Ultrastructural Alterations of Erwinia carotovora subsp. atroseptica ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2004, p. 6800–6808 0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.11.6800–6808.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 70, No. 11

Ultrastructural Alterations of Erwinia carotovora subsp. atroseptica Caused by Treatment with Aluminum Chloride and Sodium Metabisulfite Elian-Simplice Yaganza,1 Danny Rioux,2 Marie Simard,2 Joseph Arul,1 and Russell J. Tweddell1* Centre de Recherche en Horticulture, Universite´ Laval,1 and Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre,2 Que´bec City, Que´bec, Canada Received 29 March 2004/Accepted 24 June 2004

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. malfunctioning of the cell membrane and cell wall in Arthrobacter sp. PI/1-95 (8). Johnson (9) showed that the treatment of a Rhizobium sp. with aluminum (50 ␮M) induced cellular elongation similar to that observed during a treatment with mitomycin C, a compound which cross-links DNA and blocks its replication. Although several works have been carried out to elucidate the mechanism(s) by which aluminum salts and sulfiting agents exert antimicrobial activity, to our knowledge, the effect of these compounds on the microbial ultrastructure remains unknown. Thus, the objective of this study was to explore bacterial ultrastructural alterations that occur in response to exposure of bacterial cells to aluminum chloride and sodium metabisulfite. The study was carried out with E. carotovora subsp. atroseptica.

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 phosphate deprivation (21), pH effect, and inactivation of enzymes (8). A recent study also indicated that aluminum causes osmoregulative disorder apparently connected with the

MATERIALS AND METHODS Bacteria. Strain 709 of E. carotovora subsp. atroseptica was obtained from the Laboratoire de diagnostic en phytoprotection (MAPAQ, Que´bec, Canada). The bacteria were maintained on nutrient agar (NA; Difco Laboratories, Becton Dickinson, Sparks, Md.) slants at 4°C and served as stock cultures. Chemicals. Aluminum chloride, sodium metabisulfite, safranin O, bovine serum albumin, and sodium citrate (for preparing lead citrate) of high-purity grade (⬎99%) were purchased from Sigma Chemical Co. (St. Louis, Mo.). SYTOX Green nucleic acid stain was purchased from Molecular Probes (Eugene, Oreg.). Glutaraldehyde, sodium cacodylate, osmium tetroxide, and JEMBED 812 resin were from Canemco Inc. (Montre´al, Que´bec, Canada), whereas toluidine blue O, uranyl acetate, and lead nitrate (for preparing lead citrate) were from Fisher Scientific International (Fairlawn, N.J.). Salt treatments and sample processing for transmission electron microscopy. Bacterial cells were obtained by gently scraping the surface of a 16-h-old culture grown at 24°C on NA and were suspended (108 CFU/ml) in 1 ml of aluminum chloride (0.05, 0.1, or 0.2 M), sodium metabisulfite (0.05, 0.1, or 0.2 M), or 0.5% NaCl (adjusted with HCl to pH 2.5, 3.0, 3.5, or 4.0) solutions placed in micro-

* Corresponding author. Mailing address: Centre de Recherche en Horticulture, Pavillon de l’Envirotron, Universite´ Laval, Que´bec City, Que´bec G1K 7P4, Canada. Phone: (418) 656-2131, ext. 4553. Fax: (418) 656-7871. E-mail: [email protected]. 6800

