Pared Bacterias

8/3/2019 Pared Bacterias

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583CUMRIIINS, . S. & IIAHRIS, . (1956). J . gen. Microbiol. 14, 583-600

The Chemical Composition of the Cell Wall in someGram-positive Bacteria and its Possible Value as a

Taxonomic CharacterBY C. S. CUMMINS AND H. HARRIS

Departments of Bacteriology and Biochemistry, The London HospitalMedical College, London, E. 1

SUMMARY: Hydrolysates of cell-wall preparations of more than 60 strains ofcorynebacteria, lactobacilli, streptococci, staphylococci and other Gram-positivecocci have been examined by paper chromatography. A very high proportion of theamino acid moiety of the cell-wall complex could in each case be accounted for interms of 3 r 4 of the amino acids alanine, glutamic acid, lysine, diaminopimelic acid,aspartic acid and glycine. These were associated with varying combinations of sugarsand amino sugars. In general, each bacterial genus appears to have a characteristicpattern of cell-wall components, particularly in regard to the amino acids present.Variations in the relative proportions of the sugars appear to differentiate theindividual species within a genus. The possible value of cell-wall composition asa taxonomic character is discussed.It is now well established that the insoluble fraction obtained by centrifugingsuspensions of bacteria which have been mechanically disintegrated is largelycomposed of cell-wall fragments, and a number of workers have reported onchemical analyses of such fractions from several bacterial species (Mitchell &Moyle, 1951 ;Holdsworth, 1952; Salton, 1952, 1953).The present investigationwas undertaken when it was found that the cell-wall compositions of severalspecies of corynebacteria resembled each other closely, but differed markedlyfrom those of streptococci. This finding, coupled with the anomalous resultsobtained with a strain of Corynebacterium pyogenes, suggested that a moresystematic investigation of species from different bacterial genera might beprofitable. The present paper deals with the results obtained so far in a surveyof corynebacteria, lactobacilli, streptococci, staphylococci and a number ofother Gram-positive cocci.

The general method has been to grow th e organisms on a suitable medium,disintegrate the washed suspension by shaking with ballotini in a Mickledisintegrator, and purify the cell-wall (insoluble) fraction by treatment withproteolytic enzymes. These purified preparations were then hydrolysed in acidand the hydrolysates examined by paper chromatography. Some of theresults have been briefly reported previously (Cummins & Harris, 1955).

MATERIALS AND METHODSStrains of bacteria

Strains obtained from the National Collection of Type Culture (NCTC) or fromthe National Collection of Industrial Bacteria (NCIB) are listed as such, withtheir numbers. The sources of other strains were as follows:

38 G . hficrob. XIV


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584 C . S . Curnrnins and H . Harri sCorynebacterium diphtheriae (gravis, mitis and intermedius) from Dr Donald

Payne, Public Health Laboratory, Northallerton ; C . ulcerans (gelatin-liquefying, starch-fermenting) from Dr W. H. H. Jebb, Public Health Laboratory,Oxford;C .hofmanni, isolated in the Clinical Laboratory, London Hospital, fromthroat swabs; C .xerosis, isolated in the Clinical Laboratory, London Hospital,from conjunctival swabs; C . renale, obtained from Dr F. C. 0. Valentine,London Hospital (originally from Professor R. Lovell, Royal VeterinaryCollege); C . pyogenes, strains Wye 1, 2, 3 from Veterinary InvestigationCentre, Wye, Kent (Mr J. D. Paterson); strains 637 and 13081 from Dr LaneBarksdale, N.Y. University. C. huemolyticurn strain 53/W/1 was also obtainedfrom Dr Barksdale, and represents the type strain (MacLean, Liebow &Rosenberg, 1946).

Isolated in the Clinical Laboratory,London Hospital, from a throat swab. Streptococci of other Lancefields groupswere obtained from the National Collection of Type Cultures. Staphylococcusaureus, strains 1, 2 and 3, isolated from nasal swabs, all coagulase-positive;S. albus strains 1, 2 and 3, isolated from nasal swabs, all coagulase-negative.These strains were picked as aureus or albus on colonial pigmentation,and were later tested for coagulsse production. S . citreus : elected from cultureof nasal swab as giving typical lemon yellow pigment, coagulase-negative.Lactobacilli spp. The strain originally examined was obtained from a sampleof yoghurt, and purified by plating out on tomato juice agar. It has not beenfurther identified. Subsequent strains were obtained from the NationalCollection of Industrial Bacteris.

Streptococcus pyogenes (group A).

IdentiBcation and naming of strainsIn most cases strains of known origin have been used, either from the

NCTC or NCIB. These were checked for purity, but not investigated further.Strains of Corynebacterium diphtheriae had been isolated from cases, and hadthe typical morphology and fermentation reactions. Strains of C . hofmanniisolated from throat swabs were accepted as such if they stained evenly withmethylene blue except for a central unstained bar, were strongly Gram-positiveand failed to ferment glucose, maltose and sucrose. The strains designatedC. xerosis were heavily barred diphtheroids, isolated from conjunctival swabs,which fermented glucose and sucrose and gave rather small dry roughcolonies. The single strain of C . auurium was a non-acid fast corynebacterium,isolated from caseating lesions in a mouse.

With two exceptions strains tire listed under the names which they borewhen received, the exceptions bleing two strains of lactobacilli. These wereoriginally received as Lactobacillus bulgaricus (NCTC 76) and L. b i jdus(NCTC2797), bu t we were informed by Dr Dorothy Wheater th at according tophysiological and serological tests (Wheater, 1955a, b ; Sharpe, 1955;Briggs,1953) these should be reclassified as L. helveticus (76) and L. fermenti (2797)respectively. With the agreement; of the Curator of the National Collection ofType Cultures, Dr Cowan, we have therefore altered the names bu t retainedthe NCTC numbers.


