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Hemagglutinin and protease of pathogenic strains of Bacteroides Melaninogenicus Rasmy, Salwa 1979

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HEMAGGLUTININ AND PROTEASE OF PATHOGENIC STRAINS OF BACTEROIDES MELANINOGENICUS  by SALWA RASMY B.Sc.(Hons.) University of Cairo A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1979 ,1  (c) Salwa Rasmy, 1979  In p r e s e n t i n g t h i s t h e s i s  in partial  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree  for  that  the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s  thesis  f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood t h a t c o p y i n g o r  of t h i s t h e s i s f o r f i n a n c i a l written  permission.  Department Of  Microbiology  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  D  DE-6  B P  75-51 1 E  a  t  e  gain s h a l l  _June_8^79_  Columbia  publication  not be a l l o w e d w i t h o u t my  ii  ABSTRACT  Bacteroides melariiribgeriicus strains 2D and K110  were  characterized  with regard to t h e i r pathogenic, c o l l a g e n o l y t i c , p r o t e o l y t i c , hemagglutinating and metabolic a c t i v i t i e s . species 13. melariinogeriicus  Both strains were members of the sub-  ss. asaccharolyticus.  They possessed a c e l l -  bound oxygen-sensitive collagenase, a cell-bound and a soluble oxygensensitive hemagglutinin (HA),  and a protease.  Both s t r a i n s produced  butyric and phenylacetic acids and were i n f e c t i v e i n guinea pigs as characterized transferred growth and  by t h e i r a b i l i t y to produce necrotic lesions and from one animal to another.  i t s growth rate was  to be  Strain 2D required hemin for  influenced by the addition of free amino  acids to the medium. The hemagglutinating and p r o t e o l y t i c a c t i v i t i e s of s t r a i n 2D were investigated  further to determine t h e i r relationship to i n f e c t i o n .  soluble HA was  reversibly i n h i b i t e d by Hg and a c t i v i t y was  presence of reducing agents. The HA was  i n the  Iodoacetic acid caused i r r e v e r s i b l e i n h i b i t i o n .  sensitive to heat and pronase treatment.  blood c e l l s (RBC)  restored  The  Treatment of the  red  with neuraminidase enhanced HA a c t i v i t y while the presence  of galactose i n the reaction mixture i n h i b i t e d i t , suggesting the involvement of galactose residues on the RBCs i n the reaction.. Adsorption of the HA RBC  followed by elution and  gel f i l t r a t i o n resulted i n the recovery of  to 50%  of the HA a c t i v i t y and a 52-fold p u r i f i c a t i o n . Protease production by _B. melariiribgenicus the growth rate of the organism. HgCl  0  and  s t r a i n 2D was  The protease was  dependent on  reversibly i n h i b i t e d by  i r r e v e r s i b l y i n h i b i t e d by iodoacetamide and  iodoacetic  acid.  iii  The enzyme was  i n s e n s i t i v e to serine protease i n h i b i t o r s and EDTA.  optimum for p r o t e o l y t i c a c t i v i t y was natural environment,  The pH  7.0, which correlates with the pH of i t s  the gingival crevice.  It i s thus c l a s s i f i e d as a  neutral sulfhydryl enzyme. A 774-fold p u r i f i c a t i o n of the c e l l u l a r protease of 2D, with a 160% recovery of a c t i v i t y , was accomplished by p r e c i p i t a t i o n with 60% ethanol, u l t r a c e n t r i f u g a t i o n and gel f i l t r a t i o n through Sephadex G-100  and Sepharose  2B i n the presence of urea. Electrophoretic analysis of the protease on SDS-polyacrylamide gels revealed four d i s t i n c t bands, each of which was with carbohydrate.  shown to be associated  In the absence of SDS only one band, which did not  migrate into the g e l , was obtained.  Any attempts to further dissociate  the protease resulted i n the loss of a c t i v i t y .  The protease was  against a z o c o l l , azocasein, casein and N,N-dimethylcasein. l i p a s e , collagenase or HA a c t i v i t i e s were detected.  active  No glycosidase,  Protein, carbohydrate  and l i p i d were detected i n the preparation. The soluble protease which amounted to 20% of the c e l l u l a r protease of s t r a i n 2D was subjected to gel f i l t r a t i o n on Sephadex G-100 single peak at the void volume.  and eluted i n a  The properties of the soluble protease were  i d e n t i c a l to those of the c e l l associated enzyme, suggesting the presence of a single p r o t e o l y t i c enzyme which was released into the culture medium with c e l l l y s i s or due to shedding of outer membrane fragments.  iv  TABLE OF CONTENTS Page ABSTRACT  i i  LIST OF TABLES  ix  LIST OF FIGURES  xi  ACKNOWLEDGEMENTS I.  xiv  INTRODUCTION A.  Bacteroides melaninogenicus  1  1.  Biochemical c h a r a c t e r i s t i c s of 13. melaninogenicus  4  2.  Pathogenic properties of 13. melaninogenicus a.  Role of B. melaninogenicus i n mixed anaerobic — a infections and periodontal diseases  10  1Q 12  B.  b.  I n f e c t i v i t y of 15. melaninogenicus  c.  Toxin production  3.  Antibiotic sensitivity  4.  Antigenic structure and s e r o l o g i c a l heterogeneity  5.  Genetic properties  6.  Lipopolysaccharide and l i p i d s of 15. melaninogenicus  ^  ^ ^  Hemagglutinating A c t i v i t y and Adherence Properties of J5. melaninogenicus 22  1.  Adherence 2S  2. C. II.  Hemagglutinin  P r o t e o l y t i c A c t i v i t y of 15. melaninogenicus  MATERIALS AND METHODS A.  Organisms  ^  Page II.  MATERIALS AND METHODS B.  C.  D.  Growth  4 8  1.  Anaerobiosis  4 8  2.  Media  4 9  3.  Continuous cultures  Protease 1.  Assays for p r o t e o l y t i c a c t i v i t y  2.  P u r i f i c a t i o n of the protease  5 1  ^  Hemagglutination 1.  Assay  2.  -Preparation of red blood c e l l s  3.  Determination of the e f f e c t s of various reagents on HA  4.  Adhesion and e l u t i o n of hemagglutinin from RBC  ^ 6 6  ^6  6 7  67  E.  Infectivity  6 8  F.  Metabolic End Product Analysis  69  G.  Collagenase Assay  H.  Protein Determination  I.  Glucose Determination  7  ^  J.  Microdetermination of Lipids  7  ^  K.  Microdetermination of Phosphorous  7  ^  L.  Glucosidase Assay  7  ^  M.  Lipase Assay  7 4  N.  Reagents and Chemicals  7 4  ^  7  72  vi  HI.  RESULTS A.  Page  Characterization of IB. melaninogenicus  7o  1.  Fatty acid production  76  2.  Collagenase a c t i v i t y  3.  Pathogenicity  ^  81 a.  Infectivity  b.  Vascular permeability  82 4.  Growth of s t r a i n 2D 13. melaninogenicus  ^ 84  a.  Hemin requirement  b.  Growth response to amino acids  87 5.  Hemagglutinin and protease a c t i v i t y of 13. melaninogenicus.  ^ 91  B.  6.  E f f e c t of washing 2D c e l l s on the HA and protease  7.  Release of periplasmic enzymes from 2D c e l l s  ^4  Soluble Hemagglutinin of 13. melaninogenicus 1. 2.  ^6  Adherence of 13. melaninogenicus 2D c e l l s to formalinized human RBC Determination of optimal conditions f o r the hemagglutinin assay  96 ^6  3.  Relationship of HA to culture age  106  4.  Effects of RBC modification on HA  1  0  6  5.  Modification of the HA  1  0  6  6.  E f f e c t of carbohydrates on HA  1  1  0  7.  S t a b i l i t y of the soluble HA  1  1  0  8.  Oxygen s e n s i t i v i t y of the soluble HA  9.  E f f e c t of s u l f h y d r y l modifiers on HA  10.  Ultracentrifugation of soluble HA  m 1  1  1  m  vii  III.  RESULTS 11.  C.  Page  P a r t i a l p u r i f i c a t i o n of soluble HA  H5  a.  Concentration of the HA  115  b.  Chromatography  115  c.  Binding to M i l l i p o r e f i l t e r s  121  Protease of 13. melaninogenicus  122  1.  Protease assays  122  2.  Relationship of protease to culture age  128  3.  Cell-bound protease of 13. melaninogenicus  128  a. b. c.  133  E f f e c t of hemin concentration on growth and protease production  134  Protease production i n the presence of succinate  134  d.  Effect of amino acids on protease production  140  e. f.  Growth rate and protease production Production of the protease at different concentrations of hemin  142  g.  Preliminary characterization of the c e l l u l a r protease of 13. melaninogenicus  h.  P u r i f i c a t i o n of the c e l l u l a r protease of  i. 4.  Effect of passaging 2D c e l l s i n guinea pigs on protease production  142  13. melaninogenicus  1^0  Characterization of the p u r i f i e d protease  184  Soluble protease of 13. melaninogenicus  202  a. b.  Demonstration of an e x t r a c e l l u l a r protease Preliminary characterization of the extrac e l l u l a r protease  202  P a r t i a l p u r i f i c a t i o n of the soluble protease  208  c.  203  viii  Page IV. V.  DISCUSSION  215  LITERATURE CITED  232  ix  LIST OF TABLES  TABLE  Title  P a  8  e  1.  Collagenase assay  71  2.  Vascular permeability test  83  3.  E f f e c t of the addition of amino acids on growth of B_. melaninogenicus ss. asaccharolyticus 2D in TYH medium  88  4.  Hemagglutinin and protease of 2D and K110  92  5.  Effect of washing 2D c e l l s on the HA  93  6.  Release of the HA from 2D c e l l s by treatment with Polymyxin B  95  Influence of culture age on the adherence of 2D to FRBC  97  7. 8.  E f f e c t of the source of red blood c e l l s on HA a c t i v i t y  101  E f f e c t of treatment of RBC on their a b i l i t y to hemagglutinate with soluble HA  109  10.  E f f e c t of aeration on soluble HA  112  11.  E f f e c t of sulfhydryl modifiers on HA  113  12.  Ultracentrifugation of soluble HA  114  13.  Characteristics of the HA eluted from  9.  RBC with urea  117  14.  Analysis of the HA p u r i f i c a t i o n procedures  118  15. 16.  E f f e c t of passaging 2D on protease a c t i v i t y E f f e c t of additions of amino acids toTYH medium on the proteolytic a c t i v i t y of B. melaninogenicus  135  17.  E f f e c t of hemin concentration on protease production  141 145  X  TABLE 18.  19.  Title  Page  Effect of reducing agents on the c e l l u l a r protease Thermoliability  of the proteolytic  147 activity  in the c e l l - e x t r a c t  149  20.  Comparison of methods for l i b e r a t i n g protease  151  21.  Ethanol p r e c i p i t a t i o n of protease from cell-extract  154  22.  P u r i f i c a t i o n of protease from B. melaninogenicus-2D..  155  23.  Chemical composition of the p u r i f i e d protease preparation Gas-liquid chromatographic analysis of fatty  185  acids i n the p u r i f i e d enzyme preparation  187  25.  Modification of the p u r i f i e d protease  192  26. 27.  Inhibition of soluble protease P a r t i a l p u r i f i c a t i o n of I5_. melaninogenicus e x t r a c e l l u l a r protease  207  24.  209  xi  LIST OF FIGURES  FIGURE 1. 2.  3. 4. 5. 6.  Title  Page  General patterns of enzyme production i n continuous culture  45  The effect of d i l u t i o n rate on the synthesis of amidase by Pseudomonas aeruginosa growing i n a chemostat under steady-state conditions  45  Gas chromatography of v o l a t i l e fatty acids produced by ]3_. melaninogenicus  78  Gas chromatography of non-volatile f a t t y acids produced by B_. melariinogenicus  80  Response of 13. melaninogenicus (strain to hemin  86  2D)  E f f e c t of glutamic acid on growth of 13. melaninogenicus  90  7.  Effect of pH on HA of B_. melaninogenicus  98  8.  Optimal erythrocyte concentration for m i c r o t i t e r hemagglutination  9.  assay  103  E f f e c t s of incubation temperature on HA  105  10.  Relationship of HA to culture age  108  11.  Sephadex G-100  120  12.  Hydrolysis of azocasein by B_. melaninogenicus  gel f i l t r a t i o n of the soluble HA  protease  125  13.  E f f e c t of azocasein concentration  127  14.  Effect of enzyme concentration on the azocasein assay  130  15.  Relationship of protease to culture age  132  16.  E f f e c t of hemin concentration on growth and protease production of 2D  137  xii  FIGURE 17.  Title  Page  Protease production i n hemin and succinate media  139  Effect of d i l u t i o n rate (D) on protease production by I3_. melaninogenicus  144  18. 19.  Gel f i l t r a t i o n of the ethanol precipitated protease  158  20.  Fractionation on Sepharose-2B  160  21.  Chromatography on t h i o l Sepharose-4B  165  22. 23.  Sepharose mercury chromatography Polyacrylamide gel electrophoresis i n tris-glycine-SDS buffer of the p u r i f i e d protease  167  24.  25.  26. 27.  28.  29. 30. 31. 32.  171  Polyacrylamide gel electrophoresis i n t r i s glycine-SDS buffer of protease fractions obtained during various steps i n the p u r i f i c a t i o n process  173  Polyacrylamide gel electrophoresis of protease fractions obtained from the d i f f e r e n t p u r i f i c a t i o n procedures  175  Polyacrylamide gel electrophoresis i n t r i s glycine buffer without SDS  178  Polyacrylamide electrophoresis of f r a c t i o n A and B stained for l i p i d s  181  Diagram of glycoprotein and protein bands on slab gels following electrophoresis of p u r i f i e d protease  183  Effect of pH on the p u r i f i e d protease a c t i v i t y  190  -''2 l ^ l b l t l  HgC  0 1 1  °f  t h e  Purified protease  ..  194  I n h i b i t i o n of the p u r i f i e d protease by iodoacetamide  196  Effect of guanidine hydrochloride, lithium chloride and NaCl on the p u r i f i e d protease  199  xiii  FIGURE  Title  Page  33.  pH optimum of the soluble protease  206  34.  Sephadex G-100 gel f i l t r a t i o n of the soluble protease  213  ACKNOWLEDGEMENTS I wish to convey my sincere gratitude to Dr. J.J.R. Campbell, whose constant  guidance and encouragement made this work possible.  His warm  fatherly attitude has helped me during the c r i t i c a l stages of the entire, program and i s g r a t e f u l l y acknowledged. I extend my appreciation and eternal thanks to my research Dr. B.C. McBride.  advisor  His invaluable academic assistance, understanding,  c r i t i c i s m and extremely amiable attitude were necessary to complete t h i s research. I also wish to thank Dr. Antonio Weerkamp and Mary Gisslow for t h e i r h e l p f u l comments and suggestions. Last but c e r t a i n l y not l e a s t , I want to record my deep f e e l i n g of gratitude to the immeasurable support and s a c r i f i c e s rendered me by my immediate family, children, husband and mother, who were always there to lean on whenever necessary.  This thesis i s dedicated  to them.  1  I.  A.  INTRODUCTION  Bacteroides melaninogenicus The role of anaerobic bacteria i n the etiology of i n f e c t i o n and their  function i n the microbial ecology of man, has recently been of increased interest (2,58,78,79,80,117,150,158,212,217,220).  Anaerobic bacteria are  present throughout the body as constituents of the normal f l o r a (58) and under certain conditions, p a r t i c u l a r l y i n compromised patients, these organisms can invade any organ of the body and cause a variety of i l l n e s s e s (56) .  A commonly encountered species i n human i n f e c t i o n i s 13_. melanino-  genicus (4,25,57,138,202,230,231) which i s frequently isolated from abscesses i n the upper half of the body. B_. melaninogenicus i s a s t r i c t l y anaerobic, Gram-negative, non-motile, non-sporulating rod with rounded or pointed ends.  The c e l l s vary i n size  and shape from small coccoid forms to long filamentous organisms. 13. melaninogenicus i s normally found i n small numbers i n the human i n t e s t i n e (25), ^on male and female external g e n i t a l i a (25,78), i n the throat (25,163), i n the o r a l cavity where i t i s found i n large numbers i n the gingival crevice (25,68,69,197,204), and i n supragingival human dental plaque (69,85,130,207).  In the gingival crevice i t may account f o r  as much as 5% of the t o t a l c u l t i v a b l e f l o r a .  I t i s also present i n the  mouths 'of dogs but i s not found i n the mouths of rodents.  It has been  isolated i n association with other bacteria from various types of c l i n i c a l infections:  tooth abscesses (25,104), soft tissue infections (158), l i v e r  2  abscesses (182), brain and lung abscesses (25,32,192), b i t e wounds and infected surgical wounds (163,231), urine from a suspected infected kidney (163), appendicitis p e r i t o n i t i s (3,4,154), and the uterus and blood i n patients with puerperal infections (188,212,220).  The organism has been  implicated i n periodontal disease (137,138,208,217,230) but this r e l a t i o n ship has not been proven.  C h a r a c t e r i s t i c a l l y , the organism i s associated  with abscesses populated by a mixture of non-sporulating anaerobes and/or f a c u l t a t i v e anaerobes (4,25,54,57,79,80,148,192,202,208). The occurrence of 13. melaninogenicus i n c l i n i c a l specimens  was  described as early as 1921, but the organism i s s t i l l often undetected i n c l i n i c a l specimens.  This may be due to improper c o l l e c t i o n of the  specimens, the lengthy delay between specimen c o l l e c t i o n and culture, f a i l u r e to use the proper culture medium and f a i l u r e to incubate the cultures under s t r i c t l y anaerobic conditions and for s u f f i c i e n t time to permit growth and pigment production. For many years,- B^. melaninogenicus has been of interest to a number of investigators, but detailed analyses concerning the biochemical properties of the organism have been impeded by d i f f i c u l t i e s i n growing i t i n pure culture (26,54).  Furthermore, i s o l a t e s which are considered to  be B_. melaninogenicus represent a heterogeneous group. Sawyer et a l .  (185) reported that although biochemical differences  existed among various strains of the organism, for example, carbohydrate fermentation patterns and menadione requirement, none of the s t r a i n differences were related.  Thus, i t was thought that B_. melaninogenicus  should remain a single species.  Later, Moore and Holdeman (155) divided  the strains of this organism into the following subspecies based on  3  c h a r a c t e r i s t i c fermentation patterns and v o l a t i l e f a t t y acids produced during growth: 13. melaninogenicus subsp. melaninogenicus B_. melaninogenicus subsp. intermedius .B. melaninogenicus subsp. asaccharolyticus However, the c l i n i c a l and epidemiological significance of this d i f f e r e n t i a t i o n among these subspecies has not yet been determined.  Several  techniques and tests, nonetheless, have been developed for routine separation of the three subspecies of I3_. melaninogenicus (94,128,196,213, 214,235) and they c l e a r l y show the substantial differences among the subspecies (93,189).  The subspecies d i f f e r from each other i n c e l l wall  composition, guanine plus cytosine content and some biochemical tests such as esculin and starch hydrolysis (189,235).  More recently, a  fluorescent antibody technique was developed (115) for the i d e n t i f i c a t i o n of 13. melaninogenicus and i t provided support for the biochemical separation of t h i s organism into the three subspecies proposed by Moore and Holdeman (155).  It also provided a rapid procedure f o r i d e n t i f i c a t i o n of these  organisms i n the c l i n i c a l laboratory as opposed to the slower biochemical methods. More recently, the subcommittee on Gram-negative  anaerobic rods of the  International Committee on Systematic Bacteriology proposed that asaccharolytic strains of ]3. melaninogenicus should be r e c l a s s i f i e d as a separate species, Bacteroides asaccharolyticus whereas the saccharolytic strains should r e t a i n their current designation (98). For the purpose of the present work, the former nomenclature, 13. melaninogenicus ss. asaccharolyticus w i l l be used.  4  1.  Biochemical Characteristics of B_. melaninogenicus I3_. melaninogenicus produces a cell-associated black pigment  when grown i n the presence of excess heme.  The usual procedure i s to  grow the organism anaerobically on blood agar medium containing laked blood.  Pigmentation begins about the t h i r d day and becomes dark o l i v e  brown i n a week, and f i n a l l y black i n 5-14 days.  Often t h i s i s the  sole c r i t e r i o n for i d e n t i f y i n g a member of the Bacteroides genus as I3_. melaninogenicus, as the c h a r a c t e r i s t i c pigmentation remains the basic c r i t e r i o n for d i f f e r e n t i a t i o n from other Bacteroides species (95). The pigment of I3_. melaninogenicus was i d e n t i f i e d as extrac e l l u l a r melanin by Oliver and wherry (163), and l a t e r claimed to be i n t r a c e l l u l a r c o l l o i d a l ferrous sulphide by Tracy (222).  Schwabacher  et a l . (187), and more recently Duerden (44) presented data showing that the pigment was hematin.  Formation of pigment  i s dependent upon c u l t u r a l  conditions such as age of the culture, presence of heme or heme-containing compounds i n the growth medium and other factors.  Therefore, the  f i n a l i d e n t i f i c a t i o n of B_. melaninogenicus should not be based on pigmentation c h a r a c t e r i s t i c s alone (203). Most strains of 13. melaninogenicus require a complex growth medium.  They have an obligate requirement for hemin and for peptides.  Many strains also have a requirement for vitamin K or a related naphthaquinone  for growth (66,119).  It was also found that a number  of biosynthetic precursors of vitamin K could act as growth promoters for 13. melaninogenicus (179). Since the black pigment  formed by the bacterium i s a hemin  d e r i v a t i v e , and the extent of production i s dependent upon the amount  5  of hemin present i n the medium (187), i t i s tempting to speculate that pigment formation represents a storage mechanism for t h i s required nutrient.  The observation that deeply pigmented colonies subcultured  from blood agar plates develop well when i n i t i a l l y transferred to heminfree media but f a i l to grow upon subsequent transfer (66) , supports t h i s view. The fact that both heme and vitamin K are required for growth of J5. melaninogenicus suggests that an electron transport system may be involved i n energy metabolism.  Rizza et^ a l .  (177) found that  99% of the cytochromes were located i n a p a r t i a l l y p u r i f i e d membrane fraction.  Whether or not the electron transport system functions i n  the metabolism of 15. melaninogenicus remains undefined. Lev and Milford (121,122) reported that vitamin K had a s p e c i f i c effect on sphingolipid biosynthesis. of 3-keto dihydrosphingosine  synthetase  I t induced the formation  (123) and thus stimulated the  synthesis of t h i s novel microbial l i p i d . Sodium succinate was found to be an additional growth factor for B_. melaninogenicus i n that i t could replace the required heme i n the presence of vitamin K, allowing good growth of the organism.  Succinate can also p a r t i a l l y replace the required  vitamin K i n the presence of heme (120).  The addition of succinate to  a medium supplemented with both vitamin K and heme increased the growth rate of the culture.  These r e s u l t s demonstrate a central r o l e for  succinate i n the metabolism of 15. melaninogenicus and suggest that there are two pathways of succinate metabolism, mediated by heme and vitamin K, respectively.  6  The r e l a t i o n s h i p between heme, vitamin K and succinate i n B_. melaninogenicus i s not understood.  It i s not known whether strains  of Ii. melaninogenicus which do not require vitamin K synthesize the molecule de novo or whether they have evolved alternate metabolic systems which do not require the molecule.  It has been reported that  growth of I5_. melaninogenicus was dependent on the presence of large quantities of succinic acid suggesting that t h i s compound was used i n energy metabolism and was not incorporated into c e l l u l a r carbon (146) . This assumption was  supported  by the observation that only 0.5%  of the  succinate carbon could be found i n the c e l l , the remainder of the metabolized  succinate was  excreted as butyrate.  It was  also found that  hemin blocked the metabolism of succinate and that the fatty acid metabolites are q u a l i t a t i v e l y similar but quantitatively d i f f e r e n t i n c e l l s grown i n hemin free succinate medium (146). Thirty-one strains of Sawyer e_t a l . (185) .  melaninogenicus were studied by  A l l the strains were a c t i v e l y proteolytic  and  collagenolytic, attacking reconstituted neutral salt-extracted collagen and g e l a t i n , and produced  H^S.  A l l the strains required or were  stimulated by hemin and when given an excess of hemin, produced black colonies (185).  None of the strains reduced n i t r a t e s , none formed  catalase and three d i s t i n c t l y d i f f e r e n t fermentative patterns were observed.  A similar study done by Courant and Gibbons (35) showed the  same r e s u l t s .  Werner and Reichertz (232) characterized ten strains of  Ji. melaninogenicus with regard to the i n a b i l i t y to produce ammonia or propionate from threonine and the lack of glutamate activity.  