J. Mol. Biol. (1977) 109, 373-391

Conformation and Molecular Organization in Fibers of the Capsular Polysaccharide from Escherichia coli M41 Mutant R. MOORHOVSE, W . T. W ~ T ~ R , STRUTHER ~RNOTT

Department of Biological Sciences, Purdue University West Lafayette, Ind. 47907, U.S.A. AND ~ . E. :BAYER

The Institute for Cancer Research, Burholme Avenue Fox Chase, Philadelphia, Penn. 19111, U.S.A. (Received 21 July 1976)

Capsular polysaccharides from Escherichia coli serotype K29 and from its two mutants, M13 and M41, have been examined b y X - r a y fiber diffraction. The chemical structure of the wild type strain has been shown to consist of hexamerie repeating units with a disaccharide side-chain linked to a polytetrasaccharide main chain (Choy et al., 1975). Diffraction patterns obtained from oriented fibers of each of the two m u t a n t s were identical to those obtained from the wild type strain polysaccharide. Similar phage specificity and antibody reactions coupled with the similarity of the diffraction patterns indicate an overall chemical identity of the polysaccharides elaborated b y the m u t a n t strains with t h a t of the p a r e n t strain. Replacing monovalent with divalent cations did not appreciably alter either the trait-cell dimensions or the intensity distribution. U p o n drying, the helix pitch and s y m m e t r y were unchanged b u t the crosssectional area of the unit cell decreased by 26%. Both of these observations were taken to suggest t h a t the cations and solvent molecules are weakly bound and distributed throughout large cages formed by the surrounding polyanion chains. Unit-cell dimensions for the two forms were a : 2.030 nm, b : 1.178 n m and c ~ 3.044 n m (wet); and a ~ 1.730 nm, b ~ 1.020 n m and c = 3"044 n m (dry); and the space group s y m m e t r y was P2~2~2~. A model with antiparallel 2-fold helices at the corner and center of the unit cell was refined simultaneously against X - r a y intensity d a t a and stereochemical restraints to a final R value of 0.26. Although difference Fourier syntheses did suggest several plausible sites for cations or solvent molecules, it was not possible to locate unambiguously such " g u e s t " molecules. T h e molecular conformation is stabilized by three intramolecular and two intermolecular hydrogen bonds per hexasaccharide repeating lmit. I t is suggested t h a t this extensively h y d r a t e d conformation m a y persist in vivo and p l a y an i m p o r t a n t role in phage recognition and penetration of the cell envelope. 1. I n t r o d u c t i o n P o l y s a c c h a r i d e s are e l a b o r a t e d b y a wide v a r i e t y of b a c t e r i a e i t h e r e x o c e l l u l a r l y as a capsule, s u r r o u n d i n g a n d a t t a c h e d t o t h e bacteria, or e x t r a c e l l u l a r l y as a slime, 25

373

374

R. MOORHOUSE

ET

AZ.

freely dispersed in the culture fluid and unattached to the microbial cells. When both capsules and slimes are produced, the chemical constitutions of their polysaecharides are usually similar (ffeanes, 1966). It is only recently that the detailed primary structures of a few of the many known bacterial polysaccharides have been determined. Several KlebsieUa K serotype capsular polysaccharides with similar compositions have been investigated (see Thurow et al., 1975). Although many K-specific capsular polysaecharides from Esd~eri~hia coli serotypes have also been isolated, only for the E. coli serotype 29 (K29 polysaccharide) capsule has the primary structure been determined (Choy et al., 1975). Extracellular polysaccharides from Leuconostoc mesenteroides strain NRRL B-512(F) (Jeanes, 1974) and Xanthomonas campestris (Jansson et al., 1975; Melton et al., 1976) are weU-characterized and have found extensive commercial applications. In general, all these polysaccharides consist of oligomeric repeating units (Sutherland, 1972) containing two to six sugar residues. The polymer may be linear or branched and is often substituted to varying extents by pyruvate ketal or 0-acetyl groups. The capsular polysaccharide from E. coli strain Bi161/42(09 :K29(A):H-) displays the serotype 29 (Choy et al., 1975) and consists of hexasaccharide repeating units (see (I) below). Pyruvate is attached to every terminal D-glucose residue and no O-acetyl residues have been detected. -+2)-~-D-IV[anlJ-(1 ~3)-fl-D,Glcp-( l ~-3)-fl-i)-GleAp-( [ -~3)-~-t)-Gal/~-( l -~-

