Release of Surface Enzymes in Enterobacteriaceae

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1967, p Copyright ) 1967 American Society for Microbiology Voi. 94, No. 6 Printed in U.S.A. Release of Surface Enzymes in Enterobacteriaceae by Osmotic Shock HAROLD C. NEU1 ANm JAMES CHOU Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York Received for publication 22 September 1967 The process of osmotic shock, which has been used to release degradative enzymes from Escherichia coli, can be applied successfully to other n*mbers of the Enterobacteriaceae. Cyclic phosphodiesterase (3'-nucleotidase), 5'-nucleotidase (diphosphate sugar hydrolase), acid hexose phosphatase, and acid phenyl phosphatase are released from Shigella, Enterobacter, Citrobacter, and Serratia strains. Some strains of Salmonella also release these enzymes. Members of Proteus and Providencia groups fail to release enzymes when subjected to osmotic shock and do not show a lag in regrowth, although they do release their acid-soluble nucleotide pools. In contrast to E. coli, release of enzymes from other members of the Enterobacteriaceae studied is affected by growth conditions and strain of organism. None of the organisms was as stable to osmotic shock in exponential phase of growth as was E. coli. Exponential-phase cells of Shigella, Enterobacter, and Citrobacter could be shocked only with 0.5 mm MgCl2 to prevent irreparable damage to the cells. These observations suggest that this group of degradative enzymes is probably loosely bound to the cytoplasmic membrane through the mediation of divalent cations. It has been reported that a number of degradative enzymes are released from Escherichia coli (30, 34) when cells are osmotically shocked. The procedure consists of incubation of the cells in a byperosmolar solution of sucrose and ethylenediaminetetraacetic acid (EDTA) followed by sedimentation of the cells and exposure to either cold water or a dilute magnesium solution. The enzymes which have been characterized thus far are alkaline phosphatase (22, 29, 34), 5'-nucleotidase (30, 33), diphosphate sugar hydrolase (10), acid hexose phosphatase (30, 34), acid phenyl phosphatase (32), cyclic phosphodiesterase (30, 34), a ribonucleic acid (RNA)-inhibited deoxyribonuclease (5, 29, 34), thymidine phosphorylase (17), and adenosine diphosphate (ADP)-glucose pyrophosphatase (23). A large number of enzymes remain within the cells after osmotic shock, in spite of a loss of 5 to 10% of the total cellular protein (30, 34) and all of the acid-soluble nucleotide pool (31). The cells are viable. Work in a number of laboratories has shown that protein functions related to transport of materials from the environment to within the protoplasmic membrane of the cell may also be released by osmotic shock. Studies have been concerned 1 Career scientist of the New York Health Research Council. with amino acid transport (36), glycoside transport (18), galactose transport (1) in E. coli, and sulfate transport (35) in Salmonella typhimurium. The exposure to EDTA has rendered cells permeable to the exit of the acid-soluble nucleotide pool and the entry of substances such as actinomycin (19, 31), puromycin (38), and nucleotides (4). The change would appear to be related to the release of lipopolysaccharide component of the cell wall (2, 20). However, the alteration of permeability appears to be separate from the change which occurs in osmotic shock, since there is no enzyme release when cells are rendered permeable by EDTA and tris(hydroxymethyl)aminomethane (Tris), as shown by Neu et al. (30, 31). In the present investigation, we have carried out a detailed examination of the process of osmotic shock for a number of the members of the Enterobacteriaceae. We have been concerned with both stationary and exponential phase organisms. The organisms studied belonged to the following groups: Shigella, Klebsiella-Enterobacter, Salmonella, Citrobacter, Serratia, Proteus, and Providencia. Osmotic shock causes the release of a group of degradative enzymes from all orgaisms except the members of Proteus and Providencia groups. This paper details the condi- 1934

2 VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1935 tions for osmotic shock in each organism, as well as studies on regrowth fo'lowing osmotic shock. MATERIALS AND METHODS Materials. Bis(p-nitrophenyl)phosphate, p-nitrophenyl phosphate, and nucleotides were obtained from commercial sources. E. coli-soluble RNA was purchased from General Biochemicals Corp., Chagrin Falls, Ohio. Penassay broth was purchased from Difco. Crystalline lysozyme (muramidase) was purchased from Worthington Biochemical Corp., Freehold, N.J. Organisms. Isolates from the diagnostic laboratory of the Presbyterian Hospital, New York, N.Y., were used. Identification was based on the methods of Edwards and Ewing (8). Salmonella typhimurium LT2, Ade 97, and Leu 126 were a generous gift of Dr. Rudner. E. coli strains were those previously described (30). Media and culture conditions. Stock cultures were maintained on Penassay slants. Low- and high-phosphate