All higher plants possess highly specific binding sites for fusicoccin, a metabolite of the fungus Fusicoccum amygdali Del. These sites are harboured in the plasma membranes and formed by a 14-3-3 protein dimer associated with the C-terminal autoinhibitory domain of H + -ATPase. We considered the fusicoccin binding to plasma membranes to be an indicator of complexation between the 14-3-3 dimer and H + -ATPase, we assessed the effect of cold stress on the interaction of these proteins in suspension-cultured sugar beet cells and protoplasts derived from these cells. In both objects, upon lowering the temperature to 0^4³C, a portion of the cytoplasmic 14-3-3 proteins became associated with the plasma membrane, which showed an increasing amount of ATPase/14-3-3 complexes and enhanced ATPase activity. Association between ATPase and 14-3-3 resulted in a several-fold rise in the H + efflux from protoplasts and intact cells. We suppose that regulation of the H + pumping under changing external conditions may be based on the interaction between H + -ATPase and the 14-3-3 proteins.z 1999 Federation of European Biochemical Societies.

Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H+ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes: the 14-3-3 content in the membranes increased 2- to 3-fold, while the H+-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H+-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H+ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins.

The characteristics of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based investigation of extremely variable bacteria such as Helicobacter pylori were studied. H. pylori possesses a very high natural variability. Accurate tools for species identification and epidemiological characterization could help the scientific community to better understand the transmission pathways and virulence mechanisms of these bacteria. Seventeen clinical as well as two laboratory strains of H. pylori were analyzed by the MALDI Biotyper method for rapid species identification. Mass spectra collected were found containing 7-13 significant peaks per sample, and only six protein signals were identical for more than half of the strains. Four of them could be assigned to ribosomal proteins RL32, RL33, RL34, and RL36. The reproducible peak with m/z 6948 was identified as a histidine-rich metal-binding polypeptide by tandem mass spectrometry (MS/MS). In spite of the evident protein heterogeneity of H. pylori the mass spectra collected for a particular strain under several cultivations were highly reproducible. Moreover, all clinical strains were perfectly identified as H. pylori species through comparative analysis using the MALDI Biotyper software (Bruker Daltonics, Germany) by pattern matching against a database containing mass spectra from different microbial strains (n = 3287) including H. pylori 26695 and J99. The results of this study allow the conclusion that the MALDI-TOF direct bacterial profiling is suited for H. pylori identification and could be supported by mass spectra fragmentation of the observed polypeptide if necessary.

Helicobacter pylori is an extra macro- and microdiverse bacterial species, but where and when diversity arises is not well-understood. To test whether a new environment accelerates H. pylori genetic changes for quick adaptation, we have examined the genetic and phenotypic changes in H. pylori obtained from different locations of the stomach from patients with early gastric cancer (ECG) or chronic gastritis (CG). Macroarray analysis did not detect differences in genetic content among all of the isolates obtained from different locations within the same stomach of patients with EGC or CG. The extent and types of functional diversity of H. pylori isolates were characterized by 2-D difference gel electrophoresis (2D DIGE). Our analysis revealed 32 differentially expressed proteins in H. pylori related to EGC and 14 differentially expressed proteins in H. pylori related to CG. Most of the differentially expressed proteins belong to the antioxidant protein group (SodB, KatA, AphC/TsaA, TrxA, Pfr), tricarbon acid cycle proteins (Idh, FrdA, FrdB, FldA, AcnB) and heat shock proteins (GroEL and ClpB). We conclude that H. pylori protein expression variability is mostly associated with microorganism adaptation to morphologically different parts of the stomach, which has histological features and morphological changes due to pathological processes; gene loss or acquisition is not involved in the adaptation process.