JOURNAL OF BACTERIOLOGY, May 1975, p. 650-659Copyright i 1975 American Society for Microbiology

Vol. 122, No. 2Printed in U.SA.

Interaction of Mg2+ with Peptidoglycan and Its Relation to thePrevention of Lysis of a Marine Pseudomonad

M. KHALIL RAYMAN' AND ROBERT A. MAcLEOD*

Department ofMicrobiology, Macdonald Campus of McGill University, and Marine Sciences Centre, McGillUniversity, Montreal, Quebec, Canada

Received for publication 7 December 1974

Intact cells of marine pseudomonad B-16 (ATCC 19855) which have beenwashed with a solution of NaCl require only 0.001 M MgSO, and 100 to 300 timesthis concentration of NaCl or KCI to prevent lysis. Conversion of intact cells tomureinoplasts, a process involving removal of the outer double-track layer (outermembrane) and the periplasmic space layer of the cell wall, approximatelydoubled the requirement for the three salts to prevent lysis. The formation ofprotoplasts from mureinoplasts by removing the peptidoglycan layer againdoubled the requirement for Na+ and K+ salts but increased the requirement forthe Mg2+ salt 200- to 300-fold. Cells of the marine pseudomonad suspended insolutions containing Mg2+ salts failed to lyse on subsequent repeated suspensionin distilled water, whereas cells presuspended in NaCl lysed immediately.Isolated envelope layers including the peptidoglycan layer, when dialyzedagainst solutions containing Mg2+ salts, retained Mg2+ after subsequent suspen-sion in distilled water. Envelope layers exposed to solutions of Na+ or K+ saltsfailed to retain these ions after exposure to distilled water. Na+ displaced Mg2+from the cell envelope layers. The results obtained indicate that the capacity ofMg2+ salts at very low concentration to prevent lysis of intact cells andmureinoplasts of this organism is due primarily to the interaction of Mg2+ withthe peptidoglycan layer of the cell wall. Ion interaction with the layers lyingoutside of the peptidoglycan layer contributes only a small amount to themechanical strength of the wall.

One of the characteristics which distinguishgram-negative marine bacteria from their ter-restrial and fresh-water counterparts is theirtendency to lyse when suspended in fresh water(15). The necessity for salts in the suspendingmedium to prevent lysis was long considered toreflect a requirement of marine bacteria for amedium of suitable osmotic pressure (12).When individual salts were tested, however, itwas observed that different salts differed intheir capacity to prevent lysis. Twice as muchKCl or NH4Cl was required to prevent lysis asNaCl or LiCl (D. B. Pratt and W. H. Riley,Bacteriol. Proc. 55:26, 1955) (17) and salts ofsuch divalent cations as Mg2+, Ca2+, or Mn2+were effective at concentrations approximately100-fold lower than those of the monovalentcations (16).

Studies with isolated cell envelopes of marinebacteria showed that soluble material of com-plex composition tended to separate from the

'Present address: Health and Welfare Canada, HealthProtection Branch, Bureau of Microbial Hazards, Ottawa,Ontario, Canada.

envelopes at low salt concentration (1, 2, 3).When cells of a marine pseudomonad weresuspended repeatedly in a concentration ofsucrose sufficient to prevent lysis, electron mi-croscopy revealed that the outer double-tracklayer (cell wall membrane) of this gram-nega-tive bacterium separated from the cells andcould be recovered as fragments in the suspend-ing medium (4). Observations such as these ledto the conclusion that inorganic ions protectmarine bacterial cells against lysis primarilythrough their capacity to interact with cell en-velope components, thereby increasing the me-chanical strength of the cell wall (3, 4). Theseobservations were confirmed and extended byDeVoe and Oginsky (5, 6) who found that thesusceptibility of cells of a marine pseudomonadto lysis in distilled water was conditioned by thesalt composition of the medium to which thecells were preexposed. Cells which had beensuspended in a MgCl2 solution failed to lyse onsubsequent suspension in distilled water, butdid lyse if Na+ was present in the solution inaddition to Mg2+. Since Na+ could displace

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Mg2+ from isolated envelopes of the marinepseudomonad, it was concluded that the twoions competed for anionic sites in the cellenvelope.Unemoto et al. (21) observed that the amount

of K+ required to prevent lysis of the marinebacterium Vibrio alginolyticus decreased as theinternal solute concentration decreased andconcluded that K+ prevented lysis by balancingthe internal osmotic pressure in the cells. Sincethe amount of Na+ required to prevent lysis didnot vary with the internal solute concentration,they concluded that Na+ in contrast to K+protected the cells against lysis by interactingwith and thereby adding to the strength of thecell envelope of the organism.