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Cell-wall composition and bacterial taxonomy 585Culture media

Streptococci, aerococci and strains of Corynebacterium pyogen es were grownon infusion broth to which had been added before sterilization 0.1 yo (w/v)sodium phosphate (Na,HPO,). To this broth was added before inoculationa sterile glucose +bicarbonate solution (10 % glucose, 10 yoNaHCO,) in theproportion of 2 ml. to 100 ml. broth. In the case of C . pyogenes growth wasimproved by the further addition of 2 yo sterile horse serum.

Staphylococci were grown on nutrient agar in Petri dishes, or in 10 x 8 in.metal trays with metal lids; but some more slowly growing strains such asMicrococcus conglomeratus were grown in nutrient broth a t 28" in conicalflasks to give good aeration.

Lactobacilli were grown in tomato juice broth (Briggs, 1953) in large screw-capped bottles containing 1 l., for 48 hr. at 37". The suspensions so obtainedwere usually dirty brown in colour, even after washing, but the cell-wallpreparations from them were pure white, as with other organisms.

Preparation of cell -wall susp ensio nsBacteria were harvested from solid media by washing off in distilled water,

or collected from liquid media by centrifugation. Pathogenic organisms werekilled by heating a t 60" for 1 hr. or2 in the case of relatively heat-resistantspecies, with formalin (0 .5 yo)overnight a t room temperature. These precau-tions were omitted in the case of non-pathogens.

Suspensions were washed twice in distilled water and the bacteria disinte-grated with ballotini (Chance no. 12) in a Mickle tissue disintegrator, using4 g. ballotini and 6 ml. bacterial suspension in each cup. Shaking was doneat Po, and the process judged complete when no intact organisms could be seenin smears stained by Gram's method. The rubber stoppers of the cups wereprotected by cellophan as suggested by Hotchin, Dawson & Elford (1952).

After shaking, the cups were centrifuged a t 1000 r.p.m. for 5-10 min. tobreak the froth, and the supernatant decanted. The ballotini were washedthree times, with a few ml. of distilled water each time, and the washingsadded t o the original supernatant. This was centrifuged a t low speed(c . 1000 r.p.m.) for a short time to remove glass beads, and then at 3000-4000 r.p.m. until the crude cell-wall fraction was deposited. This was washedonce in distilled water, resuspended in 0.05 M-phosphate buffer (pH 7.6) anddigested with crystalline trypsin and ribonuclease (Armour) a t 37". Bothenzymes were used a t 0.5 mg./ml. Digestion was continued for 2-3 hr. anda considerable decrease in opacity generally occurred during this period. Themixture was then centrifuged, the deposit washed twice in distilled water,resuspended in 0-02 N-HCl with 1 mg. crystalline pepsin (Armour)/ml. anddigested at 37' for 18-24 hr. After peptic digestion, the material was finallywashed several times in distilled water.If not hydrolysed immediately, the cell-wall preparations were preserved as

38-2


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586 C . S. Cummins and H . Harrissuspensions in distilled water +0.3yo sodium azide. Such preserved materialwas centrifuged and washed once in distilled water before hydrolysis.

Ideally, each sample should have been checked by electron microscopybefore hydrolysis, but this was impossible. However, a few samples chosen a trandom were so examined, and t,hese showed that the method gave adequatelypure material.

HydrolysisThe amount of ma,terial used for hydrolysis depended t o some extent on the

size of the sample available, but in general about 50 mg. (dry weight) was used,divided so th at two-thirds was used for the investigation of sugars, and theremaining one-third for amino acids.For sugars. Samples were liydrolysed in 2 N-H,SO, in sealed tubes in

a water bath at 100" for 2 hr. After cooling, the hydrolysates were filtered,neutralized with solid Ba(OH) , and centrifuged, and the supernatantevaporated to dryness in vacuo over P,O, .The final product was redissolved in0-2-0.25 ml. distilled water.For amino acids and hexosamines. Samples were hydrolysed in 6 N - H C ~nsealed tubes a t 100"for 8 hr., or in some cases at 105' for 18-24 hr. They were

then filtered, evaporated to dryness on a boiling water bath, and finallyredissolved in 0.2-0.25 ml. distilled water.

Various degrees of humin formation were encountered with cell-wall pre-parations from different organisins. It was most marked in the case of coryne-bacteria. The humin was filtered off before evaporation on the water bath.

ChromatographyAmino acids and amino sugars. I n all cases two-dimensional descending

chromatograms (22 x 184 in. Whatman no, 4 filter-paper) were prepared.Phenol+water (8 0 :20) in an ammonical atmosphere were used for thefirst solvent, and lutidine +wate r (65 :35) for the second. The amount ofcell-wall hydrolysate chromatographed in each instance corresponded toabout 5 mg. dry weight of whole organisms. The spots were revealed by dippingthe chromatogram rapidly through a solution of 0.2y0 ninhydrin in 9 5%acetone and 5 yowater, and after it had dried heating at 105' for 5 min. Ingeneral, the amino acids present could be readily identified from the positionand colour of the spots obtained. Where there was any uncertainty appropriatemarkers were added to the hydrolysate. Further confirmation of the identityof the spots was also obtained in certain instances by ionophoresis in buffers atdifferent pH values. The Elson and Morgan reaction and the reaction withammonical silver nitrate (Part ridge& Westall, 1948) were also used to diftreren-tiate the amino sugars from the amino acids and to confirm the identificationof glucosamine and galactosamine.