decarboxylase  Burdon (25) reported on strains of B_. melaninogenicus which  7  were highly p r o t e o l y t i c , attacking g e l a t i n , coagulated serum egg albumin and milk, but which did not ferment carbohydrates.  Later, Finegold  reported that I3_. melariiribgenicus ss asacchafolyticus hydrolyzed was  (59)  esculin,  indole negative, clotted milk, and did not possess lipase a c t i v i t y .  While differences have been found between strains with regards to enzyme a c t i v i t y , indole, e s c u l i n and starch hydrolysis, NH^  and H^S  production,  fermentation patterns and end product analysis, no c o r r e l a t i o n between any of the above and pathogenicity has been established.  We have found i n our  laboratory a very relevant c l a s s i f i c a t i o n scheme which i s based on the pathogenicity of the I3_. melaninogenicus (146).  In t h i s scheme collagenase,  protease, fatty acid production, hemagglutination related. i t was  After screening 200 new  concluded  and pathogenicity are  strains as well as strains 2D and  that i r r e s p e c t i v e of the source, the i s o l a t e s could be  separated into two groups.  The pathogenic  strains produce collagenase,  high l e v e l s of protease, butyrate and phenylacetate red blood c e l l s . phenylacetate  K110,  and they agglutinate  The non-pathogenic strains produce succinate instead of  and do not produce the other compounds.  No exceptions were  reported i n the 200 strains isolated i n the laboratory.  This suggested  that a study of some of these properties i n greater d e t a i l , would be valuable. Many strains of I3_. melaninogenicus were reported to produce a collagenase  (67,83,84,106,131).  Okuda and Takazoe (161) found that  twenty nine out of f i f t y nine strains of I3_. melaninogenicus studied had hemagglutinating  activity.  Recently, Slots and Gibbons (199)  reported  that forty seven out of forty eight asaccharolytic strains of 13. melaninogenicus agglutinated human erythrocytes, whereas none of 20 fermentative strains were a c t i v e .  It has been shown by Reichertz  8  et a l . (175)  that anaerobic Gram-negative, non-sporing rods belonging  to the genus Bacteroides were unable to degrade the amino acids valine and leucine completely and therefore accumulate isobutyric and i s o v a l e r i c acid. recent report  They also characterized  13. melaninogenicus in"a more  (232), according to the results obtained with ten s t r a i n s ,  by the production of acetic, propionic, isobutyric, butyric and e r i c acids i n peptone-yeast extract-glucose was  media.  isoval-  B_. melaninogenicus  separated from other Bacteroides strains which exhibit a similar  pattern of acid products by the r e l a t i v e l y great amount of butyrate and r  the outcome of the glutamate decarboxylase t e s t . The acid end products of I3_. melaninogenicus were also analyzed by Sawyer et_ al_. (185) who  found that saccharolytic strains  produced mostly l a c t i c , succinic and acetic acids, whereas nonsaccharolytic strains produced large amounts of propionic and  butyric  acids. It was  found that the addition of glucose to t r y p t i c a s e -  yeast extract medium did not enhance growth nor was metabolized to v o l a t i l e acid end products (228).  the glucose  Studies by  Finegold  and Barnes (59) revealed basic differences in the a c i d i c end  products  of the two was  fermentative groups of I3_. melaninogenicus.  produced by both groups, whereas n-butyric acid was  by asaccharolytic s t r a i n s , and saccharolytic s t r a i n s .  succinic acid was  Isobutyric acid produced only  produced only by  Similar r e s u l t s were obtained by Williams and  Bowden (235). Biochemical studies  (35,185) have shown that most strains of  13. melaninogenicus grow well i n sugar-free peptide^containing  broth.  9  In addition, t h i s growth decreased when the trypticase concentration the medium was  reduced suggesting  that the fermentation  of  of proteinaceous  constituents plays an important r o l e i n the metabolism of B_. melaninogenicus and that the organisms have the potential to ferment amino acids Experiments using l a b e l l e d proteins (228)  indicated that strains of  B_. melaninogenicus r e a d i l y fermented amino acids when they were present as peptides.  This suggested that peptides were more e a s i l y  transported  into the c e l l than were most free amino acids. More recently (149) , i t was  found that the addition of  individual amino acids to a trypticase-yeast extract-hemin medium affected growth rates and the f i n a l y i e l d of saccharolytic and asaccharolytic s t r a i n s of 15. melaninogenicus.  Some amino acids enhanced  growth, and others inhibited i t . The s i g n i f i c a n t stimulation of growth by certain amino acids i n the presence of t r y p t i c peptides  in this  study suggested that for some strains of 13. melaninogenicus a few amino acids are taken up as r e a d i l y as peptides.  The mechanism of growth  i n h i b i t i o n by amino acids i s not known; Lev and Milford (124) was  found that growth of I5_. melaninogenicus  inhibited by the addition of c e r t a i n monosaccharides to t r y p t i c a s e -  hemin medium.  The major i n h i b i t o r y e f f e c t of the sugar was  the t r a n s i t i o n from the l a g to logarithmic growth phase.  to prolong  They ascribed  this to an e f f e c t on enzyme induction of which the i n h i b i t i o n of 3-keto dihydrosphingosine  synthetase a c t i v i t y was  one example.  In a c t i v e l y  growing cultures, addition of sugar slowed the growth rate and did not appear to be related to the a c t i v i t y of the synthetase enzyme. also possible that other enzymes were affected by the i n h i b i t o r y  It  was  10  monosaccharides, contributing to a retardation of the growth rate. They also noted that sugars did not i n h i b i t enzyme a c t i v i t y i n v i t r o . 2.  Pathogenic Properties of B_. melaninogenicus a.  Role of B_. melaninogenicus i n mixed anaerobic infections and periodontal diseases. In the oral cavity, mixed populations of anaerobes  can be isolated from a variety of necrotic lesions, including mucous membrane abscesses, dry socket and c e l l u l i t i s .  Anaerobic  bacteria comprise a large percentage of the gingival microflora and i t i s not unreasonable to assume that they may be involved i n the  i n i t i a t i o n of pathogenic processes.  The possible importance of  anaerobic organisms in.the etiology of periodontal diseases has been noted by a number of authors (134,137,138,205,206,208,217,230). Thus, the oral f l o r a not only possesses pathogenic p o t e n t i a l , but t h i s potential i s often realized when a suitable environment i s available.  B_. melaninogenicus i s almost always present i n the  human mouth (25,68,69,197,204) and i s commonly found i n mixed anaerobic infections (4,25,148,192,202).  Antigenic components of  B^. melaninogenicus have also been demonstrated tissue (34) .  i n diseased gingival  13. melaninogenicus of oral o r i g i n has been reported  to be involved i n anaerobic infections of the lung and brain (25,32,192). In a recent report (198) , Gram-negative anaerobic rods were shown to comprise approximately 75% of the c u l t i v a b l e bacteria i n plaque which was removed from the base of deep gingival pockets of adults.  I3_. melaninogenicus constituted almost half of  11  these Gram-negative  i s o l a t e s , and most of the 13_. melaninogenicus  strains were non-saccharolytic and appeared to belong to the subspecies asaccharolyticus (198). Mixtures of pure cultures of anaerobic human gingival crevice bacteria were shown to be pathogenic when injected subcutaneously into the groin of a guinea pig (134).  Macdonal.d  extended t h i s observation to show that an i n f e c t i o n could be i n i t i a t e d by a combination of 13. melaninogenicus, two other bacteroides and a diphtheroid (135).  The p r i n c i p a l role played  by 15. melaninogenicus i n the etiology of the i n f e c t i o n was -shown by Socransky and Gibbons (205).  It was found that no i n f e c t i o n  occurred when 15. melaninogenicus was omitted from the mixture. The role of the "helper" organisms has only been p a r t i a l l y defined (66,205,206,211).  I t can be assumed that  "helper'' organisms assist anaerobes by using up the oxygen, decreasing the Eh and/or producing catalase.  Other factors that  might be involved are the a b i l i t y to force entry into tissues, resistance to host defences, the production of substances that block humoral antimicrobial action or n u t r i t i o n a l dependency (66,205,206,211).  Evidence for the l a t t e r was provided i n an  experiment where the 15. melaninogenicus used did not require vitamin K, thus the diptheroid and one of the bacteroides were omitted without a l t e r i n g the pathogenicity.  It was also reported  that the i n f e c t i v i t y of B. melaninogenicus asaccharolyticus was dependent  on a"helper" organism to produce a required growth factor  which was shown to be succinate (146).  The need for the second  12  organism could be eliminated by inoculating I3_. melaninogenicus together with agar-immobilized succinate (146). On the assumption that elucidation of the pathogenic mechanisms involved i n experimental mixed infections might provide some understanding of the mechanisms involved i n the pathogenicity of c l i n i c a l anaerobic i n f e c t i o n s , investigators became concerned with the production of p o t e n t i a l l y damaging metabolites, toxins, or other factors which would explain how 13. melaninogenicus and associated organisms were able to cause i n f e c t i o n s .  Although  Macdonald had suggested e a r l i e r that perhaps mixed infections were b a c t e r i a l l y nonspecific but biochemically s p e c i f i c i n terms of toxins, l y t i c enzymes and other damaging factors produced by the mixed population (135,136), the demonstration of the essential r o l e of IB. melaninogenicus i n the experimental system suggested that the "nonspecific" infections were, i n f a c t , dependent  on the  presence of 13. melaninogenicus and that the role of other organisms was one of supporting and enhancing the i n vivo growth of the primary pathogen  (139,205).  Evidence thus implicated  I3_. melaninogenicus as the primary pathogen i n mixed infections of soft tissues; consequently, 13. melaninogenicus was examined for pathogenic properties and i n f e c t i v i t y . b.  I n f e c t i v i t y of J3. melaninogenicus. The c r i t e r i a used for defining a t y p i c a l trans-  missible i n f e c t i o n were summarized by Socransky and Gibbons (205). A successful i n f e c t i o n occurs when:  (i)  inoculation r e s u l t s i n  v i s i b l e necrosis, either spreading or confined to a pustular abscess; ( i i ) exudate i s i n f e c t i v e when inoculated into another  13  animal. Two types of experimental mixed anaerobic infections are observed:  (1) A f a t a l , rapidly spreading necrotic i n f e c t i o n  which penetrates  the peritoneal cavity and/or perforates the skin  within 18 hours.  The animal loses h a i r and necrosis of the skin  occurs i n the abdominal region.  The f a s c i a connecting the  skin to the abdominal wall i s loosened and the cavity created becomes f i l l e d with an exudate containing b a c t e r i a , white blood c e l l s and eventually, red blood c e l l s .  (2) A walled-off  localized  abscess containing foul-smelling exudate which can be used to transmit the i n f e c t i o n to a second animal.  Successful trans-  missible infections i n animals inoculated with defined mixtures of microorganisms were reported by several investigators (4,89, 105,136,138,139,203) . It was shown (215) that a pathogenic s t r a i n of 15. melaninogenicus was i n f e c t i v e when a pure culture was injected intradermally i n rabbits and guinea pigs.  During a study on the  immunological characterization of 13. melaninogenicus (215), i t was found that a vaccine prepared by phenol treatment from one s t r a i n was so harmful that rabbits frequently died during the immunization period. Therefore, the p o t e n t i a l pathogenicity of the s t r a i n was suspected. I5_. melaninogenicus, except f o r two reported cases (2 1 5,1420 , does not possess any known capsular material or ahti-phagocytic components.  surface  However, some s t r a i n s can elaborate cell-associated  and e x t r a c e l l u l a r enzymes which may enhance their invasive properties.  These enzymes include collagenase  (69), proteases and  14  hyaluronidase  (1).  Kestenbaum (106) demonstrated a positive  c o r r e l a t i o n between collagenase a c t i v i t y and i n f e c t i v i t y for four  melaninogenicus strains i n a guinea p i g system.  Collagen  degradation i s a feature of periodontal disease (131,206) and although ]}. melaninogenicus i s the only organism indigenous to the oral cavity known to produce a collagenase (67,83,84), the relationship between collagenase production and the pathogenicity of the organism either i n o r a l lesions or i n other mixed anaerobic infections remains unclear.  Whether factors other than collagen-  ase were involved i n Kestenbaum's system i s not known. ment of a fusobacterial i n f e c t i o n i n rabbits by  Enhance-  simultaneous  i n j e c t i o n of a crude c e l l - f r e e preparation of B_. melaninogenicus collagenase was demonstrated by Kaufman (101).  Heating eliminated  both enzyme a c t i v i t y and the effects of the extract on i n f e c t i o n , suggesting that collagenase may play a r o l e i n the organism's pathogenicity.  This does not exclude the p o s s i b i l i t y that other  heat-sensitive factors such as protease or hemagglutinin may contribute to the pathogenicity of the c e l l s .  This also  suggests that endotoxin was not involved since i t s effect cannot be removed by heating. c.  Toxin production. Some mixtures of bacteria containing I3_. melanino-  genicus when injected subcutaneously  into the groin of a guinea  pig cause a rapid and spreading i n f e c t i o n (134) r e s u l t i n g i n extensive f l u i d accumulation.  A pathogenic  s t r a i n of B_. melanino-  genicus ss. asaccharolyticus was reported to possess a heat  15  sensitive toxin which induced i l e a l loops (146).  V i b r i o cholerae toxin induces a s i m i l a r  response i n the gut (168) . B.C.  f l u i d accumulation i n ligated mouse  Preliminary experiments by  McBride (personal communication) have indicated that culture  supernatants obtained  from 13. melaninogenicus ss. asaccharolyticus  possess a cholera t o x i n - l i k e a c t i v i t y as measured by the vascular permeability assay described by Craig (36).  The a c t i v i t y i s not  as strong as that of V_. cholerae and i s l o s t when the c e l l s are subcultured repeatedly.  Toxin i s injected intracutaneously into  the skin of a shaved guinea pig, and a f t e r a suitable i n t e r v a l , a blue dye i s injected i n t r a c a r d i a l l y .  The dye complexes with  serum proteins and passes through c a p i l l a r y walls i n areas where vascular permeability has been increased by toxin.  The r e s u l t i n g  blue area around the s i t e of inoculation i s then measured.  The  ' assay i s simple and s e n s i t i v e . 3.  Antibiotic Sensitivity A study on a n t i b i o t i c s u s c e p t i b i l i t y (232) showed that  13. melaninogenicus ss. asaccharolyticus was s e n s i t i v e to p e n i c i l l i n , cephalosporins, b a c i t r a c i n , c h l o r t e t r a c y c l i n , chloramphenicol, mycin and rifampicin and r e s i s t a n t to streptomycin, and neomycin.  Finegold  erythro-  c o l i s t i n , polymyxin B  (55) has found stock strains of 13. melanino-  genicus to be uniquely r e s i s t a n t to kanamycin and vancomycin and has suggested that the i s o l a t i o n of B_. melaninogenicus from heavily contaminated source material would be f a c i l i t a t e d by the incorporation of these a n t i b i o t i c s i n the media.  Loesche and Hockett (129) reported  the resistance of c e r t a i n s t r a i n s of I3_. melaninogenicus to kanamycin  16  and that the addition of t h i s a n t i b i o t i c to a culture medium f a c i l i t a t e d the primary i s o l a t i o n of the organism from source material such as dental plaque.  A more recent study (189) showed that s e n s i t i v i t y to vancomycin  and c o l i s t i n w i l l d i f f e r e n t i a t e between saccharolytic and asaccharol y t i c strains.  Most asaccharolytic strains were s e n s i t i v e to vancomycin  and r e s i s t a n t to c o l i s t i n whereas both saccharolytic groups were r e s i s t ant to vancomycin and s e n s i t i v e to c o l i s t i n . 4.  Antigenic Structure and Serological Heterogeneity The antigenic composition of bacteroides c e l l s has been shown  to be s p e c i e s - s p e c i f i c and p o t e n t i a l l y useful i n speciation (39). However, only few studies have attempted to group strains of 13. melaninogenicus on the basis of serology. protein from two ally distinct.  Weiss (230)  extracted  strains of 13. melaninogenicus which were immunologicShevky et a l . (192) reported that several strains of  I3_. melaninogenicus reacted with a single antiserum and stated that there was  "no reason to postulate the existence of a wide v a r i e t y of  s e r o l o g i c a l l y heterogeneous s t r a i n s " .  The a n t i g e n i c i t y of t h i r t e e n  strains of I3_. melaninogenicus isolated from various sources was by Courant  and Gibbons (35) who  studied  concluded that 13. melaninogenicus  strains were s e r o l o g i c a l l y heterogeneous, and seemed to represent spectrum of serotypes.  a  More recently, a fluorescent antibody procedure  was developed (115) which showed that human 13. melaninogenicus strains could be divided into three s p e c i f i c serogroups chemical subspecies already known.  according to the b i o -  The fluorescent antibody conjugates  were s p e c i f i c and no cross-reaction occurred with other anaerobes or aerobes tested.  17  Rabbits and guinea pigs immunized with IJ. melaninogenicus were examined for their humoral and c e l l u l a r antibody (162) .  The  results indicated that I5_. melaninogenicus resident i n the gingival crevice  has an a b i l i t y to induce delayed hypersensitivity with the  result that the area becomes susceptible to i n f e c t i o n by the microorganism. 5.  Genetic Properties Genetic studies on 13. melaninogenicus are scarce and no  s p e c i f i c d e t a i l s have been published concerning the genetic properties and v a r i a b i l i t y of the organism. rough variants are found (215).  Colony form i s usually smooth but Pigmentation i s also noticeably  variable being generally black, but d i f f e r e n t shades of brown can be observed.  Other properties, such as the requirement for heme and  vitamin K, hemagglutinin, collagenase and protease production might also r e f l e c t some genetic v a r i a t i o n , although t h i s has not been proven. melaninogenicus has not been found to possess plasmids nor has i t been shown to acquire plasmids from d i f f e r e n t Gram-negative species (27).  A n t i b i o t i c resistance i s not related to the presence of plasmids  (41).  Bacteriophages capable of i n f e c t i n g B_. melaninogenicus have not  been isolated (103). 6.  Outer Membrane of 13. melaninogenicus Lipopolysaccharides (LPS) are located i n the c e l l wall of  Gram-negative bacteria where they form, along with l i p i d s and proteins, the outer membrane of the c e l l . endotoxins of these organisms  They represent the 0 antigen and the  (99).  Endotoxic. LPS, consisting of three  major components, i . e . fatty acids, saccharides and sometimes bound amino  18  acids,, i s c o n s i d e r e d t o i n c l u d e t h r e e main r e g i o n s o f c o n t r a s t i n g c h e m i c a l and b i o l o g i c a l p r o p e r t i e s .  The 0 - s p e c i f i c p o l y s a c c h a r i d e  ( r e g i o n I ) , c a r r y i n g t h e main s e r o l o g i c s p e c i f i c i t y , i s l i n k e d t o t h e c o r e p o l y s a c c h a r i d e ( r e g i o n I I ) , which i s r e l a t i v e l y group s p e c i f i c . The c o r e i s l i n k e d through 2-keto-3-deoxyoctonate (KDO) t o l i p i d ( r e g i o n I I I ) termed l i p i d A (133). L i p o p o l y s a c c h a r i d e s , a l s o c a l l e d endotoxins  from a e r o b i c  Gram-negative b a c t e r i a have been t h e s u b j e c t o f d e t a i l e d f o r many y e a r s .  investigation  C o n s i d e r a b l e a t t e n t i o n has been d i r e c t e d toward  d e f i n i t i o n o f t h e c h e m i c a l s t r u c t u r e , b i o l o g i c a c t i v i t y , and immunog e n i c i t y o f these o u t e r c e l l membrane-localized  antigens  (17,99,133).  The LPS o f S a l m o n e l l a i s an i m p o r t a n t v i r u l e n c e f a c t o r ; l o s s o f i t s 0 - s p e c i f i c s i d e c h a i n s r e s u l t s i n l o s s o f v i r u l e n c e b u t has no e f f e c t on endotoxic p r o p e r t i e s .  Endotoxins  e x e r t t h e i r m u l t i p l e b i o l o g i c a l and  immunologic e f f e c t s o n l y a f t e r l i b e r a t i o n from b a c t e r i a .  Such b a c t e r i a l  and immunologic e f f e c t s become e v i d e n t a f t e r a d s o r p t i o n o f e n d o t o x i n onto t h e h o s t c e l l u l a r membrane ( 1 7 ) . L i p o p o l y s a c c h a r i d e (LPS) a n t i g e n s o f a n a e r o b i c  Gram-negative  b a c t e r i a have r e c e i v e d f a r l e s s s t u d y , because t h e importance  of t h e s e  b a c t e r i a i n c l i n i c a l i n f e c t i o n s was n o t f u l l y a p p r e c i a t e d u n t i l r e c e n t l y when t h e i r i s o l a t i o n from c l i n i c a l specimens became t e c h n i c a l l y more feasible.  Some a n a e r o b i c Gram-negative b a c t e r i a c o n t a i n LPS w h i c h i s  c h e m i c a l l y and b i o l o g i c a l l y s i m i l a r t o t h e e n d o t o x i n s of a e r o b i c b a c t e r i a (92).  However, i t i s o f i n t e r e s t t h a t B a c t e r o i d e s  and JB. m e l a n i n o g e n i c u s In  appear t o have r a t h e r u n u s u a l  fragilis  LPS(92,100).  s t u d i e s o f t h e s e two s p e c i e s , H o f s t a d has noted t h e predominance  19  of  f a t t y acids and neutral sugars with the absence of the sugars  2-keto-3-deoxyoctonate  (KDO) and heptose, which are found uniformly  i n the LPS of aerobic Gram-negative  bacteria.  The i s o l a t i o n and p u r i f i c a t i o n of the outer membrane complex of I5_. melaninogenicus subspecies asaccharolyticus was studied by Mansheim and Kasper (141).  Morphologic study by electron microscopy  disclosed the presence of a capsule and a c e l l wall structure otherwise t y p i c a l of a Gram-negative  organism.  With the use of gentle techniques  of heat, EDTA treatment, shearing, and d i f f e r e n t i a l centrifugation, the outer membrane was isolated.  A r e l a t i v e l y pure preparation was  suggested by the absence of nucleic acids and muramic acids, the existence of r e l a t i v e l y few peptide bands on SDS-polyacrylamide gel electrophoresis, morphologic studies by electron microscopy, and the presence of a single band on a sucrose density gradient (141). Fractionation by gel f i l t r a t i o n of the outer membrane after deoxycholate treatment revealed two major components.  The f i r s t consisted  primarily of a large molecular weight protein-polysaccharide complex with loosely bound l i p i d  (26%).  Antigenicity of t h i s f i r s t component was  demonstrated by agar gel d i f f u s i o n .  Analysis of the protein by SDS-poly-  acrylamide gel electrophoresis of three strains revealed s t r a i n s p e c i f i c i t y . Further p u r i f i c a t i o n of this f r a c t i o n showed that the polysaccharide component cross-reacted with antiserum to another s t r a i n of the same subspecies. This component probably represents the capsular antigen and may prove to be the basis of serogrouping. The second membrane f r a c t i o n d i f f e r e d chemically from the f i r s t f r a c t i o n and represents the lipopolysaccharide component of the  20  outer membrane.  I t consisted mainly of loosely bound l i p i d  (62%), protein  (5%) , and polysaccharide which was c l e a r l y d i s t i n c t from that of the f i r s t fraction.  Notably, t h i s component lacked 2-keto-3-deoxyoctonate, one  of the backbone components of aerobic, Gram-negative lipopolysaccharides. P u r i f i c a t i o n of the outer membrane of 13. melaninogenicus and i d e n t i f i c ation of the outer membrane antigens w i l l provide an opportunity f o r better study of the mechanisms of immunity to infections involving t h i s organism.  