Procedures have been devised to separatesequentially and isolate the various layers of thecell envelope of marine pseudomonad B-16 (8).This capability has presented us with an oppor-tunity to assess the extent of the contribution ofeach of the envelope layers to the mechanicalstrength of the wall in the presence of varioussalts.

MATERIALS AND METHODSOrganism. The organism used in this study was

marine pseudomonad B-16 (ATCC 19855, NCMB 19).This bacterium has been named Alteromonashaloplanktis by Reichelt and Baumann (19). A num-ber of variants of this organism have been recognized(11). The variant used in this study was variant 1. Themethods used to maintain the organisms have beendescribed (11).Growth conditions. The organism was grown in a

basal medium containing 0.8% nutrient broth (Difco)and 0.5% yeast extract (Difco) in a salt solutionconsisting of 0.2 M NaCl, 0.05 M MgSO4-7H,0, 0.01M KCI, and 0.1 mM FeSO,(NH4)2SO4.

Preparation of cells and cell forms for lyticstudies. Cells were prepared for lytic susceptibilitytests by suspending in and centrifuging from volumesof washing solution equal to the volume of growthmedium three times. Unless otherwise stated, thewashing solution was 0.5 M NaCl. Mureinoplasts andprotoplasts were prepared by slight modifications ofprocedures which have been described (8). Mureino-plasts were obtained by washing cells first with 0.5 MNaCl three times followed by three suspensions in andcentrifugations from 0.5 M sucrose. Protoplasts wereprepared by suspending mureinoplasts at 15 to 20 mg(dry weight) per ml in a solution containing 0.05 Mtris(hydroxymethyl)aminomethane (Tris)-hydrochlo-ride buffer (pH 8.0), 0.005 M ethylenediaminetetra-acetic acid (EDTA), and 300 ug of lysozyme per ml in0.5 M sucrose.

Lytic susceptibility tests. For studies of lyticsusceptibility, suspensions of intact cells, mureino-plasts or protoplasts were adjusted to contain theequivalent of 0.6 to 0.8 mg (dry weight) of cells per ml.A 1:25 dilution of the suspension was then made in-the

test system and the turbidity measured at 420 nmafter 5 min. The suspension was then centrifuged at10,000 x g for 15 min and the absorbancy of thesupernatant solution at 260 nm was determined.

Isolation of envelope layers. The loosely boundouter layer, the outer membrane (outer double-tracklayer), the periplasmic space (underlying) layer, thepeptidoglycan layer, and the cytoplasmic membranewere isolated essentially as described previously(8-10, 18). The periplasmic space in this organism hasbeen found not to be a void but to contain lipid,protein, and carbohydrate (including lipopolysaccha-ride [J. Nelson and R. A. MacLeod, unpublishedobservations ]) in amounts sufficient to account for 6%of the dry weight of the cells (9).

Cation-binding experiments. Solutions or homo-geneous suspensions of each of the envelope layerscontaining 2 to 6 mg (dry weight) of material weredialyzed for 24 h at 4 C against each of 0.3 M NaCl,0.05 M MgSO,, 0.01 M KCl, Complete salts solution(0.3 M NaCl + 0.05 M MgSO4 + 0.01 M KCl), ordistilled water. At the end of 24 h the cells weredialyzed for a further 48 h against distilled water.The dialyzed samples were wet ashed with HClO4

using a slight modification of a procedure describedby Sanui and Pace (20). Na+ and K+ were determinedby flame photometry, Mg2+ by atomic absorptionspectrophotometry using a Zeiss PMQ II spectropho-tometer with flame and atomic absorption attach-ments.

Determination of hexosamine. The procedureused to hydrolyze the samples and determine hexosa-mine has been described (8).

