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Physical and chemical studies of the exopolysaccharide isolated from Pseudomonas fragi ATCC 4973 Lee Wing, Phillip 1984

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P H Y S I C A L A N D C H E M I C A L S T U D I E S OF T H E E X O P O L Y S A C C H A R I D E I SOLATED FROM PSEUDOMONAS FRAGI A T C C 4973 B y P H I L L I P J L E E WING B . S c , The Un i v e r s i t y of Man i toba , 1976 B . S . A . , The Un i v e r s i t y of Man i toba, 1978 M . S c , The Un i v e r s i t y of Man i toba, 1980 A THES IS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E REQU IREMENTS OF T H E DEGREE OF DOCTOR OF PH I LOSOPHY in THE F A C U L T Y OF G R A D U A T E S T U D I E S (Depar tment of Food Sc ience) We accept th i s thes i s as conforming to the r equ i r ed s tanda rd T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A May, 1984 © Ph i l l i p Lee Wing, 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f - ^ T S - E - A ^ *5> o-r-g. The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date r<^ *A*^- \=>^>\ A B S T R A C T Pseudomonas fragi A T C C 4973, inoculated onto meat surfaces and incubated at 21°C, was examined by electron microscopy. Both scanning and transmission electron microscopy revealed an extracellular material (glycocalyx) which appeared to mediate cell-to-cell as well as cell-to-muscle attachment to the meat samples. Transmission electron micrographs of cells grown on solid medium, harvested at the early logarithmic growth phase, revealed bleb-like evaginations or protrusions on the cell surface. Stationary phase cells, however, exhibited no bleb-like evaginations. Fine extracellular material randomly distributed on the cell surface was observed. Transmission electron micrographs of bacterial cells grown in liquid medium, harvested at the early logarithmic growth phase, revealed no blebs or extracellular material. In contrast, stationary phase cells showed only few attached blebs as well as globules adjacent to the cells. The type of substrate (solid vs. liquid) influenced the expression of blebs on the surface of P. fragi cells. An association between the glycocalyx fibres and bleb-like evaginations was also noted. The exocytosic formation of blebs and globules into the immediate environment of the cell appeared to precede the formation of glycocalyx by P. fragi cells. Chemical studies on the isolated glycocalyx of P_. fragi indicated a hexosaminoglycan structure. T r i f luoroacetic acid and hydrogen fluoride were used in the hydrolysis of the polysaccharide. Quantitative amounts of the individual monosaccharides were obtained from the hydrolysed polysaccharide using preparative paper chromatography. Gas liquid chromatography and mass spectrometry were used in the verification of an N-acetyl amino sugar - iii -component. Monomeric units and substituents in the polysaccharide chain 1 13 were determined by H and C nuclear magnetic resonance spectroscopy. By methylation analysis, the polysaccharide of P. fragi was shown to consist of a linear repeating trisaccharide unit. The proposed partial hexosaminogly-can structure is: ^ >-4) D-Glucose (1—-3) amino sugar ( 1 — • ?) deoxy sugar (1 — • 2 NAc - i v -T A B L E O F C O N T E N T S P A G E A B S T R A C T ii T A B L E O F C O N T E N T S iv L I S T O F T A B L E S v i i i L I S T O F F I G U R E S ix A C K N O W L E D G E M E N T S x i i i G E N E R A L I N T R O D U C T I O N 1 P A R T 1 G R O W T H O F P S E U D O M O N A S F R A G I A T C C 4973 A N D G L Y C O C A L Y X F O R M A T I O N O N M E A T S U R F A C E S 2 I N T R O D U C T I O N 3 L I T E R A T U R E R E V I E W 4 E X P E R I M E N T A L 6 A . P r e p a r a t i o n o f A t t a c h m e n t M e d i a 6 B. P r e p a r a t i o n o f M u s c l e S a m p l e s 6 C . p H D e t e r m i n a t i o n 7 D. B a c t e r i a l C o u n t s 7 E. P r e p a r a t i o n o f M u s c l e f o r S E M 7 F. P r e p a r a t i o n o f M u s c l e f o r T E M 8 G . M i c r o t o m y 8 R E S U L T S 9 A . G r o w t h o f P. f r a g i 9 - v -B. pH 9 C. Electron Microscopy 9 DISCUSSION 27 CONCLUSION 33 PART 2 GROWTH PHASE AND CAPSULAR FINE STRUCTURE OF PSEUDOMONAS FRAGI A T C C 4973 34 INTRODUCTION 35 LITERATURE REVIEW 36 EXPERIMENTAL 40 A. Culture Conditions 40 B. Incubation on Solid Medium 40 C. Incubation in Liquid Medium 40 D. Kellenberger-Ryter (RK) Fixation 40 E. Sample Preparation 41 F. Microtomy and Electron Microscopy 42 RESULTS 43 A. Growth of P. fragi 43 B. Electron Microscopy 43 1. Cells grown on solid medium 43 2. Cells grown in liquid medium 43 DISCUSSION 54 CONCLUSION 60 PART 3 CHEMICAL COMPOSITION AND STRUCTURAL ANALYSIS OF THE EXTRACELLULAR POLY-SACCHARIDE ISOLATED FROM PSEUDOMONAS FRAGI ATCC 4973 61 INTRODUCTION 62 - vi L ITERATURE REVIEW 66 A. Monosaccharide Types 67 B. Total Hydrolysis 71 C. Sugar Determination 72 D. Methylation Analysis 74 E. Mass Spectrometry (M.S.) 76 F. Nuclear Magnetic Resonance Spectroscopy (N.M.R.) 78 1. Proton Magnetic Resonance Spectroscopy ( H N.M.R.) 79 2. 1 3 C N.M.R. Spectroscopy 80 EXPERIMENTAL 81 A. Isolation of Polysaccharide 81 B. Molecular Weight Determination 82 C. Paper Chromatography 82 D. Gas Liquid Chromatography 83 E. Gas Liquid Chromatography - Mass Spectrometry 84 F. Nuclear Magnetic Resonance Spectroscopy 84 1. Proton ( 1 H) N.M.R. Spectroscopy 84 13 2. C N.M.R. Spectroscopy 85 G. Sugar Analysis 85 H. N-Deacetylation of the Polysaccharide 85 I. Deamination of the Polysaccharide 86 J . HF Solvolysis of the Polysaccharide 86 K. Preparation of Bio-Gel P-2 Column 86 L. Reduction of Uronic Acid Carboxyl Groups in Polysaccharides 87 vii -M. Enzymatic Determination of Glucose 87 N. Methylation of Polysaccharide 87 O. Acetolysis, Hydrolysis and Derivatization of Sugars into Partially Methylated Hexitol Acetates and 2-deoxy-2-N-methylamido-Hexitol Acetates 88 RESULTS 89 DISCUSSION 110 SUMMARY 120 CONCLUSION 123 REFERENCES 124 APPENDICES 134 - viii -LIST OF TABLES TABLE PAGE 1 Rare sugar components of Pseudomonas polysaccharides 68 2 Functional group modification of sugar residues 69 3 N.M.R. data for P. fragi polysaccharide 90 4 R | Values of components of hydrolysed polysaccharide on Whatman No. 1 paper 91 5 G. l .c . analysis of alditol acetates of hydrolysed polysaccharide (2 M TFA) isolated by prep, paper chromatography 100 1 6 H N.M.R. data for hydrolysed P. fragi polysaccharide (2 M TFA) 101 7 1 H N.M.R. data for hydrolysed P. fragi polysaccharide (HF) 102 8 N.M.R. data for P. fragi polysaccharide (de-N-acetylated; deaminated) 106 1 13 9 Representative H and C chemical shifts for nuclei of polysaccharides 113 ix -LIST OF FIGURES FIGURES PAGE 1. Growth of P. fragi cells on, and pH of inoculated muscle at various storage times 10 2. (A) Scanning electron micrograph of day 1 muscle sample, inoculated with P. fragi, showing the initial formation of fibre-like glycocalyx material (G) . (B) This represents a magnifica-tion of the area enclosed in the box in micro-graph (A) 12 3. (A) Scanning electron micrograph of the external surface of £. fragi encased in a mass of pebble-like extracellular material. (B) This represents a magnification of the area enclosed in the box in micrograph (A) 14 4. Scanning electron micrograph of the external surface of P. fragi (day 5) encased in a mass of pebble-like extracellular material 16 5. Scanning electron micrograph of muscle sample (day 5) inoculated with P_. fragi. There is an intensification of the pebbling effect on the bacterium surface, as well as a coiling of glycocalyx fibres 18 6. Scanning electron micrograph of muscle sample (day 5) inoculated with P. fragi. There is an intensification of the pebbling effect on the bacterium surface, as well as a coiling of glycocalyx fibres 20 7. Transmission electron micrograph of extra-cellular material associated with P_. fragi cells. (A) and (B) are examples of the morphological forms of the extracellular material: (A) , adherent to the bacterium surface and ( B ) , amorphous in nature 23 8. Scanning electron micrograph of bleb-like protru-sions (P) in association with the glycocalyx fibres (G) 25 9. Schematic illustration of the relationship between the various forms of extracellular material as seen by means of SEM and TEM. (A) and (B) are examples of the morphological forms of the extracellular material: (A) , adherent to the bacterium surface and (B) , amorphous in nature 30 X 10. Growth characteristics of P. fragi in liquid (TSB) and on solid (TSA) media, incubated at 21 °C 44 11. Thin section of P. fragi cells grown on solid medium harvested at the early logarithmic growth phase. Blebs which contain dense granular material are present on the entire cell surface 46 12. Transmission electron micrograph of P. fragi cells grown on solid medium harvested at the stationary phase. Fine extracellular materials are randomly distributed on the cell surface.. 48 13. Transmission electron micrograph of P. fragi cells grown in liquid medium harvested at the early logarithmic phase 50 14. Thin section of stationary phase cells of P. fragi grown in liquid medium. Few attached blebs as well as globules adjacent to the cells are observed 52 15. Transmission electron micrograph of P. fragi cells grown on solid medium harvested at the early logarithmic phase of growth. Blebs as well as globules adjacent to the cells are observed 55 16. Transmission electron micrograph of P. fragi cells grown on solid medium, harvested at the early logarithmic phase of growth. Blebs as well as globules adjacent to the cells are observed. A, B, C and D depict the respective phases of exocytosis 57 17. Diagramatic representation of the bacterial cell envelope 64 18. Methylation analysis of a polysaccharide 75 19. Mass spectrum of glucitol hexaacetate component of hydrolysed polysaccharide 92 20. Mass spectrum of amino sugar (alditol acetate) component of hydrolysed polysaccharide 93 21. Mass spectrum of deoxy sugar (alditol acetate) component of hydrolysed polysaccharide 94 22. Paper chromatogram of (a) hydrolysed polysaccharide (2 M TFA) and (b) reference standards 96 23. Mass spectrum of B-neutral (alditol acetate) component of hydrolyzed polysaccharide 98 - xi -24. Mass s p e c t r u m of B-amino (alditol acetate) component of h y d r o l y s e d polysacchar ide 99 25. Mass spectrum of H F - B . Hexitol hexaacetate component of h y d r o l y s e d polysacchar ide 104 26. Mass spectrum of H F - B . 2-Acetamido-2-deoxy-penta-O-acety l -D-hex i to l component of hydro lysed polysacchar ide 105 27. Mass spectrum of 2 , 3 , 6 - t r i - O - m e t h y l - D -glucitol 107 28. Mass spectrum of 4 , 6 - d i - 0 - m e t h y l - 2 - d e o x y - 2 -N-methylacetamido hexitol 108 29. Mass spectrum of per-methylated deoxy component (alditol acetate) 109 30. Fragmentation pattern observed in the mass spectrum of 2 ,3 ,6 - t r i -O-methy l -D-g luc i to l 118 31. Fragmentation pattern observed in the mass spectrum of 4 , 6 - d i - Q - m e t h y l - 2 - d e o x y -2-N-methyl acetamido hexitol 119 - xii -LIST OF APPENDICES APPENDIX " PAGE I Infrared Spectroscopy Data , 134 II N.M.R. Spectroscopy Data 136 III Mass Spectrometry Data 150 xiii -ACKNOWLEDGEMENTS The author wishes to thank Dr. B.J. Skura, Supervisor, for the many hours of rewarding discussions and for his encouraging help and unflagging interest throughout this work. Many thanks are due to Dr. G.G.S. Dutton for his interest and helpful support and also to the members of the supervisory committee. The encouragement and support of my colleagues, especially "the group at 446", is greatfully appreciated. Finally, I wish to acknowledge Lou Lee Wing, to whom I owe all. -1-GENERAL INTRODUCTION: The ecology of bacterial spoilage of meat at both chill and room temperatures has been extensively studied (Gill and Newton, 1977, 1978, 1980; Gill, 1976). At chill temperatures, the spoilage flora of meat is compos-ed of psychrotrophs originating largely from the hides of slaughtered animals. Under humid conditions, aerobic floras are usually dominated by pseudomonads while anaerobic floras are dominated by Lactobacillus. Under aerobic condi-tions, psychrotrophic pseudomonads are the predominant spoilage flora at 20°C while at 30°C, they are displaced by species of Acinetobacter and Enterobacteriacae which included both mesophilic and psychrotrophic strains. To understand how spoilage floras develop, it is necessary to know (1) which organisms can be present; (2) the nature of the environment in which they are growing; (3) the effect on microbial growth of alterations in the environment; and (4) the interactions which occur between competing species (Gill and Newton, 1978). Since, under moist aerobic conditions, pseudomonads form a major component of the final spoilage flora, Pseudomonas  fragi ATCC 4973 was chosen as the microoganism for the study. The understanding of the mechanism of meat spoilage by microorgan-isms requires a knowledge of how microorganisms initially attach to meat surfaces. Fletcher and Floodgate (1973) suggested that a glycocalyx (extra-cellular polymeric material) may be involved in this process. An examination of the extracellular material using transmission and scanning electron micros-copy will, therefore, provide information on its morphology and ultrastructure. Chemical studies of the extracellular material, however, will provide informa-tion on its structural components and conformation. - 2 -P A R T I G R O W T H O F P S E U D O M O N A S F R A G I A T C C 4973 A N D G L Y C O C A L Y X F O R M A T I O N O N M E A T S U R F A C E S - 3 -INTRODUCTION: Growth of bacteria on meat surfaces results in the production of "off" odours and flavours. Concurrently, the formation of surface slime is usually involved in the spoilage process (Gill, 1976). Slime material aids in the attachment of microbial cells to surfaces (Firstenberg-Eden, 1981). It has been well documented in the literature that the attachment process apparently involves at least two main steps (Butler et a[., 1980; Firstenberg-Eden, 1981). The first step involves the initial stage of revers-ible sorption. This sorption is thought to be associated with hydrophobic as well as van der Waals forces (Fletcher and Loeb, 1979). The second step, or stage of "permanent adhesion", involves formation of a glycocalyx (extra-cellular polymeric material) between the bacteria and substrate (Fletcher and Floodgate, 1973). Extensive research by Costerton et aL (1978) demonstrated that microorganisms adhered to substrates by means of a mass of tangled fibres of polysaccharide, with the formation of "felt-like glycocalyx". Information on the detailed mechanism of attachment and adhesion to surfaces by microorganisms is particularly lacking. The object of this study was, therefore, to investigate the growth of Pseudomonas fragi ATCC 4973 and formation of glycocalyx on meat surfaces. - 4 -LITERATURE REVIEW: More information is available on the growth of bacteria on meat surfaces at chill temperatures than at ambient temperatures (Gill and Newton, 1980). While Notermans and Kampelmacher (1974) showed that the optimum temperature for attachment of Pseudomonas strains was ca. 21 °C, other researchers indicated that psychrotrophs compete successfully with mesophilic species at normal ambient temperatures, except with meat stored anaerobically at temperatures in excess of 20°C. Storage at 21 °C should then favour greater association of pseudomonads with the meat surface and hence may enhance glycocalyx formation during the adhesion stage of the attachment process. In a study of the ecology of bacterial spoilage of fresh meat at chill temperatures, Gill and Newton (1978) reported that the growth of bacteria on a meat surface is facilitated by the utilization of low molecular weight soluble components of the meat. Various researchers showed that no significant changes in the meat texture or quality were observed until the g bacterial numbers exceeded 10 colony forming units (c.f.u.)