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centrifuge tubes. Control bacteria were suspended in 0.5% NaCl solution (pH 7.0). After incubation (0 to 20 min) of the bacterial suspensions at 24°C, 200 ␮l of 15% glutaraldehyde (pH 7.0) in 0.1 M sodium cacodylate buffer (SCB) was added to each tube to minimize the impact of salts on bacterial ultrastructure during centrifugation, and the suspension was centrifuged (2,360 ⫻ g for 5 min at 4°C) with a Biofuge 17R centrifuge (Heraeus Sepatech GmbH, Osterode, Germany). The pellet was then fixed with a mixture of 3% glutaraldehyde, 0.2% ruthenium red, and 0.05 M CaCl2, also in 0.1 M SCB (pH 7.3), for 2.5 h at room temperature. Gels of 1 to 2 mm3 were prepared by adding 10% bovine serum albumin and 5% glutaraldehyde in SCB to the pellet. After thorough rinsing with 0.1 M SCB, the gels were postfixed with 2% osmium tetroxide in 0.1 M SCB for 2 h at room temperature, dehydrated by using a series of increasing ethanol concentrations, and embedded in JEMBED 812 resin. Thin (1-␮m) and ultrathin (90-nm) sections were observed with an Orthoplan light microscope (Leitz, Wetzlar, Germany) and a transmission electron microscope (model 300; Philips, Eindhoven, The Netherlands), respectively, after staining the sections with toluidine blue O and safranin O (23) and contrasting them with uranyl acetate and lead citrate (22). For each treatment, the test was repeated twice, and at least two blocks by replicate were examined. Effect of salt treatments on bacterial viability and incorporation of SYTOX Green nucleic acid stain. SYTOX Green nucleic acid stain is a positively charged compound (three positive charges) that was developed to evaluate the integrity of the plasma membrane of microorganisms including bacteria (24). It does not cross intact plasma membranes but readily penetrates damaged ones and binds to nucleic acids, where it induces a fluorescence emission under blue light. Bacteria were grown at 24°C in Erlenmeyer flasks (250 ml) containing 100 ml of tryptic soy broth (Difco) under agitation (150 rpm). After a 16-h growth period, bacteria were recovered by centrifugation (2,360 ⫻ g for 5 min at 4°C) and suspended (108 CFU/ml) in 1 ml of aluminum chloride (0.05, 0.1, or 0.2 M), sodium metabisulfite (0.05, 0.1, or 0.2 M), or 0.5% NaCl (adjusted to pH 2.5, 3.0, 3.5, or 4.0 with HCl) solutions kept in microcentrifuge tubes. Control bacterial cells were suspended in 0.5% NaCl solution (pH 7.0). After exposures of 5, 10, and 20 min (including centrifugation time), bacteria were recovered by centrifugation (2,360 ⫻ g for 5 min at 4°C), washed with 0.5% NaCl (pH 7.0), and concentrated by centrifugation once again. Bacteria were resuspended in 1 ml of 0.5% NaCl (pH 7.0), and an aliquot (100 ␮l) of the suspension was added to an equal volume of SYTOX Green to a final concentration of 5 ␮M. The mixture was allowed to rest for at least 5 min and then examined with a Leitz Orthoplan microscope with blue light excitation using a BP 455-490 exciter filter combined with an RKP 510 separator mirror and an LP 515 barrier filter. For each treatment, the percentage of the fluorescent bacteria (blue light) was determined based on at least 250 bacteria counted under normal light with a hemacytometer. Plate counts on NA were carried out in parallel to the SYTOX test in order to determine bacterial viability. Experiments were repeated at least twice, and each replicate was operated in duplicate.

RESULTS The typical ultrastructure of the untreated bacteria is presented in Fig. 1. Bacteria generally displayed intact cell walls with distinguishable external and internal electron-dense layers characteristic of gram-negative bacteria. The ribosomes were evenly distributed in the cytoplasm, and extracellular vesicles attached to the outer wall layer were clearly visible. Effect of aluminum chloride. The treatment of E. carotovora subsp. atroseptica with different concentrations of aluminum chloride induced significant structural alterations such as wall loosening, as judged by decreased electron density, and at times wall disruption (Fig. 2A to D). Cell voids resulting from plasmolysis and cytoplasmic aggregations were also observed (Fig. 2). Wall loosening was observed immediately after the bacteria were put in contact with the salt (0 min) at a 0.2 M concentration, and these alterations were particularly noticeable at one cellular extremity (Fig. 2A). Generally, the loosening of the walls and cellular voids occurred at opposing ends; cytoplasmic aggregations were only occasionally observable in the cells, and no extracellular vesicles were perceivable (Fig. 2A). After 5 min of contact at the 0.2 M concentration, cyto-

FIG. 1. Transmission electron micrographs of control cells of E. carotovora subsp. atroseptica. (A) Normal appearance of cytoplasm, wall, and external vesicles visible around some cells (arrowheads); bar ⫽ 0.27 ␮m. (B) Details of single cells showing vesicles (arrowheads) attached to the wall (arrows) consisting of electron-dense external and internal layers separated by an electron-lucent layer and bordered internally by the plasma membrane (small arrowheads); bar ⫽ 0.10 ␮m.

