Effect of natural modifications on the functional properties of extracellular bacterial polysaccharides

Effect of natural modifications on the functional properties of extracellular bacterial polysaccharides V. J. Morris* and M. J. Miles AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA, UK

(Received 7 August 1986) Extracellular bacterial polysacharides comprise the capsules and slimes secreted by many bacteria. Little is known about the .features of the chemical structure which are of importance in determinin9 the helical conformation and inter- or intramolecular associations of these polysaccharides. An understanding of such structure function relationships is hampered by the often complex chemical repeat units of these bacterial polysaccharides. One approach is to investigate and compare the properties of families of polysaccharides in which individual members of the 9roup show small naturally arisin9 modifications to the chemical structure. This approach is illustrated by studies which show the effects of changes in the polymer backbone, polymer side chains and non-carbohydrate substituents on polymer functionality. It is shown how such studies form a basis Jor explainin9 and optimizin9 the industrial applications of bacterial polysaccharides and .for understandin9 the natural roles of extracellular polysaccharides. Keywords: Extracellularpolysaccharides;bacterial polysaccharides;gelation; X-ray fibre diffraction

Function of extraceilular polysaccharides Extracellular bacterial polysaccharides comprise the capsules and slimes secreted by many bacteria and form an interface between the bacterial cell and its environment. Despite an extensive knowledge 1 of the chemical structures of many of these polysaccharides very little is known about their functionality. A variety of roles has been demonstrated or suggested for bacterial polysaccharides. Extracellular polysaccharides, together with the O-antigens of the lipopolysaccharides, constitute the principal immunogens and antigens of bacteria 2. Capsular polysaccharides have provided a source of successful and cost-effective vaccines z and form a basis for the search for new vaccines to certain bacterial infections 2. These polysaccharides are important virulence factors whose roles are to protect the microorganism from ingestion by phagocytes. The ability of encapsulated cells to resist ingestion, whilst nonencapsulated avirulent clones are ingested by phagocytes, is well documented 3 6. Extracellular capsules may act as bacteriophage receptor sites 7's or conversely provide a mechanism for blocking or masking bacteriophage attachment 7 9. Extracellular polysaccharides have been implicated 1°-~7 in the selective or non-selective adhesion of bacteria to surfaces which include inert surfaces, cultured human cells, mucoid epithelial layers and plant cell walls. Roles involving penetration, invasion and colonization of both plant and mammalian tissues 13,14,1 s and as receptor sites for surface located biosynthetic * To whom correspondenceshould be addressed. Presented in part at BiologicallyEngineered Polymers Conference, Churchill College,Cambridge, 21 23 July 1986. 0141 8130/86/060342-07503.00 © 1986 Butterworth & Co. (Publishers) Ltd 342

Int. J. Biol. Macromol., 1986, Vol 8, December

enzymes 19 have also been suggested. Specific molecular recognition steps in host-pathogen interactions have been proposed involving specific binding of extracellutar polysaccharides to plant lectins 2° or plant cell polysaccharides 17'1s. Extracellular polysaccharides may protect cells from dehydration ~8.2 ~ and, in the case of soil micro-organisms, promote soil adhesion and inhibit soil erosion 22. Nowadays, micro-organisms are being considered as a source of industrially useful polysaccharides 23. Production of polysaccharides by fermentation offers the potential of controlled physical and chemical properties, cost and supply. A greater understanding of the genetic control and biosynthetic routes to bacterial polysaccharide production offers the prospect of new and modified industrially useful polymers. Present industrial usage includes microencapsulation 24'25, blood plasma substitutes 23 emulsifiers23 and the replacement of traditional plant and animal polysaccharide gelling, thickening and suspending agents 23.

