Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1984_A1 L43.pdf [ 12.99MB ]
Metadata
JSON: 831-1.0096556.json
JSON-LD: 831-1.0096556-ld.json
RDF/XML (Pretty): 831-1.0096556-rdf.xml
RDF/JSON: 831-1.0096556-rdf.json
Turtle: 831-1.0096556-turtle.txt
N-Triples: 831-1.0096556-rdf-ntriples.txt
Original Record: 831-1.0096556-source.json
Full Text
831-1.0096556-fulltext.txt
Citation
831-1.0096556.ris

Full Text

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 ISOLATED FROM PSEUDOMONAS FRAGI A T C C By PHILLIPJLEE  WING  B . S c , T h e U n i v e r s i t y of M a n i t o b a , 1976 B . S . A . , T h e U n i v e r s i t y of M a n i t o b a , 1978 M . S c , T h e U n i v e r s i t y of M a n i t o b a , 1980  A THESIS SUBMITTED  IN P A R T I A L  FULFILLMENT  T H E R E Q U I R E M E N T S OF T H E D E G R E E D O C T O R OF  OF  PHILOSOPHY in  THE F A C U L T Y OF G R A D U A T E  STUDIES  ( D e p a r t m e n t of Food S c i e n c e )  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to t h e r e q u i r e d  standard  T H E U N I V E R S I T Y OF B R I T I S H May,  COLUMBIA  1984  © P h i l l i p Lee W i n g , 1984  OF  4973  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree t h a t for  understood for  Library  shall  for reference  and  study.  I  f o r extensive copying of  that  h i s or  be  her  g r a n t e d by  f i n a n c i a l gain  shall  not  be  -^TS-E-A^  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3  Date  r<^ * *^A  *5>  o-r-g.  Columbia  \=>^>\  of  make  further this  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  Department o f  the  representatives.  copying or p u b l i c a t i o n  the  University  the  p u r p o s e s may by  the  I agree that  permission  department or  f u l f i l m e n t of  advanced degree a t  Columbia,  scholarly  in partial  written  ABSTRACT  Pseudomonas incubated  at 2 1 ° C , was  transmission which the  appeared  medium,  ATCC  examined  4973,  evaginations  Transmission  or  protrusions  exhibited  randomly  distributed  micrographs  of  surfaces  and  Both scanning  and  e l e c t r o n m i c r o g r a p h s of cells g r o w n  no  on  the  bleb-like  Stationary  phase  cells,  material  on t h e cell s u r f a c e was o b s e r v e d .  Transmission  electron  growth  phase,  evaginations.  grown  in  revealed  In c o n t r a s t , s t a t i o n a r y p h a s e cells to t h e c e l l s .  influenced  the expression  association  between t h e g l y c o c a l y x  The  surface.  revealed bleb-like  extracellular  cells  adjacent  cell  phase,  on solid  Fine  bacterial  logarithmic  environment  meat  as well as c e l l - t o - m u s c l e attachment to  at t h e e a r l y l o g a r i t h m i c g r o w t h  however,  noted.  onto  by electron microscopy.  to mediate c e l l - t o - c e l l  harvested  globules  inoculated  e l e c t r o n m i c r o s c o p y r e v e a l e d an e x t r a c e l l u l a r material ( g l y c o c a l y x )  meat samples.  early  fragi  liquid no  showed The  of blebs  on  harvested  few a t t a c h e d  surface  of  b l e b s as well as  (solid P.  vs.  fragi  formation  of blebs  and  of t h e cell  appeared  to p r e c e d e  globules  liquid)  cells.  and bleb-like evaginations  exocytosic  at t h e  o r e x t r a c e l l u l a r material.  of s u b s t r a t e  the  fibres  blebs  only  type  medium,  An  was  also  into t h e immediate  t h e formation  of g l y c o c a l y x  by  P. f r a g i c e l l s . Chemical hexosaminoglycan used  on t h e isolated  structure.  glycocalyx  monosaccharides  preparative  mass s p e c t r o m e t r y  paper  of P_. f r a g i  T r i f luoroacetic a c i d a n d h y d r o g e n  in t h e h y d r o l y s i s of t h e p o l y s a c c h a r i d e .  individual using  studies  were o b t a i n e d  from  chromatography.  Quantitative the hydrolysed  Gas  liquid  indicated a  fluoride  amounts  were  of t h e  polysaccharide  chromatography  were u s e d in t h e v e r i f i c a t i o n of an N - a c e t y l amino s u g a r  and  - iii -  component.  Monomeric units and 1  were determined  by  substituents in the polysaccharide chain  13 H  and  C nuclear magnetic resonance  By methylation analysis, the polysaccharide of P. fragi was of a linear repeating trisaccharide unit. can structure is:  spectroscopy.  shown to consist  The proposed partial hexosaminogly-  ^  >-4) D-Glucose ( 1 — - 3 ) amino sugar ( 1 — • ?) deoxy sugar (1 — • 2  NAc  - iv  TABLE  OF  -  CONTENTS  PAGE  ABSTRACT TABLE  ii  OF  CONTENTS  LIST  OF  TABLES  LIST  OF  FIGURES  iv viii ix  ACKNOWLEDGEMENTS GENERAL  xiii  INTRODUCTION  PART  1  1  GROWTH OF PSEUDOMONAS FRAGI ATCC 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 MEAT SURFACES  2  INTRODUCTION  3  LITERATURE  4  REVIEW  EXPERIMENTAL  6  A.  Preparation  of Attachment Media  6  B.  Preparation  of Muscle Samples  6  C.  pH  D.  Bacterial  E.  Preparation  of Muscle f o r  SEM  7  F.  Preparation  of Muscle f o r  TEM  8  G.  Microtomy  Determination Counts  7  8  RESULTS A.  7  9 Growth  of  P.  fragi  9  - v -  B.  pH  9  C.  Electron Microscopy  9  DISCUSSION  27  CONCLUSION  33  PART 2  GROWTH PHASE AND C A P S U L A R FINE S T R U C T U R E OF PSEUDOMONAS FRAGI A T C C 4973  34  INTRODUCTION  35  LITERATURE  36  REVIEW  EXPERIMENTAL  40  A.  Culture Conditions  40  B.  Incubation on Solid Medium  40  C.  Incubation in Liquid Medium  40  D.  Kellenberger-Ryter (RK)  40  E.  Sample Preparation  41  F.  Microtomy and Electron Microscopy  42  Fixation  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 S T R U C T U R A L ANALYSIS 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 ISOLATED FROM PSEUDOMONAS FRAGI A T C C 4973  INTRODUCTION  61 62  - vi  L I T E R A T U R E 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 . ) 1.  2.  78  Proton Magnetic Resonance Spectroscopy ( H N . M . R . )  79  C N.M.R. Spectroscopy  80  1 3  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  F.  Spectrometry  84  Nuclear Magnetic Resonance Spectroscopy  84  1.  84  2.  Proton ( H ) 13 1  N.M.R. Spectroscopy  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-methylamidoHexitol Acetates  88  RESULTS  89  DISCUSSION  110  SUMMARY  120  CONCLUSION  123  REFERENCES  124  APPENDICES  134  - viii -  LIST OF T A B L E S TABLE  1 2  PAGE  Rare sugar components of Pseudomonas  polysaccharides  68  Functional group modification of sugar residues  69  3  N.M.R. data for P. fragi polysaccharide  90  4  R |  5  6 7 8  9  Values of components of hydrolysed  polysaccharide on Whatman No. 1 paper G . l . c . analysis of alditol acetates of hydrolysed polysaccharide (2 M T F A ) isolated by prep, paper chromatography 1 H N.M.R. data for hydrolysed P.  fragi  H N.M.R. data for hydrolysed P.  fragi  polysaccharide (2 M T F A ) 1  polysaccharide (HF)  N.M.R. data for P. fragi polysaccharide (de-N-acetylated; deaminated) Representative  1  H and  91 100  101 102 106  13  C chemical shifts  for nuclei of polysaccharides  113  ix -  LIST OF FIGURES FIGURES  1. 2.  3.  4.  5.  6.  7.  8.  9.  PAGE  Growth of P. fragi cells on, and pH of inoculated muscle at various storage times  10  ( A ) Scanning electron micrograph of day 1 muscle sample, inoculated with P. f r a g i , showing the initial formation of fibre-like glycocalyx material ( G ) . (B) This represents a magnification of the area enclosed in the box in micrograph ( A )  12  ( 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  Scanning electron micrograph of the external surface of P. fragi (day 5) encased in a mass of pebble-like extracellular material  16  Scanning electron micrograph of muscle sample (day 5) inoculated with P_. f r a g i . There is an intensification of the pebbling effect on the bacterium surface, as well as a coiling of glycocalyx fibres  18  Scanning electron micrograph of muscle sample (day 5) inoculated with P. f r a g i . There is an intensification of the pebbling effect on the bacterium surface, as well as a coiling of glycocalyx fibres  20  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  23  Scanning electron micrograph of bleb-like protrusions (P) in association with the glycocalyx fibres (G)  25  Schematic illustration of the relationship between the various forms of extracellular material as seen by means of SEM and T E M . (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.  11.  12.  13.  14.  15.  16.  17.  Growth characteristics of P. fragi in liquid (TSB) and on solid (TSA) media, incubated at 21 °C  44  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  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  Transmission electron micrograph of P. fragi cells grown in liquid medium harvested at the early logarithmic phase  50  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  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  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  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 Mass spectrum of amino sugar (alditol acetate) component of hydrolysed polysaccharide  93  Mass spectrum of deoxy sugar (alditol acetate) component of hydrolysed polysaccharide  94  Paper chromatogram of (a) hydrolysed polysaccharide (2 M TFA) and (b) reference standards  96  Mass spectrum of B-neutral (alditol acetate) component of hydrolyzed polysaccharide  98  20. 21. 22.  23.  92  - xi  24.  25.  26.  27.  28.  29.  30.  31.  -  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 p o l y s a c c h a r i d e  99  Mass s p e c t r u m of H F - B . Hexitol hexaacetate component of h y d r o l y s e d p o l y s a c c h a r i d e  104  Mass s p e c t r u m of H F - B . penta-O-acetyl-D-hexitol polysaccharide  105  Mass s p e c t r u m of glucitol  2-Acetamido-2-deoxycomponent of h y d r o l y s e d  2,3,6-tri-O-methyl-D107  Mass s p e c t r u m of 4 , 6 - d i - 0 - m e t h y l - 2 - d e o x y - 2 N-methylacetamido hexitol  108  Mass s p e c t r u m of p e r - m e t h y l a t e d component (alditol acetate)  109  deoxy  F r a g m e n t a t i o n p a t t e r n o b s e r v e d in t h e mass s p e c t r u m of 2 , 3 , 6 - t r i - O - m e t h y l - D - g l u c i t o l  118  F r a g m e n t a t i o n p a t t e r n o b s e r v e d in t h e mass s p e c t r u m of 4 , 6 - d i - Q - m e t h y l - 2 - d e o x y 2 - N - m e t h y l acetamido hexitol  119  - xii -  LIST OF APPENDICES APPENDIX  "  PAGE  I  Infrared Spectroscopy Data  II  N.M.R. Spectroscopy Data  136  III  Mass Spectrometry  150  Data  ,  134  xiii -  ACKNOWLEDGEMENTS  The many  hours  author wishes to thank Dr. B.J. Skura, Supervisor, for the 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 temperatures 1980;  ecology has  of bacterial spoilage of meat at both chill and  been extensively studied  (Gill  and  Newton, 1977,  room 1978,  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 tions, 20°C  floras are dominated by Lactobacillus. Under aerobic condi-  psychrotrophic while  pseudomonads are the predominant spoilage flora at  at 30°C, they  are displaced by  species of Acinetobacter  and  Enterobacteriacae which included both mesophilic and psychrotrophic strains. To understand how (1) which organisms can  spoilage floras develop, it is necessary to know  be present;  (2) the nature of the environment in  which they are growing; (3) the effect on microbial growth of alterations in the  environment;  species  (Gill  and  and  (4) the  interactions which occur  Newton, 1978).  Since,  between competing  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 isms  requires  surfaces.  of the mechanism of meat spoilage by microorgan-  a knowledge of how  Fletcher and  microorganisms initially attach to meat  Floodgate (1973) suggested that a glycocalyx (extra-  cellular polymeric material) may  be involved in this process.  of the extracellular material using transmission and  An examination  scanning electron micros-  copy will, therefore, provide information on its morphology and ultrastructure. Chemical studies of the extracellular material, however, will provide information on its structural components and  conformation.  - 2 -  P A R T  I  G R O W T H  O F  PSEUDOMONAS  G L Y C O C A L Y X  FORMATION  FRAGI ON  A T C C  M E A T  4973  A N D  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 process  been well documented in the literature that the attachment  apparently  involves at least two  Firstenberg-Eden, 1981).  main steps (Butler et a[., 1980;  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  or stage of "permanent adhesion",  Loeb, 1979).  The  second step,  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 to  surfaces by  study was,  microorganisms is particularly  lacking.  The  adhesion  object of this  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  of  for attachment  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 chill  a study of the ecology of bacterial spoilage of fresh meat at  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 was produced,  10 colony forming units (c.f.u.)/g, when ammonia  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).  value was  It was  obtained in meat undergoing  further reported that a high pH  aerobic spoilage since the pH  almost  invariably increased to 6.5 from the usual postrigor value of ca.5.5. Although by  the study of microbial meat spoilage may  be  accomplished  the enumeration of bacterial numbers as well as using subjective para-  meters for evaluation, the use of electron microscopy has been mainly explored in the last few decades. mission electron microscopy  in this area of study  Both scanning and trans-  have been used to examine the attachment of  bacteria to meat surfaces (Butler et aL,  1980;  teats of cows (Firstenberg-Eden et aL , 1979;  Firstenberg-Eden, 1981); to 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 Floodgate, 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  chosen methods (depending  on  shrinkage  (Brunk  et aL,  1981), carefully  the type of material being examined) have  been developed  to minimize distortion of cellular morphology (Whittaker and  Drucker,  Domagala et aL,  1970;  aL, 1979; Watson et aL , 1980).  1979;  Garland  et aL,  1979;  Rittenburg et  - 6 -  EXPERIMENTAL:  Bovine from (3  a  mm)  Canada to  local of  abattoir  muscle  Inc.,  minimize  were  longissimus  Don  and  prepared  Mills,  Ont.).  with  muscles,  transported  were  microbial  sterilized  dorsi  using  kGy  ice a  Aseptic  contamination  10  on  24  h  post  to  the  Hobart  to  laboratory.  were  using  obtained  Thin  slicer  maintained  irradiation.  of gamma-radiation  were  delicatessen  conditions  prior  mortem,  All  slices  (Hobart in  order  muscle  slices  a Gammacell  220  (Atomic  60 Energy  of C a n a d a L t d . ,  Kanata,  Pseudomonas Culture  Collection  agar  slants  week  intervals.  day  (ATCC,  (BBL,  All  fragi  Ont.)  ATCC  4973,  Rockville,  Cockeysville,  analyses  containing  were  performed  Preparation The  of  Attachment  attachment  medium  Preparation The  attachment Skura  treated,  medium  (1981)  incubation  gamma  of  was the  sterilized  samples  used samples  for in  in a s i m i l a r m a n n e r ,  intervals  on  trypticase  subcultured  of  0,  Type  1,  2,  at  3  soy 8-10  and  5  The sample  controlled  P.  fragi  cells  was  reconstituted  (1981).  were  fragi.  further a  was  American  samples.  containing  Samples:  P_.  the  maintained and  time  from  1974).  Media:  of Muscle  containing  4°C  at  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  B.  were  at  s t o r a g e on both inoculated and control  A.  obtained  MD)  MD)  Co (Sage,  immersed method  with sterile attachment  10 m i n  described  treatment  environment.  for  which Control  medium.  by  in  4  L  of  Yada  and  involved  the  samples  were  -  C.  pH  homogenized  each  with  Inc.,  determined  A  and  E.  2  buffer  tetroxide This  ethanol  (pH  The  (J.B.  using  (4  muscle  water  Omni  of  a  g)  the  was  in  an  resultant  EM  was  three  20  water.  Instrument silver  Co.,  for  were  Mixer  slurry  with  stomacher  Bacterial  was  (Ivan  was  then  36  mL  cut  24  h;  7.0)  Services  21°C  Inc.)  followed  by  50,  70,  80% f o r  min  periods.  Moline, (J.B.  evaporation.  Samples  o p e r a t e d a t 40  kV.  into  2  lab-blender  enumeration  EM were  before  in  0.05  5 min  All  cm  cubes  of  400  both  prior  Montreal,  0.1% (A.J.  inoculated  h.  were  further  through  each,  dilutions  point  CO2.  