Release of Surface Enzymes in Enterobacteriaceae by Osmotic Shock HAROLD C

Home , Shigella sonnei

JOURNAL OF BACTERIOLOGY, Dec. 1967, p. 1934-1945 Voi. 94, No. 6 Copyright ©) 1967 American Society for Microbiology Printed in U.S.A. Release of Surface Enzymes in Enterobacteriaceae by Osmotic Shock HAROLD C. NEU1 ANm JAMES CHOU Department ofMedicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 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 Entero- bacteriaceae 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 degrada- with amino acid transport (36), glycoside trans- tive enzymes are released from Escherichia coli port (18), galactose transport (1) in E. coli, and (30, 34) when cells are osmotically shocked. The sulfate transport (35) in Salmonella typhimurium. procedure consists of incubation of the cells in a The exposure to EDTA has rendered cells byperosmolar solution of sucrose and ethylene- permeable to the exit of the acid-soluble nucleo- diaminetetraacetic acid (EDTA) followed by tide pool and the entry of substances such as sedimentation of the cells and exposure to either actinomycin (19, 31), puromycin (38), and cold water or a dilute magnesium solution. The nucleotides (4). The change would appear to be enzymes which have been characterized thus far related to the release of lipopolysaccharide com- are alkaline phosphatase (22, 29, 34), 5'-nucleo- ponent of the cell wall (2, 20). However, the tidase (30, 33), diphosphate sugar hydrolase (10), alteration of permeability appears to be separate acid hexose phosphatase (30, 34), acid phenyl from the change which occurs in osmotic shock, phosphatase (32), cyclic phosphodiesterase (30, since there is no enzyme release when cells are 34), a ribonucleic acid (RNA)-inhibited deoxy- rendered permeable by EDTA and tris(hydroxy- ribonuclease (5, 29, 34), thymidine phosphorylase methyl)aminomethane (Tris), as shown by Neu (17), and adenosine diphosphate (ADP)-glucose et al. (30, 31). pyrophosphatase (23). A large number ofenzymes In the present investigation, we have carried remain within the cells after osmotic shock, in out a detailed examination of the process of spite of a loss of 5 to 10% of the total cellular osmotic shock for a number of the members ofthe protein (30, 34) and all of the acid-soluble nucleo- Enterobacteriaceae. We have been concerned with tide pool (31). The cells are viable. both stationary and exponential phase organisms. Work in a number of laboratories has shown The organisms studied belonged to the following that protein functions related to transport of ma- groups: Shigella, Klebsiella-Enterobacter, Sal- terials from the environment to within the proto- monella, Citrobacter, Serratia, Proteus, and plasmic membrane of the cell may also be released Providencia. Osmotic shock causes the release of by osmotic shock. Studies have been concerned a group of degradative enzymes from all or- 1Career scientist of the New York Health Research gaisms except the members of Proteus and Council. Providencia groups. This paper details the condi- 1934 VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1935 tions for osmotic shock in each organism, as well Viability was determined on serial dilutions, made as studies on regrowth fo'lowing osmotic shock. in Penassay broth, which were plated on either Pen- assay Agar or Nutrient Agar containing 0.5% NaCI. MATERIALS AND METHODS Enzyme assays. Previously published methods were Materials. Bis(p-nitrophenyl)phosphate, p-nitro- used for ribonuclease (28), deoxyribonuclease (27), phenyl phosphate, and nucleotides were obtained ,3-galactosidase (22), alkaline phosphatase (29), acid from commercial sources. E. coli-soluble RNA was phenyl phosphatase (33), 5'-nucleotidase (33), uridine purchased from General Biochemicals Corp., Chagrin diphosphate glucose pyrophosphatase (33), acid Falls, Ohio. Penassay broth was purchased from hexose phosphatase (30), and cyclic phosphodiesterase Difco. Crystalline lysozyme (muramidase) was pur- (30). Adenosine triphosphatase was measured in chased from Worthington Biochemical Corp., Free- Salmonella by incubating (0.1 ml) 2 mm adenosine hold, N.J. triphosphate, 100 mm Tris-HCl, pH 7.4, and 10 mM Organisms. Isolates from the diagnostic laboratory MgCl2 at 37 C for 20 min. The reaction was halted of the Presbyterian Hospital, New York, N.Y., were with 1 N H2SO4 and the phosphate was determined by used. Identification was based on the methods of a modification of the Fiske-SubbaRow method (9). Edwards and Ewing (8). Salmonella typhimurium LT2, Inorganic pyrophosphatase was determined by in- Ade 97, and Leu 126 were a generous gift of Dr. cubating (0.3 ml) 3 mm Na4PPi, 0.4 mt MgCI2, and Rudner. E. coli strains were those previously described 60 mm Tris-HCI, pH 912. The reaction was stopped (30). after 15 min at 37 C with 1 N H2SO4 and the phosphate Media and culture conditions. Stock cultures were was determined by a modified Fiske-SubbaRow maintained on Penassay slants. Low- and high-phos- method (9). A unit of enzyme activity is the amount phate media were used. The high-phosphate medium of enzyme that will hydrolyze 10 umoles of PPi in 1 hr. Protein was determined according to Lowry et al. (21). contained 0.04 M K2PO4, 0.022 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). RESULTS The pH was adjusted to 7.1 with NaOH. The low- phosphate medium contained 0.12 M Tris, 0.08 M Conditions for osmotic shock. The process of NaCl, 0.02 M KCl, 3 mm Na2SO4, 1 mM MgCI2, osmotic shock was affected by the media and 0.1 mM CaCl2, and 0.5% Bacto-peptone, with the pH culture conditions, as discussed in the following. adjusted to 7.4. The Tris medium was supplemented medium. Previous studies Neu and with potassium phosphate to make it 0.01 M, when Growth by noted. The content of magnesium and calcium was Heppel (30) and Nossal and Heppel (34) had altered in both media in some cases, as specified in the utilized synthetic media for the growth of cells for particular experiment. Carbon content of the media osmotic shock. Escherichia coli, Shigella sonnei, was 0.5% glucose, 0.5% glycerol, or 0.3% sodium Enterobacter-aerogenes, Salmonella typhimurium, succinate. Penassay broth (Difco) was also used. Citrobacter freundii, and Serratia marcescens Organisms were incubated at 35 C on a rapid rotary were all susceptible to osmotic shock after growth shaker, and growth was followed by change in optical in the two standard synthetic media (high and density (OD)o00 in a DU spectrophotometer (Beck- low phosphate content), Penassay Broth (Difco), man Instruments, Inc., Fullerton, Calif.). Procedure for osmotic shock. Stationary-phase cells Trypticase Soy Broth (BBL), and the standard were harvested at 16 hr after a 1% inoculum into the high- and low-phosphate media from which specified medium. Exponential cells were harvested peptone was omitted. However, the release of at an OD600 of 0.3 with a cell density of 5 X 108 cells enzymes from cells grown in Penassay Broth per milliliter. In both cases, the cells were washed with (Difco Antibiotic Medium 3) or Trypticase Soy either 0.01 M Tris-HCl (pH 7.3)-0.03 M NaCl or 0.85% Broth