Structure and function Despite the large number of suggested natural roles for extracellular bacterial polysaccharides, and the increasing industrial interest in these polymers, there have been limited studies of the molecular basis of the functional properties. It is important to establish which functional properties depend solely upon the chemical structure, and which are influenced by the helical structures adopted by the polysaccharides and the subsequent association of these helices or their interaction with other biopolymers. Certain bacteria

Bacterial polysaccharides: V. J. Morris and M. J. Miles

produce polysaccharides which are similar to, or identical to, plant polysaccharides. Examples include the bacterial synthesis of cellulose26, //(1--3) glucans 27 and alginate zs-aa, For these polymers it is possible to infer structure-function relationships from previous studies on their counterparts in the plant kingdom. In many cases the chemical repeat units of bacterial polysaccharides are complex and there are no related polymers in the plant or animal kingdom. In these cases it is necessary to identify the features of the chemical structure which are important and describe in molecular terms how they control functionality. One approach is to extend earlier studies which were performed in order to establish the need for capsules or slimes. Early workers compared and contrasted the behaviour of encapsulated and denuded cells. Thus the role of extracellular polysaccharides in enhancing bacterial virulence was established by comparing the phagocytic ingestion of encapsulated and non-encapsulated avirulent clones a-6. Similarly a mutation in E. coil K12 resulting in excess capsular polysaccharide production was found to confer 'phage resistance 9. Non-encapsulated 'phage sensitive pneumococci became resistant after transformation to encapsulated cells and cells enzymatically treated to remove capsular polysaccharide became sensitive to infection 34. An extension of such studies to all aspects of functionality using genetically altered or natural mutants in which small changes arise in the chemical structure of the extracellular polysaccharide may provide a means of determining the influence of chemical structure upon functionality. In certain cases it may be sufficient to use unrelated bacterial species which secrete similar polysaccharides. Physicochemical studies of isolated bacterial polysaccharides differing only slightly in chemical structure may be used to explain the molecular basis of changes in functionality. In general a combination of both these methods will probably be required in order to establish biological activity and to explain this in molecular terms. In the case where the function of the polysaccharide is to modify the rheology of the aqueous environment or in the case of extracted industrially useful polysaccharides the latter approach may, in itself, be sufficient to identify and explain functionality. This paper demonstrates the success of such an approach through studies on families of polysaccharides with similar chemical structures. Such studies have tended to concentrate on industrially useful polysaccharides which have readily identifiable functional properties.

Bacterial polysaccharides Bacterial alginate Alginates are the salts of alginic acid which is a structural component of the cell walls of brown algae (Phaeophyta) 35. Certain strains of Pseudomonas aeruginosa 2a-3°. and Azotobacter vinelandii 3~-a3 produce a form of alginic acid. Mucoid strains of P. aeruginosa have been found a°'a6 in association with chronic respiratory tract infections accompanying cystic fibrosis or in connection with secondary infections accompanying major surgery, burns, leukaemia or following immunosuppressive drug treatment. Bacteria of the genus Azotobacter are normally-mucoid soil micro-organisms. Bacterial alginate is, like the algal material as, a (1--*4)

linked copolymer of fl-D-mannuronic acid (M) and 0~-Lguluronic acid (G). The polymer is a block copolymer containing M and G blocks together with mixed sequences (MG) containing both uronic acids. The monomer composition and block structure depend on the growth conditions and the bacterial species. Alginic acid is biosynthesized as polymannuronic acid and the block copolymer produced by the subsequent action of a mannuron C-5 epimerase, active during biosynthesis and liberated extracellularly by the bacteria 32-3a. Alginic acid containing G blocks greater than 20 monomers in length will gel in the presence of calcium 39-41 . Gelation involves intermolecular association of G blocks via site binding of calcium ions 41. Thus the MG ratio and block distribution are important determinants of functionality. Molecular descriptions of gelation were established through studies on plant extracts. Bacterial alginate differs from its algal counterpart in containing O-acetyl groups. Davidson et al.42 found that the O-acetyl groups in alginate from A. vinelandii were associated with mannuronic acid-rich regions and suggested that they may protect mannuronic acid residues from epimerization. Recent studies 43 have confirmed the locations of the O-acetyi substituents and their suggested protective role. Thus comparative studies 43 of acetylated and non-acetylated alginic acid have demonstrated that a non-carbohydrate substituent introduced intracellularly may provide a mechanism for controlling the subsequent extracellular epimerization, and hence the MG ratio, block distribution and resultant cation binding and gelling ability of the polysaccharide. Polysaccharides from Enterobacter NCIB 11870 and Klebsiella aerogenes K54 Comparative studies of the extracellular polysaccharide XM6 produced by Enterobacter NCIB 11870 and the capsular polysaccharide produced by K. aerogenes serotype K54 (hereafter called K54) provide another and different example of the importance of noncarbohydrate substituents. XM6 disperses in water as a viscous liquid and at higher ionic strengths will form thermoreversible gels44. XM6 may be regarded 45 as a naturally occurring deacetylated form of the capsular polysaccharide K5446 (Figure la and b). K54 disperses in water as a viscous liquid but does not gel at higher ionic strength. Deacetylation of K54 results in a polysaccharide which disperses in water and does gel at higher ionic strength. Thus the presence of a single O-acetyl group on the fucosyl residue of every alternate tetrasaccharide unit 45'46 is sufficient to inhibit gelation. Comparative X-ray fibre diffraction studies 47 have been used to assess the helical conformations and the nature of any intermolecular association of XM6 and K54. XM6 yields highly crystalline X-ray diffraction patterns (Figue 2a) consistent with strong polymerpolymer interactions and gelation. Analysis of the X-ray data 47 coupled with computerized model-building calculations 48 suggests an eightfold double helical structure, sections of which can crystallize in either an orthorhombic or tetragonal crystal modification. K54 yields diffraction patterns showing diffuse layer lines with the first meridional reflection apparently on the third layer line 49. The patterns are poorly crystalline consistent with weak intermolecular interactions, poor polymerpolymer association and an inability to gel. Deacetylation of K54 results in highly crystalline