I 4 i"

1

I fl-1~-~'~1~-( 1 -e-2)-~-D-Manp

(I)

/\ 4

\/ 6 /~

HaC

C02H

E. coli K29 polysaccharide is the receptor of E. coli K phage 29, whieh has beeI1 shown to be highly specific for this capsule (Fehmel et al., 1975). For example, the phage does not adsorb to acapsular host mutants nor, in general, to related strains with capsules of different serotype and chemical structure. The phage cleaves fl-Dglucosido-(1->3)-D-glucuronic acid bonds exclusively and cannot tolerate either removal of the pyruvate ketal or reduction of the glucuronic acid residues in the substrate. Capsular polysaccharides synthesized by Klebsiella serotype K31 and two mutants, M13 and M41, of E. coli serotype K29 are the only other materials known to be depolymerized by this phage. The exact structure of the Klebsiella K31 poly-

E.

GOLI

Mdl CAPSULAR P O L Y S A C C H A R I D E

375

saccharide is not known, although it contains the same sugar monomers as the

E. coli K29 glycan (Nimmich, 1968). We have prepared well oriented and crystalline fibers of the polysaccharides from the native K29 strain as well as from the Mdl and M13 mutants. The X - r a y diffraction patterns suggest t h a t the molecular conformation and packing are the same for all and, therefore, t h a t the primary structures are essentially unchanged. This conclusion is supported b y our further observation t h a t both the wild type and the two m u t a n t strains exhibit similar phage specificity and agglutinate with antibodies against the native strain (Bayer & Thurow, 1976). Our detailed analysis of the better quality X - r a y patterns from the 1K41 strain is, because of the large size of the molecular asymmetric unit, unusual among fibrous structures. 1No branched polysaccharide structure has previously been examined in this way. 2. M a t e r i a l s a n d M e t h o d s

(a) Materials The mutant strains Mdl and M13 were obtained after treatment of wild type E. coli serotype K29 with a mutagen, ICR 191, a bisnitrogen mustard (Creeeh et al., 1972). Cultures were extracted with phenol/water (Westphal & Jann, 1965), after which lipopolysaccharide and associated nucleic acids were removed by sedimentation of the aqueous phase at 105g. Capsular polysaccharide was isolated from the supernatant by differential Cetavlon (cetyl trimethylammouium bromide) precipitation (Scott, 1960; Orskov et al., 1963). The final product was dissolved in water, reprecipitated 3 times from alcohol and lyophilized. To ensure complete removal of nucleic acid, some samples were also subjected to an RNAase treatment (Jann et al., 1966) immediately after the Cetavlon precipitation step. (b) .Fiber preparation The most successful method found for making fibers was to suspend droplets of polysaccharide solution between glass beads in an atmosphere of 0 to 30~/o relative humidity. When the droplets had almost dried out, more solution was added and the process repeated until the fiber was of the desired thickness. The fiber was then suspended under tension (10 to 20 g) at room temperature and 66O/o relative h, lmldity. Some samples were subquently dried over silica gel or equilibrated at 920/0 relative humidity. Densities of fibers were measured by flotation in a mixture of halogenated hydrocarbons.

(e) X-ray diffraction Fiber diffraction diagrams were recorded with a flat plate camera using pinholecollimated, nickel-filtered CuKa radiation from a Picker microfocus X-ray generator. Specimens were maintained at the appropriate relative humidity by passing through the specimen chamber a stream of helium that had been bubbled through a saturated solution of a suitable salt. The diffraction patterns were calibrated by dusting the specimen with calcite (characteristic spacing 0.3056 nm). The spacings of the Bragg reflections were measured on projected enlargements. The unit cell dimensions were refined by a least-squares refinement (Arnott & Hukins, 1973). Intensities were measured as areas (Am) under radial mierodensitometer traces across spots on the film. Baseline profiles were determined from radial traces on either side of each reflexion. The areas were converted to relative structures amplitudes, oFm, using the relationship

o_~ ~ ~ AmRm(tan2Om)/(1 ~- cos220m),

(1)

which provides approximate Lorentz, polarization and spot extension corrections. (Rm is the cylindrical polar radius in reciprocal space of the X-ray reflection with Bragg angle 0m.)