I n most cases after 8 hr. hydrolysis in 6 N-HCl the only ninhydrin reactingspots present corresponded to known amino acids and amino sugars, and thehydrolysis was therefore regarded as complete. There were, however, twoexceptions to this. First , in cell-wall preparations from all the lactobacilli


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Cell-wall com position and bacterial taxono my 587examined. Wi th the exception of the various strains of Lactobacillus plantaruna,hydrolysis in 6 N-HCl for 8 hr. revealed strong spots corresponding to asparticacid, glutamic acid, alanine and lysine, and also a strong unknown spot withR , values about the same as lysine in phenol +NH, and as glycine in lutidine.On further hydrolysis up to 24 hr. this spot gradually became weaker till itdisappeared almost entirely. Par i passu there occurred a progressive intensi-fication of the spots corresponding to aspartic acid and lysine, and possibly aless pronounced increase in the glutamic acid and alanine spots. The unknownmaterial was therefore regarded as a peptide relatively resistant to hydrolysis,and probably composed largely of aspartic acid and lysine. No further investi-gation of this point has yet been undertaken, but for the purposes of thepresent paper, hydrolysis of the cell-wall preparations in this group of organismswas regarded as complete after 24 hr. in 6 N-HC~t 105". Secondly, in all thecell-wall preparations examined there was found a ninhydrin reacting sub-stance, moving in phenol+NH, somewhat more slowly than either glucosamineor galactosamine, and in lutidine a t about the same rate as these amino sugars.In lutidine, and t o a lesser extent in phenol +NH,, its R, value was somewhatvariable from run to run. The colour after development with ninhydrin wasmuch the same as tha t given by glucosamine, and i t was found to react withthe Elson and Morgan reagents and with ammonical silver nitrate in the sameway as glucosamine and galactosamine. It did not correspond in chromato-graphic behaviour or in reactions to any known naturally occurring aminoacid or amino sugar. Prolongation of the hydrolysis up to 24 hr. indicated thatit was about as stable as glucosamine and galactosamine under these condi-tions, and it did not appear to be disrupted into any additional ninhydrin-reacting substances. This material seems to correspond closely in properties tothe unidentified hexosamine reported by Strange & Powell (1954) in a solublepeptide obtained from cultures of germinating spores of BacilZus subtilis,B. cereus and B. megaterium. In the rest of this paper the material will bereferred to as 'unknown "hexosamine " .Sugars. Adequate resolution of the complex mixtures of sugars encounteredin many of these cell-wall hydrolysates could not be obtained by uni-dimensional chromatography. As a routine, therefore, two-dimensionalchromatograms (2 2 x IS$ in., no. 4 Whatman filter-paper) were preparedusing phenol +water as the first solvent and lutidine +water as the second.The spots were revealed by dipping in a reagent mixture containing:aniline 2.0 ml., phthalic acid 3-3 ., acetone 95 ml., and water 5 ml. ; ollowedby heating at 105" for 5-10 min. I n general, an amount of hydrolysatecorresponding to about 20mg. dry weight of whole bacteria was used in thepreparation of each chromatogram.

The distribution of the sugars on the chromatograms was substantially asdescribed by Partridge & Westall (1948),who used phenol +NH, and collidineas their solvents. I n our experiments ammonia was omitted from the phenolrun because it was found to lead to excessive streaking of the hexose andpentose spots. The only disadvantage of performing the phenol run in neutraland not alkaline conditions was th at the amino sugars were poorly resolved. In


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588 C . S. Czlcmminsand H . Harriscomparison to the other sugars, however, these substances give a very feeblereaction with aniline hydrogen phthalate, and we relied for their identi-fication on the methods described in the previous section. The individualhexoses and pentoses could be readily identified by their relative positionson the paper and their characteristic colour reactions with aniline hydrogenphtha1a e.

No attempt a t accurate qua:ntitative evaluation of the relative proportionsof either the amino acids or sugars has been made. The relative amounts of thedifferent substances present in each case have been arbitrarily graded as+ + +, + +, +, f or trace, according to the relative sizes and intensities ofthe spots obtained.

RESULTSFor ease of presentation the results have been collected into several tables,each dealing with strains or species which form a related group, even though insome cases their classification is still uncertain. To bring out more clearly thepattern of amino acids, only the major components have been recorded, andthe other columns left blank. The only exception to this is in the comparisonbetween Staphylococcus aureus and S. aEbus,where the difference in the amountof serine appears to be significant, although it was present in amounts con-siderably smaller than were the other four amino acids.

Although dealt with more fully later, two points may be made a t the outset.First, th at the pattern of amino acid components appears to distinguish largergroups such as genera, while the species within these groups seem to be dis-tinguished by the sugars and aniino sugars which their cell walls contain. Thisis, however, only a broad distinction, and certain genera such as Streptococcusand Corynebacterium have distinguishing cell-wall sugars which are present inall members of the genus. Secondly, attention may be drawn to the distributionof the unusual amino acid a, -diaminopimelicacid (D.A.P.) originally describedby Work (see Work, 1951) in C'. diphtheriae. The present results confirm thesuggestion of Work & Dewey (1953) ha t the presence or absence of this sub-stance may be of taxonomic importance.

