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THE EXTRACELLULAR POLYSACCHARIDES OF BACTERIA

J. F. WILKINSON

Bacteriology Department, University of Edinburgh, Edinburgh, Scotland

CONTENTSI. Introduction..................................................................... 46

II. Morphological Localization............................................................... 47A. Intracellular Polysaccharide.......................................................... 47B. Cell Walls and Capsules.............................................................. 47C. Microcapsules..................................................................... 48D. Cell-Wall Polysaccharide.............................................................. 49E. Extracellular Polysaccharide.......................................................... 49F. Demonstration of Capsules ........................................................... 49

III. Chemistry and Antigenicity............................................................... 51A. Methods of Purification and Analysis .................................................. 51B. Chemistry of Homopolysaccharides .................................................... 52C. Chemistry of Heteropolysaccharides.................................................... 52D. Relationship of Antigenicity to Colony Form .. 56E. Immunochemistry of Extracellular Polysaccharides .. 56F. Typing by Infrared Spectrophotometry................................................ 57

IV. Formation by Intact Cells................................................................ 58A. The Influence of the Growth-Limiting Nutrient........................................ 58B. Formation by Washed Suspensions..................................................... 59C. Factors Influencing Carbon Assimilation into Polysaccharide ........................... 59

V. Metabolism...................................................................... 61A. Catabolism...................................................................... 61B. Anabolism of Homopolysaccharides.................................................... 63C. Anabolism of Heteropolysaccharides ................................................... 63

VI. Function................................................................................. 65A. Protection Against Phagocytosis....................................................... 66B. Protection Against Amoebic Attack.................................................... 67C. Protection Against Bacteriophage...................................................... 67D. Endotoxins and Aggressins............................................................. 68E. Protection against Desiccation......................................................... 68F. Reserve Carbon and Energy Source.................................................... 69G. To Aid in the Uptake of Ions.......................................................... 69H. To Aid in Dispersal......................................................................69I. Conclusion...................................................................... 69

VII. References................................................................................ 69

I. INTRODUCTION structures containing three or more componentBacteria, in common with all higher living or- sugars, some of which seem to occur naturally

galnisms that have been studied, contain poly- only in bacteria. Because of the importance ofsaccharides. The amount formed will vary ac- these compounds in determining virulence andcording to the organism but may rise under in the antigenic differentiation of pathogenicfavorable conditions to as much as 60 per cent organisms, much research has been carried outof the bacterial dry weight as intracellular poly- into their chemical nature, metabolism, and func-saccharide and many times the cellular dryweight as extracellular polysaccharide. There is tion. Reviews have been written on specialized

a wide range in the composition of bacterial aspects concerned mainly with intracellular and

polysaccharides from those containing single cell-wall polysaccharides, but this review at-sugars joined together by a single type of linkage, tempts to generalize our knowledge of the extra-to complex, high molecular weight branching cellular polysaccharides.

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1958] EXTRACELLULAR BACTERIAL POLYSACCHARIDES 47

II. MORPHOLOGICAL LOCALIZATION a definite external surface. But this implies an

Polysaccharides can be divided intot. understanding of the term cell wall and, as

main groups according to their morphological Tomesik (3) has pointed out in an informativelocalization: (a) Intracellular polysaccharides review of the subject, it is often difficult to dis-located inside, or as part of, the cytoplasmic tinguish where the cell wall ends and the capsulemembrane; (b) cell-wall polysaccharides forming begins.a structural part of the cell wall; and (c) extra- The following criteria are suggested for such acellular polysaccharides located outside the cell distinction: (a) Size: The cell wall is a thin layerwall. normally between 10 and 20 m;& and rarely ex-

This review is concerned mainly with the third ceeding 25 myA in thickness. It is thus resolvablegroup, except where the other two groups have only by the electron microscope. The capsule isan intimate bearing on the subject under dis- by definition visible using the light microscopecussion. However, it is first necessary to consider and must, therefore, have a thickness of at leastthe distinction between these groups. 200 m1A. Smaller capsules might be resolvable by

the electron microscope but the great shrinkageA. Intraceuar Polysacharide which occurs during the preparation of the

With the production of protoplasts from m~any specimen for the microscope grid makes recog-

organisms, the distinction between the cytoplas- nition by this method most uncertain.mic membrane and the cell wall has been clarified. (b) Structural function: The cell wall is essen-

tial for bacterial viability. It prforms a me-We may, therefore, consider the intracellular y pepolysaccharide as being characteristic of the chanical function preventing the osmotic swellingbacterial protoplast. We may further divide this and bursting of the bacterial protoplast. If it is

intracellular protoplast fraction into thr specifically destroyed (e.g., by lysozyme or

furtherpossiblegrmutation) then the cell is usually unable tofurther possiblegrous. () Cytoplasmic mem- divide. The capsule, on the other hand, can bebrane polysacchayerideto cytoaick removed without loss of cell viability. Thus,brane is a layer 5 to 10 m~u thick containig DuoanAv 4shwdttane emainly lipid and protein (1). Polysaccharide has ry ()not been demonstrated as a component, although obtained from a soil bacterium hydrolyzed thesystems synthesizing it are probably present. capsular polysaccharide of Diplococcus pneu-

.There have been moniae type 3. The cells obtained were completely(b)Poysacchrideganules free from a visible capsule and from immuno-many claims in the past for the presence of poly- chemically deetble capsularpoycid

saccharide granules in the bacterial cytoplasmbut there has been no well-substantiated case for yet they remained fully viable. Mutants can also

isolation and identification of such granules, be obtained from capsulate organisms, whichthe

a.

e f have either lost the ability to form the capsularnor any clear evidence for the presence of poly-saccharide as a partial constituent of other substance or form it as a soluble, free slime (5).granules, such as nucleus, volutin granules, and These mutants will grow in artificial media as

lipid granules. (c) Polysaccharide generally d well or better than the capsulate organisms from

tributed in the cytoplasm: Many, if not a, which they were derived.bacteria contain polysaccharides dispersed i (c) Composition: The cell wall is composed ofsolution throughout the cytoplasm. These poly- very high molecular weight components, usuallysaccharides are usually of a simple chemical type in the form of complexes between protein, lipid,(e.g., starch and glycogen) and are assumed to and polysaccharide (for a review on the chemistryact as carbon and energy reserves (2). of the cell wall, see (6)). It forms a structural

unit which is difficult to break into its componentB. CeU Walls and Capsues parts and is relatively unhydrated. On the other

Before dealing with cell-wall polysaccharides hand, the capsule is usually composed basicallyand extracellular polysaccharides, it is essential of a single molecular species, which is eitherto discuss the distinction between a cell wall and polysaccharide or protein. It is highly hydrated,a capsule. The working definition of a capsule is containing as much as 98 per cent water (5), andthat it is a covering layer outside the cell wall, it has a slimy or gelatinous consistency.demonstrable by the light microscope and having It has often been assumed that the cell wall


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48 J. F. WILKINSON [VOL. 22

and the capsule are quite distinct chemically (Lancefield group A) where the M protein, al-and immunologically. This is certainly the case though not constituting a microscopically visiblewith the majority of organisms studied. Thus capsule in cells not producing hyaluronic acid,D. pneumoniae and Klebsiella aerogenes have a apparently permeates the polysaccharide capsule,capsule which is apparently made up solely of when present, acting as an agglutinin (9). Again,the type-specific polysaccharide and water. No it may be assumed that there is a salt-like linkagecellular antigens (i.e., those formed against the between the hyaluronic acid which forms thenoncapsulate mutant) are present in the capsule, basic structure of the capsule, and the M protein.as may be seen by agglutination and antibody- To sum up, the cell-wall components and theabsorbing experiments (5). However, in some predominant capsular component are quiteorganisms, the capsule contains a component distinct, although the capsule may be permeatedwhich is characteristic of the cell wall. Thus by other compounds which combine with it in aTomcsik (3) showed that the capsule of Bacillus nonspecific manner but do not contribute tomegaterium has a complex structure. The main capsular strength. These compounds may becomponent was poly-D-glutamic acid, as in formed only at certain positions on the cellBacillus anrthracis (7), but there was another surface and give the capsule an appearance ofcomponent present, which was identified by being a complex structure, as in B. megaterium.chemical and immunological means as a poly-saccharide characteristic of the cell wall. Using C. Microcapsulesan antigenic staining method, Tomcsik showedthat~ ~.thpoyetd wa ditiue truhu A capsule has already been defined as beingvisible by light microscopy and it must thereforethe capsule, whereas the polysaccharide was

present only as transverse septa, polar condensa- haeialthicns gterthan 200cmic. Thistions, and indefinite striations on the surface of iiSarbitrary. There are many cases of sub-lght-the capsule (figure 1). The conclusion was drawn i . T aothat the capsule of B. megaterium consists of a microscopic "capsules" which are outside the cell.shfaot spaces within wall but which are under 200 mA in thickness.

polysaccharide~~frmwrteIt is proposed that these sub-llght-microscopicwhich are occupied by larger amounts of polypep- "capsulese know asmicrcpsls andcthat. . . ~~~~(capsules" be known as microcapsules and thattide. But does the cell-wall polysaccharide con- they can be distinguished from cell walls by thestitute a framework necessary for the structural following criteria: (a) Microcapsular substancesintegrity of the capsule? Cannot it rather be saidthat an excess of cell-wall polysaccharide is pro- are not eal for the men icalstability ofduced at the points of cell division, where it com- th clwallon can be ov chemically orbmew.tthe poyetd byannseii by mutation without loss of the viablity of the

stikes inkaghe simlarptod th at describ byc organism in media of normal osmotic pressure. ItJacoxlike(8)kabetween lartothapu pysacribede is essential to emphasize the osmotic pressureJacox (8) between the capsular polysaccharidle fatr sic .rtpatr bet iieiof~~ ~D.*nuoieadsvrlpoenSca. factor, since protoplasts are able to divide inlinkage is common between proteins and p media of high osmotic pressure (10); and (b)..iscom.onbetween proteinsThe main component is chemically and immuno-saccharides containing ionic groupings (e.g., logically distinct from the cell wall and, since ituronic acids, hexosamines). A similar type is exterior to the cell wall, it will normally occludecombination may occur in Streptococcus pyogenes the cell-wall antigen.

Microcapsules may be produced by normallycapsulate organisms under conditions unfavorableto capsule production since the thickness of thecell-wall covering substance can vary widelyunder different cultural conditions (see section on

" Cell Formation). Kauffmann (11) has divided theEM Polypeptide type-specific K antigens of Escherichia coli intoM Polysaccharide three groups (L, A, and B), all occluding the 0

Figure 1. The capsular structure of Bacillus antigen. Only the A antigen was said to bemegaterium as revealed by specific antibodies, capsulate, the L and B antigens being calledafter Tomcsik (3). "envelope antigens." However, Prskov (12)


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showed that if the bacteria were grown at 15 to on the composition of the structural components20 C instead of 37 C, all B antigens and many L of the cell walls of organisms from which allantigens were produced as visible capsules. There- superficial antigens have been lost by mutation,fore, in place of the term "envelope antigen," it it cannot definitely be stated whether polysac-is preferable to use capsular antigen (A), capsular charides enter into the composition of cell walls.or microcapsular antigen (B and L), and micro-capsular antigen (L). The K antigens of the E. Extracellular Polysaccharideenterobacteriaceae are probably all polysaccha- Extracellular polysaccharides occur in tworides and an example of a protein microcapsule forms: (a) Loose slime which is nonadherent tois the 1\1 antigen of S. pyogenes, which can be the cell. It imparts a sticky consistency to bac-removed from the cell by trypsin digestion with- terial growth on a solid medium or an increasedout affecting viability (13). The examples of viscosity in a liquid medium, and (b) Micro-microcapsules quoted above are all of single capsules and capsules which adhere to the cellmolecular species. Complex antigens may also wall. They have a definite form and boundary,form microcapsules. For example, the 0 antigen being only slowly removed by shaking in waterof smooth gram negative bacteria can be lost by or salt solutions. It is therefore possible to sepa-mutation without affecting cell viability, as rate microcapsules and capsules from loose slimediscussed subsequently. by centrifugation. Cells producing capsulesAlthough distinctions have been drawn be- always produce slime which is very similar to thetween the cellular cell wall and the extracellular capsular material, as discussed subsequently.microcapsule and capsule, these distinctions are In a few bacteria, a structure intermediatenecessarily arbitrary and are drawn mainly for between a capsule and slime is produced. Thus,convenience. The layers lying outside the cyto- a strain of Aerobacter cloacae gave indefinitelyplasmic membrane are closely bound together demarcated capsules which slowly disintegratedand, as in the case of B. megaterium, components and dispersed (5).from one layer may permeate another.