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FIG. 2. Effect of various concentrations of aluminum chloride (AlCl3) on the ultrastructure of E. carotovora subsp. atroseptica after different exposure times. lcw: loosened cell wall; wd: wall disruption. (A) Effect of 0.2 M AlCl3 (0 min). Aggregation of cytoplasmic materials (arrows) as well as empty areas (arrowheads) are evident in many cells. Bar ⫽ 0.25 ␮m. (B and C) Effect of 0.2 M AlCl3 after 5 min (B) and 10 min (C) of exposure. Details of a single cell (B, bar ⫽ 0.10 ␮m) and an overview at a lower magnification (C, bar ⫽ 0.31 ␮m) show intense cytoplasmic aggregation (arrows), numerous empty areas (arrowheads), and occasional cytoplasmic leakage (curved arrow). (D and E) Effect of 0.1 M (D) and 0.05 M (E) AlCl3 (10 min). Bacteria (D, bar ⫽ 0.40 ␮m; E, bar ⫽ 0.41 ␮m) show cytoplasmic aggregation (arrows), cytoplasmic leakage (curved arrow), and empty spaces (arrowheads) in several cells.

plasmic aggregations were extensive, cellular voids were dispersed in the treated cells, and cell wall rupture was evident (Fig. 2B). Cell leakage, generally observed at one cellular pole, sometimes accompanied wall disruption (Fig. 2C). These ultrastructural alterations also occurred at lower salt concentrations (0.1 and 0.05 M) after 10 min of exposure but

were less pronounced compared to the 0.2 M concentration, and again, no extracellular vesicles were noticeable (Fig. 2D and E). Effect of sodium metabisulfite. Sodium metabisulfite caused a retraction of plasma membrane and cellular voids at all concentrations (Fig. 3), but extracellular vesicles were present

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FIG. 3. Effect of various concentrations of sodium metabisulfite (Na2S2O5) on the ultrastructure of E. carotovora subsp. atroseptica showing empty spaces (arrowheads), extracellular vesicles (large arrows), and cell walls that seem mostly unaffected (open arrows). An overview (A, bar ⫽ 0.39 ␮m) and details of single cells (B, bar ⫽ 0.10 ␮m) after exposure to 0.2 M Na2S2O5 for 10 min with visible empty zones, cytoplasmic leakage (curved arrow), loosened cell wall (lcw), and wall disruption (wd) are shown. (C) Effect of 0.1 M Na2S2O5 after 10 min of exposure (bar ⫽ 0.10 ␮m) showing numerous vesicles and loosened cell wall (lcw). An overview (D, bar ⫽ 0.31 ␮m) and details of single cells (E, bar ⫽ 0.10 ␮m) 20 min after treatment with 0.05 M Na2S2O5 are shown. Extracellular vesicles remain clearly visible on these micrographs.

