that glutamine uptake occurs via a specific transport system in symport with at least one sodium ion.