media were used. The high-phosphate medium contained 0.04 M K2PO4, M KH2PO4, 0.08 M NaCl, 0.02 M NH4Cl, 3mM Na2SO4, 1 mm MgCl2, 0.1 mm CaCl2, and 0.5% Bacto-peptone (Difco). The ph was adjusted to 7.1 with NaOH. The lowphosphate medium contained 0.12 M Tris, 0.08 M NaCl, 0.02 M KCl, 3 mm Na2SO4, 1 mm MgCI2, 0.1 mm CaCl2, and 0.5% Bacto-peptone, with the ph adjusted to 7.4. The Tris medium was supplemented with potassium phosphate to make it 0.01 M, when noted. The content of magnesium and calcium was altered in both media in some cases, as specified in the particular experiment. Carbon content of the media was 0.5% glucose, 0.5% glycerol, or 0.3% sodium succinate. Penassay broth (Difco) was also used. Organisms were incubated at 35 C on a rapid rotary shaker, and growth was followed by change in optical density (OD)o00 in a DU spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). Procedure for osmotic shock. Stationary-phase cells were harvested at 16 hr after a 1% inoculum into the specified medium. Exponential cells were harvested at an OD600 of 0.3 with a cell density of 5 X 108 cells per milliliter. In both cases, the cells were washed with either 0.01 M Tris-HCl (ph 7.3)-0.03 M NaCl or 0.85% NaCl at 3 C. A sample at this stage was removed for preparation of a sonic extract. Stationary-phase cells (16-hr culture) were suspended in 20% sucrose-0.03 M Tris-HCl, ph 7.3, at 21 C at a ratio of 1 g (wet weight) to 80 ml of sucrose-tris. EDTA was added to a concentration of 1 mm, and, after 2 to 10 min of mixing, the cells were removed by centrifugation at 0 C. The pellet of cells was resuspended in water at 3 C and mixed for 5 to 10 min. The cells were removed by centrifugation. Exponential cells were also suspended in 20% sucrose-0.03 M Tris-HCl, ph 7.3; however, the EDTA concentration was 0.1 mm and the cells were resuspended in 0.5 mm MgCl2 at 3 C. Sonic extracts were prepared by use of a Branson Sonifer model LS75, with 2 min of sonic disintegration at 0 C using 15-sec bursts. Spheroplasts were prepared as described (27). Viability was determined on serial dilutions, made in Penassay broth, which were plated on either Penassay Agar or Nutrient Agar containing 0.5% NaCI. Enzyme assays. Previously published methods were used for ribonuclease (28), deoxyribonuclease (27),,3-galactosidase (22), alkaline phosphatase (29), acid phenyl phosphatase (33), 5'-nucleotidase (33), uridine diphosphate glucose pyrophosphatase (33), acid hexose phosphatase (30), and cyclic phosphodiesterase (30). Adenosine triphosphatase was measured in Salmonella by incubating (0.1 ml) 2 mm adenosine triphosphate, 100 mm Tris-HCl, ph 7.4, and 10 mm MgCl2 at 37 C for 20 min. The reaction was halted with 1 N H2SO4 and the phosphate was determined by a modification of the Fiske-SubbaRow method (9). Inorganic pyrophosphatase was determined by incubating (0.3 ml) 3 mm Na4PPi, 0.4 mt MgCI2, and 60 mm Tris-HCI, ph 912. The reaction was stopped after 15 min at 37 C with 1 N H2SO4 and the phosphate was determined by a modified Fiske-SubbaRow method (9). A unit of enzyme activity is the amount of enzyme that will hydrolyze 10 umoles of PPi in 1 hr. Protein was determined according to Lowry et al. (21). RESULTS Conditions for osmotic shock. The process of osmotic shock was affected by the media and culture conditions, as discussed in the following. Growth medium. Previous studies by Neu and Heppel (30) and Nossal and Heppel (34) had utilized synthetic media for the growth of cells for osmotic shock. Escherichia coli, Shigella sonnei, Enterobacter-aerogenes, Salmonella typhimurium, Citrobacter freundii, and Serratia marcescens were all susceptible to osmotic shock after growth in the two standard synthetic media (high and low phosphate content), Penassay Broth (Difco), Trypticase Soy Broth (BBL), and the standard high- and low-phosphate media from which peptone was omitted. However, the release of enzymes from cells grown in Penassay Broth (Difco Antibiotic Medium 3) or Trypticase Soy Broth was 10 to 15% below that of cells grown in minimal medium. Exponential-phase cells of Shigella, Enterobacter, and Citrobacter grown in the phosphate medium were not less stable than the Tris-HCI grown cells when subjected to the Tris-HCl-sucrose osmotic swelling medium. The magnesium content of the growth medium had a significant effect on the release of enzymes of organisms in the exponential phase of growth, but not on stationary-phase cells. When the Mg++ content was lowered to 0.1 or 0.01 mm, the enzymes were released into the sucrose-tris- EDTA rather than into the osmotic shock fluid. Also, there was gross damage to ribosomal RNA and viability fell to 25%. This was particularly true of Salmonella and Enterobacter strains. Age and concentration of cells. Stationary-phase

