Christian Schultz

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.

Glutamate production by Corynebacterium glutamicum: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG

We recently showed that the activity of the 2-oxoglutarate dehydrogenase complex (ODHC) in Corynebacterium glutamicum is controlled by a novel regulatory mechanism that involves a 15-kDa protein called OdhI and serine/threonine protein kinase G (PknG). In its unphosphorylated state, OdhI binds to the E1 subunit (OdhA) of ODHC and, thereby, inhibits its activity. Inhibition is relieved by phosphorylation of OdhI at threonine-14 by PknG under conditions requiring high ODHC activity. In this work, evidence is provided that the dephosphorylation of phosphorylated OdhI is catalyzed by a phospho-Ser/Thr protein phosphatase encoded by the gene cg0062, designated ppp. As a decreased ODHC activity is important for glutamate synthesis, we investigated the role of OdhI and PknG for glutamate production under biotin limitation and after addition of Tween-40, penicillin, or ethambutol. A ΔodhI mutant formed only 1–13% of the glutamate synthesized by the wild type. Thus, OdhI is essential for efficient glutamate production. The effect of a pknG deletion on glutamate synthesis was dependent on the induction conditions. Under strong biotin limitation and in the presence of ethambutol, the ΔpknG mutant showed significantly increased glutamate production, offering a new way to improve production strains.

Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases

We previously showed that the 2‐oxoglutarate dehydrogenase inhibitor protein OdhI of Corynebacterium glutamicum is phosphorylated by PknG at Thr14, but that also additional serine/threonine protein kinases (STPKs) can phosphorylate OdhI. To identify these, a set of three single (ΔpknA, ΔpknB, ΔpknL), five double (ΔpknAG, ΔpknAL, ΔpknBG, ΔpknBL, ΔpknLG) and two triple deletion mutants (ΔpknALG, ΔpknBLG) were constructed. The existence of these mutants shows that PknA, PknB, PknG and PknL are not essential in C.u2003glutamicum. Analysis of the OdhI phosphorylation status in the mutant strains revealed that all four STPKs can contribute to OdhI phosphorylation, with PknG being the most important one. Only mutants in which pknG was deleted showed a strong growth inhibition on agar plates containing glutamine as carbon and nitrogen source. Thr14 and Thr15 of OdhI were shown to be phosphorylated in vivo, either individually or simultaneously, and evidence for up to two additional phosphorylation sites was obtained. Dephosphorylation of OdhI was shown to be catalysed by the phospho‐Ser/Thr protein phosphatase Ppp. Besides OdhI, the cell division protein FtsZ was identified as substrate of PknA, PknB and PknL and of the phosphatase Ppp, suggesting a role of these proteins in cell division.

New insights into the phase behaviour of a complex anionic amphiphile: architecture and dynamics of bacterial deep rough lipopolysaccharide membranes as seen by FTIR, X-ray, and molecular modelling techniques

Abstract Using the “functional group probing capability” of FTIR together with X-ray and molecular modelling techniques, the dynamic properties, conformational structure and phase behaviour of deep rough bacterial lipopolysaccharide (Re-LPS) have been investigated. Re-LPS (Na-salt form, pH 7) exhibited a pronounced L β →L α -like main phase transition at a mid-point temperature T m of 29°C. Only lamellar phases were observed between pH 4 and 10, in the temperature range from 5 to 70°C and upon addition of divalent cations like Ca 2+ . On the basis of these findings, it is concluded that the described inverted hexagonal, or inverted micellar-like phases play no significant role under physiological conditions. The observation of precipitating aggregates at low pH and on addition of divalent cations and the pronounced dependence of phase transition temperatures and enthalpies on pH is interpreted to be due to the process of charge neutralization leading to mixed phases of strongly interacting multilamellar aggregates and rigidified polar head group regions and to the reduction of cooperativity of the acyl chain melting process. Depending on time of isothermal annealing at low temperatures, several sub-transition-like features were observed which revealed the coexistence of slightly differing acyl chain packing prior to the gel to liquid crystalline (L β →L α ) transition, including hexagonal and distorted hexagonal types of packing patterns. During the main phase transition, evidence for the breakdown of distinct associates or aggregates of Re-LPS molecules was obtained. Even at elevated temperatures, fairly far above T m , complex rearrangements within the polar head group and the interfacial region accompanied by an extra increase in acyl chain disorder were observed, indicating specific LPS-LPS interactions. The comparison of theoretical and molecular modelling results with the experimental data leads to a new understanding of the peculiarities of this complex amphiphile which enable it to stabilize the asymmetric configuration of the bacterial outer membrane and its pronounced permeation barrier towards hydrophobic agents. There are several determinants for the specific functional role of LPS: (i) the high negative charge density and the conformational rigidity (especially in the polar backbone and interfacial region); (ii) the effectively reduced length of acyl-chain segments which participate in the cooperative chain melting process; (iii) the high tendency to form strongly interacting aggregates within monolamellar or multilamellar supramolecular architectures which is favoured by divalent cation bridging; and (iv) the remarkably extended interfacial region of high electron density between the hydrophilic and hydrophobic parts of the molecule.