was 10 to 15% below that of cells grown in NaCl at 3 C. A sample at this stage was removed for minimal medium. Exponential-phase cells of preparation of a sonic extract. Stationary-phase cells Shigella, Enterobacter, and Citrobacter grown in (16-hr culture) were suspended in 20% sucrose-0.03 M the medium were not less stable than Tris-HCl, pH 7.3, at 21 C at a ratio of 1 g (wet weight) phosphate to 80 ml of sucrose-Tris. EDTA was added to a con- the Tris-HCI grown cells when subjected to the centration of 1 mm, and, after 2 to 10 min of mixing, Tris-HCl-sucrose osmotic swelling medium. The the cells were removed by centrifugation at 0 C. magnesium content of the growth medium had a The pellet of cells was resuspended in water at 3 C and significant effect on the release of enzymes of mixed for 5 to 10 min. The cells were removed by organisms in the exponential phase of growth, centrifugation. but not on stationary-phase cells. When the Mg++ Exponential cells were also suspended in 20% content was lowered to 0.1 or 0.01 mm, the sucrose-0.03 M Tris-HCl, pH 7.3; however, the EDTA enzymes were released into the sucrose-Tris- concentration was 0.1 mM and the cells were resus- EDTA rather than into the osmotic shock fluid. pended in 0.5 mM MgCl2 at 3 C. Sonic extracts were prepared by use of a Branson Also, there was gross damage to ribosomal RNA Sonifer model LS75, with 2 min of sonic disintegration and viability fell to 25%. This was particularly at 0 C using 15-sec bursts. Spheroplasts were prepared true of Salmonella and Enterobacter strains. as described (27). Age and concentration of cells. Stationary-phase 1936 NEU AND CHOU J. BAcrERIOL. cells of the organisms studied were similar to E. render E. coli permeable to release of the acid- coli. Exponential cells, particularly Enterobacter soluble nucleotide pool (31). and Shigella strains, showed partial lysis during Effect of replacement oJ sucrose. It had been osmotic shock, even with 1 mM MgCl2, if har- shown, for stationary-phase E. coli, that reduction vested in very early exponential phase. We of the concentration of sucrose below 12% caused routinely harvested cells at a cell density of about a significant decrease in the release of enzymes 5 x 108 per ml. At 5 X 107 and 108 cells per milli- (30), and that sucrose could be replaced by 0.5 M liter, internal enzyme leakage reached 30% and NaCl in exponential cells. As Table 1 shows, viability fell to 20%. Washed cells were centri- sucrose can be replaced by 0.5 M glucose, 0.5 M fuged in an SS34 head of a RC2-B Sorvall centri- NaCl, and 0.5 M Tris, but not by 0.5 M glycerol. fuge at 16,000 rev/min for 5 min, and 1 g (wet However, survival in the case of glucose and Tris weight) of cells was suspended in 80 ml of sucrose- was below 50%. The length of contact in the Tris-EDTA. When the concentration of stationary sucrose, NaCl, or glucose could be shortened to 2 cells was increased to 2 g per 80 ml, the release of min without effect. Prolongation of the contact of enzymes was decreased by less than 10%; how- cells in sucrose-Tris-EDTA beyond 10 min at 23 ever, at 3 and 4 g per 80 ml, release was cut as C merely causes degradation of RNA (9). The much as 30%: Exponential cells for all groups failure of glycerol as an agent to allow osmotic studied showed best results with no more than 1.5 shock is consistent with its penetration into cells g per 80 ml. as shown by Mitchell and Moyle (26). Wash system. The system used to wash the cells Effect of chelation. As was originally shown after harvest did not alter the release of enzymes with stationary-phase E. coli (30), all of the bac- or subsequent growth and viability of Escherichia teria studied required the presence of a chelating coli, Shigella sonnei, Enterobacter aerogenes, or agent in the initial phase. EDTA showed the Serratia marcescens. The following were used optimal activity at a concentration of 1 mm for with identical results: 0.85% NaCl, 0.01 M Tris- Shigella, Klebsiella-Enterobacter, Salmonella, Ser- HCI (pH 7.3)-0.03 M NaCI, 0.03 M Tris-HCI (pH ratia, and Citrobacter in the stationary phase. 7.3), and 0.5 M sucrose-0.03 M Tris-HCI (pH 7.8). Exponential phase cells of all of these strains Cells washed with 0.5 M sucrose-0.03 M Tris-HCl (Table 3-6) showed lysis at concentrations above did release a significant part of their surface en- 0.1 mM. Cells were treated with Dowex-50 [H+], zymes into the sucrose-Tris-EDTA in contrast to a cation exchange resin (37), for a brief period the other systems. The temperature of the wash and were then suspended in 20% sucrose and system did not affect the results when at 0 C or 21 lysozyme, providing cells that lysed on resuspen- C. More than two washes with 10 ml/g were not sion in water. These cells failed to release any necessary. enzymes into the sucrose-lysozyme medium. Simi- Effect of buffer system. Previous studies from larly, cells harvested in very early exponential our laboratory had shown that both buffer sys- phase (E. coli K-12 or Shigella sonnei) and treated tem and pH affected the altered permeability with 0.5 M sucrose, 0.03 M Tris, pH 8.0, and achieved by EDTA (31). Tris-HCI appeared to be lysozyme 20 ,ug/ml lysed on dilution in water or the best buffer system for both release and in 0.1 mM MgC92, but enzymes were not released viability. The pH range used was 7.2 to 8.0 with into the sucrose. the pH measured at 21 C, the temperature used Osmotic shock ofShigella. Shigella strains were for the osmotic swelling. Tris-maleate, 0.03 M, in a more fragile in regard to osmotic shock than any pH range of 5.1 to 7.7 released only 60% of the of the wide variety of E. coli strains tested (30). 5'-nucleotidase and 45% of the acid phenyl phos- Table 2 shows that stationary-phase cells grown in phatase of both E. coli Hfr H and Enterobacter the standard high-phosphate medium required aerogenes. Hepes, pH 7.1, released only 36%. sucrose or NaCl as the osmotic swelling medium, Glycylglycine, in a pH range of 5.7 to 7.7, re- and that the osmotic transition alone is inade- leased only 25% and NH4HCO3, pH 8.4, released quate to release the surface enzymes, because 45%, but viability was greatly lowered. Although both EDTA and Tris are needed. Stationary-phase chelation by EDTA is enhanced at a more alkaline cells require a greater osmotic transition than pH, in the case of glycylglycine and NH4HCO3 exponential cells. The inorganic pyrophosphatase, improved release of enzymes did not occur at which had been shown by Neu and Heppel (29) more alkaline values. The explanation of the and Josse (16) to be an intracellular enzyme in E. lower release of enzymes by Tris-maleate buffer coli, was used as the control enzyme for lysis, compared with Tris-HCl is unknown at present. since it is unaffected by any of the constituents of Phosphate buffer cannot be used to replace the the shock medium and is an extremely sensitive Tris in osmotic shock, since it cannot be used to assay. Simmonds (40) has recently pointed out VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1937 TABLE 1. Effect oJ various media on osmotic shock in Escherichia coli and Shigeila soinnei-