F. Demonstration of CapsulesD. Cell-IVail Polysaccharide

A wide variety of staining methods have beenCell walls are normally isolated by mechanical devised to demonstrate the presence of capsules

disruption of the cell, followed by differential in bacteria and they have been compared bycentrifugation. These procedures often yield cell Duguid (16). Unfortunately. capsules are un-walls contaminated by inner (cytoplasmic mem- reactive to normal staining procedures appliedbrane) and outer (microcapsules and capsules) without preliminary mordanting and are easilycell materials. Thus Salton (14) showed that the distorted by fixing. They rely on the presence oftype-specific AI protein of S. pyogenes was present at least 90 per cent of water to retain theirin purified cell-wall preparations and that the rigidity and if this water is lost during fixation,monosaccharide constituents identified in similar they will shrink to a small fraction of their originalcell-wall preparations from Salmonella pullorum size. Thus cells with large capsules studied in thewere identical with those of 0 antigens of related electron-microscope after air drying generallyspecies isolated by Davies (15). No data are show a dense shrunken capsule (17). Some cellsavailable giving an analysis of the cell wall of a show good differentiation (figure 2) but even intypically capsulate organism but it seems likely these cases there is a shrinkage of the capsulethat the preparation would again be contami- compared with observations with wet films.nated by capsular material, since capsules are Probably better results would be obtained usingknown to be relatively resistant to mechanical freeze-dried cells.agitation. It is thus possible that the carbo- Two methods have been described recently forhydrate components of these isolated cell walls the specific demonstration of polysaccharideare due to the presence of adherent microcapsules capsules but both require preliminary dryingand capsules and in particular to group-specific and heat fixing. The first is the periodate-Schiffand type-specific antigens. It is probable that method of Hotchkiss (18), which is said to bethe R antigen in gram negative bacteria is a basic specific for polysaccharides. Unfortunately, notcell-wall antigen but until more data are available all polysaccharides are stained, probably due to


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Figures 2-7

All strains of Kiebsiella aerogenes grown at 35 C on the surface of solid media as described previously(5, 17). Magnification of electron micrograph X 7000. (Enlargement, 1.71X). Magnificant of light micro-graphs X 1000. (Enlargement, 3X). Figures 2, 4, and 5 originally published in the Journal of GeneralMicrobiology. We thank the copyright owners for permission to reproduce. Figures 3, 6, and 7 are byJ. P. Duguid.

Figure 2. (top left) Strain A3. Nitrogen-deficient medium for 48 hr. Unfixed and unshadowed filmby the electron microscope at 75 kv. Dark bacilli with lighter capsules surrounded by even lighter slimefenestrated by drying.

Figure 3. (top right) Strain A3. Nitrogen-deficient medium for 168 hr. India ink wet, film showingcapsules and abundant slime.

Figure 4. (center left) Strain A4. Nitrogen-deficient medium for 24 hr. Periodate-Schiff stain. Intensebipolar staining of cells and lighter staining of capsules.

Figure 6. (center right) Strain Al. Nitrogen-deficient medium for 48 hr. Suspended in antiserum forhomologous type 54. Wet film shows "specific capsular reaction" and agglutination.

Figure 6. (bottomt left) Strain A3. Nitrogen-deficient medium for 24 hr. India ink wet film. Showslarge capsules.

Figure 7. (bottomn right). Strain A3. Carbon and energy-deficient medium for 24 hr. India ink wetfilm. Shows small capsules.


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differences in the initial reaction with periodate. III. CHEMISTRY AND ANTIGENICITYThus, Duguid and Wilkinson (17) found that the Polysaccharides fall into two groups accordingcapsules of K. aerogenes were unstained with the to the number of component sugars present. Firstexception of one strain (A4) (figure 4). This are the homopolysaccharides or homoglycans inmethod also stains the cell, often giving a polar which there is only one component sugar. Thedistribution of stainable material (figure 4) second are heteropolysaccharides or heterogly-which, in gram negative bacteria, is probably due cans in which there are two or more componentto the polysaccharide component of the 0 anti- cansmwch ther or morelcomponegen. The second method of staining is the use of extracellularly pAlcian blue described by Novelli (19). Capsulesare stained blue against the bacilli counter- A. Methods of Purification and Analysisstained with basic fuchsin. Although this method The reparation of extracellular olysaccha-can be used to demonstrate capsules, its speci- rides i ppep yrdsma reasonably pure state is often a simpleficity for polysaccharide is open to doubt, since matter, particularly if they occur as loose slime.McKinney (20) found many monosaccharides The bacteria are grown under conditions whichand organic acids would also react.The bestnic metds forld capsuledemonstrai give a maximum concentration of polysaccharideThe best methods for capsule demonstration

an a muto erdto. Independ~.upo neaiesanngb.hc h and a minimum amount of degradation. Incapsule surfacnegatiseutlinednprefably when the practice, the following rules should be observed.

capsule surface is outlined, preferably when the Uetesmls yeo eimspotnsuspndedinawatey meium. Use the simplest type of medium supportingbacterium is suspended in a watery medium. growth, preferably a simple synthetic one free ofThere are two methods of general applicability. nondialyzable products and, in particular, free of(a) The India ink method: The particles of India preformed polysaccharide. Alternatively, theink are unable to penetrate the polysaccharideor protein gel of the capsule. Because of the high oganismmy e grn the sfe ofaatsolopacity~ofteepricete.roiea da agar medium. The danger that the preparation

method for the relef demonstran ofeth will be contaminated by agar polysaccharides candihdftamete myt be avoided by placing a layer of cellophane on

colorless capsules. The capsule diameter may the surface of the agar and inoculating the organ-then be determined with the aid of a micrometer isms on top of this (17). Dialyzable constituentseyepiece. The method is easy to use and is in the medium pass through the cellophane, whiledescribed in detail by Duguid (16). It may also the bacteria together with nondialyzable poly-be used to demonstrate the presence of loose saccharide remain on the surface.slime. The film is prepared from a surface culture Use conditions giving a maximal yield ofon solid medium. The slime forms an amorphous polysaccharide per cell, as described subse-gel which is only slowly permeated by India ink quently.particles and shows as an irregular white or gray Use conditions giving minimum autolysis ofmass surrounding the cells. For examples of cells, i.e., short time of incubation, low tempera-results using this method, see figures 3, 6, and 7. ture, and neutral pH. Extracellular products are

(b) Specific capsular reaction: This method then uncontaminated by intracellular products.depends upon the combination of the capsular Use conditions giving minimum degradationsubstance with its homologous antibody. The re- of polysaccharides. In the absence of enzymesaction was originally known as Neufeld's swelling breaking down the extracellular polysaccharides,reaction but comparison with the India l the main considerations are again a low tempera-method has shown that no swelling occurs (16) ture and a pH near neutrality.

After growth, the cells are harvested, centri-and that the antibody-antigen complex merely fuged, and washed. This step provides an imme-

outlmes the surface of the capsule (figure 5). diate and simple separation of intracellular,

Tomesik (3), after reviewing the reaction, sug- capsular, and microcapsular polysaccharides ingested the use of the term "specific capsular re- the centrifugate from extracellular slime poly-action." The method has the advantage of saccharide in the supernatant. Various methodsspecificity and can be used for the serological have been devised to remove the capsular poly-typing of capsulate organisms and for studies on saccharides from the cell. In a few cases, thecapsule structure. capsule is sufficiently labile to be removed by


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shaking in water, saline, or buffer solutions, but mation. Thus Jeanes et al. (22) have character-generally more drastic procedures are required ized the dextrans produced by 96 bacterial(e.g., dilute sodium hydroxide solutions, boiling strains and found differences in the percentagesat a neutral pH, or autoclaving). The dissolution of the different linkages. Although all strains hadof microcapsular polysaccharides is often more a majority (50 to 97 per cent) of 1-6 linkages,difficult still without the use of degradative there was a widely varying percentage of 1-3 andmethods, e.g., trichloracetic acid treatment. 1-4 linkages (0 to 40 and 0 to 50 per cent, re-After extraction, the polysaccharide is purified spectively).by methods which should avoid extremes of pHand temperature, tested for purity and homoge- C. Chemistry of Heteropolysaccharidesneity, and its structure analyzed. Finally, the A wide variety of bacterial extracellulartype of binding of the polysaccharide into com- heteropolysaccharides have been studied ex-plex cellular structures is determined. tensively because of their importance in classifi-

B. Chemistry of Homopolysaccharides cation and pathogenicity. The relation betweenbacterial antigenicity and the chemical groupingsThe five mai groups of bacterial extracellular on the cell surface is still obscure in most cases

homopolysaccharides, together with their com except for D. pneumoniae and the enterobac-ponent sugars and the linkages between them teriaceae. In the enterobacteriaceae, antigenicand the main groups of synthesizing bacteria, are specificity is determined by two main types ofshown in table 1. Bacterial cellulose, starch, and polysaccharide, that in the 0 or somatic antigenhyaluronic acid have properties similar to the and that in the K or capsular antigen. The maincorresponding plant and animal products. Levans characteristics of these two types may be sum-and dextrans, however, occur only in bacteria. marized as follows:Hyaluronic acid is included for convenience in Somatic 0 polysaccharide: (a) Largely boundthe homopolysaccharides, although it has two in high molecular weight complex with lipid andcomponent sugars. Chemically it can be con- protein; (b) closely bound to cell wall and usuallysidered as a simple straight chain polymer of the present as a microcapsule, although it may perme-disaccharide acetylhyalobiuronic acid (glucuronc ate any true capsule present; (c) heat-stableacid-l-6-3-N-acetylglucosamine) as distinct from antigen; (d) group-specific; (e) results in a smooththe typical branched heteropolysaccharide with colonial form in absence of the K polysaccharide;no simple repeating unit. and (f) in complex form is an endotoxin.These homopolysaccharides are characterized Capsular K polysaccharide: (a) comparatively

by having often a very high molecular weight low molecular weight and mainly noncomplexed;and in being highly polydisperse (21). Those with (b) not bound to cell wall but present as extra-branched chains have a fairly wide range of cellular capsule or slime; (c) heat-labile antigen;composition, according to the conditions of for- (d) type-specific; (e) results in mucoid colonial

TABLE 1 form; and (f) nontoxic aggressin.As is emphasized in the appropriate sections of

Bacterial extracellular homopolysaccharides this review, these distinctions are not invariableNam-an- Rfernc C

but can serve to distinguish the two main groups.Name and Reference Component Sugar Linkages A typical example of each will be considered in

Cellulose (23) Glucose detail in order to bring out their different natures.Starch (24) Glucose -1--4 The somatic 0 polysaccharide of Shigella dysen-

teriae. Westphal and Lideritz (27) have reviewedDextran (22) Glucose 1 -6 the various methods of preparing the 0 antigen

(-1--3-) from the cell, which include extraction with solu-(-1 ox-4-) tions of trichloracetic acid, phenol, pyridine,

Levan (25) Fructose 2 4- urea, or trypsin. These methods all give rise to a(-2-fI-1-) partial destruction of the native complex and

Hyaluronic acid Acetylhyalo- ?Morgan (28) showed that the most useful solvent(26) biuronic for Shigella dysenteriae was anhydrous diethylene-

acidglycol. After extraction and purification, the 0

* Branching linkages in parentheses. antigen represented 5 to 7 per cent of the bacterial


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dry weight. The morphological and staining graded polysaccharide which was produced incharacteristics of the extracted organism were excess by the bacteria in an uncombined form. Itlargely unchanged, showing that the bulk of the accounted for more than half the weight of thecytoplasmic material remained intact. The puri- first diethyleneglycol extract and decreased byfied 0 antigen was a very high molecular weight about half in successive extracts. It was thereforecomplex of polysaccharide (ca. 60 per cent), suggested that it formed the outermost layer oflipoprotein (ca. 20 per cent), and phospholipid the cell and that the complete antigen lay be-(ca. 10 per cent) (29). It is uncertain how far the neath it.complex is a natural molecule and how far it is The chemical and biological properties of thesean arbitrary unit joined together by weak physi- three forms are shown in table 2. The followingcal attractions, and it would be interesting to further points should be noted: (a) The unde-determine how far the proportions of the four graded polysaccharide, but not the degradedcomponents of the complex were constant when polysaccharide, combined with the lipoproteinthe organism had been grown under different to form the complex; (b) If the undegraded poly-cultural conditions. Certainly, an excess of free saccharide was hydrolyzed to the degraded formpolysaccharide is often produced (30), and it is by dilute acetic acid, there was a drop in viscosityprobable that the components are produced and about 5 per cent of an alkali-soluble non-separately at the cell membrane. Morgan and carbohydrate component was formed. ThisPartridge (31) showed that the diethyleneglycol- material was similar to the lipoprotein but had aextractable material was the complete 0 antigen, lower nitrogen content; (c) The amount of threesince no such antigen could be extracted from a component sugars of the degraded polysacchariderough variant. However, lipopolysaccharide can left 22 per cent of the material unaccounted for;be extracted by other methods from rough (d) The lipopolysaccharide, when comparedbacteria (27, 32), either because of a change in with the undegraded polysaccharide, containedthe 0 antigen resulting in a rough colony form orbecusetherouhatien s aso liopoy-a heptose component showing that the lipoid

saccaridetherougasningPasisurelsaaeio(33). contains carbohydrate units. Additional N-acetyl-The polysaccharide component of the3 glucosamine is also combined into the natural

antigen of S. dysenteriae has been analyzed in complex; (e) The endotoxic and pyrogenicdetail by Davies et al. (30). They studied it in properties were mainly dependent on the presencethree forms. First, lipopolysaccharide prepared of the lipopolysaccharide; and (f) There was afrom the antigenic complex by phenol treatment; progressive loss of antigenicity with the break-second, degraded polysaccharide prepared from down of the complete antigen. The capacity tothe antigenic complex or the lipopolysaccharide produce Forssmann heterophile antibody was

by mild acetic acid hydrolysis; and third, unde- present in the lipopolysaccharide but was com-

TABLE 2

The chemical and biological properties of the somatic antigen of Shigella dysenteriae and its degradationproducts (29, 30)

Component Sugars (per cent)Molecular Heptose LMO Pyrogenic Anti-Weight N-Acetyl- Galactose Rhanose Activityt genicity

glucosamine

Antigenic complex ca. 1 X 107 18 22 + 85 0.002 1560Lipopolysaccharide 18 26 + 80 0.002 75Undegraded poly- 950,000 22 25-26 30 370 0.05 10saccharide

Degraded polysac- 26,000 27.5 27 33 Nontoxic 5 0charide at 10

mg

* LDwI) in pg for mice.t pg to give minimum significant temperature rise (0.60/kg rabbit).t Titer for rabbits with 30 pg total dose.


