JOURNAL OF BACTERIOLOGY, Feb. 1972, p. 895-905Copyright O 1972 American Society for Microbiology

Vol. 109, No. 2Printed in U.S.A.

Isolation, Characterization, and Ultrastructureof the Peptidoglycan Layer of a Marine

PseudomonadC. W. FORSBERG,I M. KHALIL RAYMAN,2 J. W. COSTERTON,3 AND ROBERT A. MAcLEOD

Department of Microbiology, Macdonald Campus, McGill University, and Marine Sciences Center, McGillUniversity, Montreal, Quebec, Canada

Received for publication 5 November 1971

The peptidoglycan layer of a marine pseudomonad was observed by electronmicroscopy in thin sections of plasmolyzed intact cells and mureinoplasts butnot in untreated intact cells. Only fragments of this layer could be isolated bysodium lauryl sulfate (SLS) treatment of mureinoplast envelopes. Sacculus-likepeptidoglycan structures were obtained from growing cells by immediate heatinactivation of cellular autolytic enzymes and subsequent SLS, trypsin, andnuclease treatments. Recently, similar peptidoglycan sacculus-like structureshave been obtained by adding SLS to the growing culture and treating the iso-lated particulate material with nucleases. Thin-sectioned and negativelystained preparations of whole cell peptidoglycan showed compressed profiles ofcell-shaped sacculi. Peptidoglycan prepared by SLS treatment of mureinoplastenvelopes had a similar composition to that prepared from whole cells. Themajor amino sugars and amino acids in the peptidoglycan component were glu-cosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid inthe molar ratios 1.18:1.24:1.77:1.00:0.79. Forty-five per cent of the e-aminogroups of diaminopimelic acid were cross-linked. The peptidoglycan was esti-mated to account for about 1% of the cell dry weight.

The peptidoglycan component of the cellwall has been identified as a dense-staininglayer 3 to 8 nm wide in thin sections of a largenumber of gram-negative bacterial species (12).This dense layer has not been detected insections of marine pseudomonads (3, 4, 6),even when Murray's technique was applied inhis own laboratory (4).The peptidoglycan is isolated and purified

by removing other cell wall components. In thecase of Salmonella, in one of the proceduresused, sodium lauryl sulfate (SLS)-treated cellwalls prepared from heat-inactivated cellswere treated with hot 45% phenol and subse-quently with proteolytic enzymes (25). Thisleft the peptidoglycan component of the cellwall in the form of a bag-shaped macromole-cule which has been referred to as a mureinsacculus (26). The application of similar proce-

I Present address: National Institute for Medical Re-search, Mill Hill, London, N.W.7, England.

2 Present address: Cell Biology Group, Faculty of Medi-cine, University of Toronto, Toronto, Ontario, Canada.

3Present address: Department of Biology, University ofCalgary, Calgary, Alberta, Canada.

dures has permitted the isolation of peptido-glycan from other gram-negative bacteria (13),and detailed analyses of the material havebeen made (16, 19).

Studies in this laboratory have provided asystem for the removal of the outer layers ofthe cell wall of a marine pseudomonad (9).When this gram-negative bacterium waswashed free of medium components with 0.5 MNaCl and subsequently suspended in 0.5 Msucrose, the outer three layers of the cell wallseparated from the cell leaving a rod-shapedcell form which has been referred to as a mur-einoplast (9). The mureinoplast, which con-tained all the muramic acid and diaminopi-melic acid in the cell, could be converted to aprotoplast lacking these peptidoglycan compo-nents by treatment with lysozyme.The present report shows that, under certain

circumstances, the peptidoglycan layer can bedetected by electron microscopy in thin sec-tions of cells and mureinoplasts of the marinepseudomonad. The peptidoglycan has beenisolated as a sacculus, and its composition, ex-tent of cross-linkage, and contribution to the

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FORSBERG ET AL.

cell dry weight have been established.

MATERIALS AND METHODSOrganism and culture maintenance. The

marine pseudomonad used in these studies and re-ferred to as B-16 has been classified by the TorryResearch Group, Aberdeen, Scotland, as a Pseudo-monas species type IV. The organism is deposited inthe collection of marine bacteria maintained by theTorry Research Station as NCMB 19 and in theAmerican Type Culture Collection as ATCC 19855.The methods used to maintain the culture and toobtain large populations of cells in the logarithmicphase of growth have been described (24). For thepreparation of peptidoglycan by method 4, SLS wasadded directly to the growing culture, the ferrousammonium sulfate ordinarily added to the growthmedium (24) being omitted.Production of mureinoplasts. The procedure

used to prepare mureinoplasts has been presentedpreviously (9). A modified method which is quickerhas been published (8).Preparation of plasmolyzed cells. The cells were

separated from the medium by centrifugation (4 C)and washed three times in a salts solution similar incomposition to that in the growth medium. The cellswere then suspended in 0.5 M sucrose at 25 C (9).