/g, when ammonia was produced, and decreases in carbohydrate, free amino acids and nucleo-tides were observed (Ockerman et al., 1969; Ingram and Dainty, 1971). Ingram and Dainty (1971) further reported on the changes in bacterial numbers as related to biochemical changes. They observed that 7 2 when bacterial numbers exceeded 10 c.f.u./cm , the meat had a distinct off-odour, and at the same time, began to show the onset of slime formation, 8 2 i.e. when the numbers reached ca. 10 c.f.u./cm . It was also difficult to draw any general conclusions about the changes in concentration of soluble constituents to be expected in spoiling meat, since the problem is usually - 5 -complicated by the possibility of changes in the concentration of many of these compounds due to autolysis. Various studies have been reported on the relationship between pH and the number of bacterial colony forming units on meat surfaces (Turner, 1960; Rogers and McCleskey, 1961). It was further reported that a high pH value was obtained in meat undergoing aerobic spoilage since the pH almost invariably increased to 6.5 from the usual postrigor value of ca.5.5. Although the study of microbial meat spoilage may be accomplished by the enumeration of bacterial numbers as well as using subjective para-meters for evaluation, the use of electron microscopy in this area of study has been mainly explored in the last few decades. Both scanning and trans-mission electron microscopy have been used to examine the attachment of bacteria to meat surfaces (Butler et aL, 1980; Firstenberg-Eden, 1981); to teats of cows (Firstenberg-Eden et aL , 1979; Notermans et aL, 1979); to broiler carcass skin (Thomas and McMeekin, 1980); to epithelial cell surfaces of cattle (McCowan et aL, 1978); as well as the study of cell morphology and ultrastructure (Jones et aL, 1969; Marshall et a[., 1971; Fletcher and Flood-gate, 1973; Bayer and Thurow, 1977). Electron microscopy provides an understanding of the ultrastruc-tural morphology as well as function of procaryotic cells from the observation of cell shapes and surface-associated components. Although all commonly used dehydration methods used in sample preparation have their drawbacks and may result in artifactual shrinkage (Brunk et aL, 1981), carefully chosen methods (depending on the type of material being examined) have been developed to minimize distortion of cellular morphology (Whittaker and Drucker, 1970; Domagala et aL, 1979; Garland et aL, 1979; Rittenburg et aL, 1979; Watson et aL , 1980). - 6 -E X P E R I M E N T A L : B o v i n e l o n g i s s i m u s d o r s i m u s c l e s , 24 h p o s t m o r t e m , w e r e o b t a i n e d f r o m a local a b a t t o i r a n d t r a n s p o r t e d on ice to t h e l a b o r a t o r y . T h i n s l i c e s (3 mm) of m u s c l e w e r e p r e p a r e d u s i n g a H o b a r t d e l i c a t e s s e n s l i c e r ( H o b a r t C a n a d a I n c . , D o n M i l l s , O n t . ) . A s e p t i c c o n d i t i o n s we re m a i n t a i n e d in o r d e r to m in im ize m i c r o b i a l c o n t a m i n a t i o n p r i o r to i r r a d i a t i o n . A l l m u s c l e s l i c e s w e r e s t e r i l i z e d w i th 10 k G y o f g a m m a - r a d i a t i o n u s i n g a Gammace l l 220 ( A t o m i c 60 E n e r g y o f C a n a d a L t d . , K a n a t a , O n t . ) c o n t a i n i n g C o ( S a g e , 1974 ) . P s e u d o m o n a s f r a g i A T C C 4973 , o b t a i n e d f r o m t h e A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( A T C C , R o c k v i l l e , M D ) w e r e m a i n t a i n e d on t r y p t i c a s e s o y a g a r s l a n t s ( B B L , C o c k e y s v i l l e , M D ) at 4 ° C a n d was s u b c u l t u r e d at 8-10 week i n t e r v a l s . A l l a n a l y s e s w e r e p e r f o r m e d at t ime i n t e r v a l s o f 0 , 1, 2, 3 a n d 5 d a y s t o r a g e on b o t h i n o c u l a t e d a n d c o n t r o l s a m p l e s . A . P r e p a r a t i o n o f A t t a c h m e n t M e d i a : T h e a t t a c h m e n t med ium c o n t a i n i n g P. f r a g i ce l l s was r e c o n s t i t u t e d a c c o r d i n g to t h e m e t h o d o f Y a d a a n d S k u r a ( 1 9 8 1 ) . B. P r e p a r a t i o n o f M u s c l e S a m p l e s : T h e gamma s t e r i l i z e d s amp l e s we re i m m e r s e d f o r 10 min in 4 L o f a t t a c h m e n t med ium c o n t a i n i n g P_. f r a g i . T h e m e t h o d d e s c r i b e d b y Y a d a a n d S k u r a (1981) was u s e d f o r f u r t h e r s amp le t r e a t m e n t w h i c h i n v o l v e d t h e i n c u b a t i o n o f t h e s amp les in a c o n t r o l l e d e n v i r o n m e n t . C o n t r o l s amp l e s w e r e t r e a t e d , in a s im i l a r m a n n e r , w i th s t e r i l e a t t a c h m e n t m e d i u m . - 7 -C . p H D e t e r m i n a t i o n : A f t e r e a c h i n c u b a t i o n t i m e , a 5 g r e p r e s e n t a t i v e m u s c l e s a m p l e was h o m o g e n i z e d w i t h 5 m L d i s t i l l e d - d e i o n i z e d w a t e r in a n O m n i M i x e r ( I v a n S o r v a l l I n c . , N o r w a l k , C T ) . T h e p H o f t h e r e s u l t a n t s l u r r y was t h e n d e t e r m i n e d w i t h a F i s h e r A c c u m e t p H / i o n m e t e r ( M o d e l 2 3 0 ) . D . B a c t e r i a l C o u n t s : A r e p r e s e n t a t i v e s a m p l e ( 4 g ) w a s b l e n d e d w i t h 36 m L s t e r i l e 0.1% ( w / v ) p e p t o n e f o r 2 min u s i n g a C o l w o r t h s t o m a c h e r l a b - b l e n d e r 400 ( A . J . S e w a r d a n d C o . , L o n d o n , E n g l a n d ) . B a c t e r i a l e n u m e r a t i o n o f b o t h i n o c u l a t e d a n d c o n t r o l s a m p l e s was c a r r i e d o u t a f t e r i n c u b a t i o n at 21 ° C f o r 48 h . E . P r e p a r a t i o n o f M u s c l e f o r S E M : M u s c l e t i s s u e s w e r e c u t i n t o 2 x 2 cm c u b e s p r i o r to f i x a t i o n w i t h 2.5% g l u t a r a l d e h y d e ( J . B . EM S e r v i c e s I n c . , M o n t r e a l , Q u e . ) in 0 . 0 5 M p h o s -p h a t e b u f f e r ( p H 7 . 0 ; 24 h ; 4 ° C ) . S a m p l e s w e r e r i n s e d t w i c e in 0 . 0 5 M p h o s p h a t e b u f f e r ( p H 7 . 0 ) a t 2 1 ° C b e f o r e f u r t h e r f i x a t i o n w i t h 1% o s m i u m t e t r o x i d e ( J . B . E M S e r v i c e s I n c . ) in 0 . 0 5 M p h o s p h a t e b u f f e r ( p H 7 . 0 ) f o r 1 h . T h i s s t e p w a s f o l l o w e d b y d e h y d r a t i o n t h r o u g h a g r a d e d s e r i e s o f a q u e o u s e t h a n o l s o l u t i o n s : 5 0 , 7 0 , 80% f o r 5 min e a c h , 90% f o r t w o 10 min p e r i o d s a n d 100% f o r t h r e e 20 min p e r i o d s . A l l e t h a n o l d i l u t i o n s w e r e m a d e w i t h d i s t i l l e d -d e i o n i z e d w a t e r . S a m p l e s w e r e c r i t i c a l p o i n t d r i e d in a P a r r - b o m b ( P a r r I n s t r u m e n t C o . , M o l i n e , I L ) u s i n g CO2. S a m p l e s m o u n t e d o n a l u m i n u m s t u b s w i t h s i l v e r p a s t e ( J . B . E M S e r v i c e s I n c . ) w e r e c o a t e d w i t h g o l d b y v a c u u m e v a p o r a t i o n . S a m p l e s w e r e v i e w e d w i t h t h e C a m b r i d g e S t e r e o s c a n 2 5 0 , o p e r a t e d at 40 k V . - 8 -F. Preparation of Muscle for TEM: Samples for transmission electron microscopy were prepared accord-ing to the method of McCowan et al_. (1978), with slight modifications. Tissues ( 5 x 5 mm) were fixed at 4 °C for 24 h with 2.5% glutaraldehyde solution in 0.05 M phosphate buffer (pH 7.0) prior to post-fixation (2 h, 21°C) in 0.015% dye [ruthenium red (Aldrich Chemical Co . , Milwaukee, Wl) or alcian blue (J.B. EM Services Inc.)] buffered with a 0.05 M phosphate solution. Samples, after 2 h in 0.05 M phosphate buffer containing 0.05% dye (DP buffer) at 21 ° C , were washed in five changes of the same solution for 1 h each, with one overnight wash included. Samples were post-fixed for 2 h in 2% osmium tetroxide in DP buffer, followed by five 1 h washes in DP buffer. Dehydration was achieved by immersion of samples in 15, 30, 50, 70, 90 and 100% ethanol. The lower concentrations of ethanol were prepared with distilled deionized water. Samples, following dehydration, were washed in two changes of propylene oxide (J.B. EM Services Inc.) for 15 min each, infiltrated with a 1:1 mixture of propylene oxide and Epon 812 (J.B. EM Sevices Inc.) for 16 h, then embedded in 100% Epon 812. G. Microtomy: Tissues were sectioned on a "Porter-Blum" MT-2 ultramicrotome (Ivan Sorvall Inc., Norwalk, C T ) , mounted on 3 mm copper grids, then stained with uranyl acetate and lead citrate. Electron microscopy of all specimens was performed with a Zeiss EM-10 transmission electron microscope at an accelerating voltage of 80 kV. - 9 -RESULTS A. Growth of P. fragi: 5 6 ? At day 0 there were between 10 and 10 P. fragi c.f.u./cm on the muscle surface. No bacteria were detected in control samples at day 0 or day 5. P. fragi grew rapidly on the muscle surface during the initial 3 days of the storage trial followed by a modest rate of growth during the remainder of the study (Fig.1). B. pH: Pseudomonas fragi caused a marked increase in pH of inoculated muscle from pH 6 (day 0) to pH 9 (day 5). No pH changes were observed in the control muscles (Fig. 1). C. Electron Microscopy: Scanning electron micrographs of the muscle samples as early as day 1 of the incubation period revealed the initial formation of a fibre-like extracellular material (glycocalyx) extending from the surface of the microor-ganism (Fig. 2). At higher magnifications, a pebble-like extracellular mater-ial on the outer surface of the microorganisms could be seen (Fig.3). The more detailed sequential stages of fibre-like extracellular material formation could not be differentiated during the examination of day 1, 2 or 3 samples. Muscle samples examined after a 5-day incubation period showed an intensifi-cation of the pebbling effect on the bacterial surface, in addition to a coiling of the glycocalyx fibre to form a matted mass of extracellular material (Figs. 4, 5 and 6). - 10 -F i g . 1 G r o w t h o f P. f r a g i c e l l s o n , a n d p H o f i n o c u l a t e d m u s c l e at v a r i o u s s t o r a g e t i m e s . ( • ) c o n t r o l - s t e r i l e s amp le ( p H ) ( # ) s amp le i n o c u l a t e d w i th P. f r a g i ( p H ) ( • ) s amp le i n o c u l a t e d w i th P. f r a g i ( g r o w t h ) A l l p o i n t s r e p r e s e n t t h e a v e r a g e of t h r e e t r i a l s . - II -- 12 -Fig. 2 (A) Scanning electron micrograph of day 1 muscle sample, inoculated with P. f r a g i , showing the initial formation of fibre- l i k e glycocalyx material ( G ) . Bar = 10 urn. (B) This represents a magnification of the area enclosed in the box in micrograph ( A ) . Bar = 5 um. - 14 -Fig. 3 (A) Scanning electron micrograph of the external surface of P. fragi encased in a mass of pebble-like extracellular material. Bar = 1 urn. (B) This represents a magnification of the area enclosed in the box in micrograph (A) . Bar = 1 jjm. - 16 -Scanning electron micrograph of the external surface of P. frag (day 5) encased in a mass of pebble-like extracellular material Bar = 1 pm. - 18 -F i g . 5. S c a n n i n g e l e c t r o n m i c r o g r a p h o f m u s c l e s a m p l e ( d a y 5) i n o c u l a t e d w i t h P. f r a g i . T h e r e is an i n t e n s i f i c a t i o n o f t h e p e b b l i n g e f f e c t o n t h e b a c t e r i u m s u r f a c e , as wel l as a c o i l i n g o f g l y c o c a l y x f i b r e s . B a r = 1 urn. - 20 -Fig. 6 Scanning electron micrograph of muscle sample (day 5) inoculated with P. fragi. There is an intensification of the pebbling effect on the bacterium surface, as well as a coiling of glycocalyx fibres. Bar = 1 /jm. - 22 -No marked morphological differences were observed in samples stained with ruthenium red or alcian blue when observed with the transmis-sion electron microscope. Ruthenium red, however, gave a slightly better contrast of structures. Since both ruthenium red and alcian blue are specific for acidic polysaccharides, electron dense areas were not limited only to the extracellular material but also to material present in the meat sample capable of forming complexes with the dye. TEM, combined with ruthenium red and alcian blue staining, demonstrated various forms of extracellular material associated with attachment. Two examples of the morphological forms of extracellular material are shown in Fig. 7. A similar pattern of glycocalyx localization and morphology was reported in a study of acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces (Fletcher and Floodgate, 1973). Scanning as well as transmission electron micrographs revealed bleb-like evaginations or protrusions on the surface of P. fragi after a 5-day incubation period (Fig. 8). A close association of these blebs to the site of attachment of extracellular fibres to the bacterial surface was also observed (Fig. 8). - 23 -Fig. 7 Transmission electron micrograph of extracellular material associated with P. fragi cells. (A) and (B) are examples of the morphological forms of the extracellular material: (A) , adherent to the bacterium surface and (B), amorphous in nature. Bar = 1 urn. - 25 -Fig. 8 Scanning electron micrograph of bleb-like protrusions (P) in asso-ciation with the glycocalyx fibres (G). Bar = 0.5 ^im. - 27 -DISCUSSION: Notermans and Kampelmacher (1974), showed that the optimum temperature for attachment of Pseudomonas strains was ca. 21 ° C . Ingram and Dainty (1971) reported that at higher temperatures respiration of meat tissue was much greater so that there was likely to be less available oxygen in tissue near the surface on which bacteria grew. Very thin samples were used in this study to maintain an aerobic environment. The growth of P. fragi on meat surfaces incubated at 21 ° C followed the classical growth pattern with a lag period of approximately 3 days. Under aerobic conditions, psychrotrophic pseudomonads accounted for 60% of the spoilage flora at 20°C (Gill and Newton, 1980). P. fragi, being versatile in its ability to grow at 21 ° C , is also representative of psychrotrophic spoil-age microorganisms. The type of biochemical changes occurring, particularly at inter-mediate temperatures (15°-25°C) has not been adequately documented. Contradictory reports on the correlation between pH and the number of microorganisms present on meat surfaces are found in the literature (Turner, 1960; Rogers and McCleskey, 1961). Furthermore, it has long been evident that a high pH value may exist in muscle for physiological reasons quite unconnected with bacteriology (Bate-Smith, 1948). Nevertheless, the sharp increase in pH of inoculated muscle was due to the rapid growth rate of P. fragi and the probable increased production of amines and ammonia (Jay, 1972). This was verified by analysis of both control and inoculated samples. In considering the morphology of the extracellular material as seen under the SEM, the structured organization of the fibrous extracellular components appears to indicate that this material may not be the result of - 28 -stretching of extracellular polymers when microorganisms are adjacent to each other, as reported by Fletcher and Floodgate (1973). Rather, it suggests an actual outgrowth of long fibrous material which participates in bridging microorganisms to each other or to the substrate. An examination of the bacterium surface for the determination of specific site(s) for outgrowth of extracellular material revealed a random pattern of interconnecting glycocalyx material. However, a close study of various micrographs suggested a polar site for outgrowth of the extracellular fibres from the cells of P. fragi (Figs. 5, 6). Since polysaccharide material may be involved in adhesion, ruthen-ium red or alcian blue was used for TEM studies. Although various resear-chers (Behnke, 1968; Behnke and Zelander, 1970) have used alcian blue as a means of improving fixation or increasing electron density of acidic polysac-charides, ruthenium red fixation, in this study, gave increased contrast of structures. This may be due to the homogeneous staining with the ruthenium red-osmium tetroxide combination (Dierichs, 1979). Although the specificity of ruthenium red for acidic polysaccharides is widely accepted, an alternative interpretation of the electron density which appears in extracellular spaces between cells treated with ruthenium red may be associated with protein as protein-polysaccharide complexes. In several ways, ruthenium red and alcian blue are similar. Both are metal-containing polyvalent basic dyes which precipitate similar polyanions (Scott et a[., 1964) and may be described as being reactive primarily by electro-static or ionic forces. The charge distribution over the ruthenium red molecule, however, is different from, and probably higher than, that of alcian blue which has fewer localized charges (Luft, 1971a). - 29 -Th r ee mechanisms have been p roposed to exp la in the spec i f i c i t y of ru then ium red ( L u f t , 1971a; 1971b). The f i r s t hypothe t i ca l mechanism, wh i ch was r e f e r r ed to as a " ca ta l y t i c mode l " , implies a c lose f i t between ru then ium red and some g roups of g l y can cha ins ( e . g . h y d r o x y l g r o u p s ) . The ru then ium red is ox id i zed b y O s 0 4 to ru then ium b r own , and the r u t h e n -ium b rown in t u r n ox id i zes the g l y can to wh ich i t is b ound . The ru then ium red t hus func t i ons as a ca ta l ys t in tha t one molecule of ru then ium red may be able to reduce severa l molecules of OsO^ to e l e c t ron -dense inso lub le p r o d u c t s . The second " s e l f - p r opaga t i n g " model p rov i des an a l te rna t i ve exp lanat ion in that the ox ida t ion of the g l y can subs t r a t e generates a new g roup wh ich b inds another ru then ium red molecule and so f o r t h . T h e last model o r "cha in react ion mechanism" a r r e s t s the reduc t ion of OsO„ at osmate 4 anion ins tead of promot ing i ts reduc t i on to lower ox ida t ion p roduc t s so that the e lec t ron dens i t y resu l t s f rom format ion of a l ayered complex . B l anque t (1976a, 1976b) also p roposed a mechanism wh i ch emphas ized the in i t ia l f o rma-t ion of a s tab le c y c l i c osmic ac id d i e s te r bond g i v i n g r i se to " co l l o i da l - l i ke " osmium d i ox i de , wh i ch in t u r n associates w i th ru then ium red to p roduce the e l e c t ron -dense pos i t i ve ma rke r . T EM ve r i f i e d conc l u s i ons , made f rom scann ing e lec t ron m i c rog raphs , that t h i n ex t r a ce l l u l a r mater ia l was adheren t to the bacter ia l c e l l . SEM and TEM revea led pebb l i ng on the su r f ace of the P_. f r ag i ce l l s wh ich may be due to the co r r uga t ed morpho logy of the bacter ia l cel l wall as well as dehyd ra t i on ef fects d u r i n g sample p repa ra t i on fo r e lec t ron m ic roscopy . No ev idence was ava i lab le to s uppo r t a re la t ionsh ip between the amorphous subs tance as seen in t ransmiss ion e lec t ron m i c rog raphs and the matted mass of ex t r ace l l u l a r mater ia l ob se r ved in s cann ing e lec t ron m i c r og raphs . However , b y means of a schemat ic i l l u s t ra t i on ( F i g . 