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and the cytoplasm appeared as normal as that of the untreated cells (Fig. 3). Cell wall loosening was more evident after 10 min of exposure of the organism to higher concentrations of 0.2 M (Fig. 3B) and 0.1 M (Fig. 3C) sodium metabisulfite. Cellular leakage and wall disruptions were also observed, at times, at the 0.2 M concentration (Fig. 3A and B). The ultrastructural changes were less pronounced at 0.05 M after 20 min of exposure than at higher concentrations (Fig. 3D and E); cell walls remained generally clearly visible, and the cytoplasm appeared normal. Effect of pH. Aluminum chloride solutions are strongly acidic. In order to verify whether the observed effects of aluminum chloride were due to aluminum toxicity or acidic pH, the influence of pH on E. carotovora subsp. atroseptica ultrastructure was also evaluated. Bacteria exposed to pH 3.0 (Fig. 4A), pH 3.5, or pH 4.0 for 10 min showed no apparent ultrastructural alterations, while some bacteria exposed for 10 min to pH 2.5 displayed slight cytoplasmic aggregation (Fig. 4B and C). As in the control bacteria, voids in the cytoplasm and empty cells with ruptured walls were rarely observed. At all pH conditions tested, the extracellular vesicles remained visible (Fig. 4). Effect of the treatments on the incorporation of SYTOX Green. Typical results of epifluorescence after the incorporation of SYTOX Green by dead cells are presented in Fig. 5. Clearly fluorescent dead cells with damaged plasma membranes were numerous after aluminum treatment (0.2 M, 10 min) (Fig. 5A), whereas those with unaltered membranes were undistinguishable. In the case of the untreated bacteria (Fig. 5B), only a few cells (⬍5%) were fluorescent, a level corresponding to the number of bacteria appearing abnormal in transmission electron microscopy. The small smear of fluorescence surrounding the bacteria, which slightly affected their resolution, was due to the bacterial motion in the mounting solution occurring during the long exposure times necessary to photograph the fluorescence. The effect of aluminum chloride, sodium metabisulfite, and pH treatments on the incorporation of SYTOX Green and on bacterial mortality is presented in Fig. 6 and 7. The mortality rate of the bacteria exposed to sodium metabisulfite at 0.05, 0.1, or 0.2 M was 100% after 5 min, while the rate of fluorescent bacteria after exposure to this salt ranged from 4.5% (0.05 M, 5 min) to 32% (0.2 M, 10 min), with the corresponding ratio of mortality to fluorescent bacteria (M/F) ranging from 3.1 to 22.1 (Fig. 6). An M/F ratio of unity would signify a concurrent occurrence of bacterial mortality and plasma membrane damage severe enough to allow the penetration of SYTOX stain into the cell. On the other hand, high M/F ratio values suggest the occurrence of bacterial mortality with plasma membrane damage either little or too weak to allow stain absorption. The mortality of bacteria exposed to aluminum chloride increased with the salt concentration and exposure time (Fig. 6). At a 0.2 M concentration, 100% mortality and 90% fluorescent bacteria were recorded after 10 min of exposure, with an M/F ratio of 1.1. At a 0.05 M FIG. 4. Effect of pH conditions on the ultrastructure of E. carotovora subsp. atroseptica after 10 min of exposure. (A) An apparently unaltered bacterial cell displays perceivable extracellular vesicles (large arrows) and a very well-defined cell wall (open arrow) after treatment at pH 3.0. Bar ⫽ 0.11 ␮m. An overview (B, bar ⫽ 0.41 ␮m)

and details of single cells (C, bar ⫽ 0.11 ␮m) after exposure to pH 2.5 reveal extracellular vesicles (large arrow), mostly unaffected cell walls (open arrows), and slight cytoplasmic aggregation in a few cells (arrows).

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FIG. 5. Fluorescence of bacterial cells after treatment of approximately 250 bacteria with SYTOX stain. (A) Intense yellow-green fluorescence is evident in most bacterial cells treated with aluminum chloride at 0.2 M (10 min), indicating an alteration of their plasma membranes, whereas the few nonfluorescent cells are not visible in this micrograph. (B) Control bacteria (isotonic solution) displaying only a few isolated fluorescent cells.

concentration, the mortality increased from 42% after 5 min of exposure to 88% after 20 min of exposure, with the M/F ratio decreasing from 7.9 to 1.6. Although the mortality reached 100%, the rate of fluorescent bacteria reached only 11.5%

after 10 min of exposure to 0.1 M aluminum chloride, with an M/F ratio of 8.7. Exposure of bacteria to pH 3.0, 3.5, 4.0, and 7.0 caused only low levels of both mortality and fluorescence, while exposure to pH 2.5 resulted in high levels of mortality

FIG. 6. Effect of salt treatments (Al chloride, aluminum chloride; Na metabisulfite, sodium metabisulfite) on the rate of dead (■) and fluorescent (䊐) cells of E. carotovora subsp. atroseptica obtained from a 16-h-old culture in tryptic soy broth. The numbers in brackets above the bars indicate the ratio of bacterial mortality to the rate of fluorescent bacteria.