Characterization and Identification of Micro-Organisms by FT-IR Spectroscopy and FT-IR Microscopy

A large series of physical techniques has been tested for the characterization of microbial cells. These techniques include gas chromatographic (GC) and high-performance liquid chromatographic (HPLC) techniques, gel electrophoresis, pyrolysis mass spectroscopy, fluorescence and chemiluminescence techniques, flow cytometry, impedance measurements, microcalorimetry, circular intensity differential scattering, nuclear magnetic resonance, and infrared as well as Raman spectroscopy (for review see Nelson, 1991). All these efforts have provided additional biochemical and structural information, which might -in some areas -be helpful for taxonomic characterizations.

In vivo study of the state of order of the membranes of Gram-negative bacteria by Fourier-transform infrared spectroscopy (FT-IR)

Temperature‐induced order/disorder transition profiles were obtained from the membranes of intact Gram‐negative bacterial cells by FT‐IR analysis of the frequency shifts of the acyl chain methylene symmetric stretching band as a monitor. Cells grown at different temperatures yielded distinct transition profiles. At the individual growth temperatures, however, the nearly alike frequency values indicated a very similar ‘state of order’ of the bacterial membranes. The FT‐IR data were complemented by GC analysis of whole cell fatty acid composition. The FT‐IR data obtained in vivo gave direct evidence of the adaptation of the ‘state of order’ and ‘fluidity’ of bacterial membranes to varying growth temperatures.

Thermally induced hydrogen exchange processes in small proteins as seen by FTIR spectroscopy

Fourier‐transform infrared (FTIR) spectroscopy has been used to study the thermally induced exchange characteristics of those backbone amide protons which persist H‐D exchange at ambient conditions in ribonuclease A, in wild type ribonuclease T1 and some of its variants, and in the histone‐like protein HBsu. The H‐D exchange processes were induced by increasing the thermal energy of the protein solutions in two ways: (i) by linearly increasing the temperature, and (ii) by a temperature jump. To trace the H‐D exchange in the proteins, various infrared absorption bands known to be sensitive to H‐D exchange were used as specific monitors. Characteristic H‐D exchange curves were obtained from which the endpoints (TH/D) of H‐D exchange could be determined. The H‐D exchange curves, the TH/D‐values and the phase transition temperatures Tm were used to estimate the structural flexibility and stability of the given proteins. It is suggested that time‐resolved FTIR spectroscopy can be used to determine global stability parameters of proteins.

The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.

Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. Deep rough mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a border region model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the ternary system ReLPS/DMPEd54/PLL(500) showed the features of a phase segregation phenomenon not affecting the transition temperatures of the pure lipid components, the ternary system composed of ReLPS/DMPEd54 and OmpF exhibited phase transition curves that were characterized by an unspecific (DMPEd54/OmpF) and a strong and unique (ReLPS/OmpF) type of lipid-protein interaction. Furthermore, semiquantitative estimations supported the supposition that OmpF might be able to induce bilayer asymmetry in preformed symmetrical ReLPS/DMPEd54 vesicles.

The FHA domain of OdhI interacts with the carboxyterminal 2-oxoglutarate dehydrogenase domain of OdhA in Corynebacterium glutamicum

OdhI (uniprotkb:Q8NQJ3) physically interacts (MI:0915) with OdhA (uniprotkb:Q8NRC3) by pull down (MI:0096)

OdhI dephosphorylation kinetics during different glutamate production processes involving Corynebacterium glutamicum

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase complex was shown to be controlled by the phosphorylation of a 15-kDa protein OdhI by different serine/threonine protein kinases. In this paper, the phosphorylation status and kinetics of OdhI dephosphorylation were assessed during glutamate producing processes triggered by either a biotin limitation or a temperature upshock from 33°C to 39°C. A dephosphorylation of OdhI in C. glutamicum 2262 was observed during the biotin-limited as well as the temperature-induced glutamate-producing process. Deletion of pknG in C. glutamicum 2262 did not affect the phosphorylation status of OdhI during growth and glutamate production phases triggered by a temperature upshock, though a 40% increase in the specific glutamate production rate was measured. These results suggest that, under the conditions analyzed, PknG is not the kinase responsible for the phosphorylation of OdhI in C. glutamicum 2262. The phosphorylation status of OdhI alone is, as expected, not the only parameter that determines the performance of a specific strain, as no clear relation between the specific glutamate production rate and OdhI phosphorylation level was demonstrated.