9 ) , an attempt was made to re late the two - 30 -Fig. 9 Schematic illustration of the relationship between the various forms of extracellular material as seen by means of SEM and TEM. (A) and (B) are examples of the morphological forms of the extracellular material: (A) , adherent to the bacterium surface and (B), amor-phous in nature. - 32 -e x t r a c e l l u l a r ma te r i a l t y p e s . W iebe a n d C h a p m a n (1968) f o u n d t h a t c e r t a i n n u t r i t i o n a l a n d p h y s i o l o g i c a l c o n d i t i o n s i n d u c e d t h e f o r m a t i o n o f b l e b s o n t h e ce l l wall o f some p s e u d o m o n a d s . D u t s o n et a L (1971) s t a t e d t h a t s i n c e it was p o s s i b l e t h a t b a c t e r i a l p r o t e o l y t i c a c t i v i t y was r e s p o n s i b l e f o r m y o f i b r i l l a r d i s r u p t i o n , t h e e n z y m e s may be s e c r e t e d i n to b l e b s on t h e b a c t e r i a l s u r f a c e a n d la te r f o r m g l o b u l e s . T h e g l o b u l e s may t h e n r e l e a s e t h e i r c o n t e n t s in to t h e m u s c l e t i s s u e s u r r o u n d i n g t h e b a c t e r i a . B o e t h l i n g (1975) r e p o r t e d t h a t p r o t e a s e s a n d o t h e r e x o e n z y m e s a r e u s u a l l y r e p r e s s e d un t i l t h e late e x p o n e n t i a l p h a s e o f g r o w t h . O t h e r w o r k e r s ( T a r r a n t et a L , 1971; D a i n t y et a L , 1975) h a v e b e e n u n a b l e to d e m o n s t r a t e p r o t e o l y s i s un t i l s p o i l a g e is wel l a d v a n c e d . T h e s e f i n d i n g s may e x p l a i n t h e l a ck o f b l e b - l i k e e v a g i n a t i o n s o n samp les o t h e r t h a n t h o s e i n c u b a t e d f o r 5 d a y s . T h e c l o s e p h y s i c a l a s s o c i a t i o n a n d t h e t ime s e q u e n c e o f t h e a p p e a r -a n c e o f t h e e x t r a c e l l u l a r f i b r e s a n d b l e b s r e i n f o r c e p r e v i o u s s t u d i e s ( C o s t e r -t o n et a L , 1978) w h i c h s u g g e s t t h a t t h e e x t r a c e l l u l a r p o l y m e r i c mate r i a l a i d s i n : ( a ) p o s i t i o n i n g t h e b a c t e r i a to t he s u b s t r a t e s u r f a c e ; ( b ) c h a n n e l l i n g v a r i o u s n u t r i e n t s t o w a r d s t h e b a c t e r i a ; a n d ( c ) c o n c e n t r a t i n g a n d c o n s e r v i n g d i g e s t i v e e n z y m e s r e l e a s e d b y t h e b a c t e r i a . - 33 -C O N C L U S I O N : Growth of P. f r ag i on meat su r faces was not supp re s sed at in te rme-diate tempera tu res ( c a . 2 1 °C ) , however , the inocu lated muscle samples showed an inc rease in p H . Ex t r a ce l l u l a r mater ia l was v i sua l i z ed by u s i ng both SEM and T E M . Th i s mater ia l appeared to mediate ce l l - t o - ce l l as well as ce l l - to -musc le a t t a ch -ment to the muscle samples. The format ion and local izat ion of b lebs in assoc iat ion wi th the ex t r a ce l l u l a r f i b r e s may rep resen t the stages p r i o r to spo i lage of meat by h y d r o l y t i c enzymes of P. f r a g i . - 34 -PART 2 GROWTH PHASE AND CAPSULAR FINE STRUCTURE OF PSEUDOMONAS FRAGI ATCC 4973 - 35 -INTRODUCTION: As mentioned in the previous section, it is known that the mechan-ism of attachment of microorganisms to meat surfaces involves two consecutive stages; however, a detailed understanding of the latter attachment stage is still a subject of debate. During the spoilage of meat by P. fragi cells, an extracellular material was produced. Although transmission and scanning electron microsopy were used in the examination of the extracellular material, additional information is needed in order to understand the development and role of this material during bacterial growth. Little or no information is available on the stages of formation or fine structure of the extracellular material from P. fragi cells. The object of this study was to investigate the growth phase and capsular fine structure of Pseudomonas fragi ATCC 4973 when grown on solid and in liquid media. Since extracellular material was reported to be involved in the attachment of microorganisms to surfaces (Costerton et a[., 1978; Firstenberg-Eden, 1981), it would be valuable to know which phase of the bacterial cell growth is related to extracellular material formation as well as the determination of events associated with the attachment process. This information may be useful in explaining the mechanism of spoilage of foods by psychrotrophic microorganisms. - 36 -LITERATURE REVIEW: During the second phase of attachment of microorganisms to sur-faces, the microorganisms adhere by means of a mass of tangled fibres of polysaccharide that extend from the bacterial surface and form the "glycoca-lyx" that surrounds the cell. The fibres of the glycocalyx may not only position the microorganism but also may conserve and concentrate the diges-tive enzymes released by the bacteria and direct them against the host cell (Costerton et aL , 1978). Although the term glycocalyx is often used synonymously with extraneous or extracellular coats, Costerton et aL (1981) defined the bacterial glycocalyx as those polysaccharide-containing structures of bacterial origin lying outside the integral elements of the outer membrane of gram-negative cells and the peptidoglycan of gram-positive cells. Glycocalyces are subdivid-ed into two types. 1. S layers composed of a regular array of glycoprotein subunits at the cell surface. 2. Capsules composed of a fibrous matrix at the cell surface that may vary in thickness and may accurately be described by the following nonexclusive descriptors: (a) rigid - a capsule sufficiently structurally coherent to exclude particles (e.g. India ink); (b) flexible - a capsule sufficiently deformable that it does not exclude particles; (c) integral - a capsule that is normally intimately associated with the cell surface; - 37 -(d) peripheral - a capsule that may remain associated with the cell in some circumstances and may be shed into the menstruum in others. Various researchers have reported on the structure and function of the cell envelope (Costerton, 1970; Costerton et ah, 1974) and glycocalyx (Bennett, 1963; Ito, 1969; Costerton et al., 1981) o f gram-negative bacteria. More specifically, Costerton (1979) recently reviewed the role o f electron microscopy in the elucidation o f bacterial structure and function. Other reviews (Hollenberg and Erickson, 1973; Holt and Beveridge, 1982) have noted the development o f advanced techniques which greatly simplifed the task in the examination o f cell ultrastructure. Surface structures (e.g. glycocalyces) have been ignored, to some degree, because they are not easily preserved and resolved by microscopy and because many bacteria lose their glycocalyces on subculture Fjn vitro. Methods are currently available, however, for the stabilization and microscopic demonstration of structures such as glycocalyces and for their sustained production in m vitro cultures (Costerton et aL, 1981). By means o f electron microscopy, using numerous staining tech-niques, various bacterial glycocalyces have been studied (Behnke and Zelan-der, 1970; Fletcher and Floodgate, 1973; Bayer and Thurow, 1977; Reid and Brooks, 1982). Ruthenium red as well as alcian blue were considered effec-tive in staining acidic polysaccharides associated with glycocalyx material (Fletcher and Floodgate, 1973). Cassone and Garaci (1977), however, repor-ted that it was unnecessary to use special stains for preservation o f the delicate filamentous strands o f Klebsiella capsules. Other researchers have - 38 -also shown that ruthenium red deforms the bacterial capsule into thick bun-dles (Bayer and Thurow, 1977). Studies by Schmid et a[. (1981) showed that emphasis has been laid on the crucial importance of fixation and dehydra-tion procedures for the preservation of capsular micromorphology, whereas optimum growth conditions have been neglected. The fine structure of the bacterial capsule was affected by culture conditions, whereas the fixation techniques contributed mainly to the maintenance of integrity of cell wall, cytoplasmic membrane, DNA and ribosomes. By using osmium tetroxide as sole fixative under "RK conditions", Schmid et aJL (1981) found that this method was suitable to preserve capsular material, and that ruthenium red proved to be unnecessary; it injured and concealed capsular fine structures by amorphous precipitation of the capsular material. Although the early stages of exopolysaccharide synthesis have been investigated, little is known about the final stages of polysaccharide synthesis - the transfer of the oligosaccharide chains from their attachment to isoprenoid lipid carrier to a possible surface receptor and extrusion from the cell surface (Sutherland, 1979). It was further suggested that there may even be differences in the release of polysaccharides from cells of different physiological states. The outer membrane of gram negative bacteria is also more readily lost from bacteria under some conditions than others. This is likely to affect the attachment and release of exopolysaccharides as well as affecting their final purity. Sutherland (1979) also reported that in some microorganisms, exopolysaccharide is secreted continuously during growth, whereas in others, exopolysaccharide production is a feature of only the logarithmic and stationary phases. Williams and Wimpenny (1977), how-ever, demonstrated the formation of exopolysaccharide material during the stationary phase of growth of a pseudomonad. - 39 -Bleb-like evaginations or protrusions, inter alia, have long been noticed on the surfaces of several gram negative bacteria (Dutson et a[., 1971; Baechler and Berk, 1974; Maclntyre et a L , 1980). The significance of these blebs has been investigated by Wiebe and Chapman (1968), who reported that the increased surface area produced by the formation of the blebs may be of some importance in the ecology of the microorganisms in terms of either physical attachment of cells to a particle surface, or as an increased surface area for the initial acquisition of substrates. Bayer (1967) suggested that the evaginations of the cell walls after osmotic shocking may be the result of the escape of cytoplasm through pores (sites of growth) in the micropeptide cell wall layer, thereby expanding the more elastic overlying outer wall layer. Burdett and Murray (1974) suggested that the blebbing process, as well as being related to septation, may be due to the overproduction of lipid during rapid growth. The inability of the bacterial cell to produce sufficient lipoprotein to cement the outer membrane to the attached peptidoglycan layer during growth was further suggested. Other functions of the blebbing phenomenon in gram negative bacteria were reviewed by Russell (1976), who reported on the production of vesicles as a means of enclosing enzymes which then played a role in pathogenesis. This hypothesis supports earlier studies by Dutson et ah (1971) who noted that enzymes may be secreted into blebs on the bacterial surface, and later form globules. - 40 -EXPERIMENTAL: A. Culture Conditions: Pseudomonas fragi A T C C 4973 was grown (21 ° C , 24 h) in 200 mL trypticase soy broth (TSB) (BBL, Cockeysville, MD) in an oscillatory (100 rpm) water bath. Cell numbers were estimated by determining the turbidance at 600 nm with a Beckman Model DB spectrophotometer. Surface plating on trypticase soy agar (TSA) (BBL, Cockeysville, MD) was used for enumeration of P. fragi (21 °C ; 24 h) . B. Incubation on Solid Medium: A 24 h culture (0.01 mL) was plated on TSA and incubated at 21 ° C . Colonies were washed off after an incubation period of 4 h (early log phase cells) and after 24 h (stationary phase cells). Duplicate samples of two trials were harvested and were immediately fixed prior to further sample preparation. C. Incubation in Liquid Medium: P. fragi was inoculated into TSB and incubated at 21 ° C . On the basis of the growth curve, the samples of most interest were those obtained at 4 h (early logarithmic growth phase) and 24 h (stationary growth phase). Bacterial cells were removed after 4 and 24 h incubation. Duplicate samples of two trials were harvested and were immediately fixed prior to further sample preparation. D. Kellenberger-Ryter (RK) Fixation: The modified procedure of Schmid et aL (1981) was used for the - 41 -fixation and preparation of cells harvested from TSA, prior to investigation by transmission electron microscopy. In this study, RK buffer is synonymous with Michaelis buffer (Kellenberger and Ryter, 1958). The initial step consisted of washing the agar surface with 5 mL TSB. One millilitre of osmium tetroxide, 1% (w/v) in RK buffer, was added to the cell suspension and the mixture was left at room temperature for 10 min, prior to centrifuga-tion (4,000 x g; 10 min). The resulting pellet was immediately covered with 1 mL of 1% (w/v) Os0 4 in RK buffer and 0.1 mL TSB and incubated for 18 h at room temperature. The same fixation procedure as outlined for cells grown on solid medium was used for cells grown in liquid medium, with the omission of the washing step. E. Sample Preparation: The cell suspensions in OsO—RK-TSB buffer were filtered through 0.4 urn membrane filters (Millipore Corp., Bedford, MA) in order to separate the cell pellet from the supernatant fluid. The cell pellet was then carefully agitated with a mixture of melted 2% (w/v) agar in RK buffer. This proce-dure facilitated the fixing of the agar-infiltrated cell pellet on the membrane filter surface. Samples (1 cm cube) were cut and immersed into 2% (w/v) uranyl acetate (J.B. EM Services Inc., Montreal, Que.) in RK buffer at room temperature for 2 h. Dehydration was achieved by immersion of samples in aqueous solutions of 30, 50, 70 and 95% acetone for 15 min each followed by two changes of 100% acetone for 15 min each. The samples were infiltrated with acetone: Epon 812 (J.B. EM Services Inc.) (3:1, 1:1 and 1:3, 60 min each), Epon 812 (2 x; 45 min) followed by a two step polymerization at 45°C for 12 h and then at 65°C for 36 h. - 42 -F. Microtomy and Electron Microscopy: Embedded cells were sectioned with a "Porter-Blum" MT-2 ultra-microtome (Ivan Sorvall Inc., Norwalk, C T ) , mounted on 3 mm copper grids, then stained with uranyl acetate and lead citrate. Electron microscopy of all specimens was performed with a Zeiss EM-10 transmission electron microscope operated at an accelerating voltage of 80 kV. - 43 -RESULTS: A. Growth of P. fragi: Growth characteristics of P. fragi in liquid (TSB) and on solid (TSA) media were determined by monitoring cell numbers at timed intervals up to 40 h. Similar growth patterns were observed for P. fragi on TSA and in TSB (Fig. 10). P. fragi was in the early logarithmic phase of growth within 4 h incubation on TSA plates and in the stationary phase of growth by 24 h incubation on TSA at 21 ° C . B. Electron Microscopy: 1. Cells grown on solid medium: Transmission electron micrographs of bacterial cells harvested at the early logarithmic growth phase revealed bleb-like evaginations or protru-sions on the cell surface. The blebs, which contain dense granular material, appear to be present on the entire surface of the P. fragi cells (Fig. 11). Stationary phase cells, however, exhibited no bleb-like evaginations. Fine extracellular material randomly distributed on the cell surface was observed (Fig. 12). 2. Cells grown in liquid medium: Transmission electron micrographs of bacterial cells harvested at the early logarithmic growth phase revealed no blebs or extracellular material (Fig. 13). In contrast to early logarithmic phase cells, stationary phase cells seemed to be shrunken in appearance and revealed only few attached blebs as well as globules adjacent to the cells. No extracellular material was observed associated with cells at the stationary phase of growth (Fig. 14). - 44 -Fig. 10 Growth characteristics of P. fragi in liquid (TSB) ( • ) and on solid (TSA) ( • ) media, incubated at 21 °C. All points represent the average of three trials. INCUBATION TIME Ch) - 46 -Fig. 11 Thin section of P. fragi cells grown on solid medium harvested at the early logarithmic growth phase. Blebs which contain dense granular material are present on the entire cell surface. Bar = 1 urn. - 48 -Fig. 12 Transmission electron micrograph of P. fragi cells grown on solid medium harvested at the stationary phase. Fine extracellular mater-ials are randomly distributed on the cell surface. Bar = 1 urn. - 50 -Fig. 13 Transmission electron micrograph of P. fragi cells grown in liquid medium harvested at the early logarithmic phase. Bar = 1 pm. - 52 -F i g . 