JOURNAL oF BACTioLOY, Oct. 1969, p. 403-410Copyright ( 1969 American Society for Microbiology

Mechanism of Optical Effects in Suspensionsof a Marine Pseudomonadt

TIBOR I. MATULA' AND ROBERT A. MACLEODDepartment of Microbiology, Macdonald College of McGill University, and Marine Sciences

Center, McGill University, Montreal, Quebec, Canada

Received for publication 24 June 1969

When cells of a marine pseudomonad washed free of medium components with0.05 M MgSO4 were suspended in solutions containing 200-mM concentrations ofvarious salts, there was an immediate increase in optical density (OD), followed bya slow decrease. The decrease following the initial increase, but not the increase it-self, could be prevented by omitting K+ from or by adding metabolic inhibitors tothe suspending solution. With NaCl, the initial increase in OD rose to a maximumas the salt concentration was increased to 200 mm and then declined at 500 mM.There was a corresponding decrease in intracellular fluid volume to a minimum at200-mM NaCl and then a rise. When the increased OD produced by NaCl was main-tained, the internal Na+ and Cl- could be shown to have reached essentially thesame concentration in the cells as in the medium. Thus, the OD changes could nothave been due to osmotic effects. No evidence was obtained of a salt-induced aggre-gation of nuclear material. The OD of suspensions of isolated cell envelopes in-creased in response to increases in NaCl concentration in the absence but not in thepresence of 0.05 M MgSO4. The data was interpreted to indicate that the salt-inducedincreases in OD occurring in suspensions of the cells resulted from an interaction ofsalts with components of the cell envelope, causing contraction of the envelopesand shrinkage of the cells.

Turbidity changes occur in suspensions ofgram-negative bacteria when the solute concen-tration of the suspending medium is increasedby the addition of electrolytes or nonelectrolytes.The changes can often be separated into twophases. The first is an increase in turbidity whichis complete within seconds. The second, whichmay or may not occur depending on the species(6), is a slow decrease in turbidity which followsthe initial increase. These effects, which havebeen observed by a number of workers (1, 2, 6,14-16), have been ascribed generally to aninitial rapid decrease in size of the cells caused bythe sudden increase in osmotic pressure in thesuspending medium followed by a slow restora-tion of the cells to normal size as the solutescome into equilibrium across the osmotic barrier.Gram-positive bacteria ordinarily do not show

1From a thesis submitted by Tibor I. Matula in partial fulfill-ment of the requirements for the Ph.D. degree at McGill Univer-sity, May 1967. A preliminary report of these findings was pre-sented at the Annual Meeting of the American Society forMicrobiology 1-5, May 1966 at Los Angeles, Calif. The paper wasissued as Macdonald College Journal Series no. 592.

Present address: Division of Biosciences, National ResearchCouncil, Ottawa, Ontario, Canada.

such optical effects, although after incubation inphosphate buffer followed by washing in distilledwater, the cells of a number of species of gram-positive bacteria became susceptible to opticalchanges comparable to those of gram-negativeorganisms (6, 9). Our interest in the mechanismof these optical effects was aroused when weobserved that suspensions of a marine pseudo-monad showed optical changes typical of thoseof other gram-negative bacteria upon the addi-tion of NaCl to the suspending medium. Sinceprevious studies had indicated that the cyto-plasmic membrane of the marine pseudomonadpresented no osmotic barrier to NaCl (19), itseemed unlikely that osmotic effects could ac-count for the optical changes produced in sus-pensions of this organism. This paper providesevidence that the first-phage optical changes insuspensions of this organism are due to aninteraction of salts with components of the cellenvelope, whereas the second-phase changes re-quire K+ and are under metabolic control.