3 1936 NEU AND CHOU J. BAcrERIOL. cells of the organisms studied were similar to E. coli. Exponential cells, particularly Enterobacter and Shigella strains, showed partial lysis during osmotic shock, even with 1 mm MgCl2, if harvested in very early exponential phase. We routinely harvested cells at a cell density of about 5 x 108 per ml. At 5 X 107 and 108 cells per milliliter, internal enzyme leakage reached 30% and viability fell to 20%. Washed cells were centrifuged in an SS34 head of a RC2-B Sorvall centrifuge at 16,000 rev/min for 5 min, and 1 g (wet weight) of cells was suspended in 80 ml of sucrose- Tris-EDTA. When the concentration of stationary cells was increased to 2 g per 80 ml, the release of enzymes was decreased by less than 10%; however, at 3 and 4 g per 80 ml, release was cut as much as 30%: Exponential cells for all groups studied showed best results with no more than 1.5 g per 80 ml. Wash system. The system used to wash the cells after harvest did not alter the release of enzymes or subsequent growth and viability of Escherichia coli, Shigella sonnei, Enterobacter aerogenes, or Serratia marcescens. The following were used with identical results: 0.85% NaCl, 0.01 M Tris- HCI (ph 7.3)-0.03 M NaCI, 0.03 M Tris-HCI (ph 7.3), and 0.5 M sucrose-0.03 M Tris-HCI (ph 7.8). Cells washed with 0.5 M sucrose-0.03 M Tris-HCl did release a significant part of their surface enzymes into the sucrose-tris-edta in contrast to the other systems. The temperature of the wash system did not affect the results when at 0 C or 21 C. More than two washes with 10 ml/g were not necessary. Effect of buffer system. Previous studies from our laboratory had shown that both buffer system and ph affected the altered permeability achieved by EDTA (31). Tris-HCI appeared to be the best buffer system for both release and viability. The ph range used was 7.2 to 8.0 with the ph measured at 21 C, the temperature used for the osmotic swelling. Tris-maleate, 0.03 M, in a ph range of 5.1 to 7.7 released only 60% of the 5'-nucleotidase and 45% of the acid phenyl phosphatase of both E. coli Hfr H and Enterobacter aerogenes. Hepes, ph 7.1, released only 36%. Glycylglycine, in a ph range of 5.7 to 7.7, released only 25% and NH4HCO3, ph 8.4, released 45%, but viability was greatly lowered. Although chelation by EDTA is enhanced at a more alkaline ph, in the case of glycylglycine and NH4HCO3 improved release of enzymes did not occur at more alkaline values. The explanation of the lower release of enzymes by Tris-maleate buffer compared with Tris-HCl is unknown at present. Phosphate buffer cannot be used to replace the Tris in osmotic shock, since it cannot be used to render E. coli permeable to release of the acidsoluble nucleotide pool (31). Effect of replacement oj sucrose. It had been shown, for stationary-phase E. coli, that reduction of the concentration of sucrose below 12% caused a significant decrease in the release of enzymes (30), and that sucrose could be replaced by 0.5 M NaCl in exponential cells. As Table 1 shows, sucrose can be replaced by 0.5 M glucose, 0.5 M NaCl, and 0.5 M Tris, but not by 0.5 M glycerol. However, survival in the case of glucose and Tris was below 50%. The length of contact in the sucrose, NaCl, or glucose could be shortened to 2 min without effect. Prolongation of the contact of cells in sucrose-tris-edta beyond 10 min at 23 C merely causes degradation of RNA (9). The failure of glycerol as an agent to allow osmotic shock is consistent with its penetration into cells as shown by Mitchell and Moyle (26). Effect of chelation. As was originally shown with stationary-phase E. coli (30), all of the bacteria studied required the presence of a chelating agent in the initial phase. EDTA showed the optimal activity at a concentration of 1 mm for Shigella, Klebsiella-Enterobacter, Salmonella, Serratia, and Citrobacter in the stationary phase. Exponential phase cells of all of these strains (Table 3-6) showed lysis at concentrations above 0.1 mm. Cells were treated with Dowex-50 [H+], a cation exchange resin (37), for a brief period and were then suspended in 20% sucrose and lysozyme, providing cells that lysed on resuspension in water. These cells failed to release any enzymes into the sucrose-lysozyme medium. Similarly, cells harvested in very early exponential phase (E. coli K-12 or Shigella sonnei) and treated with 0.5 M sucrose, 0.03 M Tris, ph 8.0, and lysozyme 20,ug/ml lysed on dilution in water or in 0.1 mm MgC92, but enzymes were not released into the sucrose. Osmotic shock ofshigella. Shigella strains were more fragile in regard to osmotic shock than any of the wide variety of E. coli strains tested (30). Table 2 shows that stationary-phase cells grown in the standard high-phosphate medium required sucrose or NaCl as the osmotic swelling medium, and that the osmotic transition alone is inadequate to release the surface enzymes, because both EDTA and Tris are needed. Stationary-phase cells require a greater osmotic transition than exponential cells. The inorganic pyrophosphatase, which had been shown by Neu and Heppel (29) and Josse (16) to be an intracellular enzyme in E. coli, was used as the control enzyme for lysis, since it is unaffected by any of the constituents of the shock medium and is an extremely sensitive assay. Simmonds (40) has recently pointed out