Isolation of peptidoglycan. Two procedures wereused initially to isolate peptidoglycan from intactcells. In one, cells were treated with SLS directly. Inthe other, growing cells were immediately pouredinto boiling 0.5 M NaCl before being treated withSLS.

In the first procedure, cells in the logarithmicphase of growth were harvested by centrifugation at10,000 x g for 10 min and washed three times at 4 Cby suspension in and centrifugation from volumes of0.5 M NaCl equal to the volume of the growth me-dium. Washed cells equivalent to 3.0 g dry weightwere suspended in 200 ml of 0.5 M NaCl, and 50 mlof a 25% aqueous solution of SLS was added drop-wise with stirring. Stirring was continued for 6 hr,after which the suspension was centrifuged at 73,000x g for 2 hr at 20 C. The supernatant solution wasremoved and centrifuged under the same conditions.The material which sedimented in the two centrifu-gations was pooled and suspended in 100 ml of 0.5 MNaCl. Sufficient SLS was added to give a final con-centration of 5%. The mixture was stirred at 20 C for3 hr, and the double centrifugation procedure wasrepeated. The colorless gel obtained was washeduntil free from detergent by repeated suspension inglass-distilled water and centrifugation at 73,000 x gfor 1 hr.

In the second method, the medium containingcells in the logarithmic phase of growth was takenfrom the shaker and poured immediately into anequal volume of boiling 0.5 M NaCl. Heating wascontinued for 10 min, after which the suspension wascooled and centrifuged at 16,000 x g for 20 min. Thecells were washed twice by suspension in and cen-trifugation from volumes of 0.5 M NaCl equal to thevolume of the growth medium. The cells weretreated with SLS, and the insoluble residue was

freed from the detergent in the same way as in thefirst procedure. The washed residue from 3.0 g dryweight of cells was suspended in 100 ml of 5 mM tris-(hydroxymethyl)aminomethane (Tris)-hydrochloridebuffer (pH 8.0) containing 100 ,g of trypsin per ml(Calbiochem, A grade). The mixture was stirred at20 C for 20 min, at the end of which period the pre-viously turbid suspension was completely translu-cent. The suspension was centrifuged at 73,000 x gfor 1 hr, and the gelatinous material which sedi-mented was suspended in 100 ml of a solution con-taining 5 mM MgSO4, 5 mm Tris (pH 8.0), 20 ;&g ofdeoxyribonuclease per ml, and 10 Mg of ribonucleaseper ml. The suspension was incubated at 20 C for 20min, after which it was centrifuged at 73,000 x g for1 hr. The material which sedimented was washedthree times by suspension in and centrifugation fromdistilled water.A third procedure involved the isolation of pepti-

doglycan from mureinoplast envelopes. Mureino-plasts were prepared from intact cells of the marinepseudomonad by procedures described previously (9).Envelopes were prepared from the mureinoplasts inthe manner described previously for the preparationof envelopes from intact cells of this organism (4).The mureinoplast envelopes were then treated withSLS in the manner described for the isolation ofpeptidoglycan by treating cells with SLS directly.

All of the data reported in this paper were ob-tained from peptidoglycan isolated by one or other ofthe above procedures, as indicated in the text. Morerecently, however, a fourth procedure for isolation ofpeptidoglycan from this organism has been devel-oped which is simpler than any of the others andwhich gave rise directly to a very highly purifiedpeptidoglycan in the form of an intact murein sac-culus. In this procedure, the ferrous ammonium sul-fate normally added to the growth medium wasomitted. This omission did not interfere with thegrowth of the culture since the complex mediumused contained sufficient iron present as a contami-nant to satisfy the growth requirements of the orga-nism. In the procedure developed, 30 ml of 20% SLSwas added to each 250 ml of growing culture withoutstopping the shaker, and the incubation was con-tinued for 10 min. The clarified suspensions werecentrifuged at 27,000 x g for 30 min and the pelletsobtained were washed one time in 1 M NaCl; thegelatinous pellets were suspended in 5 mM Trisbuffer (pH 8.0) containing 10 gg each of deoxyribo-nuclease and ribonuclease per ml and incubated at30 C for 60 min. The suspension was made 2% in SLSand centrifuged at 105,000 x g for 30 min. The pelletwas then washed three times in 1 M NaCl, washedthree or four times in water, and lyophilized.