MATERIALS AND METHODSCulture. The organism used (ATCC 19855), referred

to as marine pseudomonad B-16, was isolated origi-403

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nally from a marine clam and has been classified as aPseudomonas species type IV. Studies on the nutritionand metabolism of this organism have been reportedin some detail in previous communications, the mostrecent by Wong et al. (22).Medium. The culture was maintained by monthly

transfer on slants of a medium containing 0.8%Nutrient Broth (Difco), 0.5% yeast extract (Difco),and 1.5% agar in a salt solution consisting of 0.22 MNaCl, 0.026 M MgCI2, 0.01 M KCI, and 0.1 mm FeSO4-(NH4)2SO4. The liquid medium employed containedthe same constituents with the agaromitted.

Preparation of cell suspensions. Cells were grown in250 ml of liquid medium in a 2-liter flask for 16 hr at25 C on a rotary shaker and were harvested by cen-trifugation at 16,000 X g at 4 C. Unless otherwisestated, the cells were washed three times by resuspen-sion in and centrifugation from volumes of 0.05 M

MgSO4 equal to the volume of the growth medium.Preparation of cell envelopes. The cell envelopes

were prepared by a slight modification of the pro-

cedure described by Buckmire and MacLeod (4).Cells were grown and harvested as described above, butthey were washed in 0.5 M NaCl. A suspension of thecells adjusted to contain the equivalent of 33 to 35 mg(dry weight) of cells per ml in a total volume of 20 mlwas placed in a polypropylene tube and frozen byusing liquid air. The suspension was thawed at roomtemperature, diluted with 0.5 M NaCl to a volume of200 ml, and centrifuged at 3,500 X g. The supematantfluid was removed and the sedimented matter wasresuspended in 20 ml of 0.5 M NaCl. The freezing andthawing treatment was repeated once more by whichtime all the cells had lost their contrast, as indicatedby phase-contrast microscopy. The envelopes werewashed by repeated resuspension in and centrifugationfrom 0.5 M NaCl until the optical density (OD) ofthe washings measured at 260 nm had dropped es-sentially to zero. Examination of thin sections byelectron microscopy revealed large fragments ofenvelopes, some in the shape of whole cells containingvariable but small amounts of nuclear material and noevidence of ribosomes.OD changes. For these measurements, a suspension

of appropriately washed cells was added in 0.1-mlvolumes to 9.9 ml of the test solution, and the OD ofthe resulting suspension was measured at intervalswith a Coleman Junior spectrophotometer at 525 nm.

The final cell concentration in each suspension con-

tained the equivalent of 0.33 to 0.35 mg (dry weight)of cells per ml. For the measurement of OD changesin suspensions of envelopes, 0.05-ml samples ofenvelope suspension were added to 1.5-ml volumes ofthe test solution contained in 0.5-cm cuvettes. Ab-sorbancy changes were measured with a Zeiss PMQII spectrophotometer. The cell envelope suspensioncontained envelopes equivalent to 3 mg (dry weight)of cells per ml.

Intacellular fluid volume. Intracellular fluid volumewas measured by a modified thick-suspension tech-nique essentially as described previously (19), exceptthat "4C-inulin was used in place of '4C-carboxypoly-glucose for the measurement of the extracellular fluidvolume, and the cells were suspended in a solution

containing 0.1 M tris(hydroxymethyl)aminomethane(Tris) buffer (pH 7.2), 0.05 M MgSO4, and variousconcentrations of NaCl.

Intracellular Na+ and Cl. Intracellular Na+ andCl- concentrations were determined by proceduresdescribed previously (19), except that the cells weresuspended in a solution containing 0.01 M Tris buffer(pH 7.2), 0.05 M MgSO4, and concentrations of NaClas indicated in the text. Corrections were made forions present in the extracellular fluid using 14C-inulinfor the measure of the extracellular fluid volume.