4 VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1937 TABLE 1. Effect oj various media on osmotic shock in Escherichia coli and Shigeila soinnei- Method 5'-Nucleotidase Cyclic phos- phodiesterase Acid phenylphosphatase Acid hexose phosphatase Inorganic pyrophosphatase A 26) Protein E. coli Sucrose Glucose Glycerol NaCl Tris Soniic extract Shigella sonnei Sucrose Glucose Glycerol NaCI Tris Sonic extract units/ml 69 77S units/ml units/ml units/ml units/ml mg/ml a Stationary phase E. coli K 10, R 6 and Shigella sonnei were grown in the high-phosphate glycerol medium and washed with 0.85% NaCl. They were then suspended in 0.03 M tris(hydroxymethyl)- aminomethane (Tris)-HCl, ph 7.3, at 21 C (1 g/80 ml) to which a 1 M solution of equal volumes of sucrose, glucose, glycerol, NaCl, or Tris (ph 7.3) was added. Ethylenediaminetetraacetic acid (EDTA) to 1 mm was added, and they were agitated for 5 min. The cells were removed by centrifugation and resuspended in water at 3 C for 5 min. The cells were removed and the supernatant fluid was assayed. The sucrose and NaCl-treated cells showed survival of 70%; the Tris-treated cells showed 25% survival. There was no release of the chloramphenicol acetylating enzyme (35) of either organism. TABLE 2. Effect of sucrose, Tris, and EDTA on release of enzymes by osmotic shock from Shigella sonneid ~~~~5'-Nucleo- Cyclic phos- Acid hexose Inorganic Sucrose Tris ] EDTA tidase phodiesterase phosphatase phoahtase X M mm units/g units/g units/g units/g 0.5 None None None None None NaClb Sonic extract, cells ,088 a S. sonnei cells were grown to stationary phase in the high phosphate-glycerol medium. The standard osmotic shock procedure was used, except that the sucrose, tris(hydroxymethyl)aminomethane (Tris), and ethylenediaminetetraacetic acid (EDTA) were varied as noted. b Sucrose replaced by 0.5 M NaCl. the dangers of the use of f-galactosidase as the reference internal enzyme, unless the assay is modified. Table 3 summarizes a number of experiments with Shigella sonnei in exponential and stationary phase. Cells in early exponential phase show leakage of internal enzymes even when shocked with 0.1 mm MgC12. At mid-exponential phase, the cells are more stable, but 0.5 mm MgCl2 is needed to stabilize. In most cases, Shigella sonnei released more Amso material during osmotic shock than could be accounted for by the expected release of the acid-soluble nucleotide pool. Warm osmotic shock shows significantly less release of the surface enzymes but allows more degradation of the RNA. Addition of ribonuclease or deoxy-

5 1938 NEU AND CHOU J. BACTERIOL. TABLE 3. Effect of various growth conditions and osmotic shock conditions on the release of enzymes irom Shigella sonnep Acid Cyclic Acid Inorganic Fraction Temp 5'-Nucleotidase phosphahexosa phosphodiesphenylphos- phos- Ebo- Surival A 260 Protein pnod ApePrtinSrvvlda tase terase p atase phatase C unit s/g unils/g unitsig units/g units/g total mg/g % min Experiment 1l Sucrose-Tris-EDTA Mg++, 0.5 mm Mg++, 0.5 mm + RNase Mg-, 0.5 mm + DNase Ca++, 0.5 mm Sonic extract cells , Experiment 2b Sucrose-Tris-EDTA H , > 200 Mg++, 0.1 mm Mg', 0.5 mm Sonic extract cells , Experiment 3b Sucrose-Tris-EDTA Mg++), 0.5 mm MgH+, 0.5 mm 3 1, Sucrose-Tris-EDTA Mg4, 0.5 mm 3 1, Sonic extract cells , Experiment 4e Sucrose-Tris-EDTA H20 3 3, Sonic extract cells 3, ,800 Experiment 5d Sucrose-Tris-EDTA H Sonic extract cells , a S. sonnei cells were grown in the specified medium. Standard procedure for osmotic shock was followed except for the changes noted in the table. Cells of experiment 1 were harvested at an optical density (OD), at 600 mp, of Cells of experiment 3 were harvested at an OD60, of The concentrati9r1 of ethylenediaminetetraacetic acid (EDTA) of experiments 1 to 3 was 0.1 mm; in experiments 4 and Sit was 1 mm. Assays are presented as units of enzyme released per g (wet weight) of cells. Blank spaces mean no assay was performed. Abbreviations: RNase = ribonuclease; DNase = deoxyribonuclease; Tris = tris(hydroxymethyl)aminomethane. b Exponential-phase cells grown in high-phosphate, glycerol medium. c Stationary-phase cells grown in high-phosphate, glycerol medium. d Stationary-phase cells grown in Penassay broth. ribonuclease to the Mg++ shock fluid resulted in a significant decrease in viability, indicating that permeability to protein molecules is altered. CaCk2 was able to replace the Mg++ in prevention of nucleotide loss, as shown by the stable A2w0 values, but did not increase survival to normal. CoCl2 and ZnCl2 were not able to replace the MgC12. Addition of 0.1 mm CaCd2 to 0.5 mm MgCl2 failed to increase survival, to decrease nucleotide release, or to shorten furtherthe lag period before logarithmic growth is resumed. The rate of release of enzymes from Shigella sonnei was found to be essentially complete by 2 to 3 min after cold 0.5 mm MgCI2 shock (Fig. 1). Cells in exponential phase were suspended in 0.5 M sucrose, 0.03 M Tris-HCI, ph 7.3, 0.1 mm EDTA (1 g/80 ml) at 21 C for 5 min. The cells were removed by centrifugation and then rapidly resuspended in 0.5 mm MgCl2 at 3 C. One-ml portions were placed on 0.45-Iu (pore size) filters (Millipore Corp., Bedford, Mass.) and the filtrate was collected and assayed. Immediately, 5'-nucleotidase, cyclic phosphodiesterase, and acid hexose phosphatase were released, but no significant amount of inorganic pyrophosphatase was released. All members of the genus Shigella that were tested contained 5'-nucleotidase (5'-nucleotidase