14 T h i n s e c t i o n o f s t a t i o n a r y p h a s e ce l l s o f P. f r a g i g r o w n in l i q u i d m e d i u m . Few a t t a c h e d b l e b s as well as g l o b u l e s a d j a c e n t to t h e ce l l s a r e o b s e r v e d . B a r = 1 p m . -53-- 54 -DISCUSSION: Electron micrographs of cells grown on solid medium and harvested at the early logarithmic phase of growth revealed an abundance of blebbing on the surface of bacterial cells. Detailed examination of the transmission electron micrographs suggested that the blebs may arise by an exocytosic process (Figs. 15 and 16). The absence of blebs on the surface of cells, harvested at the logarithmic growth phase, grown in liquid medium may be explained by the observations of previous researchers (Schmid et a L , 1981) that different culture conditions cause different fine structural aspects. Transmission electron micrographs of cells grown on a solid medium harvested at the stationary phase revealed extracellular material, but did not reveal blebs on the cell surface. Cells of P. fragi grown in liquid medium, and harvested at the stationary phase showed the presence of bleb-like evaginations on the cell surface as well as globules, containing dense granular material similar to that in the blebs, which can be seen in close proximity to the bacterial cells. This also suggests that the globules may be formed from the surface blebs. Rowe and Gilmor (1982) reported on the ability of psychrotrophic pseudomonads to continue net multiplication at the end of the true log phase when grown in liquid medium, which perhaps reflected their nutritional versatility in being able to utilize a wide range of substrates present in the growth medium. The hypothesis based on blebs containing hydrolytic enzymes was adopted in this study in order to explain our findings. Since pseudomonads grown in liquid media may be nutritionally versatile, as stated previously, there may not be an immediate need for the production of hydrolytic enzymes - 55 -Fig. 15 Transmission electron micrograph of P. fragi cells grown on solid medium, harvested at the early logarithmic phase of growth. Blebs as well as globules adjacent to the cells are observed. Bar = 0.5.urn. -56-- 57 -Fig. 16 Transmission electron micrograph of P. fragi cells grown on solid medium, harvested at the early logarithmic phase of growth. Blebs as well as globules adjacent to the cells are observed. A, B, C and D depict the respective phases of exocytosis. - 59 -rides) into small ones that can be assimilated by the bacteria. This may not only explain the presence of few blebs on the surface of P. fragi cells grown in liquid medium, but also the "delayed" response of bleb formation, which may suggest their association with nutrient availability. The presence of only few globules adjacent to the cells may also be due to the "washing" effect of the agitated liquid medium on the surface of the cells of P. fragi. A study of growth and proteolytic activity of six strains of Pseudo-monas grown for 18 h at 28°C on three media showed that proteolytic activity of Pseudomonas was highly dependent upon the medium employed and was not necessarily associated with rapid growth (Juffs et al_., 1968). Recently, similar observations were reported by McKellar (1982) who also showed that protease activity of three strains of Pseudomonas fluorescens was inducible. A study of enzyme production of Aeromonas hydrophila suggested the possibi-lity of an inducer catabolite repression system, where, under nutritional stress, protease production was high, despite slow growth (O'Reilly and Day, 1983). These studies tend to agree with the findings of the present study concerning the observation of blebs on the surface of P. fragi cells. - 60 -C O N C L U S I O N : T h e t y p e o f med ium ( s o l i d v s . l i q u i d ) is i m p o r t a n t in t h e e x p r e s s i o n of b l e b s o n t h e s u r f a c e o f P. f r a g i c e l l s . B l e b f o r m a t i o n may t h e n be f o l l owed b y t h e e x o c y t o s i c a c t i on o f g l o b u l e f o r m a t i o n in to t h e immedia te e n v i r o n m e n t o f t h e c e l l s . W h e t h e r o r no t t h i s p h e n o m e n o n is a s s o c i a t e d w i th t h e p r o c e s s o f a t t a c h m e n t o f t h e b a c t e r i a l c e l l s to s u r f a c e s , e x o c y t o s i s a p p e a r s to p r e c e d e t h e f o r m a t i o n o f e x t r a c e l l u l a r ma te r i a l ( g l y c o c a l y x ) b y P. f r a g i c e l l s . Whi le t h e g l y c o c a l y x may be i n v o l v e d in t h e a t t a c h m e n t of m i c r o o r g a n i s m s to s u r f a c e s ( C o s t e r t o n et a L , 1978 ) , f u r t h e r s t u d i e s a r e r e q u i r e d to d e t e r m i n e t h e r e l a t i o n s h i p b e t w e e n b l e b s , g l o b u l e f o r m a t i o n a n d t h e g l y c o c a l y x . - 61 -C H E M I C A L C O M P O S I T I O N A N D S T R U C T U R A L A N A L Y S I S O F T H E E X T R A C E L L U L A R P O L Y S A C C H A R I D E I S O L A T E D F R O M P S E U D O M O N A S F R A G I A T C C 4973 - 62 -INTRODUCTION: A common feature of bacteria, fungi (yeast and mould), and higher living organisms is the production of polysaccharides. Morphologically, there are three types: intracellular polysaccharides located inside or as part of the cytoplasmic membrane; cell wall polysaccharides; and exocellular polysaccha-rides located outside the cell wall (Sandford, 1979). Although some exocellular polysaccharides are found covalently attached to the cell as a true capsule, others are secreted unattached into the growth medium. Wilkinson (1958) summarized the functions of extracellular polysaccharide as: (a) protection against phagocytosis; (b) protection against amoebic attack; (c) protection against bacteriophage; (d) recognition and immune response of a higher organism to microbial infection; (e) protection against desiccation; (f) reserve carbon and energy source; (g) an aid in uptake of ions, and (h) an aid in the dispersal of cells in liquid environments due to the presence of ionic charges. Therefore, since the functions of all individual polysaccharides cannot be uniquely assigned, it is evident that they may act as storage materials, as structural components and as protective substances. While polysaccharides are either an integral part of the cell wall, or occur as a slime or capsule as in the case of Escherichia coli, Pseudomonas and Klebsiella, bacteria can also produce other polymers in which carbohydrates are main components. One type, which comprises the lipopolysaccharides, is present in the cell wall of - 63 -gram-negative bacteria. Another type consists of the peptidoglycans of the bacterial cell wall, in which polysaccharide chains are cross-linked v[a short peptide chains and form a two-dimensional network. A third type includes the teichoic acids, which are present in cell walls and membranes of gram-positive bacteria (Kenne and Lindberg, 1983). Gram-positive microorganisms are characterized by a dense peptidoglycan layer, adjacent to the inner periplasmic layer. Gram-negative bacteria have a similar but much thinner peptidoglycan layer covered by an outer layer of lipopolysaccharide (Fig. 17). Pseudomonads include the group of gram-negative, aerobic, rod shaped bacteria which have been reported to be responsible for the spoilage of meats at refrigeration temperature. As has been suggested in Part 1, the extracellular material of P. fragi may be involved in the adhesion of the microorganisms to surfaces. Although the mechanism involved in the adhesion process has not been elucidated, a knowledge of the chemical composition of the bacterial extracellular material may provide a better understanding of the specificity (if any) of the attachment process. Polysaccharides are biopolymers composed of monosaccharide residues and, for several of them, non-carbohydrate substituents. As mentioned previously, they fulfill different functions including being responsible for immunological properties of the microoganisms. Information on the composition and conformational structure of microbial polysaccharides is needed in order to correlate their structure with their biological and physical properties. Such studies should involve the determination of components, linkages, sequences, anomeric configurations and conformation (Lindberg, 1982). Structurally, extracellular polysaccharides are distinct from other classes of bacterial cell envelope polymers in that they do not conform to a simple general model (Powell, 1979). Polysaccharides, however, can be GRAM NEGATIVE F i g . 17 Diagramatic representation of the b a c t e r i a l c e l l envelope - 65 -c l a s s i f i e d o n t h e b a s i s of t h e t y p e s of s u g a r s p r e s e n t in t h e p o l y m e r ( h o m o -p o l y s a c c h a r i d e s v s . h e t e r o p o l y s a c c h a r i d e s ) as wel l as t h e d e g r e e o f b r a n c h i n g ( l i n e a r v s . b r a n c h e d ) . F u r t h e r c l a s s i f i c a t i o n o f p o l y s a c c h a r i d e s is d e t e r m i n e d b y t h e p r e s e n c e o f r e g u l a r r e p e a t i n g u n i t s . E l u c i d a t i o n of t h e p o l y s a c c h a -r i d e s t r u c t u r e i n v o l v e s d e t e r m i n i n g t h e f o l l o w i n g c h a r a c t e r i s t i c s : ( a ) t h e n a t u r e o f t h e s u g a r r e s i d u e s a n d t h e i r p r o p o r t i o n in t h e p o l y s a c c h a r i d e ; ( b ) t h e c o n f i g u r a t i o n at t h e a n o m e r i c p o s i t i o n o f t h e s u g a r r e s i d u e s ; ( c ) t h e c h a r a c t e r i z a t i o n o f l i n k a g e s b e t w e e n s u g a r r e s i d u e s ; a n d ( d ) t h e s e q u e n c e o f t h e s u g a r s in t h e r e p e a t i n g u n i t . T h e a n a l y s i s o f p o l y s a c c h a r i d e s h a s d e a l t m a i n l y w i t h t h e p r i m a r y s t r u c t u r e L e . t h e n a t u r e a n d s e q u e n c e o f t h e c o n s t i t u e n t s u g a r s as wel l as t h e l i n k a g e s i n v o l v e d . D u e to c o v a l e n t l i n k a g e s b e t w e e n a d j a c e n t s u g a r s , t h e l a c k o f f l e x i b i l i t y r e s t r i c t s t h e r e s i d u e s to a n a r r o w r a n g e o f r e l a t i v e o r i e n t a t i o n s . T h e i s o l a t e d p o l y s a c c h a r i d e c h a i n s a r e t h e n c a p a b l e o f a d o p t i n g o n l y c e r t a i n s h a p e s ( o r s e c o n d a r y s t r u c t u r e ) w h i c h a r e d e p e n d e n t o n t h e p r i m a r y s e q u e n c e ( R e e s a n d W e l s h , 1 9 7 7 ) . T w o h i g h e r l e v e l s o f o r g a n i z a t i o n s t r u c t u r e t h e n e x i s t w h e r e ( a ) i n t e r a c t i o n b e t w e e n c h a i n s m a y r e s u l t in o r d e r e d s p e c i f i c s t r u c t u r e s ( t e r t i a r y s t r u c t u r e ) a n d ( b ) i n t e r a c t i o n m a y o c c u r b e t w e e n t h e s e s t r u c t u r e s o r w i t h o t h e r p o l y m e r s ( q u a t e r n a r y s t r u c -t u r e ) . T h e o b j e c t o f t h i s s t u d y was to i n v e s t i g a t e t h e c h e m i c a l c o m p o s i t i o n a n d s t r u c t u r e of t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e i s o l a t e d f r o m P s e u d o m o n a s  f r a g i A T C C 4973 . - 66 -LITERATURE REVIEW Pseudomonads belong to the family of microorganisms Pseudomonad-aceae. The cells of Pseudomonas are typically straight or slightly curved rods. During the exponential growth phase, cells of most species are 0.5 to 1.0 pm in diameter, and about 1.5 to 4.0 urn in length. Pseudomonas species are typically mesophilic; however, since some species grow at 4 ° C , they are classified together with true psychrophilic organisms (Palleroni, 1978). A common feature of the pseudomonads is the production of polysac-charides. Polysaccharides may be grouped into three categories, according to whether they contain acidic, neutral or amino sugars. It is also important to realize that it is the constituent sugars and acyl substituents, and the types of linkages between them, that determine the conformation and specific properties of each polysaccharide. Polysaccharides vary considerably in the complexity of their struc-ture, and in molecular size and shape. Greenwood (1952) initially reported on the various methods for the determination of molecular weight of polysac-charides. Churms (1970) used gel chromatography in the separation of polysaccharides having a broad molecular weight distribution. Because mild conditions were used, the technique was particularly useful for labile biologi-cal materials. The most common methods of expressing molecular weight are number-average or weight-average M w > Although information on molecular size may be of limited direct value in the determination of composition and structure, this information is useful in distinguishing between linear struc-tures and those with a low degree of branching. This aids in the initial categorizing of the polysaccharides. - 67 -A. Monosaccharide Types When monomeric sugars are condensed, with a loss of water, they form polymers. Polymers with a chain length greater than ten are called polysaccharides. The monomer species may be (a) simple sugars, or (b) sugar derivatives e.g. amino sugars, uronic acids or ester sulphate sugars. Approximately 100 different monosaccharide components and 20 different non-sugar components have been found in polysaccharides and the numbers are increasing (Lindberg, 1982). Table 1, which gives examples of Pseudo-monas polysaccharides examined, shows the variations in the sugar components encountered within the genus. Table 2 gives examples of common functional group modification of naturally occurring sugar residues in polysaccharides. Various colourimetric tests have been developed to categorize as well as identify carbohydrate compounds. These tests serve as initial screen-ing assays prior to the use of other chemical methodology for the elucidation of the monosaccharide types. The following are among the commonly used colourimetric methods: (a) anthrone-sulphuric acid (Hodge and Hofreiter, 1962) and phenol-sulphuric acid (Dubois et aL, 1956) for neutral sugars (b) carbazole-sulphuric acid (Bitter and Muir, 1962) for uronic acids; (c) cysteine-sulphuric acid (Dische and Shettles, 1948) for 6-deoxyhexoses; and (d) p_-dimethylaminobenzaldehyde hydrochloride (Werner and Odin, 1952) for sialic acids. In the analysis of sugars liberated on hydrolysis, those with stable substituents, e.g. ethers, should be regarded as separate sugar constituents, even if substituted and nonsubstituted sugars have a common biosynthetic origin with etherification occurring at a postpolymerization stage. In addition, analysis is necessary for removable substituents, e.g. O-acyl (most commonly - 68 -Table 1 Rare sugar components of Pseudomonas polysaccharides Sugar Occurrence' Pentoses L-Xylose LPS D-threo-Pentulose LPS Hexose D-Allose EPS 6-Deoxy-D-mannose (D-rhamnose) LPS Amino sugars 2-Ami no-2,6-dideoxy-D-galactose (D-fucosamine) LPS 2,4-Diamino-2,4,6-trideoxy-D-glucose (bacillosamine) LPS Uronic acids 2-Amino-2-deoxy-L-galacturonic acid LPS 2,3-Diamino-2,3-dideoxy-D-glucuronic acid LPS 2,3-Diamino-2,3-dideoxy-D-mannuronic acid LPS 2,3-Diamino-2,3-dideoxy-L-guluronic acid LPS 3 Abbreviations: EPS - extracellular polysaccharides; LPS - lipopolysaccharides Kenne and Lindberg, 1983 - 69 -Table 2 Functional group modification of sugar residues. Functional Group Derivatives (ethers) - OH ester acetal (amines) -NH2 (esters) -COOH O-Methyl O-Acetyl Pyruvyl N-Acetyl - O C H 3 -OCOCH. "VH3 •0 / COOH •NHCOCH. -COOCH. - 70 -O-acetyl) substituents, N-acetyl, sulphate and phosphate groups, and ketals (pyruvate). Certain of these substituents can be analysed nondestructively by infrared (i.r.) and nuclear magnetic resonance (n.m.r.) spectroscopy. 1 13 The use of H and C n.m.r. would enable early recognition of unusual features which will indicate the need for suitable, chemically-based, analytical procedures (Aspinall, 1982). During the hydrolysis of glycosides, the stability of the various types of monosaccharides in hot acid must be considered. The hexosamines are the group most resistant to acid destruction. This destruction is not due to the effect of acid alone since it has been shown that if oxygen is excluded, the extent of destruction is greatly reduced (Sharon, 1975). Amino sugars found in polysaccharides are most commonly 2-amino-2-deoxyhexoses, which are present to a large extent as N-acetyl derivatives. In the determination of amino sugars, N-deacetylation can be first performed before hydrolysis, followed by deamination. This process may lead to the formation of anhydrohexoses, which can be easily estimated (Williams, 1975). Improvement of procedures for quantitative analysis of sugar residues in polysaccharide samples continues to be an important field of study. There are at least four major problems involved: (a) efficient release of monosaccharide using appropriate cleavage techniques with a minimum loss by decomposition; (b) quantitative or at least reproducible conversion to volatile deriva-tives; (c) development of columns and chromatographic techniques for effective separation of the mixture of volatile components, and (d) determination of reliable response factors for converting chart or - 71 -integrator output into molar quantities of each component (Aspinall and Stephen, 1973). B. Total Hydrolysis The nature of the sugars released on hydrolysis of the polysaccha-ride constitutes the initial step for structure determination. The conditions for total hydrolysis, without degrading the sugars present, must be determin-ed to obtain a quantitative release of the sugars. Dutton (1973) reviewed the advantages and disadvantages in the use of various acids. Albersheim et aL (1967) showed that 2 M trifluoroacetic acid (TFA), (with the same hydrolytic strength as HCI (1 IV)) and H 2S0 4 (0.5 M)) did not significantly degrade sugars under the conditions used (6-8 h, 100°C) and because of its volatility, it is easily removed. Although 2 M TFA can hydrolyse glycurono-syl linkages, the presence of an aldobio-uronic acid within the polysac-charide chain will withstand the acid treatment. This incomplete hydrolysis may give rise to discrepancies in the sugar ratio of the hydrolysate. Dutton and Yang, (1973) developed a technique which overcomes these difficulties. The polysaccharide is treated with methanolic hydrochloric acid, which cleaves most of the glycosidic linkages forming the methyl glycosides, as well as the methyl esters of the uronic acids. Treatment with NaBH4 in anhydrous methanol reduces the uronic esters to the corresponding alcohols. The mixture of methyl glycosides is then hydrolysed with 2 M TFA to give the neutral sugars. By comparison of the ratio of neutral sugars released by the 2 M TFA method vs. the methanolic HCI method, it is possible to identify the uronic acid as well as the molar proportion of the sugars present. Disaccharidic fragments which resist further fission unless more - 72 -drastic conditions are employed, are occasionally observed as minor by-pro-ducts in the hydrolysis of polysaccharides containing 2-acetamido-2-deoxy-hexose. Hydrolytic removal of the N-acetyl group may partially occur prior to the cleavage of the neighbouring hexosaminyl bond; the positively charged ammonium group produced is then very effective in hindering the hydrolysis of the adjacent glycosidic link (Baer, 1969). The free amino sugar is decom-posed during hydrolysis with acid. It could, however, be isolated as the N-acyl derivative by treatment with anhydrous hydrogen fluoride (HF). This treatment, during which glycosidic linkages are cleaved but amide linkages remain intact, has proved to be of general value in studies of polysaccharides containing N-acylamino sugars. Anhydrous hydrogen fluoride has been extensively used for the deglycosylation of glycoproteins (Mort and Lamport, 1977). The acid cleaves all the linkages of neutral and acidic sugars within 1 h at 0°C while leaving peptide bonds and glycopeptide linkages of amino sugars intact. More severe treatment with anhydrous hydrogen fluoride (3 h at 23°C) cleaves the O-glycosidic linkages of amino sugars; but the N-glycosidic linkages still remain intact. Because of the low boiling point of HF (19°C) and its extreme toxicity, the acid is usually handled in an enclosed system. Excellent reviews by Lenard, (1969) and Mort, (1983) have also included methods for handling HF, with emphasis on the application to carbohydrates. C. Sugar Determination Paper chromatography (Kowkabany, 1954; Block et aL, 1958 and Macek, 1963) and thin layer chromatography (Wing and BeMiller, 1972) were commonly used for the characterization and quantitation of sugars released - 73 -by the hydrolysis of polysaccharides. More recently gas liquid chromatogra-phy (g.l.c.) as well as high performance liquid chromatography (HPLC) have been utilized for the determination of sugars. The use of gas-liquid chroma-tography in carbohydrate analysis has been extensively reviewed by Dutton (1973, 1974), however, due to the ease in sample preparation for HPLC analysis, the latter technique is becoming more popular (Palmer, 1975; Conrad and Palmer, 1976). Carbohydrates are not sufficiently volatile to be used for gas liquid chromatography and must, therefore, be converted into volatile com-pounds. The disadvantage in using trimethylsilyl ethers (Sweeley et aL, 1963), however, was the formation of four derivatives (aand ft pyranosides and a and @ furanosides). The derivatization of sugars to the alditol acetates simplified the chromatogram (Gunner et aL, 1961). Sawardeker et aL (1965) investigated several column packings and found that an organosilicone polyester (ECNSS-M) gave good separations of common alditol acetates ranging from glycerol to glucitol which had the highest retention time. More recently, various column types which are capable of a comparable or even better separation of carbohydrate components have become available commercially. The application of capillary columns in the gas liquid chromatogra-phic analysis of oligosaccharides has been reported by Geyer and co-workers (1982). Various columns with phases of different polarity and selectivity have been employed. It was shown that capillary columns with Dexsil 410, SE-30 or OV 101 can be successfully used to separate peracetylated neutral and amino sugars. - 74 -D. Methylation Analysis The aim of methylation is to achieve etherification of all the free hydroxyl groups in the polysaccharide. The simplest and most convenient method for methylation of polysaccharides and other carbohydrates is probably the one developed by Hakomori (1964) (Fig. 18). Etherification of polysaccharides is, therefore, dependent on a sufficient degree of ionization of hydroxyl groups to achieve alkoxide forma-tion with enhanced nucleophilicity toward the alkylating agent, usually methyl iodide or dimethyl sulphate. Effective reaction is also dependent on the polysaccharide being soluble in a convenient polar solvent. The completeness of methylation of polysaccharides can be ascertained (a) by methoxyl deter-mination, if a sufficient quantity of the methylated derivative is available for microanalysis or (b) more simply by the absence of O-H stretching vibrations in the i.r. spectrum (Aspinall, 1982). Structural analysis of complex carbohydrates by methylation has been greatly advanced by the application of g.l.c.