Inulin and "Na space. Inulin space in a packed cellpreparation was taken as the extracellular fluid vol-ume, determined as described previously (19) butusing '4C-inulin in place of 14C-carboxypolyglucose.The cells were suspended in the same medium used todetermine the intracellular ion concentrations. "Naspace was determined on another sample of the samecell suspension used to measure the inulin space. "2Na(specific activity about 22 mc/mM) was introduced asthe chloride salt into the cell suspension at a concentra-tion of 0.015 ,uc/ml. The cells were separated by cen-trifugation and the supernatant fluid was removed.The radioactivity of the supernatant fluid and of theseparated cells was determined by using a well-typescintillation counter. From the radioactivity of theseparated cells, it was possible to calculate the volumeof supernatant fluid to which this 21Na activity cor-responded. This volume was considered to be the"2Na space.Na+ determination. For the determination of Na+,

all samples were digested by a modification of theprocedure of Sanui and Pace (18). Samples weredigested by heating first with nitric and then withperchloric acid in Vycor-brand Kjeldahl flasks untilthe samples became colorless, after which the residualperchloric acid was removed by evaporation underpartial vacuum. Na+ analyses were performed by usinga flame photometer attachment for a Zeiss PMQ IIspectrophotometer.

Cl determination. Samples were prepared andanalyzed for Cl- by essentially the procedures ofWilson and Ball (21). Packed-cell preparations weremixed with an excess of a standard solution of AgNOs;concentrated HNO3 was added and the mixture washeated for 45 min over a bath of boiling water. In thecase of supernatant solutions, a standard solution ofAgNO3 was added directly to the samples to beanalyzed, followed by concentrated HNO3. The C17content of the cells and of the supernatant solutionswas determined by back titrating the excess AgNO3with a standard solution of NH4SCN, using FeSO4(NH)2SO4 as indicator.

Radioactivity measurements. For the measurementof 14C activity, 0.1-ml volumes of the various solutionswere pipetted onto circles of Whatman no. 1 filterpaper which were then dried. Each filter-paper discwas placed in a vial and covered with a liquid scintil-lation mixture (5); radioactivity was measured in aPackard Tri-Carb liquid scintillation spectrometer(Packard Instrument Co., Inc., Downers Grove, Ill.)."Na measurements were made with a Picker well-typescintillation counter. Care was taken to maintainconstant the geometry of all samples. In all cases, a

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MECHANISM OF OPTICAL EFFECTS 405

total of at least 10,000 counts was accumulated on

each sample to reduce the probable counting error toless than 1%.

RESULTSOptical effects produced by various solutes.

When equal samples of a suspension of marinepseudomonad B-16 that had been washed andsuspended in 0.05 M MgSO4 were added to equalvolumes of solutions differing in solute compo-

sition, the resulting suspensions were found tovary in OD, depending on the kind and con-

centration of the solutes present. In these ex-

periments, the final suspensions were made in a

basal, buffered salt solution to which variousadditional solutes were added. Two distinctphases of OD change could be distinguished(Table 1). The first phase was an initial rapidincrease in OD, complete within seconds aftersuspension of the cells. The second phase was amuch slower decrease in OD which occurred afterthe peak OD had been reached. It is evident thatthe salts of various monovalent cations and su-crose were effective in producing a response. Inthe case of NaCl, the maximal increase in ab-sorbancy was obtained at a concentration of 200mM. MgSO4 was also effective in producing aresponse (Table 2). It is of interest that com-binations of MgSO4 and NaCl produced agreater initial increase in turbidity than didMgSO4 alone.The rapid increase in turbidity of suspensions

of the marine pseudomonad in response to theaddition of solutes followed by a slow decreaseis typical of the response obtained in suspensionsof a number of gram-negative bacteria (1, 2,14, 15, 17).Requirement for K+ for the second-phase opti-

cal change. The basal salt mixture used as asuspending medium in these initial studies con-tained K+ and Mg++ at levels optimal for thegrowth of this organism (12, 13). Omission ofthe basal level of KCI from the suspending solu-tion had no effect on the first-phase opticalchange or on the increase in absorbancy of thesuspension on addition of solutes, but it almostcompletely eliminated the second-phase opticalchange, the subsequent slow decrease in ab-sorbancy after the initial increase (Table 3).Other experiments have shown that omission ofthe basal level of Mg++ from the suspendingsolution had no effect on either phase of theoptical response to added solutes.