6 VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1939 E IL A I,; I 'nuwleotidose 5 nor was the acid hexose phosphatase. In general, only 50% of the cyclic phosphodiesterase activity and adenosine triphosphatase activity were released. A number of other Salmonella strains were studied. S. montevideo, S. derby, S. oranienberg, / and S. manhattan all released 50 to 70% of their ; cyclic phosphodiesterase, 5'-nucleotidase, and /25 acid phenyl phosphataseactivities with less than./cyc phb@sptioesterase = 10% of their inorganic pyrophosphatase released. Acid Homose Phosp*losbe These Salmonella strains showed poor conversion to spheroplasts. Osmotic shock of Citrobacter freundii and Serratia marcescens. Both of these organisms re '-* I---50 leased enzymes after osmotic shock (Table 5) in Minutes a manner analogous to E. coli. The fact that Serratia released F] IG. 1. Rate of release of 5-nucleotidase, cyclic esting because, enzymes as Repaske by osmotic (37) showed, shock is it interdoes phos, phodiesterase, and acid hexose phosphatase during osmcitic shock of Shigella sonnei. Shigella sonnei grown not undergo sigmficant lysis by means of the to e; xponential phase were subjected to osmotic shock EDTA-iysozyme method. This again points out by 0..5 mm MgCI2 and were poured on Millipore filters the importance of the osmotic transition. at the noted intervals. Details are in the text. In each Osmotic shock in Klebsiella aerogenes. A wealth case, release of the enzyme represents 90% of the of data is available about the lethal effect of chillactiv,ity of a sonic extract of the cells. The amount of ing (cold shock) on bacteria (14, 24). Much work inorgranic pyrophosphatase released represents only 4% has been done (43, 44) on Aerobacter aerogenes of thrat present in a sonic extract. (Enterobacter aerogenes). The great variation in capsule and cell wall that exists in this group from of S'higella is identical to the diphosphate hexose Klebsiella pneumoniae to Enterobacter aerogenes hydirolase; Neu, in preparation), acid phenyl phos- made it difficult to generalize as we had about E. phaltase, cyclic phospl-odiesterase (cyclic phos- coli. Stationary-phase Klebsiella pneumoniae cells pho( diesterase of Shigella is identical with its released only 50% of their acid phenyl phosphaucleotidase; Neu, in preparation), and acid tase, 5'-nucleotidase, and cyclic phosphodihexc)se phosphatase. These enzymes, as in the esterase activities. Enterobacter aerogenes species case of S. sonnei (Group D), were surface en- in stationary phase released 70 to 80% of these zymles in S. dysenteriae (Group A) and S. flexneri three enzymes, but less than 5% of the inorganic (GrcDup B). pyrophosphatase (Table 6). In the case of exposmotic shock in Salmonella. Pardee has shown nential-phase Enterobacter aerogenes (Table 6), 0. that Salmonella typhimurium releases a sulfate- the release of enzymes was 50% with cold water bindling protein when subjected to osmotic shock shock, but survival was only 37%. With warm 0.5 (35). We found that release of enzymes from Sal- mm MgCl2 osmotic shock, the release of enzymes moniella was extremely dependent on prior growth was only 20%. We studied a large number of conclitions and the strain tested. Exponential- Enterobacter strains, increasing the Mg++ and phasie Salmonella typhimurium cells grown in Ca++ of the growth medium, using 0.05 mm eithe-r the low- or high-phosphate medium, but EDTA, and shocking with 1.0 mm MgCl2. In all cont;aining 0.01 mm Mg++, 0.1 mm Ca++, when cases in the exponential cells, there was leakage of subji ected to osmotic shock, released 58% of the ultraviolet absorbing material to excess of the surfetce 5'-nucleotidase into the sucrose-tris- acid-soluble nucleotide pool, and viability was ED1PA, but survival was only 25%. Cells grown poor. In exponential-phase Enterobacter strains, in 1 mm Mg++, 0.1 mm Ca++ released 3% of the we frequently noted that cells released up to 25% 5'-nLucleotidase into the sucrose-tris-edta and of the 5'-nucleotidase, cyclic phosphodiesterase, 24% into the 0.5 mm Mg++ shock fluid with a and acid phenyl phosphatase into the suspending surv: ival of 69%. The majority of Salmonella sucrose-tris before EDTA was added. In no case, typh, imurium strains contain negligible amounts however, were enzymes released into the washing of t} he Co++-stimulated 5'-nucleotidase. Table 4 medium to which cells were transferred from the shovvs the release of cyclic phosphodiesterase, growth medium. This is in contrast to the observaphenyl phosphatase, and adenosine triphos- tion of Cowie and McClure (6) that the amino acid phat:ase. Ribonuclease was not released by shock acid pool can be so removed from E. coli.