StreptococciThe results detailed in Table 1 show that the 8 strains examined form

a homogeneous group, as far as cell-wall composition is concerned, and thedistinguishing components appe,ar to be the methylpentose, rhamnose, and theamino acids alanine, glutamic acid and lysine. A point of considerableinterest is the difference in cell-wall composition between the two group Dstrains, a difference which is of approximately the same degree as that betweeneither of them and, for example, the strain of group F. Salton (1953) as alsoexamined the cell walls of strain. 6782 (group D), and his results agree closelywith the present findings, except that he did not detect mannose in hydroly-sates of his preparation. Both Salton, and also McCarty (1952 , b ) found thatthe polysaccharide part of the cell wall of Streptococcus pyogenes (groupA) was


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Glycine . . . . . . . . . . . . . . .Serine . . . . . . . . Serine . . . . . . . . . . . . . . .

Diaminopimelic . . . . . . . .acid

+ + + + + + + +Diaminopimelic + + + + + + + + . . . . . . .+ + + + + + + +cid+ + + + + + + ++ + + + + + + +ysine + + + + + + + + + + + + + + ++ + + + + + +Lysine . . . . . . . + + + + + + ++ + + + + + + +

Glutamic acid + + + ++ + + + + + + +Alanine ++ + + + + + + + -!i+ + + + + + + + 8:Aspartic acid . . . . . . . . cds.r(

+ + + + + + + + + + + + + + +Glutamic acid + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + +

Aspartic acid . . . . . . . . . . . . . . .5nknownHesosamine + + + + c + + + + + + + + + + + + + + +nknownHexosamine

Galactosamine I I + + I + + -8G Galactosamine I 1 I I + + I + + + + + + + +.r(I + + + + + + +

I I I + l + 6 I I

Glucosamine

x* @ Mannose Y)

Glucose Glucose I I + I + + I&+:++;+ I1 + 1 + 1 I + ;alactose Galactose + + + + + + + ++ + + + + + + l l l l l l l+ ++ + + + + + ++ + + + + + + +hamnose

Arabinose+ + + + + + +Rhainnose I I I I I I I + + + + + + + +

I I I I I I I I + + + + + + + ++ + + + + + + +rabinose + -t- + 4-+ + + + I I I I I I IhWd8.r)


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590 C . S. Curnrnins and H . Harrismade up of rhamnose and glucosamine, and Salton noted that D.A.P. wasabsent from the strain of S . pyogenes he examined, but t ha t alanine, glutamicacid and lysine were present in larger amounts than other amino acids.

C'ory nebacteriaThe first eight species in Table 2 (representing fifteen strains) make up

a group whose characteristic cell-wall sugars appear to be arabinose andgalactose. Although rhamnose was present in the st rain of Corynebacteriuirim u r i u m it does not seem to ha,ve any more significance in this case th an th epresence of an approximately equal amount of mannose, when the pat tern ofcomponents as a whole is consildered. The distinguishing amino acids of theseeight species are alanine, glutamic acid and D.A.P., which were the majoramino acid components in all of them. Diaminopimelic acid has alreadybeen identified in hydrolysates of whole C. diphtheriae by Work (1951), andHoldsworth (1952) has shown that almost the whole of i t is present in thecell-wall fraction of this species. From the same fraction, Holdsworth alsoobtained an oligosaccharide containing arabinose, galactose and mannose inthe ratio 3 :2 :1, and the results given for C. diphtheriae in Table 2, althoughonly roughly quantitative, agree well with these proportions. The four strainsof C. diphtheriae examined included two mitis, one intermedius and one gravisstrain, but no difference in cell-wall composition was detected between thedifTerent cultural types.

The cell-wall compositions of Corynebacterium pyogenes and C. haemolyticum(Table 2) are obviously similar to one another, but differ both in sugar andamino acid composition from the other corynebacteria since they containneither arabinose nor galactose, and lysine appears as a major component,while D.A.P. is absent. On the other hand, rhamnose was present in both cases,and this, together with the fact that alanine, glutamic acid and lysine werethe major amino acid components, suggests very strongly that these organismsare related to the streptococci in view of the results already detailed in Table 1 .Six strains of C . pyogenes have been examined, and the results are included inTable 2. Taking into account the rather arbitrary method of estimating theamounts of constituents, these s ix strains showed surprisingly little variationin cell-wall composition, except for the apparently complete absence of man-nose in strain 13081. However, this sugar was present only in traces in the cellwalls of other strains, and the difFerence seems hardly significant.

Staphylococci, aerococci, sarcina and rnicrococciThe results in Table 3 epresent the cell-wall compositions in a number of

catalase-positive species of Gram-positive cocci, and several points of interestare evident. Firs t, there is a group in which the major amino acids of the cellwall are alanine, glutamic acid, lysine and glycine, but in which no distinctivesugar appears. This group is represented by the cultures named Staphylococcusaureus, S . albus, S . citreus, Sa rc ina lutea and Micrococcus luteus (9 strains in all),and within it there seems to be a division between the strains of Staphylococcus


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- *0

+ + + + ++ + + + +Glycine + + + + + . . . . . . .