Demonstration of serologic c r o s s - r e a c t i v i t y between  capsular antigens may form the basis for serogrouping within the species By melaninogenicus. Recently, the lipopolysaccharide component was isolated from the outer membrane complex of 13. melaninogenicus ss. asaccharolyticus (142) by gel chromatography using sodium deoxycholate (NaD), a disaggregating detergent, i n the running buffer. was composed of loosely bound l i p i d less than 5% protein.  The LPS  (62%) and carbohydrate (32%) , with  Glucose, galactose, and glucosamine were the  major sugars as detected by gas-liquid chromatography (GLC). and KDO were not observed by colorimetric analysis.  Heptose  Long chain fatty  acid analysis by GLC disclosed an unusual pattern; $-0H myristic acid, a common component of aerobic Gram-negative LPS, was absent.  Further-  more, two unknown peaks, which may be c y c l i c or odd chain f a t t y acids, were detected. ]3_. melaninogenicus LPS preparation did not induce skin reactions i n rabbits when administered i n doses of up to 1 mg, compared to Salmonella typhi endotoxin which e l i c i t e d a positive reaction i n doses of 12.5 yg. L i t t l e or no endotoxic a c t i v i t y was demonstrated.  21  These findings are compatible with previously noted observations on LPS i n anaerobic bacteria (90,100), a l l of which stand i n sharp contrast to the widely known biologic a c t i v i t y of aerobic Gram-negative LPS. This may  explain the r a r i t y of septic shock i n patients infected with  anaerobic organisms.  I t i s i n t r i g u i n g to speculate that the unusual  pattern of fatty acids i n the l i p i d A, may be responsible for the b i o l o g i c impotence of the LPS of 13. melaninogenicus  (145).  The  presence  of capsular polysaccharide contaminating the phenol/water-extracted was reported (142), and may  explain p a r t i a l l y the serologic heterogeneity  which has been described previously i n studies of the LPS of this (91).  LPS  organism  The factors within the LPS which determine serologic a c t i v i t y  are extremely complex.  Characterization (142) of a r e l a t i v e l y  homogeneous LPS may make further investigation of the pathogenic mechanisms and immune response to Ii. melaninogenicus  somewhat more c l e a r .  The l i p i d s and related compounds i n the c e l l envelopes of ]3_. melaninogenicus were studied by two groups of investigators. Parker and White (167) and Rizza e_t a l . (178) reported that nearly half of the phospholipids isolated from B_. melaninogenicus lipids.  The two major phosphosphingolipids have been characterized as  ceramide phosphorylethanolamine (CPG).  are phosphosphingo-  (CPE) and ceramide phosphorylglycerol  The finding of phosphosphingolipids i n bacteria i s exceedingly  rare, although another anaerobe, Bacteroides ruminicola, has been reported to contain an ethanolamine-containing sphingolipid  (133).  22  It was also found that the l i p i d composition of 13. melaninogenicus was similar to that of other Gram-negative bacteria i n that part of the extractable fatty acids was present as phospholipid and that phosphatidyl ethanolamine was the predominant d i a c y l phospholipid. 13. melaninogenicus i s unusual i n that i t contains only a small amount of non-extractable fatty acids which are usually found to be associated with the polysaccharide, and i n the absence of (3-hydroxy f a t t y acids usually found i n l i p i d A of the outer membrane of Gram-negative bacteria.  The diacylphospholipids of 13. melaninogenicus consist of phos-  phatidylethanolamine,  phosphatidyl g l y c e r o l , phosphatidyl serine,  phosphatidic acid and c a r d i o l i p i n .  I t has been reported that these  bacteria do not contain g l u c o l i p i d s (167).  Vitamin  isoprenologues  make up the bulk of neutral l i p i d s (178) which represent a small portion of the extractable f a t t y acids.  The l i p i d s seem to be l o c a l i z e d i n the  membrane f r a c t i o n of I3_. melaninogenicus along with cytochrome c. pholipid found i n the supernatant membrane fragments (178). amount of vitamin  The pho  f r a c t i o n might represent very small  There was not much change i n the t o t a l  and phospholipid i n the membrane of 13. melanino-  genicus grown with d i f f e r e n t l e v e l s of protoheme supplementation i n the medium (178). Hemagglutinating 1.  A c t i v i t y and Adherence Properties of B. melaninogenicus  Adherence Adherence has recently been found to be an important ecologic-  a l factor i n s p e c i f i c disease processes  (52,62,70,143,236).  The  23  a b i l i t y of many organisms to adhere to a particular surface of their host i s one requirement  for colonization and i n some cases f a c i l i t a t e s  the invasion of that host (73).  It has been recognized that V i b r i o  cholerae adhere to the i n t e s t i n a l mucosa (61) and that the i n a b i l i t y to do so results i n a reduction i n virulence (63,186).  E l l e n and  Gibbons demonstrated that v i r u l e n t strains of Streptococcus pyogenes adhered well to e p i t h e l i a l c e l l s whereas an avirulent s t r a i n lacked this a b i l i t y (51). Adherence i s also an important ecological determinant which influences the colonization of bacteria i n environments subject to the flow of l i q u i d s (73).  It was  shown, as early as 1954,  that c e r t a i n  o r a l b a c t e r i a l species p r e f e r e n t i a l l y colonize d i f f e r e n t s i t e s within the mouth (112).  Gibbons has shown that t h i s s e l e c t i v e colonization  of o r a l bacteria i s correlated with selective b a c t e r i a l adherence (70).  Despite the recognition of the selective adherence of oral  bacteria, there i s limited information concerning the mechanism of adherence. of  Tentative conclusions as to the nature of the adherence  bacteria were based on either d i r e c t microscopic examination or on  the r e s u l t s of enzymatic, physical and chemical pretreatments  (125,209).  However, one must be c a r e f u l interpreting these data because of the gross nature of the various treatments.  It was postulated that for some  organisms, adherence i s mediated by proteinaceous surface components (51,52,125), while for others a l i p i d component may be involved (71.159).  There are also reports of b a c t e r i a l adherence to e p i t h e l i a l  c e l l s with the possible involvement of neuraminic acid receptors on the host c e l l s (209), and i n other cases the possible involvement of  24  carbohydrate and teichoic acid moieties on the b a c t e r i a l c e l l s (64). Adherence can generally be c l a s s i f i e d according t o : (i) (ii)  attachment to a body surface, i . e . buccal epithelium attachment to other bacteria, i . e . plaque.  or tooth  Attachment  involves  a s p e c i f i c , unique receptor s i t e on the b a c t e r i a l c e l l surface and a corresponding s i t e on the substrate to which the organism adheres. The r e l a t i v e l y high proportions of I3_. melaninogenicus ss. asaccharolyticus found i n periodontal pockets (198) and the potential of this organism to s y n e r g i s t i c a l l y produce mixed anaerobic infections i n experimental animals (72,134,139,205), suggest that i t may play an important r o l e i n the etiology and pathogenesis of periodontitis.  L i t t l e i s known about the parameters which influence the  colonization of 13. melaninogenicus i n periodontal pockets or i n other s i t e s of the mouth, and l i t t l e information i s available about the mechanisms of B_. melaninogenicus retention and prevalence i n the oral cavity.  Recently, Slots and Gibbons  (199) and Edwards (50) i n i t i a t e d  studies to determine the a b i l i t y of 13. melaninogenicus ss. asaccharol y t i c u s to attach to various oral surfaces, and to evaluate the r o l e that adherence might play i n i t s o r a l and subgingival colonization. They found that I3_. melaninogenicus c e l l s suspended  i n phosphate-  buffered saline adhered well to buccal e p i t h e l i a l c e l l s and to the surfaces of c e r t a i n Gram-negative bacteria that are prominent i n human dental plaque.  They also reported that of forty eight asaccharolytic  strains of B_. melaninogenicus, f o r t y seven agglutinated human erythrocytes (199).  Their data indicated that certain Gram-positive  organisms found i n dental plaque possess receptors for the attachment  25  of  13. melaninogenicus c e l l s and that these receptors are d i f f e r e n t  from those present on buccal e p i t h e l i a l c e l l s and erythrocytes. 2.  Hemagglutinin Macromolecules  which react with s p e c i f i c components of red  blood c e l l membranes leading to agglutination of these c e l l s are not uncommon among certain b a c t e r i a l species. For organizational purposes, the l i t e r a t u r e survey of b a c t e r i a l hemagglutination i s considered separately from studies using c e l l s and surfaces other than erythrocytes. Although recent reviews on b a c t e r i a l adherence have often overlooked the l i t e r a t u r e on b a c t e r i a l hemagglutination, there i s no evidence to suggest that adherence of bacteria to c e l l s other than erythrocytes i s a d i f f e r e n t phenomenon from b a c t e r i a l hemagglutination (72,73).  In fact, studies  using hemagglutination assume that t h i s phenomenon i s an index of b a c t e r i a l attachment  and, at least i n some cases, adherence to  e p i t h e l i a l c e l l s has exactly the same c h a r a c t e r i s t i c s as b a c t e r i a l hemagglutination (160). to  Therefore, one way of studying adherence i s  evaluate the hemagglutinating a c t i v i t y of an organism using model  adherence systems of erythrocytes as r e l a t i v e l y simple, well-characterized natural surfaces. The a b i l i t y of c e r t a i n bacteria to agglutinate red blood c e l l s was demonstrated  as early as 1955  (45).  In l a t e r studies, a great  deal of significance was placed on the r o l e of p i l i i n hemagglutination (28,48,49,72,160,191).  From studies using p i l i a t e d and non-piliated  enterobacteria, i t appeared that the presence of p i l i did not always seem to be necessary i n order for hemagglutination to occur (46,47,224). It was concluded that there are several d i f f e r e n t c h a r a c t e r i s t i c s  26  between a p i l i a t e d and a non-piliated hemagglutination reaction. Among these there are differences i n agglutination range of erythrocyte species, e l u t i o n of bacteria from erythrocytes at high temperature, and i n h i b i t o r y e f f e c t s of D-mannose (46,47,224).  Although  much work has been done on the adherence of o r a l bacteria c e l l s and other surfaces, l i t t l e a c t i v i t y of the organisms.  to e p i t h e l i a l  has been done on the hemagglutination  Recently,  i t was reported  that  Streptococcus sanguis, Streptococcus m i t i s , and Actinomyces viscosus a l l agglutinate human red blood c e l l s (180).  Studies on the hemagglutin-  ating properties of anaerobic bacteria have been minimal.  Okiida and  Takazoe (161) found that twenty nine of f i f t y - n i n e strains of I3_. melaninogenicus studied had hemagglutinating a c t i v i t y that seemed to be mediated by surface p i l i .  They suggested that these structures might  also mediate the attachment of B_. melaninogenicus c e l l s to o r a l mucosa. Slots and Gibbons (199) reported that forty-seven of forty-eight asaccharolytic strains of 13. melaninogenicus representing  fresh  i s o l a t e s from subgingival plaque and t o n s i l l a r swabbings agglutinated human erythrocytes, whereas none of twenty fermentative  s t r a i n s , which  included reference cultures of the subspecies intermedius and melaninogenicus were active.  Electron microscopy indicated that both  asaccharolytic and fermentative  strains possessed p i l i .  These workers  also found that the non-hemagglutinating strains of ]3. melaninogenicus containing p i l i  attached well to buccal e p i t h e l i a l c e l l s .  Thus, no  clear r e l a t i o n s h i p exists between the hemagglutinating a c t i v i t y of strains of 13. melaninogenicus and their a b i l i t y to attach to buccal epithelial cells.  The observations  further suggested that several  27  types of p i l i exist on d i f f e r e n t strains and subspecies of B_. melaninogenicus C.  (199) .  Proteolytic A c t i v i t y of B_. melaninogenicus It has been known for many years that many microorganisms  appreciable amounts of proteolytic enzymes.  produce  The b a c t e r i a l proteases are  instrumental i n the degradation of complex protein substrates to amino acids and peptides i n nature.  The water soluble products with lower  molecular weights are assimilable, thus supporting c e l l growth. Studies on protease enzymes are not always easy to undertake.  The  reason for t h i s may be that some proteolytic enzymes are unstable and susceptible to autodigestion; consequently, p u r i f i c a t i o n procedures must be carried out with great care under defined conditions.  A few procedures  have been shown to be useful with the majority of enzyme systems and these have been used routinely.  These procedures include the following:  f r a c t i o n a l p r e c i p i t a t i o n by pH changes (5), f r a c t i o n a l denaturation by heat, f r a c t i o n a l p r e c i p i t a t i o n by s a l t s ( i l ) , f r a c t i o n a l p r e c i p i t a t i o n with organic solvents (9), f r a c t i o n a l adsorption (166), column chromatography (5,11) and c r y s t a l l i z a t i o n (9).  The sequence i n which several or a l l of these steps  are used i s determined by the enzyme under investigation. There are many published reports on the p u r i f i c a t i o n of b a c t e r i a l proteases.  The following covers a few selected examples concerning the  p u r i f i c a t i o n of c e l l u l a r and e x t r a c e l l u l a r proteases produced by d i f f e r e n t organisms. The e x t r a c e l l u l a r protease of Pseudomonas maltophilia  was p a r t i a l l y  p u r i f i e d by ammonium sulfate p r e c i p i t a t i o n and chromatography on Sephadex G-75 and Bio-rex 70.  Gel electrophoresis revealed minor impurities (16).  28  The cell-bound protease of Bacteroides amylophilus H18  was  liberated from  the mechanically ruptured c e l l envelopes by n-butanol treatment and p u r i f i e d 80-fold by  (NH^^SO^ p r e c i p i t a t i o n , electrophoresis  and  was  gel  f i l t r a t i o n through Sephadex G-200 (15). Once a proteolytic enzyme i s discovered and interesting to investigate  i t s chemical and  partially purified, i t is  physical properties;  Inform-  ation i n these areas i s important i n order to c l a s s i f y the enzyme and determine i t s function in vivo.  to  In the past, proteolytic enzymes have  been c l a s s i f i e d by several c r i t e r i a .  One  c l a s s i f i c a t i o n grouped proteo-  l y t i c enzymes into the categories pepsin-like, t r y p s i n - l i k e or cathepsinlike.  This system i s based mainly on the pH optimum of the enzyme.  E x t r a c e l l u l a r proteases from microorganisms have been c l a s s i f i e d into three groups by their pH optima i . e . , acid, neutral and  alkaline proteases  (81).  Bergmann (10) proposed a system which grouped proteolytic enzymes according to t h e i r action on synthetic  substrates.  A system such as t h i s t e l l s  the  investigator something of the mode of action of a p a r t i c u l a r enzyme but t h i s method also has  i t s limitations.  Enzymes of d i f f e r e n t o r i g i n s  may  produce e s s e n t i a l l y the same action on synthetic  substrates but may  differ  i n t h e i r r e a c t i v i t y towards natural  Another system was  proposed  substrates.  (6) which divided the proteolytic enzymes into categories based on t h e i r behaviour toward a number of proteolytic i n h i b i t o r s . those mentioned have t h e i r inherent shortcomings. p a r t i c u l a r enzyme was  For example, i f a  c l a s s i f i e d as t r y p s i n - l i k e , one might assume that i t  possessed a l l the other chemical and i n r e a l i t y i t did not.  Systems such as  physical properties  of t r y p s i n whereas  It i s necessary therefore to characterize  proteolytic enzyme i n as many ways as  possible.  the  29  Characterization of a protease should include the determination of the s i t e of hydrolytic attack on natural substrates, the action of the enzyme on various synthetic substrates, the nature of the reactive s i t e of the enzyme, the response to i n h i b i t o r s , the pH and the temperature s t a b i l i t y , as well as the electrophoretic properties and the molecular weight of the enzyme. Generally,  i f the enzyme under investigation shows a s p e c i f i c i t y l i k e ,  or i s affected by one of the i n h i b i t o r s of the well-known and thoroughly studied groups of enzymes, i t i s preliminary referred to as belonging to t h i s class of enzymes.  I t should be noted, however, that some p r o t e o l y t i c  enzymes do not f a l l into the major categories which are based on mechanism of action rather than o r i g i n of physiological action.  These categories  include four p r i n c i p a l classes of enzymes. The f i r s t of such main groups of enzymes i s the serine proteases, which are distinguished by a serine residue i n the active s i t e (86).  A common  test for these enzymes i s the i n h i b i t i o n of t h e i r hydrolase a c t i v i t y by the reaction of this serine residue with diisopropylphosphorofluoridate  (DFP).  Examples include enzymes i s o l a t e d from ]}_. s u b t i l i s and related strains ( s u b t i l i s i n s ) as well as p r o t e o l y t i c enzymes isolated from organisms such as Streptomyces griseus The  (227).  second group of proteolytic enzymes are dependent on sulfhydryl  groups for t h e i r c a t a l y t i c a c t i v i t y . usually achieved by mild reducing  Activation of these enzymes i s  agents such as cysteine, s u l f i d e and  s u l f i t e , which l i b e r a t e a free t h i o l group on the enzyme. conformational change i s associated with reduction  (8).  L i t t l e , i f any, Optimum a c t i v a t i o n  was found to occur upon simultaneous application of a t h i o l compound such as cysteine or thioglycolate and a heavy metal-binding agent l i k e EDTA (108),  30  or by the addition of 2,3-dimercaptopropanol,  a compound which combines the  functions of both a reducing agent and a metal binder (210).  The enzymes  are reversibly inactivated i n the presence of a i r and can be  reactivated  by addition of reducing agent. 2+ Cu  2+ , Hg  Heavy metal ions such as Cd^ , Zn^ , +  +  Fe^ , +  2+ and Pb  are i n h i b i t o r y .  The metal inactivated enzymes can be  t o t a l l y reactivated by addition of a reducing agent and a chelating  agent.  The readily reversible formation of a stable inactivate complex with mercury has been u t i l i z e d as a useful step i n the p u r i f i c a t i o n of the well known sulfhydryl enzyme, papain (21,108). act as sulfhydryl enzyme i n h i b i t o r s .  A l l sulfhydryl-binding  reagents can  Thus, p-chloromercuribenzoate forms a  stable complex with the enzyme and can serve for t i t r a t i o n of the free-SH group (60).  Iodoacetic acid or iodoacetamide also react with the free  sulfhydryl group, causing thereby i r r e v e r s i b l e i n a c t i v a t i o n Papain was  (109,190).  found to react with the chloromethyl ketones of phenylalanine  and lysine (TPCK and TLCK) with t o t a l loss of a c t i v i t y (233).  In t h i s case,  the reagents act s p e c i f i c a l l y on the active sulfhydryl group of the enzyme rather than on the imidazole group of p a r t i c u l a r h i s t i d y l residues as they do i n the case of trypsin and chymotrypsin, and thus the i n a c t i v a t i o n of papain i s a stoichiometric  reaction.  enzymes are the streptococcal  Examples of b a c t e r i a l sulfhydryl  proteinases (53,114).  Another group of proteolytic enzymes includes  the a c i d i c proteases.  The presence of proteolytic enzymes with a pH optimum i n the acid pH range (pH 1-5) has been reported i n a v a r i e t y of microorganisms where they occur both i n t r a c e l l u l a r l y and e x t r a c e l l u l a r l y . (C. acetobutylicum and C_. butyricum)  Several strains of  Clostridium  (225) and L a c t o b a c i l l i (18) have been  shown to produce weak proteinase a c t i v i t y with an acid pH optimum.  31  There i s also the group of metal proteinases which includes enzymes s p e c i f i c for releasing Nl^-terminal amino acids, such as aminopeptidase-P. ' This enzyme i s an exopeptidase cleaving the bond between any  N-terminal  amino acid residue and a following proline residue (241) and i s isolated from Escherichia c o l i .  Thermophilic  B a c i l l u s stearothermophilus  aminopeptidase-APl, produced by  s p l i t s a l l amino acids from the amino  end of a polypeptide; p r e f e r e n t i a l l y hydrolyzing peptides  containing  leucine, v a l i n e , and those with aromatic amino acid residues. The following are some reported examples of protease characterization. The s u b t i l i s i n s are a l k a l i n e proteases of broad s p e c i f i c i t y produced by strains of B_. s u b t i l i s .  Three of these enzymes have been studied i n  considerable d e t a i l and are probably the best known of a l l microbial proteases.  The s u b t i l i s i n s are s p e c i f i c a l l y and s t o i c h i o m e t r i c a l l y  inactivated by DFP  (144) , i n d i c a t i n g that they are serine proteases.  s t r i k i n g feature of the s u b t i l i s i n s may  The  be that their sequences bear no  s i g n i f i c a n t r e l a t i o n s h i p to those of the pancreatic serine proteases, whereas t h e i r status as serine proteases c a r r i e s the obvious implication that the active s i t e s are i n some way pancreatic enzymes.  similar i n structure to those of the  It i s nevertheless clear that there are considerable  differences i n the s p e c i f i c i t i e s of the s u b t i l i s i n s and the pancreatic serine proteases.  Studies of the hydrolysis of ester substrates  (76)  suggest that, i n contrast to the high s p e c i f i c i t y of chymotrypsin and trypsin, the s u b t i l i s i n s have rather low  specificity.  A number of i n t e r e s t i n g DFP-sensitive proteases  from s t r a i n s of  Staphylococcus aureus have been studied by Drapeau and co-workers (42)  and  are reported to have a high s p e c i f i c i t y for glutamyl and aspartyl residues.  32  The  staphylococcal  enzymes thus do not resemble any of the previously known  serine proteases. Streptococcal  proteinase i s a sulfhydryl enzyme which i s elaborated by  group A streptococci.  I t i s excreted into the medium as a zymogen which i s  transformed into an active enzyme by proteolysis followed by reduction. Both the zymogen and the enzyme contain only a single half-cystine residue per molecule (53,114).  The reduced, active enzyme can be r e a d i l y inactivated  by reagents known to react with sulfhydryl groups, such as iodoacetic  acid,  2+ iodoacetamide, p-chloromercuribenzoate, Hg Streptococcal  , and atmospheric oxygen.  proteinase thus appears to be a c l a s s i c sulfhydryl enzyme.  There are comparatively few reports of e x t r a c e l l u l a r proteases from Gram-negative bacteria, and fewer s t i l l of DFP-sensitive proteases.  A  survey of published work indicated that the Gram-negative bacteria known to secrete e x t r a c e l l u l a r proteases are l a r g e l y confined to the pseudomonads. This s i t u a t i o n i s also true of exoenzymes i n general, and may be a r e f l e c t ion of the difference i n complexity of the c e l l envelope between Gramnegative and Gram-positive bacteria (170,82).  Several of the proteases from  Gram-negative bacteria appear to be metallo-proteases (152,16,157,151). Extensive studies on the enzymes produced by Aeromonas proteolytica showed that the organism excretes two proteolytic enzymes, an endopeptidase and an amino peptidase.  Both enzymes are metal proteinases which are  inactivated by EDTA and possess molecular weights of 34,800 and 29,500 respectively  (107).  Boethling  (16) described  the p u r i f i c a t i o n and  properties of a protease from Pseudomonas maltophilia which i s an EDTAsensitive a l k a l i n e serine protease.  Nakajima and co-workers (157) described  an a l k a l i n e protease of Escherichia  f r e u n d i i which was sensitive to EDTA and  33  had a molecular weight of 45,000. (151) resembled  The protease enzymes from Serratia sp.  the s u b t i l i s i n s i n that they have alkaline pH optima with  casein as substrate; i n other respects, however, they were not s i m i l a r . Protease production by Gram-negative anaerobic bacteria has not been extensively investigated.  