376

R.

MOORHOUSE

ET

AL.

(d) Model building and refinement Molecular models having the appropriate helical s y m m e t r y a n d pitch, and pyranose rings in the standard (An'herr & Scott, 1972) C1 chair conformation were generated using a linked-atom description (Arnott & Wonacott, 1966) with all bond lengths and angles held constant, including the bond angles at the glycosidic oxygens which were maintained at 116.5 ~. This left the glycosidic bridge conformation angles in the main chain and the branches, and the side group (hydroxymethyl, carboxyl and methyl) orientations as explicit variables. Sh~ce we could fred no accurate structure determination of a derivative of 4,6oO-(lcarboxyethylidene)-fl-D-ghmose, we generated a hypothetical model of the additional ring produced by the fusion of pyruvate to glucose using the Arnott & Scott (1972) procedure and the average bond lengths and angles listed b y them. The pyruvate carboxyl was placed in the axial configuration at the q u a t e r n a r y carbon atom as proposed for the pyruvic acid ketal that occurs in the extracellular polysaccharide from Xanthomonas campestris (Gorin et al., 1967). The 1,3 dioxane " p y r u v a t e " ring was refined to minimize (eqn (2)), with the bond. angles and conformation angles as explicit variables. The bond angles were tied elastically to standard values b u t no explicit restrictions were placed on the values of the conformation angles. The atom labeling for the asylmnetric unit is shown in Figttre 1. For positioning the model in the trait cell, 2 additional parameters were used to define the relative axial orientation (/~) and translation (w) of the chains. The introduction of each countercatiou or oxygen atom representing a water molecule required an additional 3 variables. At each stage in the modeling a n d refinement of the structure we sought to minimize the q u a n t i t y ~2 in a least-squares fashion (eqn (2)). =

X

~m(oF~--F,,,) s + S Z ~j + X~,~Gj~.

(2)

The first summation in fJ ensures o p t i n m m agreement between the observed (oF~) and calculated ( F ~ ) X - r a y structure amplitudes. The second ensures the optimization of non-covalent interatomic interactions. The third imposes, by the method of Lagrange undetermined multipliers, the exact constraints we have chosen. Detailed explanation of eqn (2) and examples of its use for other polysaccharide structure analyses have been given by Gusset al. (1975), Winter et al. (1975) and Smith & Arnott (1977). Atomic scattering factors for calculating structure factors were calculated using the method and values given in International Tables for X-ray Crystallography (1974). These were then modified using a method similar to that of A r n o t t & Hukins (1973) to approximate the effect of disordered water occupying the voids in the (mit cell (eqn (4)).

f'(p) ----f(p)-- (lOda/rS)r r

:

3(sinx--xcosx)/x a.

(4) (5)

Here f ' is the water-weighted scattering factor, f the normal scattering factor ; p is the reciprocal space radius of the reflection given by 2sin0/A, where 0 is the Bragg angle and h the wavelength of the radiation; d is the v a n der Waals' radius of the scattering atom (H, 0.12 n m : C, 0.17 n m : O, 0.14 urn) ; r is the effective radius of a water molecule (0-20 ran). Then r is the scattered diffraction amplitude in the direction of p for a 1-electron scatterer uniformly distributed in a sphere of radius s, given b y eqn (5) (James, 1965}, where x 2~sp. For heavy atoms which have hydrogen atoms bonded to them, f was taken as the sum o f f for the atom and nf for the n hydrogen atoms, and d was increased such t h a t (4/3)~d 3 was the volume occupied by the group as a whole, allowing for the intersection of the v a n der Waals' spheres. For this, bond lengths were assumed as follows: H - - C , 0-109 n m ; H - - O , 0.096 nm. =

(e) Location of cation and water molecules Difference electron density maps were calculated using phases derived from the polyanion structure a n d normal atomic scattering factors, rather t h a n the water-weighted scattering factors pre~dously used. I n the ease of overlapping reflections, the observed

E.