Analysis of peptidoglycan. Samples of peptido-glycan were hydrolyzed in 4 N HCl for 4 hr at 105 C.Hexosamine was determined by the method of Cessiand Piliego (5) with D-glucosamine as the standard.Recovery experiments established that 10.0 X 0.1%of the glucosamine and 13.1 + 1.2% of the muramicacid were destroyed by the hydrolysis procedureused. Muramic acid and the amino acids were deter-mined by applying portions of the acid-hydrolyzedsamples evaporated to dryness in a rotary evaporator

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PEPTIDOGLYCAN OF A MARINE PSEUDOMONAD

and taken up in 0.02 N sodium citrate buffer (pH 2.2)to the column of a precalibrated Beckman Spincoamino acid analyzer. The methods for total carbohy-drate and phosphorus determination have been out-lined (10).

Determination of the per cent peptidoglycan.Two methods were used to estimate the per centpeptidoglycan: direct isolation and calculation basedon the amount of muramic acid present in the cellenvelope of the organism.

For the estimation by direct isolation, peptido-glycan was isolated as quantitatively as possiblefrom a known weight of cells by the second isolationmethod, and the results were expressed as a per centof the salt-free dry weight of the cells.

For the estimation by calculation, use was madeof data reported previously (10) on the amount ofmuramic acid in the envelopes and the contributionof the cell envelope to the salt-free dry weight of thecells. These data, together with information obtainedin the present study on the ratio of the peptido-glycan components in the peptidoglycan, permittedan estimation of the per cent peptidoglycan in thesalt-free dry weight of the cell. For this calculationthe muramic acid was assumed to be N-acetylated.

Determination of N-terminal amino acids. N-terminal amino acids were determined by a modifi-cation of the method of Sanger as described byGhuysen et al. (11). The amount of mono-N-dinitro-phenyl-diaminopimelic acid (mono-DNP-DAP)formed was determined spectrophotometrically withe-N-DNP-lysine as a standard. Since it was estab-lished that the hydrolysis conditions used destroyed17% of the e-N-DNP-lysine, this value was used tocorrect for the mono-DNP-DAP found in the pepti-doglycan sample.

Preparation of di-DNP-DAP. A slight modifica-tion of the method of Salton (21) was used to pre-pare di-dinitrophexyl-diaminopimelic acid (di-DNP-DAP) from DAP.

Conversion of mono-DNP-DAP to di-DNP-DAP. The water-saturated butanol extract con-

taining what was presumed to be mono-DNP-DAPwas treated with 1-fluoro-2, 4-dixitrobenzene(FDNB) reagent under conditions similar to thosedesigned to convert DAP to di-DNP-DAP. The reac-

tion product was dissolved in ammoniacal acetone.Chromatography. All plates for thin-layer chro-

matography were prepared with Silica Gel G to a

thickness of 0.25 cm. For the separation of e-DNPamino acids and mono-DNP-DAP, the thin-layerchromatogram was developed at 20 C in a solventmixture of benzyl alcohol-chloroform-methanol-water-concentrated NH40H, 30:30:30:6:2 (11). Forthe separation of a- and di-DNP amino acids, thethin-layer chromatogram was developed sequentiallyin the same direction by using first a solvent systemcomprising the upper phase of a 1: 1 (w/v) mixture ofn-butanol-1% NH40H at 20 C. The plate was thendried and developed at 4 C in chloroform-methanol-acetic acid, 85:14: 1 (11).

Electron microscopy. Preparations to be nega-

tively stained were mixed with an equal volume offreshly prepared sodium zirconium glycolate con-

taining 2.0% zirconium oxide (National Lead Co.,

897

TAM Division, Niagara Falls, N.Y.) which had beenadjusted to pH 6.8. A drop of this mixture was

placed on a Formvar-coated grid for 30 sec and thenblotted off. Drops of preparations to be shadowedwere held on Formvar-coated grids for 30 sec andblotted off, and the grids were shadowed at an angleof approximately 25 to 30° by using fine platinumwire coiled tightly around tapered carbon rods.The fixative solution used for materials which

were to be embedded contained 5.0% glutaraldehyde[70% glutaraldehyde under noble gas (Ladd Indus-tries, Burlington, Vt.)] in 0.2 M phosphate buffer atpH 6.2, with the addition of the same solutes inwhich the material was suspended. The preparationswere prefixed for 1 hr at 20 C by the addition of one

part of the fixative to nine parts of the material insolution. The preparations were then centrifuged,enrobed in agar (18), and held in the fixative solu-tion for 2 hr at 20 C.

