Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.     

Changes in membrane fatty acid composition of Pediococcus sp. strain NRRL B-2354 in response to growth conditions and its effect on thermal resistance.

Membrane fatty acid composition and thermal resistance (D value) of Pediococcus sp. were determined for mid-exponential-phase (ME) and stationary-phase (ST) cells grown in tryptic soy broth (TSB) and tryptone-glucose-yeast extract (TGY) at 28 and 37 degrees C. As the cells entered the stationary phase of growth, the unsaturated fatty acid, C18:1 n11c, produced during the exponential phase of growth was converted to its cyclic form, C19:0 Delta9c. This shift in membrane fatty acid composition was accompanied by an increase in the D values of this bacterium. Data from this study suggest that the membrane fatty acid composition of Pediococcus sp. is dependent on the growth conditions and that membrane fatty acid composition plays a critical role in thermal resistance. Thermal inactivation curves of Pediococcus sp. cells grown in TGY at 28 degrees C indicated the presence of a cell population that is heterogeneous in thermal resistance. The growth of this bacterium in TGY at 37 degrees C and in TSB at 28 and 37 degrees C resulted in cell populations that were uniform in thermal resistance with a lag time for thermal inactivation. Thermal inactivation curves of ME and ST cultures were similar. The data presented here suggest that the cell population's uniformity of thermal inactivation is independent of the growth phase of the culture.     

Low temperature-induced cell surface membrane vesicle shedding is associated with DNA fragmentation

Temperature shift conditions of 0 degree to 22 degrees C or 0 degree to 37 degrees C induce the formation and shedding of membrane vesicles (MV) from P815 tumor cell surfaces. When the MV shedding process takes place at 22 degrees C it occurs without changes in cell surface membrane permeability, whereas at 37 degrees C, changes in permeability to 51Cr and trypan blue do occur, thus mimicking the lymphocyte-mediated lytic process of tumor cells [1]. The present studies demonstrate that nuclear DNA fragmentation also occurs in both 0 degree to 22 degrees C and 0 degree to 37 degrees C temperature shifts but cell surface membrane permeability to DNA fragments occurs only in the latter condition, i.e., 0 degree to 37 degrees C. The microtubule-stabilizing agent deuterium oxide (D2O) inhibited the MV shedding process, the changes in membrane permeability, and DNA fragmentation. When P815 cells which had been induced to shed MV by the 0 degree to 22 degrees C temperature shift were labeled with 51Cr and used as targets for alloimmune lymphocytes, they were found to be as susceptible to T-cell lysis as control P815 cells. This result indicates that the lytic effect of alloimmune T lymphocytes can be exerted at the target cell surface membrane level independently of nuclear DNA fragmentation.     

The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.     

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.     

Ion selectivity of temperature-induced and electric field induced pores in dipalmitoylphosphatidylcholine vesicles

Temperature and electric field are known to alter the permeability of the bilayer membrane in phospholipid vesicles. A study of cation selectivity of these membrane pores is reported for multilamellar liposomes (MLV) and unilamellar large vesicles (ULV, 95 +/- 5 nm diameter) of dipalmitoylphosphatidylcholine (DPPC). The permeability of ULV to Rb+ was 1.0 X 10(-6) micrograms/s at 22 degrees C and increased to 1.1 X 10(-5) micrograms/s at the gel to liquid-crystalline transition temperature (Tm) of the bilayer, at 42 degrees C. The permeability of ULV to Rb+ continued to increase beyond the Tm and reached 1.0 X 10(-4) micrograms/s at 56 degrees C, a 100-fold increase over the permeability at 22 degrees C. In contrast, the permeability of ULV to Na+ showed a local maximum of 6.0 X 10(-6) micrograms/s at 42 degrees C and decreased at temperatures higher or lower than the Tm. For MLV, the permeability to both Rb+ and Na+ peaked dramatically at the phase transition temperature, 42 degrees C, and subsided at lower and higher temperatures. When ULV were exposed to an electric field, the permeability to Rb+, Na+, and sucrose surged at a field strength of 30 kV/cm; 30 kV/cm can induce a transmembrane potential of 210 mV. In ULV, the electrically perforated lipid bilayer exhibited selectivity for Rb+ over Na+ only at a narrow electric field range, between 31 and 33 kV/cm. For MLV, no well-defined breakdown voltage was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles.

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37 degrees C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20 degrees C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20 degrees C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freeze-thaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Temperature dependence of K(+)-channel properties in human T lymphocytes.