COLI

M41 C A P S U L A R

POLYSACCHARIDE

377

06

FIO. 1. The hexasaccharide of M41 polysaccharide showing the atom labeling. A is D-mannose, B is D-glucose, C is D-glucuronate, D is D-galactose, E is D-mannose, and F is 4,6-O-(1-carboxyethylene)-D-glucose. The parentheses are omitted from the atom names for clarity. intensity was divided equally among the contributing planes. Trial atoms were placed a t the positions of the major positive peaks suggested b y the difference maps a n d the atomic environments investigated. Atoms making at least one plausible a t t r a c t i v e interaction (hydrogen bond) to a suitable a t o m on the polyanion a n d with no l m a c c e p t a b l y short contacts were included in subsequent refinement cycles (Arnott et al., 1976).

3. D i f f r a c t i o n Patterns a n d U n i t C e l l s T h e diffraction p a t t e r n s (e.g. Fig. 2) from fibers m a i n t a i n e d a t 9 2 % r e l a t i v e h u m i d i t y can be i n d e x e d on t h e basis o f a r e c t a n g u l a r l a t t i c e w i t h u n i t cell d i m e n s i o n s (and s t a n d a r d d e v i a t i o n s ) a ---- 2.030(6)nm, b -----1-178(4)nm, c = 3.044(9)nm. T h e s y s t e m a t i c absences for t h e (h00), (0k0), a n d (00/) reflections w h e n h, k or 1 is o d d

378

R.

MOORHOUSE

ET

AL~

:FIG. 2. I)iffTaction pattern from 2-fold E. coli M41 polysaccharide. Two chains pass through the orthorhombic unit cell with dimensions a ~ 2"03 nm, b ~ 1.178 nm, and c (fiber axis) = 3-04 nm. The meridional direction is vertical and the sample was tilted from a direction normal to the beam by 9~. suggest t h a t the s y m m e t r y of the unit cell is orthorhombic with the space group P212121. This s y m m e t r y dictates t h a t the unit cell contains 4n hexasaceharide repeating units. I f we let n equal 1 and assume t h a t the chemical repeating unit contains two sodium ions (for charge balance), then the calculated density is 1.00. Any larger value of n would result in a calculated density value substantially larger t h a n the experimentally determined value of 1.48. I t is likely therefore t h a t the molecules are 2-fold helices in which the hexasaceharide p r i m a r y structural unit is also the helically repeating unit, and t h a t each unit cell contains two antiparallel molecules arranged to produce the observed P212~21 symmetry. I f we attribute the difference between observed and calculated densities to the presence of interstitial water molecules, then there are approximately 115 such molecules in each unit cell or approximately five water molecules per monosaccharide residue. Anionic mono- and oligosaeeharides often crystallize as di- or trihydrates (Cook & Bugg, 1973,1975; Gould et al., 1975), while density measurements for other anionic polysaeeharides suggest the presence of three to seven water molecules per hexose unit (Atldns et al., 1974; Winter et al., 1975). When samples are X-rayed in a d r y helium atmosphere after prolonged (100 h) drying over silica gel, there is no change in the general intensity distribution, including the systematic absences, nor in the c dimension of the unit cell. However, a is reduced

E.