5'-Nucleo- Acid phenyl- Cyclic phos- Acid hexose Inorganic Method phodies- pyrophos- A 26) Protein tidase phosphatase terase phosphatase phatase

units/ml units/ml units/ml units/ml units/ml mg/ml E. coli Sucrose 69 11.2 26 8.6 8 0.730 0.18 Glucose 77 11.7 30 10.4 5 0.587 0.21 Glycerol S 0.3 2 0.8 6 0.290 0.08 NaCl 56 10.3 21 7.5 5 0.720 0.17 Tris 52 14 31 13 10 0.880 0.27 Soniic extract 40 11.5 28.1 12.2 445 1.6 Shigella sonnei Sucrose 36 7.2 12.6 22 9 0.427 0.15 Glucose 45 19 10.7 27.7 4 0.364 0.15 Glycerol 1 0.3 0.2 0.4 2 0.137 0.05 NaCI 41 24 19.2 30 7 0.405 0.16 Tris 47 23 16.6 27 6 0.582 0.17 Sonic extract 23 23 16 27 425 1.7 a Stationary phase E. coli K 10, R 6 and Shigella sonnei 11-23 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 0 0 32 27 16 0.5 None 1 0 1.8 12 27 15 0.5 0.03 None 32 6.4 120 24 18 0.5 0.03 1 322 268 716 22 22 0.25 0.03 1 90 100 218 20 26 None 0.03 1 0 0 10 20 25 NaClb 0.03 1 464 368 800 24 23 Sonic extract, cells 243 231 800 3,088 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 the cells are more stable, but 0.5 mM MgCl2 is reference internal enzyme, unless the assay is needed to stabilize. In most cases, Shigella sonnei modified. released more Amso material during osmotic shock Table 3 summarizes a number of experiments than could be accounted for by the expected re- with Shigella sonnei in exponential and stationary lease of the acid-soluble nucleotide pool. Warm phase. Cells in early exponential phase show osmotic shock shows significantly less release of leakage of internal enzymes even when shocked the surface enzymes but allows more degradation with 0.1 mM MgC12. At mid-exponential phase, of the RNA. Addition of ribonuclease or deoxy- 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'-Nucle-otidase phospha-hexosa phodies-phos- phenyl-phos- phos-Ebo-SurivalApePrtinSrvvlA 260 Protein pnodda tase terase p atase phatase

