JOURNAL OF BACTERIOLOGY, Apr. 1986, p. 72-770021-9193/86/040072-06$02.00/0Copyright © 1986, American Society for Microbiology

Vol. 166, No. 1

Critical Micelle Concentrations of Lipoteichoic AcidsA. J. WICKEN,1* J. D. EVANS,' AND K. W. KNOX'

School of Microbiology, University ofNew South Wales, Kensington, New South Wales 2033,' and Institute ofDentalResearch, United Dental Hospital, Surrey Hills, New South Wales 2010,2 Australia

Received 4 November 1985/Accepted 15 January 1986

Purified lipoteichoic acids (LTAs) from several gram-positive organisms have been shown, by methodsinvolving spectral changes of an added merocyanine dye probe, to have critical micelie concentrations in therange of 1 to 10 jig/ml, suggesting that acylated LTAs in their monomer forms may represent the majorconfiguration of extracellular LTAs in bacterial culture fluids. The critical micelle concentrations obtained didnot differ markedly with degree of carbohydrate substitution of the polymers. The significance of these findingsin relation to the biological properties of LTA is discussed.

Lipoteichoic acid (LTA) is the generic name given to agroup of structurally related amphipathic molecules oramphiphiles found as cell membrane components in a widerange of gram-positive bacteria. The hydrophilic portion ofthe molecule is typically a 1,3-phosphodiester-linked poly-mer of glycerophosphate, variously substituted in the C-2position of the glycerol residues with sugars in glycosidiclinkage and D-alanine in ester linkage. The hydrophobicregion of the molecule, linked covalently through thephosphomonoester end of the polymer, is generally either aglycolipid or phosphatidylglycolipid. This lipid "end" isintercalated with the upper half of the bilayer of the cellmembrane and provides a membrane anchor for the mole-cule. LTA is also found as a soluble excreted product incultures of gram-positive bacteria (29, 35).

In aqueous solution, amphipathic molecules oramphiphiles tend to form micellar aggregates to occludewater from the hydrophobic regions of their molecularstructure. LTAs similarly form high-molecular-weight aggre-gates or presumptive micelles in aqueous solution, as judgedby their behavior on gel chromatography (9, 29, 35). Treat-ment with detergents reduces the high-molecular-weightaggregates to what is presumed to be a monomeric form (9).In any solution of an amphiphile, the micellar form mustcoexist with monomer. The critical micelle concentration(CMC) is a physical measurement of the concentration ofmonomer at which micellar aggregation commences. Inother words, below the CMC an amphiphile exists entirely inthe monomeric form. The micellar form will become moredominant as the concentration is increased above the CMC,while the monomer concentration remains approximatelyconstant at its maximum value set by the CMC. For mem-brane lipids, such as phospholipids, CMCs are of the orderof 10-10 M (14, 27), indicating very low concentrations ofmonomer in aqueous solution. LTAs, however, have a muchlarger hydrophilic component, the polyglycerophosphatechain, than most lipids and therefore might be expected toexhibit significantly higher CMCs than membrane lipids.High concentrations of monomer could be an importantfactor in determining biological properties of the molecule.Most CMC measurements depend on detecting the

marked changes in the properties of amphiphile solutions astheir concentration is increased through the CMC or theequally marked changes in the electronic spectra of indicatorprobe molecules in response to changes in polarity of their

* Corresponding author.

environment (10, 25, 26). Merocyanine dyes are among themore thoroughly characterized of the various types of com-pounds used as indicators of solvent polarities. With changein solvent polarities, their electronic spectra can show verylarge shifts in their absorption bands or intensity of fluores-cence which in turn are presumed to be due to changes in thebalance of benzenoid and quinonoid valence structure of theprobe (4). The merocyanine dye most commonly used insuch studies is 4-[2-(1-methyl-1,4,dihydropyridin-4-ylidene)ethylidene] cyclohexa-2-5-dien-1-one, which showsa shift in the UV-visible absorption spectrum towards longerwavelength as the solvent polarity decreases (5). Morerecently, an amphipathic merocyanine dye has been synthe-sized in which the methyl group has been replaced by ahexadecyl chain (8), thereby increasing its hydrophobiccharacter and favoring its solubilization by micelles. Studieswith this probe showed upward wavelength shifts in itsabsorption spectrum in solutions of various detergents as theconcentrations of the latter were increased from below toabove their known CMCs (8). It was suggested that themerocyanine dye probe was solubilized by micelles so thatthe hydrocarbon chain penetrated the hydrophobic core ofthe micelle while the merocyanine head group resided in thesame microenvironment as the head groups of the surfactantconstituting the micelle (8).