+ +. . . . . . . .iaminopimelic , . + +acid + ++ + + + + + + + + +Lysine + + + + + + + + + ++ + + + + + + + + ++ + + + + + + + + + + +Glutaniic acid + + + + + +-l ++ + + + + + + + + + + ++ + + + + + + + + + + +Alanine + + + + + + + + + + + ++ + + + + + + + + + + +. . . . . . . . . . . .Aspartic acid

Mannose I I + I I I + I I & 6 i]Glucose I I + 1: + + + + + I I+alactose 1 I I 1 I + I I + + ; +

Rhamnose I I I I 1 I I I I I I I+ +Arabinose I I I I I I I I I I + ++ +


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C . S . Cummins and H . Harrisaureus and 5'. albus, which hatve a moderate amount of serine but no hexosesor pentoses, and the other three strains which contain no serine bu t have oneor more hexoses in the cell wall. There may also be a significant differencebetween aureus and albus strains in respect of the amount of serine present.Both of these tentative subdivisions require confirmation by the examinationof a far larger number of strains,

The second group of strains which can be distinguished among those whosecell-wall compositions are set lout in Table 3 are the four strains of Aerococcusand probably also the strain labelled Micrococcus conglomeratus, Williams,Hirch & Cowan (1953), n defining Aerococcus as a new bacterial genus,described i t as being intermediate in many ways between Staphylococcus andStreptococcus. This is borne out by the cell-wall composition of these fourstrains, the essential pattern of which differs from Streptococcus only in th atrhamnose is absent, and from Staphylococcus only in not containing glycine.

Thirdly, there are the two strains labelled Micrococcus rhodochrous andM. cinnabareus. These had been rejected on morphological grounds by Shaw,Stitt & Cowan (1951) nd Cowan (personal communication) from their collec-tion of Gram-positive, catalase-positive cocci, and this has been confirmed byus, since both cultures show diphtheroid forms several p. long, particularly inthe case ofM . cinnabareus. It is interesting to see th at by cell-wall compositionthese two strains would clearly fall into the genus Corynebacterium (Table 2),and the results in each case are so alike as to suggest that they may be twostrains of the same species.

LactobacilliThe results of cell-wall analyses of 7 strains (the first 7 in Table 4) how tha t

this genus appears to be characterized by aspartic acid, alanine, glutamic acidand lysine as the major amino acids of the cell wall, but has no distinguishinghexose or pentose. The 2 strainis of Lactobacillus cme i and L. delbrueckii havea rather more complicated cell-wall structure than the others ;galactosamineas well as glucosamine is present, both strains contain rhamnose, and thestrain L. casei has also a small amount of arabinose. However, their aminoacid pattern is the same as the other 5 trains under discussion, and all 7 seemto form a homogeneous group vvith the characters mentioned above.

The 4 strains of Lactobacillus plantarum , on the other hand, differ from theother strains of lactobacilli in three ways so far as the amino acid pattern intheir cell walls is concerned: they lack aspartic acid and lysine and containD.A.P. Diaminopimelic acid was found by Work & Dewey (1953) n thestrain of L. l an tarum which they examined, although they did not identify i tas a cell-wall component since they examined hydrolysates of whole organisms.

IDISCUSSIONGeneral !nature of the cell wall

The present series of results represents the analyses of cell-wall compositionin nearly 60 strains, and is large enough to enable some general conclusionsto be drawn as to the nature of the cell wall in Gram-positive bacteria.


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Serine . . . . . . . . . . .Diaminopimelic + + + +acid . + + + ++ + + +. . . . . .

+ + + + + + ++ + + + + + +ysine+ + + + + + + * - *+ + + + + + + + + + ++ + + + + + + + + ++ + + + + + + + + + +lutamic acid ++ + + + + + + + + + ++ + + + + + + + + ++ + + + + + + + + + +hnine ++ + + + + + ++ + + + + + ++ Aspartic acid + + + + + + + * * - *3Y$

90u + + + + + + + + + + +nknown.cy 'Hexosaminect0-8 Galactosamine I I I I I + + I I I I'*.0 Glucosamine + + + + + + + + + + +8

Rhamnose I I I 1 I +I I I I


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594 C . S . Cummins and H . HarrisCharacteristically the cell-wall material is very tough and extremely insolublein a wide variety of solvents. I t is made up to a large extent of sugar and aminoacid components and presumably, therefore, falls into the class of r nuco ids ormucosubstances (Kent& Whitehouse, 1955). A similar conclusion was reachedby Salton (1952, 1953) as a result of his findings in a smaller series of fiveGram-positive and two Gram-negative species. One remarkable feature is theapparent simplicity of the ammo acid patterns encountered, in contrast to thefindings in other mucoids or in proteins.

Sugars and amino sugars were present in every preparation except thowfrom Staphylococcus aureus and S . albus, in whose cell walls no hexoser orpentoses were detected, but these can be regarded as limiting cases. Glucos-amine was invariably found, as was the unknown hexosamine-like substancealready described in the section on methods. The exact nature of this substanceis still obscure. Galactosamine, on the other hand, was present in only aboutone-third of the species examined. *4mong he sugars one or more of the threehexoses, glucose, galactose and mannose, seemed to be almost invariablypresent; arabinose and rhamiiose occurred less frequently and had a morerestricted range. Ribose has not been detected in cell-wall hydrolysates exceptin the case of some strains of co rynebacteria. The amount was usually small andvaried considerably in different preparations from the same strain, beingentirely absent from some. Holdsworth (1952)did no t detect ribose in the cellwall of Corynebacteriurn diphtheriae, and Salton (1953) did not find it in thewall of any of the seven species of bacteria he examined. It seems mostlikely, therefore, that the preparations in which ribose was present had notbeen adequately purified.

A very high proportion of the amino acid moiety of the cell-wall complexcould in each case be accounted for in terms of three or four of the followingamino acids : alanine, glutamic acid, lysine, a, 6-diaminopimelic acid, asparticacid and glycine. Of these alanine and glutamic acid were invariably presentas major components. The others were found to be characteristic major com-ponents of the cell walls of some organisms bu t not of others.

