JOURNAL OF BACTERIOLOGY, Mar. 1972, p. 1273-1283Copyright ® 1972 American Society for Microbiology

Vol. 109, No. 3Printed in U.S.A.

Osmotic-Sensitive Mutant of Salmonellatyphimurium

DORA N. ANTONN'

Departamento de Radiobiologia, Comisibn Nacional de Energia Atbmica, Buenos Aires, Argentina

Received for publication 29 October 1971

A strain (DA82) having peculiar osmotic properties was isolated in Salmonellatyphimurium. The mutant shows increased elasticity of its cell wall and makesspherical instead of elongated cells, regardless of the osmolality of the medium.The strain withstands dilution in distilled water without disruption or deathand grows normally in 0.1 molal NaCl broth (240 milliosmol), but it dies expo-

nentially in low-osmolality broth (40 milliosmol). Addition of salts or sucroseinstantly stops death and allows growth and cell division to proceed. Death isnot due to lysis because this appears at later times and at a much lower rate.Osmotic inactivation is temperature-dependent: higher death rates occur athigher incubation temperatures. Inhibition of protein synthesis by chloram-phenicol (20 gg/ml) prevents osmotic death. At 37 C and at lower tempera-tures, the phenomenon of osmotic death is transient. After a variable interval,growth of the osmotic-sensitive strain resumes. It is assumed that the strain'sosmotic behavior is due to membrane defectiveness. The membrane disfunc-tion and the wall defect shown by the strain may be consequences of a singlegenetic alteration or the results of independent mutations.

The involvement of the membrane in impor-tant cell functions such as permeation, elec-tron transport, various macromolecularsyntheses, deoxyribonucleic acid (DNA) repli-cation, and cell division is at present widelyrecognized (27); yet, very little is known of themechanisms underlying membrane action orthe consequences that alterations in its struc-ture have on these diverse activities.The genetic approach to these problems has

lately been opened by the isolation and studyof several types of mutants. Some of them,such as fatty acid (11, 30) or glycerol (19) mu-tants, are unable to synthesize a membranecomponent and are therefore dependent onits exogenous provision for the normal consti-tution of the membrane. Others are defectivein functions ascribed to or connected with themembrane: conditional cell division mutants(12), colicin-tolerant (21), or chlorate-resistant(4) mutants. For some of these, reports onstructural or functional alterations of the mem-brane have appeared (5, 13, 28).

This paper describes a strain of Salmonellatyphimurium that shows peculiar osmoticproperties. Data presented indicate that thisstrain may be a novel kind of membrane-defective mutant.

I Career investigator of the Consejo Nacional de Investi-gaciones Cientificas y Tenicas, Argentina.

MATERIALS AND METHODSBacterial strains. The strains used are deriva-

tives of S. typhimurium LT2.Strain DA82 [his W1824 hisO1242 strA (P22)] ap-

peared as a his+ transductant in a transductionalcross with DA78 [hisW1824 hisOG203 strA (P22)1 asrecipient and his01242 as donor. No mutagens wereused in the procedure. Strain DA18 [his W1824his01812 strA (P22)] and strain DA23 [hisW1824his01242 strA (P22)] are derivatives of SB564 [his-W1824 hisE35 strA (P22)]. Strain DA78 is a deriva-tive of strain DA18. The construction of strainsDA18, DA23, and DA78 will be described elsewhere(Ant6n, manuscript in preparation). Strain DA88[his W1824 his01242 strA (P22) ] was casually isolatedin a culture of DA23 treated with diethylsulfate andpenicillin. DA88 differs from DA23 in that it makesrounded cells.

Media. Downshock medium (NoS-broth) was nu-trient broth (Difco) with no NaCl added (40 millios-mol). Both the ready-made nutrient broth and oneprepared by using Difco components and formula(peptone, 5 g; beef extract, 3 g; distilled water, 1liter) were employed with the same results. Com-plete medium (S-broth) was nutrient broth (Difco)containing 0.1 molal (m) NaCl (240 milliosmol).Minimal medium was the E medium of Vogel andBonner (33) with 0.5% dextrose (211 milliosmol). Insome experiments, medium M9 (1) with 5 g of NaClper liter and no carbon source was employed as abuffer. All the dilutions for viable counts were madein 0.1 m NaCl; samples of the dilutions were placedin 2.5 ml of molten soft agar (S-broth containing

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0.7% Difco agar) and poured on S-agar (S-broth with1.5% Difco agar) plates.