In this study we have utilized the spectral properties of theamphipathic hexadecyl-merocyanine dye as well as thewater-soluble cationic dyes toluidine blue and acridine or-ange to determine the CMC of LTAs differing in their degreeof carbohydrate substitution. We have demonstrated thatvalues of the order of 10-' M are obtained. These indicatefree monomer LTA concentrations in the range of 1 to 10,ug/ml, where biological activity of LTA is most usuallydemonstrated.

MATERIALS AND METHODS

LTAs. Purified LTA preparations either were available orwere extracted from organisms used in previous studies:Lactobacillus casei NIRD R094 (30, 33), Streptococcusmutans BHT (18), Lactobacillusfermentum NCTC 6991 (30,32), Streptococcus lactis ATCC 9936 (34), and Streptococcusfaecalis ATCC 9790 (17). In each case the LTA preparationswere obtained by hot phenol extraction (31) of stationary-phase cultures grown with 2% (wt/vol) glucose as describedin the literature cited above. Crude extracts were purified byrepeated chromatography on columns of Ultrogel AcA 22(LKB, Stockholm) essentially as described in earlier

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CRITICAL MICELLE CONCENTRATIONS OF LIPOTEICHOIC ACIDS

TABLE 1. CMCs of LTAs of differing composition at 20°C in 0.005 M MES buffer (pH 6.0)

LTA CMC concn

CompositionSource Mole ratiob of phosphorus- % Protein ,g/ml M (10-7)a

glucose-galactose-D-alanine

L. casei NIRD R094 1.00:0.08:0.04:0.17 3.7 1.8 3.1S. mutans BHT (S. rattus) 1.00:0.14:0.00:<0.02 4.5 1.9 2.8L. fermentum NCTC 6991 1.00:0.06:0.12:0.38 0.65 3.3 4.4S. lactis ATCC 9936 1.00:0.12:0.53:0.15 2.8 3.6 6.9S.faecium ATCC 9790 1.00:1.02:0.00:0.25 3.4 8.1 6.0

a Calculated on the basis of a previously proposed average chain length of 27.5 glycerophosphate residues (7) with the exception of S. lactis, where the chainlength has been calculated to be 17 (34).

b Includes glycolipid carbohydrate components.

publications (31, 34). All LTA preparations were analyzed forphosphorus (1), glucose (15), galactose (21), D-alanine (12),and protein (22). Relevant analytical data are given in Table1.CMCs. The hexadecyl-merocyanine dye 4-[2-(1-

hexadecyl- 1 ,4-dihydropyridin-4-ylidene)ethylidene]cyclohexa-2-5-dien-1-one was the kind gift of P. J. Derrick,University ofNew South Wales. Toluidine blue and acridineorange were obtained from Sigma Chemical Co., St. Louis,Mo.; sodium dodecyl sulfate was obtained from PierceChemical Co., Rockford, Ill.; Triton X-100 came fromCalbiochem, La Jolla, Calif. Visible spectra of the variousdyes were determined at 20°C in 5 mM MES (mor-pholineethanesulfonic acid) buffer (Sigma) (pH 6.0) contain-ing different concentrations of LTA or detergent, using aBeckman DU-8 recording spectrophotometer. For themerocyanine dye, 200 ,ul of a 10-4 M solution in methanolwas added to 1,300 ,ul of reaction mixture, resulting in a finaldye concentration of 1.33 x 10-5 M. The water-solublecationic dyes were used at 0.0002% (wt/vol) in 5 mM MESbuffer; this formulation resulted in active dye concentrationsbetween 10-5 and 10-6 M.