Int. J. Biol. Macromol., 1986, Vol 8, December

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Bacterial polysaccharides: V. J. Morris and M. d. Miles

diffraction patterns (Figure 2b) characteristic of XM6. This would seem to suggest a change in helical conformation upon deacetylation. However, the meridional spacings in the XM6 and K54 patterns remain unchanged and it is the layer line spacing which appears to change upon deacetylation. At present it is believed 47 that both K54 and XM6 adopt a relatively rigid double helical structure. The K54 diffraction patterns are considered47'4a to be poorly resolved patterns of the characteristic XM6 pattern. Thus acetylation does not appear to alter the helical conformation but controls intermolecular association and crystallization of

3)~ DGIcp ( I ~ ) ( X D G I c p A(1~3) (~LFucp (1 1

segments of the polysaccharide chains and hence gelation. The mechanism by which acetyl groups inhibit molecular association and gelation remains to be established. It is interesting to note that K54 is a member of a group of polysaccharides for which alkali treatment, resulting in deacetylation, is claimed 5° to enhance adsorption of the polysaccharide to erythrocyte cell surfaces. The present set of experiments could be expanded by including studies on the related polysaccharides51 53 secreted by E. coli (Figure lc and d). Such studies would permit investigation of the effects of changes in the linkages of backbone residues and/or their anomeric configuration plus studies of the effect of changes in the branch or the backbone-side chain linkage. It would be particularly interesting to observe the effects of deacetylation on the E. coli K28 polysaccharide.

?

C

a

~DGIcp

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~ t

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Gellan gum and S-130 Gellan gum provides an example of the dramatic effect of O-acetyl groups on the intermolecular association and gelation of a polysaccharide. This anionic heteropolysaccharide secreted by Pseudomonas elodea has a linear tetrasaccharide chemical repeat unit 54'55 (Figure 3d). Under appropriate conditions aqueous dispersions of gellan form thermoreversible56 or thermoirreversible gels. The native form of the polysaccharide contains on average one O-acetyl group per repeat unit which is

4] C(DGlcp ( I ~ 4 ) ~ D C I c p A ( I~3)O(LFucp( 1 1

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Figure 1 Chemical repeat units for the extracellular polysaccharides from (a) Enterobacter (NCIB 11870), (b) K. aerogenes serotype K54, (c) E. coil K27, (d) E. coli K28

a

Figure 3 Chemical repeat units for the extracellular polysaccharides from (a) P. elodea--(gellan gum), (b) Alcaligenes (ATCC 31555)--(S-130)

L

.......................

b

Figure 2 X-rayfibre diffraction pattern obtained for (a) XM6 and (b) deacetylated Klebsiella K54 polysaccharide. The fibre axis is vertical. Wavelength 0.154 nm, relative humidity 98 ~o