Bacteroides amylophilus was reported to produce  protease(s), active at pH 7.0, which was neither induced nor repressed by a wide range of nutrients.  The protease was synthesized by exponentially  growing organisms and 20% was liberated into the growth medium.  The  cell-bound protease was completely accessible to the protein substrate (13). The major function of e x t r a c e l l u l a r proteinases and other hydrolytic enzymes i s most reasonably a n u t r i t i o n a l one which evolved to allow the microorganism  growing i n i t s natural environment to u t i l i z e complex non-  d i f f u s i b l e substrates as a source of nutrients.  In addition to the  n u t r i t i o n a l r o l e , e x t r a c e l l u l a r proteinases of the genus B a c i l l u s are thought to be required for sporulation (37) and thus c l e a r l y have an i n t r a c e l l u l a r function i n a s p e c i f i c developmental  process.  Limited proteolytic degradations are responsible for the induction of b i o l o g i c a l a c t i v i t i e s , as i n the formation of b i o l o g i c a l l y active enzymes from their inactive precursors.  The i n i t i a l step i n proteolytic de-  gradation might be the opening of one or a few exposed peptide bonds, or the s p l i t t i n g of a small amount of unfolded protein i n equilibrium with the native protein.  An example of induction of b i o l o g i c a l a c t i v i t y by  limited proteolysis i s the conversion of the e x t r a c e l l u l a r zymogen of Group A streptococci to the active enzyme (53,127).  The r o l e of a protease  34  i n natural a c t i v a t i o n of Clostridium botulinum neurotoxin has been reported by Bibhuti et^ a l . (12).  The  s p e c i f i c t o x i c i t y of the simple protein  increases during incubation of the culture. progenitor  Since the conversion of  toxin to the more toxic form can be accomplished with t r y p s i n ,  one mechanism for the natural a c t i v a t i o n of progenitor  toxin would be through  the action of a suitable enzyme(s) produced by the culture.  One  such  enzyme i s a protease with t r y p s i n - l i k e s p e c i f i c i t y which activates  progenitor  toxin obtained from young cultures of the same proteolytic type B s t r a i n (38). C a t a l y t i c processes involving s p e c i f i c enzymes in the membtane may involved i n secretion of exoenzymes. membrane of E_. c o l i was  reported  be  A protease located i n the outer  to cleave a protein located i n the  plasmic membrane, the respiratory enzyme n i t r a t e reductase.  cyto-  This cleavage  i s accompanied by s o l u b i l i z a t i o n of the enzyme (140). The  importance of i n t r a c e l l u l a r proteolytic a c t i v i t y i n the physiology  of the b a c t e r i a l c e l l has been implicated and might include roles i n : protein turnover leading to continued regeneration of l a b i l e proteins, increased  p r o t e o l y t i s during c e l l d i v i s i o n , proteolytic maturation of  proteins, and p r e f e r e n t i a l breakdown of s t r u c t u r a l l y altered (169).  proteins  It has been shown that the autolysin of Streptococcus f a e c a l i s  i s present i n an inactive form i n the c e l l wall but i s activated by a neutral proteinase;  and  that the active form of the autolysin i s asociated  with recently synthesized  wall (193).  35  In the past few years, considerable  importance has been placed upon  proteolytic enzymes as tools for studies of the structure of proteins for  and  investigation of hydrolysis products which possess b i o l o g i c a l a c t i v i t y .  This i s exemplified diphtheria toxin.  i n the case of the structure and the biochemistry  of  The toxin molecule i s released from the b a c t e r i a l c e l l  as a single polypeptide  chain having two non-overlapping cystine bridges.  The toxin contains a protease s e n s i t i v e s i t e which i s r e a d i l y hydrolyzed y i e l d two sulfhydryl linked polypeptides.  One  polypeptide  to  i s responsible  for binding the toxin to i t s target c e l l ; the second hydrolysis product i s responsible for inducing the biochemical system of the c e l l . presumed to represent  l e s i o n i n the protein synthesizing  A sequence containing three arginine residues  is  an exposed loop i n the intact molecule since i t i s  abnormally sensitive to p r o t e o l y t i c attack.  Short treatment with proteases  with t r y p s i n - l i k e s p e c i f i c i t y yielded two large peptides, an amino-terminal fragment A and a carboxyl-terminal  fragment B (74) , which f a c i l i t a t e d  further studies on the structure and c h a r a c t e r i s t i c s of the toxin molecule. Bacteroides melaninogenicus has been shown to possess p r o t e o l y t i c a c t i v i t y but this a c t i v i t y has not been characterized.  An organism  dependent on peptides for growth (228) might be expected to be a c t i v e l y proteolytic.  It has been reported that strains K110  and CR2A have a limited  a b i l i t y to ferment free amino acids, but the organisms can more r e a d i l y d i s s i m i l a t e peptides  (228) .  Many strains of B_. melaninogenicus have been  observed by Sawyer et_ a l . (185)  to be p r o t e o l y t i c , and the organisms appear  to grow well i n culture media without carbohydrate supplementation, suggesting  that the fermentation  of proteinaceous  constituents play an important  role i n the metabolism of B_. melaninogenicus.  Hausman and Kaufman have  found c a s e i n o l y t i c a c t i v i t y associated with a p a r t i c u l a t e f r a c t i o n from  36.  the autolysate supernatant of 13. melaninogenicus (84).  Gibbons reported  that forty-two  of forty-seven strains of ]3_. melaninogenicus l i q u i f i e d  gelatin (35).  On the other hand, Oliver and Wherry (163)  and Cohen  (32)  found that their strains fermented a number of carbohydrates but did not attack g e l a t i n . Hydrolysis of proteinaceous substrates by B_. melaninogenicus was reported by Weiss (230), Schwabacher et a l . (187) and Pulverer  also  (173).  Burdon (26) reported that his asaccharolytic strains of B_. melaninogenicus were highly p r o t e o l y t i c , attacking g e l a t i n , coagulated and milk.  serum, egg albumin  The protease found i n these organisms i s not l i k e l y to be  collagenase  since  casein was  attacked.  Gelatin, the denatured form of  collagen, i s generally susceptible to a number of proteases which are incapable of attacking native collagen.  Therefore,  the  collagenases  constitute a class of unique proteases capable of attacking native collagen which i s resistant to other p r o t e o l y t i c enzymes. I3_. melaninogenicus may  elaborate more than one protease, as a c t i v i t y  has been demonstrated i n the washed c e l l s as well as i n the supernatant. The c e l l u l a r and soluble. proteases of B_. melaninogenicus which gelatin  hydrolyze  are also active against a number of protein substrates including  a z o c o l l , casein, azocasein, and N,N-dimethylcasein. the rate of dye released from the dye-protein p r o t e o l y t i c a c t i v i t y i n the sample. than quantitative.  For the a z o c o l l assay,  conjugate r e f l e c t s the  This assay i s usually q u a l i t a t i v e rather  Casein, having many d i f f e r e n t p o t e n t i a l l y .susceptible  bonds, i s generally used as a protein substrate for enzymes having unknown, undefined, or broad substrate s p e c i f i c i t i e s . determination  of the amounts of TCA  The assay depends on the  soluble peptides liberated from the  37  casein substrate by the enzyme as detected by measuring absorption at 280 nm.  This assay i s not extremely sensitive, and i s therefore not  useful for measuring small amounts of proteolytic a c t i v i t y .  Among the  reasons for i t s r e l a t i v e i n s e n s i t i v i t y , the most important seems to be i t s high background cleavages.  reading.  The assay also f a i l s to measure a l l bond  Hydrolysis of small numbers of bonds i n such an assay would be  expected to result i n larger peptides, proportionally greater numbers of which would, because of their s i z e , be precipitated by t r i c h l o r o a c e t i c acid and not be distinguished from uncleaved protein.  In addition, because  the assay r e l i e s upon the absorbance of soluble peptides at 280 nm and such absorbance varies from one peptide to the next, equal degrees of proteolysis by d i f f e r e n t enzymes do not r e s u l t i n the same increment of increased absorbance.  Another assay, the dimethylcasein assay, depends on the  conversion of primary amino groups into dimethyl-amino  groups, a change  which does not affect many properties of the substrate protein but does prevent i t s reaction with trinitrobenzene-sulfonic acid (TNBS), a sensitive reagent for the determination of protein amino groups.  The proteolytic  a c t i v i t y i s followed by determining, with TNBS, the new amino groups produced after hydrolysis.  The low background values obtained with  N,N-dimethylcasein r e s u l t s i n greater s e n s i t i v i t y and accuracy not possible with the unmodified casein. 14 The use of  C-labeled N,N-dimethylcasein as a substrate for determining  t o t a l proteolytic a c t i v i t y offers several advantages over other methods. The assay i s more sensitive than spectrophotometric procedures.  The  labeled substrate i s stable and can be stored for a long period of time; the assay i s rapid and i s not affected by the presence of large concentrations of peptides or amino acids i n the sample to be assayed.  38  Another assay i s based on the s o l u b i l i z a t i o n of a covalently linked chromophore from a modified i s azocasein.  protein.  An example of this type of substrate  After incubation with the enzyme, the unhydrolyzed protein i s  precipitated and hydrolysis products containing coupled dye are spectrophotometrically.  The  quantitated  important point i s that the absorption maximum  of the covalently linked chromophore i s d i f f e r e n t than that of chromophores contaminating the enzyme preparation. In 1962,  i n a review that has become a landmark i n the f i e l d , Pollock  (170) defined an e x t r a c e l l u l a r enzyme as one that "exists i n the medium around the c e l l s , having originated from the c e l l without any a l t e r a t i o n to c e l l structure greater than the maximum compatible with the c e l l ' s normal processes of growth and reproduction".  Externalization of enzymes  could be accomplished either by active secretion during logarithmic growth or unintentionally (170) as a r e s u l t of c e l l l y s i s , aging and leakage during division. A consideration of the possible mechanisms involved i n secretion of proteins must necessarily be related to the nature of the membrane. Costerton e_t a l . (33) summarized evidence suggesting  that various protein  molecules, both s t r u c t u r a l membrane proteins and enzymes, are inserted into the membrane basic phospholipid b i l a y e r . May  and E l l i o t t reported that a protease was  c e l l s apparently  as i t was  i n t r a c e l l u l a r accumulation. molecules were ever present  synthesized  since there was no  They speculated  significant  that none of the enzyme  i n a completed form inside the c e l l membrane  but rather that the nascent polypeptide membrane as i t was  secreted from I5_. s u b t i l i s  synthesized  chain was  extruded through the  to take up i t s t e r t i a r y structure with  39  enzyme a c t i v i t y only on the outside (147). A s p e c i f i c hypothesis to explain protein excretion, the signal hypot h e s i s , has been developed.  An elongation of peptide chain on membrane-  bound ribosome r e s u l t s i n discharging the nascent chain across the membrane; the signal sequence for excretion i s then removed from the polypeptide  chain  by p r o t e o l y t i c cleavage, which was reported to be i n the outer membrane f r a c t i o n i n E_. c o l i (97) . The presence of large pores through the outer membrane of P_. aeruginosa was reported by Hancock et^ a l . (82) .  The organism secretes three proteases  into the medium and has been shown to possess membrane-bound peptidases, thus the larger pores would permit entry of quite large peptides  into the  periplasmic space, rendering them susceptible to peptidases, whereas the e x t r a c e l l u l a r proteases may be involved i n the i n i t i a l processing of proteins i n the environment (82). The release of lipopolysaccharide-phospholipid-protein  complexes from  E_. c o l i has been observed f o r growing and stationary phase c e l l s .  The  outer membrane fragments were p r e f e r e n t i a l l y released from those regions where newly synthesized  proteins are inserted into the outer membrane (164).  Membrane bound structures have been found to be associated with exoenzyme produced by 13. l i c h e n i f o r m i s .  After protoplast formation the  enzyme i s found associated with v e s i c l e s (183,184). The outer layers of the c e l l envelope, p a r t i c u l a r l y i n Gram-neative bacteria, would also pose a b a r r i e r to exoprotein enzymes found outside of the cytoplasmic  secretion.  Certain  membrane are not released into the  medium but are bound to the outer layers of the c e l l envelope (33,127).  )  40  The l o c a t i o n of enzymes i n bacteria has been determined by a v a r i e t y of techniques.  Many enzymes now  are thought to be external to the c e l l  membrane, as judged by c r i t e r i a such as a v a i l a b i l i t y to substrates and i n h i b i t o r s , e l u t a b i l i t y by nondamaging solvents, i n h i b i t i o n by s p e c i f i c antibodies and release by osmotic shock or by such compounds as polymyxin B. Preparation of protoplasts i n s t a b i l i z i n g media with measurement of enzymes released, indicated the location of the liberated enzyme outside the permeability b a r r i e r i n the intact c e l l (183).  The outer membrane layer  contains charged moieties but i t i s not yet clear what forces are involved in determining  i f a molecule w i l l remain bound to the c e l l , either i n  association with mucopeptide (194) , with various components i n the periplasmic space (30) , with lipopolysaccharide or protein of the outer membrane (96) or to be released into the menstruum (127). properties of the enzyme such as hydrophobicity  Clearly, the  and charge w i l l have a  bearing on the l o c a t i o n of proteins r e l a t i v e to the cytoplasmic membrane. Studies on the a l k a l i n e phosphatase (APase) of Pseudomonas aeruginosa by Ingram e_t a l . (96) showed that a c e r t a i n percentage of the enzyme was complexed with -lipopolysaccharide which was Phosphatase i s located i n three areas: wall surface, and the periplasmic space. may  also released during secretion.  the culture f i l t r a t e , the outer  cell  The r e s u l t s suggest that APase  become associated with, and bound to, a c e l l wall f r a c t i o n which contains  LPS and l i b e r a t i o n of the complex from the outer wall may  be accomplished  by mechanical shearing forces developed during growth. C e l l suspensions of Micrococcus sodonensis secrete seven to ten individual proteins including an a l k a l i n e phosphatase and a protease. The appearance of enzyme a c t i v i t i e s i n the e x t r a c e l l u l a r medium was  found  41  to be dependent on the co-secretion of at least one of several polysaccharides  (19).  A functional membrane-bound enzyme, the galactosyl  transferase system of Salmonella typhimurium, was  reconstituted i n v i t r o  from p u r i f i e d components including lipopolysaccharide,  phosphatidyl  ethanolamine and enzyme protein (181). MacGregor reported a p r o t e o l y t i c a c t i v i t y which was washed membrane preparations  (140).  found i n extensively  This membrane-bound protease was  found  to be responsible for the cleavage and s o l u b i l i z a t i o n of n i t r a t e reductase enzyme from the cytoplasmic membrane of E_. c o l i (140) .  Regnier and Thang  (174) reported that at least 50% of the protease a c t i v i t y found i n E_. c o l i i s associated with the membrane.  This membrane-bound protease was  found to  have many c h a r a c t e r i s t i c s i n common with t r y p s i n . As many microorganisms are known to produce e x t r a c e l l u l a r p r o t e o l y t i c enzymes, several studies have been carried out on the regulation of the production  of e x t r a c e l l u l a r proteases by Gram-positive bacteria, e s p e c i a l l y  Bacillus strains.  However, only a few detailed reports have appeared on  Gram-negative bacteria (77). differences i n the way production  (234).  Among these organisms there are marked  i n which environmental factors a f f e c t enzyme  In general, induction, end product i n h i b i t i o n and  catabolic repression have been implicated i n the regulation of the synthesis of these enzymes.  An e f f i c i e n t regulatory control has been  described by Tanaka and Tuchi (218) organism the production  for V i b r i o parahaemolyticus.  of a protease was  In t h i s  induced by amino acids and  subject to catabolite repression by e a s i l y metabolizable  carbon  was  sources.  42  Repression of protease synthesis by amino acids has been widely reported as an example of end product repression i n bacteria of the genera B a c i l l u s , Serratia and Arthobacter  (77), but does not appear to occur i n  a l l Gram-negative organisms that have been studied (218) . The production  of microbial c e l l u l a r and e x t r a c e l l u l a r enzymes has been  investigated extensively i n batch cultures, but applications of continuous culture techniques i n these studies have not been widespread (22,77).  In  the studies that have been reported, the production of c o n s t i t u t i v e and inducible enzymes followed one of two considered  general patterns  (Fig. 1), and  that the r e l a t i o n s h i p between enzyme production  i t is  and growth rate  depends on the c h a r a c t e r i s t i c s of the regulatory mechanism involved For c o n s t i t u t i v e enzymes where the rate of enzyme production  (22).  is a  function of the product of c e l l concentration and growth rate, the r e l a t i o n ship between the rate of enzyme production and d i l u t i o n rate for such enzymes i s l i n e a r (Fig. 1A).  This has been observed for the p e n i c i l l i n a s e s pro-  duction by B a c i l l u s licheniformis by Wouters and Buysman (239) and for several other enzymes (31,40).  An example of this i s seen (Fig. 2)  where the content of amidase per c e l l of Pseudomonas aeruginosa r i s e s to a peak as the growth rate ( d i l u t i o n rate) i s increased and the content then f a l l s as the growth rate i s increased beyond this point  (31).  A non-linear r e l a t i o n s h i p between rate of enzyme production  and  d i l u t i o n rate i s sometimes found for inducible enzymes (Fig. IB).  Such a  behaviour i s p a r t i c u l a r l y apparent when organisms producing such enzymes are grown under conditions where the inducer i s the growth-limiting strate (40).  sub-  The advantages of continuous culture are undeniable i n f i e l d s  of microbial biochemistry  and metabolism (23,219), and chemostat experiments  43  have made a valuable contribution i n the elucidation of mechanisms of enzyme regulation.  Moreover, the unique growth conditiions provided by  chemostasis have contributed to our understanding of those processes which allow microbes to adapt to changing n u t r i t i o n a l and other environmental conditions. The chemostat offers p o s s i b i l i t i e s which are absent i n any closed culture system.  During growth i n batch culture microbes  continuously  change t h e i r environment as a r e u s l t of consumption of nutrients and accumulation of waste products, therefore, the morphological and metabolic properties of the c e l l s are apt to change during the growth period  (226).  In the chemostat c e l l s can be grown i n steady states at any of a whole range of growth rates.  In addition, i t i s usually possible to make any  substrate growth-limiting.  Thus, the n u t r i t i o n a l status of an organism  as well as i t s growth rate can be varied at w i l l .  Once a steady rate i s  reached, neither the properties of the culture nor those of the environment undergo further change. conditions are stable.  The culture then has become time-independent, and Therefore,  experiments with the chemostat are highly  reproducible and are ideal f o r studying the properties of an organism as a function of growth rate. Growth rate of a culture can be varied by changing the d i l u t i o n rate, the t o t a l c e l l mass of the culture remaining the same.  However, at  extremely low growth rates, a proportion of the c e l l s may become non-viable and the growth y i e l d , Y, which i s defined as the c e l l mass (m) produced by the metabolism of unit mass of the substrate, may decrease.  A lower c e l l  mass at lower d i l u t i o n rates i s to be expected only when growth i s limited by the substrate whose metabolism supplies energy.  FIGURE 1.  General patterns of enzyme production i n continuous culture.  A.  Constitutive enzymes whose synthesis only depends on c e l l concentration.  B.  Inducible enzymes.  FIGURE 2.  The effect of d i l u t i o n rate on the synthesis of amidase by  Pseudomonas aeruginosa  growing i n a chemostat under steady-state conditions.  46  I n t h e c a s e o f c h e m i c a l l y complex media, where t h e r e i s an u n s a t i s f a c t o r y vagueness about t h e c o n c e n t r a t i o n  of medium c o n s t i t u t e n t s , i t can be assumed  t h a t growth i s u l t i m a t e l y r e s t r i c t e d by t h e e x h a u s t i o n other  o f some one s u b s t a n c e ,  substances r e m a i n i n g i n s u f f i c i e n t c o n c e n t r a t i o n n o t t o a f f e c t t h e  growth r a t e on t h e i r own a c c o u n t .  For example, i n t h e case of  13. m e l a n i n o g e n i c u s , w h i c h r e q u i r e s a complex medium f o r growth due t o i t s o b l i g a t e r e q u i r e m e n t f o r hemin and p e p t i d e s , hemin can thus be s i n g l e d o u t and c a l l e d t h e " l i m i t i n g  substrate".  I n c o n t i n u o u s c u l t u r e s , t h e r a t e o f b a c t e r i a l growth may be r e g u l a t e d by c o n t r o l l i n g t h e r a t e of n u t r i e n t a d d i t i o n .  The r a t e of n u t r i e n t a d d i t i o n  i s u s u a l l y e x p r e s s e d as t h e d i l u t i o n r a t e D, w h i c h i s t h e volume o f n u t r i e n t added h r The  expressed as a f r a c t i o n o f t h e volume of t h e v e s s e l .  d i l u t i o n r a t e w i l l d e t e r m i n e t h e l e n g t h of t i m e t h a t a b a c t e r i a l  c e l l w i l l . r e m a i n i n t h e chemostat and i n t h e absence o f b a c t e r i a l c e l l division dx where x i s t h e number o f b a c t e r i a p r e s e n t i n t h e v e s s e l .  In fact, b a c t e r i a l  c e l l d i v i s i o n i s o c c u r r i n g as d e f i n e d by  -Sr where K i s t h e growth c o n s t a n t .  Any change i n b a c t e r i a l c o n c e n t r a t i o n  would be d e f i n e d by r a t e o f change i n c e l l c o n c e n t r a t i o n = r a t e of growth - r a t e of d i l u t i o n O  I  ^ = K x - D x dt  •  47  A chemostat, run at one d i l u t i o n rate, soon establishes steady state conditions:  there i s no change i n b a c t e r i a l numbers.  This indicates that  b a c t e r i a l growth exactly balances the bacteria l o s t by d i l u t i o n : Kx = Dx or K = D When the d i l u t i o n rate approaches the maximum growth rate and eventually exceeds i t , more bacteria are washed out than are produced by c e l l and the b a c t e r i a l concentration  f a l l s (87).  division  48  II.  A.  MATERIALS AND METHODS  Organisms B_. melaninogenicus  from Dr. P.A.  subspecies asaccharolyticus s t r a i n K110 was  Mashimo.  This i s a collagenolytic s t r a i n o r i g i n a l l y isolated  by Macdonald and co-workers (134) from a patient with diagnosed melaninogenicus  obtained  subspecies asaccharolyticus s t r a i n 2D was  gingivitis.  isolated i n the  laboratory from a gingival scraping taken from an individual with periodontal  disease.  The gingival sample was streaked on freshly poured blood  agar plate and incubated at 37°C i n a ^ " . ^ i C ^  (85:10:5) atmosphere.  Black colonies were repeatedly subcultured on the same medium u n t i l a pure culture was obtained. procedures  The i s o l a t e was characterized according to standard  (94) . Neither s t r a i n K110 nor 2D required vitamin K for  growth. B.  