C units/g unils/g unitsig units/g units/g total mg/g % min Experiment 1l Sucrose-Tris-EDTA 21 48 0 15 0.05 90 Mg++, 0.5 mM 3 550 90 234 190 24 36 0.13 84 Mg++, 0.5 mM + RNase 3 676 120 241 185 96 45 0.10 45 Mg-, 0.5 mM + DNase 3 500 100 281 170 64 36 0.09 47 Ca++, 0.5 mm 3 610 80 271 160 88 35 0.08 50 Sonic extract cells 800 400 338 440 2,800 1.4 Experiment 2b Sucrose-Tris-EDTA 21 160 101 64 22 0.08 H20 3 960 320 1,600 420 0.38 1 >200 Mg++, 0.1 mM 3 964 320 800 177 0.35 8.3 150 Mg' , 0.5 mM 3 744 316 640 160 0.20 76 90 Sonic extract cells 400 192 2,820 1 .27 60 Experiment 3b Sucrose-Tris-EDTA 21 88 23 88 22 24 0.10 Mg++), 0.5 mM 21 624 100 274 610 144 0.21 11 MgH+, 0.5 mM 3 1,648 230 374 450 112 0.18 15 Sucrose-Tris-EDTA 3 200 11 73 30 25 0.11 Mg4, 0.5 mM 3 1,424 147 383 330 80 0.14 25 Sonic extract cells 848 266 394 2,432 51 1.17 Experiment 4e Sucrose-Tris-EDTA 21 100 17 8 22 28 H20 3 3,176 354 352 90 67 76 Sonic extract cells 3,226 430 401 2,800 Experiment 5d Sucrose-Tris-EDTA 21 190 0 40 0.09 H20 3 870 85 312 290 400 88 0.12 35 Sonic extract cells 550 82 330 446 4,160 2.36 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 0.32. Cells of experiment 3 were harvested at an OD60, of 0.27. The concen- trati9r1 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 Cells in exponential phase were suspended in 0.5 a significant decrease in viability, indicating that M sucrose, 0.03 M Tris-HCI, pH 7.3, 0.1 mM EDTA permeability to protein molecules is altered. CaCk2 (1 g/80 ml) at 21 C for 5 min. The cells were was able to replace the Mg++ in prevention of removed by centrifugation and then rapidly re- nucleotide loss, as shown by the stable A2w0 values, suspended in 0.5 mm MgCl2 at 3 C. One-ml but did not increase survival to normal. CoCl2 and portions were placed on 0.45-Iu (pore size) filters ZnCl2 were not able to replace the MgC12. Addi- (Millipore Corp., Bedford, Mass.) and the filtrate tion of 0.1 mm CaCd2 to 0.5 mM MgCl2 failed to was collected and assayed. Immediately, 5'-nu- increase survival, to decrease nucleotide release, cleotidase, cyclic phosphodiesterase, and acid or to shorten furtherthe lag period before loga- hexose phosphatase were released, but no sig- rithmic growth is resumed. nificant amount of inorganic pyrophosphatase The rate of release of enzymes from Shigella was released. sonnei was found to be essentially complete by 2 All members of the genus Shigella that were to 3 min after cold 0.5 mm MgCI2 shock (Fig. 1). tested contained 5'-nucleotidase (5'-nucleotidase VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1939 nor was the acid hexose phosphatase. In general, only 50% of the cyclic phosphodiesterase activity 5 I0. 5.'nuwleotidose and adenosine triphosphatase activity were released. A number of other Salmonella strains were studied. S. montevideo, S. derby, S. oranienberg, E / and S. manhattan all released 50 to 70% of their 61 ; cyclic phosphodiesterase, 5'-nucleotidase, and A IL /25 acid phenyl phosphatase activities with less than I ./Cyc phb@sptioesterase = 10% of their inorganic pyrophosphatase released. These Salmonella strains showed poor conversion ,; Acid Homose to spheroplasts. Phosp*losbe Osmotic shock of Citrobacter freundii and Ser- ratia marcescens. Both of these organisms re- 5 - --'-* I---50 leased enzymes after osmotic shock (Table 5) in Minutes a manner analogous to E. coli. The fact that Ser- ratia released enzymes by osmotic shock is inter- F] IG. 1. Rate of release of 5-nucleotidase, cyclic esting because, as Repaske (37) showed, it does phos,phodiesterase, and acid hexose phosphatase during osmcitic shock ofShigella sonnei. Shigella sonnei grown not undergo sigmficant lysis by means of the to e;xponential phase were subjected to osmotic shock method. This again points out by 0..5 mM MgCI2 and were poured on Milliporefilters EDTA-iysozymethe 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 chill- activ,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 phospha- ucleotidase; Neu, in preparation), and acid tase, 5'-nucleotidase, and cyclic phosphodi- hexc)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 expo- 0.smotic shock in Salmonella. Pardee has shown nential-phase Enterobacter aerogenes (Table 6), 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 observa- acid phenyl phosphatase, and adenosine triphos- tion of Cowie and McClure (6) that the amino phat:ase. Ribonuclease was not released by shock acid pool can be so removed from E. coli. 1940 NEU AND CHOU J. BACTERiOL.