dried  Samples  Services  Inc.)  viewed  with  were the  fixation  in 0 . 0 5  M  rinsed  twice  in  fixation  with  1%  buffer  a graded  90% f o r  to  Que.)  M phosphate  ethanol  critical  using  2  Samples  dehydration  were  IL)  x  Inc.,  4°C).  at  sterile  SEM:  EM S e r v i c e s  Samples  paste  sample  230).  blended  Colworth  England).  (J.B.  (pH  solutions:  deionized  pH  representative  pH/ion meter (Model  sample  of M u s c l e  7.0;  buffer  step  100% f o r  min  tissues  glutaraldehyde  phosphate  5 g  s a m p l e s w a s 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 a t 21 ° C f o r 48  Muscle  phate  CT).  London,  Preparation  2.5%  a  Counts:  for  Co.,  time,  distilled-deionized  representative  and control  with  mL  Norwalk,  peptone  Seward  h.  5  Bacterial  (w/v)  incubation  with a F i s h e r A c c u m e t  D.  -  Determination:  After  Sorvall  7  two  were in  series  10 m i n made a  mounted coated  (pH  with  Cambridge  phos-  0.05  7.0) of  by  and  distilled(Parr  aluminum gold  for 1  aqueous  periods  with  M  osmium  Parr-bomb on  with  stubs  vacuum  Stereoscan  250,  - 8-  F.  Preparation of Muscle for T E M : 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  21°C)  in 0.015% dye [ruthenium  (pH 7.0) prior to post-fixation (2 h,  red (Aldrich  blue (J.B.  solution.  Samples, after 2 h in 0.05 M phosphate buffer containing 0.05% dye  buffer)  Inc.)]  Wl)  or alcian  (DP  EM Services  Chemical C o . , Milwaukee,  buffered with a 0.05 M phosphate  at 21 ° C , were washed in five changes of the same solution for 1  h each, with one overnight wash included. in  2% osmium tetroxide  buffer.  Dehydration  in DP buffer,  was achieved  70, 90 and 100% ethanol.  followed by five 1 h washes  by immersion of samples in 15, 30, 50,  Samples, following dehydration, were washed  in two changes of propylene oxide (J.B. with  a 1:1  in DP  T h e lower concentrations of ethanol were prepared  with distilled deionized water.  infiltrated  Samples were post-fixed for 2 h  mixture  EM Services  Inc.) for 15 min each,  of propylene oxide and Epon 812 (J.B.  EM  Sevices Inc.) for 16 h, then embedded in 100% Epon 812.  G.  Microtomy: Tissues  (Ivan  Sorvall  were  Inc.,  sectioned  Norwalk,  on a  "Porter-Blum"  MT-2 ultramicrotome  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 k V .  - 9 -  RESULTS  A.  Growth of P. fragi: At day  0 there were between 10  the muscle surface. or day  5.  No  5  and  bacteria were detected  10  6  P. fragi c.f.u./cm  ?  on  in control samples at day 0  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  muscle from pH  6 (day 0) to pH  a marked increase in pH  9 (day 5).  No pH  of inoculated  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 extracellular material (glycocalyx) extending ganism (Fig. 2).  of a fibre-like  from the surface of the microor-  At higher magnifications, a pebble-like extracellular mater-  ial on the outer surface of the microorganisms could be seen (Fig.3). more detailed sequential stages of fibre-like extracellular material  The  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 intensification 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 -  Fig.  1  G r o w t h o f P. f r a g i storage times. ( • ( # ( •  cells o n ,  and  pH  ) control - sterile sample ( p H ) ) s a m p l e i n o c u l a t e d w i t h P. f r a g i ) s a m p l e i n o c u l a t e d w i t h P. f r a g i  All points represent the average  of  inoculated  (pH) (growth)  of t h r e e  trials.  m u s c l e at  various  - II  -  - 12 -  Fig. 2  ( A ) 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 of d a y 1 muscle sample, i n o c u l a t e d with P. f r a g i , s h o w i n g t h e initial formation of f i b r e - l i k e g l y c o c a l y x material ( G ) . B a r = 10 urn. ( B ) T h i s r e p r e s e n t s a m a g n i f i c a t i o n of t h e a r e a e n c l o s e d in t h e box in m i c r o g r a p h ( A ) . B a r = 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 -  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 of the e x t e r n a l s u r f a c e of P. f r a g ( d a y 5) e n c a s e d in a mass of p e b b l e - l i k e e x t r a c e l l u l a r material B a r = 1 pm.  -  Fig.  5.  18  Scanning electron micrograph with P. fragi. T h e r e is a n o n t h e b a c t e r i u m s u r f a c e , as B a r = 1 urn.  -  of muscle sample ( d a y 5) inoculated intensification of the p e b b l i n g effect well as a c o i l i n g of g l y c o c a l y x f i b r e s .  - 20 -  Fig. 6  Scanning electron micrograph of muscle sample (day 5) inoculated with P. f r a g i . 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 transmission  electron  microscope.  contrast of structures.  Ruthenium  red,  however,  gave a slightly  better  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.  T E M , combined with ruthenium red and  alcian  various  blue  associated  staining, with  extracellular  demonstrated  attachment.  material  are  Two  shown  in  examples Fig. 7.  forms of  of  the  extracellular  material  morphological forms of  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 ( F i g . 8). attachment (Fig.  8).  of extracellular  A close association of these blebs to the site of fibres to the bacterial surface was also observed  - 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) ciation with the glycocalyx fibres ( G ) . Bar = 0.5 ^im.  in asso-  - 27 -  DISCUSSION:  Notermans temperature  for  and Dainty  (1971)  and  Kampelmacher  attachment  of  (1974),  Pseudomonas  showed  strains  was  reported that at higher temperatures  that  the  optimum  ca. 21 ° C .  Ingram  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. the  classical  Under  growth  fragi on meat surfaces incubated at 21 ° C followed  pattern  with  a  lag  period  of  approximately  3 days.  aerobic conditions, psychrotrophic pseudomonads accounted for 60% of  the spoilage flora at 20°C (Gill and Newton, 1980).  P. f r a g i , being versatile  in its ability to grow at 21 ° C , is also representative of psychrotrophic spoilage microorganisms. The mediate  type  of biochemical changes o c c u r r i n g , particularly  temperatures  Contradictory  reports  (15°-25°C) on  the  has  not  correlation  been  adequately  between  pH  and  at  documented.  the  number of  microorganisms present on meat surfaces are found in the literature 1960; that  Rogers a  high  and McCleskey, 1961). pH  value  may  exist  unconnected with bacteriology increase fragi 1972).  Furthermore,  in muscle for  (Bate-Smith,  (Turner,  it has long been evident  physiological reasons  1948).  inter-  Nevertheless,  the  quite sharp  in pH of inoculated muscle was due to the rapid growth rate of  and  the  probable  increased  production  of  amines and ammonia  P.  (Jay,  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  components appears to indicate that this material  the  fibrous  extracellular  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.  bacterium surface for the determination  An  examination of the  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 ium red or alcian blue was chers (Behnke, 1968;  used for TEM  Behnke and  be involved in adhesion, ruthenstudies.  Although various resear-  Zelander, 1970) have used alcian blue as a  means of improving fixation or increasing electron density of acidic polysaccharides, 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  which appears in extracellular spaces between cells treated with red may  or  molecule,  alcian blue are similar.  and  ionic  may  forces.  however,  be described The  charge  In  Both are metal-  polyvalent basic dyes which precipitate similar polyanions  et a[., 1964) static  ruthenium  be associated with protein as protein-polysaccharide complexes.  several ways, ruthenium red and containing  density  (Scott  as being reactive primarily by electrodistribution  is different from, and  over  probably  the  higher  alcian blue which has fewer localized charges (Luft, 1971a).  ruthenium  red  than, that of  - 29 -  Three  mechanisms  ruthenium  red  which  referred  was  ruthenium The  (Luft,  red  and  ruthenium  h a v e been p r o p o s e d to e x p l a i n t h e s p e c i f i c i t y  1971a;  to  as  a  1971b).  The  "catalytic  some g r o u p s  r e d is o x i d i z e d  of  model",  glycan  by O s 0  4  first  hypothetical  implies  chains  a close  (e.g.  thus  be  functions  able  to  reduce  products.  The  explanation  in  group  which  model o r  as  a catalyst  several  second that  binds  "chain  the  between groups).  to r u t h e n i u m b r o w n , a n d t h e r u t h e n -  of  OsO^  "self-propagating"  another  fit  The  ruthenium  in t h a t one molecule of r u t h e n i u m  molecules  oxidation  mechanism,  hydroxyl  ium b r o w n in t u r n o x i d i z e s t h e g l y c a n to w h i c h i t is b o u n d . red  of  the  ruthenium  of  model  glycan  red  to  electron-dense provides  substrate  molecule  red  and  an  may  insoluble alternative  generates  so f o r t h .  r e a c t i o n m e c h a n i s m " a r r e s t s t h e r e d u c t i o n of O s O „  a  new  The  last  at osmate  4  anion the  instead  electron  of  promoting  density  results  i t s r e d u c t i o n to lower o x i d a t i o n from formation  of  products  a layered complex.  so t h a t Blanquet  (1976a,  1976b) also p r o p o s e d a mechanism w h i c h e m p h a s i z e d t h e i n i t i a l f o r m a -  tion  a stable  c y c l i c osmic a c i d d i e s t e r  dioxide,  w h i c h in t u r n  of  osmium  bond g i v i n g  r i s e to  associates with ruthenium  "colloidal-like"  r e d to p r o d u c e  the  electron-dense positive marker. T E M v e r i f i e d c o n c l u s i o n s , made f r o m s c a n n i n g e l e c t r o n extracellular  that  thin  TEM  r e v e a l e d p e b b l i n g on t h e s u r f a c e of t h e P_. f r a g i c e l l s w h i c h may be d u e  to t h e c o r r u g a t e d effects  during  available in  was a d h e r e n t to t h e b a c t e r i a l c e l l .  sample  preparation for electron microscopy.  electron  observed  SEM  and  m o r p h o l o g y of t h e b a c t e r i a l cell wall as well as d e h y d r a t i o n  to s u p p o r t a r e l a t i o n s h i p  transmission  material  material  micrographs,  in  between the amorphous  micrographs  scanning  electron  and  the  matted  micrographs.  No e v i d e n c e s u b s t a n c e as  mass  of  However,  was seen  extracellular b y means  a s c h e m a t i c i l l u s t r a t i o n ( F i g . 9 ) , an a t t e m p t was made to r e l a t e t h e two  of  - 30 -  Fig. 9  Schematic illustration of of extracellular material and (B) are examples of material: ( A ) , adherent phous in nature.  the as the to  relationship between the various forms seen by means of SEM and T E M . (A) morphological forms of the extracellular the bacterium surface and ( B ) , amor-  - 32 -  extracellular material Wiebe physiological some  bacterial  form  may  The  surrounding  other  Other  unable  These  to  findings  into  workers  explain  lack  their  1971;  until  nutritional  on  the  since  it  the  late  Dainty  spoilage  possible  and the  that  later muscle  proteases  exponential et  aL,  is  well  evaginations  of  disruption,  into  reported  and  wall  was  surface  contents  bleb-like  cell  myofibrillar  bacterial  until  aL,  of  that  (1975)  repressed et  blebs  for  the  release  proteolysis the  on  certain  of  stated  Boethling  (Tarrant  demonstrate may  then  usually  that  responsible  blebs  bacteria. are  (1971)  was  may  found formation  aL  activity  globules the  the  et  secreted  exoenzymes  growth.  been  Dutson  be  (1968)  induced  proteolytic  globules.  tissue  of  Chapman  conditions  enzymes  and  and  pseudomonads.  that the  types.  phase  1975)  have  advanced. on  samples  of the  appear-  other than those incubated for 5 days. The ance ton  of et  the aL ,  close  physical  extracellular 1978)  which  association  fibres suggest  and that  and  blebs the  the  time  reinforce  sequence  previous  extracellular  studies  (Coster-  polymeric material  aids  in: (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 h e s u b s t r a t e  (b)  channelling various  (c)  concentrating bacteria.  and  nutrients towards conserving  surface;  the bacteria;  digestive  enzymes  and released  by  the  - 33 -  CONCLUSION:  G r o w t h of P. f r a g i on meat s u r f a c e s was not s u p p r e s s e d at i n t e r m e diate  temperatures  (ca. 21°C),  however,  the  inoculated  muscle  samples  s h o w e d an i n c r e a s e in p H . Extracellular  material  was  visualized  T h i s m a t e r i a l a p p e a r e d to mediate c e l l - t o - c e l l ment  to  the  association  muscle  with  the  samples.  The  extracellular  by u s i n g both SEM and  as well as c e l l - t o - m u s c l e a t t a c h -  formation  fibres  may  TEM.  and  localization  represent  s p o i l a g e of meat b y h y d r o l y t i c e n z y m e s of P. f r a g i .  the  of  stages  blebs  in  prior  to  - 34 -  PART 2  GROWTH PHASE AND CAPSULAR FINE S T R U C T U R E OF PSEUDOMONAS FRAGI A T C C 4973  - 35 -  INTRODUCTION:  As mentioned in the previous section, it is known that the mechanism of attachment of microorganisms to meat surfaces involves two stages; however, a detailed understanding still a subject of debate. extracellular  material  consecutive  of the latter attachment stage is  During the spoilage of meat by P. fragi cells, an  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 available on  the  stages  bacterial growth.  of formation  Little or no information is  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  in liquid media. the  Since extracellular material was reported to be involved  attachment of microorganisms to surfaces  Firstenberg-Eden,  (Costerton  et a[.,  1978;  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  information may  of events associated with  the attachment process.  This  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 "glycocalyx"  that surrounds the cell.  The fibres of the glycocalyx may not only  position the microorganism but also may conserve and concentrate the digestive 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 microscopy reviews noted  (1979) recently reviewed the role o f electron  in the elucidation o f bacterial  (Hollenberg  structure and function.  and Erickson, 1973; Holt and Beveridge,  the development o f advanced  techniques  Other  1982) have  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  because many  and resolved by microscopy  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 Zelander, 1970; Fletcher and Floodgate, 1973; Bayer and Thurow, 1977; Reid and Brooks, 1982). tive  in staining  Ruthenium red as well as alcian blue were considered effecacidic  polysaccharides associated with  (Fletcher and Floodgate, 1973). ted  glycocalyx material  Cassone and Garaci (1977), however, repor-  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 dles  (Bayer  and  Thurow,  1977).  Studies  by  Schmid et a[.  (1981)  bun-  showed  that emphasis has been laid on the crucial importance of fixation and d e h y d r a tion  procedures  optimum growth bacterial  the preservation  for  conditions have been neglected.  capsule  was  affected  techniques  contributed  cytoplasmic  membrane,  sole fixative method was  of capsular micromorphology,  under  mainly DNA  "RK  by to  and  culture  proved to be unnecessary;  whereas  the  the maintenance of integrity  conditions",  suitable to preserve  The fine structure of the  conditions,  ribosomes.  By  fixation  of cell  wall,  using osmium tetroxide  Schmid et aJL  capsular  whereas  material,  (1981)  as  found that  this  and that ruthenium  red  it injured and concealed capsular fine  structures  by amorphous precipitation of the capsular material. Although been  investigated,  synthesis  the  early  little  is  - the transfer  stages  of  exopolysaccharide  known about the final  synthesis  stages of  have  polysaccharide  of the oligosaccharide chains from their  attachment  to isoprenoid lipid carrier to a possible surface receptor and extrusion from the  cell  may  surface  even  be  (Sutherland,  differences  in  different physiological states. is  also  This  more readily  is  likely  1979). the  to affect the attachment  growth, whereas the  polysaccharides  under  from  there  cells  of  some conditions than  others.  