Growth 1.  Anaerobiosis Liquid or agar cultures were usually incubated i n anaerobic  jars (Torball, Torsion Balance, C l i f t o n , New flushed with U:  CO2  anaerobic glove box  Jersey) evacuated  and  (95:5) (Canadian Liquid A i r , Montreal) or i n an (Coy Manufacturing, Ann Arbor, Michigan) containing  an atmosphere of N^tH^'.CO^ (85:10:5) at a temperature  of 37°C.  Humidity  in the glove box was controlled between 45 and 55% with desiccated s i l i c a gel.  It was  found that contamination problems were minimized  i f the humidity was kept at 45%.  Oxygen l e v e l s i n the chamber were  49  monitored every two days with a trace-oxygen analyzer (Lockwood and McLorie, Inc., Horsham;, Pa.). considered to be acceptable.  An oxygen l e v e l of two to f i v e ppm  was  In some instances, organisms were  cultured i n pre-reduced media i n stoppered tubes or flasks using conventional anaerobic techniques as described by Holdeman and Moore (94). 2.  Media a.  Trypticase-Yeast-Hemin (TYH) medium. Liquid cultures of !B. melaninogenicus were maintained  in medium containing trypticase, 17 mg/ml; yeast extract  (Difco),  3 mg/ml; NaCl, 5 mg/ml; K^HPO^, 2.5 mg/ml and hemin, 5 ug/ml. pH was adjusted to pH 7.0 with HC1 or NaOH. was made up by d i s s o l v i n g 0.05 100 ml d i s t i l l e d water. b.  The  The hemin solution  gm of hemin i n 1 ml 1 N NaOH and  This solution was stored  at 4°C-  Basal medium. The basal medium consisted of trypticase (17 g/1);  yeast extract (3 g/1); K HP0 2  4  (2.5 g/1) and NaCl (5 g/1).  I3_. melaninogenicus requires hemin for growth but apparently retains enough of the compound to sustain growth through one transfer i n hemin-free l i q u i d media (146). hemin-deficient medium.  In order to obtain c e l l s i n a  state i t was necessary to grow them once in basal  C e l l s obtained from the basal medium would not grow when  subcultured a second time i n hemin-free media.  These  organisms  w i l l be referred to as hemin-depleted I5_. melaninogenicus.  50  c.  Supplemented media. When needed, the following supplements were added  separately or together to either the TYH medium or the basal medium: sodium thioglycolate, 0.05%; glucose, 0.1%; hemin (Sigma), 10, 5, 2.5, 2, 1.5, 1 and 0.5 mg/ml; succinic acid (Eastman), 0.1% or 0.25%; single L-isomers of amino acids (Sigma), 0.5% and cysteine, 0.1-0.5%.  In a l l cases, the compounds were added to the medium  before autoclaving and the pH was adjusted to 7.0 with either HC1 or NaOH. 3.  Continuous Cultures The continuous culture was performed i n an anaerobic chamber  (Coy Manufacturing, Ann Arbor, Michigan) containing an atmosphere of N :H :C0 2  2  2  (85:10:5) at a temperature of 37°C.  The chemostat consisted  of a glass vessel equipped with a rubber stopper which was punctured by three stainless s t e e l tubes (2 mm I.D.).  One tube supplied gas, another  growth medium, and the other carried away the effluent or overflow. growth flask had a working volume of 100 ml.  The culture was  The  stirred  vigorously by a mechanically-driven teflon-covered s t i r r i n g bar.  The  addition of medium was controlled by a p e r i s t a l t i c pump equipped with v a r i a b l e speed control.  The cultures were supposed to be i n steady  state after eight changes of the medium. The d i l u t i o n rate, D, was determined by measuring the output of culture from the chemostat.  Then  D = output (hr ) working volume 1  51  C.  Protease 1.  Assays for P r o t e o l y t i c A c t i v i t y a.  The azocasein assay. The azocasein substrate was prepared by d i s s o l v i n g  2 g of azocasein i n 100 ml of phosphate buffered saline (PBS) i n a b o i l i n g water bath.  The solution was stored at -20°C.  The reaction  mixture contained from 0.1-0.5 ml of either a washed c e l l suspension (A^^Q=1.0), or soluble protease, and was made up to a 1 ml volume with PBS containing 50 mM 3-mercaptoethanol  ($ME).  The reaction  components were pre-incubated at _37°C for 15 min before the addition of 1 ml of azocasein substrate and incubation was then continued f o r 60 min at 37°C.  The reaction was terminated and unhydrolyzed  protein precipitated by the addition of 2 ml of 10% t r i c h l o r o a c e t i c acid (TCA).  The a c i d i f i e d solution was mixed and incubated at room  temperature for 20 min and f i l t e r e d through Whatman No. 1 f i l t e r paper.  Absorbance at 370 nm of the f i l t r a t e s was determined.  Control lacking enzyme was included i n a l l assays. Azocasein hydrolysis was also measured on agar plates. One gram of agar (Difco) was dissolved i n 50 ml b o i l i n g PBS pH 7.0 and mixed with 50 ml of a 2% azocasein-PBS solution.  The solution  was heated to 80°C and 30 ml of molten agar were poured into each plate.  Three mm holes were made i n the agar plates and 20 y l  of the enzyme solution was placed i n the holes and the plates were incubated at 37°C.  Enzyme a c t i v i t y was demonstrated by the  presence of a clear zone around the holes.  The logarithm of  the diameter of the zones was d i r e c t l y proportional  52  to the actual enzyme a c t i v i t y .  BME  ( f i n a l concentration 50  mM)  was added to the 13. melaninogenicus culture supernatant or enzyme preparation.  Samples containing known amounts of trypsin were  included i n each assay. b.  The azocoll assay. The reaction mixture for the azocoll assay contained:  Tris-HCl buffer (0.05M, pH 7.2), 4.8 ml; BME i n the same buffer (50 mM),  0.2 ml; and supernatant from sedimented  or enzyme preparation, 0.5 ml.  culture  The reaction components were pre-  incubated at 37°C for 15 min before addition of 20 mg of a z o c o l l . Incubation was continued at the same temperature i n a shaking water bath.  Two m i l l i l i t e r samples were removed at various time i n t e r v a l s ,  c h i l l e d i n i c e , and f i l t e r e d to remove insoluble substrate.  The  amount of s o l u b i l i z e d chromophore was determined by measuring the absorb ance of the f i l t r a t e at 520 c.  nm.  The casein assay. The substrate for the casein assay was prepared by  d i s s o l v i n g 1 g of casein i n 100 ml of 0.1 M phosphate buffer (pH 7.4), and heating for 15 min i n a b o i l i n g water bath. reaction mixture contained 2.5 ml casein, 1.0 ml BME  The  (50 mM)  phosphate buffer, 0.5 ml of supernatant from a sedimented  in  culture  or a 10 times concentrated c e l l suspension, and phosphate buffer to 5.0 ml.  The mixture was incubated at 37°C and the reaction  terminated by the addition acid.  of 5.0 ml of 10% t r i c h o l o r o a c e t i c  After 20 min at room temperature the contents of the tubes  were f i l t e r e d and the A „ of the f i l t r a t e s determined. o n  53  d.  The d i m e t h y l c a s e i n assay. The d i m e t h y l c a s e i n s u b s t r a t e was prepared by t h e  method o f L i n e_t^ a l .  ( 1 2 6 ) , l y o p h i l i z e d and s t o r e d a t -20°C.  The r e a c t i o n m i x t u r e c o n t a i n e d 0.5 ml o f t h e enzyme and 1 ml o f 0.1% d i m e t h y l c a s e i n i n PBS pH 7.0 and was i n c u b a t e d f o r 1 h r a t 37°C.  The r e a c t i o n s were t e r m i n a t e d by immersing t h e  samples i n a b o i l i n g water b a t h . 0.1%  One m i l l i l i t e r of a s o l u t i o n o f  t r i n i t r o b e n z e n e s u l f o n i c a c i d (TBS) and 1 ml o f a 4% sodium  b i c a r b o n a t e s o l u t i o n , pH 8.5, were added t o each sample and t h e m i x t u r e s were i n c u b a t e d i n t h e d a r k f o r 30 min a t 50°C.  After incu-  b a t i o n 1 m l o f 10% sodium d o d e c y l s u l f a t e (SDS) s o l u t i o n and 0.5 ml of a 1 N HC1 s o l u t i o n were added t o each sample, and t h e absorbance at 340 nm was determined  r e l a t i v e t o a blank incubated w i t h a l l of  t h e components p r e s e n t i n t h e sample except a c t i v e enzyme. e.  The r a d i o c h e m i c a l assay. 14 A  C - l a b e l e d N , N - d i m e t h y l c a s e i n was used a s t h e  s u b s t r a t e i n t h i s r a d i o c h e m i c a l assay. prepared as d e s c r i b e d by L i n et^ a l .  N , N - d i m e t h y l c a s e i n was  (126) and was l a b e l e d w i t h  by a d d i t i o n o f 0.5 mCi [ ^ C ] formaldehyde  (specific  activity  44.3 mCi/mM, I n t e r n a t i o n a l Chemical and N u c l e a r Corp.) d i l u t e d w i t h 14 3 ml 37% formaldehyde s o l u t i o n . The C-labeled N,N-dimethylcasein had a s p e c i f i c a c t i v i t y o f 0.02 yCi/mg p r o t e i n . The s u b s t r a t e used i n t h e f o l l o w i n g procedure  (43) was prepared by m i x i n g 300 mg  14 [  C ] N , N - d i m e t h y l c a s e i n w i t h 2 gm c o l d Hammersten  quality casein  i n 60 ml d i s t i l l e d w a t e r , and d i s s o l v i n g t h e s u s p e n s i o n by r a i s i n g t h e s o l u t i o n pH t o 12.0 w i t h 1 N NaOH.  The pH o f t h e s o l u t i o n was  54  then lowered to 7.0 with 1 N HC1, and the concentration of casein was adjusted to 2% and the molarity of the T r i s buffer to 0.01 M at pH 8.0. The reaction mixture contained from 0.01 to 0.1 ml protease sample i n a t o t a l volume of 0.5 ml.  The solution was  buffered by addition of 0.01 ml of 1 M Tris-HCl, pH 8.0. One ml of p r e c i p i t a t i n g reagent (8% t r i c h l o r o a c e t i c acid) was added to one of two tubes containing i d e n t i c a l amounts of enzyme l y before the addition of substrate.  This tube served to determine  14 the background of non-precipitable  immediate-  14 C.  [ C] casein substrate  (0.5 ml) was added to a l l tubes, and the samples were incubated for 30 min at 37°C.  At the end of incubation, 1 ml of 8% t r i c h l o -  roacetic acid was added to the samples containing active enzyme, and a l l tubes were incubated for 30 min at 37°C to ensure complete p r e c i p i t a t i o n of protein.  The protein precipitate was removed by  f i l t r a t i o n through Whatman No. 1 f i l t e r paper, and the f i l t r a t e was extracted with two additions of 1 ml ethyl ether. removal of ether, the aqueous phase was l e f t at room overnight to remove a l l traces of ether. 14 (0.4 ml) was assayed for f.  After temperature  Ether extracted f i l t r a t e  C i n a s c i n t i l l a t i o n counter,  The hemoglobin assay. Hemoglobin (Hb) was denatured i n alkaline urea  solution by suspending 2.0 g Hb i n 50 ml d i s t i l l e d water, adding 36 g urea, 8 ml 1 N NaOH and d i l u t i n g with d i s t i l l e d water to 80 ml.  The mixture was allowed to stand for 30-60 min at room  temperature  before adding 10 ml of 1 M boric acid solution.  After  thorough shaking, the pH was adjusted to 7.5 with 1 N HC1 and the  55  suspension was diluted to 100 ml with d i s t i l l e d water and then centrifuged at 4000 x g for 15 min. i n the assay was  The substrate concentration  6.7 mg Hb/ml reaction mixture;  incubation  temperature was 37°C and the substrate solution was equilibrated to 37°C before the assay. was  Five percent t r i c h l o r o a c e t i c acid  added to p r e c i p i t a t e the unhydrolyzed  c i p i t a t e was g.  (TCA)  proteins and the pre-  f i l t e r e d and A gQ of the f i l t r a t e measured, 2  Determination  of esterase a c t i v i t y .  Hydrolysis of the amino acid esters benzoylarginine ethyl ester (BAEE) and acetyltyrosine ethyl ester (ATEE) was determined by the procedure of Prestidge, Gage and Spizizen (172). A c t i v i t y against t o s y l arginine methyl ester (TAME) was determined by the method of Hummel as described by Walsh (229).  A solution  -3 containing 10  M of each substrate was  HC1 buffer pH 7.8  containing 0.01  prepared  M CaC^-  Assays were performed  i n quartz cuvettes which were held at 30°C i n a compartment.  The reference solution was  i n 0.1 M T r i s -  thermostatted  prepared by mixing 0.5  ml  of substrate solution and 0.5 ml of the same buffer i n a cuvette. One half ml of substrate solution and 0.4 ml of buffer were placed i n the assay cuvette.  After 5 min incubation, the absorbance at  247 nm of the two cuvettes was balanced.  At zero time, 100 y l of  the enzyme preparation were added to the assay cuvette, mixed thoroughly  for 5 sec and the difference i n absorbance was  for a period of about 15 min.  The rate of increase of absorbance  was d i r e c t l y proportional to the concentration of the enzyme.  A c t i v i t y was  recorded  standard  calculated from the slope of the l i n e a r  56  portion of the reaction curve.  One unit was equal to the hydro-  l y s i s of 1 micromole of substrate per min.  BAEE hydrolysis was  indicated by an increase i n absorbance at 254 nm.  The reaction  mixture contained 1.9 ml of enzyme preparation i n buffer; 0.3 ml of 2 M glycine-NaOH  buffer (pH 9.0); 0.8 ml of 0.68 mg of BAEE  in 0.01 M Tris-HCl (pH 8.0).  ATEE hydrolysis was measured as the  decrease i n absorbance at 237 nm, i n a reaction mixture composed of 1.7 ml of enzyme preparation i n buffer; 0.3 ml of 2M glycineNaOH buffer (pH 9.0) and 1 ml of 0.5 mg of ATEE per ml i n 0.01 M Tris-HCl (pH 8.0) 2.  P u r i f i c a t i o n of the Protease Unless indicated otherwise, a l l steps i n the p u r i f i c a t i o n  of the enzyme were carried out at 4°C. a.  Preparation of b a c t e r i a l c e l l s for enzyme assays. C e l l s intended for use i n enzyme assays were harvested  by centrifugation at 12,000 x g for 10 min, washed twice with PBS, resuspended  and standardized to an absorbance of 1.0 at 660 nm i n  the assay\ buf f er. b.  Preparation of culture supernatants for enzyme assays and column chromatography. C e l l s were harvested from 48 hr cultures by  centrifugation.  The supernatant was concentrated to 1/10 the  o r i g i n a l volume by one of the following procedures:  Amicon u l t r a -  f i l t r a t i o n using a PM-10 membrane; freeze-drying; or f l a s h evaporation. Ammonium sulfate p r e c i p i t a t i o n was also used to concentrate the protease a c t i v i t y of the culture supernatant.  57  c.  Preparation of c e l l - e x t r a c t for enzyme assays and protease p u r i f i c a t i o n . B a c t e r i a l cultures were prepared by inoculating 4  l i t r e s of standard medium with a 2% inoculum of a 24 hr culture of B_. melaninogenicus  s t r a i n 2D and incubated anaerobically at  37°C for 48 hr. Upon removal from the anaerobic chamber, cultures were centrifuged at 16,000 x g for 15 min.  The c e l l s were washed  twice with 0.1 M PBS (pH 7.0) and resuspended  i n the same buffer.  C e l l s were then broken by one of the following procedures. (i)  The French pressure c e l l Harvested and washed c e l l s from four  l i t r e s of 48 hr cultures were resuspended  i n 80 ml PBS  containing 10 yg each of protease free deoxyribonuclease and ribonuclease enzymes.  The c e l l s were then passed  through the French press (American  Instrument  Co., Inc.) 2  three times at a pressure of 20,000 p s i ( l b / i n ). The broken c e l l suspension was centrifuged at 121,000 x g for 1 hr using the A-321 rotor of the International ultracentrifuge. (ii)  The M i n i - M i l l  Ten ml of a washed c e l l suspension ( A =5) were mixed with 15 gm of glass beads, placed i n ooO the M i n i - M i l l (Gifford-Wood, Inc.) and s t i r r e d at 4°C.  for 15 min  The disintegrated c e l l s plus l i q u i d were separated  from the glass beads by f i l t r a t i o n using a coarse sintered glass f i l t e r .  The beads were washed with PBS; the  58  f i l t r a t e and the washings were made up to 20 ml with PBS and centrifuged at 121,000 x g for 1 hr. (iii)  Sonication Ultrasonic disintegration was attained by  the use of a Biosonik model sonicator (Bronwill S c i e n t i f i c , Co.) tuned to provide maximum power. The c e l l suspension was placed i n an ice-cooled bath, and treated with ultrasound u n t i l microscopic examination revealed that the majority of c e l l s were broken, d.  Release of c e l l bound HA and protease from 2D c e l l s . C e l l bound HA and protease were released from  15. melaninogenicus (i)  s t r a i n 2D using two methods: Release of periplasmic enzymes by osmotic shock C e l l s were harvested and washed with 0.01 M  Tris-HCl (pH 7.3)- 0.03 M NaCl.  Washed c e l l s were  suspended i n 0.03 M Tris-HCl (pH 7.3) at a r a t i o of 1 g • c e l l s (wet weight) to 40 ml buffer.  An equal volume of  1 M sucrose i n 0.03 M Tris-HCl was added.  The suspension  was made 1 mM with respect to EDTA and mixed at 21°C for 10 min.  The c e l l s were removed by centrifugation at 0°C.  The pellet of c e l l s was resuspended  i n cold d i s t i l l e d  water and incubated at 4°C for 10 min.  The mixture was  centrifuged and the supernatant osmotic shock f l u i d was assayed for HA and protease.  59  (ii)  Release of periplasmic enzymes by treatment with Polymyxin B Twenty ml  of a washed c e l l suspension  which had been mixed with 5 ml of 0.4 mg/ml Polymyxin B in 0.1 M PBS  (pH 7.0) were incubated at 37°C for 60 min  and then centrifuged at 12,000 x g for 10 min.  The  supernatant was assayed for HA and protease a c t i v i t y . e.  Ethanol p r e c i p i t a t i o n . Forty ml of c e l l extract were cooled to -10°C i n an  ethanol-dry ice bath and 60 ml of cold absolute ethanol were added over a period of 20 min with continuous gentle s t i r r i n g . The temperature of the bath was maintained at -10°C.  The mixture  was centrifuged at 12,000 x g for 10 min and the p e l l e t  resuspended  to the o r i g i n a l volume i n PBS. f.  Chromatographic (i)  procedures.  Gel f i l t r a t i o n Proteinases, concentrated from the culture  supernatant and precipitated with ethanol from the c e l l extract, were chromatographed on a Sephadex G 100 _  column  (62 x 1.6 cm) equilibrated with PBS and/or PBS containing 6 M urea. volume.  The sample volume was 2-4% of the t o t a l column Columns were run at 4°C. Protease was also chromatographed on  columns of Sepharose 2B and Sepharose 4B (28.3 x 1.6 equilibrated with PBS 0.1%  SDS.  cm)  or PBS containing 6 M urea and/or  60  (ii)  Ion-exchange chromatography Protease, concentrated from culture  supernatant or precipitated with 60% ethanol  from the  c e l l - e x t r a c t was applied to DEAE and CM-Sephadex ion exchange columns equilibrated i n PBS (pH 7.0), 0.05 0.5 M Tris-HCl (pH 8.4) and 0.1 - 0.5 M phosphate buffer (pH 7.4); and protein was eluted with a l i n e a r NaCl gradient i n the d i f f e r e n t buffers.  In some instances,  PBS containing 0.1% SDS was used to e q u i l i b r a t e and elute both ion exchange columns. (iii)  Activated thiol-Sepharose-4B Activated thiol-Sepharose-4B was swollen  and additives were removed i n PBS pH 7.0 (200 ml/g powder).  The column (10 x 1.6 cm) was equilibrated with  0.1 M phosphate buffer, deaerated to avoid the oxidation of free t h i o l groups, and containing 0.1 M NaCl and 1 mM EDTA to remove trace amounts of heavy metal ions.  Five  ml of the dialyzed, ethanol treated sample i n PBS (pH 7.0) was added to the column and eluted with the same e q u i l i b r a t i o n buffer.  Low flow rates were used during  sample application and elution (5 ml/hr) and 1 ml fractions were c o l l e c t e d . r a t i o n of reducing  The lowest possible concent-  agent, 10 mM L-cysteine  i n PBS  (pH 7.0), was used to elute coupled proteins.  61  (iv)  Organomercurial agarose An agarose mercury column (30 ml volume)  which s e l e c t i v e l y bound t h i o l containing molecules was prepared according to the procedure of Sluyterman and J. Wijdenes  (200) by activating the Sepharose-4B with  cyanogen bromide.  The activated agarose was quickly  washed with cold 0.1 M sodium bicarbonate at pH 9.0 and resuspended at 0°C.  i n 10 volumes of dimethyl sulfoxide (DMS0)  Six grams of p-aminophenylmercuric  dissolved i n 100 ml DMSO was added.  acetate  After gentle  s t i r r i n g for 20 hr at 0°C, the suspension was warmed to 35°C, f i l t e r e d and washed 4 times at 37°C with 20% DMSO to remove the free mercurial compound. resuspended  The agarose was  i n 0.1 M ethylenediamine, adjusted to pH 8.0  and gently s t i r r e d .  After storage overnight at room  temperature, the agarose was washed, packed into a column and reacted with 2-nitro-5-mercaptobenzoic acid i n order to eliminate a l l residual reactive groups of the activated agarose. In order to test protein binding, a column of 10 ml volume was used.  A 2% solution of papain i n 50  mM sodium acetate pH 5.0 containing 0.1 M KC1, 0.5% butanol, 10% DMSO, 1 mM EDTA and 10 mM Na S0 2  3  (standard  buffer) was passed through the column u n t i l the absorbance at 280 nm of the effluent equaled the absorbance of the sample applied to the column.  The column was washed  62  free of unbound protein with standard buffer.  The  papain was eluted with standard buffer containing 0.5 mM  HgCl . 2  (v)  Octyl-Sepharose CL-4B A column (1.6 x 11 cm) of Octyl-Sepharose  CL-4B was equilibrated with PBS containing 1M NaCl.  An  ethanol precipitated sample of the c e l l extract containing 1M NaCl was added to the column.  The  hydrophobic-  a l l y bound proteins were eluted i n a 20-50% gradient of ethylene glycol i n PBS. g.  Gel electrophoresis (i) performed  Polyacrylamide gel (10%) electrophoresis was as described by Nagai  M T r i s , 1.92 M glycine, 0.1%  et a l . (156), with  SDS buffer (pH 8.3)  v e r t i c a l gel plate apparatus.  0.25  in a  Twenty to f i f t y y l of the  protein samples (20 yg protein) were applied to the stacking gel.  Samples were electrophoresed at a constant  current of 40 mA with constant water cooling. concentration of SDS  (BioRad) was 0.1%  The  final  i n both stacking  and running gels and i n the running buffer.  Samples were  prepared i n a s o l u b i l i z a t i o n mixture containing 0.125 T r i s (pH 6.8), 4% SDS, 10% $ME, bromophenol blue as a marker.  20% glycerol and  The gels  Coomassie b r i l l i a n t blue i n 30%  methanol / 10% acetic acid for 5-12 10% acetic acid.  0.01%  The samples were denatured  by heating for 2 min i n a b o i l i n g water bath. were stained with 0.2%  M  hr and destained i n  63  (ii)  Polyacrylamide gel electrophoresis was also  performed as above without SDS i n the stacking and running gels, running buffer and samples.  In t h i s system  the samples were not heated prior to electrophoresis. (iii)  Glycoproteins were detected by staining the  gels with the c a t i o n i c carbocyanine dye " S t a i n s - a l l " . (SA)  (110).  The gels were fixed and SDS removed i n 25%  isopropanol.  A stock solution of SA 0.1% (w/v) i n  formamide was stored at 4°C i n a brown b o t t l e for a maximum of 6 weeks.  Five m i l l i l i t e r s of the stock  solution were d i l u t e d with 20 ml formamide, 100 ml i s o propanol and 275 ml of the t r i s - g l y c i n e buffer without SDS and the pH adjusted were stained overnight isopropanol  to 8.5 with 1 N NaOH.  The gels  i n the dark and destained with 10%  for 18-36 hr at room temperature.  The gel  was checked c a r e f u l l y to ensure that the purple SA had not deteriorated due to SDS, pH or l i g h t .  The glyco-  proteins stained blue and the proteins red by t h i s procedure. (iv)  Lipoprotein electrophoresis Lipoproteins were prestained p r i o r to  electrophoresis.  Acrylamide gels of 3%, 5% and 7% were  prepared i n 0.18 M T r i s - c i t r a t e buffer pH 9.0 without SDS. The running buffer was 0.065 M T r i s - 0.018 M borate buffer pH 9.0.  The t r i s - c i t r a t e buffered-stain was  prepared by d i s s o l v i n g 25 mg Sudan black B i n 24.4 ml  64  e t h y l e n e g l y c o l and 0 . 6 2 5 m l o f buffer for  (pH 9 . 0 ) .  1 hr  60°C  t h r o u g h Whatman N o . 1 p a p e r  4°C r e f r i g e r a t o r .  i n c u b a t e d w i t h 50 u l  of  Fifty  ul  of  the buffered  3 0 m i n was u s e d a s a l i p o p r o t e i n h.  tris-citrate  The s o l u t i o n was i n c u b a t e d a t  and f i l t e r e d  stored at  0.5 M  Gas c h r o m a t o g r a p h i c a n a l y s i s o f  rabbit  s t a i n at  and  plasma  37°C  for  standard,  carbohydrates  in  glycoproteins. Neutral chromatographic  s u g a r s and h e x o s a m i n e s w e r e a n a l y z e d b y  procedure d e s c r i b e d by P o r t e r  (171).  amino s u g a r s were r e l e a s e d f r o m g l y c o p r o t e i n or h y d r o l y s i s w i t h Dowex 5 0 - X 2 ( H ) +  deamination of hexoses. NaBH^,  resin,  ionization detector  g l a s s c o l u m n was u s e d .  c o n s i s t e d of  with  acetates.  A Bendix-Series  and a 6 f t  programmer,  U - s h a p e d , 1/4  The c o l u m n p a c k i n g  a  in  material  3% E C N S S - M o n 1 0 0 / 1 2 0 mesh g a s - c h r o m Q ( A p p l i e d  Science Laboratories, I n c . ) . f l o w r a t e o f 40 m l min  The c a r r i e r  were i n j e c t e d  at  as an i n t e r n a l  fifty  standard.  to  of  a  flow to each d e t e c t o r  respectively.  