Correlation of absorbancy changes with changesin intracellular fluid volume. The optical changeswhich occur in bacterial suspensions on additionof solutes are assumed to be manifestations eitherof changes in size of the organism or of different

TABLE 1. Effect ofvarious solutes on the OD changesoccurring in suspensions of cells of marine

pseudomonad B-16

OD at minutes in sus pensionbSolutea Concn(mm)

0.5 10 60

None ..0.325 0.280 0.270NaCI 50 0.360 0.310 0.305NaC C 100 0.385 0.295 0.300NaC.200 0.420 0.350 0.330NaC C 500 0.400 0.400 0.370KC.200 0.435 0.360 0.320LiC1. 200 0.440 0.390 0.370NH4Cl.200 0.430 0.380 0.365Sucrose. 400 0.380 0.350 0.335Raffinose.400 0.330 0.305 0.290

a Added to a salt Solution containing 0.01 MTris buffer (pH 7.2), 0.05 M MgSO4, and 0.01 MKC1.

b Measured at 525 nm. The experiment wasstarted by adding equal samples of a washed sus-pension of cells to cuvettes containing the testmedium.

TABLE 2. Effect of different concentrations ofMgSO4 on the OD changes in suspensions of

cells of the marine pseudomonad

solutes (mo)f OD at minutes in suspension

MgSO4 NaCl 0.5 10 30 60

1 0 0.250 0.235 0.220 0.22050 0 0.350 0.320 0.300 0.295100 0 0.375 0.355 0.340 0.335200 0 0.405 0.395 0.365 0.365

1 200 0.430 0.385 0.360 0.36050 200 0.450 0.395 0.360 0.365100 200 0.445 0.385 0.360 0.360200 200 0.460 0.405 0.400 0.395

a Suspending medium also contained 0.01 MTris buffer (pH 7.2) and 0.01 M KCI.

degrees of plasmolysis of the cells (1, 2, 6).Bernheim (2) concluded that OD changes insuspensions of Pseudomonas aeruginosa corre-sponded to changes in size of the cells as observedby electron microscopy of shadowed prepara-tions. Lehninger (10) obtained a good correlationbetween OD change and volume change meas-ured gravimetrically in rat liver mitochondria.

In the present study, changes in OD of sus-pensions of the marine bacterium were relatedto cell volume measured directly. The results inFig. 1 show that as the salt concentration in-creased to 200 mm the intracellular fluid volume

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TABLE 3. Effect ofK+ on OD changes in suspensionsof cells of marine pseudomonad B-16

OD at minutes in suspensionSolute" (MM) KCI

0.5 10 60

None + 0.330 0.280 0.270- 0.325 0.325 0.325

NaCl.. 200 + 0.435 0.340 0.325200 - 0.440 0.430 0.420

KCI. 200 + 0.430 0.360 0.320200 - 0.430 0.355 0.315

LiCl.. 200 + 0.440 0.390 0.370200 - 0.440 0.430 0.415

Sucrose.... 400 + 0.380 0.350 0.335400 - 0.380 0.375 0.355

a Added to a suspending medium containing0.01 M Tris buffer (pH 7.2) and 0.05 M MgSO4.KCI added where indicated at 0.01 M.

i 0.80- 0.45

pq ~~~~~~~2

~0.75

a 0.70- :.35 m

0 0.655

-.30

~0.60

0.55 L--- .25

; 0 o 100 150 200 500z

NaCI mM.

FiG. 1. Correlation between OD changes in suspen-sions of marine pseudomonad B-16 and changes inintracellular fluid volume in response to NaCl in thesuspending medium. Curve I represents intracellularfluid volume; curve 2, OD after 0.5 min; and curve 3,OD after 60 min.

decreased. Above the 200-mM NaCl concentra-tion, the intracellular fluid volume increasedslightly. The turbidity of the suspension showedan opposite effect, increasing with increasingsalt concentration to 200 mm NaCl and thendecreasing. The turbidity values obtained ini-tially remained essentially unchanged after 60min of incubation. This result was obtained sinceK+ was omitted from the suspending mediumin this experiment. Thus, the increase in OD ofthe suspension of marine pseudomonad B-16

corresponded to a decrease in intracellular fluidvolume of the cells, and hence to a decrease inthe size of the cells. Since it has been calculatedthat the swelling or contraction caused by theuptake or release of water from particles of thedimensions and composition of bacteria causes achange in light absorbancy which is inverselyproportional to the two-thirds power of the vol-ume of the particle (8), one may reasonablyconclude that the increase in OD of suspensionsof the marine pseudomonad in the presence ofNaCl is due to a decrease in the volume of thecells.

