The structure of bacterial cellulose

BIOCHIMICA ET BIOPHYSICA ACTA

VOL. 3 (1949)

527

THE STRUCTURE OF BACTERIAL CELLULOSE by KURT M ~ H L E T H A L E R *

Laboratory of Physical Biology, Experimental Biology a**d Medicine Institute, National I~tstitutes oJ Health, Bethesda 14, 3Iavyland (U.S.A.)

I NTRODUCTION

Electron microscopic studies x have shown that the cellulose from several different plants occurs as fibres of a diameter of ca 25 ° A. How these fibres are built up and why they have so constant a diameter is still unknown and the investigation of such questions is made especially difficult by the fact that the cellulose is produced within the cells of these plants. They could be studied in thin section but the necessary techniques are not yet sufficiently developed to make this possible. It therefore has seemed more profitable to evade these difficulties by choosing for study an organism which builds cellulose extra-cellularly. As long ago as 1886 A. J. BROWNs described a bacterium which produced a solid membrane when growing on a carbohydrate-rich medium. He found that these membranes were soluble in ammoniacal copper hydroxide and gave reducing sugars when hydrolysed with sulphuric acid. Since he knew that cotton gave these reactions he called the organism Bacterium xylinum (following PLINY who used the word xylium for cotton). Later investigations by VAN WISSELINGHa and HIBBERI4 demonstrated that the material of these membranes is indeed cellulose. HIBBERT found that membrane is produced only in a culture medium containing hexose sugars, their anhydrides or substances which the organism can readily convert into hexoses. MARK AND VON SUSICHs confirmed the identity of bacterial cellulose with cotton cellulose by producing well-oriented x-ray diagrams from stretched bacterial m e n , fanes that were the same as those from cotton. The original electron micrographs of FRANZ AND SCHIEBOLD6 showed that these bacterial membranes corsist of a thick network of fibres and ribbons. They concluded that the ribbons had a thickness of ca ioo A, a breadth of ca 5000 ,~ and that they were composed of smaller fibres of ca 2oo A diameter. The fact that the cellulose fibrils in the walls of plant cells have nearly the same size now raises the question of whether these are indeed primary structural units of cellulose itself. The present paper describes results obtained in attempts to examine this question using electron micrographs of bacterial cellulose. EXPERIMENTAL

Our previous work has shown (FREY-WYsSLING AND MOHLETHALER~, MOHLETHALER 8) that liquid media are best for making electron microscopic preparations of " Special Fellow of the,National Institutes of Health. Permanent Address: Pflanzenphysiologisches I n s t i t u t dcr Eidg. Techn. Hochschule, Zfirich, Switzerland.

Re/erences p. 535.

528

K. ML:HLETHALER

VOL. 3 (1949)

bacterial cellulose. We have accordingly grown Bacterium x y l i n u m for this purpose in WATSR.~tAN'S sucrosebeer solution. This is prepared by addi~ag 4 ° grams of sucrose to a liter of beer. A portion of such a medium was inoculated by dropping into it a small piece of membrane from an old culture. After a short time slimy fibres extended from this inoculum towards the surface. When they reached it they spread as a fine waterclear skin which quickly thickened and after a few days was too tough to be torn or cut without altering its natural texture. To make preparations for electron microscopy the thin, early film was lifted from the culture medium with a glass microscope slide, washed several times in water and dried, still on the slide. Pseudoreplicas like those used for studying the growth of bacteriophage on an agar surface (EDWARDS A.','D WYCKO~'V9) have been made of these. This has been done by pouring collodion diluted with amyl acetate over the surface and draining until dry. The resulting fihn was floated off onto a water surface, cut into pieces, mounted on grids in the usual fashion and shadowed with either chromium or palladium. It should be emphasized that though these preparations are made as if they were replicas they do not replicate the cellulose film which remains embedded in the collodion and is itself observed in the microscope. In some instances a different kind of preparation was made for microscopy by removing a bit of the bacterial colony with a platinum needle, suspending it in water, drying a droplet of this suspension on a collodion covered screen and shadowing. RESULTS

The young rapidly growing bacteria have characteristic shapes as seen in the electron microscope. They are 2-4 / * long and are flattened on one end (Fig. I). Midportions of the bacteria have been coated with an amorphous homogeneous substance which increased with bacterial age and eventually embedded the organisms (Fig. 2). Cellulose fibres have begun to form after this has happened. They appeared first as short-fibred thickenings distributed here and there in the amorphous material (Fig. 3)- Long fibres have rapidly grown from these thickenings, seemingly at the expense of the capsular substance. As Fig. 3 indicates this development of cellulose has not taken place within a bacterium or from its surface, but rather at some distance from the organism. This suggests that cellulose results from a sort of enzymatically induced polymerization of the embedding slime that can accordingly be thought of as a kind of cellulose-precursor. As can be seen from Fig. 4 the cellulose fibres have had from the very first a diameter of ca 25 ° A. The individual strands were separated from one another by the amorphous substance which decreased as the cellulose developed and had nearly vanished from older colonies (Figs 5 and 6). The separate fibrils have intertwined to form tangled masses, and in this way built tough membranes which could be torn apart only with difficulty. Where the slimy substance has been lacking, several individual fibres have often been seen lying side by side (Fig. 7) to give the bands already described by FRANZ AND SCHIEBOLD.These bands have commonly been twisted about one another into rope-like structures, but it could not be determined with certainty whether this expressed the original arrangement or was caused by the drying. The piece of membrane shown in Fig. 8 shows this phenomenon especially well. Transverse band structures resembling those observed by FRANZ AND SCHIEBOLDare also discernible here, but it is probable that they are the consequence of mechanical damage. Relerences p. 535.