We have used whole-cell patch clamp to determine the temperature dependence of the conductance and gating kinetics of the voltage-gated potassium channel in quiescent, human peripheral blood T lymphocytes. Threshold for activation, steady-state inactivation, and the reversal potential are the same at 22 degrees and 37 degrees C. However, the time-constants for activation, inactivation, deactivation, and release from inactivation are quite sensitive to temperature, changing by at least a factor of five in each case over this range of temperatures. The onset of cumulative inactivation at 22 degrees and 37 degrees C reflects the time-course of deactivation. Peak outward current is approximately twofold greater at 37 degrees C than at 22 degrees C; this increase is also manifest at the single channel level. Energies of activation for conductance, activation, inactivation, deactivation, and release from inactivation are 8.2, 22.1, 25.0, 36.2, and 42.2 kcal/mol, respectively. No new channels were observed at 37 degrees C, and there was no evidence for alteration of the K+ conductance by putative modulators at 22 or 37 degrees C.     

Inactivation of calcium uptake by EGTA is due to an irreversible thermotropic conformational change in the calcium binding domain of the Ca(2+)-ATPase.

Calcium uptake by rabbit skeletal sarcoplasmic reticulum (SR) is inhibited with an effective inactivation temperature (TI) of 37 degrees C in EGTA with no effect on ATPase activity. Since the Ca-ATPase denatures at a much higher temperature (49 degrees C) in EGTA, this suggests that a small or localized conformational change of the Ca-ATPase at 37 degrees C results in inability to accumulate calcium by the SR. Using a fluorescent analogue of dicyclohexylcarbodiimide, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]-carbodiimide (NCD-4), the region of the calcium binding sites of the SR Ca-ATPase was labeled. Steady-state and frequency-resolved fluorescence measurements were subsequently performed on the NCD-4-labeled Ca-ATPase. Site-specific information pertaining to the hydrophobicity and segmental flexibility of the region of the calcium binding sites was derived from the steady-state fluorescence intensity, lifetime, and rotational rate of the covalently bound NCD-4 label as a function of temperature (0-50 degrees C). A reversible transition at approximately 15 degrees C and an irreversible transition at approximately 35 degrees C were deduced from the measured fluorescence parameters. The low-temperature transition agrees with the previously observed break in the Arrhenius plot of ATPase activity of the native Ca-ATPase at 15-20 degrees C. The high-temperature transition conforms well with the conformational transition, resulting in uncoupling of Ca translocation from ATP hydrolysis as predicted from the irreversible inactivation of Ca uptake at 31-37 degrees C in 1 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The enumeration of Bacteroides fragilis group organisms from sewage and natural waters

A membrane filtration technique has been developed for the enumeration of Bacteroides fragilis group (BFG) organisms from sewage and natural waters. The method uses the agar medium of Wilkins and Chalgren supplemented with gentamicin, penicillin, aesculin and ferric ammonium citrate. Membrane filters with 0.22 micron pores were significantly more efficient than those with 0.45 micron pores in the isolation of BFG. A preliminary incubation period of 4 h at 30 degrees C prior to 44 h at 37 degrees C yielded significantly higher numbers of BFG than direct incubation at 37 degrees C for 48 h.     

Effects of p-chloromercuribenzene sulfonate on water transport across the marsupial erythrocyte membrane

The effects of exposure of red blood cells (RBC) of three species of marsupial to a mercury-containing sulfhydryl-modifying reagent, p-chloromercuribenzene sulfonate (PCMBS), on the water diffusional permeability ( P (d)) of their membranes were monitored by using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. For koala ( Phascolarctos cinereus), RBC the maximal inhibition was reached at 37 degrees C in 60 min with 1 mmol.l(-1) PCMBS or in 15-30 min with 2 mmol. l(-1) PCMBS. In contrast, in the case of red kangaroo ( Macropus rufus) or swamp wallaby ( Wallabia bicolor) RBC, maximal inhibition required an incubation of 90 min at 37 degrees C with 2 mmol.l(-1) PCMBS. For the RBC of all three species the value of maximal inhibition was very high, being 50-70% when measured at 25 degrees C, 60-80% at 30 degrees C and 60-70% at 37 degrees C. The lowest values of P (d) appeared to be around 2 x 10(-3)-3 x 10(-3) cm.s(-1) in the temperature range of 25-37 degrees C. The mean value of the activation energy of water diffusion ( E (a,d)) was approximately 20-25 kJ.mol(-1) for control and approximately 40 kJ.mol(-1) for PCMBS-inhibited RBCs. These results show that marsupial RBC have a basal permeability to water similar to that previously reported for human RBC, but a higher value of the PCMBS-inhibitable water permeability. This indicates that the higher water permeability of marsupial RBC compared with human RBC is associated with a higher fraction of protein-mediated water permeability.     