The preparations were washed five times in phos-phate buffer and then postfixed in the same buffercontaining 2.0% osmium tetroxide for 1 hr at room

temperature. After this, the preparations were

washed five times in Veronal-acetate buffer (pH 6.2),stained in a 1.0% solution of uranyl acetate for 1 hr,washed five times in the same buffer, dehydrated inan acetone series, and embedded in Vestopal (9).

Sections were cut by use of a Porter-Blum ultra-microtome and were stained with uranyl acetate andlead citrate (20). All preparations were photographedwith an AEI EM-6B electron microscope.

RESULTS

Forsberg et al. (9) have presented chemicalevidence to show that the mureinoplasts de-rived from the marine pseudomonad ATCC19855 retain the peptidoglycan layer of the cellwall. We have noted that in mureinoplastsarising from cells in which some degree ofplasmolysis has occurred, vesicular fragmentspresumably arising from the cytoplasmicmembrane are confined by a very delicateelectron-dense layer to spaces immediatelyadjacent to the mureinoplast (Fig. 1 and 3,arrows). This layer could only be resolvedwhere it was separated from the cytoplasmicmembrane. This electron-dense layer, which isextremely thin (<2 nm), is difficult to measure

accurately because its thickness appears to bebelow the resolution of the electron microscopeusing sectioned material. We probably see itonly when it extends through the section at an

angle which serves to increase its apparentwidth. This layer can also be resolved (Fig. 2,arrow) in whole cells when factors such as

septum formation and plasmolysis combine toseparate it both from the double-track layer ofthe cell wall and from the cytoplasmic mem-

brane. The layer has not been detected in pro-toplasts prepared from plasmolyzed cells (24).When mureinoplast envelopes were treated

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FIG. 1. Electron micrograph of a mureinoplast of the marine pseudomonad showing membranous vesiclesconfined in a "cap" at one end of the cell form by a very thin electron-dense layer (arrow). The bar in thisand in all subsequent electron micrographs indicates 0.1 um.

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PEPTIDOGLYCAN OF A MARINE PSEUDOMONAD

with SLS to produce murein sacculi, shad-owing revealed that the structures obtainedwere distinctly fragmented and the originalshape of the cell was not discernible. Simi-larly, intact sacculi were not obtained if wholecells were washed three times in 0.5 M NaCl at4 C prior to isolation of peptidoglycan (Fig. 4).When steps were taken to destroy autolyticenzymes by introducing growing cells very rap-idly into boiling 0.5 M NaCl, subsequent treat-ment with SLS gave rise to cell-shaped sacculi(Fig. 5). The rough and distinctly raised ap-pearance of these sacculi indicates that theydo not consist solely of the thin peptidoglycanlayer, and sections of the same material (Fig.6) show that a very large amount of the cyto-plasm of the cell remains after the SLS treat-ment. No double-track profiles can be seen inthese thin sections, but it is clear that this wasnot a clean preparation of the peptidoglycanlayer.When sacculi from heat- and SLS-treated

cells were digested with trypsin, shadowedpreparations showed a very thin structurewhich still contained some adherent material(Fig. 7). The sacculus was so thin that itslimits could be seen only with difficulty (ar-rows). When the same material was thin-sec-tioned, profiles of the electron-dense peptido-glycan layer could be seen with contaminatingfibrillar material both within the profiles andwithout (Fig. 8).When the sacculi after digestion with

trypsin were further treated with deoxyribonu-clease and ribonuclease, shadowed prepara-tions showed thin sacculi with very smallamounts of adherent material. Sections of thematerial treated with nucleases showed com-pressed profiles of the electron-dense peptido-glycan layer with no evidence of contami-nating material (Fig. 9). Negative staining ofthe same preparation showed cell-shaped sac-culi with small amounts of contaminatingmaterial (Fig. 10). Because the remaining con-taminants are not electron-dense in the sec-tioned material, it is not evident whether thematerial lies on the inside or the outside of thepeptidoglycan layer.Chemical composition of peptidoglycan.