VOI.. 3

(i949)

STRUCTURE OF BACTERIAL CFLLULOSE

529

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STRUCTURE OF BACTERIAL CELLULOSE

This work was carried out in the laboratory I wish to thank

for many

535

o f D r R A L P H W . G. W Y C K O F F , w h o m

helpful discussions.

SUMMARY Electron microscopic o b s e r v a t i o n s s h o w t h a t t h e cellulose b u i l t by Acetobacterium .~?,lim.n occurs in t h e form of fibres h a v i n g a b o u t t h e s a m e d i a m e t e r (ca 250 :~,) as t h a t of t h e fibrils of cc llulo%e Iron: t h e cell walls of n u m e r o u s p l a n t s . In Bacterium xylinum t h e s e s t r a n d s are s y n t h e s i z e d out,'ide t h e cells a n d c a n therefore be e x a m i n e d especm]ly well u n d e r t h e electron microscope. T h e b a c t e r i a first secrete a s t r u c t u r a l l y h o m o g e n e o u s s l i m y s u b s t a n c e within which, a f t e r a s h o r t t i m e , t h e cellulose fibres can be seen forming. T h i s secreted s u b s t a n c e a p p e a r s to be a precursor of cellulose whose p o l y m e r i z a t i o n proceeds o u t s i d e t h e o r g a n i s m s p r e s u m a b l y with t h e help of a n e n z y m e . In older c u l t v r e s t h e cellulose s t r a n d s h a v e b e c o m e so i n t e r t w i n e d .is to build a rigid framework.

R~st~t~ I.es o b s e r v a t i o n s faites au microscope 61ectronique m o n t r e n t que la cellulose s y n t h 6 t i s 6 e par

Bacterium xylinum se pr6sente sous forme de fibres a y a n t b. peu pr6s le m~3me d i a m 6 t r e (environ 25o ]~) q u e cehfi d e s fibrilles de cellulose c o n s t i t u a n t lcs parois cellulaires de n o m b r e u x v6g6taux. Chez Bacterium xylinum, ces fibres s o n t s y n t h 6 t i s 6 e s h l'ext6rieur des cellules et p e u v e n t par cons6q u e n t 6tre e x a m i n 6 c s p a r t i c u l i ~ r e m e n t I a c i l e m e n t au microscope 61ectronique. I.es bact6ries s e c r 6 t e n t d ' a b o r d u n e s u b s t a n c e m u c i l a g i n e u s e homog6ne, au sein tie laquelle on p e u t voir se former apr~s u n t e m p s court, les fibres de cellulose. La s u b s t a n c e ainsi secr6t6e est u n p r 6 c u r s e u r de la cellulose d o n t la p o l y m 6 r i s a t i o n se faitb, l ' e x t 6 r i e u r d e s o r g a n i s m e s , et p r o b a b l e m e n t sous l'action d ' u n e e n z y m e . Darts les c u l t u r e s 5.g6es, les fibres de cellulose s o n t t e l l e m e n t entrelac6es q u ' e l l e s c o n s t i t u e n t un e n t r e l a c rigide.

ZUSAMMENFASSUNG B e o b a c h t u n g e n m i t Hilfe des E l e k t r o n e n m i k r o s k o p s lassen e r k e n n e n , d a s s die dtLrch Bacterium xylinum s y n t h e t i s i e r t e Cellulose a u s F a s e r n b e s t e h t , die ungefiihr d e n gleichen D u r c h m e s s e r h a b e n (ca. 250 .~.) wie die F a s e r n der ZellwS.nde vieler Pflanzen. Bei Bacterium xylinum w e r d e n diese 1;asern a u s s e r h a l b der Zelle s y n t h e t i s i e r t u n d k 6 n n e n d e s h a l b besond~rs leicht im E l e k t r o n e n m i k r o s k o p beobachtet werden. Die B a k t e r i e n s c h e i d e n z u e r s t eine schleimige h o m o g e n e S u b s t a n z a u s u n d n a c h kurzer Zeit k a n n m a n s c h e n , wie sich i n n e r h a l b dieser S u b s t a n z die Cellulosefasern bilden. ])iese S u b s t a n z ist ein Vorl/iufer der Cellulose d e r e n P o l y m e r i s a t i o n sich a u s s c r h a l b der O r g a n i s m e n , w a h r s c h e i n l i c h u n t e r d e m Einfluss eines E n z y m s vollzieht. In £1teren K u l t u r e n sind die Cellulosefasern so verzweigt, d a s s sie ein steifes N e t z w e r k bilden.

REFERENCFS K..~,[i,~HLETHALER, Biochim.. Biophys. Acta, 3 (I949) I5. A. J. BROWN, J. Chem. Soc. London, 49 (1886) 172 a n d 432 . (-. v. WlSSELINGH, Chem. Zentr., 21 (1917) 522. H. HIBBERT, Science, 71 (193 o) 419. H. MARK Am) G. v. SuSICH. Z. physik. Chem., (B) 4 (I929) 431. 6 E. FRANZ AND E. SCHIEBOLD, ] . makromol. Chem., i (1943) 4-16. 7 A. FREY-WYsSLING AND 1(. ~']~L'III.ETHALER,.]. Polymer Sci., I (1946) 172-i74. s K. MOHLETHALER, Die makromol. Chemie, (B)., (1948) 143-171. 9 0 . F. EDWARDS AND I¢. \V. G. \VvcKOFF, l?roc. Soc. Exptl Biol. Med., 64 (1947) 16. 1 2 a 4

Received

February

5th, 1949