Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Different Modes of Diaminopimelate Synthesis and Their Role in Cell Wall Integrity: a Study with Corynebacterium glutamicum

In eubacteria, there are three slightly different pathways for the synthesis of m-diaminopimelate (m-DAP), which is one of the key linking units of peptidoglycan. Surprisingly, for unknown reasons, some bacteria use two of these pathways together. An example is Corynebacterium glutamicum, which uses both the succinylase and dehydrogenase pathways for m-DAP synthesis. In this study, we clone dapD and prove by enzyme experiments that this gene encodes the succinylase (M_r = 24082), initiating the succinylase pathway of m-DAP synthesis. By using gene-directed mutation, dapD, as well as dapE encoding the desuccinylase, was inactivated, thereby forcing C. glutamicum to use only the dehydrogenase pathway of m-DAP synthesis. The mutants are unable to grow on organic nitrogen sources. When supplied with low ammonium concentrations but excess carbon, their morphology is radically altered and they are less resistant to mechanical stress than the wild type. Since the succinylase has a high affinity toward its substrate and uses glutamate as the nitrogen donor, while the dehydrogenase has a low affinity and incorporates ammonium directly, the m-DAP synthesis is another example of twin activities present in bacteria for access to important metabolites such as the well-known twin activities for the synthesis of glutamate or for the uptake of potassium.     

Remote stimulation by heat induces characteristic membrane-potential responses in the veins of wild-type and abscisic acid-deficient tomato plants

The systemic induction of proteinase inhibitor genes in tomato plants is either mediated by fast electrical signals or alternatively by chemical messengers. In the present study we analyzed the pathway of the electrical signal. The question of which cell types are involved in this pathway of long-distance signaling within plants is still controversial. To identify these we inserted microelectrodes into the veins of tomato leaves (Lycopersicon esculentum Mill. cv. Moneymaker). A newly developed computer program and microcomputer interface enabled us to position these microelectrodes inside the vein with an accuracy of 1 μm. Due to this precision in positioning we were able to demonstrate that the pathway of the electrical signal is not restricted to a specific tissue type, e.g. the phloem. In particular, the entire vein contributes to the propagation of the electrical wave along the plant. Therefore, an apoplastic contribution to the long-distance signal transduction mechanism appears most likely. To furthermore investigate the involvement of cis-abscisic acid (ABA) in this long-distance signal transduction pathway, ABA-deficient tomato mutants (Lycopersicon esculentum cv. Sitiens) were used in comparison to the wild type. Significant differences between the membrane-potential relaxation kinetics of the wild type and the mutants could be detected. Wild-type tomato plants exhibited six characteristic classes of membrane-potential relaxation kinetics following heat treatment. In contrast, the ABA-deficient mutants were more restricted in terms of their relaxation upon heat stimulation. The responses in the membrane potential of all cells within a vein consisted of only three categories. In conclusion, ABA did not affect all cells within the vein in a similar manner. Single cells exhibited different response patterns to systemic heat application in the presence of ABA. Moreover, ABA had a pronounced effect on the resting potentials of individual cells within the veins of tomato. Received: 1 July 1997 / Accepted: 16 January 1998     

l-Isoleucine Production with Corynebacterium glutamicum: Further Flux Increase and Limitation of Export

The synthesis of l-isoleucine with Corynebacterium glutamicum involves 11 reaction steps, in at least five of which activity or expression is regulated. We used four genes and alleles encoding feedback-resistant enzymes (Fbr) in various combinations to assay flux increase through the sequence. During strain construction, the order of genes overexpressed was important. Only when ilvA(Fbr) was first overexpressed could hom(Fbr) be introduced. This succession apparently prevents the toxic accumulation of biosynthesis intermediates. The best strain constructed (SM13) was characterized by high-level expression of hom(Fbr), thrB, and ilvA(Fbr). With this strain a yield of 0.22 g of l-isoleucine per g of glucose was obtained, with a maximal specific productivity of 0.10 g of l-isoleucine per g (dry weight) per h. In strain SM13, with the high metabolite flux through the reaction sequence, effects on (i) other enzyme levels, (ii) time-dependent variations with process time, and (iii) concentrations of cytosolic intermediates were quantified. Most importantly, the intracellular l-isoleucine concentration is always higher at all process times than the extracellular concentration. The intracellular concentration rises to 110 mM, whereas extracellularly only 60 mM is accumulated. Also the immediate l-isoleucine precursor 2-ketomethyl valerate accumulates in the cell. Therefore, in the high-level l-isoleucine producer SM13, the export of this amino acid is the major limiting reaction step and therefore is a new target of strain design for biotechnological purposes.     