Apart from these major components, other amino acids were encountered inrelatively smaller amounts in some of the preparations. We have never had anydifficulty in deciding whether to call an amino acid a major or a minor com-ponent in any particular case, hu t the significance of the minor components israther difficult to assess. They do not seem to be constant from one sample toanother, and have generally been present only in traces except in the case ofsome preparations from corynebacteria examined early in the series, whichshowed moderately strong spots for lysine, serine, glycine, aspartic acid, valineand the leucines, as well as the characteristic major spots for alanine, glutamicacid and D.A.P. These corynebatcterial preparations also contained a variableamount of ribose, and might legitimately be regarded as being contaminatedby cytoplasmic remains. In other cases there were frequently no races , forexample, in hydrolysates from Staphylococcus aureu s and Lactobacillus plan-tarum. It is not possible to decide at the moment whether these minor aminoacid components are part of the cell-wall complex proper, or whether they


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Cell-wall compositionand bacterial taxonomy 595represent the remains of surface protein layers as exemplified by the M antigensof streptococci, or cytoplasmic remnants in slightly impure cell-wall suspensions.

The individual amino acids found are common ones, except for D.A.P., whichhas so far been described only in bacteria or their products (Work, 1951,1955).The distribution of this substance among a variety of micro-organisms includingbacteria, fungi, yeasts and protozoa was surveyed by Work & Dewey (1953)'who examined hydrolysates of whole organisms. We have not noted anydiscrepancies between the distribution of D.A.P. as described by these workersin whole organisms of various species, and our own findings on separated cellwalls and i t seems possible th at the bulk of the D.A.P. in Gram-positive bacteriais situated in the cell wall.

In the species we have examined, the cell walls contain either D.A.P. or lysineas a major component, but not both in similar quantities. This perhaps sug-gests th at they have similar structural functions. Lysine can be formed fromD.A.P. by the action of D.A.P. decarboxylase which is fairly widely distributedin bacteria (Dewey, 1954; Work, 1955), and i t might have been expected t ha tthe decarboxylase would be found in those cases in which lysine and not D.A.P.was a major cell-wall component. There is, however, no such simple relation-ship, since some organisms which have lysine in their cell walls seem to bedevoid of decarboxylase activity, while others which have D .A.P. apparentlycontain the enzyme (Work, 1955).

No account has been taken here of lipid components which may be presentin the cell walls of any of the species examined. It would seem from the findingsof Salton (1953) that this type of substance forms at most a relatively smallpart of the cell wall in Gram-positive bacteria. Fo r example, he found 1.2 yototal lipid in the cell walls of Micrococcus lysodeilcticus, and 2.6 yo in those ofBacillu s subtil is . In both cases the lipid was firmly bound, and did not seem tobe completely liberated until after several hours hydrolysis in 6 N-HCl a t 100".Proteolytic enzymes are without effect on the cell-wall material as a whole,bu t may remove surface protein components such as the M antigens of strepto-cocci (Salton, 1953). The removal of this material with trypsin made nodifference to the appearance of the cell wall in electron micrographs,and did notseem to alter its physical properties, but Salton noted that the amino acidconstitution was simpler after treatment with trypsin, and that in particularsulphur-containing and aromatic amino acids were no longer present. Cum-mins (1954) ound that a superficial protein antigen in a strain of Coryne-bacterium diphtheriae appeared to be destroyed by pepsin, and it was thisobservation, together with Salton's findings in the case of the M antigen, thatled us to adopt the routine use of trypsin followed by pepsin in the purificationof the cell-wall material, in the hope of obtaining as simple a pattern of com-ponents as possible.

Cell-wall composition and bacterial taxonomyThe qualities necessary for a good taxonomic character have recently been

discussed in some detail (Report of Discussion Meeting on the Principles ofMicrobial Classification, 1955), and the chemical composition of the bacterial


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596 C . S . Cummins and H . Harriscell wall appears to fulfil in imany respects the necessary requirements. Thepreparation of the purified cell-wall fractions, although somewhat laborious, isnot technically difficult, and the components present can be accuratelyidentified, if necessary, by comparison with synthetic standards. Cell-wallcomposition is also in our experience a stable character, unaffected by varia-tions in culture media or conditions of growth, and this point is underlined bythe close agreement between the present results and those of other workers.Our findings in the case of Stn:ptococcus aecalis (NCTC6782), for example, arevirtually identical with those of Salton (1953)with the same strain, and thereis the same degree of correspondence in the case of Sarcina Zutea, although inthis instance different strains were used.

In an at tempt to test the stability of cell-wall composition under alteredmetabolic conditions, a strain of Staphylococcus aureus was ' rained ' overa period of several months by growth in gradually increasing concentrationsof penicillin in broth. The highly resistant strain so obtained would grow in thepresence of 5000 units penicillin/ml., bu t its cell-wall composition was unalteredwhen compared with th at of the original culture. It should be pointed out,however, that this resistant strain differed from those described by Gale &Rodwell (1948) nd by Bellamy & Klimek (1948))n th at i t still retained thecoccal form, and was still Gram-positive although somewhat irregularly so ; talso grew both aerobically and anaerobically. The resistant strains describedby the authors quoted appeared in stained films as pleomorphic Gram-negative bacilli, and would not grow anaerobically. They may therefore haveundergone a more fundamental alteration than the strain described in thepresent paper.