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542 J. F. WILKINSON [voL. 22

pletely absent in the two polysaccharide prepara- Other linkages existed but it was difficult totions. assess their importance because of probable

Other somatic 0 antigens of gram negative incomplete methylation in the products ofbacteria. The other 0 antigens of gram negative hydrolysis of the methylated polysaccharide.bacteria and, in particular, those of the families Although these chemical studies as well asEnterobacteriaceae and Parvobacteriaceae, are antigenic studies (5) indicated that these fourprobably similar to S. dysenteriae in being lipo- preparations were similar, differences werepolysaccharide-protein complexes. In most cases, found in their physical properties and theirhowever, only the lipopolysaccharide has been ability to adsorb onto red blood corpuscles.isolated using trichloracetic acid extraction. The Thus, sedimentation and diffusion data (38)properties of these compounds in general have indicated that while the slime polysaccharidesbeen reviewed by Westphal and Lideritz (27). were elongated molecules of molecular weightK antigen of K. aerogenes type 64. All naturally about 1.5 X 106, A3 capsular polysaccharide

occurring strains of K. aerogenes are mucoid and studied prior to purification had a molecularcapsulate and recently their extracellular capsu- weight of over 1.0 X 108. This high value waslar polysaccharides have been shown to fall into lowered on purification to a figure similar toa large number of antigenic types analogous to that of the slime polysaccharides and probablythose of D. pneumoniae (34). K. aerogenes type represents an initially incomplete breakdown54 was selected for an exhaustive chemical of the polysaccharide gel during disintegrationstudy because of the knowledge of the factors of the capsule. Macpherson et al. (39) foundinfluencing its polysaccharide production (17). differences in the ability of these polysaccharides,Four preparations of the type-specific polysac- after prior treatment with sodium hydroxide,charide were obtained (35, 36). Two were the to inhibit hemagglutination by the influenzacapsular and slime polysaccharides of a typical group of viruses. While both polysaccharidesstrain A3. The third was strain A3 "S1" slime from strain A3 inhibited hemagglutination at apolysaccharide. This strain was a naturally concentration of 2.5 ,ug per ml, Al polysac-occurring mutant of strain A3 which no longer charide required a concentration of 333 gg performed a capsule but produced all the extra- ml, and A3 "S1" slime polysaccharide wascellular type-specific polysaccharide as a highly completely noninhibitory. Although all fourviscous slime. The fourth was a strain Al cap- polysaccharides were adsorbed onto the redsular polysaccharide. This was a naturally oc- blood cell surface, there was a relationshipcurring strain with type 54 specificity but much between the degree of inhibition and the loweringlarger capsules (about twice the capsule di- of the surface charge, probably owing to ioniza-ameter of strain A3). tion of the uronic acids of the polysaccharide.

All polysaccharides contained glucose, fucose, This indicates a difference in the physical statesand a uronic acid as the three component sugars of various antigenically similar polysaccharidesin essentially the same molecular proportions, in closely related strains and a difference in theshowing a chemical identity between capsules availability of carboxyl groups.and slime. They contained about 48 per cent The question arises as to the state of theglucose, 10 per cent fucose, and 29 per cent polysaccharide in the capsule which, althoughuronic acid. Further structural studies on the stable to water and salt solutions, may vary inslime polysaccharide of strain A3 "S1" (37) size according to the nature of the surroundingshowed that the uronic acid was glucuronic acid medium. If strain Al is grown on a solid medium,and that the molecule was highly branched. the capsules are shrunken because of exposureThe following sugar residues were definitely to the atmosphere, and if the cells are thenpresent: suspended in water, saline, or India ink, there is

Gp-1 4Gp-1 ;2-Fu-1 4-(or 5-) Fu-1.. nearly a tenfold expansion (5). Similarly, un-| published experiments by the author showed3 that the capsule diameter is dependent upon

the amount of salt present in the surroundingmedium. Thus, strain Al grown under nitrogen-

(Gp = D-glucopyranose, Fu = -fucofuranose). deficient conditions gave an average capsule


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diameter of about 10 ,u when a suspension in TABLE 3distilled water was mixed with an equal volume The percentages of the component sugars of theof India ink but only about 7 ,u with a corre- extracellular type-specific polysaccharides of fivesponding suspension in 10 per cent NaCl and Klebsiella strains (36)India ink. Calculations of the concentration oftype-specific polysaccharide in strain A3 from Component Sugars lKebsiella Typethe data of Wilkinson et al. (5) give values of 8 26 29 54 57about 1.4 per cent in cells with large capsulesand 1.0 per cent in cells with small capsules. Glucose 22 35 0 50 0At these concentrations, the capsular polysac- Galactose 51 26 31 1 21charide might be expected to be a fairly stable Fucose 0 0 0 10 0gel which would vary in size according to the Uronic acid 25 17 25 29 28amount of salt present in the surrounding me- -._____acid_2 17

_ 25 _29 _2dium, on which depends the degree of neutrali-zation of the negative charge on the polysac-charide molecule by positive ions. The molecules Eacellular yehioide o gam postiein this gel should be equally distributed through-out. Assuming a 1 per cent solution of polysac- any general types of antigens occur in gram

charide of molecular weight 1.5 X 106, the positive bacteria. Polysaccharides may occur as

average distance between the center of mole- type-specific- capsules or slime analogous tocules would be about 65 mn~z, which provides a gram negative K antigens, as in the well known

rough estimate of the permeability of the capsule pneumococcal types. These type-specific Poly-shape and saccharides of D. pneumoniae are haptens, being

subject to variation according to the combineasacmlxnnie n h eldistribution of molecules. Slime polysaccharide Dubos (40) has shown that this complex canmay either come gradually into solution by achange in the capsular polysaccharide at the be broken down by ribonuclease and he there-surface of the capsule or continually diffuse fore assumed that it is a ribonucleic acid-polysac-out through the capsule. Mutation to produce charide compound. However, it is difficult toonly slime, as in strain A3 "Sl", might have determine how far the enzyme preparation wasresulted in a different type of folding of the specific for ribonucleic acid and it is also possiblepolysaccharide chain so that there were less that the complex is a nonspecific combinationcarboxyl groups available. produced during the preparation of the antigen.No evidence is available on the nature of any A typical lipopolysaccharide-protein 0 antigen

complex in the cell where polysaccharide is complex is unlikely to occur in gram positivecombined with a noncarbohydrate component. bacteria, since cell-wall preparations from these

Other K antigens in gram negative bacteria, organisms which contain group-specific antigensTypical K antigens occur in most groups of have only a small percentage of lipid comparedgram negative bacteria, although they are more with those from gram negative organisms (14).abundant in some groups (e.g., Klebsiella) than Instead, a polysaccharide-protein group-specificothers. They have a wide range of composition, antigen is often present which may be a trueaccounting for their antigenic specificity, as cell-wall component. Guex-Holzer and Tomesikseen in the analyses of five kiebsielia strains (41) studied this antigen in Bacillus M and foundshown in table 3. the haptenic polysaccharide to contain gluco-

In some bacteria, factors in the environmentmay lead to a greater degree of antigenic van- s galactosamine, and another component.ation in the 0 antigen than in the K antigen so This polysaccharide was produced in excessthat the terms group-specific and type-specific over that combined to give a mucoprotein andare not invariable. It must be further empha- the excess permeated the D-glutamic-acid poly-sized that not all antigens fall clearly into one peptide capsule. In the absence of a capsule, itof the two classes discussed above but that might form a microcapsular layer analogousintermediate classes exist (e.g., Vi antigen of to that of the free 0 polysaccharide in S. dys-Salmonella species). enteriae.


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D. Relationship of Antigenicity to Colony Form negative bacteria (table 4). Various attempts

Ideally, a correlation exists in gram negative have been made to extend the gram negativebacteria between the presence of the K, 0, and nomenclature to all bacteria (42) but, in spite ofR antigens and colonial form on solid media, the convenience of such uniformity, the differentas shown in table 4. nomenclature for the pneumococcus is still

It is therefore possible to get an idea of anti- commonly used. In this review a uniform no-genic character by studying colonial form on a menclature will be adopted.suitable medium. However, the relationship is The presence of additional protein antigensnot invariable and the following points should on the cell surface will alter colony form. Thusbe borne in mind. in S. pyogenes, the matt form possesses the MThe hydrophilic K antigen confers a mucoid protein but no type-specific polysaccharide.

form to a culture only when organisms are Variants without the M protein are usuallygrown under conditions suitable for its produc- referred to as glossy but should be classified astion, as discussed subsequently. Various degrees smooth.of mucoidness will exist according to the medium E. Immunochemistry of Extracellularused (5, 17). Further, a colony may become Polystharyfdemucoid in the absence of a type-specific antigenbecause of the production of an extracellular Since polysaccharides are the major com-homopolysaccharide such as dextran. This can pounds determining the antigenic specificity ofoften be avoided by the absence from the medium the surface of most living cells and since theyof the oligosaccharides required for homopoly- are simpler in composition than the other largesaccharide synthesis. group of naturally occurring antigens-theThe possession of an 0 antigen in the absence proteins-much work has been done on their

of a K antigen does not necessarily give a smooth chemical specificity. The evidence suggestsculture. Various factors contribute to the rough- that the combination between a polysaccharideness of a colony, among the most important of and its homologous antibody does not occurwhich is the mode of division. If a microorganism over a large area of the polysaccharide surfaceproduces long chains instead of single cells that but rather in an area corresponding to an oligo-separate immediately after division, a rough saccharide of not more than six monosaccharidecolony will be formed. Presumably, the 0 antigen units, usually at the end of a polysaccharideaids separation after cell division. However, chain (43).many bacterial strains are intermediate between The type-specific polysaccharides of D.a typically rough and typically smooth colony pneumoniae have been most studied immuno-type. chemically and type 2 (TSP 2) will be used as

In gram positive bacteria and, in particular, an illustration of immunochemical analysis.in the pneumococcus, nomenclature is not in It is made up of units of glucose, glucuronicline with that generally accepted for gram acid, and rhamnose (44) and Heidelberger and

Adams (45) have shown that the homologousTABLE 4 antibody exhibits a series of cross reactions

The relationship between colony form and antigens which fall into various groups according to thepresent component sugar which is antigenically active.