RESULTS

Comparative lytic susceptibility of intactcells, mureinoplasts and protoplasts. Whencells of marine pseudomonad B-16 are washedfree of medium components with 0.5 M NaClthe loosely bound outer layer of the cells isremoved (8). Since the cells are fully viableafter this treatment and in the form used inprevious lytic studies (17) such cells were con-sidered to be intact and served as the startingpoint in the present investigation.The comparative lytic susceptibility of intact

cells, mureinoplasts, and protoplasts in thepresence of NaCl was compared by determin-ing the concentration of NaCl at which amarked decrease in turbidity of suspensions ofthe cell forms and increase in ultraviolet (UV)absorbance of the corresponding supernatantsolutions occurred (Fig. 1). The results showthat only slightly more NaCl was required toprevent the lysis of mureinoplasts than intactcells. Since to produce mureinoplasts, the twoouter layers of the cell wall must be removed, itis evident that these two outer layers contributelittle, in the presence of NaCl, to the preventionof lysis of the cells. Protoplasts, on the other

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FIG. 1. Comparative lytic susceptibility of intact cells, mureinoplasts, and protoplasts of marine pseudomo-nad B-16 in the presence of NaCI. (A) Change in percent transmission (turbidity) of suspensions of: curve 1,intact cells; curve 2, mureinoplasts; curve 3, protoplasts. (B) UV absorbance of the supernatant solutionsobtained from suspensions corresponding to curves 1, 2, and 3 in Fig. 1A.

hand, required appreciably more NaCl to pre-vent them from lysing than did the other twocell forms. Since mureinoplasts are converted toprotoplasts by removing peptidoglycan from thesurface of the mureinoplasts (8), the increase inthe requirement for NaCl to prevent the lysis ofthe protoplasts can be ascribed to the loss of thepeptidoglycan layer.Previous studies have shown that more KCl

than NaCl is required to prevent the lysis ofintact cells of various marine bacteria (17, 21).

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The results, using turbidity measurements (Fig.2A), indicate that this applies for each of thecell forms of B-16 tested. Changes in the UVabsorbance of the supernatant solutions, notshown, confirmed this observation.The effect of sucrose concentration on the

lysis of the various cell forms is shown in Fig.2B. Removing the various cell wall layers in-creased the concentration of sucrose required toprevent the decrease in turbidity of suspensionsof the various cell forms by a surprisingly small

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KCI (M) SUCROSE (M)FIG. 2. Comparative lytic susceptibility of intact cells (curve 1), mureinoplasts (curve 2) and protoplasts

(curve 3) in the presence of (A) KCI and (B) sucrose as measured by following decrease in percentage oftransmission (turbidity) of suspensions of the cell forms as the solute concentration is lowered.

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INTERACTION OF Mg2+ WITH PEPTIDOGLYCAN

amount. Though not shown, the concentrationsof sucrose at which decreases in turbidity oc-curred were found to correspond to the concen-trations at which increases in UV absorbance ofthe supernatant solutions took place. The re-sults indicate that lower osmolar concentrationsof sucrose than either NaCl or KCl are requiredto prevent the lysis of the various cell forms,particularly the protoplasts.Mg2+ and other divalent cations prevent lysis

of intact cells of the marine pseudomonad at0.01 of the concentration of salts of the mono-valent cations (16). The results in Fig. 3 showthat mureinoplasts required only slightly morebut protoplasts required 200- to 300-fold moreMg2+ than did intact cells to prevent lysis. Inthe case of protoplasts the amount of Mg2+and Na+ needed to prevent lysis was nearlythe same.Conversion of mureinoplasts to

protoplasts. In the absence of EDTA andTris-hydrochloride buffer the conversion of mu-reinoplasts to protoplasts by lysozyme required5 h of incubation to reach approximately 90%completion. In the presence of EDTA andTris-hydrothloride buffer conversion was essen-tially complete in 20 min. This observationsuggested that even though the three outerlayers of the cell wall had been removed in theformation of the mureinoplasts there was still abarrier to the action of lysozyme on the peptido-glycan layer of these forms that was removableby adding EDTA and Tris-hydrochloride buffer.

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To examine this possibility, the hexosaminecontent of the cells was determined after vari-ous treatments. The results (Table 1) show thatmost (81%) of the hexosamine was lost when thecells were converted to mureinoplasts. Another8% was lost, however, after treatment of themureinoplasts with EDTA in the presence of

TABLE 1. Hexosamine content of intact cells andrelated forms of marine pseudomonad B-16 after

various treatments

Hexosaminea

Cell form Treatment A B

(%) (%)Intact cells Washed three time 3.35 ± 0.02 100

in 0.5 M NaClMureinoplasts As intact cells 0.63 ± 0.06 18.8

followed by threewashes in 0.5 Msucrose

Mureinoplasts As in mureinoplasts 0.28 ± 0.06 8.3followed by incu-bation in EDTA-Tris-hydrochloridefor 1 h

Protoplasts As in mureinoplasts 0.06 ± 0.03 1.7followed by incu-bation in EDTA-Tris-hydrochloridelysozyme for 15to 45 min

a In column A hexosamine is expressed as a percentage ofthe dry weight of the intact cells. In column B hexosamine isexpressed as a percentage of the total present in the intactcells.