a column temperature  t e m p e r a t u r e was i n c r e a s e d l i n e a r l y Two h u n d r e d  g a s was h e l i u m a t  H y d r o g e n and a i r  w e r e 50 m l m i n ^ and 70 m l m i n \  min.  acid  chromatographed  2500 g a s c h r o m a t o g r a p h e q u i p p e d w i t h a t e m p e r a t u r e hydrogen flame  by  2,5-anhydro-  H e x o s e s and 2 , 5 - a n h y d r o h e x o s e s w e r e t h e n r e d u c e d  alditol  and  glycopeptides  t h e r e s i n bound hexosamine t o n e u t r a l  as the c o r r e s p o n d i n g n e u t r a l  diameter  Neutral  f o l l o w e d by n i t r o u s  a c e t y l a t e d w i t h a c e t i c a n h y d r i d e and  the  S a m p l e s (2  ul)  1 5 0 ° and e l u t e d a s  the  200° a t  a rate  nanomoles of m y o i n o s i t o l  of  3°  per  i n 50 u l was u s e d  65  i.  L i p i d analysis. The l i p i d s were extracted from the p u r i f i e d  protease preparation (0.3 mg protein) by adding 3 ml of chloroform and 6 ml of methanol, mixing and incubating at room temperature for 10 min. The solution was extracted with a mixture of 3 ml of CHC1 and 3 ml of 0.74% KC1. 3  The lower CHC1 layer was removed 3  and the methanol layer was reextracted with 12 ml of H^O-saturated CHCl^.  The combined CHCl^ fractions were evaporated to dryness  under a stream of N , resuspended i n 0.5 ml acetone and evaporated 2  to dryness, the procedure was repeated and the residue dissolved in CHC1 and stored at -20°C. 3  Phospholipids were analyzed by the thin-layer chromatographic method described by Yavin (240) using the l i p i d s extracted from the p u r i f i e d protease sample.  Phosphorous assay was performed  on the developed spots of phospholipids, after tracing around the spots and scraping them into acid-washed  screw capped tubes.  For f a t t y acid analysis, the extracted l i p i d was fractionated by thin-layer chromatography to remove the free f a t t y acids (20), and the neutral l i p i d s were eluted from the plate and saponified with 15% K0H i n methanol at 70°C for 1 hr. The solution was a c i d i f i e d with H„S0. and extracted three times with 2 4 equal volumes of pentane.  The pooled pentane extracts were dried  under nitrogen and methylated with BF^/methanol reagent.  The  methylated f a t t y acids were extracted into pentane and then separated by gas-liquid chromatography using a column of 10% diethylene glycol succinate on 60-80 mesh chromosorb G at 160°C.  66  The f a t t y acid methyl esters were i d e n t i f i e d by comparison of their retention times with those of known standards. D.  Hemagglut inat ion 1.  Assay C e l l s from a 48 hr culture were harvested by centrifugation,  washed twice i n PBS and resuspended 660 nm of 1.0.  i n PBS to give an absorbance at  Growth liquor obtained from the centrifugation of a  48 hr culture was also assayed.  Hemagglutination was measured i n  m i c r o t i t e r plates by adding 0.025 ml of a 2.5% suspension of formalinized human red blood c e l l s (FRBC) i n PBS to 0.025 ml of a 2-fold d i l u t i o n of the sample. 25 mM 3ME.  serial  The samples were diluted i n PBS containing  Results were recorded after 30 min incubation at 37°C.  The HA a c t i v i t y was recorded as the reciprocal of the highest d i l u t i o n showing complete hemagglutination (no erythrocyte p e l l e t formation) and was considered as the HA t i t e r .  In some instances, HA was measured i n  test tubes (100 x 12 mm) by the addition of 0.2 ml of a 2.5% RBC suspension to 0.2 ml culture supernatant or b a c t e r i a l c e l l suspension. The mixture was agitated gently for 10 min at room temperature.  Samples  possessing hemagglutinating a c t i v i t y usually clumped the RBC within 10 min.  The extent of clumping was assessed v i s u a l l y and scored on a  0 to 44 basis. 2.  Preparation of Red Blood C e l l s Hemagglutination was assayed with formalinized and non-  formalinized human red blood c e l l s (RBC).  Fresh human RBC obtained from  the Canadian Red Cross Blood Transfusion Service were washed three times with PBS at 4°C and used as a 2.5% suspension i n the same buffer.  67  Formalinized human RBC were prepared by resuspending 25 ml of washed packed c e l l s i n 200 ml PBS pH 7.2.  F i f t y ml of formalin was  placed i n a d i a l y s i s tube and this was submerged i n the c e l l suspension, and the mixture was gently agitated at 20°C. formalin  After 4 hr, the remaining  was transferred from the d i a l y s i s sac to the c e l l suspension  and t h i s mixture was s t i r r e d slowly for 18 hr. free of formalin with 0.9% NaCl.  C e l l s were washed  The c e l l s were stored at 4°C as a  25 percent suspension i n PBS containing 0.02% sodium azide. 3.  Determination of the E f f e c t s of Various Reagents on HA Equal volumes of the culture supernatant and the reagent being  tested were incubated at 37°C for 30 min, and then assayed for HA a c t i v i t y by the microtiter method.  A control using PBS instead of the  culture supernatant was always assayed i n p a r a l l e l with the samples. In some instances, the effect of a reagent on HA was measured by s e r i a l l y d i l u t i n g the sample i n PBS containing an appropriate concentr a t i o n of the reagent. Treatment  of RBC with d i f f e r e n t enzymes and reagents was done  by mixing two volumes of the desired concentration of the test compound i n PBS with one volume of PBS washed packed c e l l s .  The RBC  suspension was incubated at 37°C for 1 hr, and the c e l l s were washed three times and resuspended 4.  to 2.5% i n PBS.  Adhesion and Elution of Hemagglutinin from RBC Ten ml' of formalinized RBC (25%) were mixed with 20 ml of culture  supernatant and incubated at 4°C for 30 min.  The RBC suspension was  centrifuged and the supernatant assayed by the microtiter test f o r the presence of unadsorbed hemagglutinin.  The RBCs with the adsorbed HA  68  were resuspended i n 10 ml of PBS-urea (8M) and shaken for 30 min at 37°C.  The RBC were removed by centrifugation and the supernatant was  assayed for HA after d i a l y s i s against PBS. E.  Infectivity C e l l s used for inoculation of animals were harvested from blood agar 9  plates or from broth cultures and resuspended to 10 buffered saline, pH 7.0.  cells/ml i n phosphate-  Guinea pigs weighing 150 to 200 g were shaved  on the abdomen and injected subcutaneously with 0.5 ml of either i n v i t r o cultured c e l l s or exudate aspirated from an infected guinea pig. animals were observed for up to four weeks. positive i n f e c t i o n were:  The  The c r i t e r i a for evaluating a  ( i ) the presence of an abscess (pustular or  n e c r o t i c ) ; ( i i ) the t r a n s m i s s i b i l i t y of the disease.  The l a t t e r was  demonstrated by i n j e c t i n g material aspirated from a l e s i o n into a second animal to produce a similar pathology.  Exudate was aspirated from infected  guinea pigs using a s t e r i l e disposable syringe while the animal was under l i g h t ether anaesthesia.  The exudate was examined for microbial contaminat-  ion by plating on blood agar and incubating i t anaerobically and aerobically. The vascular permeability assay was performed following the method of Craig (36) with concentrated culture supernatant from a 48 h culture of melaninogenicus. control.  P u r i f i e d cholera  toxin (10 ug/ml) served as a test  The toxin was donated by the National Institute of Health  (Bethesda, Maryland).  Test samples of 0.1 ml were injected intracutaneously  into the skin of shaved guinea pigs i n duplicate. were made on each guinea pig.  Six to nine injections  After 20-24 hr, f i l t e r e d Evans Blue dye  (5% i n saline) was injected i n t r a c a r d i a l l y , (0.1 ml/100 g body weight).  The  diameter of the r e s u l t i n g blue area around the s i t e of inoculation was then  69  measured.  The control was s t e r i l e medium concentrated 10 times i n a D i a f l o  ultrafiltration F.  apparatus.  Metabolic End Product Analysis 1.  Preparation of Samples V o l a t i l e and non-volatile f a t t y acids were analyzed by gas  l i q u i d chromatography (GLC) as described by Holdeman and Moore (94).. For the analysis of v o l a t i l e f a t t y acids, culture supernatants were a c i d i f i e d to pH 2 or below with 50% aqueous IL^SO^, and the v o l a t i l e f a t t y acids were extracted into ether.  The ether was then dried with  anhydrous MgSO^ and 15 y l was injected into the chromatographic  column.  For the analysis of non-volatile f a t t y acids, 1.5 ml of the culture supernatant a c i d i f i e d as described above was methylated  by  addition of either 2 ml of methanol or 1 ml of b o r o n t r i f l u o r i d e methanol, 14% w/v  (Applied Science Laboratories).  The tubes were  stoppered and incubated overnight at room temperature.  The methylated  acids were extracted into 0.5 ml chloroform and 15 y l samples were analyzed by GLC.  Known standards of v o l a t i l e and methylated  fatty  acids were prepared with each set of samples. 2.  Operating conditions of the gas chromatograph Samples were analyzed i n a Bendix model 2500 gas chromatograph  (Canadian Dynamics, Vancouver, B.C.) i o n i z a t i o n detector. the oven temperature v o l a t i l e acids.  equipped with a hydrogen flame  The c a r r i e r gas flow was set at 90 ml min  and  at 120°C for v o l a t i l e acids and 125°C for non-  Column packing material was prepared by mixing 10 gm of  acid washed chromosorb W (60-80 mesh) ( J . Manville Co.) with 1.1 g of Resoflex LAC-1-R296 dissolved i n 20 ml of chloroform.  The mixture  was  70  G.  mixed g e n t l y u n t i l  t h e CHCl-j h a d e v a p o r a t e d and was t h e n p a c k e d i n t o  6'  tube.  x 1/4"  U-shaped  Collagenase Assay C o l l a g e n a s e was m e a s u r e d a s d e s c r i b e d b y G i s s l o w and M c B r i d e  A c i d - s o l u b l e c o l l a g e n was e x t r a c t e d b y G a l l o p and S e i f e r filtration.  from f r e s h f e t a l  (65) , e x c e p t t h a t  calf  (75).  s k i n as d e s c r i b e d  p a r t i c u l a t e matter  was r e m o v e d b y  L y o p h i l i z e d c o l l a g e n was s t o r e d i n s t o p p e r e d f l a s k s a t  T h i s m a t e r i a l was s o l u b i l i z e d i n 0 . 0 1 % c o l d a c e t i c a c i d a t of  a  a  -20°C.  concentration  2 m g / m l , and t h e pH a d j u s t e d t o 8 . 5 b y t h e a d d i t i o n o f 1 M K^HPO^ a n d was 14  then acetylated w i t h a c e t i c -  C-anhydride i n benzene.  The l a b e l e d  was t h e n a c i d i f i e d w i t h g l a c i a l a c e t i c a c i d and t h e a c e t y l a t e d dialyzed against cold d i s t i l l e d  water  t o remove  c o l l a g e n was l y o p h i l i z e d and s t o r e d a t  14  collagen  C-acetic acid.  -20°C u n t i l needed.  mixture  14  The  The  C-  substrate  was p r e p a r e d b y s o l u b i l i z i n g t h e l y o p h i l i z e d , a c e t y l a t e d c o l l a g e n i n 0 . 0 1 % acetic  a c i d at  a c o n c e n t r a t i o n of  A t y p i c a l reaction mixture genicus collagenase i s incubated for appropriate tube  for  microfuge  of  the r e a c t i o n mixture Inc.),  for  (model 1 5 2 , Beckman I n s t r u m e n t s ,  5  4°C.  and c y s t e i n e w e r e  the s u b s t r a t e . was a d d e d t o of  a  At  the  "microfuge"  0.04 N phospho-  The s a m p l e s w e r e l e f t  1 0 m i n and t h e n c e n t r i f u g e d  at  c o l l a g e n a s e a n d I3_. m e l a n i n o -  c o n t a i n i n g 50 y l  2 N HC1.  overnight  Enzyme, b u f f e r  20°C b e f o r e a d d i t i o n o f  time 0.1 ml of  a c i d and 50 y l  C_. h i s t o l y t i c u m  shown i n T a b l e 1 .  (Beckman I n s t r u m e n t s ,  tungstic ature  15 m i n a t  for  1 mg/ml by s t i r r i n g  at  room  temper-  min i n a Beckman-Spinco  Inc.).  One h u n d r e d y l  of  14 s u p e r n a t a n t was a n a l y z e d f o r contained buffer  C.  The c o n t r o l  sample f o r  o r u n i n o c u l a t e d medium i n p l a c e o f  cells.  each assay  the  71  TABLE I  Collagenase Assay  Volume (ml)  Components  B. melaninogenicus 14  C_. histolyticum  Control  0.2  0.2  0.2  Tris-HCl Buffer (0.05 M, pH 7.2) )5 M) with C a C l (0.005  0.1  0.2  0.2  .1 Cysteine (0.05 M)'  0.1  C-collagen (0.1% i n 0.01% acetic acid)  2  Collagenase  0.1  (30 units/ml) 2  Cell  0.1  suspension  0.1  "''Cysteine hydrochloride was made up to 0.05 M concentration i n 0.05 M Tris-HCl buffer and neutralized by adding 5 N NaOH. C e l l s were resuspended i n PBS pH 7.0 to give an A  6 6 Q  = 10.  72  H.  Protein Determination Protein was determined by the method of Lowry et^ a l . (132) using bovine  serum albumin as a standard. I.  Hexose  Determination  Total hexoses were measured by the anthrone assay (223) using glucose as a standard. J.  Microdetermination of L i p i d s Lipids were quantified according to the procedures described by Pande  and Parvin (165) : (1)  Ultramicro method for 2-12 Vg l i p i d  (2)  Micro method for 20-140 ug l i p i d  (3)  Semimicro method for 170 Vg to 1.33 mg  lipid  An aliquot of the l i p i d solution to be analyzed was oxidized with acid dichromate.  The reaction was followed by d i r e c t colorimetry (micro method),  and by an iodometric colorimetry (ultramicro method). In the ultramicro method, 1.0 ml of 0.034% (w/v) potassium dichromate i n 97% s u l f u r i c acid was added to 2-12 Vg dried l i p i d sample. tube was also included which did not contain any l i p i d .  A control  The tubes were  placed i n a b o i l i n g water bath for 15 min and then cooled; 9.0 ml water was added to a l l the tubes, contents were mixed w e l l , and 0.5 ml of these solutions was added to 4.5 ml C d ^ - s t a r c h reagent.  The reagent blank was  prepared by adding 0.5 ml 3.6 N s u l f u r i c acid to 4.5 ml Cdl^-starch reagent. The color i n t e n s i t i e s were read against the reagent blank at 575 In the micro method, 2.0 ml 0.15%  potassium dichromate i n 96%  nm. (w/v)  s u l f u r i c acid was added to tubes containing 0 (two blank tubes required) to 140 ug solvent-free l i p i d .  After heating and cooling as described above,  73  4.5 ml water was added and the solutions were re-cooled after mixing; 0.1 ml freshly prepared aqueous 20% Na2S0^.7 1^0 the dichromate i n one of the blanks. reduced blank at 440 nm.  (w/v) was added to reduce  A l l tubes were read against the  The unreduced blank tube serves as a control,  showing the amount of dichromate present i n i t i a l l y . K.  Microdetermination of Phosphorous Total free and organic phosphorous was determined by the procedure of  Ghen et^ al.(29).  Samples were placed i n a c i d - a l k a l i cleaned t h i c k walled  glass tubes and 4 drops of concentrated H^SO^  were added.  The tubes were  heated over bunsen flame u n t i l white fumes of sulphur t r i o x i d e appeared. Two drops of perchloric acid (72%) were then added and the samples heated u n t i l l i q u i d s became c l e a r , and then cooled and volumes were adjusted to 25 ml i n a volumetric flask.  Standards containing up to 8 Jig phosphorous  and a blank containing only water (4 ml) were used.  Four ml of a freshly  prepared reagent containing 1 volume 6 N H^SO^, 2 volumes d i s t i l l e d water, 1 volume 2.5% ammonium molybdate and 1 volume 10% ascorbic acid were added and the tubes incubated at 37°C for 1 hr.  The samples were allowed to  cool to room temperature and absorbance at 820 nm against the blank was measured. L.  Glucosidase Assay Alpha-glucosidase and 6-glucosidase a c t i v i t i e s were determined with  p-nitrophenyl a-D-glucoside (a-PNPG) and p-nitrophenyl 3-D-glucoside (B-PNPG) (Calbiochem) as substrates, respectively.  The enzyme a c t i v i t y was  assayed i n the p u r i f i e d protease preparation and with known glycosidic enzymes purchased from Miles Laboratories, Inc.  The reaction mixture for  each assay contained 0.2 ml of PBS, pH 7, enzyme (0.2 to 1.0 ml) and  74  d i s t i l l e d water to a f i n a l volume of 3.0 ml.  This mixture was incubated  at 37°C for 5 min prior to the addition of substrate.  The reaction was  i n i t i a t e d by the addition of 1.0 ml of 10 mM prewarmed solution of the appropriate substrate i n PBS (pH 7.0). placing the tubes i n an i c e bath and  The assay was terminated  by  adding 1.0 ml of 0.2 M Na2C0.j.  Appropriate controls lacking enzyme or substrate were included with each assay.  The hydrolysis of substrate was monitored at 400 nm i n a Perkin  Elmer-Hitachi spectrophotometer model 124. M.  Lipase Assay The l i p a s e a c t i v i t y i n the p u r i f i e d protease preparation was determined  by a modification of the method of Huggins, Charles and Lapides as described by Winters (237), which i s based on the amount of p-nitrophenol released during the hydrolysis of p-nitrophenyl acetate (Eastman Organic Chemicals, Rochester, N.Y.).  The reaction mixture consisted of 4 ml of 0.06 M phosphate _3  buffer, pH 7; 0.5 ml of enzyme preparation; and 1.0 ml of 4 x 10 strate i n the same buffer.  M sub-  After incubation at 30°C for 1 hr, the absorbance  at 410 nm was read against a reagent blank. N.  Reagents and Chemicals The following reagents and chemicals were purchased from Sigma Chemical  Company:  t r y p s i n , papain, neuraminidase Type VI, C_. histolyticum collagen-  ase, wheat germ l i p a s e , pronase, BSA, d i t h i o t h r e i t o l , trinitrobenzenesulfonic acid, sodium borohydride, tosyl-L-arginine methyl ester-HCl (TAME), benzoylarginine ethyl ester (BAEE), and acetyltyrosine ethyl ester (ATEE). The following were purchased from Calbiochem:  tosyl-L-phenylethyl  chloromethyl ketone (TPCK), phenyl methyl sulfonyl f l u o r i d e (PMSF), a z o c o l l and azocasein.  75  The following were purchased from Difco:  hemoglobin, D-mannose,  D-fructose, D-galactose, sucrose, L-arabinose and D-mannitol.  Acrylamide  and 3-mercaptoethanol were purchased from Eastman. F i c o l l , Sephadex G-100, Sepharose 2B, Sepharose 4B, activated  thiol-  Sepharose 4B, octyl-Sepharose CL-4B, DEAE-Sephadex A-50 and CM-Sephadex C-50 were purchased from Pharmacia  (Montreal).  Mixed glycosidases (T_. cornutus) were purchased from Miles Laboratories, Inc.  A l l other chemicals used were Fisher reagent grade (Fisher S c i e n t i f i c  Company). The r e s u l t s of the present investigation may be divided into three sections.  In the f i r s t , the preliminary i d e n t i f i c a t i o n of 1J. melaninogenicus  strains i s reported.  The second section deals with the p a r t i a l character-  i z a t i o n and p u r i f i c a t i o n of the soluble hemagglutinin.  The l a s t section  presents the techniques used i n attempt to purify and characterize the soluble and cell-bound protease(s) of I3_. me 1 aninogenicus.  76  III.  A.  RESULTS  Characterization of ]3. melaninogenicus The purpose of this part of the thesis i s to describe experiments  which were carried out to characterize B_. melaninogenicus s t r a i n s 2D and K110 and further to investigate s t r a i n 2D which was used i n t h i s study. 1.  Fatty Acid  Production  The f i r s t step i n characterizing the organisms'was  .  to subdivide them on the basis of the a c i d i c end products produced during growth.  Organisms were cultured for 48 hr i n TYH medium  and the supernatants analyzed  for v o l a t i l e and non-volatile f a t t y  acids by gas chromatography (Fig. 3 and 4). The peaks were i d e n t i f i e d by comparing their retention times with those of standards.  Both  K110 and 2D strains produced a c e t i c , propionic, i s o v a l e r i c , i s o b u t y r i c , and butyric acids i n addition to an unknown compound which was shown to be phenylacetic acid by mass spectroscopy and gas chromatography (Susan Jensen, personal communication).  The r e s u l t s indicated that  both s t r a i n s correspond to the subspecies known as ss. asaccharolyticus.  melaninogenicus  The presence of phenylacetic acid i n cultures of  15_. melaninogenicus ss. asaccharolyticus had not been noted previously and should provide a useful t o o l i n i d e n t i f y i n g t h i s organism i n mixed infection.  FIGURE 3. Gas chromatography  of v o l a t i l e f a t t y acids produced by  ]}. melaninogenicus V o l a t i l e f a t t y acids were extracted from a c i d i f i e d culture supernatant. a  = acetic acid;  p = propionic  iso b  = isobutyric acid;  iso v  = i s o v a l e r i c acid  acid;  b = butyric acid;  78  Retention Time (min)  FIGURE 4. Gas chromatography of non-volatile f a t t y acids produced by 15. melaningenicus. Methylated f a t t y acids were extracted from a c i d i f i e d culture supernatant. £ = l a c t i c acid pa = phenylacetic acid  Retention Time (min)  81  2.  Collagenase A c t i v i t y J3. melaninogenicus has been reported to possess a c e l l bound  collagenase which i s believed to be associated with pathogenicity (67,83,84).  In order to determine i f collagenase was associated with  K110 and 2D s t r a i n s , 48 hr cultures were harvested and c e l l s assayed 14 for collagenase by incubating them with strains possess a c e l l bound collagenase.  C-collagen. Both K110 and 2D Unlike other microbial c o l l -  agenases, the 13. melaninogenicus enzyme i s oxygen sensitive and i s stimulated by reducing agents. No soluble collagenase was detected i n the culture supernatant of either organism. The s p e c i f i c a c t i v i t y of the c e l l bound collagenase was expressed as the ug of collagen s o l u b i l i z e d per hr by 1 ml of c e l l s (Absorbance^ . = 1.0). B. melaninogenicus s t r a i n K110 had a s p e c i f i c 660 — ° r  a c t i v i t y of 122 pg/hr/O.D.  Strain 2D 13. melaninogenicus had a s p e c i f i c  a c t i v i t y of 140 ug/hr/O.D. 3.  Pathogenicity a.  Infectivity. _B. melaninogenicus s t r a i n 2D was tested f o r i t s  a b i l i t y to produce an i n f e c t i o n i n the guinea pig model system. Cells from a 48 hr l i q u i d culture were washed, resuspended i n s t e r i l e PBS and injected into the groin of a 200 g guinea p i g . Within 18 hr the animal developed symptoms of a rapidly spreading i n f e c t i o n : darkening of skin and loss of h a i r i n the thoracic area and accumulation of a large volume of f l u i d .  Material  aspirated from the animal was dark i n colour, watery and foul-smelling, and when examined by phase-contrast microscopy  82  appeared to contain a pure culture of  melaninogenicus along  with red blood c e l l s indicating that the organism had invaded the c i r c u l a t o r y system.  Culture of the exudate on blood agar  plates confirmed that i t contained a pure culture of genicus .  melanino-  Fatty acid analysis revealed that acetic, isobutyric,  butyric and phenylacetic acids were present.  These acids are  produced by I5_. melaninogenicus i n i n v i t r o culture.  The trans-  missible nature of the i n f e c t i o n was proven by using the exudate to infect another guinea p i g .  S t e r i l i z i n g the exudate by auto-  claving resulted i n a loss of i n f e c t i v i t y .  Strain 2D was thus  one of the few I3_. melaninogenicus strains capable of producing an i n f e c t i o n without the support of other organisms (139,215).  The  i n f e c t i o n produced symptoms similar to those described i n the l i t e r a t u r e for infections produced by CR2A (139).  Strain K110  f a i l e d to produce i n f e c t i o n when pure cultures were injected into guinea pigs but was i n f e c t i v e i n mixed culture (146).  Generally,  successful infections resulted when 200-250 g guinea pigs were infected instead of larger guinea pigs and when 48 hr c e l l s were used instead of 24 hr c e l l s . b.  Vascular permeability. F i l t r a t e s from stationary phase cultures of  IJ. melaninogenicus strains 2D and K110 were s t e r i l i z e d by f i l t r a t i o n through  a  0.45 u m i l l i p o r e f i l t e r and concentrated 10 times by  u l t r a f i l t r a t i o n through an Amicon PM-10 membrane.  The concent-  rated culture f i l t r a t e s were tested to determine i f they would increase vascular permeability by i n j e c t i n g 0.2 ml intracutaneously  83  Table 2. Inoculum Cholera toxin  Vascular Permeability Test  Blueing diameter (mm) 10  2D  7  K110  4  Control  0  The test samples were injected intracutaneously, i n a random sequence, into the shaved backs of three guinea pigs i n duplicates, and s i x i n j e c t i o n s were made on each guinea p i g . Evans Blue (5% i n saline) was injected i n t r a c a r d i a l l y , (0.1 ml/100 g body weight), about 20-24 hr a f t e r i n j e c t i o n of samples. The dye was disseminated throughout the vascular system almost immediately, and the guinea pigs were s a c r i f i c e d after 10 min.  84  i n t o each o f two  guinea p i g s .  The c o n t r o l was  c o n c e n t r a t e d 10 times i n the same way A f t e r 24 h r , a b l u e dye was c a r d i a c puncture.  