Characteristic Designation Glucose. Although it has been known for manyyears that bacterial dextrans gave cross reactions

Antigens present K, R o, R R with many antipneumococcal sera, includingor those against TSP2, the basis of these reactions

K, 0, R has been studied only recently (46). A searchAntigenic designa- K 0 R was made for other glucose polymers containing

tion similar linkages to dextran (1-6 with 1-4 andColony form Mucoid Smooth Rough 1-3 side linkages). Synthetic polyglucoses whichColony designation M S R contain a variety of linkages, with 1-4, 1-6, andDiplococcus no- Smooth Rough 1-4-6 predominant, also showed cross reactionsmenclature with TSP2 antibody, as did a number of glyco-


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gens which contain 1-4-6 glucose branch points. the V-determinant rhamnose linkages. TheThese results confirm the chemical studies of mutation A -+ V had apparently resulted in theButler and Stacey (44), which showed that all removal of some N-acetylglucosamine groups,the glucose in TSP2 was in the form of 1-4-6 demonstrating that a change in only a section ofbranch points. a whole polysaccharide molecule may cause a

Glucuronic acid. The precipitation of TSP2 considerable change in antigenic properties.antibody by gum arabic and related polysac- Rhamnose. A cross reaction exists betweencharides was due to the common possession of TSP2 and rhamnose-containing polysaccharides,glucuronic acid. Glucuronic acid-containing such as the C fraction of a human strain of thepolysaccharides precipitated much more anti- tubercle bacillus and karaya gum.body than those in which the cross reaction is Thus the antigenic specificity is partly adue to glucose or rhamnose. Up to 40 per cent reflection of the presence of the componentof the total TSP2 antibody may be precipitated sugars of the polysaccharide. However, theby gum arabic, whereas polysaccharides con- proportion of antibodies reacting with the partialtaining glucose or rhamnose fail to give values specificities described above did not exceed 20higher than 5 per cent. The importance of the per cent and 50 per cent in the two sera studiedposition and availability of the determinant (45). Therefore, a proportion of antibodiesgroups is shown by the fact that a mild acid require more than the occurrence of the singlehydrolysis of gum arabic resulted in a reactivity component sugars of TSP2 for specific precipita-with TSP2 antibody at a several hundredfold tion. It may be assumed that an oligosaccharidehigher dilution. The hydrolysis removed anti- side chain with the component sugars in thebody-blocking arabinose units which were correct order is required for complete precipita-attached to glucuronic acid. Thus the acid-stable tion. But many bacterial extracellular polysac-glucuronic acid groups became end groups, a charides probably have a branched, highlycharacteristic they have in common with TSP2. complex structure with different types of sideIt is evident that end groups are the prime de- branches and we may assume that there will beterminants of immunological specificity. more than one antibody produced against theThe importance of external groupings and different side chains. Some will have a specificity

the way in which antigenic units may be blocked against a single sugar but others will be directedin the interior of a polysaccharide molecule are against an oligosaccharide and will thus have afurther illustrated by the work of McCarty greater degree of specificity. Immunochemistry(47). He compared the immunochemistry of the is able to tell us something of the type of sugarsgroup A specific carbohydrate of S. pyogeucs and linkages involved. At the same time, it mustwith that of the V carbohydrate from a variant be emphasized that in practice most antibodieswhich had lost the group A antigen. The two are specific against the homologous polysac-polysaccharides both contained rhamnose and charide when studies are carried out at lowN-acetylglucosamine but indifferent proportions, concentrations. It is only when much higherwith a higher percentage of rhamnose in the V concentrations of other polysaccharides arecarbohydrate. Acid or specific enzymatic hy- used that cross reactions occur. For example,drolysis was used to determine the reason for the klebsiella group can be adequately typedthe quite different antigenicity of the two poly- on the basis of their type-specific polysaccharides.saccharides. The determinant group in V car- Yet many contain identical sugars (table 3),bohydrate activity resided in a rhamnose- probably with similar linkages, and cross re-rhamnose linked oligosaccharide. When group A actions would probably be observed at highcarbohydrate was treated with a specific hy- concentrations. Thus, the immunological specific-drolytic enzyme, reactivity with A-antibody ity of bacterial polysaccharides is a practicalwas lost while reactivity with V-antibody was rather than an absolute one and most crossgained. The evidence suggested that side chains reactions will not show themselves under theof N-acetylglucosamine were serving as the conditions used for routine testing.determinants of group A activity. They maskedunderlying rhamnose-rhamnose linkages, which F. Typin by Infrared Spectrophotometrywere removed by the A enzyme, thus exposing Polysaccharides give infrared absorption


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spectra characteristic of the sugars present and of a suitable supply of other nutrients, theseof the linkages between them. Levine et al. carbon intermediates are metabolized into new(48) showed that it was possible to distinguish cell material, the bulk of which is in the form ofbetween 57 types and subtypes of D. pneumoniae high molecular weight components, i.e., protein,by the infrared spectra of the purified type- fat, and polysaccharide. The proportion of thespecific polysaccharides and also to gain some various cell constituents will vary, often be-idea of the chemical composition by the presence tween wide limits, with the nature and concentra-of amide, carboxylate, or acetyl bands in the tion of the nutrients present and the physicalspectrum. Obviously it would be impracticable conditions of growth.to use this method of typing as an alternativeto the serological method if complete purification A. The Influence of the Growth-Limiting Nutrientof the extracellular polysaccharide were neces- In the absence of toxic factors, the growth of asary. However, Levine et al. (49) simplified the bacterial culture is usually stopped by a de-method with klebsiella types and showed that ficienc in some nutrient, the nature of whichalthough whole cells gave a spectrum too com-

wywllinfluence the amount of polysaccharideplex to allow separation of types, crude poly- present in the stationary-phase cell. Duguid and

saccharide preparations, which were satisfactory Wilkinson (17) studied the effect of variousfor typing purposes, could be obtained in a few . . .hor fro aga plte inuae ov .ih, growth-limiting nutrients on polysaccharide

Aswith ther

pneumococcus infbared tpinghto a production by K. aerogenes type 54. The or-Aswrth the pneumococcus, infrared typing toa ganism was chosen because of its relativelylarge.tnprestg simple growth requirements and the large amounthas the advantage of not requiring a large of polysaccharide formed. If the carbon andnumber of specific sera. When infrared spectro-photmetesbeome orereadly aailaleenergy source (lactose) was the growth-limitingphotometers become more readily available, nuret thr wa

. rdcino... . ~nutrient, there was a minimum production ofthe method should have considerable practical polysaceharide which resulted in a mnuand theoretical importance in future studies of capsule diameter and mucoidness on solidbacterial surface structures. medium. If, however, the level of the nitrogen

IV. FORMATION BY INTACT CELLS source in the medium (an ammonium salt) wasgradually lowered until it became limiting, the

During the growth of a heterotrophic bac- amount of polysaccharide produced per cellterium, the organic substances which act as rose to a level (figure 8). This increasecarbon and energy sources are metabolized into was reflected in both the extracellular andcarbon intermediates, into sources of utilizable intracellular polysaccharides, in the mucoidnessenergy, and into waste products. In the presence of the culture, and in the capsule diameter

(figures 6 and 7). When the experiment wasz S 0 ColsWktC 2 repeated with decreasing concentrations of the

4 . Polysacharde Z sulfur or the phosphorus source, similar resultsi ° i0 Production w were obtained. In all cases, polysaccharide pro-.S8\duction, as measured by the polysaccharide:ni-aa\_ trogen ratio of the cells, reached a maximum.

ff2- \\ \ °41 The value of this maximum varied according|\ to the growth-limiting nutrient (32 for nitrogen-s deficient, 40 for phosphorus-deficient, and 17

for sulfur-deficient cultures). The reason forGo0 , these maxima is unknown, since they were

0 I 2 reached in cultures where carbohydrate waso Concntriation of Nitroggn Source still present, the majority of the bacilli were

mg ml (NH4) 4 viable, and the conditions were nutritionallyFigure 8. The influence of various degrees of suitable for.olsaccharide sythesis. The ratenitrogen deficiency during growth, on polysac- . y w

charide production, capsule diameter, and mucoid- of polysaccharide synthesis was highest duringness of culture in Klebsiella aerogenes type 54. the logarithmic phase and diminished progres-After Duguid and Wilkinson (17). sively thereafter. However, the major part of


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the production occurred after the end of the will depend upon the organism and substratelogarithmic phase (about 95 per cent in nitrogen- used.limited cultures). It appeared that the excess Assimilation can be totally inhibited by agentsof the carbon source remaining at the end of the such as sodium azide or dinitrophenol whichlogarithmic phase was utilized for polysac- uncouple the production of energy-rich phosphatecharide synthesis during the stationary phase, bonds. This occurs under conditions which leavean assumption confirmed by experiments with the rate of metabolism unaffected or even in-washed suspensions, discussed subsequently. creased, and a complete breakdown to wasteNot all deficiencies result in greatly increased products may be obtained.

polysaccharide synthesis. Thus, a potassium The extent of glucose assimilation into poly-deficiency caused only a small increase in the saccharide was studied by Wilkinson and Starkpolysaccharide:nitrogen ratio and the capsule (52), using a slime-forming variant of K. aerogenesdiameter of K. aerogenes (50), suggesting that type 54. The oxygen consumption correspondedpotassium is essential for synthesis. Since much to 65 per cent of that required for a completepolysaccharide is produced with a deficiency oxidation of glucose to carbon dioxide and water,of nitrogen, sulfur, or phosphorus, it is probable suggesting that in the absence of any furtherthat these substances are not required for poly- waste products the amount of glucose assimi-saccharide synthesis and that any substance lated was 35 per cent. The extracellular slimewhich is not directly required in this way, if it polysaccharide amounted to 30 per cent of theacts as a growth-limiting nutrient, should give assimilated glucose while the remainder wasincreased polysaccharide production per cell. present as cellular polysaccharide (at least 25Growth experiments of this type emphasize per cent) and lipid. The corresponding figurethe importance of knowing the growth-limiting for the assimilation of glucose into extracellularnutrient when dealing with stationary-phase slime polysaccharide by the same organismcultures since the morphological and metabolic under growth conditions was 7 per cent (5), thecharacteristics of these cultures will vary pro- smaller figure probably being due to additionalfoundly under different deficiencies. assimilation into nitrogenous products. The

figure of 35 per cent for the efficiency of con-B. Formation by Washed Suspensions version of glucose into polysaccharide and

The growth experiments outlined above sug- lipid by washed suspensions is low in view ofghest hairowth bxper possibedto oba pol- the energy changes that might be expected ingest that it should be possible to obtain poly-suhacneiondtremtbeao-saccharide synthesis in the absence of compounds su era sion anere mustbe aon-

cotinn nirgn sufr an phophru siderable wastage of energy in the form of heat

but in presence of a carbon and energy source unless there is some inorganic storage compoundandof potassium ions. U nde tese conios such as metaphosphate. However, no evidencethe synthess of protenm and mostgowth ors for the synthesis of metachromatic materialthesynthesisof proteinandmostgrowthf could be obtained under the conditions used inwill be inhibited so that the main assimilation the assimilation experiment.products will be those containing oniy carbon,hydrogen, and oxygen, i.e., polysaccharides and .Factrs Influencing Carbon Assimilation intolipids. The general process of carbon assimilationhas been reviewed by Clifton (51) and the find- Polysaccharideings may be summarized as follows. Nature of the carbon source. Two types ofDuring the metabolism of an organic sub- extracellular polysaccharides must be dis-

strate by a bacterium, part of the substrate is tinguished, first, those which require certainassimilated into higher molecular weight com- specific carbon sources for their formation and,pounds (rarely analyzed but assumed to be second, those which are synthesized to varyingpolysaccharide and lipid), while the other part is degrees from any utilizable carbon source.broken down to waste products (usually as- In the first case, the products are all homo-sumed to be carbon dioxide and water, although polysaccharides and are formed by the action of ain many cases other small molecular weight single enzyme on a specific oligosaccharide. Thewaste products probably occur). specificity of the carbon source is a reflection ofThe percentage of the substrate assimilated the specificity of the synthesizing enzymes,


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TABLE 5 lipid production, presumably because of anFree energy of oxidation and percentage assimilation increased concentration of the necessary sugarof various carbon sources by Escherichia coli (5s, 54) donors. Bernheimer (56) studied the rate of

production of the type 3 specific polysaccharideAssimilation (Per mnn

-AP Cent C Assimilated) (TSP3) of D. pneumoniae in washed suspensionsC Source -^A~Atom* previously treated with the specific TSP3-

Washed Young destroying enzyme to remove the capsularcells, cultures

polysaccharide. Considerable variation in theLactose 122,930 47 55 rate of TSP3 production from different carbo-Arabinose 118,383 40 64 hydrates was found. Glucose gave the highestGlucose 117,216 45 59 activity whereas fructose, cellobiose, maltose,Glycerol 105,689 20 67 galactose, and glucosamine gave reduced activi-Lactate 78,699 30 40 ties in that order. Unfortunately, no attempt wasSuccinate 68,503 30 41 tie that the or to themptiwarFumarate 68,368 25 43 madetoadaptthe organisms to theparticularPyruvate 63,080 30 44 sugar before the experiment so that the resultsI__________-I- - may partially reflect differences in rates of*-,F of oxidation to C00 + H20 adaptation.

No. of C atoms per molecule In summary, the carbon source will determineboth the amount of assimilation and the assimi-

which are discussed subsequently, and the yield lation product. This effect is mainly broughtis often close to the theoretical calculated for about by differences in the steady state con-the enzyme. centration of intermediates during metabolism.