and release of exopolysaccharides as  Sutherland  exopolysaccharide  is  (1979) also reported that in  secreted  continuously  during  in others, exopolysaccharide production is a feature of only  logarithmic and stationary  ever,  of  suggested that  The outer membrane of gram negative bacteria  lost from bacteria  microorganisms,  was further  release  well as affecting their final purity. some  It  demonstrated  the  phases.  formation  of  Williams and Wimpenny exopolysaccharide  stationary phase of growth of a pseudomonad.  material  (1977), during  howthe  - 39 -  Bleb-like noticed  on  the  evaginations  surfaces  1971; Baechler and Berk,  or  of several  protrusions,  inter  gram negative  1974; Maclntyre et a L ,  alia,  have long been  bacteria 1980).  (Dutson  et  a[.,  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.  layer,  thereby  Burdett  expanding  and Murray  (1974)  the  more elastic  overlying  outer  wall  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), reported  on  which then  the  production of  on  as  played a role in pathogenesis.  studies by Dutson et ah blebs  vesicles  the  a means of  who  enclosing enzymes  This hypothesis supports earlier  (1971) who noted that enzymes may be secreted into  bacterial  surface,  and  later  form  globules.  - 40 -  EXPERIMENTAL:  A.  Culture Conditions: Pseudomonas fragi  trypticase  soy broth  rpm) water bath.  (TSB)  ATCC  4973 was  grown  (BBL,  Cockeysville,  (21 ° C , 24 h)  MD)  in 200 mL  in an oscillatory (100  Cell numbers were estimated by determining the turbidance  at 600 nm with a Beckman Model DB spectrophotometer. Surface  plating on trypticase soy agar ( T S A )  (BBL,  Cockeysville,  MD) was used for enumeration of P. fragi ( 2 1 ° C ; 24 h ) .  B.  Incubation on Solid Medium: A  21 ° C .  24 h  culture  (0.01  mL)  was  plated on T S A  and incubated  at  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  basis of the growth c u r v e ,  into T S B  and incubated at 21 ° C .  the samples of most interest were those obtained  at 4 h (early logarithmic growth phase) and 24 h (stationary Bacterial cells were removed after 4 and 24 h incubation. of  two  trials  were  On the  harvested  and  were  immediately  fixed  growth phase).  Duplicate samples 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. with  Michaelis  buffer  In this study, RK buffer is synonymous  (Kellenberger and Ryter,  consisted of washing the agar  surface with 5 mL  1958). TSB.  The initial  step  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 centrifugation (4,000 x g; 10 min). 1 mL of 1% (w/v) O s 0  4  The resulting pellet was immediately covered with  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  microtome (Ivan Sorvall  were  Inc.,  sectioned  Norwalk,  with  CT),  a "Porter-Blum"  MT-2 ultra-  mounted on 3 mm copper g r i d s ,  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. Growth  (TSA)  TSB  within  characteristics  media were determined  up to 40 h. in  fragi: of  by  P.  fragi  in  liquid  (TSB)  and on solid  monitoring cell numbers at timed  intervals  Similar growth patterns were observed for P. fragi on T S A and  (Fig.  10).  P.  fragi  was  4 h incubation on T S A  in the early  logarithmic phase of  growth  plates and in the stationary phase of growth  by 24 h incubation on T S A at 21 ° C .  B.  Electron Microscopy: 1.  Cells grown on solid medium: Transmission  the early  electron  micrographs of  bacterial  phase  extracellular  cells,  material  however,  fragi cells ( F i g .  exhibited no bleb-like evaginations.  11). Fine  randomly distributed on the cell surface was observed  12).  2.  Cells grown in liquid medium: Transmission  the early ( F i g . 13). cells  at  The blebs, which contain dense granular material,  appear to be present on the entire surface of the P.  (Fig.  harvested  logarithmic growth phase revealed bleb-like evaginations or p r o t r u -  sions on the cell surface.  Stationary  cells  electron  micrographs  of  bacterial  cells  harvested  at  logarithmic growth phase revealed no blebs or extracellular material In  contrast  to  early  logarithmic  phase  seemed to be shrunken in appearance and  blebs as well as globules adjacent to the cells.  cells,  revealed  stationary only few  phase  attached  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 ( T S B ) ( • ) and on solid ( T S A ) ( • ) 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.  -  Fig. 12  48  -  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. 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 -  Fig.  14  T h i n section of s t a t i o n a r y phase medium. Few attached b l e b s as cells are o b s e r v e d . Bar = 1 pm.  cells well  of as  P. fragi globules  g r o w n in l i q u i d a d j a c e n t to t h e  -53-  - 54 -  DISCUSSION:  Electron micrographs of cells grown on solid medium and harvested at the on  early  the  logarithmic phase of growth revealed an abundance of blebbing  surface  electron  of bacterial  micrographs  process (Figs. harvested  explained by  suggested  15 and 16).  at the  cells.  Detailed examination of the transmission  that  the  blebs may arise  The absence of blebs on the  logarithmic growth  phase,  grown  by  an exocytosic  surface of cells,  in liquid medium may be  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 reveal and  phase revealed extracellular material, but did not  blebs on the cell surface.  harvested  at  the  stationary  Cells of P. phase  fragi grown in liquid medium,  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  versatility  in  liquid  medium,  which  perhaps  reflected  their  nutritional  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. grown  in liquid media may be nutritionally  versatile,  Since pseudomonads 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 may  suggest  only  few  their  globules  association adjacent  "delayed" with  response of bleb formation, which  nutrient  availability.  The  presence  to the cells may also be due to the  of  "washing"  effect of the agitated liquid medium on the surface of the cells of P. f r a g i . A study of growth and proteolytic activity of six strains of Pseudomonas 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  similar observations protease activity  with  rapid  growth  were reported by  of three  (Juffs  et  al_.,  1968).  Recently,  McKellar (1982) who also showed that  strains of Pseudomonas fluorescens was inducible.  A study of enzyme production of Aeromonas hydrophila suggested the possibility  of  stress, Day,  an  inducer  protease  1983).  catabolite  production  repression was  high,  system, despite  These studies tend to agree  where, slow  under  growth  nutritional  (O'Reilly  and  with the findings of the present  study concerning the observation of blebs on the surface of P. fragi cells.  - 60 -  CONCLUSION:  The of by  type  blebs on the the  surface  exocytosic  of  the  of  attachment  of  formation  of  the the  cells.  of  action  Whether  glycocalyx  surfaces  of medium  the  or  (solid  P.  fragi  of not  bacterial  (Costerton  be et  the relationship between  Bleb  formation  this  phenomenon  cells  to  material  involved  aL,  liquid)  cells.  globule  extracellular may  vs.  1978),  in  surfaces,  is  important  formation into is  the  the  further  may  exocytosis by  P.  fragi  studies  required  followed  environment the  process  to  precede  appears  of  blebs, globule formation and the  be  with  attachment are  expression  then  immediate  associated  (glycocalyx)  in t h e  cells.  While  microorganisms to  glycocalyx.  to  determine  - 61 -  CHEMICAL  COMPOSITION  EXTRACELLULAR PSEUDOMONAS  AND STRUCTURAL  POLYSACCHARIDE  FRAGI  ATCC  4973  ANALYSIS OF  ISOLATED  FROM  THE  - 62 -  INTRODUCTION:  A living  common  organisms  are t h r e e  rides  is t h e p r o d u c t i o n  types:  cytoplasmic  f e a t u r e of b a c t e r i a , f u n g i ( y e a s t a n d m o u l d ) , a n d h i g h e r  intracellular  membrane;  cell  of polysaccharides.  polysaccharides  wall  polysaccharides others  are found  are secreted  into  and exocellular polysaccha-  1979). A l t h o u g h  covalently attached  unattached  there  located i n s i d e o r as p a r t of t h e  polysaccharides;  located o u t s i d e t h e cell wall ( S a n d f o r d ,  Morphologically,  to t h e cell  the growth  some e x o c e l l u l a r  as a t r u e  medium.  capsule,  Wilkinson  (1958)  summarized t h e f u n c t i o n s of 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 a s : (a) p r o t e c t i o n a g a i n s t  phagocytosis;  ( b ) p r o t e c t i o n a g a i n s t amoebic a t t a c k ; (c) protection against (d) recognition and  bacteriophage;  immune  response of a higher  organism  to microbial  infection; (e) p r o t e c t i o n a g a i n s t d e s i c c a t i o n ; (f)  reserve carbon and energy  source;  ( g ) an a i d in u p t a k e of i o n s , a n d ( h ) an  a i d in t h e d i s p e r s a l  p r e s e n c e of ionic Therefore, uniquely  since  assigned,  are e i t h e r  an i n t e g r a l  in  t h e case  produce type,  which  and  as  polymers  environments  that  they  may  polysaccharides a c t as s t o r a g e  protective substances.  p a r t of t h e cell wall, o r o c c u r  of Escherichia coli,  other  of all i n d i v i d u a l  i t is e v i d e n t  components  in l i q u i d  d u e to t h e  charges.  the functions  structural  of cells  While  cannot  be  materials, as  polysaccharides  as a slime o r c a p s u l e as  Pseudomonas a n d K l e b s i e l l a , b a c t e r i a c a n also  in w h i c h  carbohydrates  a r e main  c o m p r i s e s t h e l i p o p o l y s a c c h a r i d e s , is p r e s e n t  components. in t h e cell  One  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.  the teichoic acids, which are present positive bacteria (Kenne and are  characterized  periplasmic  layer.  by  a  A third type includes  in cell walls and  Lindberg,  1983).  dense peptidoglycan  membranes of gram-  Gram-positive microorganisms layer, adjacent  to the  inner  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  microorganisms to surfaces.  Although the mechanism involved in the adhesion  process has  be  involved  in the adhesion of the  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  previously, they fulfill  different functions  substituents.  including being  As  mentioned  responsible for  immunological properties of the microoganisms. Information on the composition and  conformational  structure of microbial polysaccharides  to correlate their structure with their biological and Such  studies  should  involve  the  determination  of  is needed in order physical properties.  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  Fig.  17  NEGATIVE  D i a g r a m a t i c r e p r e s e n t a t i o n o f the b a c t e r i a l c e l l envelope  classified  on  the  polysaccharides (linear by  vs.  the  basis vs.  ride structure  the  65  -  types  of  sugars  heteropolysaccharides)  branched).  presence  (a)  of  -  of  Further  regular  as  classification  repeating  present well of  units.  as  in  the  the  polymer  degree  polysaccharides Elucidation  of  determining the following  characteristics:  nature  of  and  the  sugar  residues  their  is  branching determined  the  involves  the  of  (homo-  polysaccha-  proportion  in  the  polysaccharide; (b) the configuration (c)  at t h e a n o m e r i c p o s i t i o n of t h e s u g a r  the characterization of linkages  ( d ) the s e q u e n c e of t h e s u g a r s  The structure  analysis  Le.  the  linkages  the  lack  the  involved.  of  only  shapes  certain  sequence  structure ordered occur  Due  flexibility The  then  between  isolated (or  (Rees  to  covalent the  secondary and  where  Welsh, (a)  the  or  to  chains  structure)  a  narrow  are  then  between  other  as  range  (b)  of  well  as  sugars,  of  relative  of  adopting  dependent  chains  polymers  primary  adjacent  levels  and  the  sugars  capable  are  higher  structure)  with  and  with  between  which  Two  interaction  mainly  constituent  linkages  1977).  residues;  unit.  dealt  residues  (tertiary  structures  has  of  polysaccharide  structures  these  sequence  restricts  exist  specific  and  sugar  in t h e r e p e a t i n g  polysaccharides  nature  orientations.  primary  of  between  residues;  on  the  organization  may  result  interaction  (quaternary  in may  struc-  ture). The and  structure  fragi A T C C  object of  4973.  the  of  this  study  extracellular  was  to  investigate  polysaccharide  the  isolated  chemical from  composition Pseudomonas  - 66 -  L I T E R A T U R E REVIEW  Pseudomonads belong to the family of microorganisms Pseudomonadaceae. rods.  The  cells  of  Pseudomonas are typically  straight or slightly  curved  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. are typically mesophilic; however,  Pseudomonas species  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 polysaccharides.  Polysaccharides  may  be grouped into three  categories,  to whether they contain acidic, neutral or amino sugars. to realize that it is the constituent  sugars and acyl  according  It is also important  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 s t r u c ture,  and in molecular size and shape.  Greenwood (1952)  initially  reported  on the various methods for the determination of molecular weight of polysaccharides.  Churms  polysaccharides  (1970)  having  used  gel  chromatography  a broad molecular weight  in  the  distribution.  separation  of  Because mild  conditions were used, the technique was particularly useful for labile biological materials.  The most common methods of expressing molecular weight  number-average  or weight-average M  w >  are  Although information on molecular  size may be of limited direct value in the determination of composition and structure,  this  tures  those  and  information is useful  in distinguishing between  with a low degree of branching.  categorizing of the polysaccharides.  This aids  linear  struc-  in the  initial  - 67 -  A.  Monosaccharide Types When monomeric sugars are condensed, with a loss of water, they  form  polymers.  Polymers with  polysaccharides.  a chain  length greater than ten are called sugars,  or (b)  sugar derivatives e.g. amino sugars, uronic acids or ester sulphate  sugars.  Approximately  The monomer species may be (a) simple  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  well as identify carbohydrate compounds.  to categorize as  These tests serve as initial screen-  ing assays prior to the use of other chemical methodology for the elucidation of the monosaccharide types. colourimetric 1962)  methods:  The following are among the commonly used  (a) anthrone-sulphuric  and phenol-sulphuric  acid  (Hodge  and Hofreiter,  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 Sugar  polysaccharides 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 2,3-Diamino-2,3-dideoxy-D-glucuronic acid 2,3-Diamino-2,3-dideoxy-D-mannuronic acid 2,3-Diamino-2,3-dideoxy-L-guluronic acid  3  Abbreviations:  EPS - extracellular  polysaccharides;  LPS - lipopolysaccharides  Kenne and Lindberg, 1983  LPS LPS LPS LPS  - 69 -  Table 2  Functional group modification of sugar residues.  Functional Group  Derivatives  (ethers) - OH  O-Methyl  -OCH3  ester  O-Acetyl  -OCOCH.  acetal  Pyruvyl  "V 3 H  •0 (amines) - N H 2 (esters) -COOH  N-Acetyl  /  COOH  •NHCOCH. -COOCH.  - 70 -  O-acetyl) substituents, N-acetyl, sulphate and (pyruvate). by  Certain of these substituents can be analysed  infrared (i.r.) and 1  The  use  of  phosphate groups, and  nuclear  ketals  nondestructively  magnetic resonance (n.m.r.) spectroscopy.  13 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  types of monosaccharides in hot acid must be considered. are  the  group most resistant to acid destruction.  due  to the effect of acid alone since it has  The  various  hexosamines  This destruction is not  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 before  of amino sugars, N-deacetylation  hydrolysis, followed  by  deamination.  can be first performed  This process may  lead to the  formation of anhydrohexoses, which can be easily estimated (Williams, 1975). Improvement residues  of  in polysaccharide  procedures samples  for  quantitative  continues  to be  an  analysis  of  sugar  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 determined to obtain a quantitative release of the sugars. the advantages and et  aL  Dutton (1973) reviewed  disadvantages in the use of various acids.  (1967) showed that 2 M  hydrolytic strength as HCI  trifluoroacetic acid (TFA),  (1 IV)) and  H S0 2  4  Albersheim  (with the same  (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. syl  linkages,  the  presence  charide chain will withstand  Although 2 M TFA  of an  aldobio-uronic  the acid treatment.  can hydrolyse  glycurono-  acid within the  polysac-  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  methanol  of the  reduces  the  uronic  acids.  Treatment with  uronic  esters  to the  corresponding  mixture of methyl glycosides is then hydrolysed neutral sugars. the 2 M TFA  By  NaBH  in anhydrous  4  alcohols.  with 2 M TFA  The  to give the  comparison of the ratio of neutral sugars released  by  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-products in the hexose.  hydrolysis of polysaccharides  containing 2-acetamido-2-deoxy-  Hydrolytic removal of the N-acetyl group may  to the cleavage of the neighbouring  partially occur prior  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 decomposed during N-acyl This  hydrolysis with acid.  derivative by treatment,  linkages  treatment  during  remain  which  intact, has  It could, however, be isolated as the  with  anhydrous  hydrogen  glycosidic linkages  proved  to be  are  of general  polysaccharides containing N-acylamino sugars.  fluoride (HF).  cleaved value  but  amide  in studies of  Anhydrous hydrogen fluoride  has been extensively used for the deglycosylation of glycoproteins (Mort and Lamport, sugars linkages  1977).  within  1  The h  of amino  acid cleaves  at 0°C sugars  while intact.  all the  linkages of neutral and  leaving peptide More severe  bonds and  treatment  acidic  glycopeptide  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. boiling handled  point of HF in an  (19°C) and  enclosed  system.  Because of the low  its extreme toxicity, the Excellent reviews by  acid is usually  Lenard, (1969) and  Mort, (1983) have also included methods for handling HF,  with emphasis on  the application to carbohydrates.  C.  Sugar Determination Paper chromatography  Macek, 1963)  (Kowkabany, 1954;  Block et aL,  and thin layer chromatography (Wing and  commonly used for the characterization and  1958  and  BeMiller, 1972) were  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 compounds.  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  highest  retention  time.  ranging  from  glycerol  More  recently,  to glucitol  various  column  which  had the  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 chromatographic analysis of oligosaccharides has been reported by Geyer and co-workers (1982).  Various columns  have been employed. SE-30 or OV  with phases of different polarity and  It was  selectivity  shown that capillary columns with Dexsil 410,  101 can be successfully used to separate peracetylated neutral  and amino sugars.  - 74 -  D.  Methylation Analysis The  hydroxyl  aim of methylation is to achieve etherification of all the free  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 formation 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 determination, 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  accompanied by  containing 2-deoxy-2-acetamido sugars.  N-methylation  This is  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, per-methylated  polysaccharide  positively charged  is greatly  1973).  affected  by  Hydrolysis of the  the  methyl amido hexosyl (CH^-NR) residue.  formation  of a  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 recovery  of  methylated  amino sugars.  Stellner  may and  greatly affect the co-workers  further  -  75  -  POLYSACCHARIDE i) base  ii) Mel  0 Me 2  i) hydrolysis ii) reduction  Mt6  d  CHO—CH£H  iii) acetylation r-OAc MeO-  pOAc -OAc  pOAc  i-OAc  -OAc MeO-OAc AcOhOAc -OMe -OMe OMe  -OAc -OAc -OAc -OAc  1  MeOAcO-  -OMe -OMe  -OMe  Q.I.C—m.s. Fig.  18  Methylation  analysis  of a  polysaccharide  - 76 -  reported on the effective use of acetolysis in obtaining a satisfactory yield of 2-deoxy-2-N-methyl-amido  E.  hexoses.  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.  electron  Carbohydrate derivatives rarely give molecular ions in  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 fragmentation of organic molecules under electron impact, and differentiation of the resulting particles by use of the mass-to-charge (m/e) ratio involves subjecting  the compound  Ionization  under  investigation  of the molecule causes  to a beam  of electrons ( « 70 eV).  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  The use of combined  and Chizhov, 1966, Lonngren  and Svensson,  1974).  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 or ketene fragments Mass  (m/e  42) from  these primary fragments.  The  60)  intensities of the  decrease with increasing molecular weight (Bjorndal et aL, 1970).  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.Kern, s.  Although isomeric alditol acetates having the same structure but differ-  ent configuration are practically indistinguishable, the presence of substituents 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 lar, g.l.c.-m.s.  be readily examined by m.s. and in particu-  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 fragmentation, 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  a methoxyl-bearing  and an acetoxyl-  cations, and (c)  ions  from  cleavage  between  bearing carbon atom with marked preference for the methoxyl-bearing species to carry the positive charge. Ions  formed  however,  by  are  fragmentation  scission  between  generally pathways  of  low  the  two  acetoxyl-bearing  abundance  (Bjorndal  for a specific compound are,  the preference in scission as outlined below: I H-C-OCH \ H-C-OCI-U \  Primary to give (m/e 60)  /  fragment  1967).  therefore,  The  dictated by  /  H-C-OAC  ions undergo a series of subsequent eliminations  secondary fragments, including losses by ^-elimination of acetic acid  60) or methanol but  hyde,  H-C-OAC  aL,  atoms,  H-C-OAC  3  H-C-OCHi 3  et  carbon  (m/e  32),  losses by  CV-elimination of acetic acid  (m/e  not of methanol, and losses via cyclic transition states of formalde-  methoxymethyl  acetate,  or  be noted that mass spectrometry partially  methylated  methyl-D-galactose  acetoxymethyl  Finally,  will not distinguish between  alditol acetates. and  acetate.  it should  diastereomeric  The alditol acetates of 2,3,4,6-tetra-O-  2 , 3 , 4 , 6 , -tetra-O-methyl-D-glucose  will  give  similar  included  routine  mass spectra, although the intensities of the peaks may v a r y .  F.  Nuclear Magnetic Resonance Spectroscopy ( N . M . R . ) The  uses  of  n.m.r.  spectroscopy have not only  applications for analysis of the composition of mixtures and the monitoring of reactions, ences.  but  also  the  elucidation  of structure  and conformational  The application of this technique to simple carbohydrates  prefer-  (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. n.m.r. instrumentation has well  as  the  Dramatic progress in the development of  resulted  utilization of pulsed  in increased operating frequencies as  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. spectrum  In order to eliminate interference in the  by the numerous hydroxyl groups present, a good quality deuterium  oxide (preferably 99.95 atom%) must be used. due  to residual water (HOD  peak are often obtained. the spectrum  signal) as well as substantial side bands of the The  HOD  peak usually appears  in the region of  associated with the anomeric protons ( 5 4.5-5.5).  the chemical shift of the HOD the pH  Nevertheless, a strong peak  peak may  be accomplished  A change in  by either altering  of the solution or more preferably by recording the spectrum  elevated temperature.  The  at an  latter procedure not only aids in reducing visco-  sity but also has a dispersing effect on the sample.  - 80 -  The on  H  the number  presence  spectrum  of s u g a r s  present  o r absence  6-deoxy Rosell  n.m.r.  sugars  in t h e r e p e a t i n g u n i t a n d may  of 1 - c a r b o x y - e t h y l i d e n e  (De B r u y n  and Jennings,  of a p o l y s a c c h a r i d e p r o v i d e s information  (Garegg  et a L , 1980),  et a L , 1976), a n d acetate g r o u p s  ( S a v a g e , 1980;  1983).  An  examination  with t h e v a l u e of t h e c o u p l i n g c o n s t a n t  enable for  the differentiation  both  pyranosyl  of t h e chemical  ( J ) between  of t h e anomeric  (Bundle  acetal  reveal t h e  a n d Lemieux,  (g)  shift  along  H-1 a n d H-2 ( J - „ ) may 1 / <-  n a t u r e o f t h e l i n k a g e s ( QL o r jg  )  1976) a n d f u r a n o s y l ( S t e v e n s a n d  F l e t c h e r , 1968) s u g a r s . Acetone  is u s u a l l y  p r i o r to n.m.r. a n a l y s i s . ride be  should  masked  can  be  added  acetone,  by the standard. removed  as an  internal  standard  However, initial s p e c t r a of an u n k n o w n p o l y s a c c h a -  be r u n without  readily  to t h e sample  since a substituent O-acetyl group  may  A c e t o n e has t h e a d v a n t a g e of b e i n g volatile ( i t  from  t h e sample) a n d its chemical  shift  is v i r t u a l l y  unaffected b y variations in temperature. 13  2.  C N.M.R. S p e c t r o s c o p y 1  Whereas t h e F T mode may be a d v a n t a g e o u s f o r o b t a i n i n g  H spectra,  13 its u s e is m a n d a t o r y f o r abundance  C  n.m.r. s p e c t r o s c o p y  (1.1%) a n d i n t r i n s i c a l l y  low s e n s i t i v i t y  b e c a u s e of t h e low n a t u r a l 13 of the  C  nucleus.  A s in  1 the  case  study  of  n.m.r.  of  H  n.m.r. s p e c t r o s c o p y ,  carbohydrate  spectroscopy  structures,  (Usui  et a L ,  numerous  reviews  conformation 1973; N u n e z  are available f o r the 13 and interaction by C  e t a L , 1977; J e n n i n g s a n d  13 Smith, acid berg  1980).  C  N.m.r.  spectroscopy  has also  been  used  to s t u d y  sialic  p o l y s a c c h a r i d e a n t i g e n ( B h a t t a c h a r j e e et a L , 1t975), g l y c o p r o t e i n s ( B l u m and Bush,  1982) as well  G o r i n , 1981; B r a d b u r y  as simple p o l y s a c c h a r i d e s ( B e r r y et a L , 1977;  a n d J e n k i n s , 1984).  - 81 -  EXPERIMENTAL:  A.  Isolation of Polysaccharide: Pseudomonas fragi ATCC 4973 was  tripticase soy broth in an oscillatory (100 liquid culture (10 mL)  was  inoculated  grown (21°C, 24 h) in 200  rpm)  water bath.  onto (30 x 38 cm)  soy agar which were then incubated for 3 days at 21 °C. was  harvested, followed by  w/v).  Irvine, CA)]  the  viscous  and  added to absolute ethanol.  water and  further  The  polysaccharide material.  dissolution in 4 M water and  lawn of bacteria  with a 10%  Further  fraction) was  resulting precipitate was  collected  dissolved in  solution of cetyl trimethylammonium  bromide (CETAVLON) (Aldrich Chemical Co., ted  The  using a type 45 Ti rotor,  (polysaccharide-containing  treated  trays of tripticase  [Beckman Model L3-50 (Beckman Instrument  at 60,000 x g for 5 h (4°C)  supernatant  subsequent  the addition of phenol (to a concentration of 1%  After ultracentrifugation  Inc.,  The  mL  Milwaukee, Wl), which precipita-  purification of this fraction involved  NaCl, reprecipitation in ethanol, followed by dissolution in  dialysis to remove residual  NaCl.  The  dialysed  solution, after  freeze drying, yielded a polysaccharide component. An to  increase  alternative method of polysaccharide isolation was the  total polysaccharide  yield.  The  isolation procedure  performed according to the method of Westphal & Jann (1965). 400  mL  of harvested P.  incubated 65°C, was 10°C,  at  65°C.  An  was  Approximately  fragi cells including the extracellular material were equal volume of 90%  (w/v)  phenol, preheated  then added with vigorous stirring for 15 min.  the emulsion was  used in order  centrifuged  to  After cooling to  at (1,000 x g) for 30 min which resulted  in the formation of three layers: a water layer, a phenol layer and an insoluble 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 substances. T h e dialysed solution was then centrifuged (1,000 x g) remove any traces  of insoluble material.  The supernatant  and yielded mainly  polysaccharide material.  in order to  was freeze dried  Nucleic acids were removed by  incubation with ribonuclease (Sigma Chemical C o . , St.  Louis, MO) as d e s c r i b -  ed by Westphal and Jann (1965). 1 H both  n.m.r.  procedures  spectroscopy of the  was  used  to  determine  native  polysaccharides  similarity  isolated  in polysaccharide  by  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  according to the procedure of Churms (1970).  Cape  Town,  South  Africa)  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  paper and the following solvent systems (all (a)  ethyl acetate-pyridine-water  (b) ethyl  acetate-acetic  with  Whatman  No.1  filter  v/v):  (8:2:1) - basic solvent;  acid-formic  acid-water  (18:3:1:4)  -  acidic  solvent.  Chromatograms were visualized b y : (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  (1962).  were  prepared  according  to the  method of  Hough  and  Jones  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 No.1  paper chromatography was  filter  paper  eluted  (18:3:1:4).  Strips  about  with  a  suitable  spray  with  ethyl  carried out using Whatman  acetate-acetic  acid-formic  acid-water  4 cm wide were cut from the edges and  reagent  to  reveal  the  position of  the  treated  sugars.  By  reference to these strips, areas containing each component were cut from the main body of the chromatogram. section  (shredded)  The sugar was then eluted from each paper  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. phase  for the analysis  silicone) Ont.).  of alditol acetates  on Supelcoport The  temperature  (100 - 120 mesh) profile used was  The stationary  was 3% SP 2340 (75% cyanopropyl (Supelco  Canada L t d . ,  Oakville,  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,  ionization detectors  and coupled to a Hewlett-Packard  was  capillary  also  phase  was  used. used  A  throughout  column the  (0.20  analyses.  mm i.d.)  equipped  with  flame  Model 3390A integrator with SE-30  Hydrogen was  the  stationary  carrier  gas.  - 84 -  The  injector  temperature  and  detector  temperatures  profile used was  were  maintained  at  100°C to 250°C at 10 C ° / m i n .  260°C.  The  Molar ratios of  alditol acetates and hence the relative molar proportions of sugars in mixtures were  calculated  sugar.  using  the  detector  These response factors  ionization  detector,  by  response  factors  were determined by  injecting  several  alditol  (R  g  factors)  for  each  calibration of the flame  acetate  mixtures  of  known  composition into the chromatograph. Although encountered  as  phthalic esters  contaminants  in  (used extensively as plasticisers)  g.l.c.  analyses,  mass spectral  may be  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; ionisation current  - 200 u A ; inlet temperature - 240°C;  ion source temperature -  220°C.  F.  Nuclear Magnetic Resonance Spectroscopy: -i  1.  Proton ( H) N.M.R. Spectroscopy: Proton  instrument.  To  magnetic eliminate  resonance spectra were run on a Bruker interference  in  the  spectrum  by  the  WH-400  numerous  hydroxyl groups present, a number of exchanges were made with D^O (Stohler was  Isotope then  Chem.,  dissolved  from the anomeric  Waltham, in  MA),  99.9% D,,0  region (to  followed by and any  5 4.18,  freeze d r y i n g .  residual  HOD was  The  sample  shifted away  9 0 ° C ) by determining the spectrum at  - 85 -  elevated temperature. concentrations  Due to the viscous nature of the sample, only low  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 v/v).  borohydride  and acetylated  with  pyridine/acetic anhydride (1:1,  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 temperature.  The solution was  diluted with water  (3 mL)  and freeze d r i e d .  The  product was dissolved in water (2 mL) and reduced with sodium borohydride (50 mg)  for 2 h.  The  reaction  evaporated to dryness.  The  mixture was acidified with acetic acid  H n.m.r.  and  spectrum of the deaminated polysac-  charide was obtained.  J.  HF Solvolysis of the Polysaccharide: The  polysaccharide  (100  mg)  was  treated  with  anhydrous  mL) at ambient temperature with stirring for 3 h using Kel-F valves, and pipes.  