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FIG. 3. Comparative lytic susceptibility of intact cells, mureinoplasts, and protoplasts of marine pseudomo-nad B-16 in the presence of MgSO4 as measured by following (A) the decrease in turbidity of suspensions of thecell forms and (B) the increase in UV absorbance of the corresponding supernatant solutions. Curve 1, intactcells; curve 2, mureinoplasts; curve 3, protoplasts.

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Tris-hydrochloride buffer.Treatment of mureinoplasts with EDTA-

Tris-hydrochloride increased by only a smallamount the concentrations of NaCl, KCl, andsucrose required to prevent lysis of these cellforms. With these solutes, the decreases inturbidity obtained as the solute concentrationwas lowered were paralleled by increases in theextent of release of UV-absorbing material. Inthe case of MgSO4, treatment of mureinoplastswith EDTA-Tris-hydrochloride increased by a

similar small amount the concentration of thissolute required to prevent lysis of this cell formas measured by release of UV-absorbing mate-rial (Fig. 4B). As shown in Fig. 4A, however, inthe case of the treated mureinoplasts, the de-crease in the turbidity of the suspensions as theMgSO, concentrations were lowered was more

than proportional to the release of UV-absorb-ing material.

Effect of the composition of the washingsolution on the lytic susceptibility of intactceils. Cells of marine pseudomonad B-16 andother marine bacteria (5, 21), when washed freeof medium components with 0.5 or 1.0 M NaCl,lyse when subsequently suspended in distilledwater. Studies in our laboratories (T. I. Matula,Ph.D. thesis, McGill University, 1967) andelsewhere (5) have shown, however, that if cellsof the organisms are washed with a solution of aMg2+ salt, the cells do not lyse on subsequentsuspension in distilled water. Lysis of Mg2+washed cells did occur, however, if the cells weresuspended in a solution of NaCl before suspen-sion in distilled water (5).The effect of the composition of the salt

solution to which the cells are preexposed on the

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lytic susceptibility of cells of marine pseudomo-nad B-16 was examined. In these experimentsthe cells of the organism were first washed freeof medium components with Complete saltssolution, a solution similar to that used for thepreparation of the growth medium. The washedcells were then suspended in and centrifugedfrom a test solution three times after which theywere suspended repeatedly in distilled water.When the test solution was 0.5 M NaCl (Fig. 5),the suspensions maintained a constant turbid-ity after each successive suspension in the NaClsolution and there was a negligible release ofUV-absorbing material. When these cells weretransferred to distilled water there was a de-crease in turbidity of the suspension and a

corresponding increase in the UV absorption ofthe supernatant solution, indicating that lysisof the cells had occurred.When the test solution was the Complete

salts solution (Fig. 6), subsequent suspensionin distilled water did not cause a large decreasein turbidity or release of UV-absorbing material.There was instead a decrease in turbidity to a

value which remained nearly constant over fivesuccessive suspensions of the cells in distilledwater and a small loss of UV-absorbing materialwhich decreased on each suspension. Observa-tion of the cells by phase contrast microscopyafter the successive suspensions in distilledwater revealed that the cells were still intact butthat they had lost much of their opacity. Afterthe cells had been suspended five times indistilled water they were resuspended in 0.5 MNaCl. On the first suspension there was a

release of UV-absorbing material though littlechange in optical density. Subsequent suspen-

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INTERACTION OF Mg2+ WITH PEPTIDOGLYCAN

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NUMBER OF SUSPENSIONSFIG. 5. The effect of suspending cells of marine

pseudomonad B-16 successively in 0.5 M NaCI anddistilled water on the turbidity (percentage of trans-mission of each suspension) (open bar) and the UVabsorbancy (absorbance [260 nm ]) of the supernatantsolution obtained from each suspension (solid bar).