In the second case, there will be a considerable Influence of other nutrients. The influence ofvariation in the amount of assimilation from nutrient factors on the rate of extracellularvarious carbon sources. Siegel and Clifton polysaccharide (type-specific polysaccharide) for-(53, 54) studied the extent of assimilation in the mation by washed suspensions has been shownpresence of various organic acids and carbohy- by Bernheimer (56), using D. pneumoniae typedrates, using both young growing cultures and 3, and by Wilkinson and Stark (52), using K.washed suspensions of Escherichia coli. As aerogenes type 54. Under ideal conditions, ashown in table 5, they found that young cultures high rate of polysaccharide synthesis was ob-gave a higher efficiency of assimilation than tained, D. pneumoniae producing an amountwashed cells, a difference particularly marked of type-specific polysaccharide correspondingwith substrates such as glycerol. There was also a to about half the dry weight of the cells in 1 hr.considerable difference in the percentage of The experiments, summarized in table 6, re-assimilation between different carbon and energy vealed the following influences upon the ratesources, which was not always related to the of extracellular polysaccharide synthesis: (a)free energy of oxidation. The important factor in confirmation of growth experiments, a sourcein determining the extent of assimilation was the of nitrogen or sulfur was not necessary; (b)molecular structure of the substrate and the potassium ions and, to a lesser extent, magnesiumintermediates produced during oxidation. These ions were stimulatory; (c) calcium ions wereexperiments give no clue as to the nature of the required by K. aerogenes for maximal productionassimilation products or the variation in these of extracellular slime, although they had littleproducts with different carbon sources. Such a effect upon cellular polysaccharide synthesis;variation has been found by Dagley and Johnson (d) phosphate ions were inhibitory for K.(55) who studied lipid and polysaccharide pro- aerogenes but were stimulatory for D. pneumoniae;duction by cultures of E. coli in liquid aerated the difference may reside in the possible pres-media containing varying amounts of glucose ence of phosphate in D. pneumoniae polysac-and acetate. The presence of acetate stimulated charide or a more efficient removal of phosphatelipid production and depressed polysaccharide in the washing of the gram positive organism;production, presumably because of an increased and (e) CO2 was without effect.concentration of acetyl coenzyme A and thus of It is possible that other nutrients may befatty acids. Glucose, on the other hand, stimu- required for the synthesis of polysaccharideslated polysaccharide production and depressed containing specialized groupings. Thus, a nitro-


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19581 EXTRACELLULAR BACTERIAL POLYSACCHARIDES 61

gen source would be required for the synthesis TABLE 6of hexosamine-containing polysaccharides. There The influence of the suspending medium and the gashave been many claims in past literature that phase on polysaccharide production by Klebsiellapathogenic capsulated bacteria show their aerogenes and Diplococcus pneumoniaemaximum capsule production when grown in Constituents of Suspending Polysaccharide

animal tissues. Although large quantities of Medium Gas Production'exopolysaccharides are undoubtedly produced

PhiMg Ca P AC KASDPE

by organisms grown in vivo, no quantitative -studies have been carried out and many of the + + + + - 02 100 100 100results are due to the selection of capsulate cells + + + + - 02+CO2 101 101in vivo or of noncapsulate cells in vitro. Further, + + + + - N2 36 36 25many body fluids contain a relative excess of + + + + - 02 111 13glucose which will lead to an increased polysac- + + - + - 02 18 37 44charide production compared with normal lab- + + + - + 02 83 88 63oratory media. + + + ++ 02s 103 122

Influence of oxygen. The effect of oxygen onpolysaccharide synthesis is generally related * Figures for production taking value for firstto the metabolic character of the bacterium and line as 100. KAC = K. aerogenes cellular polysac-the relative amounts of energy produced under charide (52); KAS = K. aerogenes extracellularaerobic and anaerobic conditions. In D. pneu- slime polysaccharide (52); DPE = D. pneumoniaemoniae and K. aerogenes, anaerobic polysac- type 3 specific extracellular polysaccharide (56).charide production was from 25 to 40 per cent ofthe aerobic value (table 6). Hestrin and Schramm the pH of a culture near neutrality to obtain(57), studying cellulose production by freeze- maximum polysatcharide production. Bern-dried cells of Acetobactr xylinum, found a marked heimer (56), studying the effect of pH on theeffect of oxygen tension with no production production of type 3 specific polysaccharide by aanaerobically, suggesting that synthesis occurred washed suspension of D. pneumonie found ansolely at the liquid-air interface in an undis- appreciable amount of polysaccharide formedturbed culture of the organism. between pH 5.0 and 8.0 with an optimum aboutturbedulturef the oganism.6.5 Hestrin and Schramm (57), studying cellu-

Influence of temperature. Temperature is oftenlos poutinand xylinum , andyin

critically important in determining the mucoid- lose production by A.sylinum, and Wilkinsonness of a culture and the extent of extracellular and Stark (52), studying polysaccharide pro-polysaccharide formation, irrespective of its duction by R. aerogenmes type 54, obtained aeffect on growth and general metabolism. Morgan much flatter pH optimum over a range of aboutand Beckwith (58) found increased mucoidness one pH unit on either side of neutrality.of colony in members of the Enterobaceriaceae v. METABOLISMwhen grown at low temperatures of incubation.Wilkinson et al. (5) made a quantitative study A. Catabolismof polysaccharide production in E. coli after The enzymic degradation of polysaccharidesgrowth at 37 C and at 15 to 20 C in nitrogen- has been reviewed by Manners (59). The en-deficient media. Large differences in the total zymes concerned, in common with all thoseamount of polysaccharide formed at different breaking down extracellular high moleculartemperatures were noted in some strains, e.g., weight polymers to which the cell is impermeable,strain A 102 produced 0.2 mg slime polysac- into small permeating units, are hydrolytic andcharide per mg total nitrogen after growth at often adaptive. They are largely irreversible and37 C compared with a figure of 5.3 mg at 15 cannot serve for polysaccharide synthesis.to 20 C, in spite of the total growth and cellular Those most studied have been homopolysac-polysaccharide production being almost identical. charases and, in particular, amylases, hyaluroni-On the other hand, extracellular polysaccharide dases, and cellulases. Heteropolysaccharasessynthesis in K. aerogenes was relatively un- have been only rarely described and seem to beaffected by temperature (17). very restricted in occurrence. They have been

Influence of pH. It is important to maintain generally obtained from organisms isolated after


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soil enrichment with the particular polysac- determined by chemical estimation or capsule-charide under investigation, e.g., type 3 specific size measurements. There was, however, a signifi-pneumococcus polysaccharase (4) and group- cant rate of endogenous respiration, which wasspecific streptococcus polysaccharases (47), all probably due to metabolism of the intracellularfrom Bacillus species. polysaccharide (table 6). This inability of most

Bacteria are generally incapable of breaking organisms to catabolize their own extracellulardown their own extracellular polysaccharides, polysaccharide is probably an appreciable factormucoid variants usually remaining mucoid long in governing soil structure. The porosity of soilafter autolysis of the cells. Thus, unpublished is largely determined by the presence of microbialresults from this laboratory (Liston and Wilkin- extracellular polysaccharides which persist inson) have shown that K. aerogenes was unable the soil for a considerable period (60).to break down or utilize its own extracellular The inability of a bacterium to catabolize itstype-specific polysaccharide. Purified prepara- own extracellular polysaccharide may be due totions of type 54 polysaceharide (36) would not the irreversibility of the synthesizing system,act as a source of carbon and energy for growth to the inability of the excreted polysaccharideat 30 C for 72 hr in the presence of an aerated to return to the synthesizing system, or to bothliquid medium containing 0.1 per cent glucose, factors. Many homopolysaccharide-forming en-0.5 per cent ammonium sulfate, and other zymes are irreversible, particularly if the aglyconeessential nutrients (17). The results, given in produced from the glycoside donor (see nexttable 7, show no significant difference in the section) is removed. It is unknown whetheramount of growth with or without the addition heteropolysaccharide-synthesizing systems areof 0.1 per cent polysaccharide, whereas 0.2 per reversible but, by analogy to protein synthesis,cent glucose gave nearly double the growth. they will not be if a template is involved. It isNor was there any significant decrease in the also probable that most extracellular polysac-amount of added polysaccharide, even after 10 charides are formed inside the cell or on the innerdays, although there should have been sufficient surface of the cytoplasmic membrane, wherebacteria present to allow for a possible polysac- suitable intermediates are available. They arecharase-producing mutant. Further washed- then excreted through the cytoplasmic mem-suspension experiments gave no evidence of brane by an unknown mechanism which maybreakdown of the extracellular polysaccharide as well be irreversible.

The only clearly substantiated case of anTABLE 7 organism catabolizing its own extracellular

Growth of Klebsiella aerogenes type 54 on media polysaccharide is in S. pyogenes. Faber andcontaining glucose and type 54 polysaccharide co Rosendal (61) showed that group A streptococci

carbon and energy sources fell into four subgroups according to the pro--________________- - duction of hyaluronic acid and hyaluronidase:

Total Growth FmalTotal (a) 1 strain produced neither; (b) 30 strainsCarbon and Energy Source jgg N/ml Polysaccharide/ g/ml produced only hyaluronidase; (c) 12 strains

None0.10produced only hyaluronic acid as evidenced by

None 0.1 0 the stability of the polysaccharide in cultures

Glucose 31 51 incubated for as long as three weeks; and (d)Glucose and polysac- 31 510 35 strains produced both. In these strains, hy-charide aluronic acid was produced in the early stages

Glucose and polysac- 27 570 of growth and later disappeared as the amountcharidet of hyaluronidase increased. MacLennan (62)

Glucose$ 58 98 showed that this type of organism, which gives amucoid colony in the early stages of growth due* Estimated initial anthrone reading - 5 to the possession of hyaluronic acid capsules,

;&g/ml. produced nonmucoid variants which synthesizedt Incubated for 10 days. Others all incubated roucedunonmuc as which sythsz3 days. roughiy equal amounts of hyalurondase but no$ 0.2 per cent glucose. Concentration of other hyaluronic acid.

carbon sources all 0.1 per cent. These results suggest that the production of


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the hyaluronic acid synthesizing system and C. Anabolism of Heteropolysaccharideshyaluronidase are quite unrelated. Many bacteria There is no direct evidence upon the pathwayare capable of producing hyaluronidase, e.g., of bacterial heteropolysaccharide synthesis orS. pyogenes, Staphylococcus aureus, Clostridium upon the glycoside-donors involved, and allperjringens, and D. pneumoniae, whereas the attempts to obtain synthesis in cell-free extractsynthesizing system is confined to S. pyogenes. have failed. This lack of evidence also holds for

B. Anabolism of Homopolysaccharides plant and animal heteropolysaccharides (e.g.,plant gums and blood-group substances). The

The enzymes concerned in the synthesis of question arises as to whether such synthesis is,homopolysaccharides have been much reviewed in fact, enzymatic or whether the failure to find(e.g., 63) and will be dealt with only briefly. enzymes is because of their nonexistence.The mechanism in all cases is probably a trans- The ability to synthesize a particular hetero-glycosidation as follows: polysaccharide is a stable genetic character

Reaction between a glycoside donor under appropriate growth conditions, indicating(G -O-}X), in which the sugar unit (Gl) is that deoxyribonucleic acid (DNA) provides thejoined to an aglycone (X) by a glycoside bond, initial replica for synthesis. This is borne out byand the transglycosidase (E) to give an enzyme- the transformation of antigenic type and there-carbohydrate complex, i.e., fore of the ability to synthesize a particular

type-specific polysaccharide, by purified prepa-G1{)-X + E * G10-E + X. rations of DNA (42). The transforming DNA

is reproduced in the cells after transformation,The free aglycone is produced and at the sametime the free energy of the glycoside bond is presumably as part of the genetic material.preserved in the enzyme complex. For the The phenomenon appears to be a general one forsynthesis of extracellular starch, the glycoside proteins as well as polysaccharides in trans-donors and enzymes are maltose (amylomaltase) formable organisms and it is evident that DNA

andsucros (amylosucase); fordextran, they is involved in protein and polysaccharide syn-and sucrose (deramylosucrase); r . . thesis. The specificity of protein synthesis isare sucrose (dextran sucrase) and dextrins (dex-tran dextrinase); for levan, they are sucrose and controlled by ribonucleic acid (RNA) through a

raffinose (levan sucrase). The enzyme concerned multitemplate system, i.e.,in the bacterial synthesis of cellulose has been DNA --+ RNA -- protein.shown to require uridine diphosphoglucose as aglycoside donor (64) and, by analogy to plant If DNA is also required for heteropolysac-tissues (64a), uridine polyphosphate sugars are charide synthesis, the mechanism might requireprobably involved in the bacterial synthesis of the intermediate formation of protein enzymes,hyaluronic acid. as in homopolysaccharide synthesis, i.e.,

Reaction between the enzyme complex and a DNA -- RNA -- protein -- polysaccharide.glycoside acceptor (an oligosaccharide or poly-saccharide) to give a product with one extra Alternatively, a template system might be in-sugarunit, i.e., volved without the intermediate formation of

protein enzymes, as proposed by Stacey (65)(Gl--O)R-Gl + Gl-O-E -* (Gl-O),+1--Gl and Wilkinson et al. (35), i.e.,

+E.DNA -- polysaccharide

Again, the free energy of the glycoside bond is or DNA -+ X -- polysaccharidepreserved. The two equations may be combined: or DNA -- RNA polysaccharide

Eor DNA > RNA .- X polysaccharide.