(70LI

M41 C A P S U L A R

POLYSACCHARIDE

379

to 1"73 nm and b to 1.02 nm, which is a reduction of 26~/o in the area of the base of the unit cell. The changes produced on drying are easily reversed by rehumidification. Apparently the molecular conformation survives drying with little change and the substantial quantity of water which fills out the crystal unit cell at 92% relative humidity is not firmly bound. This makes our later conclusion that most of the water molecules are not ordered in the crystal lattice unexceptional. When sodium is replaced by calcium there is no detectable change in the diffraction patterns. This supports the idea that only the polyanions are organized in a regular fashion, a n d that the small components (cations and water molecules) are, for the most part, disordered. The K29 and lV[13 polysaccharides give essentially the same patterns as the i~I41 samples, suggesting that there is no change in basic primary structure among these materials and justifying our use of the primary structure determined for the K29 polysaeeharide in our analysis of the best diffraction patterns, which were obtained with the M41 polysaecharide. 4. Structure Analysis (a) Polyanion conformation and packing A first approximation to the polyanion structure was prepared by setting the glycosidic bridge conformation angles of the main and branch chains at values corresponding to the centers of allowed regions in the "hard sphere" linkage map for appropriate disaceharides (Rees, 1969), by setting the hydroxymethyl oxygen atoms gauche to both 0(5) and C(4), one oxygen of the ghicuronate carboxylate group cis to 0(5), and one oxygen of the pyruvate carboxylate group cis to O(4)F. This structure was then refined to produce a 2-fold helical model of pitch 3.044 nm and with minimum steric compression using equation (2) but excluding the terms involving X-ray structure amplitudes. This preliminary molecular model, whose parameters are given in Table 1, was maintained as a rigid body in the P212121 unit cell and initial values of the packing parameters/z and w (see Materials and l~Iethods, section (e)) were obtained such that the contribution to ~ (eqn (2)) from overshort contact distances between atoms of different chains was minimized. A crystal model consisting of polyanion chains was then refined by minimizing ~ (eqn (2)) with molecular and packing parameters as variables. Initially R = ~1 oFm--Fm ]/~, oF,, was 0"56 and R" = ~. wm (oFm -- Fro)2/ m

r m0F~ was 0.59. After refinement R and R" became 0.26 and 0.31, respectively. The conformation angles of this refined model are given in Table 1. The observed and calculated structure amplitudes may be compared in Table 2. The atomic coordinates of the hexasaceharide unit are given in Table 3, and the molecular conformation is shown in Figure 3. (b) Location of cations and water molecules The behavior of the i~41 polysaeeharide fibers (particularly the readily reversible shrinkage of the intermolecular distances accompanying dehydration and the insensitivity of the crystal structure to changes in the type of cation present) is reminiscent of hydrated fibrous polyanion systems like DNA and polyeytidylie acid (Arnott et al., 1976), where only the polyanions are ordered and the cations and water molecules behave like a liquid filling the spaces between polymer molecules. The response of

380

R.

M O O R H O U S E E T AZ,. TABLr 1

Final values for the refined conformation angles of the capsular polysaccharide from mutant M41, E. coli serotype K29 Conformation angle

RefinedValue (~(Initial)

Comment

8[Col)A, O2) linkage between residues F and E are comparable with those in ~-sophorose (161 ~ 98~ Walkinshaw, 1975). In the main chain the angles at the ~(1 ->3) linkage between residues A and B have values (76 ~ 106 ~ comparable with those (88 ~ 97 ~ in a galactotriose containing the same linkage (Walkinshaw, 1975). At the ~(1 ->2) linkage between residues D and A comparisons with accurate singlecrystal studies of small molecules are available only for 0[Cr C(1)D, O(2)A, C(2~A], w h i c h h a s a v a l u e o f 69 ~ i n o u r s t r u c t u r e , 60 ~ i n s t r o n t i u m 4-O-(4-deoxy-fl-L-threoh e x - 4 - e n o s y l ) - c c - D - g a l a c t u r o n a t e ( G o u l d et al., 1 9 7 6 ) , 62 ~ i n ~ - ~ - t r e h a l o s e ( B r o w n et al., 1 9 7 2 ) , 79 ~ i n O-(4-O-methyl-~-D-glucuronate)-(1-2)-O-fl-D-xylose-(1-4)-D-xylose (Moran