HF  (7  vessels  The HF was vented off and complete removal was ensured.  The  resulting hydrolysed residue was then treated with 1 M acetic acid ( 1 0 0 ° C , 3 h).  The acetic  acid was  roto-evaporated  and a portion of the  spotted onto paper and developed in acidic and basic solvents.  sample  was  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 L a b . , Mississauga, O n t . ) . 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  distilled water.  P-2  (200  - 400 mesh)  was  allowed to swell  overnight  in  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  sugars.  acid  residues  in the  polymer to their  corresponding  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  nate  (0.1  Milwaukee,  allowed  neutral  g)  to  [Aldrich  proceed  Chemical  while  Co.,  maintaining  pH  4.75  Wl].  until  The  sulfo-  reaction  hydrogen  ion  was  uptake  ceased (30 - 60 min), then (8 mL) 2 M sodium borohydride was added dropwise at 20°  - 25°C over a 1 h period.  the dropwise addition of 4 M HCI  The pH was maintained at 6.8 with  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  Hakomori (1964).  analysis  was  The methylated  followed by freeze drying  performed material  was  according recovered  to  the  either  method of by  dialysis  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-2N-methylamido-Hexitol Acetates: The procedure as described by Stellner  the analysis  of the partially methylated sugars.  et aL  (1973) was used in  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 sulphuric  acid mixed with 95 mL of glacial acetic acid),  night.  The reaction  and heated at 80°C  over-  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 anion  exchanger,  Que.)  and  washed  under a nitrogen NaBH^. ated  1R-45  (acetate form)  with methanol.  The  (Sargent-Welch Sci. residue was  cm) of weak  C o . , Montreal,  evaporated  to  dryness  stream,  dissolved in 0.2 mL water and then reduced with  The mixture was  acidified with glacial acetic acid and roto-evapor-  with intermittent  in the flask.  addition of methanol.  Acetylation  dride: pyridine (1:1, v : v )  was  A white salty  residue was  accomplished by the addition of acetic  at 100°C for 1 h.  left  anhy-  - 89 -  RESULTS The initial yield of capsular polysaccharide isolated by the cetyltrimethylammonium  bromide  tively  the  less  than  (Cetavlon)  precipitation  polysaccharide yield  procedure  was  quantita-  isolated by the Westphal  method.  1 H n.m.r. indicated  spectra of polysaccharides from each isolation procedure, however, a  high  degree  of  similarity.  Gel-permeation  chromatography  on  Sepharose 4B gel gave a single 1.0 X 10 dalton peak. 7  1 The  400-MHz  showed a doublet at ratio and  of 1:1. an  H  n.m.r.  These were assigned to the  N-acetyl  )  '  n  t  n  e  of  the  5 1 . 4 0 and a sharp singlet at  group  respectively.  anomeric region, at 5 5.26 (1H, 2  spectrum  ratio of 1:1:1.  2  ),  polysaccharide  5 2.05 in the approximate  methyl  Three  native  group of a deoxy  signals  5 4.87 (1H,  sugar  were observed in 2  ),  the  and 5 4.60 (1H,  Other signals were observed at 5 4.46 and 5 4.32  (Table 3). Table 3 gives a summarized account of signals observed in the 13  spectrum  of  polysaccharide group  of  the  native  showed  a deoxy  polysaccharide.  signals  sugar  (19.5  which ppm)  The  C  indicated  the  and  an  13  C  spectrum of  the  native  presence  the  methyl  N-acetyl  of  (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  acetate derivatives of the respective sugars.  were characteristic  of the  alditol  Table 3  N.M.R. d a t a f o r P. f r a g i p o l y s a c c h a r i d e H - n.m.r. d a t a  Carbohydrate  ^  Native polysaccharide  a  J  i2  Integral  (Hz)  proton  Assignment  p.p.m.  Assignment  5..26  S  1 .0  4,.87  S  1 .0  4,.60  B  1 .0  53 .5  C-2 o f amino s u g a r  4..46  B  1 .0  22 .9  CHj o f a c e t a t e  4,.32  6  1 .0  19 .5  C-6 o f deoxy s u g a r  2 .05  S  3 .0  CH  1..40  8  3 .0  C-6 o f deoxy sugar  Chemical s h i f t r e l a t i v e t o i n t e r n a l s u l f o n a t e (DSS)  acetone;  k C h e m i c a l s h i f t i n p.p.m. d o w n f i e l d from Me.Si, from DSS B = broad;  C - n.m.r. D a t a  S = singlet  102 .6 H-l o f amino s u g a r  3  0 configuration  100 .2 100 .1  ofjtecetate  8 2.23 d o w n f i e l d from  4,4-dimethyl-4-silapentane-l-  relative to internal  acetone;$31.07 ppm d o w n f i e l d  - 91 -  Table 4  R .  Q I C  Values of components of hydrolysed polysaccharide on Whatman  N o . l paper  Hydrolysed Polysaccharide (2 M T F A )  a  b  R  . glc  3  Ninhydrin  A (disaccharide)  0.45  +  B-amino sugar  0.86  +  B-neutral sugar  1.00  -  C (deoxy sugar)  1.78  -  13  Relative to glucose Reaction 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)  IM  -  m  m  ... L..  SSI  -;  IT*  i i 'ff  | i1" i i -r-r-r-r  1  T  T  I  |  i  i  i-1  |  t  t  t  >~|  J75  i  i  i  t  |  r  i  i  i  Ill  | i n VO  lit  1*1  l4i.|Lj|,.Il. h  r  |H  44  ll  r IM  lift  I2t  I  1  |iiL,L  tl?  17*  III  l*»  I, l, I  "I  1I1-i  IM  m/e  fig.  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 hydrolysed polysaccharide.  component  of  I i  Ii.. I..  1  |  i,.. L A i  311  2M  131  i"  37*  -i—pt-T-r  i  32t  3M  i  |  I  i  i  i  )  - r  I  I  |  I  I  I  I  |  III  371  127  1(9  •4  71  ft'  102  IM  217  4u • »»  T — r IB*  I7»  2 M  22*  >M  m/e Mass s p e c t r u m of hydrolysed  o f amino sugar (alditol polysaccharide.  acetate)  component  Relative  Intensity  s 5 s :  .  •! I I I I I 5 1  T  I— 1  o  s  H i P>  i  tr oi ^ O O (D O rt 01 H h  ro O Hi  0  M  JEL.  01 ro  0) o o x o  r  tr  &) 01 H. C H-  CD Hi  3 \ ro  si  0) M  i  H-  rt O 0>  o ro  rt  0)  rt  ro  ' I  o o 3 tl  S  o  3  ro rt  «4  -  *6  -  J  =  % 2  !  - 95 -  Paper chromatography (18 h ) , sate of the slowly,  polysaccharide  inter  alia.  revealed  using the basic solvent, of a hydroly-  the  presence of a spot which  Uronic acid sugars would be retained  migrated  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 as  1 H  n.m.r.  spectroscopy.  polysaccharide  showed  no  G.l.c.  change  analysis  in  the  of  molar  the  carbodiimide-reduced  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 separated  into  (80 mg) was hydrolysed (2 M T F A , 100°, 18 h) and  components  main bands were visualised. gram mg)  and  by  preparative  chromatography.  The components A(10.8 mg),  were identified as the  disaccharide,  paper  released  chromatography  upon hydrolysis  permitted  recovery  of  of the polysaccharide.  B(31.5  combined glucose  and amino sugar, and deoxy-sugar portion, respectively (Fig. 22). tive  Three  Each band was eluted from the paper chromato-  subsequently freeze d r i e d .  and C(16.9 mg)  paper  Prepara-  about 74% of the  sugars  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  Since band tions,  g.l.c. B,  Component A  could  not  be  detected  by  g.l.c.-m.s..  on the paper chromatogram ( F i g . 22), consisted of two f r a c -  B-amino (an amino sugar component) and B-neutral (glucose),  separation  by  preparative  individual  components.  paper  chromatography  A portion of the  permitted  isolation  B-neutral component was  further of  the  reduced  -  —  Origi  96  -  "I  -OriginJ —ClcNBCl -GalNHCl —ManNHCl  —A  (Oisacc)  V"~r{B amino f.—r.\h n e u t r a l  ..glucose  -fucose 6-d«oxy-glucose •C (deoxy)  (a)  hydrolysed  Solvent:ethyl  Fig.  22  polysaccharide  acetate:acetic  (b)  reference  standards  acid:formic acidrwater  (18:3:1:4)  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 T F A ) a n d ( b ) r e f e r e n c e s t a n d a r d s .  - 97 -  with N a B H m.s.. the  and acetylated.  4  The alditol acetate was then analysed by g . l . c . -  F i g . 23 shows the mass spectrum of this component. spectrum  hexaacetate.  with  known  references  indicated  the  A comparison of  presence  of  a  hexitol  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 n i n h y d r i n .  The alditol acetate  derivative of fraction B-amino was analyzed by g . l . c . - m . s .  ( F i g . 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  purified fraction  liquid  chromatogram of  the  alditol  acetate  C showed two peaks corresponding to  derivative  and P  3  of  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. Bio-Gel were  The hydrolysate,  when applied to a  P-2 column, was separated into two major fractions.  assayed  using  the  method of  Park  and  Johnson  The components  (1949).  Two  main  fractions HF-A (28.5 mg) and HF-B (39.7 mg) were obtained, i H tabulated  in  n.m.r.  analysis  Table 7.  Paper  of HF-A  and HF-B  revealed signals which  chromatography of fraction  the component consisted of glucose and an amino sugar.  HF-B  showed  This was  are that  verified  by reduction of the sample with NaBH^ and acetylation to form the corresponding alditol  acetate  derivative.  G.l.c.-m.s.  of the alditol acetate  derivative  Relative Intensity %  ? -»  •  I  1—u  -1  Ul  .en  o s  Hi D) CO tr cn a cn H >3 O CD M O ^< rt cn H CD C fL g  •a  O O Hi M ^< W cn i T3  •a -5  0) 3  O CD O C rt OJ H H 0) H- — I' CD —  * 51 CD  -iji  *  • 0) M  a  rt O •00  n  CD rt  0>  rt CD  o O 3 T3 O 3 CD 3 rt  2 2  a-  3  Sw T  '  '  «  '  |  5*  -  86  -  >1  •p  •H  180.8 n  43  r  w c  4-»  c CJ  >  id  58.864 144 102  60  %j  771  69. 100  ICE  150 130  208  230  m/e  Fig.  24 M a s s s p e c t r u m o f B - a m i n o ( a l d i t o l hydrolysed polysaccharide.  acetate)  component  of  111744. 10.  - 100 -  Table 5  G . l . c . analysis of alditol acetates of hydrolysed polysaccharide (2 M T F A ) isolated by prep, paper chromatography.  T  (Peaks) P., P P  2  3  b Column A SP 2340  a  Column B SE-30  0.22  0.02  (glucose)  1.00  1.00  (deoxy sugar)  1.28  1.28  (amino sugar)  1.50  1.11  c  Retention time relative to that of the alditol acetate derivative of glucose. b  c  programme: 195°C for 4 min, and then 2 C°/min to 260°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 T F A )  Carbohydrate  3  J  Disaccharide  B  B-neutral  B-amino C  M  Integral proton  0  Assignment  5. 23  4  0.4  5. 01  S  1.0  4. 64  B  0.6  1. 41  B  5. 41  S  5. 23  4  5. 21  S  4. 64 1. 40  8 8  Glc J _ C-6 of deoxy sugar  5. 23  4  Glc  a  4. 94  S  4.,63  B  Glc  J3_  4..50  B  1.,46  B  5. 42  S  H-1 of amino sugar  5.,22  S  H-1 of amino sugar  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  1.,92  S  3.0  1..41  8  3.0  H-1 of amino sugar  Glc  a  H-5 of deoxy sugar 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 9 0 ° 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  HF-A  5.24 4.65 4.29 2.05 1.40  B 8 triplet s 6  HF-B  5.23 5.12 5.02 4.63 2.09 2.05  4 s s 8 s s  a  -] 2  J  (Hz)  Assignment  H-1 of glc H-1 of amino sugar H-1 of amino sugar H-1 of glc \ acetyl groups  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  (Fig. 25) and 2-acetamido-2-deoxy penta-O-acetyl hexitol ( F i g . The spectroscopy (Table  de-N-acetylated to  complete  removal  of  was  analysed  the  N-acetyl  26). by  H  group  n.m.r. (§2.05)  8). A  60%  polysaccharide. 1 The  confirm  polysaccharide  hexaacetate  H n.m.r.  recovery The  was  achieved  de-N-acetylated  after  polysaccharide  was  of  then  the  native  deaminated.  spectrum of the deaminated polysaccharide showed the disap-  pearance of an anomeric signal ( 5 4.78) (Table G.l.c.-m.s.  spectra  of  are shown in Figs. 27, 28 and 29. by i.r.  deacetylation  spectroscopy (Appendix  I).  8).  per-methylated  hydrolysed  polysaccharide  The degree of methylation was determined  dp  IOOK* I0I744O  H -OtlOS.O  >i 4J •H U) C <U  •p c  144 o  IV  » 7  43 103  > •H +J (0 rH  « M V  Fig.  25  IlAL I0O  Mass  IM  14  ICO  IUT'11, . .T IM  MO  340  MO  m/e  s p e c t r u m o f HF-B.  hydrolysed  300  271  Hexitol  polysaccharide.  hexaacetate  component o f  SI1MOO  dP  Of •09I31.4  >i 4J •H  to  C <D •P C  O  144  > •H •P (0 •H CU  103 41  •0  tOO  IIO  140  ISO  100  200  >20  *40  MO  T. 3JO  940  m/e  Fig.  26  Mass s p e c t r u m component  of  o f HF-B. hydrolysed  2-Acetamido-2-deoxy-oenta-0-acetyl-D-hexitol polysaccharide.  Table 8  N.M.R. d a t a f o r P. f r a g i p o l y s a c c h a r i d e ( d e - N - a c e t y l a t e d ; d e a m i n a t e d ) 13,  H - n.m.r. d a t a a A  Carbohydrate  De-N-acetylated polysaccharide  De-N-acetylated deaminated polysaccharide  1.2 (Hz)  Integral  Assignment  p.p.m.  Assignment  102 .6  configuration  proton  5..26  S  1,.0  4..78  S  1..0  4..52  B  1..0  97 .8  C-l  4..34  8  1..0  55 .4  C-2 o f amino s u g a r  1..40  8  3..0  19 .5  C-6 o f deoxy s u g a r  5.31  H-l o f amino s u g a r  C-6 o f deoxy s u g a r  4..54  8  2 .0  4..34  8  1 .0  4..24  4  3..0  1..41  8  3..0  o f amino s u g a r  s h i f t r e l a t i v e to internal  acetone,  CH, o f de5;xy s u g a r  Chemical  b  Chemical s h i f t i n p.p.m. d o w n f i e l d from Me S i , r e l a t i v e t o i n t e r n a l from DSS S = singlet  99 .9  1.0  a  broad;  n.m.r. Data  6 2.23 d o w n f i e l d from DSS a c e t o n e , 31.07 ppm d o w n f i e l d  BASE rVE: 43 RICt 973824. lee.e-,  43  162568. 19.  116 >1  -p  •H M  C  0) +J  c  158  59. e H  > •H  74 129  QJ  JI  56  8  7  l j l  142  178  JL  58  188  188  196  212  238 242 258 • 4 • l » |• • • • • i 258 IJ  158  288  m/e Fig.  28  Mass  spectrum o f 4,6-di-0-methyl-2-deoxy-2-N-methylacetamido  hexitol.  272  -  109  -  - 110 -  DISCUSSION  Complex formation between lon)  and  between  various  polysaccharides  cetyltrimethylammonium bromide (Cetav-  has  been  used  to exemplify the  carboxy-containing polysaccharides and quaternary  A wide range of acidic polysaccharides with quaternary ammonium salts.  reaction  ammonium salts.  have been isolated y_[a  precipitation  The mechanism of the Cetavlon precipitation  reaction involves salt formation due to charge interaction between the quaternary ammonium salt and the carboxyl residues present on the polymer chains (Scott,  1955).  quaternary  Scott (1960)  ammonium  later  compounds  reported on the precipitation with  proteins.  reaction of  Consequently,  the  lower  yield of polysaccharide material obtained from the Cetavlon method as compared to  the  Westphal  residues  in  method  the  may  be  polysaccharide  explained chain.  by  the  absence  Amino acid  of  uronic  contaminants,  acid  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  superior contain  yield  of  no acidic  polysaccharide. units.  The  (Westphal and Jann, isolated  1965) gave a  polysaccharide  This conclusion is based on the  appears  results  to  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  that the  reaction  gave  is not specific for  a positive uronic acid.  result,  it should be noted  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 charides  is a simple technique,  there  are  a few  of reduced monosac-  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  may  1963).  of  Bishop  sugar  alcohol  and co-workers  acetates  be  suggested four  possible  (Bishop  et  aL,  possible reactions which may  occur during g . l . c . analysis of carbohydrate derivatives: (a)  deamidation;  (b) change in size of the sugar r i n g ; (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 pretreatment of the  latter;  by the tubing used for the column; or by the  solvent  used for injection of the sample. G.l.c. the  presence  analysis of the hydrolysed polysaccharide (2M T F A )  of four components, however,  data for three peaks Lje. origin  (Table 5).  chromatography  P ^ , Pg and P^.  G.l.c.-m.s. in  that  component (deoxy sugar)  the  g.l.c.-m.s.  Peak  showed  analysis only  gave  was retained close to the  data agreed with the data obtained by paper  polysaccharide contains  and an amino sugar.  glucose, a fast  moving  The isolation and purification  of the fast moving component by preparative paper chromatography followed by g . l . c . the  deoxy  analysis indicated that both peaks sugar component.  and P  Conclusive verification  3  were associated with  of the  identity  amino sugar as well as the deoxy component was not achieved by  of the  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  hydrolysis  acid  hydrolysis.  the  of  It  then  polysaccharide  (2  appears  M TFA)  that  may  the  long  duration  have led to  of  inconsistent  molar ratios of each component, which could be explained b y : (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, 1  provided by spectral methods, L_e.  