7 1940 NEU AND CHOU J. BACTERiOL. TABLE 4. Release of enzymes from Salmonella strains by osmotic shocka 5'-Nucle- Cyclic Acid Acid Adenosine Strain Growth stage otidace phospho- hexose phenyl- rienospte diesterase phosphatase phosphatase S. typhimurium, Stationary S. typhimurium, LT2 Log S. typhimurium, Ade 97 Log S. typhimurium, Leu 126 Log S. montevideo Stationary S. derby Stationary S. heidelberg Stationary a Various Salmonella strains were grown in the high-phosphate medium with glycerol as the carbon source. They were subjected to osmotic shock by the standard procedures for either stationary or exponential phase cells. Results are given as per cent of enzyme found in a sonic extract of cells. Blank spaces indicate no assay was done. Survival in the case of stationary cells averaged 80%; in the case of exponential cells 70%. Less than 6% of the inorganic pyrophosphatase was released in any case. Release of protein amounted to 4% of the total protein of intact cells. Release of acid-soluble nucleotide material equaled the perchloric acid-soluble pool of intact cells. Expt C. freundii Experiment lb Sucrose-Tris-EDTA H20 Mg+, 0.5 mm Sonic extract cells Experiment 2c Sucrose-Tris-EDTA H20 Sonic extract cells S. marcescens Experiment lb Sucrose-Tris-EDTA H20 Mg+, 0.5 mm Sonic extract cells Experiment 2e H20 Sonic extract cells TABLE 5. Osmotic shock of Citrobacter and Serratiaa E-4 C '-Nucleotidase unitslg 160 2,080 1,800 2, ,000 3, ,000 1,930 1,950 unitslg Cyclic phosphodiesterase Ribonuclease unilsig 140 1, , Inorganic pyrophosphatase unitslg 120 1, , , , ,500 A 260 (total) Protein mg/ml Viability a Cells of Citrobacter freundii or Serratia marcescens were grown to either mid-exponential phase or stationary phase in the high-phosphate medium with glycerol. Cells were subjected to osmotic shock by the standard procedures. Data are given as total units of enzyme released. Blank space indicates no assay was performed. bexponential-phase cells. c Stationary-phase cells. Proteus and Providencia. A number of strains of Proteus mirabilis, Proteus vulgaris, and Providencia were subjected to osmotic shock. With a variety of methods in both stationary and exponential cells there was no release of 5'- nucleotidase, cyclic phosphodiesterase, nor the acid phenyl phosphatase. Preliminary experiments show that the 5'-nucleotidase and 3' nucleotidase of Proteus strains have the same characteristics and molecular size as the E. coli, Shigella, and Enterobacter enzymes. Low Mg+, Ca++ content of the growth medium and 1 mm EDTA could not release these enzymes. From 2 to 4% of the total soluble protein was released by osmotic shock and yielded a number of faint bands on application of the concentrated shock

8 VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1941 TABLE 6. Osmotic shock of Enterobacter aerogenesa Fraction Temp 5'-Nucleotidase Cyclic phos- Acid phenyl- Inorganic pyro- Viability phodiesterase phosphatase phosphatase c Experiment Ib Sucrose-Tris-EDTA H Mg Mg Experiment 2c Sucrose-Tris-EDTA H Experiment 3d H Mg aenterobacter aerogenes cells were grown in the specified media and subjected to osmotic shock by the two standard methods. Data are given as per cent of enzyme activity released. Blank spaces indicate assays were not performed. bexponential-phase cells, phosphate medium. c Stationary-phase cells, phosphate medium. d Stationary-phase cells, Penassay broth. fluid to acrylamide disc gel electrophoresis and subsequent straining with amido black. No enzymatic activity has yet been defined. Growth of cells after osmotic shock. Nossal and Heppel (34) showed that exponential-phase E. coli subjected to osmotic shock showed a marked lag in resumption of growth when Mg++ was not used in the shock medium. Figure 2 shows that the same situation occurs in Shigella sonnei, Citrobacter freundii, and Serratia marcescens. Attempts to abolish the water lag of regrowth by use of NaCl, ZnCI2, or CoCl2 were unsuccessful. Addition of these salts to MgCl2 did not cause a further decrease of the lag period. Part of this lag is undoubtedly due to the fact that a significant number of cells is irrevocably damaged by the EDTA treatment and the cold shock. Lysis of these cells accounts for the fall in OD seen in the first 40 min of incubation. Part of this lag in growth is thought to be due to the release of transport proteins. Proteus mirabilis, which does release its acid-soluble nucleotide pool but none of the degradative enzymes, shows no lag on cold water shock (Fig. 2). The situation with Enterobacter aerogenes io less clear (Fig. 3). Cells exposed to osmotic shock in the absence of prior exposure to EDTA also showed a lag that was overcome by MgC92. This suggests that the osmotic transition to which the Enterobacter has been exposed has damaged its permeability control mechanisms. This is under investigation. Growth in the lag period. Heppel had reported (15) that E. coli was extremely sensitive to harmful agents during the lag period. We were able to confirm this only partially in Shigella sonnet (Table 2) or Enterobacter-aerogenes. After osmotic shock with 0.5 mm MgC12, 20 jug/ml of pancreatic ribonuclease, 10,ug/ml of pancreatic deoxyribonuclease, and 25 jig/ml of lysozyme were added separately to the shocked cells. These cells showed only a slightly greater lag than the control MgC12 shocked cells. But plate survival was decreased by 25 to 50%. These results suggest to us that, although all of the cells release their enzymes, the increased permeability is a damage phenomenon occurring in only a fraction of the population. Studies are in progress to answer this question. General observations on osmotic shock. Although we