sion of the cells in distilled water caused an

additional release of UV-absorbing materialand a further reduction of the optical density ofthe suspension.A picture essentially identical to that shown

in Fig. 6 was observed if cells were suspendedthree times in a test solution consisting of 0.05M MgSO, solution rather than Complete saltssolution before suspension in distilled water.Separate experiments also established that cellsafter first being exposed either to Completesalts solution or to 0.05 M MgSO, were stillintact after as many as nine subsequent suspen-sions in distilled water.Capacity of cell envelope layers to bind

cations after exposure to distilled water. Theprevious experiments showed that cells whichhad been exposed to a solution containing a

Mg2+ salt remained intact after subsequentrepeated suspensions in distilled water, whereasthose exposed to NaCl did not. To determinewhether this phenomenon could be related tothe ions bound to the various layers of the cellenvelope after exposure to distilled water, the

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FIG. 6. The effect of suspending cells of marinepseudomonad B-16 successively in Complete saltssolution, H20, 0.5M NaCI, and H20 on the turbidityof each suspension and the UV absorbancy of thesupernatant solution obtained from each suspension.Designations as in Fig. 5.

following experiment was performed. The vari-ous layers of the cell envelope were separatedand isolated. Since the procedures used toobtain the layers would be expected to disturbthe normal ionic relationships in the cell enve-lope, the layers after isolation were first equili-brated with salts by dialyzing against Completesalts solution before exposing the layers todistilled water. The effect of equilibrating thelayers against the various individual compo-nents of the Complete salts solution beforeexposure to distilled water was also tested.The results in Table 2 show that when the

layers of the cell envelope were equilibratedwith Complete salts solution all the layersexcept the cytoplasmic membrane containedappreciable amounts of Mg2+ after subsequentexposure to distilled water. When the layerswere exposed to MgSO, alone at the concentra-tion present in the Complete salts solution, theamount of Mg2+ which remained bound afterdialysis of the layers against distilled water wasnot appreciably different from that of the layersequilibrated with Complete salts solution. Lay-ers equilibrated with solutions of Na+ or K+followed by dialysis with distilled water con-tained very little of either ion and even lessMg2+ than layers equilibrated with distilledwater. This latter observation is particularlyevident in the case of both the loosely boundouter layer and the peptidoglycan layer whichstill contained appreciable amounts of residualMg2+ after isolation.The results obtained with the loosely bound

outer layer and the peptidoglycan layer indicate

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that both Na+ and K+ can displace Mg2+ fromthese layers. Since there was insufficient resid-ual Mg2+ in the other layers after isolation todetermine whether monovalent cations coulddisplace Mg2+, the outer double-track layer andthe underlying or periplasmic space layer afterisolation were preloaded with Mg2+ by equili-brating with 0.05 M MgSO4 before testing forthe capacity of Na+ to displace the Mg2+. Theresults in Table 3 show that the Mg2+ bound toeach of the two layers could indeed be displacedby dialysis with NaCl. The Na+ which replacesthe Mg2+ is largely removed, however, by subse-quent dialysis against distilled water. Previousstudies have shown that when cell envelopes ofthis organism in NaCl are diluted with waterthe pH of the suspending medium rises (3).Thus, at low Na+ concentrations, Na+ is re-placed by H+ as the counterion in the envelopelayers.When peptidoglycan was preloaded with

Mg2+, the amount of Mg2+ still associated withthe layer after 48 h of dialysis against distilledwater was determined to be 0.41 ± 0.04 ,molper mg (Table 2). The results in Fig. 6 show thatwhen intact cells of the organism were exposedto a solution containing Mg2+ the cells were stillintact after five subsequent suspensions in dis-tilled water. When isolated peptidoglycan waspreloaded with Mg2+ and subjected to suspen-sion in and centrifugation from distilled waterfive times, the amount of Mg2+ still associatedwith the peptidoglycan was found to be 0.37 4

0.02 ,umol per mg.

TABLE 3. Capacity ofNa+ to displace Mg'+ from theouter double-track and underlying (periplasmicspace) layers of marine pseudomonad B-16

Dialysis with 0.3 M NaCla Na+Mgg +(;MO,/mg) fiumoV/mg)

Before dialysisOuter double-track layer 0.12 0.22Underlying (periplasmic 0.16 0.35

space) layer

After dialysisOuter double-track layer 0.12 0.01Underlying (periplasmic 0.12 0.01

space) layer

a Before dialysis with 0.3 M NaCl the layers wereequilibrated with 0.05 M MgSO,, dialyzed againstdistilled water to remove excess MgSO4 and analyzedfor Na+ and Mg2+. The layers were then dialyzedagainst 0.3 M NaCl fpllowed by dialysis againstdistilled water to remove excess NaCl and againanalyzed for Na+ and Mg2+.