Fluorescence spectra of merocyanine dye solutions in thesame buffer were determined with a Perkin-Elmer MPF-44Bfluorescence spectrophotometer at an excitation wavelengthof 400 nm. Dye concentration was set at 10-6 M for thesemeasurements.

RESULTSFluorescence properties of merocyanine dye. The

merocyanine dye was shown on excitation at 400 nm toexhibit a fluorescence maximum at 732 nm in aqueous buffer.Fluorescence is sensitive to the nature of the molecularenvironment, and solubilization of fluorescent probes intomiceiles increases the relative intensity of fluorescence withan accompanying shift to a lower wavelength of maximnumemission (10). In the presence of sodium dodecyl sulfate orTriton X-100 the fluorescence spectrum of the merocyaninedye showed a shift from 732 to 510 nm with increase inintensity as the concentrations of the detergents were in-creased above their known CMCs (8 x 10-3 and 2.4 x 10-4M, respectively). The fluorescence spectrumt of the dye inthe presence of L. fermentum LTA (20 ,ug/ml) showed asimilar shift in wavelength and relative intensity of emission(Fig. 1). A plot of relative intensity of fluorescence at 510 nmagainst LTA concentration is shown in Fig. 2. The strongincrease in fluorescence intensity occurs over a narrowconcentration range which would include the CMC (10).Extrapolation of the straight-line portion of the curve indi-cated a presumed CMC of the order of 1.5 jig of LTA per ml

(Fig. 2, A). Alternatively, calculation of the midpoint of therapid change in fluorescence intensity gave the higher valueof 4.3 ,g/ml for the CMC (Fig. 2, B).

Absorption spectra of merocyanine dye. In the presence ofvarious detergents and below pH 7.0, the merocyanine dyehas been reported to exhibit maximum absorption in theregion of 330 to 360 nm at concentrations below the CMC ofthe detergents and around 390 nm above the CMC (8). InMES buffer at pH 6.0 and in the presence of variousconcentrations of Triton X-100, the respective absorptionmaxima were found to be 355 and 395 nm. A plot of thedifference in absorption at these two wavelengths againstTriton X-100 concentration is shown in Fig. 3. Extrapolationof the two straight-line portions of the curve results in anintersection corresponding with the known CMC of TritonX-100, i.e., 2.4 x 10- M (26).

In the presence of increasing concentrations of L.fermentum LTA, the merocyanine dye showed a broadabsorption peak with a shift in the wavelength of theabsorption maximum from 355 to 380 nm (Fig. 4). A plot ofthe differences in absorption at these wavelengths againstLTA concentration (0 to 40 ,ug/ml) gave a curve (Fig. 5)shaped similarly to that obtained with Triton X-100. Theintersection of extrapolations to the straight-line portions ofthe curve gave a value for the CMC of 3.3 ,ug/ml or 4.4 x10-7 M.Absorption spectra of toluidine blue and acridine orange.

G)acc)

0

ci)

0L)0)

0)

L-

co

7a:

nmWavelength

FIG. 1. Relative fluorescence at 20°C, after excitation at 400 nm,of 10-6 M merocyanine dye in 5 mM MES buffer (pH 6.0) (H20) andof 20 jig of L. fermentum LTA per ml in the same buffer (LTA).

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74 WICKEN ET AL.

Toluidine blue and acridine orange are water-solublecationic dyes and would be expected to interact ionicallywith the negatively charged polyglycerophosphate chains ofLTAs; both dyes stain LTA in gels (A. J. Wicken and K. W.Knox, unpublished data). Although these dyes would not beexpected to penetrate the hydrophobic region of an LTAmicelle, it was of interest to determine whether the onset ofLTA micelle formation had an effect on the absorptionspectrum of the dyes. In the presence of increasing concen-trations of L. fermentum LTA, both dyes showed a down-ward shift in the wavelengths of the maxima of peaks ofabsorption, from 635 to 552 nm for toluidine blue and from491 to 460 nm for acridine orange. Plots of differences inabsorption at these pairs of wavelengths against LTA con-centration gave curves similar to that obtained for themerocyanine dye and intercepts corresponding to 3.2 ,ug/mlor 4.3 x 1i-O M for toluidine blue and 3.9 ,g/ml or 5.2 x10-7 M for acridine orange. These values are in closeagreement with the CMC of this LTA as determined with themerocyanine dye.CMCs of a range of LTAs. Of the various methods used to