Intracellular Na+ and CR concentration. It isgenerally assumed that the initial increase inturbidity of a suspension of cells upon additionof NaCl is due to an osmotic phenomenon.NaCl is assumed to be a nonpenetrating or slowlypenetrating solute which causes the cells toshrink by increasing the osmotic pressure of themedium. Previous studies have shown that Na+assumes the same concentration inside the cellsof this organism as prevails in the suspendingmedium (19). To establish whether this stillapplied under the conditions used in the presentstudies and to determine the disposition of theanion involved, the intracellular Na+ and C17concentrations in the marine pseudomonad weredetermined when the cells were suspended in thepresence and in the absence of added NaCl(Table 4). In the absence of added NaCl, therewas a small amount of Na+ and C1- present as acontaminant in the suspending medium. Underthese circumstances, there was more Na+ in thecells than in the medium. This small amountrepresents Na+ tightly bound to the cells (R. C.Gordon and R. A. MacLeod, unpublished data).Under the same conditions, Cl- was also some-what higher in the cells than in the medium.When NaCl was added to the suspension at aconcentration of 200 mm, the concentration ofNa+ in the cells became very nearly the same asin the medium. The Cl- concentration in thecells was somewhat less than in the medium.

Further support for the conclusion that anNaCl gradient is not maintained across themembrane of this organism was obtained bymeasuring the volume penetrated by 22Na ('2Naspace) in cell preparations of the marine pseudo-monad. In this experiment, the volume occupiedby 22Na in a packed-cell preparation was com-pared with the total fluid volume and with theextracellular fluid volume as determined by usingYC-inulin. The results (Table 5) show thatwhen no unlabeled NaCl was added to thesuspending medium the 22Na space exceeded thetotal fluid volume, suggesting that some "Nawas exchanged for Na+ bound to the cells. When

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TABLE 4. Intracellular Na+ and Cl- concentrations in cells of marine pseudomonad B-16 in the presenceand in the absence of NaCla

Suspending mediUmb Intracellular concn

NaCl added Na+ found Cl found Nat Cl-

0 0.1 + 0.0 14.6 0.0 1.0 + 0.1 29.0 :1 0.9

200 204 4 1.0 222 4 2.0 219 1 4.0 175 4 8.0

a Values are expressed as millimolar units. Analyses represent the average and average deviations oftriplicate determinations.

b Contained, in addition, 0.01 M Tris buffer (pH 7.2) and 0.05 M MgSO4.

NaCl was added at a level of 200 mm, the amountof bound Na+ became negligible compared tothe total Na+ present. The 22Na space deter-mined under these conditions was quite similarto the total available fluid space in the cell prep-

arations.Effect of metabolic inhibitors. An energy-de-

pendent water extrusion mechanism has beendetected in mitochondria (11), chloroplasts, andphotosynthetic bacteria (16). If such a mecha-nism were present in the marine pseudomonadand required salts for activation, the initialdecrease in intracellular fluid volume whichoccurs upon the addition of salts could be ex-plained. As a test for the operation of such a

system, the effect of metabolic inhibitors on theproduction of optical changes by salts wasexamined. The results (Table 6) show that noneof the inhibitors tested prevented the initialincrease in turbidity which occurred upon theaddition of 200 mm NaCl to the suspension.The subsequent slow decrease which couldoccur because K+ was present, however, wasprevented or retarded by the presence of theinhibitors. In a further effort to influence theextent or rate of the initial increase in turbidityupon the addition of salts, the cells were pre-incubated with the inhibitors in the basal sus-

pending medium for 1 hr before adding the 200mm NaCl. No change in response was obtained.

