JOURNAL OF BACTERIOLOGY, Sept. 1985, p. 1158-11660021-9193/85/091158-09$02.00/0Copyright C 1985, American Society for Microbiology

Vol. 163, No. 3

Effect of Changes in the Osmolarity of the Growth Medium onVibrio cholerae Cells

ANURADHA LOHIA,' SABITA MAJUMDAR,' ANADI N. CHATTERJEE,2 AND JYOTIRMOY DASl*Biophysics Division, Indian Institute of Chemical Biology, Calcutta 700 032,1 and Organon Research Center,

Calcutta 700 014,2 India

Received 8 April 1985/Accepted 17 June 1985

The rate and extent of lysis of Vibrio cholerae cells under nongrowing conditions were dependent on theosmolarity of the growth medium. Gross alterations in cellular morphology were observed when V. chokraecells were grown in media of high and low osmolarity. The rate of lysis of V. cholerae cells under nongrowingconditions increased after treatment with chloramphenicol. Chloramphenicol-treated V. cholerae 569B cellsshowed formation of sphaeroplast-like bodies in medium of high osmolarity, but not in low osmolarity. Changesin the osmolarity of the growth medium also regulated the expression of the outer membrane proteins. Thisregulation was abolished if V. cholerae cells were grown in Pi-depleted medium. Analysis of the lytic behaviorand composition of outer membrane proteins of an osmotically fragile mutant strain revealed a similardependence on the osmolarity of the growth medium.

Vibrio cholerae, the etiological agent of cholera, is anoninvasive, gram-negative pathogen which colonizes thesmall intestine, presumably by the interaction of specificbacterial cell surface components as well as nonspecifichydrophobic interactions with the intestinal mucosa (12).Excretion of cholera toxin from its cellular location has beenshown to be unique to V. cholerae, since cholera toxinsynthesized in Escherichia coli cells was not excreted andaccumulated in the periplasmic space as is the heat-labileenterotoxin (LT) of E. coli (26). In contrast, when the E. coliLT was synthesized in V. cholerae cells, the product wasexported out (24). It has been postulated that this behavior ofV. cholerae may be due to an unusual organization of itsenvelope components (15). Studies with mutants have sug-gested that changes in the cell surface may also play a role inthe virulence of V. cholerae (2).

Studies on the composition of the outer membrane pro-teins (11, 15), lipopolysaccharide (LPS) (29, 30), phos-pholipids (28), and various surface properties, such as hy-drophobicity and charge density (12), of V. cholerae cellshave revealed differences with other gram-negative entericbacteria. It has been reported that V. cholerae cells areextremely sensitive to protein denaturants (19) and that it ispossible to isolate the outer membrane directly from wholecells of this organism, unlike other gram-negative entericbacteria, by treatment with urea (19). Furthermore, V.cholerae cells lyse rapidly in hypotonic medium outside thegrowth medium (19). The stability of bacteria in a hypotonicmedium depends on a mechanically resistant cell wall thatgives osmotic protection (9). Various factors, such as growthconditions, osmotic environment, and pH, have been recog-nized as being particularly important in regulating autolysis(33).

In E. coli, three outer membrane proteins-OmpF,OmpC, and PhoE-which are biochemically and geneticallysimilar (20, 23), have been identified as being responsible forthe passage of solutes across the outer membrane. Theirexpression is, however, regulated by growth conditions. Theosmolarity of the growth medium is a major factor indetermining the relative levels of expression of OmpF and

* Corresponding author.

OmpC proteins (13, 36). The synthesis of the PhoE protein isderepressed upon phosphate starvation (25). It has also beensuggested that the rigidity of the outer membrane-peptidoglycan layer might also depend upon the specificmajor outer membrane protein present (13).

It is in this context that the present report describes thechanges in the lytic phenotype, cellular morphology, andcomposition of the outer membrane of a hypertoxinogenicstrain (569B) of V. cholerae grown under variousosmolarities and in phosphate-depleted (LP) growth me-dium. The lytic phenotype and cellular morphology werealso affected drastically after treatment with chlorampheni-col (CAM).

MATERIALS AND METHODS

Bacteria. The wild-type strain V. cholerae 569B(prototroph, hypertoxinogenic) of Inaba serotype wasobtained from the National Institute of Cholera, Calcutta,India. E. coli C600 (SuII) and Salmonella typhimurium LT2were obtained from S. Ghosh, Bose Institute, Calcutta, India.The phage-resistant mutant DC15 of V. cholerae 569B wasobtained from D. Chakraborti, Bose Institute, Calcutta,India. The mutant strain was highly sensitive to sodiumdeoxycholate compared with the parent 569B strain.