Penicillin G (Squibb & Sons, Argentina) solutionswere prepared by aseptically dissolving the steriledrug in S-broth. Vancomycin (Eli Lilly & Co.), D-cycloserine (Mann Research Laboratory), bacitracin,chloramphenicol, nalidixic acid, and lysozyme(Sigma Chemical Co.) solutions were sterilized byfiltration through membrane filters (450-nm poresize; Millipore Corp., type HA). Ethylenedia-minetetraacetate (EDTA), disodium salt (May &Baker), acridine orange (G. T. Gurr Ltd.), and 2,3,5-triphenyltetrazolium chloride (British Drug HousesLtd.) solutions were sterilized by autoclaving.

Bacitracin was obtained from M. Dankert (Univ-ersity of Buenos Aires); chloramphenicol, fromParke, Davis & Co, Argentina; and nalidixic acid,from Winthrop Laboratories, Argentina. The 04-012.somatic antigen immune serum was a gift of J. J.Monteverde (University of Buenos Aires).

Osmolalities were measured in an Osmette Preci-sion osmometer.Growth experiments. Strains were grown over-

night at 37 C in minimal medium with shaking.After 10- or 20-fold dilution in fresh medium,shaking was continued for 3 to 4 hr. The logarithmicculture (about 2 x 108 cells/ml) was centrifuged, thepellet was rinsed with NoS-broth, and the cells wereresuspended in one-tenth the original volume ofNoS-broth. No attempts were made to eliminatecompletely salts carried over from the first medium.Experimental cultures were started by adding 0.2 mlof the NoS-broth suspension to 10-ml amounts ofprewarmed medium contained in 25-ml flasks. Thecultures were incubated with shaking in a NewBrunswick gyratory water bath shaker. Samples werewithdrawn at intervals and assayed for the numberof viable cells, and their optical density (OD) at 650nm was read in a Beckman DU spectrophotometer.Total number of cells was determined in samples ofcultures diluted in 10% formaldehyde by using aPetroff-Hausser counting chamber.

Induction of P22 prophage in the cultures wasstudied by assaying free phage in samples treatedwith chloroform.

Cell wall elasticity. Log-phase cells were grownin minimal medium (supplemented with 50 ,ug of L-histidine/ml for strain DA78) as described above.After centrifugation, the cells were suspended in 0.1m NaCl and incubated for 2 hr at 37 C to avoidchanges produced by residual growth. They werethen centrifuged twice and resuspended in double-distilled water, and equal amounts of the suspensionwere diluted in double-distilled water and in solu-tions containing increasing molalities of NaCl. TheOD (at 650 nm) of these suspensions was read in aGilford modified Beckman DU spectrophotometer.The difference between OD at each concentration(ODm) and OD in distilled water (ODw), expressed asper cent of the latter: 100 x (ODW - ODni)/ODW wascalled "relative optical change" and plotted againstmolality of the corresponding NaCl solution.

Sensitivity tests. Serial dilutions of the test sub-stance were made in S-broth, and 0.9-ml sampleswere dispensed in small test tubes. The strains,

grown to log phase in S-broth, were diluted in thesame medium to about 107 bacteria/ml, and 0.1 mlof the dilution was inoculated in each tube. Thetubes were incubated at 37 C with shaking overnight.Growth was estimated either visually or by OD read-ings at 650 nm.To test lysozyme sensitivity, the cells were grown

in S-broth or minimal medium with shaking untillog phase. The cells were then sedimented, washed,and suspended in sterile 0.067 M phosphate buffer(pH 8.0). After the viable count and OD of the sus-pension were assayed, half received 40 Ag oflysozyme/ml, and the other half received the samevolume of water. Both samples were incubated at 37C for 30 min without shaking, and viability and ODwere then reassayed.

Electron microscopy. The preparative method ofKellenberger et al. (15) was used. Epon 812 served asembedding medium. Sections were cut in an LKBultramicrotome with a diamond knife. The sectionswere collected on uncoated 400-mesh copper gridsand stained first with uranyl acetate and then withlead citrate (23). They were examined in an RCAEMU-3F or a Philips 300 electron microscope.