The insoluble residue remaining after treat-ment of mureinoplast envelopes with SLS wasanalyzed (Table 1). Glucosamine, muramicacid, alanine, glutamic acid, and diaminopi-melic acid were the major components andwere present in a molar ratio very close to 1: 1:

2: 1: 1. Most other amino acids were either notdetected or were present in only traceamounts. Exceptions to this were leucine andisoleucine as well as glycine, serine, and as-partic acid. If it is assumed that the aminosugars were N-acetylated, then the total re-covery of specific peptidoglycan componentsin this analysis was 77.2%.The murein sacculi isolated from heat-

treated intact cells by SLS and enzyme treat-ment were also analyzed (Table 2). The com-ponents specific to peptidoglycan were presentin amounts and ratios similar to those ob-tained in the preparation isolated from murein-oplasts. Except for glycine, however, aminoacids which had been present in appreciableamounts in the previous preparation werepresent in smaller amounts in this one. Therecovery of specific peptidoglycan componentsin this experiment was 106%.As mentioned above, a simpler and more

direct method of isolating peptidoglycan fromintact cells of this organism has been devel-oped more recently. It involves the direct ad-dition of SLS to a growing culture of the orga-nism. This step inactivated autolytic enzymesand at the same time solubilized most othercomponents of the cell. A very highly purifiedpeptidoglycan could readily be isolated after alimited number of additional manipulations.Analysis showed this material to contain only0.1% carbohydrate and 0.04% phosphorus, sim-ilar quantities in the same proportions of glu-cosamine, muramic acid, alanine, glutamicacid, and diaminopimelic acid as reported forpeptidoglycan from mureinoplast envelopes(Table 1), but even less contaminating aminoacids. Electron microscopy indicated that thepeptidoglycan was present in the form of anintact murein sacculus.

Contribution of peptidoglycan to wholecell dry weight. By direct isolation it was es-timated that the peptidoglycan layer ac-counted for 1.2% of the whole cell dry weightor 6% of the cell wall dry weight of this marinepseudomonad. By calculation based on themuramic acid content of the cell envelope andassuming the muramic acid to be present inthe cell envelope in the N-acetylated form, thepeptidoglycan was estimated to contribute0.9% to the whole cell dry weight. The averageof the two estimations is thus 1.05%.Determination of extent of cross-linking.

Samples of lysozyme-dissolved peptidoglycanwere tested for free amino groups. To correct

FIG. 2. Electron micrograph of a plasmolyzed whole cell. The electron-dense peptidoglycan layer is re-solved where it has formed a septum (arrow) and where the cytoplasmic membrane has pulled away due toplasmolysis.

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FIG. 3. Electron micrograph of a mureinoplast showing the very thin electron-dense peptidoglycan layer asit traverses a bay formed in the cell by plasmolysis. Note that this layer confines vesicular elements whichpresumably have been derived from the cytoplasmic membrane.

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PEPTIDOGLYCAN OF A MARINE PSEUDOMONAD

for the contribution of free amino groups bylysozyme, a parallel determination was carriedout on a solution of lysozyme at the same con-centration as was used to dissolve the peptido-glycan. Table 3 shows that, after correctionsfor the contribution by lysozyme and for de-struction due to hydrolysis, the amount of a-amino groups free to react with the FDNBreagent and subsequently extractable with drydiethyl ether was 0.04 qmole per mg of pepti-doglycan. This was not a significantly highfigure and could be due to breaks in the mur-amyl-alanine bonds, as suggested by Kolen-brander and Ensign (14). The only componentin the water-saturated butanol extracts arisingfrom peptidoglycan was found to be mono-DNP-DAP. This was established by showingchromatographically that it had a migrationrate different from e-N-DNP-lysine and that,after dinitrophenylation, the migration ratecorresponded to that of synthetically prepareddi-DNP-DAP (21). The mono-DNP-DAP ac-counted for 0.45 ,umole per mg of peptido-glycan. Thus, in 1.0 mg of peptidoglycan, 0.45Amole of amino groups on the D-carbon ofmeso-DAP were uncross-linked and free toreact with FDNB reagent. From this resultand from the amino acid analysis, the extentof cross-linking in this organism would be 45%.

