Metabolic quenching of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>: efficiency of methods and impact of cold shock

Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself. A rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites is worthwhile. This can be achieved by applying of cold methanol quenching combined with reproducible, fast, and automated sampling. Unfortunately, quenching the metabolism by a sharp temperature shift leads to what is known as cold shock or the cell-leakage effect. In the present work, we applied a microstructure heat exchanger to analyze the cold shock effect using Corynebacterium glutamicum as a model microorganism. Using this apparatus together with a silicon pipe, it was possible to assay the leakage effect on a timescale starting at 1 s after cooling cell suspension. The high turnover rates not only require a rapid quenching technique, but also the correct application. Moreover, we succeeded in showing that even when the required appropriate setup of methanol quenching is not used, the metabolism is not stopped within the required timescale. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we ensured fast and reliable metabolic inactivation during all steps.     

MreC and MreD balance the interaction between the elongasome proteins PBP2 and RodA

Rod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a change in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the “on state” of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.     

Ureolytic activity of Nitellopsis obtusa (Characeae)

The decomposition of urea by Nitellopsis obtusa from Characeae was investigated. The intact cells were exposed to the inhibition by two typical urease inhibitors: boric acid and fluoride ion, used as a criterion to define if urease or UAL-ase is responsible for the ureolytic activity of the algae. It was found that boric acid and fluoride ion are simple competitive and slow-binding competitive inhibitors of Nitellopsis obtusa enzyme respectively, which is the response characteristic of urease. The inhibition constants equal to 2.3 and 0.1 mM for boric acid and fluoride ion, when compared to those of jack bean urease, indicate that in the observed kinetic behaviour of Nitellopsis obtusa urease partake transport processes taking place in the intact cells.     

Linear Discriminant Functions for Stationary Time Series

Optimal classification rules based on linear functions which maximize the Chernoff distance, or the Morisita distance, or the Kullback-Leibler distance are studied here. We obtain an expression for the optimal linear discriminant function and show that the resulting linear procedure belongs to the Anderson-Bahadur admissible class. For the comparison of discriminant rules we use some index which is the measure of the accuracy of a given class of discriminant procedures. The asymptotic form of the discriminant function is also studied.