-m.s. to the identification of methylated sugars and by improved methylation procedures. The methyla-tion procedure according to Hakomori (1964), gives successful per-methylation of complex carbohydrates containing 2-deoxy-2-acetamido sugars. This is accompanied by N-methylation of the acetamido group which is unavoidable, regardless of the method of methylation, although the degree of N-methylation varies according to the method (Stellner et aL, 1973). Hydrolysis of the per-methylated polysaccharide is greatly affected by the formation of a positively charged methyl amido hexosyl (CH^-NR) residue. This factor as well as the destruction of these compounds by contact with any metal tubing or other surface during the g.l.c.-m.s. procedure may greatly affect the recovery of methylated amino sugars. Stellner and co-workers further - 75 -POLYSACCHARIDE i) base ii) Mel d Mt6 0 2Me i) hydrolysis ii) reduction CHO—CH£H iii) acetylation r-OAc MeO-pOAc -OAc -OAc MeO--OAc AcO--OMe -OMe hOAc OMe 1 i-OAc pOAc -OAc -OMe -OAc MeO--OAc -OMe -OAc AcO--OMe Q.I.C—m.s. F i g . 18 M e t h y l a t i o n a n a l y s i s o f a p o l y s a c c h a r i d e - 76 -reported on the effective use of acetolysis in obtaining a satisfactory yield of 2-deoxy-2-N-methyl-amido hexoses. E. Mass Spectrometry (M.S.) The use of mass spectrometry for the structural determination of a new sugar provides information on the class to which it belongs. Mass spectrometry, as commonly practised, uses electron impact most frequently as the ionization mode. Carbohydrate derivatives rarely give molecular ions in electron impact spectra, although molecular weights may often be inferred from various fragment ions. Several ion sources can be used, which can give rise to different modes of m.s., viz. electron impact (e.i.), chemical ionization ( c i . ) , field ionization (f.i.) and field desorption (f.d.). However, detailed structural information is best obtained from electron impact spectra. Mass spectrometry (m.s.) of organic compounds based on fragmenta-tion of organic molecules under electron impact, and differentiation of the resulting particles by use of the mass-to-charge (m/e) ratio involves subject-ing the compound under investigation to a beam of electrons ( « 70 eV). Ionization of the molecule causes decomposition to smaller fragment ions. Usually one electron is eliminated, resulting in the formation of a positively charged ion i.e. the "molecular" or "parent" ion (M +). Subsequent fragmen-tation and rearrangement of the molecular ion give rise to "daughter" ions. The application of mass spectrometry to the structural analysis of carbohydrate derivatives has been reviewed by various researchers (Finan et aL, 1958, Kochetkov and Chizhov, 1966, Lonngren and Svensson, 1974). The use of combined g.l.c.-m.s. in which the components from the chromato-graphic column are introduced directly into the ionization chamber of the mass spectrometer, has led to new methods for the qualitative and quantitative analysis of the mixture of sugars. The main peaks of the mass spectrum - 77 -correspond to ions formed by primary fission between two adjacent carbon atoms in the chain and to those arising by elimination of acetic acid (m/e 60) or ketene (m/e 42) from these primary fragments. The intensities of the fragments decrease with increasing molecular weight (Bjorndal et aL, 1970). Mass spectrometry may then be employed for two distinct purposes: the analysis of component sugars of polysaccharides, and the analysis of partially methylated alditol acetates in order to determine or verify linkage positions. (a) The analysis of component sugars of polysaccharides: Hydrolysis of a polysaccharide followed by the formation of the alditol acetates provides derivatives which can be easily analyzed by g.Ke-rn, s. Although isomeric alditol acetates having the same structure but differ-ent configuration are practically indistinguishable, the presence of substitu-ents such as deoxy-groups may be determined by fragmentation patterns as a result of cleavage stability of certain groups (Stortz et aL , 1983a, 1983b). (b) Analysis of partially methylated alditol acetates: Alditol acetate derivatives of components obtained from methylation analyses of polysaccharides may be readily examined by m.s. and in particu-lar, g.l.c.-m.s. Numerous articles have been published on mass spectrometry of partially methylated alditol acetates (Bjorndal et al., 1967; 1970). Although the ionization of the partially methylated alditol acetates results in fragmenta-tion, no molecular ions are seen. Primary fragment ions from partially methylated alditol acetates arise by CY-cleavage with, in general, preferred formation of: (a) ions of lower molecular weight; (b) ions from cleavage between two methoxyl-bearing carbon atoms, - 78 -with no marked preferences between the two methoxyl-bearing cations, and (c) ions from cleavage between a methoxyl-bearing and an acetoxyl-bearing carbon atom with marked preference for the methoxyl-bear-ing species to carry the positive charge. Ions formed by scission between the two acetoxyl-bearing carbon atoms, however, are generally of low abundance (Bjorndal et a L , 1967). The fragmentation pathways for a specific compound are, therefore, dictated by the preference in scission as outlined below: I H-C-OCH 3 \ H-C-OCI-U \ H-C-OAC H-C-OCH- / H-C-OAC / H-C-OAC i 3 Primary fragment ions undergo a series of subsequent eliminations to give secondary fragments, including losses by ^-elimination of acetic acid (m/e 60) or methanol (m/e 32), losses by CV-elimination of acetic acid (m/e 60) but not of methanol, and losses via cyclic transition states of formalde-hyde, methoxymethyl acetate, or acetoxymethyl acetate. Finally, it should be noted that mass spectrometry will not distinguish between diastereomeric partially methylated alditol acetates. The alditol acetates of 2,3,4,6-tetra-O-methyl-D-galactose and 2,3,4,6, -tetra-O-methyl-D-glucose will give similar mass spectra, although the intensities of the peaks may vary. F. Nuclear Magnetic Resonance Spectroscopy (N.M.R.) The uses of n.m.r. spectroscopy have not only included routine applications for analysis of the composition of mixtures and the monitoring of reactions, but also the elucidation of structure and conformational prefer-ences. The application of this technique to simple carbohydrates (Kotowycz - 79 -and Lemieux, 1973), oligosaccharides and polysaccharides (Hall, 1964; Coxon, 1972a, 1972b; Hall, 1974) have been extensively reviewed. N.m.r. spectroscopy, as with most other physical-chemical methods, enables the examination of a polymer without modifying or degrading it and then recovering the material intact. Dramatic progress in the development of n.m.r. instrumentation has resulted in increased operating frequencies as well as the utilization of pulsed Fourier transform (FT) technique which affords a large enhancement of sensitivity. 1. Proton Magnetic Resonance Spectroscopy ( H N.M.R.) 1 H n.m.r. spectroscopy is widely used in the analysis of polysac-charides. Although the application of the technique to high molecular weight complex molecules such as polysaccharides suffers from a number of limitations inherent with increases in molecular size and complexity, solutions to these problems were proposed by Hall (1974). The preparation of aqueous solutions of polysaccharides involves deuteration of all labile hydrogen atoms by repeated exchange with deuterium oxide prior to n.m.r. analysis. In order to eliminate interference in the spectrum by the numerous hydroxyl groups present, a good quality deuterium oxide (preferably 99.95 atom%) must be used. Nevertheless, a strong peak due to residual water (HOD signal) as well as substantial side bands of the peak are often obtained. The HOD peak usually appears in the region of the spectrum associated with the anomeric protons ( 5 4.5-5.5). A change in the chemical shift of the HOD peak may be accomplished by either altering the pH of the solution or more preferably by recording the spectrum at an elevated temperature. The latter procedure not only aids in reducing visco-sity but also has a dispersing effect on the sample. - 80 -The H n.m.r. spectrum of a polysaccharide provides information on the number of sugars present in the repeating unit and may reveal the presence or absence of 1-carboxy-ethylidene acetal (Garegg et a L , 1980), 6-deoxy sugars (De Bruyn et a L , 1976), and acetate groups (Savage, 1980; Rosell and Jennings, 1983). An examination of the chemical shift ( g ) along with the value of the coupling constant (J) between H-1 and H-2 ( J - „) may 1 / <-enable the differentiation of the anomeric nature of the linkages ( QL or jg ) for both pyranosyl (Bundle and Lemieux, 1976) and furanosyl (Stevens and Fletcher, 1968) sugars. Acetone is usually added to the sample as an internal standard prior to n.m.r. analysis. However, initial spectra of an unknown polysaccha-ride should be run without acetone, since a substituent O-acetyl group may be masked by the standard. Acetone has the advantage of being volatile (it can be readily removed from the sample) and its chemical shift is virtually unaffected by variations in temperature. 13 2. C N.M.R. Spectroscopy 1 Whereas the FT mode may be advantageous for obtaining H spectra, 13 its use is mandatory for C n.m.r. spectroscopy because of the low natural 13 abundance (1.1%) and intrinsically low sensitivity of the C nucleus. As in 1 the case of H n.m.r. spectroscopy, numerous reviews are available for the 13 study of carbohydrate structures, conformation and interaction by C n.m.r. spectroscopy (Usui et a L , 1973; Nunez et a L , 1977; Jennings and 13 Smith, 1980). C N.m.r. spectroscopy has also been used to study sialic acid polysaccharide antigen (Bhattacharjee et a L , 1t975), glycoproteins (Blum-berg and Bush, 1982) as well as simple polysaccharides ( B e r r y et a L , 1977; Gorin, 1981; Bradbury and Jenkins, 1984). - 81 -EXPERIMENTAL: A. Isolation of Polysaccharide: Pseudomonas fragi ATCC 4973 was grown (21°C, 24 h) in 200 mL tripticase soy broth in an oscillatory (100 rpm) water bath. The subsequent liquid culture (10 mL) was inoculated onto (30 x 38 cm) trays of tripticase soy agar which were then incubated for 3 days at 21 °C. The lawn of bacteria was harvested, followed by the addition of phenol (to a concentration of 1% w/v). After ultracentrifugation [Beckman Model L3-50 (Beckman Instrument Inc., Irvine, CA)] at 60,000 x g for 5 h (4°C) using a type 45 Ti rotor, the viscous supernatant (polysaccharide-containing fraction) was collected and added to absolute ethanol. The resulting precipitate was dissolved in water and further treated with a 10% solution of cetyl trimethylammonium bromide (CETAVLON) (Aldrich Chemical Co., Milwaukee, Wl), which precipita-ted polysaccharide material. Further purification of this fraction involved dissolution in 4 M NaCl, reprecipitation in ethanol, followed by dissolution in water and dialysis to remove residual NaCl. The dialysed solution, after freeze drying, yielded a polysaccharide component. An alternative method of polysaccharide isolation was used in order to increase the total polysaccharide yield. The isolation procedure was performed according to the method of Westphal & Jann (1965). Approximately 400 mL of harvested P. fragi cells including the extracellular material were incubated at 65°C. An equal volume of 90% (w/v) phenol, preheated to 65°C, was then added with vigorous stirring for 15 min. After cooling to 10°C, the emulsion was centrifuged at (1,000 x g) for 30 min which resulted in the formation of three layers: a water layer, a phenol layer and an insol-uble layer. The water layer was collected and the phenol layer was extracted - 82 -with a further 400 mL of water. The combined water extracts were dialysed, 3-4 d , against tap water to remove phenol and low molecular weight substan-ces. The dialysed solution was then centrifuged (1,000 x g) in order to remove any traces of insoluble material. The supernatant was freeze dried and yielded mainly polysaccharide material. Nucleic acids were removed by incubation with ribonuclease (Sigma Chemical Co . , St. Louis, MO) as describ-ed by Westphal and Jann (1965). 1 H n.m.r. spectroscopy of the native polysaccharides isolated by both procedures was used to determine similarity in polysaccharide types. All analyses were repeated at least three times. B. Molecular Weight Determination: Determination of the molecular weight of the P. fragi polysaccharide was performed by Dr. S. Churms (Univ. of Cape Town, South Africa) according to the procedure of Churms (1970). A 0.9 x 60 cm Sepharose 4B column was eluted with 1 M NaCl at a flow rate of 15 mL/h. C. Paper Chromatography: Paper chromatography was performed with Whatman No.1 filter paper and the following solvent systems (all v/v): (a) ethyl acetate-pyridine-water (8:2:1) - basic solvent; (b) ethyl acetate-acetic acid-formic acid-water (18:3:1:4) - acidic solvent. Chromatograms were visualized by: (1) heating at 110°C for 5-10 min. after being sprayed with p_-anisidine hydrochloride in aqueous 1-butanol, or - 83 -(2) dipping successively in silver nitrate - sodium hydroxide - sodium thiosulphate. All solvents were prepared according to the method of Hough and Jones (1962). Amino sugar component was observed by spraying the chromatograms with 0.25% (w/v) ninhydrin in 1-butanol, followed by heating at 100°C for 10 min. Preparative paper chromatography was carried out using Whatman No.1 filter paper eluted with ethyl acetate-acetic acid-formic acid-water (18:3:1:4). Strips about 4 cm wide were cut from the edges and treated with a suitable spray reagent to reveal the position of the sugars. By reference to these strips, areas containing each component were cut from the main body of the chromatogram. The sugar was then eluted from each paper section (shredded) with distilled water, concentrated in_ vacuo, and freeze-dried. D. Gas Liquid Chromatography: The g . l . c . analysis of alditols was conducted with a Hewlett-Packard 5700A gas chromatograph fitted with flame ionization detectors. The stationary phase for the analysis of alditol acetates was 3% SP 2340 (75% cyanopropyl silicone) on Supelcoport (100 - 120 mesh) (Supelco Canada L td . , Oakville, Ont . ) . The temperature profile used was 195°C for 4 min, followed by a programmed increase at 2 C°/min up to 260°C which was then maintained for 32 min. A Varian Model 3700 gas chromatograph, equipped with flame ionization detectors and coupled to a Hewlett-Packard Model 3390A integrator was also used. A capillary column (0.20 mm i.d.) with SE-30 stationary phase was used throughout the analyses. Hydrogen was the carrier gas. - 84 -The injector and detector temperatures were maintained at 260°C. The temperature profile used was 100°C to 250°C at 10 C°/min. Molar ratios of alditol acetates and hence the relative molar proportions of sugars in mixtures were calculated using the detector response factors (R g factors) for each sugar. These response factors were determined by calibration of the flame ionization detector, by injecting several alditol acetate mixtures of known composition into the chromatograph. Although phthalic esters (used extensively as plasticisers) may be encountered as contaminants in g . l . c . analyses, mass spectral data enable the differentiation of these contaminants from sugar derivatives (Fales et a l . , 1971; Dudman and Whittle, 1976). E. Gas Liquid Chromatography - Mass Spectrometry: All mass spectra were recorded using a NERMAG R10-21 instrument connected to a VARIAN VISTA 6000 capillary gas chromatograph. The following experimental conditions were used: electron energy - 70 eV; ionisa-tion current - 200 uA; inlet temperature - 240°C; ion source temperature -220°C. F. Nuclear Magnetic Resonance Spectroscopy: -i 1. Proton ( H) N.M.R. Spectroscopy: Proton magnetic resonance spectra were run on a Bruker WH-400 instrument. To eliminate interference in the spectrum by the numerous hydroxyl groups present, a number of exchanges were made with D^O (Stoh-ler Isotope Chem., Waltham, MA), followed by freeze drying. The sample was then dissolved in 99.9% D,,0 and any residual HOD was shifted away from the anomeric region (to 5 4.18, 90°C) by determining the spectrum at - 85 -elevated temperature. Due to the viscous nature of the sample, only low concentrations of polysaccharide could be transferred to the sample tube. The Fourier transform mode was then used. Acetone (62.23) was added to the sample and served as the internal standard. A sample was also analysed without acetone in order to prevent masking of any substituent O-acetyl group present. 