& Richards,

1973). T h e

fl(1 ->3) l i n k a g e

between

residues

C and

D has

a

The cited angles (~1, ~b2) are defined as r : 0[O(5~, C(1), O(,), Cr a n d ~2 : 0[C(1), O3) linkage between residues B and C are compared with the corresponding quantities (--115 ~ 145 ~ in ~-laminaribiose (Walkinshaw, 1975), there is a notable difference between the values of ~[C(3), 0(8)c, Cr O(~)s] which may be caused by the propinquity of the large branch chain. (b) Side.chain conformations The orientations of the four unique hydroxymethyl side chains are all different. 0r approximately eclipses O(~)A, while 0r is distorted from a trans orientation relative to Ocs)B in the direction of eclipsing Cr O(s)D is oriented approximately gauche to O(S)D and trans to C(4)D, and 0(6>~.is nearly gauche to both O(5)~. and C(4)~.. Similar variations in the orientation of crystaUographically independent hydroxymethyl substitutents has been observed in a variety of oligo- and polysaccharides including raffmose (Berman, 1970), planteose (Rohrer, 1972), ~-cyclodextrin hexahydrate (Manor & Saenger, 1972), amylose (Zugenmaier & Sarko, 1976) and calcium hyaluronate (Winter & Arnott, 1977). Sincein each of the last three structures all of the

E. GOLI M41 CAPSULAR POLYSACCHARIDE C0RN~R

387

CB~rrER

FIG. 4. A model of sodium co-ordlnation by the M41 mutemt polysemcharide, viewed down the y axis. Filled circles are sodium ions; striped circles are water molecules. Polya~ion atoms involved: (i) on corner strands (1) O(6)Fon the strand whose axis is displaced 1.178 nm out of the page from that shown. (2) Oo; (4) Ocelot; (8) O~6~c. (il) ~5) O~3~r; (6) O~>~; (7) O~p. The atom notation is that used in Fig. i.

h y d r o x y m e t h y l side-chains are in chemically identical environments, intermolecular packing effects would seem to play a major role in determining the orientation of this side chain. In one instance, residue D, the possibility of forming an intramolecular hydrogen bond involving the O~s~ plays an important role in determining the sidechain orientation. The conformation of the glucuronosyl carboxylate group is similar to t h a t observed in other polysaccharide structures (Guss et al., 1975; Winter et al., 1975) with 0r almost exactly eclipsing Hcs)c. The pyruvate carboxylate is oriented such t h a t O~sb~F eclipses 0c4)~. (c) Hydrogen bonding The hydrogen bonds within and between molecules are depicted in Figures 3 and 5. Their lengths are given in Table 4. Of the 15 potential hydrogen bond donors in each asymmetric unit, only five have been explicitly identified as such. Presumably the remainder bind to the water molecules which are abundant in the unit cell. Two modeling experiments support this explanation. :First, of the ten potential hydrogen-bond donors not participating in hydrogen bonds within or between polysaccharide chains in our model, nine are less than 0.42 n m distant from possible accepter atoms on other polyanion chains. I t is conceivable that each of these pairs is actually representative of a hydrogen bond bridge utilizing a single water molecule situated between the chains. Secondly, if we introduce our molecular model into the smaller unit eel], observed at low humidity, and refine the orientation and translation

5,044nm

(a) 2.0~nm

H'/' ~nm

FIG. 5. Views uf the refined o r t h o r h o m b i e st,rueture of M 4 ] polysaeeharide, (a) View along the (010) direction showing 2 corner and 1 center chains.

(b) View along the (001) direction (helix axis); the corner chains are emphasized,

E. COLI M41 C A P S U L A R P O L Y S A C C H A R I D E

389

TABLE 4

Stereochemical features

of the M41 capsular polysac~haride crystal structure

A. Hydrogen bonds Donor

Acceptor

Distance (nm)

Angle~ (~

O(3)^

O(4)F

0"283

90"5

O(~)a

O(6)v

0.260

127.7

O(3~E

O(s)F

0"305

104"5

O(4)D O(4)~.

