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 ⌬pknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a ⌬odhI 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 K i 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.Increasing numbers of eukaryotic-like serine/threonine protein kinases found in bacteria implicate that they play important roles in cell signaling, but their targets and specific functions are largely unknown (1). The genome of the important human pathogen Mycobacterium tuberculosis encodes 11 members of this protein family (2). Among these, protein kinase G (PknG) 2 gained particular interest because it was reported to inhibit phagosome-lysosome fusion, thus allowing for intracellular survival of mycobacteria. Deletion of the pknG gene in Mycobacterium bovis BCG resulted in lysosomal localization and mycobacterial cell death in infected macrophages. PknG was detected in the cytosol of infected macrophages and was therefore suggested to interfere with host cell signaling pathways (3). A pknG deletion mutant of M. tuberculosis displayed decreased viability upon infection of immunocompetent mice but also reduced growth in vitro (4), implying that PknG function is not restricted to the pathogenic life style. This is supported by the fact that genes encoding PknG homologs are not only present in pathogenic mycobacteria but also in all other members of the suborder Corynebacterineae with known genome sequence, i.e. species of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus, as well as in Streptomyces species. To determine the function of PknG, we chose Coryneb...

Under nitrogen starvation conditions, Corynebacterium glutamicum was found to take up methylammonium at a rate of 20 +/- 5 nmol.min-1.(mg dry weight)-1. 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 Km of 44 +/- 7 microM and was completely inhibited by the addition of 10 microM 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 (Mr = 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.

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 pl range 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 pl 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 pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.

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 x 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 microM, 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].

Gram-positive soil bacterium Corynebacterium glutamicumuses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (Km , 48 μM) to ectoine (Km , 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (Km , 63 μM) to betaine (Km , 333 μM) and proline (Km , 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.