13 2. C N.M.R. Spectroscopy: Samples were usually dissolved in 50% D£> to give a deuterium lock. Acetone (31.07 ppm) was used as internal standard. G. Sugar Analysis: Polysaccharide samples (100 mg) were hydrolysed in 2 M trifluoro-acetic acid (20 mL) at 100°C for 18 h. The hydrolysates were reduced with sodium borohydride and acetylated with pyridine/acetic anhydride (1:1, v/v). The resulting alditol acetates were analyzed by g.l.c. Concurrently, samples (2 mL) of the hydrolysates were withdrawn and examined by paper chromatography using both acidic and basic solvent systems. H. N-Deacetylation of the Polysaccharide: The method of Lindberg et a[. (1981) was used. The polysaccharide (15 mg) was dissolved in water (2.5 mL) and sodium hydroxide (200 mg) and thiophenol (1 drop) were added. The solution, in a serum vial, was stirred for 15 h at 80°C, neutralized with 2 M HCI, dialysed, and centrifuged (1,000 x g, 15 min). The polysaccharide (8.6 mg) was recovered from the superna-tant solution by freeze drying. A sample was exchanged (3 x) with D2O 1 and the H n.m.r. spectrum was obtained. - 86 -I. Deamination of the Polysaccharide: The method of Lindberg et ah (1981) was used. The N-deacetylat-ed polysaccharide (8.6 mg) was treated with a mixture of 33% aqueous acetic acid (1 mL) and 5% aqueous sodium nitrite (1 mL) for 1 h at room tempera-ture. The solution was diluted with water (3 mL) and freeze dried. The product was dissolved in water (2 mL) and reduced with sodium borohydride (50 mg) for 2 h. The reaction mixture was acidified with acetic acid and evaporated to dryness. The H n.m.r. spectrum of the deaminated polysac-charide was obtained. J . HF Solvolysis of the Polysaccharide: The polysaccharide (100 mg) was treated with anhydrous HF (7 mL) at ambient temperature with stirring for 3 h using Kel-F valves, vessels and pipes. The HF was vented off and complete removal was ensured. The resulting hydrolysed residue was then treated with 1 M acetic acid (100°C, 3 h). The acetic acid was roto-evaporated and a portion of the sample was spotted onto paper and developed in acidic and basic solvents. The remaining sample was then dissolved in water (5 mL) and applied to a column (100 x 5 cm) of Bio-Gel P-2 (Bio-Rad Lab., Mississauga, Ont . ) . Fractions (5 mL) eluted from the column were collected and the reducing power of the sample was measured using the ferricyanide method of Park and Johnson (1949). K. Preparation of Bio-Gel P-2 Column: Bio-Gel P-2 (200 - 400 mesh) was allowed to swell overnight in distilled water. The column (5 x 100 cm) was packed by gravity sedimenta-tion and left overnight. The column was eluted with distilled water and the - 87 -flow rate was set at w 0.5 mL/min. The void volume was determined by using blue dextran. L. Reduction of Uronic Acid Carboxyl Groups in Polysaccharides: The method of Taylor and Conrad (1972) was used for the reduction of the uronic acid residues in the polymer to their corresponding neutral sugars. The polysaccharide (20 mg) was dissolved in water (10 mL) and the solution was adjusted to pH 4.75 with 0.1 N HCI, prior to the addition of solid 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-g-toluene sulfo-nate (0.1 g) [Aldrich Chemical C o . , Milwaukee, Wl]. The reaction was allowed to proceed while maintaining pH 4.75 until hydrogen ion uptake ceased (30 - 60 min), then (8 mL) 2 M sodium borohydride was added drop-wise at 20° - 25°C over a 1 h period. The pH was maintained at 6.8 with the dropwise addition of 4 M HCI in order to minimize foaming. The reaction mixture was made acidic in order to destroy any remaining sodium borohydride. The solution was dialysed against running tap water for 2 d and freeze dried. M. Enzymatic Determination of Glucose: Samples were analysed for the presence of D-glucose using commer-cially available test kits (Boehringer Mannheim, Dorval, Que.) N . Methylation of Polysaccharide: Methylation analysis was performed according to the method of Hakomori (1964). The methylated material was recovered either by dialysis followed by freeze drying in the case of polysaccharides, or by extraction with chloroform for oligomers. - 88 -O. Acetolysis, Hydrolysis and Derivatization of Sugars into Partially Methylated Hexitol Acetates and 2-deoxy-2- N-methylamido-Hexitol Acetates: The procedure as described by Stellner et aL (1973) was used in the analysis of the partially methylated sugars. The per-methylated residue (5 mg) was dried in a reactor vial with Teflon-lined screw cap, to which was added (0.3 mL) 0.5 N sulphuric acid in 95% acetic acid (5 mL of 10 N sulphu-ric acid mixed with 95 mL of glacial acetic acid), and heated at 80°C over-night. The reaction mixture was then mixed with 0.3 mL water and heated to 80°C for an additional 5 h. The hydrolysate was passed through a column ( 1 x 6 cm) of weak anion exchanger, 1R-45 (acetate form) (Sargent-Welch Sci. C o . , Montreal, Que.) and washed with methanol. The residue was evaporated to dryness under a nitrogen stream, dissolved in 0.2 mL water and then reduced with NaBH^. The mixture was acidified with glacial acetic acid and roto-evapor-ated with intermittent addition of methanol. A white salty residue was left in the flask. Acetylation was accomplished by the addition of acetic anhy-dride: pyridine (1:1, v :v) at 100°C for 1 h. - 89 -RESULTS The initial yield of capsular polysaccharide isolated by the cetyltri-methylammonium bromide (Cetavlon) precipitation procedure was quantita-tively less than the polysaccharide yield isolated by the Westphal method. 1 H n.m.r. spectra of polysaccharides from each isolation procedure, however, indicated a high degree of similarity. Gel-permeation chromatography on Sepharose 4B gel gave a single 1.0 X 10 7 dalton peak. 1 The 400-MHz H n.m.r. spectrum of the native polysaccharide showed a doublet at 5 1 . 4 0 and a sharp singlet at 5 2.05 in the approximate ratio of 1:1. These were assigned to the methyl group of a deoxy sugar and an N-acetyl group respectively. Three signals were observed in the anomeric region, at 5 5.26 (1H, 2 ) , 5 4.87 (1H, 2 ) , and 5 4.60 (1H, 2 ) ' n t n e ratio of 1:1:1. Other signals were observed at 5 4.46 and 5 4.32 (Table 3). 13 Table 3 gives a summarized account of signals observed in the C 13 spectrum of the native polysaccharide. The C spectrum of the native polysaccharide showed signals which indicated the presence of the methyl group of a deoxy sugar (19.5 ppm) and an N-acetyl (22.9 ppm) group. Signals in the anomeric region of the spectrum were not easily discernable. Paper chromatography of a hydrolysate (2 M T F A , 100°C, 18 h) of the native polysaccharide showed the presence of glucose, an amino sugar, a fast moving component (deoxy sugar) and a disaccharide fraction (Table 4). Gas liquid chromatography analysis of the hydrolysed polysaccharide showed three major peaks, with the molar proportion of glucose:amino sugar: deoxy sugar as 1:0.5:0.5. Mass spectral data of the major peaks showed m/e 103, 115, 187, 217, 289, 361; m/e 84, 144, 318 and m/e 101, 153, 187, 275 and 375 (Figs. 19, 20 and 21) which were characteristic of the alditol acetate derivatives of the respective sugars. Table 3 N.M.R. data f o r P. f r a g i polysaccharide H - n.m.r. data C - n.m.r. Data Carbohydrate ^ J i 2 Integral Assignment p.p.m. Assignment (Hz) proton Native 5. .26 S 1 .0 102 .6 0 c o n f i g u r a t i o n polysaccharide 4, .87 S 1 .0 H-l o f amino sugar 100 100 .2 .1 4, .60 B 1 .0 53 .5 C-2 of amino sugar 4. .46 B 1 .0 22 .9 CHj o f acetate 4, .32 6 1 .0 19 .5 C-6 of deoxy sugar 2 .05 S 3 .0 CH 3 ofjtecetate 1. .40 8 3 .0 C-6 of deoxy sugar a Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; 8 2.23 downfield from 4,4-dimethyl-4-silapentane-l-sulfonate (DSS) k Chemical s h i f t i n p.p.m. downfield from Me.Si, r e l a t i v e to i n t e r n a l acetone;$31.07 ppm downfield from DSS B = broad; S = s i n g l e t - 91 -Table 4 R . Values of components of hydrolysed polysaccharide on Whatman Q I C No.l paper Hydrolysed Polysaccharide (2 M TFA) R . 3 glc Ninhydrin 1 3 A (disaccharide) 0.45 + B-amino sugar 0.86 + B-neutral sugar 1.00 -C (deoxy sugar) 1.78 -aRelative to glucose bReaction with 0.25% (w/v) ninhydrin in 1-butanol, followed by heating at 100°C for 10 min. (see text) Solvent: ethyl acetate:acetic acid:formic acid:water (18:3:1:4) I M - m m IT* SSI -; i i 'ff | i 1 " i i -r-r-r-r 1 ... L.. J75 T T I | i i i -1 | t t t >~| i i i t | r i i i | 1*1 l i t I2 t lh4i.|Lrj|,.I|l.H 44 r I M ll I 1 |iiL,L III l i f t 1 7 * l * » I M m/e I l l t l ? i n I, l, 11- I Ii.. I.. i , . . I "I I i i 1 | i L A -VO f i g . 19 Mass s p e c t r u m o f g l u c i t o l h e x a a c e t a t e component o f h y d r o l y s e d p o l y s a c c h a r i d e . 2 M 311 i " - i — p t - T - r 131 37* 371 32t 3 M I I I i i | I i i i ) - r I I | I I I I | •4 71 102 I M 4 127 u 1(9 f t ' 217 T — r • »» IB* I7» 2 M 22* > M m/e Mass s p e c t r u m o f amino s u g a r ( a l d i t o l a c e t a t e ) component o f h y d r o l y s e d p o l y s a c c h a r i d e . R e l a t i v e I n t e n s i t y % s 5 s : . = 2 ! I—1 o s Hi P> tr oi ^ hO O (D O rt H •! I I I I I 01 ro O 0 Hi M 01 ro 0) o o x o tr &) 01 H. C H-CD Hi 0) M H-rt O 5 1 T i JEL. r 3 \ ro s i i 0> o ro rt 0) rt ro ' I o o 3 t l o 3 ro r t S J « 4 - *6 -- 95 -Paper chromatography (18 h), using the basic solvent, of a hydroly-sate of the polysaccharide revealed the presence of a spot which migrated slowly, inter alia. Uronic acid sugars would be retained at the origin on paper chromatography using basic solvents. The modified uronic acid carbazole reaction (Bitter and Muir, 1962) gave a positive reaction. The colourimetric method for determination of hexuronic acids (Dische, 1962), however, gave a negative result. Carbodiimide-reduction of the polysaccharide using the method of Taylor and Conrad (1972) was performed, followed by g . l . c . analysis as well 1 as H n.m.r. spectroscopy. G. l .c . analysis of the carbodiimide-reduced polysaccharide showed no change in the molar proportion of any of the 1 sugars. The H n.m.r. spectrum of the reduced polysaccharide showed similar patterns to that of the native polysaccharide. Polysaccharide (80 mg) was hydrolysed (2 M T F A , 100°, 18 h) and separated into components by preparative paper chromatography. Three main bands were visualised. Each band was eluted from the paper chromato-gram and subsequently freeze dried. The components A(10.8 mg), B(31.5 mg) and C(16.9 mg) were identified as the disaccharide, combined glucose and amino sugar, and deoxy-sugar portion, respectively (Fig. 22). Prepara-tive paper chromatography permitted recovery of about 74% of the sugars released upon hydrolysis of the polysaccharide. The ensuing area of study was then the verification and further identification of these components. A portion of component A was reduced with NaBH^, acetylated and analysed by g . l . c . Component A could not be detected by g . l . c .-m.s . . Since band B, on the paper chromatogram (Fig. 22), consisted of two frac-tions, B-amino (an amino sugar component) and B-neutral (glucose), further separation by preparative paper chromatography permitted isolation of the individual components. A portion of the B-neutral component was reduced - 96 -— O r i g i "I —A (Oisacc) V"~r{B amino f.—r.\h neutral •C (deoxy) -Origin J —ClcNBCl -GalNHCl —ManNHCl ..glucose -fucose 6-d«oxy--glucose (a) h y d r o l y s e d p o l y s a c c h a r i d e (b) r e f e r e n c e s t a n d a r d s S o l v e n t : e t h y l a c e t a t e : a c e t i c a c i d : f o r m i c a c i d r w a t e r ( 1 8 : 3 : 1 : 4 ) F i g . 22 P a p e r c h r o m a t o g r a m o f (a) h y d r o l y s e d p o l y s a c c h a r i d e (2 M TFA) a n d (b) r e f e r e n c e s t a n d a r d s . - 97 -with NaBH 4 and acetylated. The alditol acetate was then analysed by g. l .c .-m.s.. Fig. 23 shows the mass spectrum of this component. A comparison of the spectrum with known references indicated the presence of a hexitol hexaacetate. The remainder of the B-neutral fraction was tested enzymatically for the presence of glucose. A change in the absorbance of the test solution enabled a positive identification of D-glucose in component B-neutral. The spot corresponding to B-amino on the paper chromatogram was initially detected by its positive reaction with ninhydrin. The alditol acetate derivative of fraction B-amino was analyzed by g. l .c .-m.s. (Fig. 24). Since 1 H n.m.r. spectroscopy showed that the component B-amino was de-N-acetyla-ted, deamination was then performed according to the procedure of Lindberg et a[. (1981). The resulting product was reduced, acetylated and examined by g . l . c .-m.s . , which revealed a hexitol hexaacetate. The gas liquid chromatogram of the alditol acetate derivative of purified fraction C showed two peaks corresponding to and P 3 as shown 1 in Table 5. The H n.m.r. spectra of the four fractions, A , B-amino, B-neutral and C were obtained (Table 6). HF solvolysis of the polysaccharide yielded a mixture of monosac-charide and oligosaccharide components. The hydrolysate, when applied to a Bio-Gel P-2 column, was separated into two major fractions. The components were assayed using the method of Park and Johnson (1949). Two main fractions HF-A (28.5 mg) and HF-B (39.7 mg) were obtained, i H n.m.r. analysis of HF-A and HF-B revealed signals which are tabulated in Table 7. Paper chromatography of fraction HF-B showed that the component consisted of glucose and an amino sugar. This was verified by reduction of the sample with NaBH^ and acetylation to form the correspon-ding alditol acetate derivative. G. l .c .-m.s. of the alditol acetate derivative -1 U l o s Hi D) CO tr cn a cn H > 3 O CD M O <^ rt cn H CD C fL g T3 O O Hi M <^ W cn i 0 ) 3 O CD O C rt OJ H H 0 ) H- I—' CD — • 0 ) M a rt O n CD rt 0> rt CD o O 3 T3 O 3 CD 3 rt Relative Intensity % ? I * 51 CD * a--» • 1 — u .en •a •a -5 - i j i •00 2 2 3 Sw T ' ' « ' | 5 * - 86 ->1 •p •H w c 4-» c CJ > id 180.8 n 43 r 111744. 10. 58.8-64 60 %j 69. 144 102 ICE 100 150 208 230 771 3  m/e F i g . 24 Mass s p e c t r u m o f B-amino ( a l d i t o l a c e t a t e ) component o f h y d r o l y s e d p o l y s a c c h a r i d e . - 100 -Table 5 G. l . c . analysis of alditol acetates of hydrolysed polysaccharide (2 M TFA) isolated by prep, paper chromatography. T a b c Column A Column B (Peaks) SP 2340 SE-30 P., 0.22 0.02 P 2 (glucose) 1.00 P 3 (deoxy sugar) 1.28 (amino sugar) 1.50 1.00 1.28 1.11 Retention time relative to that of the alditol acetate derivative of glucose. b programme: 195°C for 4 min, and then 2 C°/min to 260°C c programme: 100°C initial, then 4 C°/min to 250°C - 101 -Table 6 H N.M.R. data for hydrolysed P. fragi polysaccharide (2 M TFA) Carbohydrate 3 J M Integral 0 proton Assignment Disaccharide 5. 23 4 0.4 5. 01 S 1.0 H-1 of amino sugar 4. 64 B 0.6 1. 41 B B 5. 41 S 5. 23 4 Glc a 5. 21 S 4. 64 8 Glc J _ 1. 40 8 C-6 of deoxy sugar B-neutral 5. 23 4 Glc a 4. 94 S 4. ,63 B Glc J 3 _ 4. .50 B 1. ,46 B B-amino 5. 42 S H-1 of amino sugar 5. ,22 S H-1 of amino sugar C 8. ,48 S 5. ,24 2 0.4 H-1 of deoxy sugar 4. ,64 8 0.6 H-1 of deoxy sugar 4. .27 triplet H-5 of deoxy sugar 1. ,92 S 3.0 1. .41 8 3.0 C-6 of deoxy sugar For an explanation of disaccharide, B, B-neutral, B-amino and C, see text. Chemical shift relative to internal acetone; 5 2.23 downfield from DSS. Spectrum recorded at 90°C. Accurate integrals could not be obtained for some of the signals. B = broad; S = singlet - 102 -Table 7 H N.M.R. data for hydrolysed P. fragi polysaccharide (HF) a Carbohydrate A J-] 2 Assignment (Hz) HF-A HF-B 5.24 B 4.65 8 4.29 triplet 2.05 s 1.40 6 5.23 4 5.12 s 5.02 s 4.63 8 2.09 s 2.05 s H-1 of glc H-1 of amino sugar H-1 of amino sugar H-1 of glc \ acetyl groups a chemical shift relative to internal acetone; 8 2.23 downfield from DSS. Spectrum recorded at ambient temperature. s = singlet; B = Broad - 103 -of sample HF-B showed the presence of two components, hexitol hexaacetate (Fig. 25) and 2-acetamido-2-deoxy penta-O-acetyl hexitol (Fig. 26). The de-N-acetylated polysaccharide was analysed by H n.m.r. spectroscopy to confirm complete removal of the N-acetyl group ( § 2 . 0 5 ) (Table 8). A 60% recovery was achieved after deacetylation of the native polysaccharide. The de-N-acetylated polysaccharide was then deaminated. 1 The H n.m.r. spectrum of the deaminated polysaccharide showed the disap-pearance of an anomeric signal ( 5 4.78) (Table 8). G. l .c .-m.s. spectra of per-methylated hydrolysed polysaccharide are shown in Figs. 27, 28 and 29. The degree of methylation was determined by i.r. spectroscopy (Appendix I). dp >i 4J •H U) C <U •p c > •H +J (0 rH « IOOK* I0I744O H -OtlOS.O 43 103 M V 144 IlAL I V o » 7 I0O I M 14 IUT'11, ICO I M 300 MO 340 MO . .T 271 m/e F i g . 25 Mass s p e c t r u m o f HF-B. H e x i t o l h e x a a c e t a t e component o f h y d r o l y s e d p o l y s a c c h a r i d e . dP >i 4J •H to C <D •P C > •H •P (0 • H CU SI1MOO Of •09I31 .4 41 O 144 103 •0 tOO IIO 140 ISO 100 200 >20 *40 T . MO 3JO 940 m/e F i g . 