injected  s t e r i l e medium  as the c u l t u r e s u p e r n a t a n t . i n t o the t e s t guinea p i g s by  The diameter of the b l u e a r e a s around  the  site  of i n o c u l a t i o n of the d i f f e r e n t samples r e p r e s e n t e d the e f f e c t these samples on v a s c u l a r p e r m e a b i l i t y i n the t e s t (Table  of  animals  2). I t was  f a c t o r which was  found t h a t B_. melaninogenicus  produced  a  r e l e a s e d from the c e l l s and which e x h i b i t e d  b i o l o g i c a l p r o p e r t i e s s i m i l a r to V i b r i o cholerae e n t e r o t o x i n . B o i l i n g of the c o n c e n t r a t e d c u l t u r e supernatant d e s t r o y e d the b l u e i n g f a c t o r s u g g e s t i n g t h a t i t was 4.  a heat s e n s i t i v e  protein.  Growth of S t r a i n 2D 15. melaninogenicus a.  Hemin requirement. S t r a i n 2D of I5_. melaninogenicus has an  requirement hemin was  f o r hemin.  followed.  The response of 2D t o v a r y i n g amounts of  The r e s u l t s a r e shown i n F i g . 5.  seen t h a t growth of the b a c t e r i u m was  to 2.5  be  yg hemin per ml.  grew i n a medium f r e e of hemin f o r one g e n e r a t i o n but  when t r a n s f e r r e d t o f r e s h medium l a c k i n g hemin, l i t t l e o c c u r r e d whereas s u b c u l t u r e to hemin c o n t a i n i n g media resulted  I t may  r o u g h l y p r o p o r t i o n a l t o the  hemin c o n c e n t r a t i o n over a range o f 0.25 The organism  obligate  i f any  growth  always  i n good growth. The organism  of hemin as low as 0.2 h i g h e r than 20  yg/ml medium; c o n c e n t r a t i o n s o f hemin  yg/ml had no f u r t h e r enhancing e f f e c t on the growth  of the organism. the growth of  grew i n the presence of a c o n c e n t r a t i o n  2D.  A d d i t i o n o f g l u c o s e to the TYH medium i n h i b i t e d  FIGURE 5 Reponse of J3. melaninogenicus (Strain 2D) to Hemin. A 0.1 ml inoculum of 24 hr hemin-depleted 2D culture was used to inoculate 10 ml each of media containing d i f f e r e n t of hemin.  Growth was measured (A,,,.) after 24 hr incubation ooU  at 37°C.  concentrations anaerobically  yg Hemin/ml medium  87  b.  Growth response t o amino a c i d s . The  a d d i t i o n o f glutamic  a c i d t o c u l t u r e s of 2D i n  TYH medium r e s u l t e d i n a marked i n c r e a s e d  i n growth r a t e and t o t a l  cell  y i e l d when compared t o unsupplemented medium (Table 3) .  The  growth response o f 2D was dependent on the c o n c e n t r a t i o n  of glutamic  a c i d over t h e range o f 0.2 t o 4 mg/ml ( F i g . 6 ) .  Enhancement o f growth a l s o o c c u r r e d  when L - s e r i n e , L - a s p a r a g i n e ,  L-methionine and L - p r o l i n e were added t o TYH medium. Addition of L-cysteine, DL-valine, L - h i s t i d i n e , L - t r y p t o p h a n , g l y c i n e or L - a r g i n i n e t o TYH medium produced marked i n h i b i t i o n o f t h e growth o f s t r a i n 2D. i n h i b i t i o n o f growth.  L - l e u c i n e caused 66%  Growth was not a f f e c t e d by the a d d i t i o n o f  L - l y s i n e or L - p h e n y l a l a n i n e  t o the TYH medium.  The d i f f e r e n c e i n  i n h i b i t i o n by amino a c i d s when comparing 24 and 40 h r c u l t u r e s (Table  3) c o u l d be due t o the d i f f e r e n t times a t which the  c u l t u r e s reach  the s t a t i o n a r y phase o f growth.  L - c y s t e i n e , adjusted  A d d i t i o n of  t o pH 7.0 by NaOH, t o a growing c u l t u r e o f 2D  i n TYH medium (28 h r ) caused i n h i b i t i o n o f growth (no f u r t h e r i n c r e a s e i n A,,, was 660  detected).  A l t h o u g h glutamic  a c i d or a s p a r a g i n e caused  enhancement o f growth o f 2D i n TYH medium they were unable t o r e p l a c e hemin i n t r y p t i c a s e y e a s t - e x t r a c t medium.  5.  H e m a g g l u t i n i n and P r o t e a s e A c t i v i t y o f 13. m e l a n i n o g e n i c u s ]3. m e l a n i n o g e n i c u s s t r a i n s K110 and 2D produce both a s o l u b l e  and  a c e l l bound h e m a g g l u t i n i n (HA). They a g g l u t i n a t e - r a p i d l y w i t h  88  T a b l e 3.  E f f e c t o f the a d d i t i o n o f amino a c i d s on Growth o f I5_. melaninogenicus s s . asaccharolyticus  Amino a c i d added  2D i n TYH medium  Concentration (mM)  A, 6  6  0  24 h  40 h  -  0.38  0.98  Glutamic a c i d  34  0.75  1.3  L-serine  47.6  0.58  1.2  L-asparagine  37.8  0.62  1.1  L-methionine  33.5  0.5  1.1  L-proline  43.4  0.48  1.1  L-cysteine  41.3  0.03  0.04  L-valine  42.7  0.02  0.06  L-tryptophan  24.5  0.04  0.06  L-histidine  32.2  0.04  0.05  Glycine  66.6  0.03  0.1  L-arginine  28.7  0.03  0.04  L-leucine  38.1  0.12  0.18  L-lysine  34.2  0.4  0.98  L-phenylalanine  30.3  0.36  1.0  None  "  FIGURE 6  E f f e c t o f g l u t a m i c a c i d on growth of 13. m e l a n i n o g e n i c u s . TYH media c o n t a i n i n g i n c r e a s i n g c o n c e n t r a t i o n s o f g l u t a m i c  acid  were i n o c u l a t e d w i t h 2D and growth response was r e c o r d e d a t 24 h r .  Percent Increase i n A  91  human red b l o o d  cells.  Both s t r a i n s produce s o l u b l e and are a c t i v e against and  azocasein.  a number of s u b s t r a t e s  Table 4 presents  genicus and  to g r e a t e r  organism was and  of g r e a t e r  cellular  C e l l s from a 4 day  The  C e l l s on  C e l l s and  g g l u t i n i n and  amounts of HA  the HA  protease.  HA  can be  this  Protease by c e n t r i f u -  agent ( T a b l e 5 ) .  the p r o c e s s r e p e a t e d s i x  HA  s a l i n e i n the presence of  In the meantime  c o u l d not be  the HA  t i t e r of the  e l u t e d from c e l l s h a r v e s t e d  r e p l a c e the PBS  i n r e l e a s i n g the c e l l - b o u n d  BME HA.  cells  from a  or c y s t e i n e c o u l d The  not  presence of  agent i n the washing b u f f e r r e s u l t e d i n b e t t e r e l u t i o n of  from 2D  cells  than u s i n g o n l y b u f f e r .  A n a e r o b i c e l u t i o n of 2D  the HA  i n the a n a e r o b i c chamber gave the same r e s u l t s .  When  the  the s o l u b l e HA  mM).  s e q u e n t i a l l y e l u t e d from the b a c t e r i a l  T r i s b u f f e r c o n t a i n i n g 25 mM  u s i n g PBS  and  c e n t r i f u g a t i o n and  24 h r c u l t u r e .  HA  protease,  s u p e r n a t a n t from each washing were assayed f o r hema-  remained the same.  reducing  and  of the o r i g i n a l volume of PBS-8ME (50  c e l l s by washing w i t h phosphate b u f f e r e d a reducing  s t r a i n of 1$. melanino-  o l d c u l t u r e were h a r v e s t e d  resuspended i n 1/10  c e l l s were c o l l e c t e d by  times.  of the 2D  proteases.  E f f e c t o f Washing 2D  g a t i o n and  and  chosen f o r d e t a i l e d s t u d i e s of the s o l u b l e h e m a g g l u t i n i n  s o l u b l e and 6.  c e l l u l a r HA  K110.  pathogenicity  i t s production  which  including a z o c o l l , casein  the s o l u b l e and  p r o t e a s e a c t i v i t i e s of both 2D and Due  c e l l u l a r protease(s)  HA  eluted  from  2D  cells  i n c u l t u r e s u p e r n a t a n t s , no  was  compared  to  d i f f e r e n c e s were n o t i c e d  92  T a b l e 4.  H e m a g g l u t i n i n and P r o t e a s e o f 2D and K110  Hemagglutinin  Strain Soluble Titer/ml  1280  2D  K110  640  Protease  C e l l bound Titer/ml/1 A  20480  5120  660  C e l l bound  Soluble Units/ml  Units/ml/1 A  7.2  28.4  5.4  20.0  The HA a c t i v i t y was assayed by the m i c r o t i t e r method u s i n g washed resuspended 2D and K110.  i n PBS  cells  (A.,- = 1.0) and the supernatant from a 48 h r c u l t u r e o f 660  F o r m a l i n i z e d human RBC were used f o r the HA assay.  The p r o t e a s e  was assayed i n both c e l l s and c u l t u r e supernatant u s i n g c a s e i n as s u b s t r a t e .  660  T a b l e 5.  E f f e c t of washing 2D c e l l s on the HA  HA t i t e r o f Supernatant  Sample Growth  liquid  HA t i t e r of P e l l e t  64  256 -  F i r s t wash  64  256  Second wash  64  256  T h i r d wash  32  256  F o u r t h wash  32  256  F i f t h wash  4  256  S i x t h wash  16  256  A ten-times-concentrated  s u s p e n s i o n o f f o u r days o l d 2D c u l t u r e  was washed s i x times w i t h 25 mM p e l l e t were assayed  $ME i n PBS, and t h e washing and the  f o r HA by the m i c r o t i t e r  plate.  94  between the two i n r e s p e c t t o optimum pH, s t a b i l i t y and the e f f e c t o f i n h i b i t o r s on the h e m a g g l u t i n i n . 100,000 x g  resulted  C e n t r i f u g a t i o n of the e l u t e d HA a t  i n the r e c o v e r y of most of the HA i n the pellet;,  which i m p l i e s t h a t the r e l e a s e of HA from the c e l l s by washing might be due to s t r i p p i n g o f f of b i t s o f the o u t e r membrane of the 2D  cells.  P r o t e a s e was not e l u t e d from 2D c e l l s by e i t h e r procedure s u g g e s t i n g t h a t the p r o t e a s e enzyme may be more t i g h t l y bound to the c e l l s i s the c e l l u l a r 7.  than  HA.  R e l e a s e o f P e r i p l a s m i c Enzymes from 2D C e l l s S i n c e the c e l l u l a r HA and p r o t e a s e of B_. m e l a n i n o g e n i c u s a r e  a c c e s s i b l e to RBC and h i g h m o l e c u l a r weight it  i s p r o b a b l e t h a t both a r e l o c a l i z e d  membrane.  substrates respectively,  e x t e r n a l t o the c y t o p l a s m i c  An attempt was made t o determine i f t h e HA and p r o t e a s e  were l o c a t e d i n the p e r i p l a s m i c space, by osmotic shock treatment, and by treatment w i t h the membrane-disrupting  a n t i b i o t i c polymyxin  B.  Only 10-18% of the c e l l u l a r p r o t e a s e was r e l e a s e d by e i t h e r procedure , t h e r e f o r e the enzyme cannot  be c o n s i d e r e d as p e r i p l a s m i c .  The HA  seemed t o be l e s s t i g h t l y bound t o t h e 2D c e l l s than t h e p r o t e a s e , c o r r e l a t i n g w i t h the r e s u l t s o b t a i n e d from e l u t i o n of the HA and p r o t e a s e by s u c c e s s i v e washings o f the c e l l s .  The r e l e a s e o f the HA  from the c e l l s by polymyxin B treatment was r e l a t e d t o the time of exposure t o the a n t i b i o t i c  (Table 6).  Most of the c e l l s t r e a t e d w i t h  the a n t i b i o t i c were i n t a c t and no l y s i s o c c u r r e d as judged by m i c r o s c o p i c examination. appearance  There was a rough c o r r e l a t i o n between the  o f s o l u b l e HA and l o s s o f a c t i v i t y from the c e l l s .  There was  no d i r e c t e f f e c t by Polymyxin B on the HA i n the m i c r o t i t r e a s s a y .  T a b l e 6.  R e l e a s e o f the HA from 2D c e l l s by treatment w i t h Polymyxin B  HA t i t e r of supernatant  HA t i t e r of pellet  Time of exposure min 0  512  -  1  512  0  5  256  4  15  128  8  30  64  16  60  64  64  A 400 ml c u l t u r e of 2D I5_. melaninogenicus was h a r v e s t e d , washed and resuspended  i n 20 ml PBS w i t h 25 mM  (3ME.  was mixed w i t h 5 ml o f 0.4 mg/ml Polymyxin  5 ml of c e l l  suspension  B and i n c u b a t e d a t 37°C.  Samples were taken a f t e r 1, 5, 15, 30 and 60 min, kept i n i c e and then centrifuged.  The p e l l e t  m i c r o t i t e r method.  and s u p e r n a t a n t were assayed f o r HA by the  96  B.  S o l u b l e H e m a g g l u t i n i n o f 13. melaninogenicus 13. m e l a n i n o g e n i c u s p o s s e s s e s a c e l l - b o u n d and a s o l u b l e h e m a g g l u t i n i n ( s )  which i s thought to be c e l l a s s o c i a t e d h e m a g g l u t i n i n which has been r e l e a s e d from the c e l l s u r f a c e .  S t u d i e s were c a r r i e d out to p r o v i d e  evidence to support t h i s assumption and a t the same time to d e f i n e the adherence  p r o p e r t i e s of ]3_. melaninogenicus which might  e s t a b l i s h m e n t o f the organism i n the g i n g i v a l 1.  c o n t r i b u t e to the  crevice.  Adherence of 13. melaninogenicus 2D C e l l s to F o r m a l i n i z e d Human RBC C u l t u r e s of 2D c e l l s were t e s t e d f o r adherence  h a r v e s t i n g the c e l l s ,  r e s u s p e n d i n g them i n PBS  to RBC  (A,,.. = 1.0)  by  and m i x i n g  w i t h packed FRBC f o r 30 min a t room temperature w i t h s h a k i n g . H e m a g g l u t i n a t i o n was  determined by the s t a n d a r d t e s t tube assay and  the  samples were observed m i c r o s c o p i c a l l y f o r J3. melaninogenicus bound to By f o l l o w i n g FRBC adherence v e r s u s c u l t u r e age of J3. m e l a n i n o g e n i c u s i t was  found t h a t the adhesion to RBC  o c c u r r e d a t 24 h r ( T a b l e 7 ) .  i n g the b a c t e r i a - R B C aggregates 4 times w i t h PBS  2D, Wash-  d i d not d i s l o d g e the  microorganisms. 2.  D e t e r m i n a t i o n o f Optimal C o n d i t i o n s f o r the H e m a g g l u t i n i n Assay The e f f e c t of pH on the HA a c t i v i t y of 13. melaninogenicus  s t r a i n 2D i s shown i n F i g . 7. range of 7.0  range of pH 6.0 below pH 6.0  Optimum a c t i v i t y o c c u r r e d over a pH  to 7.5, but good a c t i v i t y c o u l d be demonstrated to 8.0.  and above pH  H e m a g g l u t i n a t i o n a c t i v i t y decreased  i n the rapidly  8.0.  Red b l o o d c e l l s from a number of animals and from humans were  RBC.  T a b l e 7.  I n f l u e n c e o f c u l t u r e age on the adherence o f 2D t o FRBC.  C u l t u r e age hrs.  Adherence w i t h FRBC checked by the t e s t tube method  % o f 2D c e l l s not a d h e r i n g to FRBC  10  0  100  16  0  100  24  +++  50  36  +++  40  48  IIII  20  60  ++++  30  The e x t e n t o f adherence was a s s e s s e d v i s u a l l y by the t e s t - t u b e HA assay, and expressed a f t e r microscopic  as t h e p e r c e n t o f non-adhering  examination.  cells  t o FRBC  FIGURE 7.  E f f e c t o f pH on HA o f 15. melaninogenicus  Supernatant o f 48 h r c u l t u r e was t e s t e d f o r HA by the m i c r o t i t e r p l a t e u s i n g 0.2 M each o f Na a c e t a t e b u f f e r a t pH 5.0-6.0; b u f f e r a t pH 6.0-7.5 and t r i s b u f f e r a t pH 7.5-9.0.  phosphate  99  pH  100  tested i n the HA assay using the microtiter plate. shown i n Table 8.  The results are  Rabbit and human RBC showed the strongest a c t i v i t y  and guinea pig the least, but a l l c e l l types were capable of hemagglutinating with B-. melaninogenicus. Formalinized RBC, prepared as mentioned  i n the Materials and Methods,  were as e f f e c t i v e i n the HA assay as were fresh RBC.  The formalinized  RBC were more stable than were non-formalinized RBC.  Furthermore, the  c e l l s retained the microscopic appearance and shape of fresh normal RBC. For the quantitative studies of the adsorption or elution of soluble hemagglutinin, formalinized c e l l s can be used advantageously i n place of normal c e l l s .  Formalinized c e l l s were found to pack more quickly and  firmly than normal c e l l s upon centrifugation so that greater accuracy was obtained i n preparing suspensions.  A stock suspension of the  modified c e l l s may be prepared and used over a period of months with assured constancy of concentration and r e a c t i v i t y . The microtiter assay was also performed using a range of RBC concentrations as shown i n F i g . 8.  I t was determined that the optimal  RBC concentration necessary to observe HA was i n the range of 2.5 to .1.25%. The e f f e c t of incubation temperature on the hemagglutination assay i s shown i n F i g . 9.  From these r e s u l t s , 37°C was chosen as  the most satisfactory incubation temperature f o r the HA assay using an incubation period of 30 min. but was very slow.  Hemagglutination occurred at 4°C,  101 T a b l e 8.  E f f e c t o f the .source o f r e d b l o o d on HA  Source  activity.  o f RBC  Guinea p i g  HA  titer  8  Rabbit  32  Frog  16  Human  64  F o r m a l i z e d human  64  Formalized rabbit  64  The m i c r o t i t e r p l a t e method was used culture  cells  to assay the HA i n a 72 h r  supernatant w i t h 2.5% washed RBC of d i f f e r e n t s o u r c e s .  102  FIGURE 8  Optimal  erythrocyte concentrations f o r m i c r o t i t e r  hemagglutination  assay.  .  Assay c o n d i t i o n s : RBC suspended i n PBS 50 mM  $ME, c u l t u r e supernatant  from a 48 h r c u l t u r e .  p l a t e s were i n c u b a t e d f o r 30 min a t 37°C. were expressed  as percentage  (pH 7.0) c o n t a i n i n g  o f packed  The m i c r o t i t e r  The c o n c e n t r a t i o n s of RBCs  cells.  103  P e r c e n t RBC  FIGURE 9.  E f f e c t s of incubation  temperature on HA.  Assay c o n d i t i o n s : 1.25% ( v o l / v o l ) RBC suspended containing  50 mM  3ME,  i n PBS  (pH 7.0)  c u l t u r e s u p e r n a t a n t from a 48 h r c u l t u r e .  m i c r o t i t e r p l a t e s were i n c u b a t e d f o r 30 min a t v a r i o u s  The  temperatures.  106  3.  Relationship of HA to Culture  Age  The appearance of c e l l u l a r and soluble hemagglutinins followed as a function of the age of the culture. grown at 37°C under anaerobic conditions.  was  The organisms were  As shown i n F i g . 10,  the  production of cell-bound HA p a r a l l e l e d the growth curve of the organism. The soluble HA t i t e r increased during the period between 34-48 hr and correlated with the increased i n f e c t i v i t y of the organism. continued  It also  to increase after growth had ceased, presumably due to l i b -  eration of c e l l u l a r HA as a r e s u l t of c e l l l y s i s . 4.  E f f e c t s of RBC Modification on HA The r e s u l t s of the treatment of RBC with various enzymes are  presented i n Table 9.  Treatment with trypsin and a-chymotrypsin appears  to have some i n h i b i t o r y e f f e c t .  However, since the i n h i b i t o r y e f f e c t s  represent only a two-fold d i l u t i o n , t h i s i s probably not  significant.  Treatment of the RBC with neuraminidase caused enhancement of the HA a c t i v i t y , which may  have been due to the unmasking of the active  receptor or binding s i t e for the HA on the RBC. and galactosidase treatments of RBC which may HA on the 5.  Pronase, s u b t i l i s i n  caused i n h i b i t i o n of the HA  activity  have been due to a l t e r a t i o n of the receptor structure for the RBC. Modification of the HA In order to obtain information concerning  the component(s) necessary for HA,  the nature of  the culture supernatant was  with several reagents and s a l t s prior to the HA assay. of s a l t s had no e f f e c t on the HA, which might suggest  treated  A number  FIGURE 10.  R e l a t i o n s h i p of HA to c u l t u r e age.  C e l l s grown on TYH medium were h a r v e s t e d , washed and i n PBS  (A.,,,, = 1.0). boo  concentration.  resuspended  C u l t u r e s u p e r n a t a n t was assayed w i t h o u t  The HA was assayed by the m i c r o t i t e r method, and the  r e s u l t s were expressed as the number o f w e l l s o f p o s i t i v e HA.  Time - hr  109 T a b l e 9.  E f f e c t o f treatment o f RBC on t h e i r ability  Reagent  to hemagglutinate w i t h s o l u b l e HA.  Concentration  Units/ mg  HA t i t e r  Trypsin Control  1 mg/ml  65  16 32  a~ chymo t r y p s i n Control  1 mg/ml  65  16 32  Pronase Control  1 mg/ml  49  4 32  Subtilisin Control  1 mg/ml  10  8 32  DNase Control  1 mg/ml  1200  32 32  Neuraminidase Control  250 yg/ml  1.0  64 16  3-galactosidase Control  250 yg/ml  4.0  4 16  Dextranase Control  250 yg/ml  4.0  32 32  Red b l o o d c e l l s were exposed to t h e d i f f e r e n t 37°C i n 0.1 M a c e t a t e b u f f e r  enzymes f o r  30 min a t  (pH 4.0) i n case o f neuraminidase,  3 - g a l a c t o s i d a s e and dextranase, and i n PBS (pH 7.0) f o r  the o t h e r  reagents i n the presence of 25 mM u s i n g 48 h r c u l t u r e s u p e r n a t a n t o f 2D I5_. m e l a n i n o g e n i c u s .  110  t h a t a g g l u t i n a t i o n of the HA and RBC action.  i s not a n  EDTA had no e f f e c t on the HA  ionic  activity.  inter-  Pretreatments  r e a g e n t s which n o r m a l l y i n t e r a c t w i t h p r o t e i n s ( t r y p s i n formalin) i n h i b i t e d  the HA  reaction.  Treatment  p e r i o d a t e a l s o i n h i b i t e d the HA r e a c t i o n .  and  w i t h sodium  These r e s u l t s g i v e some  i n d i c a t i o n t h a t p r o t e i n and/or c a r b o h y d r a t e m o i e t i e s as w e l l disulfide:  bonds may  be n e c e s s a r y f o r HA  with  to o c c u r .  as  However, t h e s e  c o n c l u s i o n s are v e r y t e n t a t i v e because of the crude and r e l a t i v e l y nons p e c i f i c n a t u r e of t h e s e 6.  treatments.  E f f e c t o f Carbohydrates  on  HA  As mentioned i n the L i t e r a t u r e Survey,  several b a c t e r i a l  r e a c t i o n s are i n h i b i t e d by i n c u b a t i o n w i t h s p e c i f i c s u g g e s t i n g t h a t these compounds may  be r e c e p t o r s .  HA  carbohydrates, The r o l e of c a r b o -  h y d r a t e s i n the Ji. melaninogenicus  HA r e a c t i o n was  a s s e s s e d by p r e -  i n c u b a t i n g the c u l t u r e supernatant  f o r 30 min w i t h v a r i o u s c a r b o h y d r a t e s .  Glucose, l a c t o s e , f r u c t o s e , rhamnose, r i b o s e , c e l l o b i o s e , f u c o s e and s u c r o s e at a c o n c e n t r a t i o n of 5% had no e f f e c t on the HA G a l a c t o s e i n h i b i t e d the HA 7.  t i t e r by  reaction.  75%.  S t a b i l i t y of the S o l u b l e HA The HA  a c t i v i t y was  o f a r e d u c i n g agent.  s t a b l e a t 4°C  f o r 3 days i n the absence  At 37°C and 20°C, t h e a c t i v i t y was  stable for  48 hr i n the absence o f a r e d u c i n g agent. H e a t i n g a t 57°C f o r 30 min d i d not i n h i b i t the HA but h e a t i n g a t 70°-80°C f o r 10 min destroyed hemagglutination. f r e e z i n g and thawing  No  or 100°C f o r 5 min  activity,  completely  change i n a c t i v i t y o c c u r r e d a f t e r  i n the absence of  BME.  Ill  •8.  Oxygen S e n s i t i v i t y o f the S o l u b l e The  e f f e c t o f oxygen on HA was  which the supernatant a e r a t i o n f o r 30 min  HA  demonstrated i n an experiment i n  from a 48 hr c u l t u r e of 2D was  p r i o r to the assay  (Table 10).  s u b j e c t e d to v i g o r o u s The HA  activity  was  c o n s t a n t l y i n h i b i t e d by 2 t i t e r s upon a e r a t i o n , but a c t i v i t y c o u l d be r e s t o r e d by the a d d i t i o n of 3-mercaptoethanol (25 mM). the HA  A d e f i n i t e increase i n  t i t e r o c c u r r e d when a r e d u c i n g agent such as (3ME "or c y s t e i n e  i n c l u d e d i n the HA  assay, a l t h o u g h  pronounced because the supernatant activity.  the e f f e c t i n t h i s case was  not  was as  c o n t a i n e d endogenous r e d u c i n g  When the c u l t u r e supernatant was  c o l l e c t e d and  assayed  i n the  a n a e r o b i c chamber, r e d u c i n g agents had no b e n e f i c i a l e f f e c t on the  HA  activity. 9.  E f f e c t s of S u l f h y d r y l M o d i f i e r s on  HA  S i n c e r e d u c i n g c o n d i t i o n s a r e r e q u i r e d f o r the HA reagents  t h a t b i n d to s u l f h y d r y l compounds were t e s t e d f o r i n h i b i t i o n  of the HA  ( T a b l e 11) .  r e v e r s e d by addingBME.  HgC^  i n h i b i t e d HA  and  i n h i b i t i o n could  I o d o a c e t i c a c i d and iodoacetamide,  a l k y l a t e s u l f h y d r y l groups, i r r e v e r s i b l y i n h i b i t e d 10.  U l t r a c e n t r i f u g a t i o n of S o l u b l e  1 h r r e s u l t e d i n the s e d i m e n t a t i o n  of 83% of the HA  amount remained i n the s u p e r n a t a n t  ( T a b l e 12).  i n the c u l t u r e s u p e r n a t a n t  be  which  the s o l u b l e  HA.  HA  U l t r a c e n t r i f u g a t i o n of the supernatant  a t 141,000 x g f o r activity.  A small  The m a j o r i t y of the  seemed to be of h i g h m o l e c u l a r weight  might be p a r t i c l e - b o u n d ; i t i s not known whether the non HA  activity,  r e p r e s e n t e d a d i f f e r e n t HA  or the HA  in a different  and  sedimenting  form.  HA  112  T a b l e 10.  (3ME was  E f f e c t of a e r a t i o n on s o l u b l e  Aeration  3ME*  -  -  16  -  +  64  +  +  64  +  -  8  added immediately  b e f o r e the  HA  assay.  HA  titer  113  T a b l e 11.  E f f e c t of s u l f h y d r y l m o d i f i e r s on  HA  HA  Concentration mM  *Inhibitors  titer  0  16  0  64  2  5  2  HgCl +BME  5  32  25  2  25  2  Control Control +  HgCl  BME  2  Iodoacetic acid  + BME  Iodoacetamide + BME  * F o r t y - e i g h t h r c u l t u r e supernatant temperature w i t h the reagent a s s a y i n g i t f o r HA.  