Gl--O-X + (GX-O).--Gl In this case, the mode of synthesis of homo-(Gl--OX.+i--Gl + X. polysaccharides and heteropolysaccharides would

In this way a linear polymer is built up, ad- be different but it is interesting to note that suchditional enzymes being required for branching a difference occurs for proteins. Whereas normalpoints. heteroproteins axe synthesized by a template


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system, a homoprotein, such as the D-glutamic fewer and, therefore, the less specific enzymesacid polypeptide capsule of B. anthracis, is involved in synthesis, the greater the degreesynthesized by enzymes (66). It is possible to of heterogeneity expected. Ultracentrifuge dataobtain indirect evidence of the type of mech- on homopolysaccharides indicate a wide rangeanism for heteropolysaccharide synthesis by in the molecular size of any single preparationconsidering the number of enzymes and, there- (21). Thus, Wilham et al. (68), using fractionalfore, genes, which might be required. precipitation methods, found both size andThe typical heteropolysaccharide, as shown structural heterogeneity in the dextran of a

previously, is a complex branched structure single bacterial species. On the other hand,containing three or four component sugars in heteropolysaccharides prepared with care toproportions not readily explicable in terms of a avoid degradative procedures are relativelysmall repeating unit. Knowledge of homopoly- monodisperse. This is particularly true of slimesaccharide synthesis makes it probable that at polysaccharides, which can be separated fromleast one enzyme will be required for each com- other products without appreciable degradation.ponent sugar linkage, together with additional Ultracentrifuge and diffusion studies of type 54enzymes for branching points. Therefore, the specific polysaccharide of K. aerogenes haveminimum number of enzymes required to syn- shown that the greater the care used in avoidingthesize a typical heteropolysaccharide containing extremes of temperature and pH during bacterialthree component sugars and one type of branch- synthesis and later purification, the greater wasing point is four. But this assumes specificity the homogeneity of the product (38). The prepa-only in the glycoside-donor in which case a wide rations had a degree of monodispersity com-range of different sized and proportioned poly- parable to that of pure proteins, suggesting thatsaccharides would result during synthesis under if synthesis is enzymatic, very numerous specificdifferent conditions. This is true even in a com- enzymes will be required. If, on the other hand,paratively simple homopolysaccharide such as synthesis is controlled by a template mechanism,dextran (22, 67). Yet there is no evidence in a monodisperse product will result irrespectivethe literature for a difference in antigenicity of cultural conditions.of the extracellular polysaccharide formed under a The considerations outlined above lead onevariety of cultural conditions except when the to expect numerous genes controlling the syn-conditions have led to a chemical degradation thesis of a heteropolysaccharide if enzymes areof the antigen. Wilkinson et al. (35) studied the involved, but only one gene if a template mech-structure of type 54 specific polysaccharide of anism is involved. In both cases, further genesK. aerogenes, produced from a variety of carbo- will be necessary for the enzymes controllinghydrates acting as the sole carbon and energy the synthesis of glycoside-donors, some of whichsources (glucose, galactose, mannitol, xylose, may not be required in other metabolic systems.rhamnose, fucose, or glucurone). The proportions Therefore, mutations resulting in the loss in theof the component sugars in the polysaccharide ability to form heteropolysaccharides shouldwere identical within experimental error. Unless always occur at a single gene locus with thethe steady state levels of the glucose, glucuronic template mechanism, but at various loci with theacid, and fucose donors were relatively inde- multienzyme mechanism. Unfortunately, littlependent of the nature of the carbon source, the evidence is available on this point with bacterialenzymes concerned in polysaccharide synthesis systems but with human blood groups there ismust be unaffected by the level of these donors. probably only a single gene involved. Thus,In the latter case, it must be assumed that the according to Wiener (69), the Rh blood groupssynthesizing enzymes have a fairly high degree are controlled by a series of eight allelomorphicof specificity, directed not solely against the genes all capable of occupying the same locus.terminal sugar of an oligosaccharide chain, and It is also theoretically possible to distinguishthis would increase the total number of enzymes between the multienzyme and template systemsinvolved. by a study of type transformation. Consider aA further question then arises. What is the multienzyme system of three enzymes, A, B,

degree of heterogeneity of a polysaccharide and C, controlled by their respective genes, a, b,produced under a given set of conditions? The and c, and involved in the synthesis of a type-


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specific polysaccharide. There will be three acid, iLysine and DL-alanine in the ratio 1:1:3.possible 0 variants from the original mucoid They suggested that the cell wall is synthesizedform according to which gene and, therefore, enzymatically from the nucleotide, which alreadywhich enzyme is lost. If this loss of an enzyme contains the basic repeating unit. Probablyresults in loss of the ability to form the particular no template is involved in the final synthetictype-specific polysaccharide, the transforming stage in this case but it is difficult to imagineprinciple from any of the three smooth mutants such a system synthesizing a more complexshould transform either of the other mutants protein, such as the M protein of S. pyogenes,back to the original mucoid form. MacLeod which may contain as many as 11 amino acidsand Krauss (70) have obtained two nonallelic (72), or an extracellular enzyme. There is also nosmooth mutants from pneumococcus type 8. evidence for the occurrence of a nucleotide-A DNA extract prepared from either smooth oligosaccharide complex which might act instrain restored the capacity to produce type 8 polysaccharide synthesis, although uridine pyro-polysaccharide in the other strain, which became phosphate compounds containing glucuronic acid,mucoid. However, the DNA factor from other glucose, and N-acetylglucosamine separatelymucoid pneumococci (types 1, 7, 14, and 18 but have been found in D. pneumoniae (73).not type 3) would also transform the smoothmutants back to the original type 8. It was VI. FUNCTIONconcluded that mutations had occurred in Extracellular polysaccharides are produced bygenes not concerned with immunological and the majority of bacteria and, unless they arechemical specificity but it is not possible at excretory products, they must have some func-present to distinguish between a loss of enzymes tion in maintaining a bacterial species in itsconcerned directly in polysaccharide synthesis environment. Certain naturally isolated bac-and a loss of enzymes concerned in glycoside- terial species are always mucoid (K. aerogenesdonor synthesis. or D. pneumoniae), yet they readily change in

Further indirect evidence for a template the laboratory to the smooth form which growssystem is as follows: as well or better in artificial media. This suggestsThe multienzyme systems for all the different that the type-specific polysaccharide is not an

type-specific polysaccharides in one bacterial excretory or waste product and that some factorspecies would stretch the range of synthetic icapabilities of such a species, even if many of f the eletiron m oi ts. imthenzymsar shaed y diferet tyes.favors the selection of mucoid varints. Similarly,The enzymes aretweeshared byediff typ pes.fic those naturally occurring gram negative bacteriaThe analogy between the range of type-specific wihaerrl uod(~. .cl)aeams

proteins (e.g., M proteins of streptococi) and which are rarely mucod (e.g., . colt) are almost

the type-specific polysaccharides suggests the tha vaiats lackin te0 tie casythti me as. that rough variants lacking the 0 antigen can

similarsynthetic formec fthan type-specificpoly often be obtained artificially. It is possible thatThe antigenic form of the type-specific poly- sm xrclua oooyacaie uhasaccharide in the pneumococcus is said to be a levans and dextrans, which are formed as "by-RNA complex (40), suggesting a synthetic system procs" of exnzm ahcto ave f unction.withNAasaninermedate, ~e.,products" of enzyme action, have no function.

But if this were so, it would be an advantage forDNA -* RNA -+ polysaccharide. an organism to metabolize a disaccharide such as

sucrose by a hydrolytic enzyme such as sucrase,It is tempting to assume that nucleotide- rather than to utilize only half the molecule with

diphosphate-sugar compounds are active as levan or dextran sucrase. Therefore, it may beglycoside donors for the final synthesis through assumed that most extracellular polysaccharidessome type of interaction with the nucleotides have a function in preserving bacteria in theirof the RNA chain. Park and Strominger (71) natural habitat. It is probable that the functionshave found a correspondence between the com- are related to two properties which the majorityposition of a uridine nucleotide, accumulated by of these polysaccharides have in common.S. aureus in the presence of penicillin, and the Firstly, they are very hydrophilic, particularlycell wall. Both contained an N-acetylamino sugar the capsular and slime polysaccharides. Sec-coupled with a peptide containing D-glutamic ondly, they give the cell surface a characteristic


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charge. Thus, K polysaccharides generally relationship in types 2, 3, and 8 pneumococcuspossess uronic acids and, therefore, give mucoid between the amount of type-specific polysac-cells a large negative charge at a normal pH charide (determined antigenically) and virulencerange, whereas 0 polysaccharides generally pos- (75); (b) Addition of an antiserum preparedsess hexosamines giving them a more positive against the type-specific polysaccharide (coupledcharge. Any functions proposed must account with horse globulin) causes both loss of virulencefor the wide structural variations of the poly- and increased sensitivity to phagocytosis (76);saccharides within a single species. Possible and (c) If animals are infected with type 3functions will now be considered. pneumococcus together with the enzyme specifi-

cally hydrolyzing its type-specific polysac-A. Protection Against Phagocytosis charide, no infection results (77). Phagocytosis

Although there are probably many factors occurs immediately the polysaccharide capsulewhich cause resistance of a microorganism to has been destroyed, although there is no effectphagocytosis, it is clear that one of the most upon viability or polysaccharide synthesizingimportant is the presence of a capsular polysac- capacity. Here specificity of the enzyme pro-charide. It is well known that virulent pneumo- vides excellent evidence for the specificity ofcocci isolated from pathological material are the substrate in protecting pneumococci againstmucoid and there is a clear correlation between phagocytosis.virulence, mucoidness of colony, and resistance A similar correlation between mucoidness andto phagocytosis. Thus smooth, nonencapsulated, virulence has been found with other bacteria.avirulent pneumococci are readily phagocytized It is probable that the type-specific polysac-when added to a suspension of leucocytes in charide must be present as a capsule and notnormal serum, whereas mucoid, capsulate, solely as loose slime to exert an antiphagocyticvirulent organisms are resistant to phagocytosis action, since Wood and Smith (78) found type 3and multiply rapidly (74). Further evidence for pneumococcus polysaccharide to be nontoxic tothe antiphagocytic action of the type-specific leucocytes. It is probable that the only way slimepolysaccharide in mucoid cultures may be sum- polysaccharides influence infection is by amarized as follows: (a) There is a quantitative combination with antibody, rendering it in-

active against the capsule. What effect, then,TABLE 8 does the capsular polysaccharide have on the

Effect of anti-M serum and hyaluronidase upon the surface of the cell so as to render it resistant tovirulence and resistance to phagocytosis of Strepto- phagocytosis? Dubos (40) stated that leucocytes

coccus pyogenes (9) are maintained at a definite distance from acapsulate pneumococcus by a sort of negative

M L D (ml) Phagocytosis chemotactic effect. It is usually assumed that thisIndex*effect is either a repulsion between the hydro-

Matt group A (M pro- philic capsule and the possibly hydrophobictein present) phagocyte surface, or a repulsion between differ-

Untreated 10-8 46 ent charges on the two surfaces. The antibody,+ anti-M serum 102 1240 by combining with the surface layer of the+ hyaluronidase 10-7 270 capsule, reduces the charge and decreases the+ anti-M serum 10-1 1700 hydrophilic character. Although this explanation+ hyaluronidase) may be partially true, it cannot be a complete

Glossy group A (M pro- one. Thus, Rothbard (9) studied the virulencetein absent)

Untreated 10-2 1560 and pathogenicity of S. pyogenes in relation to+ hyaluronidase 2210 the capsule. Matt forms of group A streptococci,

Group C (M protein ab- which possess a hyaluronic acid capsule im-sent) pregnated with M protein, are a million times

Untreated 10-7 more virulent than the corresponding glossy+ hyaluronidase 10-' variants with no M protein. Similar results were* Number of cocci phagocytized by 100 leuco- obtained by treatment with anti-M serum.