DISCUSSIONThe past inability to detect a dense-staining

layer corresponding to the peptidoglycan layerin this marine pseudomonad would now ap-pear to be due largely to the fact that thiscomponent comprises only 1.2% of the cellmass, and under normal circumstances it ad-heres closely to the outside surface of the cyto-plasmic membrane. By comparison, Esche-richia coli has been shown to contain 2% ofthis component (27) and Spirillum serpens2.4% (14).

In thin sections of E. coli and Proteus mira-bilis, the peptidoglycan layer appears to bemore closely associated with the inside surfaceof the outer double-track layer than with thecytoplasmic membrane. Furthermore, duringpreparation, the peptidoglycan layer stays as-sociated with the outer double-track layer (7,

13), and treatment with a proteolytic enzymeis necessary to separate them (2, 7, 13).

In E. coli it has been postulated that pepti-doglycan forms a bag-shaped macromoleculemonolayer surrounding the cell (26). Since anintact murein sacculus has been isolated fromthe marine pseudomonad and since this orga-nism contains about one-half as much peptido-glycan as E. coli, it is evident that the pepti-doglycan layer in E. coli must be more thanone molecule thick. It is of interest in thisconnection that vegetative cells of Myxococcusxanthus which were found to contain 0.86%peptidoglycan did not give rise to an intactmurein sacculus (27). This would suggest that,in the marine pseudomonad, the peptidoglycanmay well be present as a monolayer.The fact that an intact sacculus was not de-

rived from mureinoplasts of the organism sug-gests that, in the course of manipulationsgiving rise to mureinoplasts, some degree ofautolysis of the peptidoglycan layer has takenplace. Since the mureinoplasts maintain therod shape of the intact cell, this would suggestthat an entirely intact peptidoglycan layer isnot essential for the maintenance of the rodshape. Similarly, Myxococcus xanthus, whichappears not to have an intact murein sacculus(27), is a rod-shaped organism.The results here show that, in the marine

pseudomonad, the very delicate peptidoglycanlayer can be resolved only when it has becomeseparated from the cytoplasmic membrane oris involved in septum formation. The forma-tion of the septum by the much thicker pepti-doglycan layers of E. coli and S. serpens hasbeen described by Steed and Murray (22). TheY-shaped bifurcations of the septum seen inFig. 2 closely resemble those seen in their mi-crographs. In the case of E. coli, the identity ofthe dense-staining layer located between theouter double-track layer of the cell wall andthe cytoplasmic membrane as the peptido-glycan layer has been established by the disap-pearance of this layer on treatment with lyso-zyme (7, 18) and by the similarity betweensectioned profiles of this layer and sectionedprofiles of isolated murein sacculi from thisorganism (18). In the case of the marine pseu-

FIG. 4. Electron micrograph of a shadowed preparation of peptidoglycan sacculi produced by treatingwhole cells directly with sodium lauryl sulfate. For the isolation of peptidoglycan, growing cells were notheat-inactivated and were washed free of medium components before treatment with sodium lauryl sulfate.The sacculi isolated are seen to be fragmented.

FIG. 5. Electron micrograph of a shadowed preparation of a peptidoglycan sacculus prepared by using so-dium lauryl sulfate after cells were introduced directly from the growth medium into boiling 0.5 M NaCI. Notethat a cell-shaped sacculus was produced.

FIG. 6. Electron micrograph of a section of an embedding of the same sacculi shown in Fig. 5. Note that avery large amount of cytoplasmic material remained after the treatment with sodium lauryl sulfate.

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PEPTIDOGLYCAN OF A MARINE PSEUDOMONAD

10

FIG. 10. Electron micrograph of negatively stained sacculi from the same preparation used in Fig. 9. Notethe cell-shaped sacculi and the small amounts of filamentous adherent material.

FIG. 8. Electron micrograph of a section of an embedding of the same sacculi shown in Fig. 7. Note thatthe amount of cytoplasmic material has been markedly reduced by treatment with trypsin, so that the profileof the electron-dense peptidoglycan layer can be seen.

FIG. 9. Electron micrograph of a section of an embedding of peptidoglycan sacculi from heat-inactivatedsodium lauryl sulfate-, trypsin- and nuclease-treated cells. Note that all electron-dense contaminating mate-rial has been removed from the profiles of the peptidoglycan layers.

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FORSBERG ET AL.