measure the CMC of L. fermentum LTA, absorption spectraof the merocyanine dye appeared to offer greater precision indetermining the required parameter as well as being ahydrophobic probe. This procedure was therefore used todetermine the CMC values for a number of LTAs havingvarious degrees of carbohydrate substitution (Table 1). The

1 50.

1 25.

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._"

0

c

0

0

0

-i

1

7

5

2

.B

0 "1 '1 0 '2 0

pg LTA/mIFIG. 2. Relative fluorescence at 20°C of 10-6 M merocyanine dye

at 510 nm in 5 mM MES buffer (pH 6.0) in the presence of increasingconcentrations of L. fermentum LTA. Dotted lines represent esti-mates of the CMC: A, extrapolation of the straight-line portion ofthe curve to zero relative fluorescence (=1.5 ,ug of LTA per ml); B,concentration of LTA (4.3 Fg/ml) corresponding to the midpoint inthe rapid rise in relative fluorescence.

Li

COI'

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-.05-

-.1-

-.15-

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10 4 MolarityFIG. 3. Plot of the differences in the absorbance values obtained

at 355 and 395 nm for 1.33 x 10-1 M merocyanine dye solutions in5 mM MES (pH 6.0), at 20°C, against concentration of added TritonX-100. The intercept of extrapolations from the two straight-lineportions of the curve corresponds to the CMC of Triton X-100.

carbohydrate components of the LTAs from L. casei NIRDR094 and S. mutans BHT are restricted to the hydrophobicglycolipid part of the molecule, whereas the LTAs from L.fermentum NCTC 6991, S. lactis ATCC 9936, and S.faecium ATCC 9790 are also substituted with carbohydratemoieties along the hydrophilic polyglycerophosphate chain(Table 1). It is evident from these studies that LTAs differingmarkedly in their degree of carbohydrate substitution allshow CMCs in a relatively narrow range, namely, 2.8 x 10-to 6.9 x 0-7 M.

DISCUSSIONThe formation of micelles from constituent monomers is

generally considered to be rapid and dynamic and to involveassociation-dissociation equilibria. Accordingly, there is aconcentration below which micelles are not formed andabove that a narrow range of concentration where they arefirst detectable; then, as the concentration is increased, alladditional solute material forms micelles (26). The CMC isthus not a sharply defined physical property, since allproperties of a solution in the CMC region will vary in acontinuous manner, and the CMC is taken as some point,usually the midpoint, in the narrow range of concentrationsin which the changes are most marked. Micelles are alsousually polydisperse in the sense of there being a range ofnumbers of monomers that can form a micelle (26). In thecase of LTA micelles, some polydispersity could also resultfrom qualitative differences in the fatty acid substitution ofindividual molecules as well as in the length and secondarysubstitution of the polyglycerophosphate chain (36).

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CRITICAL MICELLE CONCENTRATIONS OF LIPOTEICHOIC ACIDS

0.5

a)C.)CcOm IL.

0

Cl)

40(

nmWavelength

FIG. 4. Visible spectra of 1.33 x 10-5 M merocyanine dye in 5mM MES buffer (pH 6.0), at 20°C, containing 0 (I), 1.0 (II), and 20.0(III) ,ug of L. fermentum LTA per ml.

These considerations are well illustrated by the shapes ofthe curves in Fig. 3 and 5, which show transition in the caseof Triton X-100 occurring over a narrower range of concen-trations than for the more polydisperse LTA. Lipopolysac-charides are another class of bacterial amphiphile which arewell characterized as being extremely polydisperse, partic-ularly with respect to the chain length of the hydrophilicportion of the molecule (11). However, application of themethods described here to a number of lipopolysaccharidesfailed to produce data that could be used to determine CMCs(data not presented).