RESULTS

Isolation of strain DA82. Strain DA82 ap-peared during the construction of the doublemutant hisW1824 his01242. Each of these twomutations produces constitutive synthesis ofthe histidine enzymes: his01242, an operatorconstitutive mutation, is linked to the histi-dine structural genes (25), whereas his W1824 isnot linked to them (3). It should be empha-sized, however, that the peculiar propertiesof strain DA82 cannot be imputed to the com-bination of regulatory mutations carried be-cause many independently isolated strains,carrying the same two mutations, were ob-tained, and none have shown the uniquephenotype described below. Thus, the osmoticphenotype of DA82 should be ascribed to amutation, or mutations, that spontaneouslyappeared during the construction of the doublemutant.

Cell morphology. In contrast to normal rod-shaped Salmonella, the cells of DA82 areround. Under the phase-contrast microscope,DA82 cells display a morphology similar tothat described for spheroplasts (17) and veryfrequently exhibit dark spots, often along theenvelope (Fig. 1).

Histidine-regulatory mutants make long fila-mentous cells on media containing a highamount (2%) of metabolizable carbon source(M. L. Murray and P. E. Hartman, manuscriptsubmitted for publication). The inhibition ofcell division that leads to filament formationin those mutants appears to be caused by over-production of the histidine enzymes coded by

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genes hisF and hisH (M. L. Murray and P. E.Hartman, manuscript submitted for publica-tion). Polar histidine mutations that reducethe level of synthesis of hisF and hisH prod-ucts normalize cell division and cell shape(M. L. Murray and P. E. Hartman, manuscriptsubmitted for publication). Strain DA82,though derepressed for the histidine enzymes,has not been observed to filament under anyconditions. Furthermore, DA82 derivativescarrying polar mutations in the histidine op-

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eron retain the spheric cell shape and ab-normal osmotic behavior of the original DA82strain. This observation supports the conclu-sion that the anomalies shown by DA82 arenot related to the derepression of the histidineoperon.With the exception of the anomalous shape,

electron micrographs of thin sections showcells that look structurally normal, with nosign of defectiveness noticeable in the cell wall(Fig. 2).

4

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FIG. 1. Phase-contrast photomicrograph of cells of strain DA82 grown on S-nutrient agar containing 1%dextrose. Final magnification, approximately x3,800.

FIG. 2. Thin section of a dividing cell of strain DA82 grown in 0.1 m NaCl-broth. Marker bar represents0.2 um.

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The data in Table 1 show that strain DA82resembles its parent, DA78, in all respectstested except in the requirement for histidine,removed at the time of construction of DA82,and in cell morphology. The positive reactionof DA82 to antiserum specific for the 04-012somatic antigens is significant since it proves

that the cell wall retains at least part of thelipopolysaccharide layer.Osmotic fragility. The cell wall is thought

to give gram-negative organisms not only theirparticular shape (34) but also their capacity totolerate wide variations in the osmotic pres-sure of the surrounding environment (8). Cellssuch as spheroplasts obtained by the action ofpenicillin contain defective walls too weak toresist the rapid influx of water that is pro-

duced upon transfer to hypotonic media(downshock, reference 6), and lysis occurs.

Since the cell shape of DA82 indicates thatit lacks a rigid cell wall, its osmotic fragilitywas investigated. DA82 logarithmic cellsgrown in minimal medium were washed andtransferred either to distilled water, M9 buffer,0.1 m NaCl solution, or S-broth. Changes inOD and number of viable cells were followed(Fig. 3). In buffer or plain 0.1 m NaCl, whichare unable to support growth but afford enoughosmotic strength for the cells to keep theirosmotic balance, a striking increase in thenumber of viable cells, corresponding to aboutthree residual divisions, occurs. The transfer todistilled water produces after long incubationa twofold reduction in the viable count. Thisdemonstrates that DA82 cells are not fragilebecause a rapid drop of at least 1,000-foldwould be expected if that were the case (17).This result, therefore, indicates that eventhough the cell wall of strain DA82 lacks therigidity necessary to maintain the normal ba-cillary shape, it still retains enough strength towithstand the cytoplasmic swelling producedby downshock.