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FIG. 7. Effect of pH on the rate of dead (■) and fluorescent (䊐) cells of E. carotovora subsp. atroseptica obtained from a 16-h-old culture in tryptic soy broth. The number in brackets above the bar indicates the ratio of bacterial mortality to the rate of fluorescent bacteria.

(100%) and fluorescence (90%), with an M/F ratio of 1.1 (Fig. 7). DISCUSSION Aluminum and bisulfite salts show antimicrobial activity against several microorganisms (1, 7, 8, 15, 16). Aluminum chloride and sodium metabisulfite at relatively low concentrations were shown to be toxic to E. carotovora (32a). A better understanding of the mode of action of these salts could lead to the development of a reliable treatment to control postharvest diseases. The results clearly showed that cells of E. carotovora subsp. atroseptica treated with aluminum chloride displayed significant structural alterations. Exposure to aluminum chloride caused the loosening of the cell wall, wall rupture, cell leakage, and cytoplasmic aggregation. Aluminum is known to cross the wall and plasma membrane in rhizobacteria and complex nucleic acids (10, 32). Aluminum can also combine with proteins (18). Consequently, the aggregated cytoplasm is likely composed of proteins and nucleic acids. Aluminum chloride solutions are strongly acidic, with the 0.05, 0.1, and 0.2 M aluminum chloride solutions having pHs of 3.5, 3.0, and 2.5, respectively. A strongly acidic environment can cause adverse effects on bacteria, including cell surface protein denaturation and alteration of membrane permeability (4) and eventually wall destabilization and breaking. In order to verify whether the observed effects of aluminum chloride were due to aluminum toxicity or pH effect, bacteria exposed to acidic solutions at pH values of 2.5, 3.0, 3.5, and 4.0 and to aluminum chloride solutions of 0.05, 0.1, and 0.2 M were compared. The exposure to 0.1 M aluminum chloride (pH 3.0) caused ultrastructural modifications (wall loosening and disruptions, cell leakage, cytoplasmic aggregation, and disappearance of extracellular vesicles), and such alterations were not observed with bacteria exposed to pH 3.0. This finding suggests that aluminum per


se is involved in the observed morphological changes. Since exposure of bacteria to pH 2.5 caused only little cytoplasmic aggregation, severe cytoplasmic aggregation is mainly attributable to aluminum for the most part and to a lesser extent to a low pH at the 0.2 M concentration. Our results provide evidence of putative mechanisms of aluminum that involve leaky cell membranes and/or structural alteration of cell walls (8). In order to further confirm a possible adverse effect of aluminum chloride on the E. carotovora subsp. atroseptica plasma membrane, bacteria exposed to aluminum chloride were treated with SYTOX Green. Bacteria exposed to 0.2 M aluminum chloride for 5 and 10 min as well as those exposed to a 0.05 M concentration for 20 min exhibited high mortality and absorbed SYTOX Green with an M/F ratio of 1.1, 1.1, and 1.6, respectively. This result suggests that either the high concentration of the salt or longer exposure time caused severe membrane damage, allowing the stain to transfer into the cells and bind to nucleic acids. Given that the exposure of the organism to pH 2.5 caused a similar effect in terms of mortality and SYTOX absorption, it is difficult to discriminate between the contributions of 0.2 M aluminum salt and a highly acidic environment in causing plasma membrane alterations. On the other hand, bacteria exposed to 0.1 and 0.05 M concentrations for 10 min or less did not absorb the stain, even though the killing rates were 38 to 100%, with an M/F ratio of ⱖ4. In particular, bacteria exposed to 0.1 M for 10 min resulted in 100% mortality, with an M/F ratio of 8.7, suggesting that membrane damage caused by aluminum chloride under these conditions was not severe enough for the stain to migrate into the cells. Nonetheless, moderate cell wall alterations occurring under these conditions appear to allow the migration of hydrated aluminum ions into the cells, leading to cytoplasmic aggregation. Ultrastructural observations of untreated bacteria showed the presence of external vesicles. Such structures, which have been previously observed in E. carotovora (5) and Erwinia amylovora (12), were reported to have a composition similar to that of the outer wall layer from which they are derived (5). They appear to play a role in cell wall turnover (33) as well as pathogenesis (5). The disappearance of these vesicles at all concentrations of aluminum chloride provides additional evidence that aluminum causes alterations in the cell wall of E. carotovora subsp. atroseptica. Sodium metabisulfite caused complete mortality of the treated bacteria at all concentrations tested within 5 min of exposure. The effect of sodium metabisulfite on E. carotovora subsp. atroseptica ultrastructure was limited to plasma membrane retraction and cellular voids at all concentrations tested, with occasional cell wall disruption and cellular leakage at the 0.2 M concentration after 10 min of exposure. Unlike aluminum-treated cells, cell walls remained generally clearly visible and extracellular vesicles were intact, more so at lower concentrations (0.1 and 0.05 M), and aggregation of cytoplasmic material occurred only occasionally. The effect of sodium metabisulfite on E. carotovora subsp. atroseptica ultrastructure cannot be due to exposure to external acidic environment since the pHs of sodium metabisulfite solutions were between 4.5 and 4.8. Furthermore, the incorporation of SYTOX stain was generally weak, and extracellular vesicles persisted at all concentrations of sodium metabisulfite and exposure times, unlike