Examination of cytoplasmic components. Whit-field and Murray (20) reported that chromatinaggregation could occur in cells of Shigelladysenteria, Bacillus cereus, and Escherichia coliupon immersion of agar blocks in which theorganisms were growing into a solution of high(1.7 M) NaCl concentration. The cells were

stained with a nuclear stain and examined with a

light microscope. At the high salt concentration,the nuclear material of the cells aggregated into a

body which looked like an axial filament. Asimilar phenomenon was observed by Johnsonand Gray (7) to occur in the marine organismAchromobacter fischeri.

TABLE 5. Comparison of the totalfluid volume andthe "Na space in packed cell preparations ofmarine pseudomonad B-16 in the presence and in

the absence of unlabeled NaCl

NaCl insuspending Total fluid vol Inulin space 2"Na spacemediurna

mmY ml ml ml

0 1.39 0.66 4.08200 1.17 0.57 1.034

a Contained, in addition, 0.01 M Tris buffer (pH7.2) and 0.05 M MgSO4.

TABLE 6. Effect of metabolic inhibitors on ODchanges in suspensions of marine

pseudomonad B-16

OD at minutes in suspensionInhibitorsa Concn (X)

0.5 10 60

Without addedNaCl

None 0.330 0.270 0.260DNP 2 X 10-4 0.345 0.360 0.325KCN 2 X 10-2 0.360 0.300 0.275NaN3 2 X 10-2 0.380 0.350 0.340PCMB 2 X 10-3 0.340 0.320 0.310HgCl2 2 X 10-6 0.320 0.325 0.325

With 0.2 mNaClNone 0.430 0.365 0.340DNP 2 X 10-4 0.440 0.405 0.400KCN 2 X 10-2 0.440 0.415 0.400NaNs 2 X 10-2 0.440 0.410 0.410PCMB 2 X 10-3 0.440 0.420 0.350HgCl2 2 X 10-6 0.430 0.430 0.370

a Added to a suspending medium containing0.01 M Tris buffer (pH 7.2), 0.05 M MgSO4, and0.01 M KC1. DNP = 2,4-dinitrophenol; PCMB =p-chloromercuribenzoate.

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The possibility was considered that the ODincrease which occurred upon the addition of200 mm NaCl might be due to chromatin ag-gregation. Staining of the nuclear material wascarried out on cells of the marine pseudomonadaccording to the procedure of Whitfield andMurray (20). No evidence of chromatin aggre-gation was obtained either in the presence orin the absence of NaCl at the concentrationtested.

Effect of composition of washing solution onoptical effects. The OD changes observed wereobtained with cells washed with 0.05 M MgSO4solution. Suspensions of cells washed in thisway showed optical responses similar to thosereported in the literature for terrestrial species.It was observed, however, that cells respondeddifferently if washed with 0.5 M NaCl or with asolution of NaCl, KCI, and MgSO4 at concen-trations of the salts permitting optimal growthof the organism. A comparison of the effect ofdifferent washing solutions on subsequent opticaldensity changes in suspensions of the marinebacterium is presented in Table 7. Equal numbersof cells washed in the three washing solutionswere placed in the basal suspending mediumcontaining various concentrations of NaCl. Cellswashed in 0.5 M NaCl or in the salt mixtureshowed a relatively small initial response toadded NaCl, as compared to cells washed in

TABLE 7. Effect of different washing solutions onOD changes in suspensions of marine pseudomonad

B-16 in response to NaCl

OD at minutes in suspensionWashing solutions NaCla

(mm)-

0.5 30 60

0.05 M MgSO4 0 0.340 0.325 0.31550 0.390 0.330 0.330100 0.450 0.370 0.360200 0.520 0.380 0.370500 0.450 0.435 0.420

0.5 M NaCI 0 0.280 0.290 0.29550 0.275 0.285 0.285100 0.275 0.305 0.310200 0.300 0.320 0.325500 0.370 0.375 0.375

0.2 M NaCl 0 0.255 0.300 0.3000.01 M KCI 50 0.260 0.305 0.3050.05 M MgSO4 100 0.275 0.325 0.325

200 0.305 0.340 0.335500 0.380 0.390 0.390

Added to a suspending medium containing0.01 M Tris buffer (pH 7.2), 0.05 M MgSO4, and 0.01M KCI.