Since cell wall rigidity is the factor whichlimits swelling in wild-type cells (26), largervolume changes would be possible for DA82 if

its wall were less rigid. To follow cell volumevariations as a function of external osmoticconcentration, advantage was taken of the factthat the refractive properties of the cell are

due to its dry matter and state of dispersion(16). Swelling produced by water intake modi-fies the dispersion and changes the refractiveindex and, consequently, the turbidity of thesuspension (reduction in OD). Tests were per-formed with DA82 and three control strains:LT2, which is the original wild-type S. typhi-murium strain; DA78, which is the LT2 deriv-ative that served as immediate parent forDA82 construction; and DA23, which likeDA82 has the double regulatory genotypehis W1824 his01242 but maintains the normalrod shape. The three control strains behavedalmost identically (Fig. 4). An increase in thesalt concentration, up to a molality of about0.2, produced a proportionate increase in theOD of the suspension. At this point a plateauwas reached corresponding to a 32% increaseover the OD in water. Further salt additionsbrought no turbidity changes until molalitygrew higher than 0.9, at which point a slow de-crease, due probably to plasmolysis, becameapparent. The response of DA82 was generallysimilar to the controls, but the initial slopewas much steeper and the maximal opticalchange was nearly twice the controls (Fig. 4).These results are consistent with DA82

having a more relaxed cell wall. This alterationwould make the cells capable of increasedvolume changes in response to external os-

motic variations, and it would preclude main-tenance of the rod shape typical of wild-typeSalmonella.Osmotic sensitivity. The results described

above clearly demonstrated the physical en-durance of DA82 under osmotic stress (Fig. 3).However, quite unexpected results suggestinganother kind of osmotic sensitivity were foundwhen DA82 cells were transferred to nutrientbroth without NaCl (NoS-broth). Cells placedin broth containing NaCl (S-broth) grow nor-

mally; however, cells stop growing when trans-

TABLE 1. Properties of strain DA78 and its derivative DA82.

Nutritional Lactose Response to Phage P22 ResponseStrain

reurmn tlzto tetmcnto 04-012 Cell shapeLysogeny Sensitivitya antiserum

DA78 Histidine5 r + s + ElongatedDA82 None -r + s + Spherical

a As both strains are lysogenic for P22, their sensitivity to this phage could not be demonstrated in conven-tional resistance tests; their sensitivity was concluded from the capacity of DA78 and DA82 to act as recipi-ents in P22-mediated transduction crosses.

bThe histidine requirement of DA78 was the marker employed for DA82 construction.

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ferred to NoS-broth, and after a short lag theybegin to die at exponential rate (Fig. 5). Thenumber of surviving cells after 5 hr of incuba-tion varies from 1 to 20% of the initial number.The decrease in viability is not caused by

lysis of the cells because the fall in colony for-mers is not accompanied by a parallel reduc-tion in OD (Fig. 5). The OD reduction after 5hr is rather small (15%); yet only 10% of thecells survive. Experiments following survival,OD, and number of visible cells confirmed thatobservation. Furthermore, no induction of theP22 prophage carried by the strain was ob-served during the inactivation process.DA82 behavior is not general for S. typhimu-

rium strains or his W-hisO double histidine-regulatory mutants. Both the wild-type strain,LT2, and two other double mutants; DA23(his W1824 his01242) and DA18 (his W1824his01812), showed normal growth when theywere transferred from minimal medium tobroth either with or without NaCl.

Arresting of osmotic death. To confirm theosmotic nature of the effects on DA82, eithersalts or sucrose were added to cells that hadbeen incubated for 150 min in NoS-broth andwere therefore suffering exponential inactiva-tion. The addition of salts or sucrose almostinstantly restored growth and cell division(Fig. 6). The optimal concentration was 0.1 mfor NaCl, and the other agents also showedtheir optimal action at the osmotically equiva-lent concentrations. However, the efficienciesvaried, i.e., the most effective cation wasNH4+, followed by Mg2+, and lastly Na+.In two experiments where NaCl was comparedwith KCI, no difference in efficiency was ob-

served between them. Only two anions werecompared: Cl- proved to be slightly more ef-fective than SO42-. Sucrose was less efficientthan any of the salts employed.The cations tested appeared to have some

specific effects on DA82 morphology and ultra-structure. As explained above, cells grown in

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FIG. 3. Effect of distilled water and saline mediaon DA82 turbidity and viability. "S-broth" is nutri-tive broth containing 0.1 m NaCI, and "0.1 m NaCI"refers to a NaCI solution in distilled water. Solidlines, viability; dashed lines, OD at 650 nm.