Bacterial taxonomy is a t present based on a number of different criteria, ofwhich those of widest application are probably morphological appearances andstaining properties, antigenic characteristics, and tests of biochemical activitywhich reflect various aspects of intermediary metabolism, mainly catabolic.Cell-wall composition can perhaps be regarded as an extension of morphologya t the biochemical level; a sort of chemical anatomy. It is probably quiteintimately connected with an.tigenic characteristics, since it is likely thatimportant cell antigens are located in the cell wall, and indeed form a majorpart of it. It seems quite clear, for example, from the results of McCarty(1952a, ) and Salton (1953) hat the cell walls of Streptococcus pyogenes, asprepared by mechanical disintegration, contain both the M protein and theC polysaccharide and since McCarty, and also Schmidt (1952), ave shown thatthe latter is composed of rhamnose and hexosamine, it is not unlikely tha t i trepresents the polysaccharide moiety of the basic cell-wall substance in thisspecies.

In the four main groups of G:ram-positive bacteria so far examined (strepto-cocci, corynebacteria, staphylococci and lactobacilli), the results of cell-wallanalysis agree well with the genera already defined by the use of other taxo-nomic characters. The differences in composition seem clear cut and easilyrecognizable, and there would be no difficulty, for example, in distinguishing bythis method a staphylococcus from a streptococcus, or a lactobacillus from


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Cell-wall compo sition and bacterial tax on om y 597a corynebacterium. Figs. 1 and 2 show, in diagrammatic fashion, the patternof constituents found in representative species of these four genera,

The general picture that emerges is one in which the amino acids present inthe cell wall seem to be characteristic of the genus, and the sugars and aminosugars seem to characterize the species within the genus, with the importantexception of arabinose and rhamnose, which appear t o have special significancein Corynebacterium and Streptococcus respectively. In most cases only onestrain of each species was examined, and the degree of species variation to beexpected is not known. Where more than one strain has been analysed, theresults have in some cases been virtually identical (e.g. Corynebacterium diph-theriae), while in others there have been definite qualitative differences in thesugar components present (e.g. Lactobacil lus pla ntar um ).If it can be accepted t hat distinctive patterns of cell-wall constituentscharacterize different genera, then any organism which differs markedly incell-wall composition from others in the genus to which it is normally assignedbecomes of particular interest. In the present investigation three series ofstrains have given anomalous results of this sort, and they are consideredbelow in turn.

In the case of Corynebacterium pyogenes and C. haemolyticum the results ofcell-wall analysis are at variance with those obtained in other corynebacteria.The findings in the 6 strains of C . pyogenes, for example, would support therejection of this species from the genus Corynebacteriumand its inclusion insteadin Streptococcus, since it contains rhamnose and not arabinose as a distinguish-ing cell-wall sugar, and has alanine, glutamic acid and lysine as major aminoacid components, bu t lacks D.A.P. The same arguments apply also to thesinglestrain of C. huemolyticum examined. There is no doubt that these organismswould be included with the corynebacteria, if morphology alone were con-sidered, although in some conditions they can be almost coccal. On the otherhand, McLean et al. (1946), in their original description of C . haemolyticum,commented on the close resemblance of th is species to Streptococcus pyog enes ,and the growth of Corynebacterium pyogenes on blood agar and in brothresembles very closely that of a P-haemolytic streptococcus. In addition, bothspecies resemble the streptococci in being catalase-negative, while othercorynebacteria are catalase-positive. It seems to us, therefore, that there aregood grounds for reconsidering the taxonomic position of C . pyogenes andC . h m o l y t i c u m , with a view to their inclusion in the genus Streptococcus.

The results of cell-wall analysis in the case of the four strains of Lactobacillusp lan tarum present a more difficult problem. The amino acid pattern in thesestrains is quite different from th at of the other lactobacilli examined, and is infact identical with the pattern found in Corynebacterium, and with th at whicha few preliminary observations suggest may also be found in the genus Bacil lus(Salton, 1953; Cummins & Harris, unpublished observations). Quite ap ar tfrom any morphological and physiological differences, however, these fourstrains of Lactobacil lus p lan taru m were distinguished from the corynebacteriaby the fact that none of them contained arabinose as a major cell-wall com-ponent, nor is there any evidence to suggest any close relationship to the


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598 C . S . Cumrnins and H . Harris

Fig. 1. Diagrams of typical chromatograms showing the amino acid and aminosugar patterns in representative species of four different genera.

Fig. 2. Diagrams of typical chromatograms showing the sugar patternsin representative species of four different genera.


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Cell-wall composition and bacterial taxonomy 599aerobic sporing bacilli. More than eighty strains of Lactobacil lus pla ntar um ,together with other species of lactobacilli, have recently been investigated byphysiological and serological methods (Wheater 1955a, b ; Briggs 1953; Sharpe,1955),and the results obtained did not suggest th at the position of L.plan tarumin this genus was anomalous (Wheater, personal communication). It isinteresting, however, to note th at Camien (1952)found D-aspartic acid to be anessential metabolite for L. brevis and L . lycopersici, while it was not requiredby a strain of L. plantarum. Furthermore, maspartic acid could not bedetected in hydrolysates of the latter species, although significant amounts ofit were found in the other lactobacilli. The significance of the unusual cell-wallcomposition of L. plan tarum may become apparent when a wider survey ofother Gram-positive bacilli has been undertaken.

With regard to the two strains labelledMicrococcus rhodochrousandM . cifina-bareus (Table a ) , reasons have already been given for thinking that theseorganisms belong to the genus Corynebacterium. It seems possible that thesame consideration might apply to various other strains a t present classifiedas Rhodococcus.