routinely used inorganic pyrophosphatase as our control enzyme to measure lysis, other enzymes were assayed to check this: glutamic dehydrogenase, adenosine deaminase, #- galactosidase, ribonuclease II, and leucine amino peptidase. We attempted to release the acid hemolysins (41) of several strains of E. coli, but none was consistently released. None of the chloramphenicol acetylating enzyme (39) of RTF strains was released. When we studied the effect of osmotic shock on altering the typespecific agglutination of Salmonella typhimurium, we noted a decreased agglutination compared to controls. However, this was only a qualitative estimation. It would agree with Buttin and Kornberg's (4) suggestion that only a part of the population is really affected by the EDTA-Tris treatment. In agreement with results seen with E. coli (30), Shigella sonnei, Enterobacter-aerogenes, and Ser-

9 1942 NEU AND CHOU J. BACrERIOL. <~~~~~~~~~~~~~~~M 0~~~~~~~~~~~~~~~~~~~~~~~~~~~O -_" _~~~~~~~~~~~~~ - j0_. _ 0.2 X-11 HIO -~~~~~~~~~~~~~~ Proteus Serratia 0.Mg* Mg_. _. 0I,/ / $u 9b lio ISo nso 210 Nkm es, FIG. 2. Growth of organisms after osmotic shock. Shigella sonnei, Citrobacter, Proteus mirabilis, and Serratia marcescens were grown to a density of 5 X I& cells per ml in the high-phosphate, glycerol medium. They were harvested, washed with 0.01 M Tris (ph 7.3)-0.03 M NaCI at 3 C, and then suspended in 20% sucrose-0.03 M Tris (ph 7.3)-0.1 mm EDTA. After 5 min at 23 C, the cells were centrifuged and the pellets resuspended in 0.5 mm MgCl2 at 3 C. After 10 min, a cell sample was 25-fold diluted into Penassay broth at 37 C and placed on a rotary shaker. A control sample of cells was used which had not been exposed to sucrose or EDTA. Optical density at 600 my. was followed in a Beckman DU spectrophotometer. ratia marcescens failed to release their ribonuclease I when subjected to osmotic shock in the stationary phase of growth. However, they did release 40 to 70% during osmotic shock in the exponential phase of growth, as occurs with spheroplasts (27). DIscussIoN It is apparent from this work that the process of osmotic shock by which enzymes are selectively released from E. coli (30, 34) has application to the other members of the Enterobacteriaceae, except for Proteus strains. The group of enzymes studied in this paper are all degradative and concerned with phosphate, nucleotide, and sugar degradation. From the acrylamide electrophoresis studies, it is apparent that other proteins are released as well. As in the case of E. coli, the acid phenyl phosphatase, 5'-nucleotidase, acid hexose phosphatase, and cyclic phosphodiesterase are not extracellular enzymes, because they are not released into the medium during growth of any of the bacteria studied. Osmotic shock of stationary-phase cells of Shigella, Enterobacter, Citrobacter, or Serratia strains grown under a variety of conditions permits the release of these enzymes with 1 mm EDTA and excellent survival of the organisms. Salmonella, however, shows great variability, depending on both the strain and growth medium. Exponential-phase cells of all organisms are more sensitive to osmotic shock. Enterobacter strains showed particular instability, even with 5 X 10-5 M EDTA and shocking with 1 mm MgC12. In general, osmotic shock of exponential-phase cells was more likely to result in irreparable damage to the cells. In all organisms, the release of enzymes was essentially complete within 2 min of contact of sucrose-treated cells with the shock medium. Since this occurred in cases in which only 50% of a certain activity was released, attempts are under way to determine if only half a population of cells is affected, or if there are two locations of an enzyme in some organisms. Preliminary experiments with Enterobacter strains have not shown any differences in regard to

10 VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1943 OD600 "GM FIG. 3. Growth of Enterobacter-aerogenes after osmotic shock. Enterobacter-aerogenes were grown to midexponential phase, harvested, and washed with 0.03 M NaCI-0.01 M Tris (ph 7.3) at 3 C. They were resuspended (I g/80 ml) in 20% sucrose-0.03 M Tris (ph 7.3) at 23 C. One sample was made 0.1 mm with EDTA and the cell; were gently agitated for 10 min. The cells were removed by centrifugation and were resuspended in 0.5 mm MgCl2, 0.1 mm MgC12, or H20 at 3 C. After 10 min. samples were removed and 25-fold diluted in Penassay broth at 37 C on a rotary shaker. Change in optical density (OD)600 was followed. Control cells had no contact with sucrose or EDTA. properties or physical characteristics of enzymes released and the fraction retained. We have consistently referred to these enzymes as surface enzymes. However, the evidence to date (29, 30, 33, 34) is still circumstantial. A recent review summarizes much of the current data (15). The rapid release, the electron microscopic evidence (7), and the ability of cells to grow on 5'-adenosine monophosphate (33) all speak for location in the "pericytoplasmic space" (25). There are, however, a number of objections to this. EDTA alone does not release the enzymes, although it makes the cell permeable to nucleotides (31), actinomycin (19), and puromycin (38). It is clear that EDTA has released about 50% of the lipopolysaccharide layer of the cell wall (1, 20). Early exponential-phase cells treated with lysozyme fail to release the enzymes into the sucrose supporting medium. Penicillin protoplasts also do not release the enzymes (33). The argument that these enzymes leak out because of size factors is invalid, because both the alkaline phosphatase (78,000) and 5'-nucleotidase (53,000) are smaller or of the same size as the