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS¹ type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.     

Long-Term Dosimetry of Solar UV Radiation in Antarctica with Spores of Bacillus subtilis

The main objective was to assess the influence of the seasonal stratospheric ozone depletion on the UV climate in Antarctica by using a biological test system. This method is based on the UV sensitivity of a DNA repair-deficient strain of Bacillus subtilis (TKJ 6321). In our field experiment, dried layers of B. subtilis spores on quartz discs were exposed in different seasons in an exposure box open to solar radiation at the German Antarctic Georg von Neumayer Station (70°37′S, 8°22′W). The UV-induced loss of the colony-forming ability was chosen as the biological end point and taken as a measure for the absorbed biologically harmful UV radiation. Inactivation constants were calculated from the resulting dose-response curves. The results of field experiments performed in different seasons indicate a strongly season-dependent trend of the daily UV-B level. Exposures performed at extremely depleted ozone concentrations (October 1990) gave higher biologically harmful UV-B levels than expected from the calculated season-dependent trend, which was determined at normal ozone values. These values were similar to values which were measured during the Antarctic summer, indicating that the depleted ozone column thickness has an extreme influence on the biologically harmful UV climate on ground.     

Speciation analysis of nickel in soil solutions and availability to oat plants

Soil solutions were collected for speciation analysis of nickel from a pot experiment with oats. Oat plants (Avena sativa L.) were grown on 3 soils differing in total amount and origin of nickel (Ni) (Luvisol, LS with 28 mg kg^-1; sludge amended Luvisol, LS+SS with 32 mg kg^-1; Cambisol, CS with 95 mg kg^-1). Results were compared with those for soil solutions obtained from corresponding unplanted pots. Separation methods were used for characterization of size and charge distribution and stability of the Ni species. In addition, short-term experiments were performed on the uptake rates of Ni by oat plants from the different soil solutions as well as from nutrient solutions with increasing concentrations of a synthetic chelator.The Ni concentrations in the soil solutions of unplanted soils increased in the order: LS5000 g mol^-1) was the predominant form, whereas in the other soils the low-molecular-size cationic and chelated Ni species (500–1000 g mol^-1) dominated in the soil solution. In the short-term uptake studies, the uptake rates of Ni from the solutions decreased in the order: nutrient solution > soil solutions, and in the latter in the order: LS>LS+SS>CS, which was inversely related to the concentrations of dissolved organic carbon in the soil solutions.The results demonstrate that Ni availability to plants is not only affected by total concentration of Ni in the soil solution and the rate of replenishment from the solid phase, but also by Ni species, which can differ considerably between soil types.     

Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum

The lysC/asd gene cluster of Corynebacterium glutamicum ATCC 13032 was cloned and sequenced. The lysC locus coding for aspartokinase consists of two in-frame overlapping genes, lysC alpha encoding a protein of 421 amino acids (Mr 44,300) and lysC beta encoding a protein of 172 amino acids (Mr 18,600). The C. glutamicum aspartokinase was purified and found to contain two proteins of Mr 47,000 and Mr 18,000. A C. glutamicum mutant expressing a feedback-resistant aspartokinase was shown to be changed in a single base pair of the lysC beta gene, leading to an amino acid exchange in the beta-subunit of the aspartokinase. In addition, the identified mutation was found to be responsible for the enhanced expression of the asd gene located downstream of lysC.     

Entwicklungsgeschichtliche Eigentümlichkeiten einigerAchnanthes-Arten (Diatomeae)

InAchnanthes lanceolata pairing occurs by chance in regard to the arrangement of the mother cells, inA. minutissima it is accomplished selectively: contact at the sides occupied by the nucleus is favoured very strongly. In contrast toA. exilis, pairing inA. minutissima occurs by detachment of both partner cells from their stalks, whileA. lanceolata has no stalks at all. The isogamous gamete fusion inA. lanceolata should be understood as a modification of the anisogamous behavior inA. minutissima andA. exilis. For the three species the same constant orientation of the primary cell in relation to the substratum and to the mother thecae is characteristic. The first epivalva always is the raphe valve. InA. lanceolata in connection with the property of the primary cell possessing two chromatophores resp. pyrenoids instead of one, the transapical heteropolarity of the rapheless valve, a specific character, changes to isopolarity. It seems that a sort of induction from the pyrenoids towards the formation of the rapheless valve is at work. InA. lanceolata the pseudoraphe shows a structure hitherto unknown.A. lanceolata fo.ventricosa Hust. is to be eliminated. Var.rostrata (Östrup)Hust. is a good taxon; two new differential characters are found.