MgSO4 solution, and a higher optical density at500 mm NaCl than at 200. Second-stage opticaleffects tended to show increases rather than de-creases in optical density with time. It is of con-siderable interest that after 60 min of incubationall suspensions appeared to be approaching thesame optical density at each level of NaCltested, irrespective of how the cells were washed.

Effects of salts on isolated envelopes. Suspen-sions of isolated envelopes of the marine pseudo-monad were tested to determine their capacityto respond optically to different concentrationsof salts (Fig. 2). With Mg++ present in thesuspending solution, somewhat higher opticaldensities were obtained in the absence than in thepresence of NaCl. When Mg++ was omitted,however, an increase in turbidity occurred withincreasing NaCl concentration, in a mannerquite comparable to the responses obtained withwhole cells.

DISCUSSION

The second phase of the optical changes oc-curring when the solute concentration of thesuspending medium is increased could be sepa-rated from the first, in the case of the marinepseudomonad, either by removing K+ from oradding metabolic inhibitors to the suspendingmedium. Thus, the first-phase change, the initialincrease in turbidity produced by added solutes,could be maintained and the factors giving riseto it could be examined.When NaCl was used to increase the OD of the

suspension, it was found that up to a concentra-tion of 200 mm the space not occupied by inulinin the suspension was reduced in proportion to

:L o.sbA

Ez

A B

1.8

.6 2 1

.2

o.4 .

1.2-

00 100 200 500 0 100 200 500

NaCl CONCENTRATION mM.

FIG. 2. OD changes in suspensions of isolated en-velopes of marine pseudomonad B-16 in response toNaCl in the presence and in the absence of MgSO4.(A) 0.05 X MgSO4 present. (B) MgSO4 absent. CurveI represents OD after 0.5 min; curve 2, OD after 10min.

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the increase in NaCl concentration. Studies ofthe comparative penetrability of inulin and su-crose into cells of this marine pseudomonad(F. L. A. Buckmire and R. A. MacLeod, un-published data) suggest that the permeabilitybarrier to inulin in these cells is the outer layerof the cell wall, whereas that to sucrose is thecytoplasmic membrane. Thus, decreases in thespace not occupied by inulin in the suspensionindicate a decrease in the volume of the wholecell rather than an increase in the degree ofplasmolysis. Cells washed with 0.05 M MgSO4,however, do show evidence of plasmolysis bothin the presence and in the absence of NaCl whenexamined by phase-contrast microscopy. Thislatter phenomenon is presently under investiga-tion.

Direct measurement of intracellular Na+ andC17 showed that the two ions had assumedessentially the same concentration inside thecells as prevailed in the medium. This confirmsearlier findings (19) and is supported by meas-urement of the 22Na space in the suspension.Thus, the extent of increase in OD which oc-curred reflected the degree to which the cells hadshrunk; yet no NaCl gradient was maintainedacross either the cell wall or the cytoplasmicmembrane. The possibility that the increase inOD could have been caused by an initial differ-ence in NaCl concentration between the insideand the outside of the cell due to a slow penetra-tion of the salt was ruled out, since, if this hadbeen the case, one would have expected a gradualdecrease in turbidity again as the salt concentra-tion reached equilibrium across the permeabilitybarrier. In the absence of K+, no such decreaseoccurred. The initial increase in turbidity wasmaintained over the period necessary to establishthat the internal Na+ and C1- had alreadyreached the concentration prevailing in themedium. Thus, NaCl could not have caused thecells to shrink by osmotic action.