TABLE 4. Release of enzymes from Salmonella strains by osmotic shocka

Cyclic Acid Acid Adenosine Growth stage 5'-Nucle-otidace phospho- hexose phenyl- rienospte Strain diesterase phosphatase phosphatase

S. typhimurium, Stationary 46 84 11 S. typhimurium, LT2 Log 64 87 68 S. typhimurium, Ade 97 Log 103 100 63 S. typhimurium, Leu 126 Log 61 83 68 S. montevideo Stationary 90 50 15 S. derby Stationary 30 45 S. heidelberg Stationary 68 27 42 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. TABLE 5. Osmotic shock of Citrobacter and Serratiaa

5'-Nucle- Cyclic Ribonu- Inorganic A 260 Expt otidase phospho- clease pyro- (total) Protein Viability E-4 diesterase phosphatase C. unitslg unitslg unilsig unitslg mg/ml C. freundii Experiment lb Sucrose-Tris-EDTA 160 16 140 120 56 0.09 H20 3 2,080 240 1,800 1,080 244 0.35 24 Mg+, 0.5 mM 3 1,800 232 950 340 124 0.16 82 Sonic extract cells 2,530 208 2,950 3,000 1.58 Experiment 2c Sucrose-Tris-EDTA 200 16 11 100 H20 3 3,000 400 40 38 104 Sonic extract cells 3,120 450 2,020 S. marcescens Experiment lb Sucrose-Tris-EDTA 80 300 48 25 0.08 H20 3 520 182 0 470 77 0.22 6 Mg+, 0.5 mM 3 400 120 0 80 33 0.04 100 Sonic extract cells 1,000 240 545 2,580 Experiment 2e H20 3 1,930 248 45 0.16 100 Sonic extract cells 1,950 383 2,500 1.75 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 nucleotidase of Proteus strains have the same of Proteus mirabilis, Proteus vulgaris, and characteristics and molecular size as the E. coli, Providencia were subjected to osmotic shock. Shigella, and Enterobacter enzymes. Low Mg+, With a variety of methods in both stationary and Ca++ content of the growth medium and 1 mM exponential cells there was no release of 5'- EDTA could not release these enzymes. From 2 nucleotidase, cyclic phosphodiesterase, nor the to 4% of the total soluble protein was released by acid phenyl phosphatase. Preliminary experi- osmotic shock and yielded a number of faint ments show that the 5'-nucleotidase and 3'- bands on application of the concentrated shock VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1941

TABLE 6. Osmotic shock of Enterobacter aerogenesa

Acid pyro- Fraction Temp 5'-Nucleotidase phodiesteraseCyclic phos- phosphatasephenyl- Inorganicphosphatase Viability

c Experiment Ib Sucrose-Tris-EDTA 0 25 13 8.3 H20 21 51 38 17 37 Mg+ 3 20 6 2 100 Mg+ 3 27 19 13 100 Experiment 2c Sucrose-Tris-EDTA 3 0 2 H20 3 88 74 73 5 100 Experiment 3d H20 3 60 90 Mg+ 3 30 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 confirm this only partially in Shigella sonnet subsequent straining with amido black. No en- (Table 2) or Enterobacter-aerogenes. After os- zymatic activity has yet been defined. motic shock with 0.5 mm MgC12, 20 jug/ml of Growth of cells after osmotic shock. Nossal and pancreatic ribonuclease, 10 ,ug/ml of pancreatic Heppel (34) showed that exponential-phase E. deoxyribonuclease, and 25 jig/ml of lysozyme coli subjected to osmotic shock showed a marked were added separately to the shocked cells. These lag in resumption of growth when Mg++ was not cells showed only a slightly greater lag than the used in the shock medium. Figure 2 shows that control MgC12 shocked cells. But plate survival the same situation occurs in Shigella sonnei, was decreased by 25 to 50%. These results sug- Citrobacter freundii, and Serratia marcescens. gest to us that, although all of the cells release Attempts to abolish the water lag of regrowth by their enzymes, the increased permeability is a use of NaCl, ZnCI2, or CoCl2 were unsuccessful. damage phenomenon occurring in only a fraction Addition of these salts to MgCl2 did not cause a of the population. Studies are in progress to further decrease of the lag period. Part of this lag answer this question. is undoubtedly due to the fact that a significant General observations on osmotic shock. Al- number of cells is irrevocably damaged by the though we routinely used inorganic pyrophos- EDTA treatment and the cold shock. Lysis of phatase as our control enzyme to measure lysis, these cells accounts for the fall in OD seen in the other enzymes were assayed to check this: glu- first 40 min of incubation. Part of this lag in tamic dehydrogenase, adenosine deaminase, #- growth is thought to be due to the release of galactosidase, ribonuclease II, and leucine amino transport proteins. Proteus mirabilis, which does peptidase. We attempted to release the acid release its acid-soluble nucleotide pool but none hemolysins (41) of several strains of E. coli, but of the degradative enzymes, shows no lag on cold none was consistently released. None of the water shock (Fig. 2). chloramphenicol acetylating enzyme (39) of The situation with Enterobacter aerogenes io RTF strains was released. When we studied the less clear (Fig. 3). Cells exposed to osmotic shock effect of osmotic shock on altering the type- in the absence of prior exposure to EDTA also specific agglutination of Salmonella typhimurium, showed a lag that was overcome by MgC92. This we noted a decreased agglutination compared to suggests that the osmotic transition to which the controls. However, this was only a qualitative Enterobacter has been exposed has damaged its estimation. It would agree with Buttin and permeability control mechanisms. This is under Kornberg's (4) suggestion that only a part of the investigation. population is really affected by the EDTA-Tris Growth in the lag period. Heppel had reported treatment. (15) that E. coli was extremely sensitive to harm- In agreement with results seen with E. coli (30), ful agents during the lag period. We were able to Shigella sonnei, Enterobacter-aerogenes, and Ser- 1942 NEU AND CHOU J. BACrERIOL.