H and  1  troscopy. cans  are  The  signals  known  to  in the  distribute typical  13  C high resolution n.m.r.  13  H and in  C n.m.r.  groups,  resonance  information  each  spec-  spectra of hexosaminoglyof  which  strictly  for the first  order  analysis  of the hexosaminoglycan spectrum are summarized in Table 9.  Both  1 H and  13 C n.m.r.  determination  as  well  useful  occupies  regions.  spectroscopy  regions  was  defined  structure  The  the most important  proved to be complementary techniques  as  the  verification  of structures.  n.m.r. spectral data of all samples are shown in Appendix The  1 H n.m.r.  in  Detailed  II.  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)  H  n.m.r.  analysis  upfield anomeric  shift  of  and the  another  de-N-acetylated  in signal from  signal of the  deshielding effect,  the  containing  a  native  CH^ group  polysaccharide  5 4.87 to 8 4.78 which  amino sugar. removal  (Table 3).  may  showed  an  be ascribed to the  Since steric hindrance will result in a  of the bulky N-acetyl group will,  result in an upfield shift of the anomeric proton.  therefore,  -  Table 9  Representative  113  -  H and  C chemical  shifts for  nuclei of polysaccharides . 3  a(ppm)  H  ~ 1 . ,5  CH C 3  CHgCON  1..8-2. ,1  C  a(ppm) ~ 15  CH C 3  CH.COH)  CH^C0 j '  20-23  2  CH C0 3  2 .0-2. . .2  2  CH(NH)  3.,0-3. ,2  CH C  CH 0  3..3-3. .5  CH 0  55-61  H-2 t o H-6  3..5-4. ,5  CH(NH)  58-61  H-5  4,.5-4. .6  CH OH  60-65  H-1 ( a x )  4..5-4. .8  C-2 t o C-5  65-75  c-x  b  80-87  3  H—C(OH)  5.2  2  38  2  3  2  HO  5..0-5, .4  C-l  (ax-O, r e d )  90-95  H-1 ( e q )  5,.3-5, .8  C-l  (eq-O, r e d )  95-98  5.9  c-l  (ax-O, g l y c )  98-103  C-l  (eq-O, g l y c )  103-106  c-l  (fur)  106-109  HC0  2  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 ; e q , e q u a t o r i a l ; r e d , r e d u c i n g ; glyc, glycosidic; fur, furanosyl. Nonanomeric  13  C involved i n glycosidic linkage.  P e r l i n and Casu, 1982  - 114 -  Preparative paper chromatography of the hydrolysed native polysaccharide (2 M T F A )  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.  signals which  spectrum of the disaccharide fraction showed three are attributable  to the disaccharide unit formed by the amino  sugar - monosaccharide fraction. may  be assigned to the  assigned to the signals). to  the  The  verified  by  an  sugar:deoxy  As would be expected, one anomeric signal  amino sugar  while the other  reducing end of the signal at 5 1.41  anomeric  position  of  examination  sugar  if a portion of the  two  signals  adjoining carbohydrate  unit  would  be  ( a and jg  suggests that the deoxy component is linked  the of  amino the  components.  sugar.  molar  A molar  This  proportion  observation of  the  was  also  glucose:amino  proportion of 0.5 may be obtained  corresponding sugars is not totally  fore, they may be linked as a disaccharide unit. detected by g . l . c . - m . s . ,  anomeric  hydrolysed.  There-  Component A could not be  possibly because of degradation owing to the  labile  nature of the deoxy component. The relatively units  small difference in  on Whatman No. 1 paper  complete  between the amino and glucose  using acidic  solvent  separation of these two components.  separated  into  B-neutral  and  B-amino  by  did not initially  Fraction B  repeated  paper  was  then  enable further  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 Although signals at  5 5.23 and  B.  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  by the  B-amino may  be explained  sugar on hydrolysis of the polysaccharide.  de-N-acetylation  of the  amino  - 115 -  The presence of signals at  5 8.48, 1.92 in the  reveals  the  complexity of component C .  enables  the  labelling of this  The C H  3  H n.m.r.  spectrum  signal ( 5 1 - 4 1 ) ,  however,  component as a deoxy sugar.  dards containing CH^ groups (6-deoxy-glucose,  fucose,  in  paper  an  results  attempt  to  characterize  the  sugar  by  stan-  rhamnose) were used  chromatography.  obtained from the analysis of the alditol acetates  C by g . l . c . - m . s .  Reference  of purified  The  fraction  suggests the possibility of fragmentation of this component.  The inconsistent mass spectral data obtained were probably representative the  decomposed,  fragmented  sponse when treated ninhydrin  are  nents as (i) sugar  with n i n h y d r i n .  Component C  gave  a negative  re-  Reactions of the other components with  summarized in Table 4.  glucose (B-neutral); (ii)  This aids in categorizing the compo-  amino sugar (B-amino); and (iii)  deoxy-  (C). The  any  compound.  of  hexosamine,  standards  on paper  commercially  chromatography,  available.  In  an  did not correspond to  attempt  to  provide  information on the sugar composition of the polysaccharide, other  additonal techniques  (HF solvolysis, methlyation analysis) were used. Many HF  solvolysis  researchers as  well  as  have the  polysaccharides  (Mort, 1983).  deglycosylation  suffers  reported  preferential  the  cleavage  successful  application  of  over  linkages  in  of  HF solvolysis was employed because enzymatic  from the  disadvantage that specific enzymes must be  obtained while chemical deglycosylation times incomplete.  on  involving periodate oxidation is some-  Depending on the conditions, HF solvolysis can give either  monomeric or oligomeric units.  For this reason, the hydrolysate  quently fractionated on a column of Bio-Gel  was subse-  P-2.  1 Analysis that  it  was  an  of  the  oligomer,  H  n.m.r.  however,  spectrum an  accurate  of fraction assignment  HF-A of  suggested the  signals  - 116 -  could not be achieved. showed  Analysis of fraction HF-B by  four anomeric signals and two  H n . m . r . spectroscopy  N-acetyl signals.  This may  indicate  the presence of two monomeric units, one being the amino sugar bearing the N-acetyl function. by g . l . c . - m . s .  HF-B  was  reduced with NaBH^, acetylated and analysed  The mass spectra obtained verified the presence of an amino  sugar and a hexose unit.  These data may suggest that the anomeric signals  ( a a n d 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 5 4.63,  respectively  by  using reference  compounds.  The shift  and  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  accompanied by Deamination  of  of  rearrangement the  amino  (Williams,  and  their  glycosides is  1975; Aspinall et a L ,  B-amino component, followed by  the formation of hexitol hexacetate. a  sugars  g.l.c.  usually  1978, 1980).  analysis  showed  This finding eliminates the possibility of  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 chromatography. Complete characterization of a permethylated polysaccharide requires identification and quantitative depolymerization.  With  not seen in electron  the  analysis of all the sugar derivatives formed on use of g . l . c . - m . s .  impact spectra taken  analysis,  at 70 eV,  molecular  ions  are  but molecular weights  can usually be obtained by extrapolation from fragment ions.  - 117 -  Methylation reduction, the  of  derivatization  formation  of  the  native  polysaccharide,  followed  by  acetolysis,  as alditol acetates and g . l . c . - m . s . analysis  2,3,6-tri-O-methyl  deoxy-2-N-methylacetamido  hexitol  glucitol  (Fig. 30),  4,6-di-0-methyl-2-  ( F i g . 31) and a component with m/e 231,  171,  143, 117, 59 ( F i g . 29).  These results suggest that glucose (4  and  N-acetylated amino sugar  (3  chain.  showed  linked)  are  present  in the  linked)  polysaccharide  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 |2  NAc  » ?) deoxy sugar (1  *  - 118 -  CH„OAc I H - C - OMe I MeO - C - H d  I  117 233  H - C - OAc I  H - C - OAc CH OMe 2  Fig. 30  233  (M  233  - acetic acid (m/e 60)  117  - formaldehyde (m/e 30)  +  45  - 117) 173 87  Fragmentation pattern observed in the mass spectrum of 2,3,6-tri-O-methyl-D-glucitol.  - 119 -  CH OAc 2  H - C  N  Ac 158  H - C - OAc  161  — —I  274  H - C - OMe H - CH C -OMe OAc 2  Fig. 31  161  - acetic acid (m/e 60)  101  158  - ketene (m/e 42)  116  116  - ketene (m/e 42)  74  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. microbial  Pseudomonas fragi has been reported to be involved in the  spoilage  of  meat  at  both  chill  and  ambient  temperatures.  The  spoilage process appears to include the attachment and adhesion of microorganisms to the mechanism  substrate. by  This  physical  The data from this  study  and  was  chemical  an attempt to examine the adhesion  analyses  of  the  bacterial  glycocalyx.  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 Costerton et a L , an acidic  to  published  reports  1978; Yada and S k u r a ,  polysaccharide  in bacterial  (Fletcher  and  Floodgate,  1973;  1982) suggesting the involvement of  glycocalyces,  this  study  has identified  the involvement of a neutral polysaccharide only. An understanding of structure-function relationships of the e x t r a cellular  material  may  spoilage of foods. charide  chain  microorganism,  may this  provide  answers  to  the  role  of  glycocalyces  in  the  Since the types of monomeric units present in the polysaccontribute then  to  implies  the that  biological state and properties of the  basic  functions  of  the  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 material.  materials  are formed prior to the formation of glycocalyx  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  by  (1981) demonstrated that  current  literature.  Yada and S k u r a ,  as suggested  proteolysis of beef muscle proteins is observable in the late logarithmic 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. f r a g i ; Since extracellular materials are produced at various stages of cell growth,  and  are  influenced  explain the various  by  the  type  of  substrate,  this  may  rates of microbial spoilage of liquid and solid  foods; Polysaccharides, constitute  the  together with O antigens of the lipopolysaccharide, principal  because of their Detailed  immunogen and  antigens  of  the  bacteria  location at the extreme outer surface of the cell.  knowledge of the  structure of the polysaccharide on the  bacterial surface could lead to the design of highly effective inhibitors 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  attachment on and detachment of microorganisms from the of foods would  aid in the  selection of an analytical  in  surfaces  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  reduce public health hazards;  improve  shelf  life  and  possibly  - 122 -  The involvement of a neutral the glycocalyx of P. calyx  and  present N-acetyl  various  in  the  and not an acidic polysaccharide in  fragi suggests that interaction between glycosubstrates  polymer.  may  be  Therefore,  dependent the  group of the amino sugar may  the polymer.  on  presence  restrict  sugar of  the  types bulky  the flexibility of  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 examination of: (a)  the nature of the blebs;  (b)  the relationship between blebs and glycocalyx;  (c)  variations  (if  any)  of  the  glycocalyx  composition when  fragi cells are grown on various media; and (d)  methods for the prevention of glycocalyx formation.  P.  - 123 -  CONCLUSION:  1.  Pseudomonas f r a g i , 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 morphological 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 A L B E R S H E I M , P., NEVINS, D.J., ENGLISH, P.D. and K A R R , A . 1967. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5:340. A S P I N A L L , G . O . 1982. Chemical characterization and structure determination of polysaccharides. In " T h e Polysaccharides". Vol.1. Aspinall, G . O . ( E d . ) . Academic Press, New Y o r k , NY. p.35. A S P I N A L L , G . O . and S T E P H E N , A . M . 1973. Polysaccharide methodology and plant polysaccharides. Organic Chem. Series ONE. 7:285. A S P I N A L L , G . O . , P R Z Y B Y L S K I , E., RITCHIE, R . G . S . and WONG, C O . 1978. Nitrous acid deamination of methylated amino-oligosaccharide glycosides. Carbohydr. Res. 66:225. A S P I N A L L , G . O . , GHARIA, M.M. and WONG, C O . 1980. Deamination of 2-amino-2-deoxy hexitols and of their per-O-methylated derivatives with nitrous acid. Carbohydr. Res. 78:275. B A E C H L E R , C A . and B E R K , R.S. 1974. Electron microscopic observations of Pseudomonas aeruginosa. Zeitschrift fur A l l g . Microbiologie. 14:267. BAER,  H . H . 1969. Oligosaccharides. In " T h e amino s u g a r s . " Jeanloz, ( E d ) . Academic Press, New Y o r k , NY. p.287.  R.W.  B A T E - S M I T H , E . C 1948. The physiology and chemistry of rigor mortis, with special reference to the ageing of beef. A d v . Food. Res. 1:1. B A Y E R , M.E. 1967. Response of cell walls of Escherichia coli to a sudden reduction of the environmental osmotic pressure. J . Bacteriol. 73:365. B A Y E R , M.E. and THUROW, H. 1977. Polysaccharide capsule of Escherichia coli: microscope study of its size, structure, and sites of synthesis. J . Bacteriol. 130:911. B E H N K E , O. 1968. Electron microscopical observations on the surface coating of human blood platelets. J . Ultrastruct. Res. 24:51. B E H N K E , O. and Z E L A N D E R , T . 1970. Preservation of intercellular substances by the cationic dye alcian blue in preparative procedures for electron microscopy. J . Ultrastruct. Res. 31:424. B E N N E T T , H.S. 1963. Morphological aspects of extracellular polysaccharides. J . Histol. Cytochem. 11:14. B E R R Y , J . M . , D U T T O N , G . G . S . , H A L L , L.D. and MACK IE, K.L. 1977. Structural studies of Klebsiella capsular polysaccharides by using natural abundance carbon-13 nmr spectroscopy. Carbohydr. Res. 53:C8.  - 125 -  B H A T T A C H A R J E E , A . K . , JENNINGS, H.J., KENNY, C . P . , M A R T I N , A . and SMITH, C P . 1975. Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis Serogroup B and C with Carbon 13 nuclear magnetic resonance. J . Biol. Chem. 250:1926. BISHOP, C . T . , COOPER, hydrate derivatives 4:2245.  F.P. and MURRAY, R.K. 1963. Reactions of carboduring gas-liquid chromatography. C a n . J . Chem.  B I T T E R , T . and MUIR, H.M. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4:330. B J O R N D A L , H . , LINDBERG, B. and S V E N S S O N , S. 1967. Mass spectrometry of partially methylated alditol acetates. Carbohydr. Res. 5:433. B J O R N D A L , H . , H E L L E R Q U I S T , C . G . , LINDBERG, B. and S V E N S S O N , S. 1970. Gas-liquid chromatography and mass spectrometry in methylation analysis of polysaccharides. Angew. Chem. Internat. Edit. 9:610. B L A K E , J . D . and RICHARDS, G . N . 1970. A critical re-examination of p r o blems inherent in compositional analysis of hemicelluloses by gas-liquid chromatography. Carbohydr. Res. 14:375. BLANQUET, P.R. 1976a. Ultrahistochemical study on the ruthenium red surface staining. I. Processes which give rise to electron-dense marker. Histochemistry. 47:63. BLANQUET, P.R. 1976b. Ultrahistochemical study on the ruthenium red surface staining. II. Nature and affinity of the electron-dense marker. Histochemistry. 47:175. B L O C K , R.J., DURRUM, E.L. and ZWEIG, G. 1958. A manual of paper chromatography and paper electrophoresis. Academic Press, New Y o r k , NY. p.170. B L U M B E R G , K. and B U S H , C A . 1982. Mass spectrometry and carbon-13 nuclear magnetic resonance spectroscopy of compounds modeling the glycopeptide linkage of glycoproteins. Anal. Biochem. 119:397. BOETHLING, R.S. 1975. Regulation of extracellular Pseudomonas maltophilia. J . Bacteriol. 123:954.  protease  secretion  in  B R A D B U R Y , J . H . and JENKINS, G . A . 1984. Determination of the structures of trisaccharides by C n . m . r . spectroscopy. Carbohydr. Res. 126:125. B R U N K , U . , C O L L I N S , V . P . and A R R O , E. 1981. The fixation, dehydration, drying and coating of cultured cells for SEM. J . Microsc. 123:121. B U N D L E , D.R. and LEMIEUX, R.U. 1976. Determination of anomeric configuration by NMR. Methods C a r b o h y d r . Chem. 7:79. B U R D E T T , I.D.S. and MURRAY, R.G. E. 1974. Septum formation in Escherichia coli -.characterization of septal structure and the effects of antibiotics on cell division. J . Bacteriol. 119:303.  - 126 -  BUTLER, J.L., VANDERZANT, C , CARPENTER, Z.L., SMITH, G.C., LEWIS, R.E. and D U T S O N , T . R . 