DISCUSSIONThe greatest change in the capacity of solutes

to prevent lysis of marine pseudomonad B-16occurred when mureinoplasts were converted toprotoplasts. The change was most evident withMgSO4. Intact cells and mureinoplasts wereprevented from lysing when as little as 0.001 to0.002 M MgSO, was present in the suspendingmedium. Protoplasts, on the other hand, needed0.3 to 0.5 M MgSO, to maintain them intact.This latter concentration was similar to theconcentrations of NaCl or KCl required for thesame purpose and suggests that all three saltsprevent lysis of protoplasts largely by balancingtheir internal osmotic pressure. Comparison ofthe concentrations of the individual salts andsucrose to prevent the lysis of protoplasts indi-cates that somewhat higher osmotic pressuresare required to prevent lysis when salts are theosmotic support than when sucrose is used. Thissuggests that the solutions of individual saltsactually contribute to the fragility of the proto-plasts. Previous studies have shown that proto-plasts of this organism are most stable inbalanced mixtures of salts, particularly those ofNa+ and Mg2+ (7). Since protoplasts suspendedin MgSO4 alone are only slightly less stablethan when suspended in equiosmolar concentra-tions of sucrose, the 200- to 300-fold increase inthe requirement for MgSO, to prevent lysiswhen mureinoplasts are converted to proto-plasts cannot be ascribed to effects of the salt onthe stability of the protoplasts. Rather, thechange in requirement can be attributed to theremoval of peptidoglycan which occurs whenmureinoplasts are converted to protoplasts. Theresults indicate a specific interaction of Mg2+with the murein layer of this organism. Previousstudies have shown that the nature of the anioninvolved does not change the requirement forcations to prevent lysis (16).

Treatment of mureinoplasts with EDTA-Tris-hydrochloride released a small amount ofhexosamine-containing material and increasedthe ease with which mureinoplasts are con-verted to protoplasts. The most likely explana-tion for these observations is that a smallamount of the complex of lipid protein andcarbohydrate found in the periplasmic space (9)remains attached to the surface of the mureino-plast, probably via divalent cation cross-links,when the outer layers of the cell wall areremoved. This residual material is able tointerfere with the ability of lysozyme to reachthe peptidoglycan layer but does not changesignificantly the lytic response of the mureino-

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plasts in the presence of Mg2+ as measured byrelease of UV-absorbing material. The changein turbidity response to Mg2+ obtained afterEDTA-Tris-hydrochloride treatment of the mu-reinoplasts suggests that the material removedmay buffer the protoplast membrane frompermeability changes which could be inducedwhen MgSO, is present alone in the medium.Loss of low molecular weight internal solutesthrough increased membrane permeabilitycould lower the refractive index of the cells andhence the turbidity of their suspensions withoutlysis actually taking place.When cells of the marine pseudomonad are

exposed to solutions containing Mg2+, but notNa+ salts, they fail to lyse on subsequentsuspension in distilled water. This confirmsobservations made with another marine bacte-rium (5). The studies with the isolated cell walllayers show that when the layers are exposed tosolutions containing Mg2+ salts they retainMg2+ on subsequent suspension in distilledwater, whereas layers exposed to Na+ or K+salts do not. Furthermore, bound Mg2+ in thelayers could be displaced by Na+ in confirma-tion of observations made previously with iso-lated envelopes of a marine bacterium (5).When Na+ displaced Mg2+ it could not beshown to have replaced Mg2+ in the cell enve-lope structures after the latter had been subse-quently suspended in distilled water. This indi-cates that Mg2+ forms complexes with cellenvelope structures which are not readily disso-ciable while Na+ forms ones which are. Whenboth types of complex are suspended in water,the Mg2+ complexes are stable while the Na+complexes hydrolyze.Although Mg2+ can bind to all of the cell wall

layers, the lytic studies with the various cellforms shows that it is the capacity of peptido-glycan to bind Mg2+ that is responsible for thecapacity of Mg2+ at very low concentration toprevent cell lysis. The structure of the peptido-glycan of this marine bacterium has been shownto be typical of that of other gram-negativebacteria (10). Although this structure contains anumber of free amino and carboxyl groups inthe peptide side chains, only those involving thecarboxyl and amino groups of the diaminopi-melic acid of one peptide side chain and thecarboxyl and amino groups of the C terminalD-alanine of an adjacent side chain are in aposition to form a bidentate chelate complexwith Mg2+ (Fig. 7). Such a complex would notonly be extremely stable but would result in anincrease in the extent of cross-linking and hencein the mechanical strength of the peptidogly-