cytes in 20 min at 37 C. Hyaluronidase treatment, however, resulted in


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only a tenfold decrease in virulence. As shown strains were sensitive. He concluded that therein table 8, there was a correlation between was no relation between slime formation andvirulence and resistance to phagocytosis. Yet resistance to amoebic attack but made no at-the M protein has not been shown to be different tempt to demonstrate the presence or absence offrom other proteins (e.g., antibodies) in hydro- capsules. Liston and Wilkinson (unpublishedphilic character or in amino acid content (72). results) studied the correlation between polysac-A further complication can be seen in group C charide production in K. aerogenes and E. colistreptococci, where loss of the hyaluronic acid strains (described in (5)) and resistance tocapsule by hyaluronidase results in a 100,000- amoebal attack. The bacteria were inoculatedfold decrease in virulence. Why does hyaluroni- as strokes onto agar plates and active amoebaedase have such widely different effects on group of a strain isolated from the soil were inoculatedA and C streptococci? at the end of each stroke. The rate of advance of

It is possible that the size of the capsule may the amoebae at room temperature was observedbe an important factor in phagocytosis. There microscopically. Smooth strains of the bacteriaare many observations pointing to the importance were rapidly attacked whereas mucoid strainsof the culture medium in determining resistance were resistant for periods as long as 4 days.to phagocytosis and, as shown previously, these Therefore, the possession of capsules or slimedifferent cultural conditions would lead to vari- provided a partial protection against amoebication in capsule diameter. Wood and Smith attack. However, it is possible that much of this(78) found that the high resistance to surface resistance is due to the colonial form preventingphagocytosis of type 3 pneumococcus, compared entry of the amoebae, as distinct from an in-to other penumococcal types, was due to the hibitory effect of a capsule in individual bacteria,possession of a larger capsule. When the capsule particularly as the difference between capsulatediameter became reduced on aging of the bac- and slime-forming strains was not great.terial population, the organisms became readilyphagocytized. However, factors other than C. Protection Against Bacteriophagecapsule size must be important, since hyaluroni- Among the Erderobacteriaceae species, thedase-treated matt group A streptococci com- receptor for bacteriophage on the cell surface ispletely lose their capsules but are still highly either a component of the microcapsule or thevirulent and are engulfed only slowly by leuco- cell wall. Weidel et al. (80) have obtained evidencecytes (9). It is also possible that an important for the presence of various layers in the cell wallfactor in governing phagocytosis is the sensitiza- of E. coli which are active against different Ttion of the bacterial surface by a substance phages, although their results can be partlypresent in nonimmune sera. Extracellular explained by the removal of different componentspolysaccharides may prevent this combination. of complex antigens. Jesaitis and Goebel (81)

B. Protection Against Amoebic Attack isolated and purified the 0 antigen of phase IIShigella sonnei which combined with and in-

Consideration of the normal environment of activated those phages to which the organismthe many encapsulated bacteria found in soil, was susceptible (E. coli phages T2, T3, T4, T6,river, and pond waters, and on vegetation, makes and T7). The antigen was a phosphorylatedit possible that bacteria-eating protozoa, among lipopolysaccharide containing glucose, galactose,them various species of Amoeba, are a consider- glucosamine, and an aldoheptose. Digestion byable factor in regulating the bacterial flora. The pancreatin resulted in inactivation of mostmode of ingestion by amoebae is by pseudopodial phages at much lower antigen concentrations,engulfment on solid surfaces, similar to surface while extraction of the lipid component by 70phagocytosis by mammalian phagocytes. It is, per cent ethanol resulted in loss of the abilitytherefore, possible that capsular polysaccharides to inactivate T4 phage which could be restored bymay protect saprophytic bacteria from amoebic adding of various lipid preparations. If theattack. It is known that protozoa are selective antigen was prepared from formol-killed bacteria,to the bacteria ingested. Singh (79) reported only T4, T7 and, to a lesser extent, T3 were in-that three slime-forming Rhizobium strains activated and there was no effect on T2 and Tg,were not eaten by amoebae but that two other suggesting that different phages are adsorbed


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onto different components of the 0 antigen. phage resistant. Thus, Kauffmann and VahlneBacteria can become resistant to phage at- (82) found that most capsulate strains of E. coli

tack in two ways. were resistant to phage and that when capsulateA loss of the surface receptor. If the surface strains were attacked, there was a proportionality

receptor is lost or changed by mutation so that between the thickness of the capsule and thephage can no longer be adsorbed, the mutant resistance. Similarly, Maxted (83) found thatbacterium will become phage resistant. Phage while phage-resistant group A streptococci wereresistance is often accompanied by an antigenic invariably mucoid and possessed a hyaluronicchange because of the alteration in the surface acid capsule, destruction of the capsule by hy-receptor molecule. Thus a mutant was obtained aluronidase rendered the cells phage sensitive.from the phase II S. sonnei discussed above, Although reports have appeared in the literaturewhich was resistant to phages Ts, T4, and T7 of phage-sensitive capsulate bacteria, it isbut sensitive to T2 and T6 (32). The extracted possible that in these instances the virus producesantigen of this variant inhibited T6 but not T2, a lytic enzyme which destroys the capsularT3, T4, and T7. Presumably some degradation structure. For example, phages active againstof the antigen had occurred, since it would not Klebsiella pneumoniae types 1 and 2 produceinactivate T2. It was different serologically from enzymes which reduce the viscosity of the cap-the parent antigen, containing only one com- sular polysaccharide and strip it from the bac-ponent sugar-an unidentified hexosamine. terial surface without destroying its immunologi-The results suggest that the polysaccharide cal specificity (84, 85).

component of the antigen was the receptor for D. Entoxim and Aggresinsphages T3, T4, and T7, a suggestion confirmedby the fact that the receptor of the phages in In most gram negative bacteria, the 0 antigenE. coli was also a lipopolysaccharide complex is an endotoxin, probably increasing invasivenesscontaining glucose, glucosamine, and D-gala-L- (27, 30, 86). The K antigen is nontoxic but freemannoheptose. Therefore, it is evident that the excess slime may act as an aggressin, probablypolysaccharide component of the microcapsular causing "immunological paralysis" of the hostO antigen can act as a receptor for phages and organism: there is neutralization of K antibody,that an important reason for the variability of which can no longer combine with the capsulethese polysaccharides is to produce phage-resist- surface and render the cell sensitive to phago-ant cells. There is no evidence available to show cytosis (87). Many bacterial polysaccharides willthat the K antigen may act as a phage receptor. also inhibit nonspecific antibacterial substances

Physical blocking of the surface receptor. Can in the serum and other body fluids. Thus lyso-the presence of a capsule protect the cell simply zyme and leukin (a substance isolated frombecause of its physical properties? Presumably leucocytes, active against gram positive bacteria)an infective phage must be able to inject its are both inhibited by acidic polymers such asDNA through the cytoplasmic membrane and, hyaluronic acid and, in the case of lysozyme, bytherefore, the main body of the phage must be pneumococcal type-specific polysaccharides andat a distance from the cytoplasmic membrane the Vi antigen (88, 89). Inhibition was probablysmaller than the length of the phage tail (rarely due to a combination between the basic proteinslonger than 150 minA). Therefore, any layer out- and the acidic polysaccharides. Another non-side the cytoplasmic membrane which is greater specific antibacterial agent is the properdinin thickness than 150 m1A and is impermeable to system, which has been shown to be inactivatedphages will act as a nonspecific phage inhibitor. by a whole series of bacterial polysaccharidesIt has already been shown that a capsule is by including neutral ones (dextrans and levans)definition greater than 150 mju in thickness and as well as acidic ones (hyaluronic acid, pneumo-that it is probably impermeable to particles of coccal type-specific polysaccharides, and Salmo-the size of a phage head (about 100 miA). An nella typhosa lipopolysaccharide endotoxin) (90).additional barrier to the phage might be the high E. Protection Against Desiccationnegative charge of the polysaccharide capsularsurface. In confirmation of this role, capsulate Saprophytic bacteria growing on vegetationbacteria have been reported to be generally or in the soil will be frequently exposed to con-


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ditions of temporary drought followed by a (2) and it is probable that they do act as reserverapid exposure to water. Because of their hygro- sources of carbon and energy.scopic nature, the extracellular polysaccharides G. To Aid in the Uptake of Ionsand particularly the capsular polysaccharides Rorem (92) has proposed that bacterialprovide a cellular buffer system preventing a extracellular polysaccharides aid in the uptaketoo rapid loss or gain of water, which would

of ions and provide for growth under conditionscause cell death. Morgan and Beckwith (58)wwhen certain ions would be growth-limiting

studied the relative resistance of mucoid and factors for bacteria not producing extracellularnonmucoid Enterobacteriaceae species to desicca- polysaccharide. He showed that the uptake oftion. Saline suspensions containing standard rubidium and phosphate ions by Leuconostocnumbers of bacteria were spread on sterile cover dextranicum, Ieuconostoc mesenteroes, andslips and stored in petri dishes at room tempera- Streptococcus salivariu8 was two to twenty timesture. At intervals, viable counts were made and greater when the organism was grown in presenceit was found that mucoid strains survived for Of sucrose (producing abundant levans andmuch longer periods than nonmucoid strains. dextrans) than in its absence. However, theMcGraw (personal communication) has shown fact that extracellular polysaccharide can com-that if yeast is dried to about 8 per cent moisture, bine with ions does not necessarily mean thatthe dryness at which it is moststable, and allowed these ions are more available to the cell thanto become moist in a closed vessel over wet those in solution, although evidence was obtainedfilter paper, the number of viable cells when the of an exchange of polysaccharide-combined ionsyeast is eventually resuspended in water is very correlated with the active metabolism of the cell.much greater than when the dried yeast is It is possible that in some cases, and particularlysuspended directly. Herrera et al. (91) have with ionic polysaccharides, the excretion of asuggested that damage to the semipermeable charged molecule through the cytoplasmiccytoplasmic membrane incurred during drying membrane may allow the entry of an equalmust be repaired before intracellular demands number of other charged ions.for water are made and it is probable that this H. To Aid in Dispersalrepair is more efficient if water is allowed to The presence of an ionic polysaccharide at thereach the cell slowly. In bacteria, therefore, bacterial surface gives the cell a charge whichthe extracellular polysaccharide may act as abuffer preventing the rapid entry of water after willgp. adsortio onto pariceso lecharge. Thus, most K polysaccharides, becausea period of desiccation. of their component uronic acids and resultant

negative charge, will aid dispersal of cells inF. Reserve Carbon and Energy Source liquid environments.

In order to prove the function of a substance I. Conclusionas a reserve carbon and energy source, it is There are thus many possible functions for anecessary to show that the substance can be bacterial extracellular polysaccharide, any onebroken down by the synthesizing organism and or many of which may provide an environmentalthat the products or energy of this breakdown can advantage to a bacterium. Some are nonspecificbe utilized by the cell for growth and maintenance functions shared by the whole range of polysac-purposes. However, it has already been shown charides. Others require a specific structure inthat bacteria are rarely capable of breaking down the polysaccharide (e.g., protection againsttheir own extracellular polysaccharides. In the phagocytosis or bacteriophage) and the selectiononly instance where this has been described (the of mutants producing different polysaccharideshyaluronic acid-hyaluronidase system in S. pyo- accounts for the wide variation in their chemicalgenes), there has been no clear demonstration and immunological properties.that the organism gains any benefit. On the VII. REFERENCESother hand, intracellular polysaccharides, such 1. MITCHELL, P., AND MOYLE, J. 1956 Osmoticas glycogen, can be rapidly broken down by cells function and structure in bacteria. In


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Bacterial anatomy, pp. 150-180. University 15. DAVIES, D. A. L. 1955 The specific polysac-Press, Cambridge, England. charides of some gram-negative bacteria.

2. LEVENE, S., STEVENSON, H. J. R., TABOR, Biochem. J., 59, 696-704.E. C., BORDNER, R. H., AND CHAMBERS, 16. DUGUID, J. P. 1951 The demonstration ofL. A. 1953 Glycogen of enteric bacteria. bacterial capsules and slime. J. Pathol.J. Bacteriol., 66, 664-670. Bacteriol., 63, 673-685.

3. ToMCSIK, J. 1956 Bacterial capsules and 17. DUGUID, J. P., AND WILKINSON, J. F. 1953their relation to the cell wall. In Bacterial The influence of cultural conditions onanatomy, pp. 41-67. University Press, Cam- polysaccharide production by Aerobacterbridge, England. aerogenes. J. Gen. Microbiol., 9, 174-189.

4. DuBos, R. J., AND AVERY, 0. T. 1931 De- 18. HOTCHKISS, R. D. 1948 A microchemicalcomposition of the capsular polysaccharide reaction resulting in the staining of poly-of pneumococcus Type III by a bacterial saccharide structures in fixed tissue prepara-enzyme. J. Exptl. Med., 54, 51-71. tions. Arch. Biochem., 16, 131-141.