0~~~~~~~~~~~~~~~~~~~~~~~~~~~O _ -_" <~~~~~~~~~~~~~~~M _~~~~~~~~~~~~~ _ - j0_. _ 0.2X-11 HIO -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0.2

Proteus Serratia

0.Mg*0IMg_. _._ __

,/120 0.6

0 0

0 /0

0.4- 0.4

0.2 0.2

$u 9b0 210 30 90 lio ISo nSo 210 Nkmes, FIG. 2. Growth oforganisms 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 andplaced 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 ribo- Osmotic shock of stationary-phase cells of nuclease I when subjected to osmotic shock in the Shigella, Enterobacter, Citrobacter, or Serratia stationary phase of growth. However, they did strains grown under a variety of conditions per- release 40 to 70% during osmotic shock in the mits the release of these enzymes with 1 mM exponential phase of growth, as occurs with EDTA and excellent survival of the organisms. spheroplasts (27). Salmonella, however, shows great variability, depending on both the strain and growth me- DIscussIoN dium. Exponential-phase cells of all organisms It is apparent from this work that the process are more sensitive to osmotic shock. Enterobacter of osmotic shock by which enzymes are selectively strains showed particular instability, even with 5 released from E. coli (30, 34) has application to X 10-5 M EDTA and shocking with 1 mm MgC12. the other members of the Enterobacteriaceae, In general, osmotic shock of exponential-phase except for Proteus strains. The group of enzymes cells was more likely to result in irreparable studied in this paper are all degradative and con- damage to the cells. In all organisms, the release cerned with phosphate, nucleotide, and sugar of enzymes was essentially complete within 2 min degradation. From the acrylamide electrophoresis of contact of sucrose-treated cells with the shock studies, it is apparent that other proteins are re- medium. Since this occurred in cases in which leased as well. As in the case of E. coli, the acid only 50% of a certain activity was released, at- phenyl phosphatase, 5'-nucleotidase, acid hexose tempts are under way to determine if only half a phosphatase, and cyclic phosphodiesterase are population of cells is affected, or if there are two not extracellular enzymes, because they are not locations of an enzyme in some organisms. Pre- released into the medium during growth of any liminary experiments with Enterobacter strains of the bacteria studied. have not shown any differences in regard to VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1943