-GIcNAc-MWNAc)-

-(kt4c-MurNAc)- IL-oloL-oI

A 0-gb DAPDAP-

NM CHNH CH~I CM

C.4 ..CMOIC-C -C c-MOO- _S,2 NH_"CHCCM -.C

oI 3s-cCHo \^/0

H C-CMloI*00

FIG. 7. The possible mechanism of interaction ofMg2+ with the peptidoglycan of marine pseudomonadB-16. (A) Diagrammatic representation of a section ofthe peptidoglycan of a gram-negative bacteriumshowing the site of cross-linking between peptidesubunits; (B) two-dimensional structure of the area ofcross-linking showing cross-bridging via peptide bondformation; (C) possible mechanism of cross-linkingbetween peptide subunits via the formation of abidentate chelate complex with Mg2+.

can. Analysis has shown that the peptidoglycanof marine pseudomonad B-16 is cross-linked bypeptide bond formation to the extent of 45%(10). The comparative lytic susceptibility ofintact cells, mureinoplasts, and protoplasts inthe presence of sucrose shown in Fig. 2B indi-cates that in the absence of Mg2+, the peptido-glycan layer of this organism has little intrinsicmechanical strength.Although there is no direct evidence for the

type of chelate complex proposed, it is ofinterest that there are enough uncross-linkedresidues of mesodiaminopimelic acid (0.45,umol/mg), hence terminal D-alanine residues, tobind in a chelate complex the amount of Mg2+(0.41 + 0.04 umol/mg) which remains tightlybound to the peptidoglycan after exhaustivedialysis with water. Furthermore, such a chelatecomplex in the peptidoglycan molecule is steri-

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INTERACTION OF Mg2+ WITH PEPTIDOGLYCAN

cally possible. Three-dimensional molecularmodels of bacterial cell wall peptidoglycanshave been constructed (13). Using an appropri-ate model it has been possible to show thatMg2+ can form a chelate complex with thepeptidoglycan of the type shown in Fig. 5.

If Mg2+ is involved in the cross-linking of thepeptide subunits of the peptidoglycan of themarine pseudomonad, it is likely also to beinvolved in the cross-linking of the peptidogly-can of other organisms where the extent ofcross-linking through peptide bond formation isless than 100%. Such cross-linking could beexpected to contribute to the mechanicalstrength of the peptidoglycan of these orga-nisms as well. In the case of the marine pseudo-monad Mg2+ binding by the peptidoglycan isrequired to prevent lysis of cells in distilledwater. For a terrestrial species Mg2+ binding bythe cell wall appeared not to be necessary toprevent lysis (5). The difference may lie in therelative amounts of peptidoglycan present inthe marine and terrestrial species. Marine pseu-domonad B-16 contains one-half as muchpeptidoglycan as is present in E. coli (10). Thusit is possible that Mg2+ cross-linking is essen-tial for cell integrity only when the amount ofpeptidoglycan present in the cell lies below acertain critical amount.

In sea water Mg2+ is present at 0.05 M and isthe 4ivalent cfttion present in greatest amo4t(14). About one-fifth as much Ca2+ is alsopresent. Previous studies have shown that diva-lent cations prevent lysis of this marine pseudo-monad roughly in proportion to their capacityto form chelate complexes (16). It is thus likelythat under natural conditions, other divalentcations besides Mg2+ are also involved in form-ing chelate complexes with the peptidoglycanlayer of this organism.

ACKNOWLEDGMENTSThis research was supported by a grant from the National

Research Council of Canada.We are indebted to M. V. Kelemen and H. J. Rogers for

constructing a model of the Mg'+ peptidoglycan complex.

LITERATURE CITED1. Brown, A. D. 1960. Inhibition by spermine of the action of

a cell-wall lytic enzyme. Biochim. Biophys. Acta 44:178-179.