TABLE 1. Amino acid and amino sugar analysis ofpeptidoglycan isolated from mureinoplast envelopes

Ratio to glu-

AminoacidAmttamic acid

Amino acid (Amolstg(,umoles/mg) hActual eo-

retical

Glucosaminea 0.838 1.07 1.00Muramic acida 0.767 0.98 1.00Alanine 1.419 1.81 2.00Glutamic acid 0.784 1.00 1.00Diaminopimelic acid 0.661 0.84 1.00Lysine 0.038 0.05Ammonia 0.499 0.64Ornithine N.D.bHistidine N.D.Arginine N.D.Aspartic acid 0.106 0.14Threonine 0.039 0.05Serine 0.080 0.10Proline N.D.Glycine 0.105 0.13Valine 0.013 0.02Methionine N.D.Isoleucine 0.172 0.22Leucine 0.235 0.30Tyrosine 0.017 0.02Phenylalanine 0.025 0.03

a Corrected for destruction during hydrolysis.b Not detected.

TABLE 2. Amino acid and amino sugar analysis ofpeptidoglycan isolated from heat-inactivated cells bytreatment with sodium lauryl sulfate and enzymes

Ratio to glu-Amino acid or amino Amt tamic acid

sugar (,umoles/mg) Theo-Actual retical

Glucosaminea 1.23 1.18 1.00Muramic acida 1.29 1.24 1.00Alanine 1.84 1.77 2.00Glutamic acid 1.04 1.00 1.00Diaminopimelic acid 0.82 0.79 1.00Aspartic acid TracebThreonine 0.02 0.02Serine 0.02 0.02Glycine 0.32 0.31Leucine Trace

a Corrected for destruction during hydrolysis.b Less than 0.01 gmole per mg.

domonad, the rod-shaped mureinoplast whichnormally appears in thin section to bebounded only by a cytoplasmic membrane,contains all the peptidoglycan in the cell wall(9). It loses these components when the rod-shaped mureinoplasts are converted to proto-plasts by treatment with lysozyme. The thin,

TABLE 3. Free amino groups of amino acids in thepeptidoglycan isolated from heat-inactivated cells

Amt (omoles/Component mg of pepti-

doglycan)

Free a-amino groups 0.04a 0.02Free e-amino groups, or mono- 0.45a ± 0.02DNP-DAP, or both

a Corrected for lysozyme and loss due to hydrol-ysis.

densely staining layer reported here has beendetected in mureinoplasts but never in proto-plasts, even when these have been plasmolyzed(24).The material isolated contained the usual

components of peptidoglycan in the ratiosfound typically to be present in other gram-negative bacteria (16). The preparations ana-lyzed contained only traces of other aminoacids. Since one of these preparations was ob-tained by direct treatment of growing cellswith SLS without the need for further treat-ment with proteases to obtain a pure peptido-glycan, it is evident that protein could not becovalently linked to the peptidoglycan of thisorganism as it is in E. coli (2). In this respectthe marine pseudomonad is similar to S. ser-pens (16), Proteus species, and Pseudomonasfluorescens (1). In addition, the very low carbo-hydrate and phosphorus contents would indi-cate that no polysaccharide or phosphorylatedpolymers were attached to this layer.The value of 45% for the extent of cross-

linking in the peptidoglycan of this marinepseudomonad compares with 30% for E. coli(23), 40% for P. mirabilis (15), and 54% for S.serpens (14). Thus, except for the amount ofpeptidoglycan present, the composition of thiscomponent in the marine pseudomonad is typ-ical of that present in other gram-negativebacteria.

LITERATURE CITED

1. Braun, V., K. Rehn, and H. Wolff. 1970. Supramolecularstructure of the rigid layer of the cell wall of Salmo-nella, Serratia, Proteus, and Pseudomonas fluores-cens. Number of lipoprotein molecules in a membranelayer Biochemistry 9:5041-5049.

2. Braun, V., and H. Wolff. 1970. The murein-lipoproteinlinkage in the cell wall of Escherichia coli. Eur. J.Biochem. 14:387-391.

3. Brown, A. D., D. G. Drummond, and R. J. North. 1962.The peripheral structures of gram-negative bacteria.II. Membranes of bacilli and spheroplasts of a marinepseudomonad. Biochim. Biophys. Acta 58:514-531.

4. Buckmire, F. L. A., and R. A. MacLeod. 1965. Nutritionand metabolism of marine bacteria. XIV. On themechanism of lysis of a marine bacterium. Can. J.Microbiol. 11:677-691.

904 J. BACTERIOL.

on May 22, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

PEPTIDOGLYCAN OF A N

5. Cessi, C., and F. Piliego. 1960. The determination ofamino sugars in the presence of amino acids and glu-cose. Biochem. J. 77:508-510.