26 Mass spectrum of HF-B. 2-Acetamido-2-deoxy-oenta-0-acetyl-D-hexitol component o f h y d r o l y s e d p o l y s a c c h a r i d e . Table 8 N.M.R. data for P. f r a g i polysaccharide (de-N-acetylated; deaminated) Carbohydrate H - n.m.r. data a A 1.2 Integral Assignment (Hz) proton 13, n.m.r. Data p.p.m. Assignment 5. .26 S 1, .0 102 .6 c o n f i g u r a t i o n 4. .78 S 1. .0 H-l of amino sugar 99 .9 4. .52 B 1. .0 97 .8 C - l of amino sugar 4. .34 8 1. .0 55 .4 C-2 of amino sugar 1. .40 8 3. .0 C-6 of 19 .5 C-6 of deoxy sugar De-N-acetylated polysaccharide De-N-acetylated deaminated polysaccharide 5.31 1.0 4. .54 8 2 .0 4. .34 8 1 .0 4. .24 4 3. .0 1. .41 8 3. .0 deoxy sugar CH, of de5; xy sugar a Chemical s h i f t r e l a t i v e to i n t e r n a l acetone, 6 2.23 downfield from DSS b Chemical s h i f t i n p.p.m. downfield from Me S i , r e l a t i v e to i n t e r n a l acetone, 31.07 ppm downfield from DSS broad; S = s i n g l e t >1 -p •H M C 0) +J c > • H QJ BASE rVE: 43 RICt 973824. lee.e-, 43 59. e H J 116 162568. 19. 74 I 56 8 7 l j l 58 158 129 142 JL 188 158 178 188 196 212 288 238 242 258 • 4 • l I J» | •••••i 258 272 m/e F i g . 28 Mass spec t rum o f 4 , 6 - d i - 0 - m e t h y l - 2 - d e o x y - 2 - N - m e t h y l a c e t a m i d o h e x i t o l . - 1 0 9 -- 110 -DISCUSSION Complex formation between cetyltrimethylammonium bromide (Cetav-lon) and various polysaccharides has been used to exemplify the reaction between carboxy-containing polysaccharides and quaternary ammonium salts. A wide range of acidic polysaccharides have been isolated y_[a precipitation with quaternary ammonium salts. The mechanism of the Cetavlon precipitation reaction involves salt formation due to charge interaction between the quater-nary ammonium salt and the carboxyl residues present on the polymer chains (Scott, 1955). Scott (1960) later reported on the precipitation reaction of quaternary ammonium compounds with proteins. Consequently, the lower yield of polysaccharide material obtained from the Cetavlon method as compared to the Westphal method may be explained by the absence of uronic acid residues in the polysaccharide chain. Amino acid contaminants, however, may be responsible for the precipitation of amino acid-polysaccharide-Cetavlon complex, due to the presence of carboxyl groups on the amino acid residues. The Cetavlon method of fractionation was initially utilized, since many researchers have indicated that an acidic polysaccharide may be involved in the secondary attachment of microorganisms to surfaces (Costerton et a l . , 1978). The second method of extraction (Westphal and Jann, 1965) gave a superior yield of polysaccharide. The isolated polysaccharide appears to contain no acidic units. This conclusion is based on the results obtained from the carbodiimide-reduction of the polysaccharide followed by g . l . c . and 1 H n.m.r. analyses. Amino sugars were also observed to migrate very slowly when developed with the basic solvent used in paper chromatography. Although the carbazole reaction gave a positive result, it should be noted that the reaction is not specific for uronic acid. Finally, the colourimetric - 111 -method for the determination of uronic acids reinforced the above findings that acidic units were not present. Although the preparation of acetyl derivatives of reduced monosac-charides is a simple technique, there are a few complications. During the reduction of sugars, borate complexes which can interfere with the acetylation reaction are formed (Blake and Richards, 1970) and as mentioned previously, decomposition of sugar alcohol acetates may be possible (Bishop et a L , 1963). Bishop and co-workers suggested four possible reactions which may occur during g . l . c . analysis of carbohydrate derivatives: (a) deamidation; (b) change in size of the sugar r ing; (c) rearrangement of acetal or ketal groups, or (d) degradative rearrangement of acetylated amino sugars. Degradative rearrangement may be caused by high column or injection port temperatures; by the types of liquid phases or inert supports or any pretreat-ment of the latter; by the tubing used for the column; or by the solvent used for injection of the sample. G. l .c . analysis of the hydrolysed polysaccharide (2M TFA) showed the presence of four components, however, g. l .c .-m.s. analysis only gave data for three peaks Lje. P ^ , Pg and P^ . Peak was retained close to the origin (Table 5). G. l .c .-m.s. data agreed with the data obtained by paper chromatography in that the polysaccharide contains glucose, a fast moving component (deoxy sugar) and an amino sugar. The isolation and purification of the fast moving component by preparative paper chromatography followed by g . l . c . analysis indicated that both peaks and P 3 were associated with the deoxy sugar component. Conclusive verification of the identity of the amino sugar as well as the deoxy component was not achieved by hydrolysis - 112 -of the polysaccharide with 2 M TFA due to degradation of the sample. G. l .c .-m.s. data also suggested the possibility of fragmentation or degradation of one of the components in the polysaccharide chain by the conditions being used during acid hydrolysis. It then appears that the long duration of hydrolysis of the polysaccharide (2 M TFA) may have led to inconsistent molar ratios of each component, which could be explained by: (a) the relative lability of one of the components, and/or (b) the relative stability of the hexosaminyl bond. In the course of this study, the most important information was 1 13 provided by spectral methods, L_e. H and C high resolution n.m.r. spec-1 13 troscopy. The signals in the H and C n.m.r. spectra of hexosaminogly-cans are known to distribute in groups, each of which occupies strictly defined regions. The typical resonance regions useful for the first order analysis of the hexosaminoglycan spectrum are summarized in Table 9. Both 1 13 H and C n.m.r. spectroscopy proved to be complementary techniques in structure determination as well as the verification of structures. Detailed n.m.r. spectral data of all samples are shown in Appendix II. 1 The H n.m.r. spectrum of the native polysaccharide revealed the 13 presence of three sugars, one containing an N-acetyl group (verified by C n.m.r. spectroscopy) and another containing a CH^ group (Table 3). H n.m.r. analysis of the de-N-acetylated native polysaccharide showed an upfield shift in signal from 5 4.87 to 8 4.78 which may be ascribed to the anomeric signal of the amino sugar. Since steric hindrance will result in a deshielding effect, the removal of the bulky N-acetyl group will, therefore, result in an upfield shift of the anomeric proton. - 113 -Table 9 R e p r e s e n t a t i v e H and C chemical s h i f t s f o r n u c l e i of p o l y s a c c h a r i d e s 3 . H a(ppm) C a(ppm) CH 3C ~ 1 . ,5 CH 3C ~ 15 CHgCON 1. .8-2. ,1 CH.COH) CH^C0 2j ' 20-23 CH 3C0 2 2 . 0-2. .2 CH(NH) 3. ,0-3. ,2 CH 2C 38 CH 30 3. .3-3. .5 CH 30 55-61 H-2 t o H-6 3. .5-4. ,5 CH(NH) 58-61 H-5 4, .5-4. .6 CH2OH 60-65 H-1 (ax) 4. .5-4. .8 C-2 t o C-5 65-75 H—C(OH) 2 5.2 c - x b 80-87 HO 5. .0-5, .4 C - l (ax-O, red) 90-95 H-1 (eq) 5, .3-5, .8 C - l (eq-O, red) 95-98 HC0 2 5.9 c - l (ax-O, g l y c ) 98-103 C - l (eq-O, g l y c ) 103-106 c - l ( f u r ) 106-109 COOH 174-175 C = 0 175-180 A b b r e v i a t i o n s : ax, a x i a l ; eq, e q u a t o r i a l ; red, re d u c i n g ; g l y c , g l y c o s i d i c ; f u r , f u r a n o s y l . 13 Nonanomeric C i n v o l v e d i n g l y c o s i d i c l i n k a g e . P e r l i n and Casu, 1982 - 114 -Preparative paper chromatography of the hydrolysed native polysac-charide (2 M TFA) indicated that the four components A, B-neutral, B-amino and C are a disaccharide, glucose, amino and a deoxy component, respectively. 1 The H n.m.r. spectrum of the disaccharide fraction showed three anomeric signals which are attributable to the disaccharide unit formed by the amino sugar - monosaccharide fraction. As would be expected, one anomeric signal may be assigned to the amino sugar while the other two signals would be assigned to the reducing end of the adjoining carbohydrate unit ( a and jg signals). The signal at 5 1.41 suggests that the deoxy component is linked to the anomeric position of the amino sugar. This observation was also verified by an examination of the molar proportion of the glucose:amino sugar:deoxy sugar components. A molar proportion of 0.5 may be obtained if a portion of the corresponding sugars is not totally hydrolysed. There-fore, they may be linked as a disaccharide unit. Component A could not be detected by g . l . c .-m.s . , possibly because of degradation owing to the labile nature of the deoxy component. The relatively small difference in between the amino and glucose units on Whatman No. 1 paper using acidic solvent did not initially enable complete separation of these two components. Fraction B was then further separated into B-neutral and B-amino by repeated paper chromatography. 1 H n.m.r. spectroscopy showed corresponding anomeric signals for both B-neutral and B-amino which were also observed in the mixed component B. Although signals at 5 5.23 and 5 4.63 enabled the identification of B-neutral as glucose, additional signals at 5 4.94 and 5 4.50 may be due to contamination by the B-amino component (Table 6). The absence of the N-acetyl signal in component B-amino may be explained by the de-N-acetylation of the amino sugar on hydrolysis of the polysaccharide. - 115 -The presence of signals at 5 8.48, 1.92 in the H n.m.r. spectrum reveals the complexity of component C. The C H 3 signal ( 5 1 - 4 1 ) , however, enables the labelling of this component as a deoxy sugar. Reference stan-dards containing CH^ groups (6-deoxy-glucose, fucose, rhamnose) were used in an attempt to characterize the sugar by paper chromatography. The results obtained from the analysis of the alditol acetates of purified fraction C by g. l .c .-m.s. suggests the possibility of fragmentation of this component. The inconsistent mass spectral data obtained were probably representative of the decomposed, fragmented compound. Component C gave a negative re-sponse when treated with ninhydrin. Reactions of the other components with ninhydrin are summarized in Table 4. This aids in categorizing the compo-nents as (i) glucose (B-neutral); (ii) amino sugar (B-amino); and (iii) deoxy-sugar (C) . The hexosamine, on paper chromatography, did not correspond to any standards commercially available. In an attempt to provide additonal information on the sugar composition of the polysaccharide, other techniques (HF solvolysis, methlyation analysis) were used. Many researchers have reported on the successful application of HF solvolysis as well as the preferential cleavage of over linkages in polysaccharides (Mort, 1983). HF solvolysis was employed because enzymatic deglycosylation suffers from the disadvantage that specific enzymes must be obtained while chemical deglycosylation involving periodate oxidation is some-times incomplete. Depending on the conditions, HF solvolysis can give either monomeric or oligomeric units. For this reason, the hydrolysate was subse-quently fractionated on a column of Bio-Gel P-2. 1 Analysis of the H n.m.r. spectrum of fraction HF-A suggested that it was an oligomer, however, an accurate assignment of the signals - 116 -could not be achieved. Analysis of fraction HF-B by H n.m.r. spectroscopy showed four anomeric signals and two N-acetyl signals. This may indicate the presence of two monomeric units, one being the amino sugar bearing the N-acetyl function. HF-B was reduced with NaBH^, acetylated and analysed by g. l .c .-m.s . The mass spectra obtained verified the presence of an amino sugar and a hexose unit. These data may suggest that the anomeric signals ( aand jS ) of the N-acetylated amino sugar are 5 5.02 and 5 5.12, since the anomeric signals for a and ft D-glucose were determined to be 5 5.23 and 5 4.63, respectively by using reference compounds. The shift in anomeric signals of the N-acetylated amino sugar (as observed in the HF-B component) compared to the de-N-acetylated amino component (as observed in the B-amino component) may be explained by the anisotropic effect in which the chemical shift of a proton is frequently modified by neighbouring functional groups (N-acetyl). The deamination of amino sugars and their glycosides is usually accompanied by rearrangement (Williams, 1975; Aspinall et a L , 1978, 1980). Deamination of the B-amino component, followed by g . l . c . analysis showed the formation of hexitol hexacetate. This finding eliminates the possibility of a 2-amino-2-deoxy-D-glucopyranose component in the polymer, since it has been shown that the deamination of 2-amino-2-deoxy-D-glucopyranose proceeds with the formation of 2,5 anhydro D-mannose derivatives (Williams, 1975). These data are also consistent with the results obtained from paper chromato-graphy. Complete characterization of a permethylated polysaccharide requires identification and quantitative analysis of all the sugar derivatives formed on depolymerization. With the use of g. l .c .-m.s. analysis, molecular ions are not seen in electron impact spectra taken at 70 eV, but molecular weights can usually be obtained by extrapolation from fragment ions. - 117 -Methylation of the native polysaccharide, followed by acetolysis, reduction, derivatization as alditol acetates and g. l .c .-m.s. analysis showed the formation of 2,3,6-tri-O-methyl glucitol (Fig. 30), 4,6-di-0-methyl-2-deoxy-2-N-methylacetamido hexitol (Fig. 31) and a component with m/e 231, 171, 143, 117, 59 (Fig. 29). These results suggest that glucose (4 linked) and N-acetylated amino sugar (3 linked) are present in the polysaccharide chain. Detailed mass spectral data of all components are shown in Appendix III. Analysis of the data obtained from these studies indicates that the polysaccharide of Pseudomonas fragi is probably composed of a trisaccharide repeating unit having the following partial structure: »4) D-GIc (1 -3) amino sugar (1 » ?) deoxy sugar (1 * |2 NAc - 118 -CH„OAc I d H - C - OMe 117 I MeO - C - H 233 I H - C - OAc I H - C - OAc CH 2OMe 45 233 (M + - 117) 233 - acetic acid (m/e 60) 173 117 - formaldehyde (m/e 30) 87 Fig. 30 Fragmentation pattern observed in the mass spectrum of 2,3,6-tri-O-methyl-D-glucitol. - 119 -161 CH 2 OAc H - C N H - C - OAc — —I H - C - OMe H - C - OAc Ac 158 274 CH 2OMe 161 - acetic acid (m/e 60) 101 158 - ketene (m/e 42) 116 116 - ketene (m/e 42) 74 Fig. 31 Fragmentation pattern observed in the mass spectrum of 4,6-di-0-methyl-2-deoxy-2-N-methylacetamido hexitol. - 120 -SUMMARY; The aerobic spoilage flora of foods is usually dominated by species of Pseudomonas. Pseudomonas fragi has been reported to be involved in the microbial spoilage of meat at both chill and ambient temperatures. The spoilage process appears to include the attachment and adhesion of microorgan-isms to the substrate. This study was an attempt to examine the adhesion mechanism by physical and chemical analyses of the bacterial glycocalyx. The data from this study suggested that glycocalyx material production was not dependent on the growth phase of the cell but on the type of substrate used. P. fragi grown on synthetic media produced a glycocalyx with a hexosaminoglycan structure. Contrary to published reports (Fletcher and Floodgate, 1973; Costerton et a L , 1978; Yada and Skura, 1982) suggesting the involvement of an acidic polysaccharide in bacterial glycocalyces, this study has identified the involvement of a neutral polysaccharide only. An understanding of structure-function relationships of the extra-cellular material may provide answers to the role of glycocalyces in the spoilage of foods. Since the types of monomeric units present in the polysac-charide chain may contribute to the biological state and properties of the microorganism, this then implies that the basic functions of physiological protection, nutrient transport, as well as cell interaction with the environment are greatly influenced by the nature of the glycocalyx. This study provided various avenues for future research in addition to a better understanding of the following: 1. Bleb-like materials are formed prior to the formation of glycocalyx material. If these blebs do contain hydrolytic enzymes then it can be assumed that the onset of food spoilage may occur at very early - 121 -stages of microbial growth and that the formation of slime is not a reliable indicator of food spoilage. Microbial degradation of food components may occur at a much earlier stage than as suggested by current literature. Yada and Skura, (1981) demonstrated that proteolysis of beef muscle proteins is observable in the late logarith-mic phase of growth of P. fragi. More sensitive assay techniques may show the onset of proteolysis at an earlier stage, which would support the hypothesis of the presence of hydrolytic enzymes in blebs of P. fragi; Since extracellular materials are produced at various stages of cell growth, and are influenced by the type of substrate, this may explain the various rates of microbial spoilage of liquid and solid foods; Polysaccharides, together with O antigens of the lipopolysaccharide, constitute the principal immunogen and antigens of the bacteria because of their location at the extreme outer surface of the cell. Detailed knowledge of the structure of the polysaccharide on the bacterial surface could lead to the design of highly effective inhibi-tors of adherence that may prove to be useful in the prevention of bacterial spoilage of foods; A better understanding of the role(s) of glycocalyx material in attachment on and detachment of microorganisms from the surfaces of foods would aid in the selection of an analytical procedure for estimating numbers and types of bacteria on the substrate surface; Futhermore, this information may be useful in designing techniques to reduce numbers of bacteria on food surfaces, to maintain them at reduced levels which could improve shelf life and possibly reduce public health hazards; - 122 -The involvement of a neutral and not an acidic polysaccharide in the glycocalyx of P. fragi suggests that interaction between glyco-calyx and various substrates may be dependent on sugar types present in the polymer. Therefore, the presence of the bulky N-acetyl group of the amino sugar may restrict the flexibility of the polymer. Although the linear chain molecule may then adopt a regular rigid chain geometry, the overall conformation is determined by the relative orientation of the individual sugars; and Since these studies represent the first of its kind in examining the physical and chemical structure of the polysaccharide of P. fragi, it may serve as the basis for future investigations in the examina-tion of: (a) the nature of the blebs; (b) the relationship between blebs and glycocalyx; (c) variations (if any) of the glycocalyx composition when P. fragi cells are grown on various media; and (d) methods for the prevention of glycocalyx formation. - 123 -CONCLUSION: 1. Pseudomonas fragi, incubated at 21 ° C on beef muscle and trypticase soy agar, produced a glycocalyx material as well as blebs possibly containing enzymes. 