OD600

0 0 0 0

"GM Houm FIG. 3. Growth of Enterobacter-aerogenes after osmotic shock. Enterobacter-aerogenes were grown to mid- exponential 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 galactoside transacetylase and chloramphenicol released and the fraction retained. acetylase. We have consistently referred to these enzymes Recent studies on the role of cations in the as surface enzymes. However, the evidence to date structure of cell walls (1) and the work on the (29, 30, 33, 34) is still circumstantial. A recent ultrastructure of lysozyme and EDTA-lysozyme review summarizes much of the current data (15). spheroplasts (3) suggests an explanation. The The rapid release, the electron microscopic chelation of divalent cations probably confor- evidence (7), and the ability of cells to grow on mationally alters the structural organization of 5'-adenosine monophosphate (33) all speak for lipopolysaccharide and lipoprotein components location in the "pericytoplasmic space" (25). of both cell wall and cytoplasmic membrane. There are, however, a number of objections to Lysozyme-treated cells are sensitive to lysis, but, this. EDTA alone does not release the enzymes, as such, retain their rod type structure (3), although it makes the cell permeable to nucleo- whereas, at the moment EDTA strikes the tides (31), actinomycin (19), and puromycin (38). lysozyme-treated cell, it alters its shape to become It is clear that EDTA has released about 50% of a spheroplast. These facts suggest to us that these the lipopolysaccharide layer of the cell wall (1, groups of enzymes are probably loosely bound to 20). Early exponential-phase cells treated with the cytoplasmic membrane through the mediation lysozyme fail to release the enzymes into the of the divalent cations. Through the use of elec- sucrose supporting medium. Penicillin protoplasts tron microscopic and antibody studies utilizing also do not release the enzymes (33). penicillin protoplasts, some of these points may The argument that these enzymes leak out be- be solved (Neu and Nisonson, in progress). The cause of size factors is invalid, because both the ability to use NaCl in place of sucrose should aid alkaline phosphatase (78,000) and 5'-nucleotidase in the identification of constituents of the cell (53,000) are smaller or of the same size as the wall which are released in osmotic shock. 1944 NEU AND CHOU J. BACTERIOL. Hardy and Kurland (13) have concluded that ethylenediaminetetraacetate-lysozyme sphero- no enzymes are convincingly a part of the ribo- plasts of Escherichia coli. J. Bacteriol. 93:427- somal structure. Our previous studies (21) showed 437. that ribonuclease I is fortuitously bound to the 4. BumN, G., AND A. KORNBERG. 1966. Enzymatic synthesis of deoxyribonucleic acid. J. Biol. 30S ribosomes. These studies show that the acid Chem. 241:5419-5427. phenyl phosphatase (42) also can be quantita- 5. CORDONNER, C., AND G. BERNARDL 1965. Lo- tively released. Thus, it appears that the associa- calization of E. coli endonuclease I. Biochem. tion of these enzymes with ribosomes has been Biophys. Res. Commun. 20:555-559. due to part of the preparation of cell extracts. 6. Cowm, D. B., AND MCCLuRE, F. T. 1959. Meta- Recent studies from a number of laboratories bolic pools and the synthesis ofmacromolecules (1, 18, 35, 36) have shown that a number of trans- Biochim. Biophys. Acta 31:236-245. port proteins are released from E. coli and Sal- 7. DoNE, J., C. D. SHOREY, J. P. LOKE, AND J. K monella cells by osmotic shock. Part of the lag PoLLAK. 1965. The cytochemical localization of alkaline phosphatase in Escherichia coli at the period in regrowth of shocked cells seems to be electron-microscopic level. Biochem. J. 96:27c- due to this. However, part may also be due to dis- 28c. organization in the cytoplasmic membrane in the 8. EDwARDS, P. R., AND W. H. EWING. 1962. Identi- absence of divalent cations. This has been sug- fication of Enterobacteriaceae. Burgess Pub- gested for the effect of EDTA on Pseudomonas lishing Co., Minneapolis, Minn. (12) and Alicaligenesfecalis (11). It will be inter- 9. FisKE, C. H., AND Y. SUBBAROW. 1925. The esting to see if similar proteins are released from colorimetric detennination of phosphorus. J. these other bacteria. Proteus and Providencia Biol. Chem. 66:375-400. strains release their acid-soluble nucleotide pool 10. GLASER, L., A. MELO, AND R. PAuL. 1967. Uridine diphosphate sugar hydrolase. J. Biol. Chem. (31) and some as yet unidentified proteins, but 242:1944-1954. no 5'-nucleotidase or 3'-nucleotidase. These or- 11. GRAY, G. W., Arm S. G. WILKSNoN. 1965. The ganisms also show no lag period after osmotic effect of ethylenediaminetetraacetic acid on the shock. Studies are underway to clarify the differ- cell walls of some gram-negative bacteria. J. ences between these organisms and other Entero- Gen. Microbiol. 39:385-399. bacteriaceae, which must reside in cell wall differ- 12. GRAY, G. W., AD S. G. WILKINSON. 1965. The ences. The studies of Weibull et al. (45) do not action of ethylenediaminetetra-acetic acid on suggest that the chemical composition of Proteus Pseudomonas aeruginosa. J. Appl. Bacteriol. cell walls is different from other Enterobac- 28:153-164. 13. HARDY, S. J. S., AND C. G. KuRLAmN. 1966. The teriaceae. relationship between poly A polymerase and These observations concerning the general ribosomes. Biochemistry 11:3676-3684. localization of this group of degradative enzymes 14. HEGARTY, C. P., AND 0. B. WEEKS. 1940. Sensi- in Enterobacteriaceae reinforces our previous ob- tivity of Escherichia coli to cold-shock during servations (29) that this system is analogous to the logarithmic growth phase. J. Bacteriol. 39: the lysosome system of mammalian cells. It also 475-484. adds evidence to our suggestion that this group 15. HEPPEL, L. A. 1967. Selective release of enzymes of enzymes exists bound to the cytoplasmic mem- from bacteria. Science 156:1451-1455. brane of gram-negative cells, and in gram-posi- 16. JossE, J. 1966. Constitutive inorganic pyrophosphatase of Escherichia coli. J. Biol. Chem. 241: tive cells, with their different cell wall, these en- 1938-1947. zymes escape as exoenzymes. 17. KAMMEN, H. 0. 1967. Thymine metabolism in Escherichia coli. Biochim. Biophys. Acta 134: AcKNowLEDmENwr 301-311. This work was supported by Public Health Service 18. KUNDIG, W., F. D. KUNDIG, B. ANDERSON, AN grant AI-06840 from the National Institute of Allergy S. RosEmAN. 1966. Restoration of active trans- and Infectious Diseases. port of glycosides in Escherichia coli by a com- ponent of a phosphotransferase system. J. Biol. LiTERAruRE Cnim Chem. 241:3243-3246. 1. ANRAxu, Y. 1967. The reduction and restoration 19. LEIVE, L. 1965. A non-specific increase in perme- of galactose transport in osmotically shocked ability in Escherichia coli produced by EDTA. cells ofEscherichia coli. J. Biol. Chem. 242:793- Proc. Natl. Acad. Sci. U.S. 53:745-750. 800. 20. LEIVE, L. 1965. Release of lipopolysaccharide by 2. ASBELL, M. A., AmD R. G. EAGON. 1966. Role of EDTA treatment of Escherichia coli. Biochem. multivalent cations in the organization, struc- Biophys. Res. Commun. 21:290-296. ture, and assembly of the cell wall of Pseudo- 21. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, monas aeruginosa. J. Bacteriol. 92:380-387. AND R. J. RANDALL. 1951. Protein measurement 3. BIRDSELL, D. C., AmN E. H. COTA-ROBLES. 1967. with the Folin phenol reagent. J. Biol. Chem. Production and ultrastructure of lysozyme and 193:265-275. VOL. 94, 1967 OSMOTIC SHOCK IN ENTEROBACTERIACEAE 1945