6. 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 a

gram-negative marine bacterium. J. Bacteriol. 94:1764-1777.

7. de Petris, S. 1967. Ultrastructure of the cell wall ofEscherichia coli and the chemical nature of its con-

stituent layers. J. Ultrastruct. Res. 19:45-83.8. De Voe, I. W., J. Thompson, J. W. Costeron, and R. A.

MacLeod. 1970. Stability and comparative transportcapacity of cells, mureinoplasts, and true protoplastsof a gram-negative bacterium. J. Bacteriol. 101:1014-1026.

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

10. Forsberg, C. W., J. W. Costerton, and R. A. MacLeod.1970. Quantitation, chemical characteristics, and ul-trastructure of the three outer cell wall layers of a

gram-negative bacterium. J. Bacteriol. 104:1354-1368.11. Ghuysen, J. M., D. J. Tipper, and J. L. Strominger.

1966. Enzymes that degrade bacterial cell walls, p.

685-699. In S. P. Colowick, N. 0. Kaplan, E. F. Neu-feld, and V. Ginsburg (ed.), Methods in enzymology,vol. 8, Academic Press Inc., New York.

12. Glauert, A. M., and M. J. Thornley. 1969. The topog-raphy of the bacterial cell wall. Annu. Rev. Microbiol.23:159-198.

13. Hofschneider, P. H., and H. H. Martin. 1968. Diversityof surface layers in L-forms of Proteus mirabilis. J.Gen. Microbiol. 51:23-33.

14. Kolenbrander, P. E., and J. C. Ensign. 1968. Isolationand chemical structure of the peptidoglycan of Spi-rillum serpens cell walls. J. Bacteriol. 95:201-210.

15. Martin, H. H. 1963. Zusammenensetzung derStutzmembran in der Zellwand von normalen Zellen

4ARINE PSEUDOMONAD 905

und Penicillin-Spharoplasten von Proteus mirabilis.Zentrabl. Bakteriol. Parasitenk. Infektionskr. Hyg.Abt. Orig. 191:409-415.

16. Martin, H. H. 1966. Biochemistry of bacterial cell walls.Annu. Rev. Biochem. 35:457-484.

17. Martin, H. H., and H. Frank. 1962. Die Mucopeptid-grund-structur in der Zellu und Gram-negativer bak-terien. Zentrabl. Bakteriol. Parasitenk. Infektionskr.Hyg. Abt. Orig. 184:306-311.

18. Murray, R. G. E., P. Steed, and H. E. Elson. 1965. Thelocation of the mucopeptide in sections of the cellwall of Escherichia coli and other gram-negative bac-teria. Can. J. Microbiol. 11:547-560.

19. Osborn, M. J. 1969. Structure and biosynthesis of thebacterial cell wall. Annu. Rev. Biochem. 38:501-538.

20. Reynolds, E. S. 1963. The use of lead citrate at high pHas an electron-opaque stain in electron microscopy. J.Cell Biol. 17:208-212.

21. Salton, M. R. J. 1961. Studies of the bacterial cell wall.VIII. Reaction of walls with hydrazine and with fluo-rodinitrobenzene. Biochim. Biophys. Acta 52:329-342.

22. Steed, P., and R. G. E. Murray. 1966. The cell wall andcell division of gram-negative bacteria. Can. J. Micro-biol. 12:263-270.

23. Takebe, I. 1965. Extent of cross linkage in the mureinsacculus of Escherichia coli B cell wall. Biochim. Bio-phys. Acta 101:124-126.

24. Thompson, J., J. W. Costerton, and R. A. MacLeod.1970. K+-dependent deplasmolysis of a marine pseu-domonad plasmolyzed in a hypotonic solution. J. Bac-teriol. 102:843-854.

25. Weidel, W., H. Frank, and W. Leutgeb. 1963. Autolyticenzymes as a source of error in the preparation andstudy of gram-negative cell wall. J. Gen. Microbiol.30:127-130.

26. Weidel, W., and H. Pelzer. 1964. Bagshaped macromole-cules-a new outlook on bacterial cell walls. Advan.Enzymol. 26:193-232.

27. White, D., M. Dworkin, and D. J. Tipper. 1968. Pepti-doglycan of Myxococcus xanthus: structure and rela-tion to morphogenesis. J. Bacteriol. 95:2186-2197.

VOL. 109, 1972

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