2. Glycocalyx appeared to mediate cell-to-cell and cell-to-muscle attachment. 3. Examination of scanning electron micrographs suggested an association between blebs and glycocalyx on the surface of P. fragi cells, grown on beef muscle. 4. Examination of transmission electron micrographs revealed various mor-phological forms of glycocalyx material. 5. The type of substrate (solid vs. liquid) influenced the expression of blebs and globules on the surface of P. fragi cells. 6. The formation of blebs preceded the onset of glycocalyx formation by P. fragi on trypticase soy agar. 7. The extracellular material isolated from P. fragi cells was composed of a hexosaminoglycan structure. 8. No acidic sugar components were detected in the polysaccharide chain. 9. The linear repeating trisaccharide unit of P_. fragi polysaccharide con-sists of D-glucose, a deoxy component and a N-acetyl amino sugar. - 124 -REFERENCES ALBERSHEIM, P., NEVINS, D.J., ENGLISH, P.D. and KARR, A. 1967. 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Microbiol. 43:905. - 134 -APPENDIX I Infrared Spectroscopy Data A p p e n d i x 1.1 I n f r a r e d s p e c t r u m o f p e r m e t h y l a t e d P. f r a g i p o l y s a c c h a r i d e - 136 -APPENDIX II N.M.R. Spectroscopy Data 100 MHz amb. temp 6 1 . 7 3 19.51 (ppm) A p p e n d i x I I .2 *"*C n . m . r . s p e c t r u m o f N a t i v e p o l y s a c c h a r i d e 100 MHz amb. temp. ( a c e tone ) 3 1 , 0 7 (ppm) 13 A p p e n d i x 11.4 C n . m . r . s p e c t r u m o f d e - N - a c e t y l a t e d p o l y s a c c h a r i d e 400 MHz 90 °C (ppm) A p p e n d i x II.9 1 H n . m . r . s p e c t r u m o f component B - n e u t r a l (ppm) Appendix II.12*H n.m.r. spectrum of component HF(A) - 150 -APPENDIX III Mass Spectrometry Data - 151 -APPENDIX 111.1 Mass Numbers of B-neutral PAGE 1 P EAK MEASURED NO. A B S O L U T E X I N T . X I N T . X T O T . N U . MASS PO INTS I N T E N S I T Y BASE NREF ION 1 3 6 2 r s , 1 3 9 7 . 0 . 1 0 . 1 0 . 0 2 3GI 35 7 8 6 2 . 0 . 6 0 . 6 0 . 2 3 2 9 8 Z9 2 2 U 5 . 0 . 2 0 . 2 0 . 1 4 294 21 8 5 8 . 0 . 1 0 . 1 0 . 0 5 2<Jil 35 5 4 0 7 . 0 . 4 0 . 4 0 . 2 6 2 B 9 43 3 6 8 2 3 . 2 . 6 2 . 6 1.1 7 2 7 3 35 3 9 6 0 . 0 . 3 0 . 3 0 . 1 8 2 7 2 43 3 1 6 2 . 0 . 2 0 . 2 0 . 1 10 261 • . 2 9 2 1 7 0 . 0 . 2 0 . 2 0 .1 11 2 6 0 35 3 8 6 8 . 0 . 3 0 . 3 0 .1 12 2 5 9 43 3 0 1 9 9 . 2.1 2 .1 0 . 9 13 2 5 7 21 1 3 1 0 . 0 .1 0 . 1 0 . 0 14 2 5 5 17 8 8 0 . 0 . 1 0 . 1 0 . 0 16 2 4 2 35 4 7 0 5 . 0 . 3 0 . 3 0 .1 21 2 3 0 29 2 6 7 0 . 0 . 2 0 . 2 0 .1 22 2 2 9 21 1 3 4 7 . 0 . 1 0 . 1 0 . 0 25 2 2 0 43 2 9 0 1 . 0 . 2 0 . 2 0 .1 26 2 1 9 35 1 3 2 4 . 0 . 1 0 . 1 0 . 0 27 2 1 8 51 5 1 5 5 . 0 . 4 0 . 4 0 .1 28 2 1 7 43 5 6 3 9 9 . 3 . 9 3 . 9 1 .6 29 2 1 3 43 5 5 9 0 . 0 . 4 0 . 4 0 . 2 3 0 2 1 2 43 7 8 1 6 . 0 . 5 0 . 5 0 . 2 31 211 35 2831 . 0 . 2 0 . 2 0 .1 32 2 1 0 25 1 5 9 2 . 0 . 1 0 .1 0 . 0 33 2 0 8 21 1 2 5 5 . 0 . 1 0 . 1 0 . 0 36 2 0 4 21 1 1 6 5 . 0 . 1 0 . 1 0 . 0 38 201 35 2 9 7 5 . 0 . 2 0 . 2 0 .1 39 2 0 0 35 5 6 8 6 . 0 . 4 0 . 4 0 . 2 4 0 1 9 9 43 4 8 9 7 . 0 . 3 0 . 3 0 .1 41 195 29 2 0 0 5 . 0 . 1 0 . 1 0 .1 43 191 43 1 0 4 4 . 0 . 1 0 .1 0 . 0 44 1 9 0 21 1 0 7 9 . 0 . 1 0 . 1 0 . 0 45 189 35 504 4 . 0 . 4 0 . 4 0 . 1 46 188 43 8181 . 0 . 6 0 . 6 0 . 2 47 187 51 8 7 6 2 4 . 6 .1 6 .1 2 . 5 49 184 25 1 6 0 3 . 0 . 1 0 . 1 0 . 0 5 0 181 43 1 2 9 0 . 0 . 1 0 . 1 0 . 0 51 179 21 1 1 7 7 . 0 . 1 0 . 1 0 . 0 53 176 35 2 2 9 2 . 0 .2 0 . 2 0 .1 54 175 43 1 3 9 0 5 . 1 . 0 1 . 0 0 . 4 56 173 35 1 3 4 6 . 0 . 1 0 . 1 0 . 0 57 172 29 2 2 2 5 . 0 . 2 0 . 2 0 . 1 58 171 43 9 8 2 8 . 0 . 7 0 .7 0 . 3 59 1 7 0 43 6 6 6 8 0 . 4 . 7 4 . 7 1 . 9 6 0 1 6 9 51 2 6 7 9 . 0 . 2 0 . 2 0 .1 61 168 43 4331 . 0 . 3 0 . 3 0 .1 62 166 17 8 4 7 . 0 . 1 0 . 1 0 . 0 65 161 43 5 1 2 5 . 0 . 4 0 . 4 0 . 1 66 160 35 1 5 3 4 . 0 . 1 0 . 1 0 . 0 67 1 5 9 43 4 2 6 2 . 0 . 3 0 . 3 0 . 1 68 158 43 2 4 4 2 5 . 1 .7 1 .7 0 . 7 69 157 43 7 2 4 3 6 . 5.1 5 . 1 2.1 71 155 35 . , 1 1 7 4 . 0 .1 0 .1 0 . 0 72 154 51 ) i ' ' 5 5 0 7 . 0 . 4 0 . 4 0 . 2 73 153 51 1 7 9 1 1 . 1 .3 1 .3 0 . 5 PAGE 2 PEAK H r A S u r r r NO. A B S O L U T E X I N T . X I N T . X T O T . NO. P O I N T S I N T E N S I T Y B A S E NREF ION 74 i s . ' 51 1 5 4 1 9 . ' 1.1 1.1 0 . 4 76 M b 43 9 4 1 9 . 0 . 7 0 . 7 0 . 3 7/ H'J 51 1 2 1 5 4 4 . 8 . 5 8 . 5 3 . 5 /n 144 35 2 4 1 4 . 0 . 2 0 . 2 0 .1 79 143 43 3 7 1 3 . 0 . 3 0 . 3 0. 1 8 0 142 4 3 4 2 4 0 . 0 . 3 0 . 3 0 .1 BI 141 51 2 2 2 5 . 0 . 2 0 . 2 0 . 1 B2 1 4 0 SI 8 9 3 7 . 0 . 6 0 . 6 0 . 3 83 139 « 5 9 7 4 7 2 4 . 5 . 2 5 . 2 2 . 2 34 138 25 861 . 0 . 1 0 . 1 0 . 0 86 136 35 1 7 9 0 . 0 . 1 0 .1 0 .1 OU 134 43 1 1 9 8 . 0 . 1 0 .1 0.0 B'J 133 43 3 0 6 8 . 0 . 2 0 . 2 0 . 1 9 0 131 51 2 2 0 1 . 0 . 2 0 . 2 0 . 1 91 130 4 3 1 2 9 6 . 0 . 1 0 . 1 0 . 0 92 1 2 9 4 3 2 0 2 4 3 . 1 . 4 1 .4 0 . 6 93 128 51 8 0 4 2 8 . 5 . 6 5 . 6 2 . 3 94 127 5 9 4 5 2 3 9 . 3 . 2 3 . 2 1 .3 95 126 5 9 3 2 6 0 . 0 . 2 0 . 2 0.1 96 123 43 3 6 9 2 . 0 . 3 0 . 3 0 . 1 97 121 51 1 9 2 4 . 0 . 1 0 . 1 0 . 1 98 118 43 2 5 6 1 . 0 . 2 0 . 2 0 . 1 99 117 51 2 0 7 8 7 . 1 .5 1 .5 0 . 6 m n 116 43 3 3 9 6 3 . 2 . 4 2 . 4 1 . 0 101 115 51 1 9 4 7 0 8 . 1 3 . 6 1 3 . 6 5 . 7 102 1 14 43 3 1 5 4 . 0 . 2 0 . 2 0 . 1 103 113 51 2 0 3 9 . 0 . 1 0 . 1 0 .1 104 112 51 7 8 5 8 . 0 . 6 0 . 6 0 . 2 105 111 5 9 1 4 9 4 7 . 1 . 0 1 . 0 0 . 4 106 1 1 0 5 9 2 5 8 4 1 . 1 .8 1 .8 0 .8 107 1 0 9 51 2 6 9 8 . 0 . 2 0 . 2 0.1 109 106 43 1 6 8 6 . 0 . 1 0 . 1 0 . 0 1 10 105 43 3 3 8 8 . 0 . 2 0 . 2 0 . 1 1 1 1 104 43 3 9 8 3 . 0 . 3 0 .3 0 . 1 112 103 43 9 7 7 6 0 . 6 . 8 6 . 8 2 . 8 113 102 43 8 7 5 0 . 0 . 6 0 . 6 0 . 3 114 101 43 1 5 9 7 6 . 1 . 1 1.1 0 . 5 115 100 43 2 3 0 4 . 0 . 2 0 .2 0 .1 116 9 9 5 9 1 5 3 2 4 . 1 . 1 1.1 0 . 4 117 9 8 5 9 2 2 5 7 8 . 1 . 6 1 .6 0 . 7 1 18 97 5 9 4 5 1 5 9 . 3 . 2 3 . 2 1 .3 1 19 9 5 51 6 9 0 4 . 0 . 5 0 . 5 0 . 2 120 9 3 43 1091 . 0 . 1 0 . 1 0 . 0 121 9 0 2 9 9 8 7 . 0 . 1 0 . 1 0 . 0 122 89 43 2 7 7 7 . 0 . 2 . 0 . 2 0 . 1 123 88 3 5 1 5 6 0 . 0 . 1 0.1 0 . 0 124 87 51 1 7 8 3 5 . 1 .2 1.2 0 . 5 125 86 71 4 2 5 6 5 . 3 . 0 3 . 0 1 .2 126 8 5 71 5 2 9 8 7 . 3 . 7 3 . 7 1 . 5 127 84 5 9 2 4 2 2 . 0 .2 0 . 2 0 . 1 120 83 71 6 1 9 8 . 0 . 4 0 . 4 0 . 2 129 82 43 3 1 5 9 . 0 . 2 0 . 2 0 . 1 130 81 S l i , 1 3 1 1 0 . 0 . 9 0 . 9 0 . 4 131 (10 4 3 * 2 1 2 0 . 0 . 1 0 .1 0 . 1 132 79 4 3 9 1 5 . 0 . 1 0 . 1 0 . 0 PAGfc 3 PC/K MEASURED NO. ABSOLUTE X INT. X INT. X TOT. fid. POINTS INTENSITY BASE NRCF ION 1.13 77 43 5909 . 0. 4 0. 4 0.2 13!. 7 5 4 3 594 3 . 0. 4 i». 4 0.2 136 74 43 1/499. 1 . 2 1 . 2 II. b 137 73 43 3t.'9B . 2 . 7 » _ 7 1 . 1 1 38 72 51 1 564 . 0. 1 if. 1 0 . 0 13<J 71 59 16147. 1 . 1 1 . ] 0.5 140 70 51 5982. 0. 4 a. 4 0.2 141 69 59 31944. 2. 2 2. 2 0.9 142 68 « SI 1 5UB6. I . 1 1 . 1 0 . 4 143 67 43 4545 . 0. 3 II. 3 e. i 144 66 43 5282. 0. 4 a. 4 a. :• 14b 62 43 1190. 0. 1 it. 1 0.tf 146 61 43 11712. 0. a u. 8 0.3 147 60 43 4 137. 0. 3 i i . 3 0.1 148 59 51 4220. 0. 3 0. 3 0. 1 ISO 57 51 4524 . 0. 3 a. 3 0.1 101 56 51 9083. 0. 6 H. 6 0.3 152 55 51 19220. 1 . 3 l . 3 0.6 153 54 43 3092. 0. 2 o. 2 0.1 1S4 53 43 1421 . 0. 1 u. 1 0.0 185 52 43 1683. 0. 1 ti. 1 D.a 157 47 59 2476. 0. 2 u. 2 0. 1 158 46 51 1294 . 0. 1 0. 1 0 . 0 159 45 71 18328. 1 . 3 l . 3 0.5 i t y 44 71 71168. 5. 0 5. 0 2. 1 161 43 87 1428160. 100. 0 100. 0 41.5*" 162 42 59 36123. 2. 5 2. 5 1 . 1 163 41 51 18168. 1 . 3 1 . 3 0.5 t - 154 -APPENDIX III.2 Mass Numbers of B-amino - 155 -MASS LIST DATA: 01/31/84 14: 00: 00 + 13: 49 SAMPLE: A-HRA 40 0. 00 MINIMA MIN : 430 * 0 MAXIMA MASS •/. RA MASS 7. RA 40 0. 24 101 1. 21 41 1. 23 102 14. 59 43 100. 00 103 2. 46 44 4. 36 104 0. 21 45 1. 25 105 0. 09 50 0. 06 108 0. 07 51 0. 13 109 0. 85 52 0. 09 110 1. 86 53 0. 16 111 0. 96 54 0. 10 112 0. 41 55 1. 13 114 4. 07 56 1. 38 115 3. 42 57 0. 84 116 0. 64 58 0. 30 117 0. 17 59 0. 99 118 0. 13 60 9. 38 122 0. 17 61 0 78 123 0. 13 62 0. 07 124 0. 17 65 0. 12 125 0. 16 67 0. 21 126 1. 88 68 1. 27 127 1. 35 69 1. 25 128 2. 66 70 0. 74 129 0. 32 71 0. 55 130 0. 24 72 2. 22 131 0. 12 73 1. 58 132 0. 62 74 0. 26 133 0. 12 75 0. 09 134 0. 13 77 0. 10 135 0. 08 78 0. 09 136 0. 11 79 0. 12 137 0. 05 80 0. 24 138 4. 07 81 0. 33 139 7. 39 82 0. 41 140 0. 76 84 35. 68 141 0. 17 85 6. 91 142 0. 3B 86 1. 49 143 0. 51 87 0. 14 -144 24. 77 88 0. 22 145 6. B3 89 0. 06 146 0. 52 90 0. 39 147 0. 08 91 0. 06 150 0. 65 92 0. 07 151 4. 13 93 0. 10 152 54 94 0. 13 153 9. 73 95 0. 08 154 0. 16 96 3. 05 155 0. 16 97 2 73 156 5. 15 98 l. 19 L57 3. 68 99 0. 29 158 0. 55 100 0. 64 159 0. 11 FB40 # 790 BASE M/E: 43 RIC: 389632. 56. MAX INTEN: 111744. •/. RA MASS 7. RA o. 07 232 0. 20 0 07 234 0. 11 l . 92 240 0. 46 o. 89 241 0. 07 3. 39 242 0. 22 0. 48 246 0. 76 0. 24 247 0. 10 0. 05 248 0. 07 0. 84 250 0. 03 0 21 253 1. 24 0. 05 254 0. 29 0. 33 256 0. 09 0. 43 258 4. 25 0. 08 259 7. 27 0. 06 260 0. 86 0. 14 261 0. 11 3. 95 270 0. 10 1. 11 271 1. 23 0. 32 272 1. 01 0. 08 273 0. 19 0. 07 274 0. 13 0. 08 276 0. 82 0. 03 277 0. 15 2. 93 278 0. 06 0. 63 281 0. 12 0. 09 283 0. 06 0. 09 288 0. 39 2. 16 2B9 0. 26 1. 13 290 0. 13 0 24 292 0. 07 0. 13 300 0. 86 0. 03 301 0. 15 0. 25 302 0. 06 0. 10 313 0. 25 0. 23 314 0. 33 0. 05 318 5. 76 0. 39 319 0. 98 1. 09 320 0. 20 1. 86 321 0. 07 0. 23 330 0. 14 0 10 331 0. 61 0. 65 332 0. 30 2. 51 333 0. 09 0. 30 334 0. 08 0. 06 348 0. 06 0. 05 360 0. 83 0. 07 361 0. 12 0. 23 373 0. 06 0. 49 374 0. 24 0. 67 375 0. 06 0. 30 MASS 162 165 168 169 170 171 172 173 174 175 179 180 181 182 183 184 186 187 188 189 190 191 192 193 194 193 197 198 199 200 201 203 204 205 207 208 210 211 212 213 214 216 217 218 219 224 226 228 229 230 231 - 156 -APPENDIX III.3 Mass Numbers of HF-B (Hexitol Hexaacetate) - 157 -tr m a LU LI li. u. . n ^ H 41 « o* iii ^ m — — <> rfi - « . « , « B t l n o i n i ? i o o n r>. »• n > n w r» o • n c • « i « o - r » ^ O N O o a "» — — — — — — — — — ntramntmn • oi — lil m <t — m -o • n n n o i« u o • n <o B n ~ m N — » > n r * o - o * r i a ) — * m o> o n — o — — . — — — — — — -. — m m m id \'\ — »r>-0fva)ajt>oo-- 158 -APPENDIX III.4 Mass Numbers of HF-B (2-Acetamido-2-deoxy-penta-0-acetyl-D-hexitol) - 159 -VJ N O fH — I J i« *• <4 0" O -41 -*j (h o n -* I D im 0) o • o n n n <n m i i — O T - r — omn)>in'?a)*a)n>.n — or* • tn 4) N CD Q i> o — — rn ci * -a N o i/ to r» o CJ . m « in o CJ — n n i* — — ~ « « Q i n o o * n a ) O D ' V M i i « n ( D ' * M 9 4 4 o a ) ' « • . • • o s M ^ i o o « c i i r ' t " » i n M D i ' - * - o c i r ! Cii * ^ « - -1 c, It; i", i r i f j o 6 a ci r x n n n « UJ n m -o t i rt - N o < c. o a- « tr rt*in«Ka)O0kO^nin«rtntnNt9chrtnrirvrt rt rt ... .- ~ rt rt — -. m r * ni r< - 160 -APPENDIX III.5 Mass Numbers of 2,3,6-tri-O-methyl-D-glucitol - 161 -MASS LIST DATA: FB41 « 337 BASE H/E 43 ©1/31/84 1 4 3 8 0 © • 9:24 RIC: 3701630. SAMPLE- MHRA 40 0. 00 MINIMA MIN INTEN 57. MAX INTEN : 401920. 447 * 0 MAXIMA MASS RA MASS X RA MASS X RA MASS X RA 40 2. 30 95 2. 36 151 0 03 207 0. 05 41 38. 22 96 0. 96 152 0 03 208 0. 02 43 100 00 97 9 82 153 0. 81 211 0. 02 44 29. 87 9B 35. 86 154 0. 15 213 0. 50 43 62. 48 99 56. 94 153 1. 03 214 0. 07 46 4. 00 100 14. 82 156 0. 90 213 0. 08 47 3. 85 101 56. 75 157 8. 68 216 0. 13 48 0. 10 102 9. 20 158 1. 03 217 0. 76 49 0. 04 103 7. 11 159 a. 44 218 0. 09 50 0 27 104 0. 69 160 0. 90 219 0. 02 51 0. 57 105 0. 43 161 26. 91 220 0. 03 52 0. 76 106 0. 05 162 2. 12 221 0. 04 S3 12. 10 107 0. 09 163 0. 30 223 0. 02 34 2. 89 108 0. 28 165 0. 04 227 0. 02 55 22. 55 109 1. 27 166 0. 05 229 0. 04 36 6. 19 111 13. 75 167 0. 16 230 0. 04 57 16. 50 113 63. 38 168 0. 05 231 0 66 58 28. 95 114 12. 68 169 0. 11 233 51. 46 59 25. 86 113 13. 54 170 0 16 234 8. 96 60 3. IB 117 73. 50 171 2. 58 233 1. 52 61 2. 61 118 17. 07 173 23. 57 236 0. 16 62 0. 09 119 3. 11 174 3. 81 237 0. 03 63 0. 13 120 0. 19 175 0. 85 239 0. 02 64 0 07 121 0. 09 176 0. 09 242 0. 02 65 1. 22 122 0. 05 177 0 04 245 0. 56 66 0. 63 123 0. 15 179 0 02 246 0. 06 67 2. 48 124 0. 72 180 0 03 247 0. 03 68 4. 99 125 5. 84 1B2 0. 05 248 0. 02 69 13. 11 126 0. 88 183 0 20 249 0. 10 70 6. 50 127 8. 63 184 0. 18 250 0. 01 71 44 14 129 45. 73 185 0 98 251 0. 01 72 5. 71 130 5. 96 1B6 0. 17 254 0. 02 73 21. 50 131 40. 83 187 0. 63 25B 0 04 74 30 73 132 3. 28 IBB 0. 15 260 0. 02 75 31 97 133 0. 49 189 1. 15 273 0. 18 76 1. 19 134 0. 04 190 0 37 274 0. 04 77 0 68 135 0. 03 191 1. 17 275 0. 02 78 0 19 136 0. 03 192 0. 12 277 1. 04 79 0 6B 137 0 05 193 0. 03 278 0. 12 81 16. 66 139 6. 04 194 0 02 279 0 03 82 2. 57 140 0. 77 195 0. 03 281 0. 02 83 7. 67 141 5. 23 197 0. 01 2B9 0. 06 85 42. 29 142 18. 03 19B 0. 08 291 1. 29 87 64. 27 143 21. 05 199 0. 23 292 0 18 88 21. 50 144 2. 23 200 0. 14 293 0 02 89 11. 62 145 1. 35 201 1. 22 303 0 02 90 0. 58 146 0. 14 202 0. 16 305 0. 05 91 0. 16 147 2. 50 203 3. 69 314 0. 02 92 0. 04 148 0. 40 204 0 33 319 0. 10 93 0. 59 149 0. 13 205 0. 07 320 0. 01 94 0 60 150 0. 03 206 0. 02 351 0. 04 - 162 -APPENDIX III.6 Mass Numbers of 4,6-di-0-methyl-2-deoxy-2-N-methylacetamiclo hexitol - 163 -MASS LIST DATA: FB41 # 726 BASE M/E: 43 01/31/84 14:38 00 • 12:42 RIC 973824. SAMPLE: MHRA 40 0. 00 MINIMA MIN INTEN 132. MAX INTEN: 162! 451 * 0 MAXIMA RA MASS X RA MASS X RA MASS X RA MASS X 40 0. 42 100 4. 68 157 1. 35 228 0. 58 41 5. 31 101 9. 26 158 51. 10 229 0. 93 42 10. 26 102 1. 17 159 4. 76 230 2. 54 43 100. 00 103 0. 40 160 O. 58 231 0. 33 44 6. 77 104 0. 38 161 6. 53 232 0. 26 45 38. 07 105 0. 10 162 0. 52 233 0. 19 46 0. 89 106 0. 10 163 0. 13 234 0. 10 47 0. 24 107 0. 15 165 0. 09 240 0 49 51 0. 24 108 0. 40 166 0. 38 241 0. 15 52 0. 18 109 0. 34 167 0. 19 242 2. 54 53 0. 67 110 2. 07 168 0. 42 243 0. 42 54 0 74 111 1. 28 169 0. 13 244 2. 14 55 3. 12 112 1. 65 170 7. 12 245 0. 28 36 7. 50 113 0. 99 171 0 93 246 0. 18 57 6. 21 114 1. 43 172 1. 70 250 0. 09 58 3. 09 113 3. 27 173 0. 30 236 0. 35 39 1. 98 116 B7. 09 174 0 16 257 0. 33 60 0. 84 117 7. 09 179 0. 51 258 1. 76 61 0. 42 118 0. 70 180 2. 57 259 0 32 65 0 25 119 0. 80 181 0 37 260 0. 08 66 0 IB 122 0. 26 182 0. 41 270 0. 47 67 0 73 123 0. 23 1B3 0. 16 271 1. 98 68 2. 11 124 2. 03 184 1. 00 272 6. SB 69 2 70 125 1. 95 185 0. 38 273 0. 84 70 2. 71 126 0. 95 186 0. 73 274 5. 80 71 4 66 127 1. 57 187 0 21 275 0. 80 72 2. 03 128 4. B7 18B 1. 18 276 1. 86 73 5. 30 129 22. 52 189 0. 13 277 0. 25 74 32. 44 130 2 46 190 0. 13 281 0. 17 75 2. 08 131 0. 52 191 0. 09 284 0. 11 77 0. 19 135 0. 10 193 0. 09 286 0. 47 78 0. 12 136 0. 15 196 3. 36 287 0. 10 79 0. 27 137 0. 24 197 0. 91 288 0. 48 80 0 30 138 0 94 198 1. 46 289 0. 11 81 0. 74 139 0. 50 199 0. 34 290 0. 12 82 1. 50 140 1. 47 200 0 70 292 0. 12 83 0. 98 141 0. 70 201 0. 27 299 0. 13 84 1. 3B 142 6. 60 202 0. 43 300 1. 65 85 2. 49 143 1. 29 203 4. 12 301 0. 33 86 2. 92 144 0. 25 204 0. 39 302 0. 08 87 6. 05 143 0. 13 207 0. 20 316 0. 72 B8 0. 84 146 0. 50 211 1. 25 317 0. 32 89 0. 69 147 0. IB 212 1. 71 318 4. 30 91 0. 14 149 0. 09 213 0. 21 319 0. 76 93 0. 31 150 0. 16 214 0. 86 320 0. 13 94 1. 02 151 0. 10 215 0 19 331 0. 21 95 0. 59 152 1. 12 216 0. 44 332 0. 60 96 0. 73 153 0 46 217 0. 13 333 0. 12 97 1. 50 154 1. 00 221 0. 11 346 0. 11 9B 7. 17 155 1. 21 226 0. 22 348 0. 08 99 6. 53 156 1. 62 227 0. 18 359 0. 09 - 164 -APPENDIX III.7 Mass Numbers of per-methylated deoxy component (alditol acetate) - 1 6 5 -MASS LIST 01/31/84 14:SB 00 • SAMPLE: MHRA 11: 11 DATA: FB41 « 639 BASE M/E 43 RIC 3653630 40 0 00 MINIMA MIN INTEN 123. MAX INTEN : 342< 449 # 0 MAXIMA MASS "/. RA MASS. X RA MASS •/. RA MASS •/. RA 40 2. IB 95 4. 11 148 0 05 207 e. 07 41 28. 63 96 1. 40 149 0 09 211 0 30 43 100. 00 97 10. 61 130 0. 04 212 0. 07 44 16 93 98 1. 84 151 0 24 213 0 20 45 69. 24 99 37. 80 152 0 17 214 0 46 46 2. 78 100 9. 06 133 0 84 215 4. 11 47 0. 74 101 9 IB 154 0 57 216 0 57 48 0 04 102 0. 75 155 10 83 217 0. 36 30 0. 19 103 0. 99 156 3. 91 21B 0 04 51 0. 64 104 0 17 157 3. 25 219 0 22 52 0 71 103 0. 42 158 0. 84 221 0 07 33 6. 35 106 0 07 159 5. 19 224 0 34 54 3. 66 107 0. 14 160 0. 71 225 0 05 55 21 37 108 0. 40 161 1. 01 228 0 66 36 16 65 109 2. 62 162 0 10 229 0 61 37 12. 26 110 0. 9B 163 0 04 230 0 68 58 47. 68 111 11. 79 164 0 07 231 12. 39 59 54. 72 112 3. 95 165 0 17 232 1. 27 60 3. 30 113 22. 94 166 0 17 233 0 40 61 3. 12 114 3. 48 167 0. 27 234 0. 05 62 0. 10 113 8 08 168 2. 58 243 0 61 63 0 14 117 75 82 169 6. 46 244 0. 10 65 1. 28 118 10. 67 170 13 15 245 0. 21 66 0. 97 119 1. 79 171 40 12 246 0. 14 67 3. 45 120 0 15 172 3. 88 247 0. 05 6B 3. 60 121 0 13 173 1. 35 248 0 10 69 11. 51 122 0 09 174 0. 19 236 1. 63 70 2. 84 123 0 68 175 0. 14 257 0. 26 71 31. 55 124 0 65 177 0. 08 258 0 05 72 3. 06 123 4 28 17B 0 07 239 0. 16 73 12. 93 126 1. B5 180 0 07 261 1. 56 74 7. 71 127 7. 98 1B2 2. 59 262 © 19 75 13 19 128 2. 15 183 " 5. 72 263 0. 09 76 0 52 129 13. 81 1B4 3. 66 264 0 16 77 0 78 130 1. 09 1B5 1. 56 271 0 70 7B 0 22 131 0 59 1B6 0 46 272 0 11 79 0 77 133 0 84 1B7 14. 26 273 0. 26 B l 11 92 134 0 07 IBS 1. 34 276 0 04 B2 6. 25 135 0. 08 189 7. 49 277 0 05 83 7 63 136 0 26 190 0. 67 2B8 0. 04 84 2. 53 137 1. 07 191 0 16 289 0 10 85 26 42 138 0 82 192 0. 09 290 0. 33 86 3. 90 139 0. 87 196 4. 09 291 0 04 87 21. 00 140 2. 29 197 0. 78 303 0. 66 88 2. 13 141 3. 80 198 1. 15 304 0 11 89 3. 23 142 4. 51 199 0. 45 305 0 18 90 0 17 143 43. 64 200 0. 28 306 1. 06 91 0 21 144 3 95 201 4 42 307 0. 13 92 0 04 145 3. 93 202 0 68 348 0 70 93 0 BO 146 0 32 203 0 88 349 0 64 94 0. B3 147 0. 54 204 0. 09 350 0 11 

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