Media. The V. cholerae strains 569B and DC15 weregrown in nutrient medium containing 1% (wt/vol) Bacto-peptone (Difco), 1% (wt/vol) Lablemco powder (Oxoid), and0.18 M NaCl at pH 8.50. This medium will be referred to asplus medium. The "minus" medium is the same as the plusmedium, except that no NaCl is added. E. coli C600 and S.typhimurium LT2 cells were grown in plus and minus mediaat pH 7.20. LP medium was prepared as described previ-ously (32). The defined medium used was minimal mediumA (22) supplemented with 1 mM MgSO4-0.01 mMCaCl2-0.001% vitamin Bl-0.2% glucose-0.01% caseinhydrolysate. The NaCl concentration of LP medium andminimal medium A was adjusted as in plus and minus media.Cell viability was assayed as CFU on nutrient agar platescontaining 1.5% agar (Difco) with or without 0.18 M NaCl.

Lysis of whole cells. V. cholerae 569B and DC15 cells in thelogarithmic phase of growth (5 x 108 to 6 x 108 CFU ml-';absorbance at 585 nm = 0.70) were harvested by centrifu-

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gation at 6,000 x g and 10°C and suspended in either 0.18 MNaCl-0.36 M sucrose-0.36 M KCl-50 mM sodium phos-phate buffer (pH 7.2)-0.36 M glycerol or distilled water. Thetemperature of incubation in distilled water and 0.18 M NaClwas either 10 or 37°C as described below. Preincubation inNaCl (0.18 or 0.5 M) or MgCl2 (5 mM) was carried out at 37or 10°C when necessary. E. coli C600 and S. typhimuriumLT2 cells in the logarithmic phase of growth were similarlyharvested and suspended in distilled water at 37°C. Thevolume of the final resuspending buffer or distilled water wasadjusted to maintain 5 x 108 CFU ml-' (absorbance at 585nm = 0.70). Lysis was monitored turbidimetrically at 585 nmin a Sicospec 100 spectrophotometer (Scientific InstrumentCo., Calcutta India).

Release of UV-absorbing material from whole cells. Tomeasure the release of intracellular acid-soluble materialupon suspension of log-phase cells in 0.18 M NaCl, sampleswere removed at various times during incubation at 10 or37°C. The cells were centrifuged, and an equal volume ofice-cold 10%o trichloroacetic acid was added to the superna-tant and centrifuged. The absorbance of the trichloroaceticacid-soluble material in the supernatant was then measuredat 260 and 280 nm. An equivalent number of cells was boiledat 100°C for 5 min, and the absorbance at 260 and 280 nm ofthe supernatant was taken as 100%.Measurement of osmolarity. The osmolarity of the plus and

minus media was measured with the Osmette S automaticosmometer (Precision Systems Inc., Sudbury, Mass.) andexpressed as milliosmoles per liter per kilogram.

Isolation of the outer membrane. The outer membrane ofV. cholerae cells was isolated by treatment with 4 M urea asdescribed previously (19).

Isolation of the peptidoglycan. The peptidoglycan, alongwith the bound lipoprotein, was isolated after the treatmentof whole cells with 4% sodium dodecyl sulfate at 100°C (4).

Analytical methods. Protein was measured by the methodof Markwell et al. (21), with bovine serum albumin as thestandard. LPS was isolated from crude cell envelope by 45%(wt/vol) aqueous phenol at 680C (37). Total carbohydratewas assayed by phenol-H2SO4 (6), with D-glucose as thestandard. Heptose was estimated by the cysteine-H2SO4method (38). Rhamnose (5), fructose (31), and phosphate (1)were assayed as previously described. Glucose was assayedafter the hydrolysis of LPS with 2 N HCl at 100°C for 2 h bya coupled glucose oxidase-peroxidase reaction (Sigma Glu-cose Assay Bulletin 510-4). Glucosamine was assayed afterthe hydrolysis of LPS with 4 N HCl for 4 h at 100°C (34).Phospholipids were extracted by chloroform-methanol-water (1:2:0.8 [vol/vol/vol]) (3) and quantified from the lipidphosphate. Phospholipids were identified by thin-layer chro-matography on Silica Gel G (Merck) plates with the solventschloroform-methanol-ammonia (65:25:4 [vol/vol/voll) andchloroform-methanol-acetone-acetic acid-water (8:2:2:2:1,[vol/vol/vol/vol/volI]). The chromatogram was developed iniodine vapor. The individual phospholipids were quantifiedfrom the lipid phosphate after elution from the silica gel bychloroform-methanol (2:1 [vol/vol]). All standard sugars andphospholipids were from Sigma Chemical Co.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Outer membrane proteins were analyzed on a 10% sodiumdodecyl sulfate-polyacrylamide gel by the method ofLaemrmli (16).

Electron microscopy. For thin-section electron micros-copy, cells were fixed first with 6% glutaraldehyde (BDH,Poole, England) in 0.125 M phosphate buffer (pH 7.20) for 14to 16 h and then with 1% osmium tetroxide (Ted Pella, Inc.,

Tustin, Calif.) in Kellenburger buffer for 16 to 20 h at roomtemperature. The fixed cells were washed for 2 h in 0.5%uranyl acetate in the above buffer (14), dehydrated withascending concentrations of ethanol, and embedded in Spurrmedium (Polysciences, Inc., Warrington, Pa.) (35) at 70°Cfor 48 h. Sections were cut with glass knives on a JEOLultramicrotome, stained with uranyl acetate and lead citrate,and examined under a JEOL 100C transmission electronmicroscope at 60 kV.

