Ultrastructural Alterations of Erwinia carotovora subsp. atroseptica Caused by Treatment with Aluminum Chloride and Sodium Metabisulfite

Abstract

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 SO 2 with little damage to the cellular membrane.Aluminum and bisulfite salts are toxic to several microorganisms including fungi (7, 15, 16) and bacteria (1, 8). Aluminum chloride and sodium metabisulfite were shown to be toxic at low concentrations against Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora (32a), gramnegative motile rod bacteria causing important economic losses in a wide variety of vegetable crops. Of particular interest, the application of these salts on potato tubers was effective in controlling soft rot caused by both bacteria (32a), a disease of high economic significance for the potato industry.Postulated mechanisms of sulfite toxicity or inhibition of microbial growth include reactions with protein disulfide groups (3,19,25), inhibition of enzyme activities by inactivating their cofactors (e.g., thiamine pyrophosphate) (31) or coenzymes (e.g., NAD) (20,25,27,29), and rapid depletion of ATP and ADP pools (14). Sulfites may also react with pyrimidine residues of nucleic acids (17, 26), which can lead to genetic damage and cell death. Mechanisms by which aluminum affects microorganisms include its binding to the cell wall causing impaired permeability (2, 9); its replacement of divalent metal complexes, chiefly Mg and Ca, in cells or cell membranes (1); and its complexion with ATP (6), DNA (32), and phosphates causing phos...

Bacterial outer membrane vesicles and the host–pathogen interaction

“…Naturally produced bacterial vesicles are discrete, closed OM blebs produced by growing cells, not products of cell lysis or cell death (Mug-Opstelten and Witholt 1978;Zhou et al 1998;Yaganza et al 2004;McBroom and Kuehn 2005). By electron microscopy, vesicles appear spherical with a bilayer membrane, electron-dense luminal content, and an average diameter of 50-250 nm, depending on the strain (Beveridge 1999).…”

Determinação colorimétrica de metabissulfito

“…O mecanismo de ação destas instituições químicas relaciona-se com a inativação de sistemas enzimáticos nos microorganismos, juntamente com o co-fator tiamina pirofosfato e a co-enzima NAD. Este efeito é capaz de exterminar estas espécies biológicas [12].…”

Significances of OMV and Extracellular Vesicle Proteomics

“…The ubiquitous process of vesiculation helps in the survival of Gram-negative bacteria in different conditions of environments [3]. Naturally produced membrane vesicles are very distinct, closed blebs of outer membrane released by budding cells, rather due to cell lysis or death [14,[62][63][64]. The utmost rate of membrane vesicle formation takes place at the end of log phase and is plentiful at the sites of cell division, as explained for B. melitensis, E. coli and Vibrio cholera [8,[20][21][22][23][24].…”