It is evident from these studies that LTA, even at concen-trations up to several micrograms per milliliter, would bepresent solely in the monomer form with the hydrophobiclipid end of the molecule exposed to the aqueous environ-ment. LTA occurs as an extracellular product in the culturefluids ofmany bacteria, in concentrations which depend bothon the organism and the growth conditions (19, 20). Thecurrent studies aid in defining the physical state of extracel-lular LTA. It has been implicit in earlier models that extra-cellular LTA associates as micelles in the external milieu,with the only extracellular monomer being the deacylatedform of the molecule (29, 35). It is now clear, however, thatacylated monomer will be a significant component of extra-cellular LTA and that in cases where the total LTA concen-tration is below the CMC, all extracellular LTA will be in theform of acylated monomer. A survey of extracellular LTAproduced by a range of oral streptococci and lactobacilli (23)showed that many strains of S. mutans-like organisms pro-

duced 20 to 40 ,ig of acylated LTA per ml of culture fluid,whereas S. mutans BHT (S. rattus) was unusual in produc-ing 320 jig/ml. Such concentrations are well above theCMCs, and in consequence the bulk of the extracellular LTAwould be present in the micellar form. With some strains ofStreptococcus sanguis, Streptococcus salivarius, and Strep-tococcus mitior, as well as some Lactobacillus spp., theconcentration of extracellular LTA was 2 to 4 ,ug/ml andtherefore close to estimated CMCs. For these organisms the

monomer form would be a very significant part of the totalextracellular LTA.LTAs have been shown to display a number of biological

properties (35, 37), many of which are exhibited at lowconcentrations of the amphiphile. For instance, maximumsensitization of erythrocytes occurred with 1 to 2 p,g of LTAper ml (23), and similarly, stimulation of lysosomal enzymerelease from macrophages was obtained at 0.3 to 0.6 ,ig ofLTA per ml (13), concentrations at which the moleculewould be entirely present as monomer. In the latter case,stimulation was only slightly increased by a 100-fold increasein LTA concentration, a situation where the micellar formwould predominate. Both of these biological propertiesinvolve interaction of the LTA molecule with eucaryotic cellmembranes. The precise mechanism ofLTA interaction withmembranes is unknown, but it is presumed to be an essen-tially hydrophobic interaction of the lipid end of the mole-cule with the target membrane since fatty acid deacylation ofLTA generally destroys biological activity (2, 35, 37). Therealso appears to be a finite number of LTA binding sites (2),which may reflect an analogous situation to the two-stagemechanism proposed for lipopolysaccharide binding to eu-caryotic membranes (6, 16). It is suggested that lipopolysac-charide first reacts with membrane protein binding sites,which promotes separation of the lipopolysaccharide micelleand allows for intercalation of the lipid ends of monomerunits into the membrane lipid bilayer. A relatively highmonomer concentration in solution would presumably facil-itate such membrane interactions by providing a high con-centration of reactive species.