RESULTSLysis of V. cholerae 569B cells grown in media of high and

low osmolarity. V. cholerae 569B cells grown in medium ofhigh osmolarity (390 milliosmoles * liter - kg-') or plus me-dium lysed rapidly when harvested and suspended in dis-tilled water at 37°C (Fig. la). The presence of impermeablesolutes, such as NaCl (0.18 M) (Fig. la) or sucrose (0.36M)-KCl (0.36 M)-50 mM sodium phosphate buffer (pH7,20), in the suspending medium prevented lysis, whereaspermeable solutes such as glycerol (0.36 M) were totallyineffective. The rate of lysis was reduced when cells in thestationary phase of growth were used compared with cells inthe exponential phase of growth and when the incubationtemperature for both exponential- and stationary-phase cellswas lowered to 10°C from 37°C (Fig. la).Under conditions in which the cell viability remained

unaltered (cells suspended in 0.18 M NaCl solution andincubated at 37°C), a significant amount of low-molecular-weight, 260-nm-absorbing intracellular material was released(Fig. la). The released material was primarily nucleic acidbreakdown products, as evidenced from the ratio of absor-bance at 260 and 280 nm of 1.9. The leakage of this materialwas much lower when cells were incubated at 10°C (Fig. la).No detectable amount of free reducing or N-terminal groupswas released in the supernatant of cells suspended in dis-tilled water containing 0.18 M NaCl, as would be expected ifautolytic muramidases or amidases were active.

Preincubation of cells with 5 mM MgCl2 at 0 or 37°Cprevented lysis and loss of cell viability upon suspension indistilled water. The effect of Mg2+ was irreversible in thesense that cells pretreated with Mg2+ ions and then exposedto 0.5 M NaCl were resistant to lysis.

In contrast to cells grown in plus medium, cells grown inmedium of low osmolarity (137 milliosmoles * liter * kg-') orminus medium were comparatively resistant to lysis whensuspended in distilled water (Fig. lb). An initial increase inthe absorbance of minus medium-grown cells suspended in0.18 M NaCl (Fig. lb) suggested plasmolysis of cells fol-lowed by a gradual deplasmolysis. Cell viability remainedunchanged under this condition for the 120 min that thisreaction was examined in the present study. The suscepti-bility of cells to lysis was dependent on the concentration ofNaCl in the growth medium up to an NaCl concentration of0.18 M, beyond which there was no further effect. E. coliC600 and S. typhimurium LT2 cells did not show anyappreciable lysis in distilled water when grown in either plusor minus medium.

Electron microscopy of cells grown in plus and minusmedia showed remarkable differences in the cell morphol-ogy. Relative to cells grown in plus medium (Fig. 2a), thecytoplasm of cells grown in minus medium was stained moreintensely (Fig. 2b and c) under identical experimental con-ditions. While the outer membrane of plus medium-growncells appeared flexible (Fig. 2a), that of minus medium-grown cells was more rigid (Fig. 2b and c). Some of theunusual features of cells grown in minus medium were the

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FIG. 1. Lysis of V. cholerae cells. V. cholerae 569B cells grown in plus (a) and minus (b) media were harvested, suspended in distilledwater or 0.18 M NaCl, and incubated at 37 or 10°C. Lysis was monitored by the fall in absorbance at 585 nm, and the release of intracellularmaterial from log-phase cells was measured by the increase in absorbance at 260 nm in the supernatant. The amount of 260-nm-absorbingmaterial released after boiling an equivalent number of cells at 100°C for 5 min was taken as 100%. (0) Log-phase cells in 0.18 M NaCl; (A)log-phase cells in distilled water; (A) stationary-phase cells in distilled water; and (l) release of 260-nm-absorbing material. Solid linesindicate incubation at 37°C, and broken lines indicate incubation at 10°C.

appearance of large nonstaining bodies in the cytosol and thepresence of amorphous regions (Fig. 2c).When cells grown in minus medium were transferred to

plus medium, within 15 min of incubation at 37°C, the wavystructure of the outer membrane was restored, the stainingintensity of the cytoplasm was reduced, and the nonstainingbodies disappeared (Fig. 3a). By 30 min of incubation in plusmedium, the cell morphology was indistinguishable fromthat grown in plus medium (Fig. 3b). Cells grown in minusmedium and suspended in distilled water containing 0.18 MNaCl showed no such changes in the appearance of the outermembrane and cytoplasm (Fig. 3c). On the contrary, thesecells showed extensive plasmolysis.

