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 en- zymes 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 phos- phatase 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%.