5. WILKINSON, J. F., DUGUID, J. P., AND 19. NOVELLI, A. 1953 New method of stainingEDMUJNDS, P. N. 1954 The distribution of of bacterial capsules in films and sections.polysaccharide production in Aerobacter and Experentia, 9, 34-35.Escherichia strains and its relation to anti- 20. MCKINNEY, R. E. 1953 Staining bacterialgenic character. J. Gen. Microbiol., 11, polysaccharides. J. Bacteriol., 66, 453-454.59-72. 21. GREENWOOD, C. T. 1952 The size and shape

6. CUMMINS, C. S. 1956 The chemical composi- of some polysaccharide molecules. Ad-tion of the bacterial cell wall. Intern. Rev. vances in Carbohydrate Chem., 7, 289-332.Cytol., 5, 25-50. 22. JEANES, A., HAYNES, W. C., WILHAM, C. A.,

7. IVkNOVICS, G., AND BRUCKNER, V. 1937 Die RANKIN, J. C., MELVIN, E. H., AUSTIN,chemische Strucktur der Kapselsubstanz M. J., CLUSKEY, J. E., FISHER, B. E.,des Milzbrandbazillus und der serologisch TSUCHIYA, H. M., AND RIST, C. E. 1954identischen spezifischen Substanz des Bacil- Characterization and classification of dex-lus mesentericus. Z. Immunititsforsch., trans from ninety-six strains of bacteria.90, 304-318. J. Am. Chem. Soc., 76, 5041-5052.

8. JACOX, R. F. 1947 A new method for the 23. BARCLAY, K. S., BOURNE, E. J., STACEY, M.,production of non-specific capsular swelling AND WEBB, M. 1954 Structural studies ofof the pneumococcus. Proc. Soc. Exptl. the cellulose synthesised by AcetobacterBiol. Med., 66, 635-638. acetigenum. J. Chem. Soc., 1954, 1501-1505.

9. ROTHBARD, S. 1948 Protective effect of 24. BARKER, S. A., BOURNE, E. J., AND STACEY, M.hyaluronidase and type-specific anti-M 1950 The structure of the starch-typeserum on experimental group A streptococ- polysaccharide synthesised from sucrose bycus infections in mice. J. Exptl. Med., 88, Neisseria perflava. J. Chem. Soc., 1950,325-342. 2884-2887.

10. MCQUILLEN, K. 1955 Bacterial protoplasts: 25. BELL, D. J., AND DEDONDER, R. 1954 Agrowth and division of protoplasts of structural re-examination of the levansBacillus megaterium. Biochim. et Biophys. formed by Pseudomonas prunicola, Wormald,Acta, 18, 458-461. and Bacillus subtilis, BG2 Fl. J. Chem.

11. KAUFFMANN, F. 1954 Enterobacteriaceae, Soc., 1954, 2866-2870.Ed. 2. Munksgaard, Copenhagen, Den- 26. WEISSMANN, B., AND MEYER, K. 1954 Themark. structure of hyalobiuronic acid and hyalu-

12. QRSKOV, F. 1956 Studies on E. coli K anti- ronic acid from umbilical cord. J. Am.gens. 1. On the occurrence of B. antigens. Chem. Soc., 76, 1753-1757.Acta Pathol. Microbiol. Scand., 39, 147-159. 27. WESTPHAL, O., AND LtDERITZ, 0. 1954

13. LANCEFIELD, R. C. 1943 Studies on the Chemische Erforschung von Lipopolysac-antigenic composition of group A hemolytic chariden gramnegativer Bakterien. Angew.streptococci. I. Effects of proteolytic en- Chem., 66, 407-417.zymes on streptococcal cells. J. Exptl. 28. MORGAN, W. T. J. 1937 The isolation andMed., 78, 465-476. properties of a specific antigenic substance

14. SALTON, M. R. J. 1953 The composition of from B. dysenteriae (Shiga). Biochem. J.,the cell walls of some gram-positive and 31, 2003-2021.gram-negative bacteria. Biochim. et Bio- 29. DAVIES, D. A. L., MORGAN, W. T. J., ANDphys. Acta, 10, 512-523. MOSIMANN, W. 1954 Preparation and


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properties of the "O" somatic antigen of tural studies on the type II pneumococcusShigella dysenteriae (Shiga). Biochem. J., specific polysaccharide. J. Chem. Soc.,56, 572-581. 1955, 1537-1541.

30. DAVIES, D. A. L., MORGAN, W. T. J., AND 45. HEIDEIBERGER, M., AND ADAMS, J. 1956RECORD, B. R. 1955 The specific polysac- The immunological specificity of type IIcharide of the dominant "O" somatic anti- pneumococcus and its separation into partialgen of Shigella dysenteriae. Biochem. J., specificities. J. Exptl. Med., 103, 189-197.60, 290-303. 46. HEIDELBERGER, M., AISENBERG, A. C., AND

31. MORGAN, W. T. J., AND PARTRIDGE, S. M. HAsSID, W. Z. 1954 Glycogen, an im-1940 The fractionation and nature of munologically specific polysaccharide. J.antigenic material isolated from Bact. Exptl. Med., 99, 343-353.dysenteriae (Shiga). Biochem. J., 34,169-191. 47. MCCARTY, M. 1956 Variation in the group-

32. GOEBEL, W. F., AND JEsAisS, M. A. 1952 specific carbohydrate of group A strepto-The somatic antigen of a phage-resistant cocci. II. Studies on the chemical basis forvariant of Phase II Shigella sonnei. J. serological specificity of the carbohydrates.Exptl. Med., 96, 425-38. J. Exptl. Med., 104, 629-643.

33. DAVIES, D. A. L. 1956 A specific polysac- 48. LEVINE, S., STEVENSON, H. J. R., AND KAB-charide of Pasteurella pestis. Biochem. J., LER, P. W. 1953 Qualitative studies of63,105-116. pneumococcal polysaccharides by infrared

34. EDWAD, P. R., AND FIFE, M. A. 1952 spectrophotometry. Arch. Biochem. andCapsule types of Klebeiella. J. Infectious Biophys., 45, 65-73.Diseases, 91, 92-104. 49. LEVINE, S., STEVENSON, H. J. R., BORDNER,

35. WILKINSON, J. F., DuDMAN, W. F., AND R. H., AND EDWARDS, P. R. 1955 TypingAsPINALL, G. 0. 1955 The extracellular of Klebsiella by infrared spectrophotometry.polysaccharide of Aerobacter aerogene8 A3 J. Infectious Diseases, 96, 193-198.(S1) (Klebsiella Type 54). Biochem. J., 50. DUGUID, J. P., AND WnIKINSON, J. F. 195459, 446-451. The influence of potassium deficiency upon

36. DUDMAN, W. F., AND WILKINSON, J. F. 1956 production of polysaccharide by AerobacterThe composition of the extracellular poly- aerogenes. J. Gen. Microbiol., 11, 71-72.saccharides of Aerobacter-Klebsiella strains. 51. CLIFTON, C. E. 1946 Microbial assimila-Biochem. J., 62, 289-295. tions. Advances in Enzymol., 6, 269-308.

37. ASPINALL, G. O., JAMIESON, R. S. P., AND 52. WILKINSON, J. F., AND STARK, G. H. 1956WILKINSON, J. F. 1956 The structure of The synthesis of polysaccharide by washedthe extracellular polysaccharide of Aero- suspensions of Klebeiella aerogenes. Proc.bacter aerogenes A3 (S1) (Klebsiella Type 54). Roy. Physical Soc., Edinburgh, 25, 35-38.J. Chem. Soc., 1956, 3483-3487. 53. SIEGEL, B. V., AND CLIFTON, C. E. 1950

38. BRYCE, W. A. J., DUDMAN, W. F., GREEN- Energy relationships in carbohydrate as-WOOD, C. T., AND WIINSON, J. F. In prep- similation by Escherichia coli. J. Bac-aration for press. teriol., 60, 573-583.

39. MACPHERSON, I. A., WILKINSON, J. F., AND 54. SIEGEL, B. V., AND CLIFrON, C. E. 1950SWAIN, R. H. A. 1953 The effect of Energetics and assimilation in the combus-Klebsiella aerogenes and Klebsiella cloacae tion of carbon compounds by Escherichiapolysaccharides on haemagglutination by coli. J. Bacteriol., 60, 585-593.and multiplication of the influenza group of 55. DAGLEY, S., AND JOHNSON, A. R. 1953 Theviruses. Brit. J. Exptl. Pathol., 34, 603-615. relation between lipid and polysaccharide

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43. KABAT, E. A., AND LESKOWITZ, S. 1955 2. Preparation of freeze-dried cells capableStructural units involved in blood group A of polymerising glucose to cellulose. Bio-and B specificity. J. Am. Chem. Soc., 77, chem. J., 58, 345-352.5159-5164. 58. MORGAN, H. R., AND BECKWITH, T. D. 1939

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60. WHISTLER, R. L., AND KIRBY, K. W. 1956 hemolytic streptococcus, group A. J.Composition and behavior of soil polysac- Bacteriol., 69, 529-535.charides. J. Am. Chem. Soc., 78, 1755-1759. 73. SMITH, E. E. B., MILLS, G. T., AND HARPER,

61. FABER, V., AND ROsENDAL, K. 1954 Studies E. M. 1957 A comparison of the uridineon the production of hyaluronidase and pyrophosphoglycosyl metabolism of capsu-hyaluronic acid by representatives of all lated and non-capsulated pneumococci. J.types of hemolytic streptococci belonging to Gen. Microbiol., 16, 426-437.group A. Acta Pathol. Microbiol. Scand., 74. ENDERS, J. F., SHAFFER, M. F., AND WU,35, 159-164. C.-J. 1936 Correlation of the behavior

62. MACLENNAN, A. P. 1956 The production of in vivo of pneumococci type III varying incapsules, hyaluronic acid and hyaluronidase their virulence for rabbits with certain dif-by group A and group C streptococci. J. ferences observed in vitro. J. Exptl. Med.,Gen. Microbiol., 14, 134-142. 64, 307-331.

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64. GLASER, L. 1957 The enzymic synthesis of polysaccharide formed in vitro. J. Exptl.cellulose by Acetobacter xylinum. Bio- Med., 92, 1-9.chim. et Biophys. Acta, 25, 436. 76. AVERY, 0. T., AND GOEBEL, W. F. 1931 The

64a. GLASER, L., AND BROWN, D. H. 1955 The immunological specificity of an antigenenzymic synthesis in vitro of hyaluronic acid prepared by combining the capsular poly-chains. Proc. Natl. Acad. Sci., U. 5., 41, saccharide of type III pneumococcus with253-260. foreign protein. J. Exptl. Med., 54, 437-447.

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70. MACLEOD, C. M., AND KRAUSS, M. R. 1956 Formenwechsels fur die Bacteriophagen-Transformation reactions with two non- Wirkung in der Coli-Gruppe. Acta Pathol.allelic R mutants of the same strain of Microbiol. Scand., 22, 119-137.pneumococcus Type VIII. J. Exptl. Med., 83. MAXTED, W. R. 1952 Enhancement of103, 623-638. streptococcal bacteriophage lysis by hyalu-

71. PARK, J. T., AND STROMINGER, J. L. 1957 ronidase. Nature, 170, 1020-1021.Mode of action of penicillin. Biochemical 84. HUMPHRIES, J. C. 1948 Enzymic activity of


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bacteriophage culture lysates; a capsule mere from pathogenic bacteria. J. Bac-lysin active against Klebsiella pneumoniae, teriol., 70, 110-112.type A. J. Bacteriol., 56, 683-693. 89. SKNES, R. C., AND WATSON, D. W. 1956

85. ADAMS, M. H., AND PIE, B. H. 1956 An Characterisation of leufkin: an antibacterialenzyme produced by a phage-host cell factor from leucocytes active against gram-system. 11. The properties of the polysac- positive pathogens. J. Exptl. Med., 104,charide depolymerase. Virology, 2, 719- 829-845.736. 90. PILEmmER, L., SCHOENBERG, M. D., BLum, L.,

86. LANDY, M., AND JOHNSON, A. G. 1955 AND WuRz, L. 1955 Interaction of theStudies on the 0 antigen of Salmonella properdin system with polysaccharides.typhosa. IV. Endotoxic properties of the Science, 122, 545-549.purified antigen. Proc. Soc. Exptl. Biol. 91. HERREA, T., PETERSON, W. H., COOPER,Med., 90, 57-62. E. J., AND PEPPLER, H. J. 1956 Loss of cell

87. ws~ov, I. 1956 "Immunological paralysis" constituents on reconstitution of active dryinduced in rabbits by a heavily capsulated yeast. Arch. Biochem. and Biophys., 68,Klebsiella strain. Acta Pathol. Microbiol. 131-143.Scand., 38, 375-384. 92. Roism, E. S. 1955 Uptake of rubidium and

88. SKARNEs, R. C., AND WATSON, D. W. 1955 phosphate ions by polysaccharide-producingThe inhibition of lysozyme by acidic poly- bacteria. J. Bacteriol., 70, 691-701.


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