Effect of sucrose and Mg2' ions in the growth medium onthe lysis of V. cholerae 569B cells. To investigate whether thelysis of plus medium-grown cells in distilled water was due tothe high osmolarity of the growth medium alone or due to thehigh ionic concentration of the growth medium, V. cholerae569B cells were grown in minus medium supplemented with0.36 M sucrose, harvested, and suspended in distilled water.It was observed that the rate and extent of lysis in distilledwater of V. cholerae 569B cells grown in minus mediumsupplemented with 0.36 M sucrose was identical to that ofcells grown in plus medium (cf. Fig. la). Furthermore, thesusceptibility of cells to lysis in distilled water was depen-dent on the concentration of sucrose in the growth mediumup to a sucrose concentration of 0.4 M, beyond which therewas no further effect. Similar results were obtained whencells grown in chemically defined growth medium containingvarious concentrations of NaCl or sucrose were examined.

It has been suggested that divalent cations bind to outermembrane components to participate in the maintenance ofits structural and functional integrity. It was, therefore,necessary to examine whether the observed lysis in distilled

water of plus medium-grown cells was due to a weakening ofthe outer membrane structure as a result of the replacementof divalent cations such as Mg2" by the preponderant Na+ inplus medium. V. cholerae 569B cells were grown in plusmedium, minus medium, and minus medium supplementedwith 0.36 M sucrose, each of which was further supple-mented with 5 mM MgC92, harvested in the log phase, andsuspended in distilled water at 37°C. Although the net extentof lysis was reduced by 10 to 20% when V. cholerae 569Bcells were grown in 5 mM MgC92-supplemented mediumcompared with the respective growth medium not supple-mented with 5 mM MgC92, no appreciable change in the rateof lysis was observed. These results indicate that althoughthe role of divalent cations cannot be completely ruled out,the observed lysis of V. cholerae 569B cells grown in plusmedium was primarily due to osmotic destabilization.

Effect of CAM on the lysis of V. cholerae 569B cells. Ingram-positive bacteria, autolysis is inhibited in the presenceof CAM (33), presumably due to the continuous synthesisand thickening of the cell wall even in the absence of proteinsynthesis. However, no such effect of CAM was observedon the lysis of E. coli cells (18). To examine the effect ofCAM, V. cholerae cells were treated with a 10 jig ml-'concentration of CAM in both plus and minus media. CAM-treated cells in plus medium showed an increased rate oflysis in 50 mM phosphate buffer (pH 7.20) compared withuntreated cells. The extent of lysis was dependent upon thetime of CAM treatment (Fig. 4a). A similar effect wasobserved when minus medium-grown cells were treated withCAM (Fig. 4b). In these cells, lysis was monitored indistilled water instead of 50 mM phosphate buffer. Interest-ingly, CAM was seeh to act as a bactericidal agent for V.cholerae 569B cells growing in plus medium (more than 99%killing in 120 min) but was bacteriostatic for cells growing in

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FIG. 2. Electron micrographs of thin section of V. cholerae 569B cells. Thin sections of V. cholerae 569B cells grown in plus medium (a)or minus medium (b and c). Abbreviations: im, inner membrane; onm, outer membrane; p, periplasm; n, nuclear material; and A, amorphousregion. Arrow shows nonstaining body. Bar, 2 uim.

minus medium. However, the absorbance of both plus andminus medium-grown cells treated with CAM remainedunaltered during 120 min of treatment.

Electron microscopy of CAM-treated cells in plus medium(Fig. Sa) showed sphaeroplast-like bodies which appear tohave lost most of their cytoplasmic constituents. This indi-cated that CAM-treated cells become osmotically fragile,hence the enhanced rate of lysis in phosphate buffer. Thismight also be the reason for their failure to grow and dividewhen plated on nutrient agar plates. Minus medium-growncells did not show such gross alterations in cellular morphol-

ogy upon treatment with CAM (Fig. 5b). For comparison,when E. coli C600 cells were treated with a 50 ,ug ml-lconcentration of CAM in plus medium and were examinedunder an electron microscope (Fig. 5c), no such structuralchanges were observed (cf. Fig. Sa). It may be noted that theMIC of CAM for V. cholerae is ca. 5 ,ug ml-', whereas thatfor E. coli is 30 ,ug ml-'.

Composition of the outer membrane of V. cholerae 569Bcells grown in plus and niinus medium. (i) Proteins. Analysisof the purified outer membrane proteins of V. cholerae 569Bby sodium dodecyl sulfate-polyacrylamide gel electrophore-

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FIG. 3. Electron micrographs of thin sections of V. cholerae 569B cells. V. cholerae 569B cells grown in minus medium were suspendedin plus medium for 15 min (a) and 30 min (b) at 37°C and 0.18 M NaCl for 15 min (c). Bar, 2 ,um.

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FIG. 4. Lysis of V. cholerae 569B cells after treatment with CAM. V. cholerae 569B cells were treated with CAM in plus medium (a) andminus medium (b). Cells were harvested at 30 min (A), 60 min (O), and 120 min (x) after addition of the antibiotic. (0) Untreated cells. Plusmedium-grown cells were suspended in 50 mM phosphate buffer (pH 7.20) and minus medium-grown cells were suspended in distilled water.Lysis was monitored as described in the legend to Fig. 1.