1980. Influence of certain processing steps on attachment of microorganisms to pork skin. J . Food Prot. 43:699. C A S S O N E , A . and G A R A C I , E. 1977. The capsular pneumoniae. C a n . J . Microbiol. 23:684. CHURMS, S. 1970. Gel chromatography Chem. Biochem. 25:13.  network  of carbohydrates.  of  Adv.  Klebsiella  Carbohydr.  C O N R A D , E . C . and PALMER, J . K . 1976. Rapid analysis of carbohydrates by high-pressure liquid chromatography. Food Technol. 30(10 ):84. C O S T E R T O N , J.W. 1970. The structure and function of the cell envelope of gram-negative bacteria. Rev. C a n . Biol. 29:299. C O S T E R T O N , J.W. 1979. The role of electron microscopy in the elucidation of bacterial structure and function. A n n . Rev. Microbiol. 33:459. C O S T E R T O N , J.W., INGRAM, J . M . and C H E N G , K.-J. 1974. Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 38:87. C O S T E R T O N , J.W., GEESEY, G.G. stick. Sci. Amer. 238:86.  and  CHENG,  K.-J.  1978. How  bacteria  C O S T E R T O N , J.W., IRVIN, R . T . and C H E N G , K.-J. 1981. The glycocalyx in nature and disease. A n n . Rev. Microbiol. 35:299.  bacterial  C O X O N , B. 1972a. Proton magnetic resonance Carbohydr. Chem. Biochem. 27:7.  I.  spectroscopy:  C O X O N , B. 1972b. Conformational analysis via nuclear spectroscopy. Methods C a r b o h y d r . Chem. 6:513.  Part  magnetic  Adv.  resonance  DAINTY, R . H . , SHAW, B . G . , DeBOER, K.A. and S C H E P S , E . S . J . 1975. Protein changes caused by bacterial growth on beef. J . A p p l . Bacteriol. 39:73. De BRjJYN, A . , A N T E U N I S , M . , De GUSSEM R. and D U T T O N , G . G . S . 1976. H NMR study of L-rhamnose, methyl CX-L-rhamnopyranoside, and 4-0-j3~ D-galactopyranosyl-L-rhamnose in deuterium oxide. Carbohydr. Res. 47:158. DIERICHS, R. 1979. Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action. Histochemistry. 64:171. DISCHE, Z. 1962. Color reactions Chem. 1:497.  of hexuronic acids.  Methods  Carbohydr.  DISCHE, Z. and S H E T T L E S , L.B. 1948. A specific colour reaction of methylpentoses and a spectrophotometric micromethod for their determination. J . Biol. Chem. 175:595.  - 127 -  D O M A G A L A , W., K A H A N , A . V . and KOSS, L. G. 1979. A simple method of preparation and identification of cells for scanning electron microscopy. Acta. C y t o l . 23:140. DUBOIS, Nl., G I L L E S , K . A . , HAMILTON, J . K . , R E B E R S , P. A . and SMITH, F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350. DUDMAN, W.F. and W H I T T L E , C P . 1976. Interference by phthalic esters in the gas-chromatographic analysis of sugars. Carbohydr. Res. 46:267. D U T S O N , T . R . , PEARSON, A . M . , PRICE, J . F . , SPINK, G . C and T A R R A N T , P.J.V. 1971. Observations by electron microscopy on pig muscle inoculated and incubated with Pseudomonas f r a g i . A p p l . Microbiol. 22:1152. D U T T O N , G . G . S . 1973. Applications of gas-liquid chromatography to carbohydrates. P a r t i . A d v . Carbohydr. Chem. Biochem. 28:11. D U T T O N , G . G . S . 1974. Applications of gas-liquid chromatography to carbohydrates. Part II. A d v . Carbohydr. Chem. Biochem. 30:9. D U T T O N , G . G . S . and Y A N G , M . T . 1973. The structure of the polysaccharide of Klebsiella K-type 5. C a n . J . Chem. 51:1826.  capsular  F A L E S , H . M . , MILNE, G.W.A. and NICHOLSON, R.S. 1971. Chemical ionization mass spectrometry of complex molecules: esters of di and tricarboxylic acids. Anal. Chem. 43:1785. FINAN, P . A . , REED, R.I. and SNEDDEN, W. 1958. The application of the mass spectrometer to carbohydrate chemistry. Chem. Ind. (London), p.1172. F I R S T E N B E R G - E D E N , R. 1981. Attachment review. J . Food Prot. 44:602.  of  bacteria  to  meat  surfaces:A  F I R S T E N B E R G - E D E N , R. , NOTERMANS, S., T H I E L , F., H E N S T R A , S., and KAMPELMACHER, E.H. 1979. Scanning electron microscopic investigations into attachment of bacteria to teats of cows. J . Food Prot. 42:305. F L E T C H E R , M. and F L O O D G A T E , G . M . 1973. An electron-microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J . Gen. Microbiol. 74:325. F L E T C H E R , M. and L O E B , G.I. 1979. Influence of substratum characteristics on the attachment of a marine pseudomonad to solid surfaces. A p p l . Environ. Microbiol. 37:67. G A R E G G , P.J., J A N S S O N , P.E., LINDBERG, B., LINDH, F., LONNGREN, J . , K V A R N S T R O M , I. and NIMMICH, W. 1980. Configuration of the acetal carbon atom of pyruvic and acetals in some bacterial polysaccharides. C a r b o h y d r . Res. 78:127. G A R L A N D , C D . , LEE, A . and DICKSON, M.R. 1979. The preservation of surface-associated microorganisms prepared for scanning electron microscopy. J . Microsc. 116:227.  - 128 -  G E Y E R , R., G E Y E R , H . , K U H N H A R D T , S., MINK, W. and STIRM, S. 1982. Capillary gas chromatography of methylhexitol acetates obtained upon methylation of N-glycosidically linked glycoprotein oligosaccharides. Anal. Biochem. 121:263. GILL,  O. 1976. Substrate limitation of bacterial growth at meat surfaces. A p p l . Bacteriol. 41:401.  J.  GILL,  O. and NEWTON, K . G . 1977. The development of aerobic spoilage flora on meat stored at chill temperatures. J . A p p l . Bacteriol. 43:189.  GILL,  C O . and NEWTON, K . G . 1978. The ecology of bacterial fresh meat at chill temperatures. Meat Science. 2:207.  GILL,  C O . and NEWTON, K . G . 1980. Growth temperatures. J . A p p l . Bacteriol. 49:315.  spoilage of  of bacteria on meat at room  GORIN, P . A . J . 1981. Carbon - 13 Nuclear magnetic resonance spectroscopy of polysaccharides. A d v . Carbohydr. Chem. Biochem. 38:13. GREENWOOD, C J . 1952. The size and shape of some polysaccharide molecules. A d v . Carbohydr. Chem. 7:289. GUNNER, S.W., JONES, J . K . N , and P E R R Y , M.B. 1961. The gas-liquid partition chromatography of carbohydrate derivatives. Part l:The separation of glycitol and glycose acetates. C a n . J . Chem. 39:1892. HAKOMORI, S. 1964. A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsufinyl carbanion in dimethyl sulfoxide. J . Biochem. (Tokyo) 55:205. HALL,  L.D. 19:51.  1964.  Nuclear  magnetic  resonance.  Adv.  Carbohydr.  Chem.  HALL,  L . D . 1974. Solutions to the hidden - resonance problem in proton nuclear resonance spectroscopy. A d v . Carbohydr. Chem. Biochem. 29:11.  HODGE, J . E . and H O F R E I T E R , B . T . 1962. Determination of reducing sugars and carbohydrates. Methods Carbohydr. Chem. 1:380. HOLLENBERG, M.J. and ERICKSON, A . M . 1973. The scanning electron microscope: potential usefulness to biologist. A review. J . Histochem. Cytochem. 21:109. HOLT,  S.C and B E V E R I D G E , T . J . 1982. Electron microscopy: its development and application to microbiology. C a n . J . Microbiol. 28:1.  HOUGH, L. and JONES, J . K . N . 1962. Chromatography: chromatography on paper. Methods C a r b o h y d r . Chem. 1:21. INGRAM, M. and D A I N T Y , R.H. 1971. Changes caused by microbes in spoilage of meats. J . A p p l . Bacteriol. 34:21.  - 129 -  ITO,  S. 1969. Structure Exp. Biol. 28:12.  and function o f the glycocalyx.  Fed.  Amer.  Soc.  JAY,  J . M . 1972. Mechanism and detection o f microbial spoilage in meats low temperatures:a status report. J . Milk Food Technol. 35:467.  in  JENNINGS, H.J. and SMITH, I.CP. 1980. Determination of polysaccharide structures with C NMR. Methods Carbohydr. Chem. 8:97. 13  JONES, H . C , R O T H , I.L. and S A N D E R S , W.M. study of a slime layer. J . Bacteriol. 99:316.  1969. Electron microscopic  J U F F S , H . S . , HAYWARD, A . C and D O E L L E , H.W. 1968. Growth and proteinase production in Pseudomonas spp. cultivated under various conditions of temperature and nutrition. J . Dairy Res. 35:385. K E L L E N B E R G E R , E. and R Y T E R , A . 1958. Cell wall and cytoplasmic membrane o f Escherichia coli. J . Biophys. Biochem. C y t o l . 4:323. KENNE, L. and LINDBERG, B. 1983. Bacterial polysaccharides. In " T h e Polysaccharides. Vol. 2. Aspinall, G . O . ( E d ) . Academic Press, New Y o r k , NY. p.287. K O C H E T K O V , N.K. and CHIZHOV, O . S . 1966. Mass spectrometry o f carbohydrate derivatives. A d v . C a r b o h y d r . Chem. 21:39. KOTOWYCZ, G. and LEMIEUX, R.U. 1973. Nuclear carbohydrate chemistry. Chem. Rev. 73:669.  magnetic  resonance  in  KOWKABANY, G . N . 1954. Paper chromatography o f carbohydrates and related compounds. A d v . Carbohydr. Chem. 9:304. L E N A R D , J . 1969. Reactions o f proteins, carbohydrates and related substances in liquid hydrogen fluoride. Chem. Rev. 69:625. LINDBERG, B., LINDH, F., LONNGREN, J . , LINDBERG, A . A . and S V E N S S O N , S . B . 1981. Structural studies of the O-specific side-chain of the lipo-polysaccharide from Escherichia coli. 055. C a r b o h y d r . Res. 97:105. LINDBERG, B. 11:409.  1982. Structural  studies of polysaccharides. Chem. Soc.  Rev.  LONNGREN, J . and S V E N S S O N , S. 1974. Mass spectrometry in structural analysis of natural carbohydrates. A d v . Carbohydr. Chem. Biochem. 29:41. LUFT,  J . H . 1971a. Ruthenium red and violet. I. Chemistry, purification, methods o f use for electron microscopy and mechanism o f action. Anat. Rec. 171:347.  LUFT,  J . H . 1971b. Ruthenium red and violet. in animal tissues. Anat. Rec. 171:369.  II.  Fine structural localization  M a c l N T Y R E , S., T R U S T , T . J . and B U C K L E Y , J . T . 1980. Identification and characterization o f outer membrane fragments released by Aeromonas sp. Can. J . Biochem. 58:1018.  - 130 -  McCOWAN, R.P., C H E N G , K.-J., B A I L E Y , C . B . M . and C O S T E R T O N , J.W. 1978. Adhesion of bacteria to epithelial cell surfaces within the reticulorumen of cattle. A p p l . Environ. Microbiol. 35:149. McKELLAR, R . C . 1982. Factors influencing the production of extracellular proteinase by Pseudomonas fluorescens. J . A p p l . Bacteriol. 53:305. MACEK, K. 1963. Sugars. In "Paper Chromatography". Hais, Macek, K. ( E d s ) . Academic Press, New Y o r k , N Y . p.289.  I.M.  and  M A R S H A L L , K . C . , S T O U T , R. and M I T C H E L L , R. 1971. Mechanism of the initial events in the sorption of marine bacteria to surfaces. J . Gen. Microbiol. 68:337. M O R T , A . J . 1983. An apparatus for safe and convenient handling of anhydrous, liquid hydrogen fluoride at controlled temperatures and reaction times. Application to the generation of oligosaccharides from polysaccharides. Carbohydr. Res. 122:315. M O R T , A . J . and LAMPORT, D . T . A . 1977. Anhydrous deglycosylates glycoproteins. Anal. Biochem. 82:289.  hydrogen  fluoride  NOTERMANS, S. and KAMPELMACHER, E . H . 1974. Attachment of some bacterial strains to the skin of broiler chickens. B r . Poult. S c i . 15:573. NOTERMANS,  S.,  FIRSTENBERG-EDEN,  R.  and  VAN  SCHOTHORST,  1979. Attachment of bacteria to teats of cows. J . Food Prot. 42:228.  J.  NUNEZ, H . A . , WALKER, T . E . , F U E N T E S , R., O'CONNOR, J . , SERI ANN I, A . and B A R K E R , R. 1977. Carbon-13 as a tool for the study of carbohydrate structures, conformations and interactions. J . Supramol. S t r u c ture. 6:535. O C K E R M A N , H.W., C A H I L L , V . R . , WEISER, H . H . , DAVIS, C . E . and SIEFKER, J . R . 1969. Comparison of sterile and inoculated beef tissue. J . Food. S c i . 34:93. O ' R E I L L Y , T . and D A Y , D . F . 1983. Effects of cultural conditions on protease production by Aeromonas hydrophila. A p p l . Environ. Microbiol. 45:1132. PALLERONI, N.J. 1978. T h e Pseudomonas Meadowfield Press, L t d . England, p.17.  group.  Patterns  of  Progress.  PALMER, J . K . 1975. A versatile system for sugar analysis via liquid chromatography. Anal. Lett. 8:215. PARK,  J . T . and JOHNSON, M.J. J . Biol. Chem. 181:149.  1949. A submicrodetermination of glucose.  PERLIN, A . S . and C A S U , B. 1982. Spectroscopic methods. In " T h e Polysaccharides". Vol.1. Aspinall, G . O . ( E d ) . Academic Press, New Y o r k , NY. p.146.  - 131 -  POWELL, D . A . 1979. Structure, solution properties and biological interactions of some microbial extracellular polysaccharides. In "Microbiol polysaccharides and polysaccharases". Berkeley, R . C . W . , Gooday, G . S . , and Ellwood, D . C . ( E d s . ) . Academic Press, New Y o r k , N Y . p.117. REES,  D . A . and WELSH, E.J. 1977. Secondary and tertiary structure of polysaccharides in solutions and gels. Angewandte Chemie (International Edition in English) 16:214.  REID, G . and B R O O K S , H . J . L . 1982. T h e use of double staining techniques for investigating bacterial attachment to mucopolysaccharide-coated epithelial cells. Stain Techno). 57:5. R I T T E N B U R G , J . H . , B A Y E R , R . C , G A L L A G H E R , M . L . and L E A V I T T , D . F . 1979. A rapid technique for preparing microorganisms for transmission electron microscopy. Stain Technol. 54:275. ROGERS, R.E. and M c C L E S K E Y , C . S . 1961. Objective ground beef. Food Technol. 15:210.  tests  for quality of  R O S E L L , K . G . and JENNINGS, H.J. 1983. Structural elucidation of the capsular polysaccharide of Streptococcus pneumoniae type 9N. C a n . J . Biochem. Cell Biol. 61:1102. ROWE, M . T . and GILMOR, A . 1982. Growth, enzyme production and changes in oxygen tension occurring during batch cultivation of psychrotrophic Pseudomonas fluorescens strain S. Milchwissenschaft. 37 (10):597. R U S S E L L , R . R . B . 1976. Free endotoxin - a review. Microbios. Lett. 2:125. SAGE,  G . 1974. Biochemical and ultrastructural changes occurring in chicken pectoralis muscle inoculated with Pseudomonas f r a g i . M.Sc. Thesis. Univ. British Columbia.  S A N D F O R D , P.A. 1979. Exocellular, h y d r . Chem. Biochem. 36:265.  microbial  polysaccharides.  A d v . Carbo-  S A V A G E , A . V . 1980. Structural investigations and bacteriophage degradations of Klebsiella capsular polysaccharides. P h . D . Thesis. Univ. British Columbia. SAWARDEKER, J . S . , S L O N E K E R , J . H . and J E A N E S , A . 1965. Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Chem. 37:1602. SCHMID, E . N . , MENGE, B. and L I C K F E L D , K . G . 1981. Capsule fine structure in thin slices of Klebsiella pneumoniae Biovar d ( R i c h a r d ) . J . Ultrastruct. Res. 75:41. S C O T T , J . E . 1955. The reaction of long-chain quaternary ammonium with acidic polysaccharides. Chem. Ind. (London), p.168.  salts  S C O T T , J . E . 1960. Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. Methods Biochem. Anal. 8:145.  - 132 -  SCOTT, J.E., Q U I N T A R E L L I , G. and D E L L O V O , M . C . 1964. The chemical and histochemical properties of alcian blue. I. The mechanisms of alcian blue staining. Histochemie. 4:73. S H A R O N , N. 1975. Glycoproteins - II isolation and characterization. In "Complex carbohydrates, their chemistry, biosynthesis and function". Sharon, N. ( E d . ) . Addison-Wesley Publ. C o . , Reading, MA. p.48. STELLNER, K., SAITO, aminosugar linkages Biophys. 155:464.  H. in  and HAKOMORI, S. 1973. glycolipids by methylation.  Determination of A r c h . Biochem.  S T E V E N S , J . D . and F L E T C H E R , H . G . 1968. The proton magnetic spectra of pentofuranose derivatives. J . O r g . Chem. 33:1799.  resonance  S T O R T Z , C . A . , MATULEWICZ, M . C . and C E R E Z O , A . S . 1983a. The mass spectra of acetylated and propanoylated aldopyranosylamines. Carbohydr. Res. 117:39. S T O R T Z , C . A . , MATULEWICZ, M . C . and C E R E Z O , A . S . 1983b. The mass spectra of acetylated and propanoylated aldofuranosylamines. Carbohydr. Res. 122:49. SUTHERLAND, I.W. 1979. Microbial Exopolysaccharides:Control of Synthesis and Acylation. In "Microbial polysaccharides and polysaccharases". Gooday, G.W. and Ellwood, D . C . ( E d s ) . Academic Press, New Y o r k , NY. p.1. SWEELEY, C C , B E N T L E Y , R., M A K I T A , M. and WELLS, W.W. 1963. Gas liquid chromatography of trimethylsilyl derivatives of sugars and related substances. J . Amer. Chem. Soc. 85:2497. T A R R A N T , P . J . V . , PEARSON, A . M . , PRICE, J . F . and LECHOWICH, R.V. 1971. Action of Pseudomonas fragi on the proteins of pig muscle. A p p l . Microbiol. 22:224. T A Y L O R , R.L. and C O N R A D , H.E. 1972. Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Biochemistry. 8:1383. THOMAS, C J . and McMEEKIN, T . A . 1980. Contamination of broiler carcass skin during commercial processing procedures: an electron microscopic study. A p p l . Environ. Microbiol. 40:133. TURNER, A. 35:386. USUI,  1960.  Assessing  meat  spoilage  in  the  laboratory.  Food  Mf.  