Houm galactoside transacetylase and chloramphenicol acetylase. Recent studies on the role of cations in the structure of cell walls (1) and the work on the ultrastructure of lysozyme and EDTA-lysozyme spheroplasts (3) suggests an explanation. The chelation of divalent cations probably conformationally alters the structural organization of lipopolysaccharide and lipoprotein components of both cell wall and cytoplasmic membrane. Lysozyme-treated cells are sensitive to lysis, but, as such, retain their rod type structure (3), whereas, at the moment EDTA strikes the lysozyme-treated cell, it alters its shape to become a spheroplast. These facts suggest to us that these groups of enzymes are probably loosely bound to the cytoplasmic membrane through the mediation of the divalent cations. Through the use of electron microscopic and antibody studies utilizing penicillin protoplasts, some of these points may be solved (Neu and Nisonson, in progress). The ability to use NaCl in place of sucrose should aid in the identification of constituents of the cell wall which are released in osmotic shock

11 1944 NEU AND CHOU J. BACTERIOL. Hardy and Kurland (13) have concluded that no enzymes are convincingly a part of the ribosomal structure. Our previous studies (21) showed that ribonuclease I is fortuitously bound to the 30S ribosomes. These studies show that the acid phenyl phosphatase (42) also can be quantitatively released. Thus, it appears that the association of these enzymes with ribosomes has been due to part of the preparation of cell extracts. Recent studies from a number of laboratories (1, 18, 35, 36) have shown that a number of transport proteins are released from E. coli and Salmonella cells by osmotic shock. Part of the lag period in regrowth of shocked cells seems to be due to this. However, part may also be due to disorganization in the cytoplasmic membrane in the absence of divalent cations. This has been suggested for the effect of EDTA on Pseudomonas (12) and Alicaligenesfecalis (11). It will be interesting to see if similar proteins are released from these other bacteria. Proteus and Providencia strains release their acid-soluble nucleotide pool (31) and some as yet unidentified proteins, but no 5'-nucleotidase or 3'-nucleotidase. These organisms also show no lag period after osmotic shock. Studies are underway to clarify the differences between these organisms and other Enterobacteriaceae, which must reside in cell wall differences. The studies of Weibull et al. (45) do not suggest that the chemical composition of Proteus cell walls is different from other Enterobacteriaceae. These observations concerning the general localization of this group of degradative enzymes in Enterobacteriaceae reinforces our previous observations (29) that this system is analogous to the lysosome system of mammalian cells. It also adds evidence to our suggestion that this group of enzymes exists bound to the cytoplasmic membrane of gram-negative cells, and in gram-positive cells, with their different cell wall, these enzymes escape as exoenzymes. AcKNowLEDmENwr This work was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. LiTERAruRE Cnim 1. ANRAxu, Y The reduction and restoration of galactose transport in osmotically shocked cells of Escherichia coli. J. Biol. Chem. 242: ASBELL, M. A., AmD R. G. EAGON Role of multivalent cations in the organization, structure, and assembly of the cell wall of Pseudomonas aeruginosa. J. Bacteriol. 92: BIRDSELL, D. C., AmN E. H. COTA-ROBLES Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme spheroplasts of Escherichia coli. J. Bacteriol. 93: BumN, G., AND A. KORNBERG Enzymatic synthesis of deoxyribonucleic acid. J. Biol. Chem. 241: CORDONNER, C., AND G. BERNARDL Localization of E. coli endonuclease I. Biochem. Biophys. Res. Commun. 20: Cowm, D. B., AND MCCLuRE, F. T Metabolic pools and the synthesis of macromolecules Biochim. Biophys. Acta 31: DoNE, J., C. D. SHOREY, J. P. LOKE, AND J. K PoLLAK The cytochemical localization of alkaline phosphatase in Escherichia coli at the electron-microscopic level. Biochem. J. 96:27c- 28c. 8. EDwARDS, P. R., AND W. H. EWING Identification of Enterobacteriaceae. Burgess Publishing Co., Minneapolis, Minn. 9. FisKE, C. H., AND Y. SUBBAROW The colorimetric detennination of phosphorus. J. Biol. Chem. 66: GLASER, L., A. MELO, AND R. PAuL Uridine diphosphate sugar hydrolase. J. Biol. Chem. 242: GRAY, G. W., Arm S. G. WILKSNoN The effect of ethylenediaminetetraacetic acid on the cell walls of some gram-negative bacteria. J. Gen. Microbiol. 39: GRAY, G. W., AD S. G. WILKINSON The action of ethylenediaminetetra-acetic acid on Pseudomonas aeruginosa. J. Appl. Bacteriol. 28: HARDY, S. J. S., AND C. G. KuRLAmN The relationship between poly A polymerase and ribosomes. Biochemistry 11: HEGARTY, C. P., AND 0. B. WEEKS Sensitivity of Escherichia coli to cold-shock during the logarithmic growth phase. J. Bacteriol. 39: HEPPEL, L. A Selective release of enzymes from bacteria. Science 156: JossE, J Constitutive inorganic pyrophosphatase of Escherichia coli. J. Biol. Chem. 241: KAMMEN, H Thymine metabolism in Escherichia coli. Biochim. Biophys. Acta 134: KUNDIG, W., F. D. KUNDIG, B. ANDERSON, AN S. RosEmAN Restoration of active transport of glycosides in Escherichia coli by a component of a phosphotransferase system. J. Biol. Chem. 241: LEIVE, L A non-specific increase in permeability in Escherichia coli produced by EDTA. Proc. Natl. Acad. Sci. U.S. 53: LEIVE, L Release of lipopolysaccharide by EDTA treatment of Escherichia coli. Biochem. Biophys. Res. Commun. 21: LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:

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