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a

FIG. 5. Electron micrographs of thin sections of V. cholerae 569B and E. coli C600 cells treated with CAM. V. cholerae 569B cells growingin plus medium (a) and minus medium (b) and E. ccli C600 cells growing in plus medium (c) were treated with CAM for 120 min each as

described in the text. 5, Sphaeroplast-like bodies. Bar, 2 p.m.

sis has revealed that the ratio of two major outer membraneproteins of molecular weights 45,000 and 30,000 was depen-dent upon the osmolarity of the growth medium. In cellsgrown in plus medium, the amount of the 45,000-molecular-weight protein was greater than the 30,000-molecular-weightprotein (Fig. 6, lane d). The relative amounts of these twoproteins were reversed when 569B cells were grown in minusmedium (Fig. 6, lane c). To examine the synthesis of outermembrane proteins under LP conditions, cells were grown inLP medium with or without 0.18 M NaCl. The 30,000-molecular-weight protein was not synthesized in cells grownin LP medium in the presence or absence of NaCl (Fig. 6,lanes a and b). However, the amount of the 45,000-molecular-weight protein synthesized was much higher inthese cells, and its synthesis was insensitive to the osmolar-ity of the growth medium. Three new proteins of molecularweights 64,000, 43,000, and 33,000 were observed in theouter membrane of LP medium-grown cells.

(ii) LPS, phospholipids, and peptidoglycan. The carbohy-drate composition of the LPS isolated from V. cholerae 569Bcells grown in plus or minus medium did not differ signifi-cantly (Table 1). The phospholipid composition of cellsgrown under the two conditions was also similar (Table 1),and the 4% sodium dodecyl sulfate-insoluble residue repre-senting the murein lipoprotein (Table 1) was synthesized in

equal amounts in plus and minus medium-grown cells. Theamount of this residue was, however, less than that observedin E. coli cells (8). No significant difference was observed inthe total yield of the outer membrane and the cell envelopewith respect to the cellular dry weight under the two growthconditions.

Characterization of an osmotically fragile mutant (DC15) ofV. cholerae 569B. Several detergent-sensitive, phage-resistant mutants of V. cholerae 569B were isolated afterN-methyl-N-nitro-N-nitrosoguanidine mutagenesis. One ofthese mutants, designated DC15, was found to be osmoti-cally fragile compared with the parent V. cholerae 569B,although no significant difference in the composition of the 0antigen was detected. The generation time of the mutantcells in plus medium was 60 min compared with 30 min forthe wild-type cells, and in minus medium the values were 90and 45 min, respectively. Cells of strain DC15 grown in plusmedium showed rapid lysis in distilled water and 50 mMTris-hydrochloride (pH 8.0) buffer, irrespective of the age ofthe culture and temperature of incubation (Fig. 7a). Unlikethe parent strain, these mutant cells lysed in 50 mM phos-phate buffer (pH 7.20). It may be noted that the minimumstabilizing concentration of NaCl for the mutant cells was0.18 M compared with 0.09 M for the wild-type cells. Themutant showed reversion of its lytic phenotype when grown

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in minus medium (Fig. 7b), although the extent of itsreversion was less than that of the wild-type cells.

Analysis of the outer membrane proteins of DC15 grownin plus medium revealed a reversion in the relative expres-sion of the 45,000- and 30,000-molecular-weight proteins(Fig. 6, lane f) compared with the wild-type cells (Fig. 6d).However, minus medium-grown DC15 cells showed almostequal amounts of the 45,000- and 30,000-molecular-weightproteins (Fig. 6, lane e). Thus, although no definite patterncan be assigned to the expression of these two majorproteins, they definitely respond to osmosensory mecha-nisms operating in V. cholerae cells.

DISCUSSIONThe present report demonstrates that the rate and extent

of lysis of V. cholerae cells are largely dependent on theosmolarity of the growth medium. The degree of protectionimparted by the peptidoglycan to the cell varies in differentgram-negative bacteria. In E. coli, although the peptidogly-can consists of a thin monolayer (9), enzymatic cleavage ofthis macromolecule is essential before the cells becomeosmotically fragile. On the other hand, halophilic bacteriaare capable of regular growth and division, despite their lowpeptidoglycan content (17). However, they need ahypertonic environment for their growth and survival. Theremoval of the protective ionic milieu from the growth orsuspension medium causes complete disintegration of suchbacteria (17). As far as its response to osmotic stress isconcerned, the behavior of V. cholerae is atypical andresembles more closely that of halobacteria or other gram-

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FIG. 6. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis profile of outer membrane proteins of V. cholerae cells. Outermembrane proteins of V. cholerae 569B cells grown in LP minusmedium (lane a), LP plus medium (lane b), complete minus medium(lane c), and complete plus medium (lane d). Outer membraneproteins of V. cholerae strain DC15 cells grown in plus medium (lanef) and minus medium (lane e). The numbers represent molecularweights of the proteins in thousands.