22. MALAMy, M., AND B. L. HORECKER. 1964. Release 34. NossAL, N. G., AND L. A. HEPPEL. 1966. The of alkaline phosphatase from cells of Escher- release of enzymes by osmotic shock from E. ichia coli upon lysozyme spheroplast formation. coli in exponential phase. J. Biol. Chem. 241: Biochemistry 3:1889-1893. 3055-3062. 23. MELO, A., AND L. GLASER. 1966. Nucleotide 35. PARDEE, A. B., L. S. PRESTIDGE, M. B. WHIPPLE, diphosphate hexose pyrophosphatases. Bio- AND J. DREYFuss. 1966. A binding site for chem. Biophys. Res. Commun. 22:524-531. sulfate transport and its relation to sulfate 24. MEYNELL, G. G. 1958. The effect of sudden chil- transport in S. typhimurium. J. Biol. Chem. ling on Escherichia coli. J. Gen. Microbiol. 19: 241:3962-3969. 380-389. 36. PIPERNO, J. R., AND D. L. OXENDER. 1966 Amino 25. MITCHELL, P. 1961. p. 581. In T. W. GoodwIN acid-binding protein released from Escherichia and 0. Lindberg [ed.], Biological structure and coli by osmotic shock. J. Biol. Chem. 241: function, vol. 2. Academic Press, Inc., New 5732-5734. York. 37. REPASKE, R. 1958. Lysis of gram-negative or- 26. MITCHELL, P., AND J. MOYLE. 1964. Osmotic ganisms and the role of versene. Biochim. function and structure in bacteria. Symp. Soc. Biophys. Acta 30:225-232. Gen. Microbiol. 6:150-180. 38. SELLIN, H. G., P. R. SRINIVASAN, AND E. BOREK. 27. NEU, H. C., AND L. A. HEPPEL. 1964. The release 1966. Studies of a phage-induced DNA meth- of ribonuclease into the medium when Escher- ylase. J. Mol. Biol. 19:219-222. ichia coli cells are converted to spheroplasts. J. 39. SHAW, W. V. The enzymatic acetylation of Biol. Chem. 239:3893-3900. chloramphenicol by extracts of R-factor-resist- 28. NEU, H. C., AND L. A. HEPPEL. 1964. Some ob- ant Escherichia coli. 1967. J. Biol. Chem. 242: servations on the "latent" ribonuclease of 687-693. Escherichia coli. Proc. Natl. Acad. Sci. U.S. 40. SIMMoNs, S., AND N. 0. TOYE. 1966. Peptidases in 51:1267-1274. spheroplasts of Escherichia coli K-12. J. Biol. 29. NEU, H. C., AND L. A. HEPPEL. 1964. On the Chem. 241:3852-3860. surface localization of enzymes in Escherichia 41. SNYDER, I. S., AmN N. A. KOCH. 1966. Production coli. Biochem. Biophys. Res. Commun. 14:215- and characteristics of hemolysins of Escherichia 219. coli. J. Bacteriol. 91:763-767. 30. NEU, H. C., AND L. A. HEPPEL. 1965. The release 42. SPAHR, P. R., AND B. R. HOLLINGWORTH. 1961. of enzymes from Escherichia coli by osmotic Purification and mechanism of action of ribo- shock and during the formation of spheroplasts. nuclease from E. coli. J. Biol. Chem. 236:823- J. Biol. Chem. 240:3685-3692. 829. 31. NEU, H. C., D. F. ASHMAN, AND T. D. PRICE. 43. STRANGE, R. E., AND F. A. DARK. 1962. The effect 1967. Effect of ethylenediaminetetraacetic acid- of chilling on Aerobacter aerogenes in aqueous Tris(hydroxymethyl)amino methane on release 29:719-730. of the acid-soluble nucleotide pool and on suspension. J. Gen. Microbiol. breakdown of ribosomal ribonucleic acid. J. 44. STRANGE, R. E., AND J. R. POSTAGTE. 1964. Bacteriol. 93:1360-1368. Penetration of substances into cold-shocked 32. NEU, H. C. 1967. The 5'-nucleotidase of E. coli. bacteria. J. Gen. Microbiol. 36:393-403. I. Purification and properties. J. Biol. Chem., 45. WEIBULL, C., W. D. BICKEL, W. T. HASKINS, in press. K. C. MILNER, AND E. Rim. 1967. Chemical, 33. NEU, H. C. 1967. The 5'-nucleotidase of Escher- biological, and structural properties of stable ichia coli. II. Surface localization and purifica- Proteus L forms and their parent bacteria. J. tion of its inhibitor. J. Biol. Chem., in press. Bacteriol. 93:1143-1159.