344

Int. J. Biol. Macromol., 1986, Vol 8, December

Bacterial polysaccharides: V. J. Morris and M. J. Miles believed s5 to be located at C6 of one of the Glcp residues. Acetylated gellan yields soft elastic gels and progressive deacetylation leads to increased brittleness of the gels 56. X-ray fibre diffraction studies5776° of the native polysaccharide yield patterns characteristic of well aligned but poorly crystalline samples. Gellan shows a threefold helical symmetry with an axial advance per chemical repeat unit approximately half the extended length of the chemical repeat unit. It remains to be established6o.6 t whether gellan forms a contracted single helix or an extended, intertwined parallel double helix. At present the double helix is possibly the favoured structure. Progressive deacetylation 57'59 results in X-ray patterns characteristic of the same threefold helix but showing evidence of enhanced intermolecular association and crystallization. Acetylation does not alter the helical conformation but controls brittleness of the gels by controlling the local crystallization of sections of the polymer chains. Alcalioenes (ATCC 31555) secretes a branched anionic heteropolysaccharide (S-130) with a chemical structure 62'63 related to that of gellan gum (Figure 3b). S-130 is partially acetylated but the location of the Oacetyl groups remains to be determined 62. Despite the similar chemical structures gellan and S-130 possess very different rheological properties 2*'64. Aqueous dispersions of S-130 show good thermal stability, coupled with a high viscosity at low shear rates and shear thinning behaviour 24,64. X-ray fibre diffraction patterns 65 of S-130 are characterized by good molecular alignment but poor lateral packing and interchain register. The simplest molecular conformation consistent with the Xray patterns would be a twofold extended ribbon structure with an axial advance per repeat unit approximately equal to the extended length of the repeat unit. Computer modelling 66 suggests that such a model is stereochemically feasible but possible alternative models have not been investigated in detail, Deacetylation does not significantly alter the X-ray patterns 65 suggesting that such treatments will not alter intermolecular association and crystallization and thus are unlikely drastically to alter the theological properties. There are no reports of attempts to debranch S-130 enzymically or chemically in order to produce a gelling polysaccharide, or of experimental conditions for which the polysaccharide will gel. Thus there is no evidence that S-130 can be forced to adopt the conformation favoured for the backbone structure alone. X-ray fibre diffraction studies of debranched S-130 would be extremely interesting as they would show whether the debranched polymer can convert to the gellan structure. Alcaligenes (ATCC 31961) produces a branched anionic heteropolysaccharide whose structure, although preliminary, has been reported 67 to consist of a gellan backbone containing a disaccharide side chain*. The rheological properties of S-1942'*'67 are more closely related to those of S-130 rather than those of gellan. There are no reports of the gelation of S-194 or modified S-194 structures. When the chemical structure of S-194 is reported in detail it will be interesting to compare the helical conformations of S-130 and S-194 * Structures for S-194 and S-88 (another gellan derivative) have been reported (Jansson, P. E., Kumar, N. S. and Lindberg, B. X l l l t h Int. Carb. Syrup. N.Y. August 1986, Abstract B85).

by X-ray diffraction. It would be useful to study the conformation and intermolecular association of debranched S-194.

Xanthan gum Xanthan gum secreted by Xanthomonas species of bacteria provides another example of the effect of side Chains upon the geometry of the poiysaccharide backbone. The polymers have a cellulose backbone substituted on alternate glucose residues with a trisaccharide side chain 68'69 (Fiffure 4a). The side chain solubilizes the normally insoluble cellulose backbone. Although the exact value of the persistence length is disputed 7°-77 it is agreed that xanthan molecules in solution are stiffer than the cellulosic backbone. The side chains are considered to modify the normal backbone geometry leading to a helical structure with fivefold symmetry 78'79. Whether xanthan exists as a single or double helix has been a matter for active debate for many years 17,72-75,80-85. The viscoelastic properties of xanthan dispersions are sensitive to preparation conditions and the ionic composition of the dispersion medium 7°. The differences between xanthan dispersions and solutions are normally attributed to aggregation although the nature of the aggregates or microgels is still a matter for discussion 7°'82'86. Despite the presence of aggregates xanthan dispersions do not form true gels. The X-ray fibre diffraction data 78'79 show aligned helices with poor lateral packing and are consistent with the absence of crystallization and gelation. Surprisingly, conditions which stabilize the xanthan helix in solution also stabilize the microgels once formed. Thus the adoption of the helical structure is important in developing microgel structures and hence determining the rheological behaviour of xanthan samples. Mixtures of xanthan with certain plant galactomannans will gel under conditions for which neither pure component alone will gel 17'87'88. Recent X-ray fibre diffraction studies 86'89 have revealed that xanthan-carob and xanthan-tara mixed gels yield unique X-ray fibre patterns (Figure 5) characteristic of xanthangalactomannan binding. Previous workers 17,87,88 had attributed gelation to an intermo ecular binding between