TABLE 1. Comparison of the LPS, phospholipids, andpeptidoglycan of V. cholerae 569B cells grown in plus and minus

mediuma

Values for V. cholerae 569B cellsCell component and parameters grown in:

Plus medium Minus medium

LPSTotal carbohydrateb 27.0 25.8Phosphateb 2.10 1.70Fructosec 1.0 1.0Heptosec 2.50 3.80Glucosec 1.53 1.44Glucosaminec 1.76 2.28Rhamnosec 0.36 0.39

PhospholipidsdPhosphatidyl ethanolamine 60 67.5Lysophosphatidyl 6 4.5

ethanolaminePhosphatidyl glycerol 29 27Cardiolipin NDe NDPhosphatidyl serine ND ND

Peptidoglycanf 0.52 0.48a LPS, phospholipids, and peptidoglycan were isolated, purified, and quan-

titated as described in the text. The numbers represent the average of threeindependent sets of experiments." Expressed as the percent dry weight of total LPS.

c Expressed as molar ratios of sugars with respect to fructose.d Expressed as percentages of total lipid phosphate.e ND, Not detected.f Expressed as the percent dry weight of whole cells.

negative bacteria whose peptidoglycan has been damaged byeither lysozyme or antibiotics. This is clearly demonstratedby the extensive lysis of V. cholerae cells when suspended indistilled water. We have found that 0.09 M NaCl or 0.2 Msucrose was necessary to prevent lysis of these cells. Thiscontrasts with the much higher concentrations of suchnonpenetrating solutes, e.g., 0.5 M sucrose, needed toprotect E. coli cells whose peptidoglycan has been damaged(8). This would indicate that the internal osmotic pressure ofV. cholerae cells is significantly lower than that of gram-negative bacteria such as E. coli. On the other hand, it couldalso be possible that the internal osmotic pressure of V.cholerae cells is similar to other gram-negative bacteria, butthe observed lysis could be due to less stable or weaker outermembranes of these cells.The rate of lysis of V. cholerae cells in hypotonic medium

is far too rapid for it to be mediated entirely by autolysins.Under nongrowing conditions, the irreversible effect ofMg2+ ions in preventing cellular lysis suggests that weakinteractions between outer membrane components may beimportant for stabilizing the cellular structure in these cells.In marine pseudomonads (27), it has been suggested thatMg2+ ions form bidentate chelate complexes between twoadjacent tetrapeptide subunits in the peptidoglycan, thusstabilizing this structure. In contrast, the protective effect ofMg2+ ions on the lysis of V. cholerae cells was much lesswhen the growth media were supplemented with 5 mMMgCl2. E. coli mutants (Ipo mipA) with altered outer mem-branes have been found to undergo blebbing when grown inlow-Mg2+ growth medium (39) and require Mg2+ ions ingrowth media to maintain the integrity of the outer mem-brane. An earlier report (19) has shown that noncovalentinteractions are important in stabilizing the cell envelope ofV. cholerae cells. Although we observed a minimal decrease

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TIME (min)FIG. 7. Lysis of V. cholerae DC15. V. cholerae DC15 cells were

grown in plus medium (a) and minus medium (b), harvested at theexponential phase, and suspended in 0.18 M NaCl (0), 50 mMsodium phosphate buffer (pH 7.20) (x), 50 mM Tris-hydrochloridebuffer (pH 8.0) (A), and distilled water (l). Cells were incubated at37°C (solid lines) or at 10°C (broken lines). Lysis was monitored asdescribed in the legend to Fig. 1.

in the extent of lysis of cells grown in Mg2' ion-sup-plemented plus and minus media compared with the samemedia not supplemented with Mg2+, the differential lyticpattern of these cells remained unaltered. No bleb formationwas observed in V. cholerae cells grown in plus medium(Fig. 2a). This suggests that although the presence of Mg2'ions under growing conditions could stabilize V. choleraecell envelopes to a certain extent, osmotic sensitizationinduced by growth in plus medium plays a major role inimparting susceptibility to lysis to V. cholerae cells.

Susceptibility of V. cholerae cells to lysis in hypotonicmedia under nongrowing conditions prompted us to examinewhether these cells could grow in a medium of low osmolar-ity. Unlike halophilic bacteria, these cells could not onlygrow in minus medium but also became relatively resistantto lysis under nongrowing conditions (Fig. lb). This result,along with the fact (Table 1) that there is no major differencein the quantitative amounts of the cell wall constituents ofcells grown in plus and minus media, suggests that theinternal osmotic pressure of V. cholerae cells possibly playsa key role in determining its inherent fragility. However, theelectron microscope studies indicate an increase in therigidity of the outer membrane of cells grown in low-osmolarity medium. Interestingly, no plasmolysis of cellswas noticed when minus medium-grown cells were shifted toplus medium after 15 min; however, extensive plasmolysis

was noted if minus medium-grown cells were resuspended in0.18 M NaCl under nongrowing conditions.

