Gliding arc discharge in the potato pathogen Erwinia carotovora subsp. atroseptica: mechanism of lethal action and effect on membrane-associated molecules.

Gliding arc (glidarc) discharge is a physicochemical technique for decontamination at atmospheric pressure and ambient temperature. It leads to the destruction of bacterial phytopathogens responsible for important losses in industrial agriculture, namely, Erwinia spp., without the formation of resistant forms. We investigated the effect of a novel optimized prototype allowing bacterial killing without lag time. This prototype also decreases the required duration of treatment by 50%. The study of the time course effect of the process on bacterial morphology suggests that glidarc induces major alterations of the bacterial membrane. We showed that glidarc causes the release of bacterial genomic DNA. By contrast, an apparent decrease in the level of extractible lipopolysaccharide was observed; however, no changes in the electrophoretic pattern and cytotoxic activity of the macromolecule were noted. Analysis of extractible proteins from the outer membrane of the bacteria revealed that glidarc discharge induces the release of these proteins from the lipid environment, but may also be responsible for protein dimerization and/or aggregation. This effect was not observed in secreted enzymatic proteins, such as pectate lyase. Analysis of the data supports the hypothesis that the plasma generated by glidarc discharge is acting essentially through oxidative mechanisms. Furthermore, these results indicate that, in addition to effectively destroying bacteria, glidarc discharge should be used to improve the extraction of bacterial molecules.

Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite.

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.

A pleiotropic reduced virulence (Rvi-) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens.

Erwinia carotovora subsp. atroseptica was mutagenized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi-) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non-motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII-ClaI complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella flexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.