Since metabolic inhibitors had no effect on theshrinkage of cells caused by NaCl, it seems un-likely that the salt had activated a type of energy-dependent contractile mechanism such as hasbeen described in mitochondria (11) and inphotosynthetic bacteria (16). Furthermore, noevidence of aggregation of nuclear material in thepresence of NaCl could be obtained. Thus, by aprocess of elimination it seemed most likely thatNaCl produced its effects through interactionwith the cell envelope of the organism. Supportfor this conclusion was obtained when it wasfound that NaCl caused increases in OD ofsuspensions of isolated cell envelopes of theorganism. Conditions were not exactly the sameas those used with whole cells, however, since in

the presence of 0.05 M MgSO4 no increase inturbidity of the envelope suspensions occurredupon the addition of NaCl.When cell envelopes of this organism are

suspended in distilled water, soluble, nondialyza-ble material appears in solution (4). The releaseof this material from the envelopes can be pre-vented by the addition of salts to the suspendingmedium. The material released at low salt con-centration consists of complex macromoleculesor aggregates of macromolecules composed ofcarbohydrate, lipid, and protein. (F. L. A.Buckmire and R. A. MacLeod, unpublished data).Evidence has been obtained that these macro-molecules are highly electronegative. Low con-centrations of Mg++ prevent the release of thesemacromolecules from the cell envelope withoutscreening all the negative charges. Thus it islikely that when cells of this organism arewashed and suspended in 0.05 M MgSO4 thereis sufficient Mg++ to form cross-links betweenthe macromolecules, but insufficient to screenthe negative charges completely. The macro-molecules might thus be expected to repel.oneanother sufficiently to stretch the envelope andcause the cells to swell. Higher concentrationsof salts could screen the rest of the negativecharges, thereby causing the envelope to contractand the cells to shrink. It is considered significantthat NaCl at a concentration of 200 mm producedmaximal shrinkage of the cells since this is theNaCl concentration optimum for preventingleakage of intracellular solutes from the cells(22) and for obtaining optimal growth of theorganism (5). Above a concentration of 200 mm,NaCl caused the cells to swell again and to leakintracellular solutes (22), and it inhibited growth(5). The reasons for this are obscure at present.The failure of isolated envelopes to duplicate

the response of whole cells to NaCl in thepresence of Mg++ may well be due to the fact thatin whole cells there is an outward thrust producedby internal osmotic pressure which could balancea tendency of Mg++ cross-links to cause envelopecontraction by mechanical means.The differences in response of suspensions of

cells to added NaCl, depending on the saltsolutions used to wash the cells, may possibly berelated to the fact that cells of this organism leaksmall molecules when suspended in 0.05 MMgSO4 but not when the suspending mediumcontains an appropriate concentration of Na+(22). Potassium, which is ordinarily present inthese cells at a concentration of 0.2 M, is lostfrom the cells when they are washed with 0.05 MMgSO4 (V. S. Srivastava, P. T. S. Wong, andR. A. MacLeod, unpublished data). As suggestedby Kuczynski et al. (9) in connection with

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MATULA AND MACLEOD

gram-positive bacteria which have lost theiramino acid pool, perhaps cells which have lostsmall molecules have a lower internal osmoticpressure and, hence, are able to contract to agater extent than cells with their intracellularsolutes intact.Other salts, like NaCl, would be expected to

cause cell shrinkage by envelope contraction inthe same manner as NaCl. Sucrose and raffinose,however, should behave differently. Since su-crose does not penetrate the cytopasmic mem-brane of this organism (F. L. A. Buckmire andR. A. MacLeod, unpublished data), its capacityto shrink the cells can be satisfactorily explainedby its osmotic action.The second-phase optical changes occurring

in suspensions of cells washed with 0.05 M MgSO4required K+ and were prevented by metabolicinhibitors. Evidence to be presented elsewherewill indicate that, as in the case of E. coli (3, 17),these changes result from the energy-dependenttransport of K+ and other metabolites, whenpresent, into the cells.

ACKNOWLEDGMENT

Thbis investigation was supported by a grant from the NationalResearch Council of Canada.

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