It has been postulated that one of the reasons for theinhibition of autolysis in CAM-treated gram-positive bacte-ria is the thickening of the cell wall (33). It has also beensuggested that E. coli cells have a mechanism that canrapidly modify the structure of newly made murein inbacteria that have stopped making protein. Thereafter, theattachment of murein hydrolases to such a murein isblocked, leading to a decreased susceptibility to autolyticdegradation (10). However, yet another group has shownthat pretreatment with CAM does not affect the rate ofshock-induced autolysis in E. coli (18). In light of suchinformation, it is intriguing that V. cholerae cells showpotentiation of lysis upon pretreatment with CAM whichacts as a bactericidal agent on cells growing in plus medium.CAM has also been shown to act as a bactericidal agent inHaemophilus influenzae cells (7), although formation ofsphaeroplast-like bodies has not been reported. It is difficultto explain the formation of such sphaeroplast-like bodiesupon treatment with CAM in plus medium. It is also difficultto explain the differential action ofCAM on V. cholerae cellsgrown under various osmolarities.The reciprocal expression of the 45,000- and 30,000-

molecular-weight proteins in plus and minus medium issimilar to that observed for OmpF and OmpC proteins in E.coli (13, 36). The outer membrane proteins from the mutant,however, show a reversal in the expression of the twoproteins in plus and minus media compared with the wildtype. It is intriguing to note the complete disappearance ofthe 30,000-molecular-weight protein in cells grown in LPconcomitant with the increase of the 45,000-molecular-weight protein. Interestingly, in E. coli K-12 mutants (20), ithas been observed that protein e functions as the major porinin the absence of OmpC and OmpF proteins. This protein ewas later identified as the PhoE protein (25), which wasexpressed under phosphate starvation in wild-type cells. It ispossible that under phosphate-limiting growth conditions inV. cholerae, a new protein of molecular weight 45,000 isexpressed which replaces the major 45,000- and 30,000-molecular-weight proteins observed in complete medium.This is further supported by the fact that the major outermembrane protein synthesized under phosphate-limitingconditions is not affected by the osmolarity of the growthmedium.

ACKNOWLEDGMENTSThis work was supported by the Council of Scientific and Industrial

Research, India.We are grateful to S. N. Dey for his help in electron microscopy

and to M. Chakraborty of Kothari Research Center for theosmometric measurements. We also thank all of the members of theBiophysics Division for their cooperation and encouragement duringthis study.

LITERATURE CITED1. Ames, B. N., and D. T. Dubin. 1960. The role of polyamine in the

neutralisation of bacteriophage deoxyribonucleic acid. J. Biol.Chem. 235:769-775.

2. Baselski, V. S., R. A. Medina, and C. D. Parker. 1979. In vivoand in vitro characterization of virulence-deficient mutants ofVibrio cholerae. Infect. Immun. 24:111-116.

3. Bligh, G. G., and W. J. Dyer. 1959. A rapid method of total lipidextraction and purification. Can. J. Biochem. Physiol. 37:911-917.

4. Braun, V., and K. Rehn. 1969. Chemical characterisation,spatial distribution and function of a lipoprotein (Murein-

VOL. 163, 1985

on Septem

ber 24, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

1166 LOHIA ET AL.

lipoprotein) of the E. coli cell wall. Eur. J. Biochem. 10:426-438.

5. Dische, Z., and L. B. Schettles. 1951. A new spectrophotometrictest for the detection of methyl pentose. J. Biol. Chem.192:579-587.

6. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.Smith. 1956. Colorimetric method for determination of sugarsand related substances. Anal. Chem. 28:350-356.

7. Feldman, W. E., and N. S. Manning. 1983. Effect of growthphase on the bactericidal action of chloramphenicol againstHaemophilus influenzae type b and Escherichia coli K-1. Anti-microb. Agents Chemother. 23:551-554.

8. Ghuysen, J.-M. 1968. Use of bacteriolytic enzymes in determi-nation of wall structure and their role in cell metabolism.Bacteriol. Rev. 32:425-564.

9. Ghuysen, J. M. 1977. Biosynthesis and assembly of bacterialcell walls, p. 463-595. In G. Poste and G. C. Nicholson (ed.),The synthesis, assembly and turnover of cell surface compo-nents. Cell surface reviews. vol. 4. North-Holland PublishingCo., Amsterdam.

10. Goodell, W., and A. Tomasz. 1980. Alteration of Escherichiacoli murein during amino acid starvation. J. Bacteriol.144:1009-1016.

11. Kabir, S. 1980. Composition and immunochemical properties ofouter membrane proteins of Vibrio cholerae. J. Bacteriol.144:382-389.

12. Kabir, S., and S. Ali. 1983. Characterization of surface proper-ties of Vibrio cholerae. Infect. Immun. 39:1048-1058.

13. Kawaji, H., T. Mizuno, and S. Mizushima. 1979. Influence ofmolecular size and osmolarity of sugars and dextrans on thesynthesis of outer membrane proteins 0-8 and 0-9 of Esche-richia coli K-12. J. Bacteriol. 140:843-847.

14. Kellenberger, E., A. Ryter, and J. Sechand. 1958. Electronmicroscope study of DNA-containing plasms. II. Vegetativeand mature phage DNA as compared with normal bacterialnucleoids in different physiological states. J. Biophys. Biochem.Cytol. 5:671-676.