2. Brown, A. D. 1961. The peripheral structures ofGram-negative bacteria. I. Cell wall proteins and theaction of a lytic enzyme system of a marine pseudomo-nad. Biochim. Biophys. Acta 62:132-141.

3. Buckmire, F. L. A., and R. A. MacLeod. 1965. Nutrition

and metabolism of marine bacteria. XIV. On themechanism of lysis of a marine bacterium. Can. J.Microbiol. 11:677-691.

4. Costerton, J. W., C. Forsberg, T. I. Matula, F. L. A.Buckmire, and R. A. MacLeod. 1967. Nutrition andmetabolism of marine bacteria. XVI. Formation ofprotoplasts, spheroplasts, and related forms from agram-negative marine bacterium. J. Bacteriol.94:1764-1777.

5. DeVoe, I. W., and E. L. Oginsky. 1969. Antagonisticeffect of monovalent cations in maintenance of cellularintegrity of a marine bacterium. J. Bacteriol.98:1355-1367.

6. DeVoe, I. W., and E. 0. Oginsky. 1969. Cation interac-tions and biochemical composition of the cell envelopeof a marine bacterium. J. Bacteriol. 98:1368-1377.

7. DeVoe, I. W., J. Thompson, J. W. Costerton, and R. A.MacLeod. 1970. Stability and comparative transportcapacity of cells, mureinoplasts, and true protoplasts ofa gram-negative bacterium. J. Bacteriol.101:1014-1026.

8. Forsberg, C. W., J. W. Costerton, and R. A. MacLeod.1970. Separation and localization of cell wall layers of agram-negative bacterium. J. Bacteriol. 104:1338-1353.

9. Forsberg, C. W., J. W. Costerton, and R. A. MacLeod.1970. Quantitation, chemical characteristics, and ultra-structure of the three outer cell wall layers of agram-negative bacterium. J. Bacteriol. 104:1354-1368.

10. Forsberg, C. W., M. K. Rayman, J. W. Costerton, and R.A. MacLeod. 1972. Isolation, characterization, andultrastructure of the peptidoglycan layer of a marinepseudomonad. J. Bacteriol. 109:895-905.

11. Gow, J. A., I. W. DeVoe, and R. A. MacLeod. 1973.Dissociation in a marine pseudomonad. Can. J. Micro-biol. 19:695-701.

12. Harvey, E. N. 1915. The effect of certain organic andinorganic substances upon light production by lumi-nouS bacteria. Biol. Bull. 29:308-311.

13. Kelekih, M. V., and . 3. Rogs. 1tt1. Three-di1es-sional molecular models of bacterial cell wall mucoptp-tides (peptidoglycans). Proc. Natl. Acad. Sci. U.S.A.68:992-996.

14. Lyman, J., and R. H. Fleming. 1940. Composition ofseawater. J. Mar. Res. 3:134-146.

15. MacLeod, R. A. 1965. The question of the existence ofspecific marine bacteria. Bacteriol. Rev. 29:9-23.

16. MacLeod, R. A., and T. I. Matula. 1961. Solute require-ments for preventing lysis of some marine bacteria.Nature (London) 192:1209-1210.

17. MacLeod, R. A., and T. I. Matula. 1962. Nutrition andmetabolism of marine bacteria. XI. Characteristics ofthe lytic phenomenon. Can. J. Microbiol. 8:883-896.

18. Martin, E. L., and R. A. MacLeod. 1971. Isolation andchemical composition of the cytoplasmic membrane ofa gram-negative bacterium. J. Bacteriol.105:1160-1167.

19. Reichelt, J. L., and P. Baumann. 1973. Change of thename Alteromonas marinopraesens (Zobell andUpham) Baumann et al. to Alteromonas haloplanktis(Zobell and Upham) comb. nov. and assignment ofstrain ATCC 23821 (Pseudomonas enalia) and strainC-Al of DeVoe and Oginsky to this species. Int. J. Syst.Bacteriol. 23:438-441.

20. Sanui, H., and N. Pace. 1959. Sodium and potassiumbinding by rat liver cell microsomes. J. Gen. Physiol.42:1325-1345.

21. Unemoto, T., T, Tsuruoka, and M. Hayashi. 1973. Role ofNa+ and K+ in preventing lysis of a slightly halophilicVibrio alginolyticus. Can. J. Microbiol. 19:563-571.

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