~l]~DClcp( l~ll),~DClcp (1~ 1

1 0DManp ( 1~11~ D GlcpA( I --2lOt OManp

/\

~\

,

/6

I

CH/'3C%CO2 -

O Acetyl

4),~ DGIco [ 1~ 4) ~'DGIcp [ ~ 1

b

? t I

LR hap ( I~ 6)/~DGlcp( I~61 DGIc,o ( I~41 DClcpA( I--2)DManp

Figure 4 Chemical repeat unit of the extracell polysaccharidcs from X. campestris--(Xanthan gum) and Acetobacter (NBI 1022)

Int. J. Biol. Macromoi., 1986, Vol 8, December

345

Bacterial polysaccharides: V. d. Morris and M. J. Miles

.

.

.

.

.

.

.

.

.

a

b

Figure 5 X-rayfibre diffractionpatterns obtained for (a) xanthan-carob mixed gel (45 ~o xanthan), (b) xanthan-tara mixed gel (50 % xanthan). The fibre axis is vertical. Wavelength 0.154 nm, relative humidity 98 o/,

the xanthan helix and unsubstituted regions of the galactomannan backbone. Recent mixing experiments 80"89 have shown that galactomannan binding, as revealed by X-ray diffraction studies, and gelation only occur if the galactomannan is mixed with xanthan under conditions which denature the xanthan helix. Such mixing experiments, coupled with qualitative analysis of the mixed gel X-ray diffraction patterns, have led to the suggestion s6"s9 that intermolecular binding involves a cocrystallization of sections of the denatured xanthan molecules with segments of the galactomannan chains. Intermolecular binding is attributed 86'89 to the stereochemical compatibility between the cellulosic and mannan backbones which permits co-crystallization. Such a molecular model for intermolecular binding permits control and optimization of gelation. The specificity of this interaction could account for similar synergistic interactions between xanthan and glucomannans 87. Partial debranching of xanthan molecules to produce 'cellulosic" blocks within the chain may provide a means of inducing gelation of xanthan solutions. Evidence for xanthan-galactomannan binding 86"s9 supports suggestions 1v'87'88 that such binding may provide a recognition step in the interaction of Xanthomonas plant pathogens with plant hosts or a specific mechanism for Xamhomonas bacterial adhesion to plant cell walls. Demonstration of xanthan galactomannan binding provided the first example of the conformational modification of one polysaccharide by a non-covalent interaction with a second polysaccharide and indicates that the properties of the backbone may reassert themselves under appropriate conditions. Acetobacter (NBI 1022) is reported 9° to produce a polysaccharide resembling xanthan but containing a pentasaccharide side chain (Figure 4b). There are no reported X-ray diffraction studies on this polysaccharide and the helical conformation is unknown. However, one would expect to be able to induce intermolecular binding with certain galactomannans and hence mixed .gel formation.

346 Int. J. Biol. Macromol., 1986, Vol 8, December

Conclusions The chemical repeat units of many bacterial polysaccharides are complex and it is difficult to predict the helical conformation of the polymer, the subsequent interactions between these helices or their interactions with other biopolymers. Studies on naturally occurring polysaccharides with similar chemical structures are starting to reveal which components within the chemical structure can markedly influence helical conformation or subsequent intermolecular association. Changes in helical conformation or intermolecular association give rise to drastic changes in polymer functionality. Only two general trends appear to have emerged at present. The helical conformation is particularly important in determining molecular stiffness and the viscosity of solutions. Gelation seems to be favoured by helical conformations which can crystallize or co-crystallize with other polysaccharides. It is hoped that a continuation of such studies will lead to the development and optimization of bacterial polysaccharides or modified bacterial polysaccharides as industrial additives. Such studies should also provide a basis for describing the in vivo roles played by capsules and slimes.

Acknowledgements The authors wish to acknowledge discussions of unpublished ~ork with E. D. T. Atkins, P. T. Attwool, C. E. Sanson, C. Upstill and K. Veluraja.

References 1 2 3

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