+.04

+.02

~~~0

coI

oLO.D0 0

n-0Cl) -.04

-.06

.

-.08

0 10 20 30 40

pg LTA/mlFIG. 5. Plot of the differences in the absorbance values obtained

at 355 and 380 nm for 1.33 x 10' M merocyanine dye solutions in5 mM MES (pH 6.0), at 20°C, against concentrations of added L.fermentum LTA. The intercept of extrapolations from the twostraight-line portions of the curve is taken as a measure of the CMC.

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76 WICKEN ET AL.

In the bacterial cell, the lipid moiety of LTA is believed tobe intercalated in the upper half of the bilayer of the cellmembrane. Serological detection of LTA at the cell surfaceof some bacterial cells and specific immunochemical labelingof thin sections of bacterial cells (28) were interpreted inearly models of LTA location as a reflection of an extensionof the hydrophilic portion of the molecule through thepeptidoglycan network of the cell wall. The later recognitionof LTA excretion during normal growth suggested thatdetection of LTA at the surface of producing cells mayreflect a transient phenomenon or binding of excreted LTAto other surface components such as proteins (35). LTAwould thus be expected to be a component of what has beendefined as the extramural or cell wall-associated glycocalyxregion of the cell (38) and could have a configuration inwhich the lipid end of the molecule is exposed to anessentially aqueous environment. Studies on streptococcihave suggested that LTA contributes to their surface hydro-phobicity (24) and that monomer LTA, through hydrophobicinteraction, plays an important role in their adhesion toeucaryotic cells (3). The present study would support both ofthese concepts.

It is now quite evident that the concentration of LTA usedin studying its biological properties will determine the phys-ical form of the molecule. Isolation and purification of LTAwill inevitably, through concentration, result in the micellarform. This, in terms of real environmental situations, mayindeed be an artifact. Thus, this study would suggest that aproperty displayed by an unsubstituted LTA at less than 2,ug/ml would be due to monomer, whereas properties ob-served only at much higher concentrations would be pre-dominantly due to the micellar form of the amphiphile.

ACKNOWLEDGMENT

This work was supported by the National Health and MedicalResearch Council of Australia.

LITERATURE CITED

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2. Beachey, E. H., J. B. Dale, W. A. Simpson, J. D. Evans, K. W.Knox, I. Ofek, and A. J. Wicken. 1979. Erythrocyte bindingproperties of streptococcal lipoteichoic acids. Infect. Immun.23:618-625.

3. Beachey, E. H., W. A. Simpson, and I. Ofek. 1981. The interac-tion of lipoteichoic acid with streptococcal proteins, serumalbumin and animal cell membranes, p. 315-325. In G. D.Shockman and A. J. Wicken (ed.), Chemistry and biologicalactivities of bacterial surface amphiphiles. Academic Press,Inc., New York.

4. Benson, H. G., and J. N. Murrell. 1972. Some studies ofbenzenoid-quinonoid resonance. Part 2. The effect of solventpolarity on the structure and properties of merocyanine dyes. J.Chem. Soc. Faraday Trans. 68:137-143.

5. Brooker, L. G. S., G. H. Keyes, and D. W. Heseltine. 1951. Colorand constitution. XI. Anhydronium bases of p-hydroxystyryldyes as solvent polarity indicators. J. Am. Chem. Soc.73:5350-5356.

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7. Cleveland, R. F., J.-V. HoltJe, A. J. Wicken, A. Tomasz, L.Daneo-Moore, and G. D. Shockman. 1975. Inhibition of bacterialwall lysins by lipoteichoic acids and related compounds.Biochem. Biophys. Res. Commun. 67:1128-1135.

8. Donchi, K. F., G. P. Robert, B. Ternai, and P. J. Derrick. 1980.A surface-active merocyanine dye as a probe of micellar envi-

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ized molecules in bile salt micelles. Aust. J. Chem. 32:31-39.11. Galanos, C. 1975. Physical state and biological activity of

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12. Grassl, N. 1974. D-Alanine, p. 1686-1689. In H. 0. Bergmeyer(ed.), Methods of enzymatic analysis, vol. 3. Academic Press,Inc., New York.

13. Harrop, P. J., R. L. O'Grady, K. W. Knox, and A. J. Wicken.1980. Stimulation of lysosomal enzyme release from macro-phages by lipoteichoic acid. J. Peridont. Res. 15:492-501.