The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 mumol inorganic phosphate/g muscle in 20 min at 20 degrees C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 degrees C for 18 h caused ectoenzyme inactivation which plateaued at 35-40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 micrograms ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by alpha-methyl D-mannopyranoside. As the temperature increased from 10 to 40 degrees C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 degrees C, with a "break point" and a clear change in slope at 35 degrees C. When treated with ConA the activity increased linearly from 10 to 40 degrees C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 degrees C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.     

Reversible and irreversible modification of erythrocyte membrane permeability by electric field

Electric fields of a few kV/cm and of duration in microseconds are known to implant pores of limited size in cell membranes. We report here a study of kinetics of pore formation and reversibility of pores. Loading of biologically active molecules was also attempted. For human erythrocytes in an isotonic saline, pores allowed passive Rb+ entry formed within 0.5 microsecond when a 4 kV/cm electric pulse was used. Pores that admitted oligosaccharides were introduced with an electric pulse of a longer duration in an isosmotic mixture of NaCl and sucrose. These pores were irreversible under most circumstances, but they could be resealed in an osmotically balanced medium. A complete resealing of pores that admitted Rb+ took approximately 40 min at 37 degrees C. Resealing of pores that admitted sucrose took much longer, 20 h, under similar conditions. In other cell types, resealing step may be omitted due to stronger membrane structures. Experimental protocols for loading small molecules into cells without losing cytoplasmic macromolecules are discussed.     

Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures?

Our primary objective was to determine if rates of fluid-phase endocytosis (FPE) were conserved in hepatocytes from organisms acclimated and adapted to different temperatures. To this aim, the fluorescent dye Lucifer yellow was employed to measure FPE at different assay temperatures (AT) in hepatocytes from 5 degrees C- and 20 degrees C-acclimated trout, Oncorhynchus mykiss (at 5 and 20 degrees C AT), 22 degrees C- and 35 degrees C-acclimated tilapia, Oreochromis nilotica (at 22 and 35 degrees C AT), and the Sprague-Dawley rat (at 10, 20, and 37 degrees C AT). FPE was also studied in rats fed a long-chain polyunsaturated fatty acid (PUFA)-enriched diet (at 10 degrees C AT). Despite being temperature dependent, endocytic rates (values in pl. cell(-1). h(-1)) in both species of fish were compensated after a period of acclimation. For example, in 20 degrees C-acclimated trout, the rate of endocytosis declined from 1.84 to 1.07 when the AT was reduced from 20 to 5 degrees C; however, after a period of acclimation at 5 degrees C, the rate (at 5 degrees C AT) was largely restored (1.80) and almost perfectly compensated (95%). In tilapia, endocytic rates were also temperature compensated, although only partially (36%). Relatively similar rates obtained at 5 degrees C in 5 degrees C-acclimated trout (1.8), at 20 degrees C in 20 degrees C-acclimated trout (1.84), and at 22 degrees C in 22 degrees C-acclimated tilapia (2.2) suggest that endocytic rates are somewhat conserved in these two species of fish. In contrast, the rate in rat measured at 37 degrees C (16.83) was severalfold greater than in fish at their respective body temperatures. A role for lipids in determining rates of endocytosis was supported by data obtained at 10 degrees C in hepatocytes isolated from rats fed a long-chain PUFA-enriched diet: endocytic rates were higher (5.35 pl. cell(-1). h(-1)) than those of rats fed a standard chow diet (2.33 pl. cell(-1). h(-1)). The conservation of endocytic rates in fish may be related to their ability to conserve other membrane characteristics (i.e., order or phase behavior) by restructuring their membrane lipid composition or by modulating the activities of proteins that regulate endocytosis and membrane traffic, whereas the lack of conservation between fish and rat may be due to differences in metabolic rate.     

Measurement temperature plays a pivotal role in the distribution of erythrocyte deformability after LPS

Reductions in red blood cell membrane deformability (RBC(D)) may perturb microcirculatory blood flow and impair tissue O(2)-availability. We investigated the effect of assay temperature on the distribution of RBC(D) in endotoxin (LPS) incubated and control RBCs. Fresh blood from healthy rats was incubated with and without the presence of LPS for 6 hrs. An index of red blood cell membrane deformability, delta, was measured via the micropipette aspiration technique at 25 degrees C and 37 degrees C at 0, 2 and 6 hrs of incubation. The ATP content of RBC was measured by the luciferin-luciferase technique. At 25 degrees C, LPS caused a significant decrease in mean delta after 2 and 6 hours incubation compared to controls (-10.0%, p=0.03 and -24.0%, p=0.03, respectively) characterized by a left shift in the distribution (skewness: -1.4). However, at 37 degrees C a significant decrease in delta was only detected after 6 hrs of LPS incubation (-13.8%, p=0.01, compared to -5.1%, p=0.7 at 2 hours) and lacked the left shifted distribution (skewness: 0.2). No significant difference in ATP content of RBCs was observed between groups. We have shown that LPS incubation results in a significant decrease in RBC(D) and that room temperature measurement of physical membrane properties may exaggerate the differences between normal and perturbed RBCs.     