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aluminum chloride. The M/F ratio was higher than 5.0, except for the 10-min exposure at the 0.2 M concentration of sodium metabisulfite, where the ratio was 3.1, and occasional cell wall disruptions were evident. This finding suggests that bacterial killing by sulfite occurs without causing much damage to cell walls and plasma membranes, but prolonged exposure of bacteria to high sulfite concentrations may ultimately lead to alterations of these structural components, permitting the absorption of SYTOX stain. Molecular SO2 is known to freely diffuse into the cell in the pH range of 3.0 to 5.0 (28, 30), and this diffusion can occur without causing any significant wall alterations. Sulfites may directly alter the structure of nucleic acids (17, 26) or damage them by cytoplasmic acidification (11). It may also be possible that sulfite-treated bacteria exhibit a subdued fluorescing response owing to the denaturation of nucleic acids. Lebaron et al. (13) observed that altered DNA could lead to an underestimation of membrane damage by SYTOX fluorescence. However, this possibility is unlikely since moderate fluorescence does occur with longer exposure to higher concentrations of metasulfite concomitant with cell wall alterations. Sodium metabisulfite was more effective and faster in killing the bacteria than aluminum chloride, and their mechanisms of action were different. Aluminum ions are hydrated in aqueous solution and are present as a complex acid ion, [Al(H2O)6]3⫹. Owing to its low charge density and acidity, the hydrated aluminum ions appear to disrupt the cell wall and diffuse into the cell and combine with enzymes and nucleic acids, as evidenced by cytoplasmic aggregation. The rapid killing of bacteria by metabisulfite is likely due to free diffusion of molecular SO2 into the cells without seriously compromising the cell wall structure. Inside the cell, the predominant species, the bisulfite ions, react with biologically important molecules (enzymes, coenzymes, and nucleic acids), rendering them inactive and causing severe stresses as evidenced by the retraction of plasma membrane. In conclusion, the exposure of E. carotovora subsp. atroseptica to aluminum chloride and sodium metabisulfite at concentrations of 0.05, 0.1, and 0.2 M resulted in high mortality. Metabisulfite was more effective and rapid in causing mortality of bacteria than aluminum chloride. Ultrastructural evidence suggests that the modes of action of these two salts are different. Bacteria exposed to 0.05, 0.1, or 0.2 M aluminum chloride showed cell wall alteration and cytoplasmic aggregation. Only bacteria exposed to either a concentration of 0.2 M aluminum chloride or prolonged exposure to 0.05 M aluminum chloride were shown to significantly incorporate SYTOX, suggesting that high aluminum concentration or longer exposure at low concentrations causes extensive plasma membrane alteration. Bacteria exposed to sodium metabisulfite did not show significant cell wall alteration or cytoplasmic aggregation. The low level of fluorescence of SYTOX-stained nucleic acids and the general preservation of extracellular vesicles after sulfite treatment suggest that bacterial mortality by sulfite occurs with little damage to the plasma membrane. It is likely transported across the membrane in the molecular SO2 form, and its main mode of inhibitory action involves its interactions with nucleic acids, proteins, and coenzymes.

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