14. Helenius, A., and K. Simons. 1975. Solubilization of membranesby detergents. Biochim. Biophys. Acta 415:29-79.

15. Huggett, A. St. G., and D. A. Nixon. 1957. Use of glucoseoxidase, peroxidase and o-dianisidine in determination of bloodand urinary glucose. Lancet ii:368-372.

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17. Kessler, R. E., A. J. Wicken, and G. D. Shockman. 1983.Increased carbohydrate substitution of lipoteichoic acid duringinhibition of protein synthesis. J. Bacteriol. 155:138-144.

18. Knox, K. W., J. L. Markham, and A. J. Wicken. 1976. Forma-tion of cross-reactive antibodies against cellular and extracellu-lar lipoteichoic aid of Streptococcus mutans BHT. Infect.Immun. 13:647-652.

19. Knox, K. W., and A. J. Wicken. 1984. Effect of growth condi-tions on the surface properties and surface components of oralbacteria, p. 72-87. In A. C. R. Dean, D. C. Ellwood, andC. G. T. Evans (ed.), Continuous culture 8: biotechnology,medicine and the environment. Ellis Horwood (Ltd.),Chichester, England.

20. Knox, K. W., and A. J. Wicken. 1985. Environmentally inducedchanges in the surfaces of oral streptococci and lactobacilli, p.212-219. In S. E. Mergenhagen and B. Rosan (ed.), Molecularbasis of oral microbial adhesion. American Society for Micro-biology, Washington, D.C.

21. Kurz, G., and K. Wallenfels. 1974. D-Galactose, UV-assay withgalactose dehydrogenase, p. 1279-1282. In H. U. Bergmeyer(ed.), Methods of enzymatic analysis, vol. 3. Academic Press,Inc., New York.

22. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 133:265-275.

23. Markham, J. L., K. W. Knox, A. J. Wicken, and M. J. Hewett.1975. Formation of extracellular lipoteichoic acid by oral strep-tococci and lactobacilli. Infect. Immun. 12:378-386.

24. Miorner, H., G. Johansson, and G. Kronvall. 1983. Lipoteichoicacid is the major cell wall component responsible for surfacehydrophobicity of group A streptococci. Infect. Immun. 39:336-343.

25. Mukerjee, P. 1967. The nature of the associated equilibria andhydrophobic bonding in aqueous solutions of associationcolloids. Adv. Colloid Interface Sci. 1:241-275.

26. Mukerjee, P., and K. J. Mysels. 1971. Critical micelle concen-trations of aqueous surfactant systems. National StandardsReference Data Series no. 36. National Bureau of Standards,Washington, D.C.

27. Tanford, C. 1978. The hydrophobic effect and the organizationof living matter. Science 200:1012-1018.

28. Van Driel, D., A. J. Wicken, M. R. Dickson, and K. W. Knox.1973. Cellular location of the lipoteichoic acids of Lactobacillusfermenti NCTC 6991 and Lactobacillus casei NCTC 6375. J.Ultrastruct. Res. 43:483-497.

29. Wicken, A. J. 1985. Bacterial cell walls and surfaces, p. 45-70.In D. C. Savage and M. Fletcher (ed.), Bacterial adhesion:methods and physiological significance. Plenum PublishingCorp., New York.

30. Wicken, A. J., K. W. Broady, A. Ayres, and K. W. Knox. 1982.Production of lipoteichoic acid by lactobacilli and streptococci

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ative studies on the isolation of membrane lipoteichoic acidfrom Lactobacillus fermenti NCTC 6991. J. Bacteriol. 113:365-372.

32. Wicken, A. J., and K. W. Knox. 1970. Studies in the group Fantigen of lactobacilli: isolation of a teichoic acid-lipid complexfrom Lactobacellus fermenti NCTC 6991. J. Gen. Microbiol.60:293-301.

33. Wicken, A. J., and K. W. Knox. 1971. A serological comparisonof the membrane teichoic acids from lactobacilli of differentserological groups. J. Gen. Microbiol. 67:251-254.

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34. Wicken, A. J., and K. W. Knox. 1975. Characterization of groupN streptococcus lipoteichoic acid. Infect. Immun. 11:973-981.

35. Wicken, A. J., and K. W. Knox. 1980. Bacterial cell surfaceamphiphiles. Biochim. Biophys. Acta 604:1-26.

36. Wicken, A. J., and K. W. Knox. 1981. Criteria of purity, p.89-94. In G. D. Shockman and A. J. Wicken (ed.), Chemistryand biological activities of bacterial surface amphiphiles. Aca-demic Press, Inc., New York.

37. Wicken, A. J., and K. W. Knox. 1983. Cell surface amphiphilesof gram-positive bacteria. Toxicon 3(Suppl.):501-507.

38. Wicken, A. J., and K. W. Knox. 1984. Variable nature of thebacterial cell surface. Aust. J. Biol. Sci. 37:315-322.

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