We are indebted to Professor C. F. Barwell, Professor F. L. Warren and Dr S. T.Cowan for reading the manuscript, and t o Dr Cowan also for much helpful advice onpoints of bacterial taxonomy ; o Dr Dorothy Wheater for information abou t physio-logical tests in the classification of lactobacilli; to Dr D. Payne, Dr W. H. H. JebbDr F. C. 0. Valentine, Dr Lane Barksdale and Mr J. D. Paterson for providingcultures for examination; to Mr R. C. Valentine, National Institute for MedicalResearch, Mill Hill, for electron micrographs; and finally to Mr B. Cohen for hisexcellent and painstaking technical assistance.

REFERENCESBELLAMY, . D. & KLIMEK, . W. (1948). Some properties of penicillin-resistantstaphylococci. J.Bact. 55, 153.BRIGGS,M. (1953). The classification of lactobacilli by means of physiological tests.

J.gen. Microbiol. 9, 234.CAMIEN, . N. (1952). Antagonisms in the utilization gf D-amino acids by lactic acidbacteria. IV. D-aspartic acid. J.biol. Chem. 197, 687.CUMMINS,C. S. (1954). Some observations on the nature of the antigens in the cellwall of Corynebacterium diphtheriae. Brit . J . exp. Path. 35,166.CUMMINS,C. S. & HARRIS,H. (1955). Differences in cell wall composition amongGram-positive cocci and bacilli. J.gen. Microbiol. 13, ii.DEWEY,D. L. (1954). The distribution of diaminopimelic acid decarboxylase amongsome organisms of the coli-aerogenes group and certain other bacteria. J.gen.Microbiol. 11, 307.GALE,E. F. & RODWELL, . W. (1948). Amino acid metabolism of penicillin-resistant staphylococci. J.Bact. 55,161.HOLDSWORTH,. S. (1952). The nature of the cell wall of Corynebacteriumdiphtheriae.Isolation of an oligosaccharide. Biochim. biophys. Acta, 9, 19.HOTCHIN,. E., DAWSON,. M. & ELFORD,. J. (1952). The use of empty bacterialmembranes in the study of the adsorption of StaphylococcusK phage upon itshost. Brit. J. exp . Path. 33,177.KENT,P. W. & WHITEHOUSE, . W. (1955). Biochemistry of the Amino Sugars.London : Butterworth.39 G. Microb. X IV


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600 C . S . Czcmmins and H . HarrisMACLEAN,. D., LIEBOW, . A. & ROSENBERG,. A. (1946). A hemolytic coryne-bacterium resembling Corytaebacterium ovis and Corynebacterium p yogenes inman. J . infect. Dis. 79 , 69,MCCARTY,M. ( 1 9 5 2 ~ ) . he lysis of group A hemolytic streptococci by extracellularenzymes of Streptomyces albus. I. Production and fractionation of the lyticenzymes, J. exp. Med. 96 , 5155.MCCARTY,M. (1952b). The lysis of group A hemolytic streptococci by extracellular

enzymes of Streptomyces albus. TI. Nature of the cellular substrate attacked bythe lytic enzymes. J. exp. Med. 96, 569.MITCHELL,P. & MOYLE,J. (1951).The glycerophospho-protein complex envelope ofMicrococcus pyogenes. J . gen .Microbiol. 5 , 981.PARTRIDGE,. M. & WESTALL, . G. (1948). Filter paper partition chromatographyof sugars. 1. General description and application t o the qualitative analysis ofsugars in apple-juice, egg-white and foetal blood of sheep. Biochem.J.42, 238.Report of Discussion Meeting of the Society for General Microbiology on the Princi-ples of Microbial Classification, September 1954 (1955). J.gen. Microbiol. 12,314.SALTON, . R. J. (1952). Studies !of the bacterial cell wall. 111.Preliminary investi-gations of the chemical constitution of the cell wall of Streptococcus faecalis.Biochim. biophys. Acta, 8, 510.

SALTON, . R. J. (1953). Studies of the bacterial cell wall. IV. The composition ofthe cell walls of some Gram positive and Gram negative bacteria. Biochim.biophys. Acta, 10 , 512.SCHMIDT,W. C. (1952). Group A streptococcus polysaccharide: studies on itspreparation, chemical composition and cellular localization after intravenousinjection into mice. J. exp. &Zed. 95 , 105.SHARPE, . E. (1955). A serological classification of lactobacilli. J.gen. Microbiol.12 , 107.SHAW, ., STITT, . & COWAN,S. 'I?. (1951). Staphylococci and their classification.J. gen. Microbiol. 5, 1010.STRANGE,. E. & POWELL,. F. (1954). Hexosamine-containing peptides in sporesof Bacillus subtilis. B . megathleriumand B. cereus. Biochem. J . 58 , 80.

WHEATER,D. M. (1955a). The characteristics of Lactobacillus acidophilus andLactobacillus bulgaricus. J . gem Microbiol. 12, 123.WHEATER, . M. (1955b). The char,acteristicsof Lactobacillusplantarum, L . helveticusand L. casei. J.gen. Microbior!.12 , 133.WILLIAMS, . E. O., HIRCH,A. & COWAN,S. T. (1953). Aerococcus, a new bacterialgenus. J.gen. Microbiol. 8, 475.WORK,E. (1951). The isolation of a, e-diaminopimelic acid from Corynebacteriumdiphtheriae and Mycobacterium tuberculosis. Biochem. J . 49, 17.WORK,E. (1955). Some comparative aspects of lysine metabolism. In Amino Acidmetabolism, p. 462, ed. McElroy & Glass. Baltimore: Johns Hopkins Press.WORK,E. & DEWEY,D. L. (1953). The distribution of a, s-diaminopimelic acidamong various micro-organism,s. J. gen. Microbiol. 9, 394.

(Received' 10 Novem,ber 1955)

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