O(3)r O(4)D

0"284 0.273

99"2 120.0

Comment Intramolecular Intramolecular Intramolecular Corner-center Corner-corner

B. Short intramolccular contacts Atom 1

Atom 2

O(sm 045)0 O(5)c O(8)^ O(sa)r O(8,)F

C(3)c C(3)D H(4)D O(5~^ H(4)F H(Sb)F

O(eb)F H(a)A

C(9)F H(3)F

Separation (nm)

Limiting value (nm)

0"26 0'26 0"21 0'25 0"18 0"20 0"26

0"27 0"27 0"22 0"26 0"22 0"22 0"27

0"17

0"19

-~The cited angle is defined as the angle (P, D, A) where A is the acceptor atom, D is the donor, and P is the non-hydrogen atom covalently bonded to D. of the polyanion in such a manner as to minimize overshort non-bonded interactions, it is possible to convert several of these "bridges" into direct intermolecular hydrogen bonds. The poorer crystallinity observed in the low humidity form, while preventing a more rigorous refinement, m a y arise from the existence of energetically similar, but incompatible competing hydrogen-bond networks, resulting in an overall decrease in packing regularity. N o t a b l y absent are hydrogen bonds across individual glycosidic bridges except for the 0(3)~. - - - O(5)r bond across the (1->2) linkage in the branch chain. These features, common in m a n y polysaccharide structures, are replaced in this structure b y intramolecular hydrogen bonds, 0 ( 3 ) ^ - - - O ( 4 ) r and 0r which link nonadjacent residues. Neighboring molecules are well-separated by the water in the structure and make few close contacts. Parallel chains separated along the b cell edge b y 1.178 nm are linked by 0 ( 4 ) ~ . - - - 0 ( 4 ) D hydrogen bonds. Adjacent antiparallel chains are linked by O(3)F - - - 0(4)D hydrogen bonds. (d) Biological considerations The X - r a y diffraction from samples of K29, M41 and M13 suggests t h a t their structures are essentially identical. This conclusion is supported b y the similar phage, agglutination and immunodiffusion reactions (Bayer & Thurow, 1976). The highly specific bacteriophage 29 (Fehmel et al., 1975), which is active on all of these, requires the presence of both the p y r u v a t e ketal and glueuronic acid residues. Fehmel et al. (1975) imply t h a t the ketal should be linked equatorial/equatorial to the sugar. This 26

390

R. M O O R H O U S E

BT

AL.

phage has been found to depolymerize only one other capsular polysaccharide of t h e 82 heterologus bacterial glycans tested (Fehmel eta/., 1975): t h a t from Klebsiella K31 which is known to contain the same sugar monomers as the K29 glycan (Nimmieh, 1968). These highly hydrated polysaccharides are attached to the exterior of the cell i n vivo and form a capsule or contribute to long loose strands t h a t project radially. In the case of the temperature-sensitive mutant 1K13, Bayer & Thurow (1976) have found that its long strands originate over the adhesion sites of inner and outer membrane, from which they protrude radially or form coiled structures at the cell surface. We might expect that molecular secondary structure and organization in these strands and coils would be very similar to the one we have elucidated in highly hydrated fibers. The biological role of the capsular polysaccharide is mnltifold: it prevents bacteriophages specific for cell wall receptors from infecting the cells; it also protects the bacteria from phagocytosis by macrophages (Liideritz et al., 1968). On the other hand, the capsule has also become the receptor for capsule-specific bacteriophages such as phage 29. This may involve the interaction of several phage 29 spike tips with one or more polysaccharide strands (Fehmel et al., 1975), the virus glucanase subunits moving along the polymer strands, cleaving the glycosidic linkages which opens a path for the phage head and provides energy for the movement. Some particular conformational aspect of the polysaccharide strands may, during capsule penetration, then direct the phage to a particular site (the root of the strand at the adhesion site between the inner and outer membrane) on the cell surface where it ejects its nucleic acid (Bayer, 1974). The work at Purdue was supported by grants to one of us (S. A.) from the Public

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Conformation and molecular organization in fibers of the capsular polysaccharide from Escherichia coli M41 mutant.

J. Mol. Biol. (1977) 109, 373-391 Conformation and Molecular Organization in Fibers of the Capsular Polysaccharide from Escherichia coli M41 Mutant R...
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