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0.5 1.0 1.5 2.0 molalityFIG. 4. Turbidity changes of several bacterial suspensions as a function of NaCI molality in the sus-

pending fluid. Relative optical change stands for the difference between OD (650 nm) at each molality andOD in distilled water, expressed as per cent of the latter.

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FIG. 5. Effect of low-osmolality growth mediumon DA82 viability and turbidity. NoS-broth is nu-trient broth without NaCI, and S-broth is nutrientbroth containing 0.1 m NaCI. Solid lines, viability;dashed lines, OD at 650 nm.

,N

nutrient broth containing 0.1 m NaCl showedno anomalies other than their round shape(Fig. 2). However, a small proportion of thecells grown in 0.1 m NH4C1 broth had a some-what normal rod shape (Fig. 7), and thin sec-tions showed normal structures with few butwell defined membrane invaginations in someof the cells. The most striking effect was ob-served when 0.1 m MgCl2 broth was employed(the osmolality was in this case approximately100 milliosmol higher than in the first two).The cells kept the spherical shape, but in thinsections an extraordinary proliferation of themembrane was evident (Fig. 8). All along thecell periphery the membrane showed a sinuousprofile with some folds reaching deep into thecytoplasm. In some cells, a large cluster of ves-icles, resembling vesicular mesosomes of gram-positive bacteria (24), appeared near the edge.Factors influencing osmotic death. Sur-

vival after 5 hr was variable depending on theinitial number of cells: the higher the initialcell density, the higher the survival. Further-more, the phenomenon was transient: afterlong incubation, the surviving cells recoveredand growth started again. The duration of thelag before the cells recuperated depended alsoon the initial number of cells.

rrinutes

FIG. 6. Effect of addition of salts or sucrose on osmotic inactivation of strain DA82 in NoS-broth. DA82cells were grown and washed as described in the text. At 0 time, portions were inoculated in 0.1 m NH4CI-broth and in NoS-broth. After 150 min, 10-ml samples from the NoS-broth culture were withdrawn andadded to concentrated NoS-broth solutions of the osmotic agents. Volumes were calculated so as to obtainosmotically equivalent final concentrations for all the agents. Viable counts and OD (650 nm) readings ofthese cultures were corrected for the change in volume produced by the addition.

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FIG. 8. Cell of DA82 grown in 0.1 m MgCI2-broth. Marker bar represents 0.2 um.

Cells surviving nutritional downshock werenot resistant revertants. When they were re-grown in minimal medium until log phase andthen transferred back to NoS-broth, they dis-played the same sensitivity as the original cul-ture. Therefore, recovery from osmotic sensi-tivity appears to result from the adaptation ofthe surviving cells to the poor osmotic condi-tions.

The osmolality of the medium used to growthe cells prior to the transfer appears to influ-ence their behavior in NoS-broth: higher osmo-lalities gave higher lethalities after thetransfer. More important than the absolutedrop in osmolality was, however, the finalosmotic level. When DA82 cells were grown inbroth having a high osmolality, about 1,000milliosmol, and then transferred to 200 mil-

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liosmol broth, there was no death and growthcontinued without a lag. On the other hand,when cells grown in 800 milliosmol broth wereput in NoS-broth (40 milliosmol), so that thedecrease in osmolality was almost the same asin the first case, strong growth inhibition andcell death occurred.Temperature also affected DA82 osmotic

performance. Experiments similar to that de-scribed in Fig. 5 were carried out at 25, 30, 37,and 43 C. It was observed that the rate ofdeath depended on the temperature of incuba-tion in NoS-broth, the rate increasing withtemperature. It was also found that, eventhough growth rate in S-broth was about thesame at 37 and 43 C, the rate of death in NoS-broth was much higher at 43 C.At no temperature was killing complete. It

may be assumed that part of the populationwas in a state refractory to osmotic death.Moreover, the cells that survived killing at 37C and lower temperatures were capable ofachieving the condition that allowed growth toproceed in NoS-broth. In contrast, 43 C survi-vors did not appear to be able to undergo thenecessary change because no recovery was everobserved at 43 C. However, since spontaneousrecovery was found to be faster at lower tem-peratures, one might assume that recovery at43 C was too slow to be detected under theconditions employed.Action of chloramphenicol and nalidixic

acid. The appearance of osmotic death re-quires protein synthesis; chloramphenicol ad-dition at the time of downshock in NoS-brothprevents cell death (Fig. 9). The increase inNoS-broth osmolality produced by chloram-phenicol added (20 ,ug/ml) was negligible;therefore, its action could not be ascribed tosimple osmotic protection.