15. Kelley, J. T., and C. D. Parker. 1981. Identification and prelim-inary characterization of Vibrio cholerae outer membrane pro-teins. J. Bacteriol. 145:1018-1024.

16. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

17. Larsen, H. 1967. Biochemical aspects of extreme halophitism.Adv. Microbial Physicol. 1:97-132.

18. Leduc, M., R. Kasta, and J. van Heoenoort. 1982. Induction andcontrol of autolytic system of Escherichia coli. J. Bacteriol.152:26-34.

19. Lohia, A., A. N. Chatterjee, and J. Das. 1984. Lysis of Vibriocholerae cells: direct isolation of the outer membrane fromwhole cells by treatment with urea. J. Gen. Microbiol. 130:2027-2033.

20. Lugtenberg, B., R. Van Boxtel, C. Verhoef, and W. Van Alphen.1978. Pore protein e of the outer membrane of Escherichia coliK12. FEBS Lett. 96:99-105.

21. Markwell, M. A. K., S. M. Haas, L. L. Bieber, and N. E.Tolbert. 1978. A modification of the Lowry procedure to sim-plify protein determination in membrane and lipoprotein sam-

pies. Anal. Biochem. 87:206-210.22. Miller, J. H. 1972. Experiments in molecular genetics. Cold

Spring Harbor Laboratory, Cold Spring Harbor, N.Y.23. Mizuno, T., M. Y. Chou, and M. Inouye. 1983. A comparative

study on the genes for three porins of the Escherichia coli outermembrane: DNA sequence of the osmoregula*d Omp C gene.J. Biol. Chem. 258:6932-6940.

24. Neill, R. J., B. E. Ivins, and R. K. Holmes. 1983. Synthesis andsecretion of the plasmid-coded heat-labile enterotoxin of Esch-erichia coli in Vibrio cholerae. Science 221:289-291.

25. Overbeeke, N., and B. Lugtenberg. 1980. Expression of outermembrane protein e (Pho E protein) of Escherichia coli K12 byphosphate limitation. FEBS Lett. 112:229-332.

26. Pearson, G. D. N., and J. J. Mekalanos. 1982. Molecular cloningof Vibrio cholerae enterotoxin genes in Escherichia coli K12.Proc. Natl. Acad. Sci. U.S.A. 79:2976-2980.

27. Rayman, M. K., and R. A. Macleod. 1975. Interaction of Mg2+with peptidoglycan and its relation to the prevention of lysis ofa marine pseudomonad. J. Bacteriol. 122:650-659.

28. Raziuddin, S. 1976. Effect of growth temperature and cultureage on the lipid composition of Vibrio cholerae 569B (Inaba). J.Gen. Microbiol. 94:367-372.

29. Raziuddin, S. 1977. Characterisation of lipid A and polysaccha-ride moieties of lipopolysaccharide from Vibrio cholerae.Biochem. J. 167:147-154.

30. Raziuddin, S., and T. Kawasaki. 1976. Biochemical studies onthe cell wall lipopolysaccharides (0-antigens) of Vibrio cholerae569B (Inaba) and El tor (Inaba). Biochim. Biophys. Acta431:116-126.

31. Roe, J. K. 1934. A colorimetric method for the determination offructose in blood and urine. J. Biol. Chem. 107:15-22.

32. Roy, N. K., R. K. Ghosh, and J. Das. 1982. Repression of thealkaline phosphatase of Vibrio cholerae. J. Gen. Microbiol.128:349-353.

33. Shockman, G. D. 1965. Symposium on the fine structure andreplication of bacteria and their parts. IV. Unbalanced cell-wallsynthesis, autolysis and cell-wall thickening. Bacteriol. Rev.29:345-358.

34. Strominger, J. L., J. T. Park, and R. E. Thompson. 1959.Composition of the cell wall of Staphylococcus aureus: itsrelation to the mechanism of action of penicillin. J. Biol. Chem.234:3263-3268.

35. Spurr, A. R. 1969. A low viscosity epoxy resin embeddingmedium for electron microscopy. J. Ultrastruct. Res. 26:31-43.

36. Van Alphen, W., and B. Lugtenberg. 1977. Influence of osmolar-ity of the growth medium on the outer membrane protein patternof Escherichia coli. J. Bacteriol. 131:623-630.

37. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharidesextraction with phenol-water and further applications of thisprocedure. Methods Carbon Chem. 5:83-91.

38. Wright, B. E., and P. A. Rebers. 1972. Procedure for determin-ing heptose and hexose in lipopolysaccharides. Modification ofthe cysteine-sulphuric acid method. Anal. Biochem. 49:307-319.

39. Yen, D. W., and H. C. Wu. 1978. Physiological characterizationof an Escherichia coli mutant altered in the structure of mureinlipoprotein. J. Bacteriol. 133:1419-1426.

J. BACTERIOL.

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ber 24, 2020 by guesthttp://jb.asm

.org/D

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