Influence of the biophysical and biochemical environment on the kinetics of pore formation by Cry toxins

The effect of Bacillus thuringiensis toxins on the permeability of the luminal membrane of Manduca sexta midgut columnar epithelial cells is strongly influenced by several biophysical and biochemical factors, including pH, ionic strength, and divalent cations, suggesting an important role for electrostatic interactions. The influence of these factors can differ greatly, however, depending on the toxin being studied, even for closely related toxins such as Cry1Ac and Cry1Ca. In the present study, the possibility of using temperature changes as a tool for controlling the rate and extent of pore formation in midgut brush border membrane vesicles was evaluated. Lowering temperature gradually decreased the rate of pore formation, but had little effect on the permeability of vesicles previously incubated with toxin at room temperature. The formation of new pores, following incubation of the vesicles with toxin, could thus be almost abolished by rapidly cooling the vesicles to 2 degrees C. Using this approach, changes in the rate of pore formation could be more easily distinguished from alterations in the properties of the pores formed, thus allowing a more detailed analysis of the kinetics and mechanism of pore formation.     

RecD plays an essential function during growth at low temperature in the antarctic bacterium Pseudomonas syringae Lz4W

The Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W has been used as a model system to identify genes that are required for growth at low temperature. Transposon mutagenesis was carried out to isolate mutant(s) of the bacterium that are defective for growth at 4 degrees but normal at 22 degrees . In one such cold-sensitive mutant (CS1), the transposon-disrupted gene was identified to be a homolog of the recD gene of several bacteria. Trans-complementation and freshly targeted gene disruption studies reconfirmed that the inactivation of the recD gene leads to a cold-sensitive phenotype. We cloned, sequenced, and analyzed approximately 11.2 kbp of DNA from recD and its flanking region from the bacterium. recD was the last gene of a putative recCBD operon. The RecD ORF was 694 amino acids long and 40% identical (52% similar) to the Escherichia coli protein, and it could complement the E. coli recD mutation. The recD gene of E. coli, however, could not complement the cold-sensitive phenotype of the CS1 mutant. Interestingly, the CS1 strain showed greater sensitivity toward the DNA-damaging agents, mitomycin C and UV. The inactivation of recD in P. syringae also led to cell death and accumulation of DNA fragments of approximately 25-30 kbp in size at low temperature (4 degrees ). We propose that during growth at a very low temperature the Antarctic P. syringae is subjected to DNA damage, which requires direct participation of a unique RecD function. Additional results suggest that a truncated recD encoding the N-terminal segment of (1-576) amino acids is sufficient to support growth of P. syringae at low temperature.     

Fate of Salmonella montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine.

A study was undertaken to determine the survival patterns of Salmonella montevideo G4639 on and in tomatoes during storage and the efficacy of chlorine treatment on inactivation of the pathogen. The population of S. montevideo on the surfaces of inoculated tomatoes stored at 10 degrees C did not change significantly (P < 0.05) throughout an 18-day storage period. Significant increases in population occurred within 7 days and within 1 day when tomatoes were stored at 20 and 30 degrees C, respectively. A significantly higher number of cells was taken up by the core tissue of tomatoes tempered at 25 degrees C when the tomatoes were dipped in a suspension at 10 degrees C compared with the number taken up when the tomatoes were dipped in cell suspensions tempered at 25 or 37 degrees C. Populations remained constant throughout subsequent storage for 8 days at 10 degrees C, regardless of the temperature differential between tomatoes and the dip suspension. Storage of tomatoes at 20 degrees C, however, resulted in significant increases in populations of S. montevideo. Populations of the pathogen on the surfaces and in the core tissues of tomatoes were significantly reduced by dipping for 2 min in a solution containing 60 or 110 ppm (60 or 110 micrograms/ml) chlorine, respectively; however, treatment in solution containing 320 ppm chlorine did not result in complete inactivation. Populations of S. montevideo remained unchanged in chopped tomatoes stored at 5 degrees C for 216 h (9 days) but increased significantly after storage for 96 or 22 h at 20 or 30 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)