Nalidixic acid, an inhibitor of DNA syn-thesis, affects DA82 similarly in NoS-brothand in S-broth (Fig. 9). In both cases, therewas a fast descent in the number of viablecells; this action was more intense than thatcaused by the low osmolality of NoS-brothalone. Since nalidixic acid shows bactericidalactivity only on actively metabolizing cells (7),these results indicate that the function neces-sary for nalidixic acid lethality remains activein cells undergoing osmotic death.

Sensitivity to antibiotics. Because the in-creased elasticity shown by the DA82 cell wall,as well as its peculiar osmotic behavior, impli-cates the envelope as a basic abnormal prop-erty of the strain, experiments were directed tocheck the response of DA82 to some antibioticswhose action is exerted on the cell wall or themembrane.

The antibiotics employed were penicillin,which inhibits the cross-linking reaction in thesynthesis of cell wall murein (31); D-cycloser-ine, which interferes with the synthesis of thedipeptide D-alanyl-D-alanine (32) and resultstherefore in the production of a defectivemurein; vancomycin and bacitracin, whichinterfere at different steps in the utilization ofthe membrane lipid intermediates partici-pating in the transfer of new units to thegrowing cell wall (2, 29). The last two anti-biotics have almost no effect on whole gram-negative cells but are fully active on their "invitro" murein synthesizing system. This evi-dence has lead to the conclusion that gram-negative walls hamper the access of thosedrugs to the inner sensitive target (31).The experiments, conducted as described

above, showed that DA82 growth was com-pletely inhibited by five IU of penicillin/ml or20 ,tg of D-cycloserine/ml. These results indi-cate that DA82 sensitivity to both drugs is in-creased because higher concentrations (penicil-lin, 15 IU/ml; D-cycloserine, 40 ,ug/ml) wererequired to suppress growth of the wild-typeand several control strains.With respect to vancomycin and bacitracin,

DA82 proved to be as resistant as the wildtype to the action of 200 ,ug of vancomycin/mlor 150 ,gg of bacitracin/ml. However, these andlower concentrations were found to be inhibi-tory for another strain, DA88, which is derivedfrom the same parental strain as DA82(SB564), and also makes round cells. It istherefore concluded that the DA82 cell wallalteration does not make accessible the sites ofthe envelope where the bacitracin- and vanco-mycin-sensitive enzymes are located.

Sensitivity to some drugs and lysozyme.The sensitivity of DA82 to some other sub-stances was investigated. No differences withwild-type behavior were found with respect tosensitivity to triphenyltetrazolium chloride (upto 5 mg/ml), acridine orange (up to 100 ,lg/ml),and EDTA (up to 5 x 10- 3 M).

Gram-negative organisms are resistant tothe action of lysozyme unless they are firstsensitized by special treatments that allowlysozyme to traverse the lipopolysaccharidebarrier (22). Sensitivity to lysozyme of un-treated DA82 was checked: like wild-typecells, logarithmic DA82 cells, grown either inS-broth or minimal medium, were completelyresistant to the action of the enzyme when ei-ther turbidity or viability was measured.