T . , Y A M A O K A , N . ^ M A T S U D A , K., TUZIMURA, K. , SUGIYAMA, H. and S E T O , S. 1973. C nuclear magnetic resonance spectra of glucobioses, glucotrioses, and glucans. J . Chem. S o c , Perkin T r a n s . 1:2425.  WATSON, L.P., McKEE, A . E . and M E R R E L L , B.R. 1980. Preparation of microbiological specimens for scanning electron microscopy. In: SEM/1980 II, SEM, Inc., AMF O'Hare, IL 60666, p.45.  - 133 -  WERNER, I. and ODIN, L. 1952. Presence of sialic acid in certain glycoproteins and in gangliosides. Acta. Soc. Med. Upsalien. 57:230. WESTPHAL, O. and J A N N , K. 1965. Bacterial lipopolysaccharides: Extraction with phenol-water and further applications of the procedure. Methods C a r b o h y d r . Chem. 5:83. W H I T T A K E R , D.K. and D R U C K E R , D . B . 1970. Scanning electron microscopy of intact colonies of microorganisms. J . Bacteriol. 104:902. WIEBE, W.J. and C H A P M A N , G . B . 1968. Fine structure of selected Pseudomonas and Achromobacter. J . Bacteriol. 95:1862. WILKINSON, J . F . 1958. The extracellular polysaccharides of bacteria. iol. Rev. 22:46.  marine Bacter-  WILLIAMS, J . M . 1975. Deamination of carbohydrate amines and related compounds. A d v . Carbohydr. Chem. Biochem. 31:9. WILLIAMS, G. and WIMPENNY, J . W . T . 1977. Exopolysaccharide production by Pseudomonas NCIB 11268 grown in batch culture. J . Gen. Microbiol. 102:13. WING,  R.E. and BeMILLER, J . N . 1972. Quantitative thin layer chromatography. Methods C a r b o h y d r . Chem. 6:54.  Y A D A , R.Y. and S K U R A , B.J. 1981. Some biochemical changes in sarcoplasmic depleted, intact beef muscle inoculated with Pseudomonas f r a g i . J . Food Sci. 46:1766. Y A D A , R.Y. and S K U R A , B.J. 1982. Scanning electron microscopy study of Pseudomonas fragi on intact and sarcoplasm-depleted bovine longissimus dorsi muscle. A p p l . Environ. Microbiol. 43:905.  - 134 -  APPENDIX I Infrared Spectroscopy Data  Appendix  1.1  Infrared  spectrum  of  permethylated  P.  fragi  polysaccharide  - 136 -  APPENDIX  II  N.M.R. Spectroscopy Data  100 MHz amb. t e m p  61.73  19.51  (ppm) Appendix II.2  *"*C n . m . r .  spectrum o f  Native  polysaccharide  100 MHz amb. t e m p .  (acetone) 31,07  (ppm)  A p p e n d i x 11.4  13  C n.m.r.  spectrum o f  de-N-acetylated  polysaccharide  400 MHz 90°C  (ppm) A p p e n d i x II.9  1  H n.m.r.  spectrum o f  component  B-neutral  (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 PEAK NU. 1 2 3 4 5 6 7 8 10 11 12 13 14 16 21 22 25 26 27 28 29 30 31 32 33 36 38 39 40 41 43 44 45 46 47 49 50 51 53 54 56 57 58 59 60 61 62 65 66 67 68 69 71 72 73  1  PAGE MEASURED MASS 362 3GI 298 294 2<Jil 2B9 273 272 261 260 259 257 255 242 230 229 220 219 218 217 213 212 211 210 208 204 201 200 199 195 191 190 189 188 187 184 181 179 176 175 173 172 171 170 169 168 166 161 160 159 158 157 155 154 153  NO. POINTS  ABSOLUTE INTENSITY  rs, 35 Z9 21 35 43 35 43 • .29 35 43 21 17 35 29 21 43 35 51 43 43 43 35 25 21 21 35 35 43 29 43 21 35 43 51 25 43 21 35 43 35 29 43 43 51 43 17 43 35 43 43 43 35 . , 51 51  )  i  1397. 7862. 22U5. 858. 5407. 36823. 3960. 3162. 2170. 3868. 30199. 1310. 880. 4 705. 2670. 1347. 2901. 1324. 5155. 56399. 5590. 7816. 2831 . 1592. 1255. 1165. 2975. 5686. 4897. 2005. 1044. 1079. 504 4 . 8181 . 87624. 1603. 1290. 1 177. 2292. 13905. 1346. 2225. 9828. 66680. 2679. 4331 . 847. 5125. 1534. 4262. 24425. 72436. 1174. ' ' 5507. 17911.  X  INT. BASE 0. 1 0.6 0.2 0.1 0.4 2.6 0.3 0.2 0.2 0.3 2.1 0.1 0.1 0.3 0.2 0.1 0.2 0.1 0.4 3.9 0.4 0.5 0.2 0.1 0.1 0.1 0.2 0.4 0.3 0.1 0.1 0.1 0.4 0.6 6.1 0.1 0.1 0. 1  0.2  1 .0 0. 1 0.2 0.7 4.7 0.2 0.3 0.1 0.4 0.1 0.3 1.7 5.1 0.1 0.4 1.3  X  INT. NREF 0.1 0.6 0.2 0. 1 0.4 2.6 0.3 0.2 0.2 0.3 2.1 0. 1 0.1 0.3 0.2 0.1 0.2 0. 1 0.4 3.9 0.4 0.5 0.2 0.1 0.1 0.1 0.2 0.4 0.3 0.1 0.1 0. 1 0.4 0.6 6.1 0.1 0.1 0. 1 0.2 1 .0 0.1 0.2  0.7  4.7 0.2 0.3 0.1 0.4 0. 1 0.3 1.7 5. 1 0.1 0.4 1.3  X TOT. ION 0.0 0.2 0. 1 0.0 0.2 1.1 0. 1 0.1 0.1 0.1 0.9 0.0 0.0 0.1 0.1 0.0 0.1 0.0 0.1 1.6 0.2 0.2 0.1 0.0 0.0 0.0 0.1 0.2 0.1 0.1 0.0 0.0 0. 1 0.2 2.5 0.0 0.0 0.0 0.1 0.4 0.0 0. 1 0.3 1.9 0.1 0.1 0.0 0. 1 0.0 0. 1 0.7 2.1 0.0 0.2 0.5  PEAK  NO. 74 76  7/ /n  79  80 BI B2 83 34 86 OU  B'J  90 91 92 93 94 95 96 97 98 99 mn 101 102 103 104 105 106 107 109 1 10 1 1 1 112 113 114 115 116 117 1 18 1 19 120 121 122 123 124 125 126 127 120 129 130 131 132  2 HrASurrr  NO.  POINTS  is.' Mb  H'J  144 143 142 141 140 139 138 136 134 133 131 130 129 128 127 126 123 121 118 117 116 115 1 14 113 112 111 110 109 106 105 104 103 102 101 100 99 98 97 95 93 90 89 88 87 86 85 84 83 82 81  (10 79  «  51 43 51 35 43 43 51 SI 59 25 35 43 43 51 43 43 51 59 59 43 51 43 51 43 51 43 51 51 59 59 51 43 43 43 43 43 43 43 59 59 59 51 43 29 43 35 51 71 71 59 71 43 i 43* 43  S  l  ABSOLUTE INTENSITY  ,  15419.' 9419. 121544. 2414. 3713. 4240. 2225. 8937. 74724. 861 . 1790. 1198. 3068. 2201 . 1296. 20243. 80428. 45239. 3260. 3692. 1924. 2561 . 20787. 33963. 194708. 3154. 2039. 7858. 14947. 25841. 2698. 1686. 3388. 3983. 97760. 8750. 15976. 2304. 15324. 22578. 45159. 6904. 1091 . 987. 2777. 1560. 17835. 42565. 52987. 2422. 6198. 3159. 13110. 2120. 915.  X  INT. BASE 1.1 0.7 8.5 0.2 0.3 0.3 0.2 0.6 5.2 0. 1 0.1 0. 1 0.2 0.2 0.1 1 .4 5.6 3.2 0.2 0.3 0.1 0.2 1.5 2.4 13.6 0.2 0.1 0.6 1 .0 1.8 0.2 0.1 0.2 0.3 6.8  0.6  1 . 1 0.2 1 . 1 1.6 3.2 0.5 0.1 0.1 0.2 . 0.1 1.2 3.0 3.7  0.2  0.4 0.2 0.9 0.1 0.1  X  INT. NREF 1.1 0.7 8.5 0.2 0.3 0.3 0.2 0.6 5.2 0. 1 0.1 0.1 0.2 0.2 0.1 1.4 5.6 3.2 0.2 0.3 0.1 0.2 1.5 2.4 13.6 0.2 0.1 0.6 1.0 1.8 0.2 0.1 0.2  0.3  6.8  0.6 1.1  0.2  1.1 1.6 3.2 0.5 0.1 0.1 0.2  0.1  1.2 3.0 3.7 0.2 0.4 0.2 0.9 0.1 0.1  X TOT. ION 0.4 0.3 3.5 0.1 0. 1 0.1 0.1 0.3 2.2 0.0 0.1  0.0  0.1 0.1 0.0 0.6 2.3 1.3  0.1  0.1 0.1 0. 1 0.6 1 .0 5.7 0.1 0.1 0.2 0.4  0.8 0.1  0.0 0. 1 0. 1 2.8 0.3 0.5 0.1 0.4 0.7 1.3 0.2 0.0 0.0 0.1 0.0 0.5 1.2 1 .5 0. 1 0.2 0. 1 0.4 0.1 0.0  PAGfc  3  PC/K fid.  MEASURED  1.13 13!. 136 137 1 38 13<J 140 141 142 143 144 14b 146 147 148 ISO 101 152 153 1S4 185 157 158 159  77 75 74  ity  161 162 163  73 72 71 70 69 68 67 66 62 61 60 59 57 56 55 54 53 52 47 46 45 44 43 42 41  NO. POINTS  ABSOLUTE INTENSITY  43 43 43 43 51 59 51 59 « SI 43 43 43 43 43 51 51 51 51 43 43 43 59 51 71 71 87 59 51  5909 . 594 3 . 1/499. 3t.'9B . 1 564 . 16147. 5982. 31944. 1 5UB6. 4545 . 5282. 1190. 11712. 4 137. 4220. 4524 . 9083. 19220. 3092. 1421 . 1683. 2476. 1294 . 18328. 71168. 1428160. 36123. 18168.  t  X INT. BASE  X INT. NRCF  X TOT. ION  0. 4 0. 4 1 .2 2 .7 0. 1 1 .1 0. 4 2. 2 I .1 0. 3 0. 4 0. 1 0. a 0. 3 0. 3 0. 3 0. 6 1 .3 0. 2 0. 1 0. 1 0. 2 0. 1 1 .3 5. 0 100. 0 2. 5 1 .3  0. 4 i».4 1 .2  0.2 0.2  » _ 7 if. 1 1 .]  a. 4  2. 2 1 .1 II. 3 a. 4 it. 1 u. 8 ii. 3 0. 3 a. 3 H. 6 l. 3 o. 2 u. 1 ti. 1 u. 2 0. 1 l. 3 5. 0 100. 0 2. 5 1 .3  b 1 . 1  II.  0.0  0.5  0.2 0.9 0.  4  e. i  a. :• 0.tf 0.3 0.1 0. 1 0.1 0.3 0.6 0.1 0.0 D.a  0. 1 0.0  0.5 2. 1 41.5*" 1 . 1 0.5  - 154 -  APPENDIX III.2 Mass Numbers of B-amino  - 155 -  MASS LIST 01/31/84 14: 00: 0 0 + 13: 49 SAMPLE: A-HRA 40 430 MASS  *  0. 00 MINIMA 0 MAXIMA MASS •/. RA  40 0. 24 41 1. 23 43 100. 00 44 4. 36 45 1. 25 50 0. 06 51 0. 13 52 0. 09 53 0. 16 54 0. 10 55 1. 13 56 1. 38 57 0. 84 58 0. 30 59 0. 99 60 9. 38 61 0 78 62 0. 07 65 0. 12 67 0. 21 68 1. 27 69 1. 25 70 0. 74 71 0. 55 72 2. 22 73 1. 58 74 0. 26 75 0. 09 77 0. 10 78 0. 09 79 0. 12 80 0. 24 81 0. 33 82 0. 41 84 35. 68 85 6. 91 86 1. 49 87 0. 14 88 0. 22 89 0. 06 90 0. 39 91 0. 06 92 0. 07 93 0. 10 94 0. 13 95 0. 08 96 3. 05 97 2 73 98 l. 19 99 0. 29 100 0. 64  101 102 103 104 105 108 109 110 111 112 114 115 116 117 118 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 -144 145 146 147 150 151 152 153 154 155 156 L57 158 159  DATA: FB40 # 790  MIN :  56.  BASE M/E: 43 RIC: 389632. MAX INTEN:  111744.  7. RA  MASS  •/. RA  MASS  7.RA  1. 21 14. 59 2. 46 0. 21 0. 09 0. 07 0. 85 1. 86 0. 96 0. 41 4. 07 3. 42 0. 64 0. 17 0. 13 0. 17 0. 13 0. 17 0. 16 1. 88 1. 35 2. 66 0. 32 0. 24 0. 12 0. 62 0. 12 0. 13 0. 08 0. 11 0. 05 4. 07 7. 39 0. 76 0. 17 0. 3B 0. 51 24. 77 6. B3 0. 52 0. 08 0. 65 4. 13 54 9.73 0. 16 0. 16 5. 15 3. 68 0. 55 0. 11  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  o. 07 0 07 l . 92 89 o. 3. 39 0. 48 0. 24 0. 05 0. 84 0 21 0. 05 0. 33 0. 43 0. 08 0. 06 0. 14 3. 95 1. 11 0. 32 0. 08 0. 07 0. 08 0. 03 2. 93 0. 63 0. 09 0. 09 2. 16 1. 13 0 24 0. 13 0. 03 0. 25 0. 10 0. 23 0. 05 0. 39 1. 09 1. 86 0. 23 0 10 0. 65 2. 51 0. 30 0. 06 0. 05 0. 07 0. 23 0. 49 0. 67 0. 30  232 234 240 241 242 246 247 248 250 253 254 256 258 259 260 261 270 271 272 273 274 276 277 278 281 283 288 2B9 290 292 300 301 302 313 314 318 319 320 321 330 331 332 333 334 348 360 361 373 374 375  0. 20 0. 11 0. 46 0. 07 0. 22 0. 76 0. 10 0. 07 0. 03 1. 24 0. 29 0. 09 4. 25 7. 27 0. 86 0. 11 0. 10 1. 23 1. 01 0. 19 0. 13 0. 82 0. 15 0. 06 0. 12 0. 06 0. 39 0. 26 0. 13 0. 07 0. 86 0. 15 0. 06 0. 25 0. 33 5. 76 0. 98 0. 20 0. 07 0. 14 0. 61 0. 30 0. 09 0. 08 0. 06 0. 83 0. 12 0. 06 0. 24 0. 06  - 156 -  APPENDIX III.3 Mass Numbers of HF-B (Hexitol Hexaacetate)  -  157  -  tr ma  .n  LI li. u. LU  o o  ^  41 « o* iii ^ m — — < > rfi - « . « , « B t n o i n i ? i  H  l  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 —  IJ  • o  i« * • <4 0" O -41 -*j (h o n -*  n n n <n m  ID  im  0) o  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  . m « in o  CJ  —nn  « Q i n o o * n a ) O D ' V M i i « n ( D ' * M 9  i* — — ~ «  4 4 o a ) ' «  •.••osM^ioo«ciir't"»inMDi'-*-ocir! * ^ « -1 c, It; i", i r i f j  Cii  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)O0 O^nin«rtntnNt9chrtnrirvrt rt rt ... .~ rt rt -. m r * ni r< k  —  CJ  - 160 -  APPENDIX III.5 Mass Numbers of 2,3,6-tri-O-methyl-D-glucitol  - 161 -  MASS LIST ©1/31/84 1 4 3 8 0 © SAMPLE- MHRA 40 447 MASS  *  •  0. 00 MINIMA 0 MAXIMA RA MASS  40 2. 30 41 38. 22 43 100 00 44 29. 87 43 62. 48 46 4. 00 47 3. 85 48 0. 10 49 0. 04 50 0 27 51 0. 57 52 0. 76 S3 12. 10 34 2. 89 55 22. 55 36 6. 19 57 16. 50 58 28. 95 59 25. 86 60 3. IB 61 2. 61 62 0. 09 63 0. 13 64 0 07 65 1. 22 66 0. 63 67 2. 48 68 4. 99 69 13. 11 70 6. 50 71 44 14 72 5. 71 73 21. 50 74 30 73 75 31 97 76 1. 19 77 0 68 78 0 19 79 0 6B 81 16. 66 82 2. 57 83 7. 67 85 42. 29 87 64. 27 88 21. 50 89 11. 62 90 0. 58 91 0. 16 92 0. 04 93 0. 59 94 0 60  95 96 97 9B 99 100 101 102 103 104 105 106 107 108 109 111 113 114 113 117 118 119 120 121 122 123 124 125 126 127 129 130 131 132 133 134 135 136 137 139 140 141 142 143 144 145 146 147 148 149 150  9:24 MIN  DATA: FB41 « 337 57.  INTEN  BASE H/E 43 RIC: 3701630. MAX INTEN : 401920.  X RA  MASS  X RA  MASS  X RA  2. 36 0. 96 9 82 35. 86 56. 94 14. 82 56. 75 9. 20 7. 11 0. 69 0. 43 0. 05 0. 09 0. 28 1. 27 13. 75 63. 38 12. 68 13. 54 73. 50 17. 07 3. 11 0. 19 0. 09 0. 05 0. 15 0. 72 5. 84 0. 88 8. 63 45. 73 5. 96 40. 83 3. 28 0. 49 0. 04 0. 03 0. 03 0 05 6. 04 0. 77 5. 23 18. 03 21. 05 2. 23 1. 35 0. 14 2. 50 0. 40 0. 13 0. 03  151 152 153 154 153 156 157 158 159 160 161 162 163 165 166 167 168 169 170 171 173 174 175 176 177 179 180 1B2 183 184 185 1B6 187 IBB 189 190 191 192 193 194 195 197 19B 199 200 201 202 203 204 205 206  0 03 0 03 0. 81 0. 15 1. 03 0. 90 8. 68 1. 03 a. 44 0. 90 26. 91 2. 12 0. 30 0. 04 0. 05 0. 16 0. 05 0. 11 0 16 2. 58 23. 57 3. 81 0. 85 0. 09 0 04 0 02 0 03 0. 05 0 20 0. 18 0 98 0. 17 0. 63 0. 15 1. 15 0 37 1. 17 0. 12 0. 03 0 02 0. 03 0. 01 0. 08 0. 23 0. 14 1. 22 0. 16 3. 69 0 33 0. 07 0. 02  207 208 211 213 214 213 216 217 218 219 220 221 223 227 229 230 231 233 234 233 236 237 239 242 245 246 247 248 249 250 251 254 25B 260 273 274 275 277 278 279 281 2B9 291 292 293 303 305 314 319 320 351  0. 05 0. 02 0. 02 0. 50 0. 07 0. 08 0. 13 0. 76 0. 09 0. 02 0. 03 0. 04 0. 02 0. 02 0. 04 0. 04 0 66 51. 46 8. 96 1. 52 0. 16 0. 03 0. 02 0. 02 0. 56 0. 06 0. 03 0. 02 0. 10 0. 01 0. 01 0. 02 0 04 0. 02 0. 18 0. 04 0. 02 1. 04 0. 12 0 03 0. 02 0. 06 1. 29 0 18 0 02 0 02 0. 05 0. 02 0. 10 0. 01 0. 04  - 162 -  APPENDIX III.6 Mass Numbers of 4,6-di-0-methyl-2-deoxy-2-N-methylacetamiclo  hexitol  -  MASS LIST 01/31/84 14:38 00 • 12:42 SAMPLE: MHRA 40 451 MASS  *  0. 00 MINIMA 0 MAXIMA MASS X RA  40 0. 42 5. 31 41 42 10. 26 43 100. 00 44 6. 77 45 38. 07 46 0. 89 0. 24 47 51 0. 24 52 0. 18 53 0. 67 54 0 74 3. 12 55 36 7. 50 57 6. 21 58 3. 09 39 1. 98 60 0. 84 61 0. 42 65 0 25 66 0 IB 67 0 73 68 2. 11 69 2 70 70 2. 71 71 4 66 72 2. 03 5. 30 73 74 32. 44 75 2. 08 77 0. 19 0. 12 78 79 0. 27 0 30 80 0. 74 81 82 1. 50 83 0. 98 84 1. 3B 85 2. 49 86 2. 92 87 6. 05 0. 84 B8 89 0. 69 91 0. 14 93 0. 31 94 1. 02 0. 59 95 96 0. 73 97 1. 50 9B 7. 17 99 6. 53  100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 113 116 117 118 119 122 123 124 125 126 127 128 129 130 131 135 136 137 138 139 140 141 142 143 144 143 146 147 149 150 151 152 153 154 155 156  163  -  DATA: FB41 # 726  MIN INTEN  132.  BASE M/E: 43 RIC 973824. MAX INTEN: 162!  X RA  MASS  X RA  MASS  X RA  4. 68 9. 26 1. 17 0. 40 0. 38 0. 10 0. 10 0. 15 0. 40 0. 34 2. 07 1. 28 1. 65 0. 99 1. 43 3. 27 B7. 09 7. 09 0. 70 0. 80 0. 26 0. 23 2. 03 1. 95 0. 95 1. 57 4. B7 22. 52 2 46 0. 52 0. 10 0. 15 0. 24 0 94 0. 50 1. 47 0. 70 6. 60 1. 29 0. 25 0. 13 0. 50 0. IB 0. 09 0. 16 0. 10 1. 12 0 46 1. 00 1. 21 1. 62  157 158 159 160 161 162 163 165 166 167 168 169 170 171 172 173 174 179 180 181 182 1B3 184 185 186 187 18B 189 190 191 193 196 197 198 199 200 201 202 203 204 207 211 212 213 214 215 216 217 221 226 227  1. 35 51. 10 4. 76 O. 58 6. 53 0. 52 0. 13 0. 09 0. 38 0. 19 0. 42 0. 13 7. 12 0 93 1. 70 0. 30 0 16 0. 51 2. 57 0 37 0. 41 0. 16 1. 00 0. 38 0. 73 0 21 1. 18 0. 13 0. 13 0. 09 0. 09 3. 36 0. 91 1. 46 0. 34 0 70 0. 27 0. 43 4. 12 0. 39 0. 20 1. 25 1. 71 0. 21 0. 86 0 19 0. 44 0. 13 0. 11 0. 22 0. 18  228 229 230 231 232 233 234 240 241 242 243 244 245 246 250 236 257 258 259 260 270 271 272 273 274 275 276 277 281 284 286 287 288 289 290 292 299 300 301 302 316 317 318 319 320 331 332 333 346 348 359  0. 58 0. 93 2. 54 0. 33 0. 26 0. 19 0. 10 0 49 0. 15 2. 54 0. 42 2. 14 0. 28 0. 18 0. 09 0. 35 0. 33 1. 76 0 32 0. 08 0. 47 1. 98 6. SB 0. 84 5. 80 0. 80 1. 86 0. 25 0. 17 0. 11 0. 47 0. 10 0. 48 0. 11 0. 12 0. 12 0. 13 1. 65 0. 33 0. 08 0. 72 0. 32 4. 30 0. 76 0. 13 0. 21 0. 60 0. 12 0. 11 0. 08 0. 09  - 164 -  APPENDIX III.7 Mass Numbers of per-methylated deoxy component (alditol acetate)  -  1 6 5 -  MASS LIST 01/31/84 1 4 : S B 00 • 11: 11 SAMPLE: MHRA 40 0 00 MINIMA 449 # 0 MAXIMA MASS MASS. "/. RA 40 95 2. IB 41 28. 63 96 43 100. 00 97 44 16 93 98 45 69. 24 99 46 2. 78 100 47 0. 74 101 48 0 04 102 30 0. 19 103 51 0. 64 104 52 0 71 103 33 6. 35 106 54 3. 66 107 55 21 37 108 36 16 65 109 37 12. 26 110 58 47. 68 111 59 54. 72 112 60 3. 30 113 61 3. 12 114 62 0. 10 113 63 0 14 117 65 1. 28 118 66 0. 97 119 67 3. 45 120 6B 3. 60 121 69 11. 51 122 70 2. 84 123 71 31. 55 124 72 3. 06 123 73 12. 93 126 74 7. 71 127 75 13 19 128 76 0 52 129 77 0 78 130 7B 0 22 131 79 0 77 133 Bl 11 92 134 B2 6. 25 135 83 7 63 136 84 2. 53 137 85 26 42 138 86 3. 90 139 87 21. 00 140 88 2. 13 141 89 3. 23 142 90 0 17 143 91 0 21 144 92 0 04 145 93 0 BO 146 94 0. B3 147  DATA: FB41 « 639  MIN INTEN X RA 4. 11 1. 40 10. 61 1. 84 37. 80 9. 06 9 IB 0. 75 0. 99 0 17 0. 42 0 07 0. 14 0. 40 2. 62 0. 9B 11. 79 3. 95 22. 94 3. 48 8 08 75 82 10. 67 1. 79 0 15 0 13 0 09 0 68 0 65 4 28 1. B5 7. 98 2. 15 13. 81 1. 09 0 59 0 84 0 07 0. 08 0 26 1. 07 0 82 0. 87 2. 29 3. 80 4. 51 43. 64 3 95 3. 93 0 32 0. 54  123.  BASE M/E 43 RIC 3653630 MAX INTEN : 342<  •/. RA  MASS  •/. RA  148 0 05 149 0 09 130 0. 04 151 0 24 152 0 17 133 0 84 154 0 57 155 10 83 156 3. 91 157 3. 25 158 0. 84 159 5. 19 160 0. 71 161 1. 01 162 0 10 163 0 04 164 0 07 165 0 17 166 0 17 167 0. 27 168 2. 58 169 6. 46 170 13 15 171 40 12 172 3. 88 173 1. 35 174 0. 19 175 0. 14 177 0. 08 17B 0 07 180 0 07 1B2 2. 59 183 " 5. 72 1B4 3. 66 1B5 1. 56 1B6 0 46 1B7 14. 26 IBS 1. 34 189 7. 49 190 0. 67 191 0 16 192 0. 09 196 4. 09 197 0. 78 198 1. 15 199 0. 45 200 0. 28 201 4 42 202 0 68 203 0 88 204 0. 09  207 211 212 213 214 215 216 217 21B 219 221 224 225 228 229 230 231 232 233 234 243 244 245 246 247 248 236 257 258 239 261 262 263 264 271 272 273 276 277 2B8 289 290 291 303 304 305 306 307 348 349 350  e. 07  MASS  0 30 0. 07 0 20 0 46 4. 11 0 57 0. 36 0 04 0 22 0 07 0 34 0 05 0 66 0 61 0 68 12. 39 1. 27 0 40 0. 05 0 61 0. 10 0. 21 0. 14 0. 05 0 10 1. 63 0. 26 0 05 0. 16 1. 56 © 19 0. 09 0 16 0 70 0 11 0. 26 0 04 0 05 0. 04 0 10 0. 33 0 04 0. 66 0 11 0 18 1. 06 0. 13 0 70 0 64 0 11  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096556/manifest

Comment

Related Items