DISCUSSIONThe inability of DA82 cells to keep their

normal shape and the increased elasticity of

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their cell walls indicate that the envelope ofDA82 is in some way defective. Both the shapeand the rigidity of the cell are considered to beproperties of murein (18, 34), the polymer thatforms the inner taut layer of the cell wallclosest to the membrane (20). Defectivemurein synthesis or deposition would thusseem likely in DA82.The murein layer is not a conspicuous ele-

ment of the gram-negative wall as seen in elec-tron micrographs of thin sections, and unlessspecial care in fixation and staining is taken, itis usually not noticeable (20). It is thereforenot surprising that sections of DA82 cellsgrown in NaCl-broth at 37 C do not show ab-normalities other than in cell shape.The mode of action of penicillin has been

demonstrated to occur by the inhibition of thelast step in murein biosynthesis: the peptidecross-linking reaction (31). The sensitivity ofDA82 to penicillin would then indicate thatthe penicillin-sensitive target remains intact inDA82. The same conclusion, but applied toanother step of murein making-the synthesisof the dipeptide D-ala-D-ala (32)-could bedrawn from the strain's high sensitivity to D-cycloserine. Furthermore, DA82 appears toretain normal lipopolysaccharide since itreacts with antiserum specific for 04-012 so-matic antigens. It is also resistant to the actionof lysozyme, vancomycin, and bacitracin,which are excluded by the normal gram-nega-tive wall, and its normal sensitivity to acridineorange and several other substances suggeststhat permeability is not altered either.

Although DA82 appears to have a faultywall, the observations concerning its behaviorafter downshock delineate a syndrome clearlydifferent from the one usually ascribed to cellwall defects. DA82 cells are not osmoticallyfragile; they withstand suspension in distilledwater without disruption or death. However, inlow-osmolality growth media, they show apeculiar osmotic sensitivity manifested ingrowth inhibition and exponential death. Thisosmotic death is only shown when the cells areable to grow and synthesize proteins.The osmotic nature of DA82 sensitivity is

demonstrated by the capacity of several saltsand sucrose, in osmotically equivalent concen-trations, to stop death and to reinitiatewithout delay growth and cell division. Thefact that osmotic death is not accompanied bylysis until inactivation is well advanced arguesagainst death being attributable to impairmentof wall synthesis produced by osmotic condi-tions unsuitable for the mutant. Lysis appearslater and is probably produced by the autolyticsystems of the cells working on already dead

FIG. 9. Effect of chloramphenicol (CF) and nali-dixic acid (NA) on the viability of DA82 in NoS- andS-broth. CF was used at 20 dig/ml and NA at 10iug/ml.

bacteria.The bacterial cell possesses mechanisms that

allow it to modify its internal osmolality ac-cording to the osmotic conditions of the ex-ternal medium (8). It has been postulated (8)that the structures engaged in the regulation ofintracellular osmolality are located in the cellmembrane and are sensitive to alterations ofthe osmotic equilibrium between cytoplasmand medium. The osmotic behavior of DA82could be attributed to some kind of membranedisfunction that affects its functioning underconditions of osmotic stress.

It is not known whether the cell wall and themembrane have been affected by the samemutational event or whether two independentmutations have produced the complex pheno-type of DA82. Since some steps of murein andlipopolysaccharide biosynthesis are connectedwith the membrane (2, 14), a membrane muta-tion could easily result in cell wall defective-ness. On the other hand, the laxity of theDA82 cell wall could, perhaps, by itself pro-duce the membrane disfunction observed inthe strain if the excessive dilatation that it al-lows upon downshock affects the maintenanceof membrane integrity when growth can pro-ceed.

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The loss of viability that occurs when DA82is shifted to NoS-broth cannot be explained.The consequences of nutritional downshock onmacromolecular synthesis, permeation, andenergy flow in strain DA82 are presently beinginvestigated. Phenomena somewhat similar tothe osmotic death described in this paper havebeen reported for fatty acid auxotrophs andphospholipid mutants of Escherichia coli (9-11). The inactivation in these mutants was at-tributed to membrane-defective synthesis (11)or to conformational changes that render themembrane inactive or defective (9). Similarexplanations could be valid in the case ofDA82, but the possible action of a membrane-degradative system, induced during the tem-porary disability of the membrane and activeonly under some conditions, should also beconsidered.

ACKNOWLEDGMENTSThis investigation was supported by grants from Consejo

Nacional de Investigaciones Cientificas y Tecnicas (Argen-tina) and Liga Argentina de Lucha contra el Cancer.

The author gratefully acknowledges the hospitality andunfailing support received from Luis V. Orce and the staff ofthe Laboratory of Radiomicrobiology where this work wascarried out. Thanks are due to Elsa Ant6n for her help withthe electron microscope techniques and to Philip E. Hart-man, Mary Jane Voll, and Holman Massey for criticalreading of the manuscript.

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