BME  was  was  i n c u b a t e d f o r 30 min  i n T r i s - H C l b u f f e r , pH  8.2  a t room  before  used a t a c o n c e n t r a t i o n of 25  mM.  T a b l e 12.  HA  Ultracentrifugation  HA t i t e r  of soluble  HA  % o f t o t a l HA  A c t i v i t y p r i o r to centrifugation  2448  Pellet  2034  83.0  388  15.8  Supernatant  100  The HA was assayed by the m i c r o t i t e r p l a t e method and was expressed as HA t i t e r p e r t o t a l volume o f the sample.  115  Partial Purification a.  o f s o l u b l e HA  C o n c e n t r a t i o n o f t h e HA. Because t h e HA i s e x t r a c e l l u l a r  dilute,  and t h e r e f o r e  i t was n e c e s s a r y t o c o n c e n t r a t e i t as a f i r s t  p u r i f i c a t i o n procedures.  step i n  A number o f procedures were t e s t e d t o  determine t h e most e f f e c t i v e method.  The HA i n t h e supernatant  c o u l d be c o n c e n t r a t e d by f r e e z e - d r y i n g , by f l a s h e v a p o r a t i o n and by  (NH^^SO^ p r e c i p i t a t i o n .  I t c o u l d not be c o n c e n t r a t e d by t h e  use o f F i c o l l or by u l t r a f i l t r a t i o n through Amicon PM-10 o r XM-50 membranes due t o a d s o r p t i o n o f t h e HA t o t h e d i a l y s i s t u b i n g and to t h e u l t r a f i l t r a t i o n membranes. b.  Chromatography. i)  A f f i n i t y adsorption By d e f i n i t i o n HA b i n d s t o RBCs.  I t seemed  t h e r e f o r e p o s s i b l e t o d e v i s e an a f f i n i t y chromatography procedure which would take advantage o f t h i s c h a r a c t e r istic.  F o r m a l i n i z e d RBCs were mixed f o r 30 min a t 4°C w i t h  supernatant c o n t a i n i n g HA, and washed as d e s c r i b e d i n M a t e r i a l s and Methods. Hemagglutinin was e l u t e d most i l y w i t h 8 M u r e a c o n t a i n i n g 25 mM |3ME. i n c l u d i n g NaCl, NH C1, M g C l 4  were n o t e f f e c t i v e  2 >  satisfactor-  A number o f s a l t s  MgSO^, C a C l , ' K C l and L i C l  i n r e l e a s i n g HA.  2  A v a r i e t y of s a l t  c o n c e n t r a t i o n s (up t o 6 M) and pH c o n d i t i o n s were t e s t e d b u t none proved t o be e f f e c t i v e . G a l a c t o s e , g l u c o s e and c e l l u b i o s e d i d n o t e l u t e t h e HA.  A c e t a l d e h y d e , t r i t o n X-100,  H 0-saturated o  116  b u t a n o l as w e l l as g u a n i d i n e HC1 were not e f f e c t i v e i n e l u t i n g t h e HA from RBCs.  The c h a r a c t e r i s t i c s o f t h e  u r e a e l u a t e a r e shown i n T a b l e 13. been shown t o b i n d t o neuraminic (209).  Some b a c t e r i a have  a c i d r e s i d u e s on RBC  T h i s does not appear t o be t h e case w i t h t h e  B. melaninogenicus  HA, as t h e removal  r e s i d u e s w i t h neuraminidase  of neuraminic  i n c r e a s e d t h e a b i l i t y o f RBC  to b i n d HA, and e l u t i o n o f HA adsorbed  t o neuraminidase-  t r e a t e d RBCs w i t h 8 M u r e a was found l e s s e f f e c t i v e e l u t i o n o f adsorbed  acid  than  HA from u n t r e a t e d RBCs which might  be due t o a t i g h t e r b i n d i n g o f s o l u b l e HA t o n e u r a m i n i d a s e treated  RBC. T h i s a f f i n i t y adsorption technique  resulted  i n an 11.0 f o l d - p u r i f i c a t i o n and 50% r e c o v e r y o f  the s o l u b l e HA o f t h e supernatant treatment  (Table 14).  A  second  o f t h e RBC w i t h 8 M u r e a y i e l d e d more o f t h e HA,  but t h e r e was no i n c r e a s e i n the s p e c i f i c a c t i v i t y o f t h e e l u t e d HA.  T h i s might be due t o e l u t i o n o f p r o t e i n  c o n s t i t u e n t s from the RBC t o g e t h e r w i t h t h e HA.  A control  of RBC t r e a t e d w i t h PBS i n s t e a d of c u l t u r e supernatant  was  also eluted with 8 M urea. ii)  G e l f i l t r a t i o n on Sephadex G-100 The HA e l u t e d from RBC was  resuspended  i n PBS and a p p l i e d t o a Sephadex G-100 column  and e l u t e d w i t h PBS. T a b l e 14.  lyophilized,  The r e s u l t s a r e shown i n F i g . 11 and  Even though the HA was excluded from t h e beads  Table 13.  HA i n the supernatant  81920  Adsorbed HA to FRBC  80600  The HA e l u t e d  %  98.4  C h a r a c t e r i s t i c s o f the HA e l u t e d  Urea e l u t e d HA  40960  %  50.8  Protein content ug/ml  5600  from RBC w i t h urea was c e n t r i f u g e d  from RBC w i t h Urea  Carbohydrate content ug/ml  800  at 141,000xg f o r 1 h r .  by the m i c r o t i t e r p l a t e method and i s expressed as the t i t e r produced  Ultracentrifugation of Urea e l u t e d HA Pellet  %  31949  78  The HA was  Supernatant  9011  assayed  i n t o t a l volume o f samples.  %  22  TABLE 14.  Methods of Purification  Analysis  T o t a l HA titer  of the HA P u r i f i c a t i o n Procedures  Total Protein mg 6.8  HA t i t e r / m g protein  1.  Culture  filtrate  1024  2.  E l u t i o n from RBC  512  0.31  1652  3.  Sephadex G-100  512  0.06  - 8533  Purification factor  '% Recovery  150 11  50  57.2  50  The HA was determined by the m i c r o t i t e r p l a t e and the p r o t e i n was measured by the Lowry method (132).  FIGURE 11.  Sephadex G-100  A 59 X 1.6  g e l f i l t r a t i o n o f the s o l u b l e  HA.  cm column was used a t 5.6 ml/hr f l o w r a t e .  The v o i d  volume of the column was determined w i t h B l u e Dextran 2000, and a sample of 0.06 mg p r o t e i n o f HA e l u t e d from RBC was a p p l i e d column and 3.2  ml f a c t i o n s were c o l l e c t e d .  The o p t i c a l d e n s i t y  f o l l o w e d a t 280 nm f o r p r o t e i n d e t e r m i n a t i o n and the HA was by the m i c r o t i t e r  method.  to the was  assayed  120  F r a c t i o n Number  121  and  appeared i n the v o i d volume of the column,  recovery  was  satisfactory  and  the  good p u r i f i c a t i o n  was  achieved. The HA  from RBC  G-100  followed  The  characteristics regard  as the HA  HA had  I t was  and' 50%  the same  i n h i b i t i o n by  agents enhanced the HA  HgC^titer  of  preparation,  filters  noted t h a t HA  c u l t u r e supernatant was  soluble  of the c u l t u r e supernatant w i t h  to pH optima, s t a b i l i t y and  to M i l l i p o r e  of the  fold-purification  presence of r e d u c i n g  Binding  elution  partially purified  the p a r t i a l l y p u r i f i e d c.  and  by g e l f i l t r a t i o n through Sephadex  r e s u l t e d i n a 57.2  recovery.  The  adsorption  a c t i v i t y was  l o s t when the  passed through a M i l l i p o r e  seemed p o s s i b l e t h a t some type of b i n d i n g was  filter; it  o c c u r r i n g and  this  c o u l d be used advantageously i n p u r i f i c a t i o n . HA  adsorbs to M i l l i p o r e  f i l t e r s and  can be  eluted i  with 8 M urea. HA  A l l the r e a g e n t s used i n the attempts to e l u t e  from RBCs were t e s t e d i n the M i l l i p o r e  found to have no f i l t e r had binding  effect.  I t was  filter  a l s o found t h a t the  a f i n i t e c a p a c i t y f o r b i n d i n g HA,  interaction  or  likely  The  t h a t the b i n d i n g phenomena of HA  Millipore  f i l t e r would be advantageous i n the p u r i f i c a t i o n  but  the r e c o v e r y  o f the HA was  f o l d p u r i f i c a t i o n was relatively  low.  a  pore-size  binding.  s o l u b l e HA were c o r r e c t ; 30.4  were  Millipore  so i t was  r a t h e r than j u s t a d s o r p t i o n  speculations  system and  the  to of  accomplished,  122  C.  P r o t e a s e o f B_. melaninogenicus A c t i v e l y growing  c u l t u r e s o f B_. melaninogenicus-2D  produce p r o t e a s e ( s )  which a r e bound to the c e l l as w e l l as b e i n g f r e e i n the growth media. When measured by the a b i l i t y t o h y d r o l y z e c a s e i n , 80% of the p r o t e o l y t i c a c t i v i t y was  found t o be c e l l a s s o c i a t e d .  I t i s not known whether the  c e l l - f r e e and c e l l - b o u n d enzymes a r e the same o r whether t h e y a r e d i f f e r e n t gene p r o d u c t s . 1.  P r o t e a s e Assays The c e l l u l a r and s o l u b l e p r o t e a s e ( s ) of 15. m e l a n i n o g e n i c u s  a c t i v e a g a i n s t a number o f p r o t e i n s u b s t r a t e s i n c l u d i n g a z o c o l l , a z o c a s e i n and N , N - d i m e t h y l c a s e i n .  are  casein,  For the a z o c o l l assay, the amount o f  dye r e l e a s e d , which r e f l e c t s the p r o t e o l y t i c a c t i v i t y i n the sample, i s determined by measuring  the absorbance  u s u a l l y q u a l i t a t i v e r a t h e r than  at 520 nm.  T h i s assay i s  quantitative.  The c a s e i n assay depends on the d e t e r m i n a t i o n of the amounts of  TCA  enzyme.  s o l u b l e p e p t i d e s l i b e r a t e d from the c a s e i n s u b s t r a t e by The assay depends on measuring  the absorbance  a t 0^280'  i n most c a s e s , the c e l l - e x t r a c t or c u l t u r e f i l t r a t e used  in this  c o n t a i n e d l a r g e amounts o f m a t e r i a l s t h a t absorb a t 280 nm. was,  The  a n t  ^  study assay  t h e r e f o r e , a f f e c t e d by the presence of l a r g e c o n c e n t r a t i o n s o f  p e p t i d e s o r amino a c i d s i n the sample t o be The d i m e t h y l c a s e i n assay was a c t i v i t y was new  the  assayed.  a l s o used.  The  proteolytic  f o l l o w e d by d e t e r m i n i n g w i t h TNBS the p r o d u c t i o n o f  amino groups upon h y d r o l y s i s .  t h i s a s s a y , however  Low  b l a n k v a l u e s were o b t a i n e d w i t h  the p r e p a r a t i o n of the s u b s t r a t e and the assay  procedure i t s e l f were time consuming and not r e p r o d u c i b l e .  123  C - l a b e l e d N,N-dimethylcasein  was a l s o used as a s u b s t r a t e  f o r d e t e r m i n i n g t o t a l p r o t e o l y t i c a c t i v i t y and t h e assay was more s e n s i t i v e than t h e s p e c t r o p h o t o m e t r i c p r o c e d u r e s .  Since the azocasein  assay a l s o gave r e l i a b l e r e s u l t s but was l e s s e x p e n s i v e , t h i s was  used  based  throughout  the e x p e r i m e n t a l study.  assay'  The a z o c a s e i n assay i s  on the s o l u b i l i z a t i o n o f a c o v a l e n t l y l i n k e d chromophore from a  modified protein.  The a b s o r p t i o n maximum of t h i s s u b s t r a t e was 370 nm.  The maximum absorbance  o f t h e c u l t u r e supernatant and c e l l - e x t r a c t was  found t o occur a t a range o f wavelength or no absorbance  o c c u r r e d a t 370 nm.  between 220-320 nm and l i t t l e  T h e r e f o r e , t h e r e was no background,  the assay was r e p r o d u c i b l e , q u i c k and r e l i a b l e ; and t h e s u b s t r a t e was easy t o p r e p a r e .  The a z o c a s e i n assay proved  t o be the e a s i e s t t o perfom.  C o n t r o l v a l u e s were o b t a i n e d by assay w i t h o u t enzyme and were s u b t r a c t e d from e x p e r i m e n t a l v a l u e s . 0.1.  The absorbance  of t h e c o n t r o l never  The enzyme p r e p a r a t i o n s had no absorbance  at this  A z o c a s e i n u n i t s were d e f i n e d as t h e change i n absorbance  exceeded  wavelength. a t 370 nm per  60 min per ml a t 37°C i n the presence o f PBS (pH 7.0). The  time c o u r s e o f r e l e a s e of azoMye from t h e a z o c a s e i n by  IS_. melaninogenicus  i n t r a c e l l u l a r p r o t e a s e i s shown i n F i g . 12. The  r a t e o f r e l e a s e o f t h e chromophore was l i n e a r d u r i n g t h e e a r l y p a r t of t h e r e a c t i o n . The  e f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on t h e r e l e a s e o f dye  from a z o c a s e i n was a l s o f o l l o w e d ( F i g . 1 3 ) . A 2% s o l u t i o n o f a z o c a s e i n was  adequate t o ensure maximum enzyme a c t i v i t y . The r e l a t i o n s h i p between enzyme c o n c e n t r a t i o n and h y d r o l y s i s  FIGURE 12.  H y d r o l y s i s of a z o c a s e i n by J3. melaninogenicus  Each r e a c t i o n mixture ml; 50 mM  BME  i n PBS,  temperature was  37°C.  0.75  protease  contained: c e l l - e x t r a c t ,  2 mg  ml:  Incubating  2% a z o c a s e i n , 1 ml.  protein/0.25  125  0  20  40  60  Time - Minute  80  100  FIGURE 13.  E f f e c t of a z o c a s e i n c o n c e n t r a t i o n .  Each r e a c t i o n m i x t u r e c o n t a i n e d : c e l l - e x t r a c t , 2 mg 50 mM  protein;  (3ME i n PBS, 0.75 ml: c o n c e n t r a t i o n s of a z o c a s e i n i n 1 ml PBS  as i n d i c a t e d .  R e a c t i o n m i x t u r e s were i n c u b a t e d a t 37°C f o r 60 min.  127  r  A z o c a s e i n (mg)  128  of the s u b s t r a t e i s d e s c r i b e d i n F i g . 14.  As can be seen, t h e r e i s  a l i n e a r r e l a t i o n s h i p between the enzyme c o n c e n t r a t i o n and activity.  The  assay  proteolytic  i s s e n s i t i v e , and by i n c r e a s i n g i n c u b a t i o n t i m e s ,  s m a l l amounts of enzyme c o u l d be measured. A z o c a s e i n was  i n c o r p o r a t e d i n t o agar t o assay v a r i o u s  p r e p a r a t i o n s of p r o t e a s e , as mentioned i n the M a t e r i a l s and Methods. The  a z o c a s e i n agar assay was  u s e f u l f o r assaying protease i n  electrophoretic polyacrylamide The were unable  c e l l u l a r and  gels.  e x t r a c e l l u l a r p r o t e a s e s of B_.  t o h y d r o l y z e hemoglobin and bovine  l e a s t to the e x t e n t t h a t i t was  melaninogenicus  serum albumin  (BSA)  at  p o s s i b l e to d e t e c t h y d r o l y s i s p r o d u c t s  s p e c t r o p h o t o m e t r i c a l l y (1-3.2 mg  o f c e l l - e x t r a c t and c u l t u r e  supernatant  were i n c u b a t e d f o r 1 hr w i t h these s u b s t r a t e s a t 3 7 ° C ) . 2.  R e l a t i o n s h i p of P r o t e a s e t o C u l t u r e Age The appearance o f c e l l u l a r and  as a f u n c t i o n of the age of the c u l t u r e . 37°C under a n a e r o b i c c o n d i t i o n s . of the c e l l u l a r and organism  s o l u b l e p r o t e a s e s was The  followed  organisms were grown a t  As shown i n F i g . 15, the p r o d u c t i o n  s o l u b l e p r o t e a s e s p a r a l l e l e d the growth c u r v e of t h e  u n t i l 48 hr when a f u r t h e r i n c r e a s e of the s o l u b l e p r o t e a s e  was  noted and c o n t i n u e d as the c e l l s began t o l y s e . 3.  C e l l - b o u n d P r o t e a s e of IS. The  n a t a n t and  melaninogenicus  r e l a t i v e l y weak p r o t e o l y t i c a c t i v i t y i n c u l t u r e  f a i l u r e t o c o n c e n t r a t e the s o l u b l e p r o t e a s e without  superdecrease  i n a c t i v i t y i n d i c a t e d t h a t i t would be wise t o proceed w i t h s t u d i e s of the c e l l - b o u n d p r o t e a s e . The  c e l l u l a r p r o t e a s e of 15. melaninogenicus  i s localized  129  FIGURE 14.  E f f e c t of enzyme c o n c e n t r a t i o n on the a z o c a s e i n  Each r e a c t i o n m i x t u r e BME  i n PBS  at 37°C.  up to 1 ml;  contained:  assay.  c e l l - e x t r a c t , as i n d i c a t e d and  2% a z o c a s e i n , 1 ml.  Tubes were i n c u b a t e d  50  mM  f o r 1 hour  130  FIGURE 15»  R e l a t i o n s h i p of p r o t e a s e to c u l t u r e age.  C e l l s grown on TYH medium were h a r v e s t e d , washed and i n PBS  (A. _ = 1.0). 660 £(  concentration.  resuspended  C u l t u r e s u p e r n a t a n t was assayed w i t h o u t  C a s e i n was used as the p r o t e a s e s u b s t r a t e .  132  133  i n the c e l l  envelope, p r o b a b l y near the c e l l  a c c e s s i b l e to h i g h m o l e c u l a r weight  surface since i t i s  substrates.  Cell-bound protease  c o u l d be l i b e r a t e d by d i s i n t e g r a t i o n of a s u s p e n s i o n of b a c t e r i a l in  cells  buffer. In t h i s study, attempts were made to s e p a r a t e the c e l l - b o u n d  p r o t e o l y t i c a c t i v i t y from c o n t a m i n a t i n g c e l l u l a r m a t e r i a l and  to study  the r o l e o f the p r o t e a s e i n i n f e c t i o n s by B_. m e l a n i n o g e n i c u s . a.  E f f e c t of p a s s a g i n g 2D c e l l s i n guinea p i g s on protease production. A 10 ml sample o f a 48 hr c u l t u r e o f 2D was  and resuspended guinea p i g was sion.  a s e p t i c a l l y i n 1 ml of s t e r i l e PBS. injected  A 200  i n the g r o i n w i t h 0.5 ml of c e l l  A f t e r 24 hr the guinea p i g had a v e r y pronounced  A l a r g e amount of f l u i d animal was  had c o l l e c t e d  t r a n s f e r r e d t o a second were used  exudate was  checked  A 0.5 ml sample of the exudate ml samples of  tubes of TYH medium.  on b l o o d agar.  exudate  The and  was exudate  P u r i t y of the  A 24 hr o l d c u l t u r e of 2D i n  TYH medium i n o c u l a t e d w i t h the l a b o r a t o r y non-passaged 2D,  infection.  i n the t h o r a c i c a r e a .  guinea p i g , and 0.2  t o i n o c u l a t e two  g  suspen-  a n a e s t h e t i z e d and exudate withdrawn a s e p t i c a l l y  p l a c e d i n a s t e r i l e tube.  of  harvested  strain  as w e l l as one tube o f TYH medium i n o c u l a t e d w i t h the a f t e r passage  resuspended  i n PBS  i n the guinea p i g was  c o n t a i n i n g $ME  50 mM  h a r v e s t e d , washed i n PBS,  (A,,- = 1.0) OOU  f o r p r o t e a s e a c t i v i t y u s i n g a z o c a s e i n as s u b s t r a t e .  and  assayed  134  A f t e r 24 h r , the second guinea p i g was i n f e c t e d and pig. The  The  ml of exudate was  t r a n s f e r r e d to another  guinea  exudate appeared to be more i n f e c t i v e a f t e r passage.  procedure was  i n protease 2D  0.5  also  then repeated  p r o d u c t i o n was  as mentioned above.  a s s o c i a t e d w i t h animal  c o n t a i n i n g exudate when assayed jLn v i t r o by  substrate (Table  the  An  increase  passage of azocasein  15).  I n o r d e r to i n v e s t i g a t e the r e l a t i o n s h i p of cell-bound protease p r o d u c t i o n was ent media and b.  the  to the growth of 13. melaninogenicus,  the  protease  f o l l o w e d d u r i n g growth of the organism i n d i f f e r under d i f f e r e n t growth c o n d i t i o n s .  E f f e c t of hemin c o n c e n t r a t i o n on growth and  protease  production. J3. melaninogenicus has hemin and present  an o b l i g a t e requirement  the growth r a t e i s dependent on the amount of hemin  i n the media ( F i g . 5 ) .  The  e f f e c t of d i f f e r e n t  concentra-  t i o n s of hemin on the growth and p r o t e a s e p r o d u c t i o n of 2D determined  ( F i g . 16).  Growth and  to the hemin c o n c e n t r a t i o n up  to a l e v e l o f 2.5  yg hemin/ml  no a d d i t i o n a l e f f e c t on growth or p r o t e o l y t i c c.  Protease  i n 0.1%  yg/ml  activity.  p r o d u c t i o n i n the presence of s u c c i n a t e .  Protease 2D  was  p r o t e a s e p r o d u c t i o n were r e l a t e d  medium; i n c r e a s i n g the c o n c e n t r a t i o n of hemin beyond 2.5 had  for  a c t i v i t y was  f o l l o w e d d u r i n g growth of  s u c c i n a t e - t r y p t i c a s e medium and was  p r o t e a s e produced by  2D grown on TYH  the growth curve o f 2D  medium.  compared to F i g u r e 17  the  represents  c e l l s on hemin and on s u c c i n a t e medium, as w e l l  as the r e s p e c t i v e p r o t e a s e  a c t i v i t i e s a t d i f f e r e n t c u l t u r e ages.  135  T a b l e 15.  E f f e c t o f P a s s a g i n g 2D on P r o t e a s e A c t i v i t y  Exudates from the passage of the organism i n the f i r s t , and t h i r d were i n o c u l a t e d anaerobically  second  i n t o TYH medium and c u l t u r e s were i n c u b a t e d  f o r 24 h r , checked f o r p u r i t y , h a r v e s t e d , washed,  resuspended i n PBS azocasein assay.  (A^^Q  = 1.0), and assayed f o r p r o t e a s e by the  A 24 h r c u l t u r e of 2D was used as a c o n t r o l i n each  case. Sample  P r o t e a s e U n i t s / m l / 1 A,, oou  % increase i n Protease a c t i v i t y  n  1)  2 D cells  24  2)  *E  28.6  15  3)  *E  2  33.4  40  4)  *E  3  38.1  60  * E , E  2  and E  3  r e f e r s to c e l l s passaged 1, 2 and 3 times  ' 1  respectively.  FIGURE 16.  E f f e c t o f hemin c o n c e n t r a t i o n on growth and  protease  p r o d u c t i o n of 2D.  The A, . o f 24 h r c u l t u r e s of 2D i n d i f f e r e n t 660 rr  concentrations of  hemin i n the medium was measured and then c e l l s were h a r v e s t e d , and  resuspended to A^^Q  azocasein  assay.  =  1*0 i n PBS and assayed  washed  f o r p r o t e a s e by the  Absorbance (660  P r o t e a s e Units/A  nm)  138  FIGURE 17.  Protease production  i n hemin and s u c c i n a t e media.  Media: T r y p t i c a s e - y e a s t medium c o n t a i n i n g 0.1% as TYH medium  (as i n M a t e r i a l s and Methods) were i n o c u l a t e d w i t h  48 h r c u l t u r e , A^Q  depleted volume).  At d i f f e r e n t  = 0.8  (inoculum  c u l t u r e ages, the  measured, and the c e l l s were h a r v e s t e d , PBS  (A,,_ = 1.0)  660  660  and assayed f o r p r o t e a s e  , Hemin; O , 0.1%  Protease:  B  ,  Hemin;•  succinate. , 0.1%  a hemin  was 1% of the t o t a l media o f the c u l t u r e was  washed and resuspended i n  substrate. A^,„:#  s u c c i n a t e as w e l l  succinate.  a c t i v i t y using azocasein  as  Absorbance (660 nm)  140  There was 60% g r e a t e r p r o t e a s e a c t i v i t y i n hemin medium than i n s u c c i n a t e medium where the growth r a t e was  slower.  When 2D was i n o c u l a t e d i n t o t r y p t i c a s e medium c o n t a i n i n g hemin (10 ug/ml) and s u c c i n a t e ( 0 . 1 % ) , the growth of t h e organism and i t s p r o t e a s e  p r o d u c t i o n were the same as when the  organism was grown on hemin medium (10 ug/ml) without Therefore,  succinate.  s u c c i n a t e had no e f f e c t on the growth r a t e or the  p r o t e a s e p r o d u c t i o n o f 2D c e l l s when hemin was p r e s e n t  i n the  med ium. d.  E f f e c t of amino a c i d s on p r o t e a s e  production.  The a d d i t i o n of amino a c i d s t o TYH medium a f f e c t e d the growth r a t e and f i n a l y i e l d s o f 2D 13. m e l a n i n o g e n i c u s as w e l l as p r o t e a s e p r o d u c t i o n  ( T a b l e 16).  The amino a c i d s were added t o  the TYH medium ( f i n a l c o n c e n t r a t i o n 0.5%) and growth of 2D was followed.  C u l t u r e s were h a r v e s t e d  d u r i n g the p e r i o d of most  r a p i d growth, washed and resuspended t o A protease  a c t i v i t y assayed  u s i n g the a z o c a s e i n  =  1.0 i n PBS and substrate.  Amino a c i d s d i d n o t have any e f f e c t on the p r o t e a s e assay.  A 31-75% i n c r e a s e i n p r o t e a s e  o c c u r r e d when the amino a c i d s  p r o d u c t i o n per c e l l  L-asparagine,  glutamic  acid,  L - s e r i n e , L - p r o l i n e and L-methionine were i n c l u d e d i n the growth medium.  T h i s c o r r e l a t e s w i t h the growth enhancing p r o p e r t i e s of  these compounds  (Table 3 ) .  The amino a c i d s L - l y s i n e and  L - p h e n y l a l a n i n e , which had no e f f e c t on the growth of the organism, d i d not a f f e c t the p r o t e a s e  production.  141  Table  16.  E f f e c t o f a d d i t i o n s o f amino a c i d s to TYH medium on the p r o t e o l y t i c a c t i v i t y o f I3_. melaninogenicus  Amino a c i d  added  Concentration mM  660  Protease Units/ 1  A  660  0.38  16  34  0.75  28  L-asparagine  37.8  0.62  24  L-serine  47.6  0.58  22.5  L-proline  43.4  0.5  24  L-methionine  33.5  0.54  21  L-lysine  34.2  0.36  17  L-phenylalanine  30.3  0.34  14  L-leucine  38.1  0.12  5.8  L-cysteine  41.3  0.04  2.6  L-histidine  32.2  0.08  3.7  None Glutamic  acid  142  The  f i n d i n g o b t a i n e d i n b a t c h c u l t u r e s t h a t under  d i f f e r e n t n u t r i t i o n a l c o n d i t i o n s , the organism d i f f e r e d not o n l y i n i t s growth r a t e  but a l s o i n p r o t e a s e p r o d u c t i o n , suggested  that  i t might be v a l u a b l e t o e x p l o r e the r e l a t i o n s h i p between growth r a t e and p r o t e a s e a c t i v i t y . 13. melaninogenicus  T h i s was  accomplished  by  growing  i n c o n t i n u o u s c u l t u r e at d i f f e r e n t  dilution  rates. e.  Growth r a t e and p r o t e a s e p r o d u c t i o n . A chemostat was  designed  f o r use i n the a n a e r o b i c  chamber (see M a t e r i a l s and Methods) f o r the c o n t i n u o u s c u l t u r e o f 2D,  and p r o t e a s e a c t i v i t y was  assayed at d i f f e r e n t d i l u t i o n r a t e s  under steady s t a t e c o n d i t i o n s of growth. the amount of p r o t e a s e per c e l l  i n c r e a s e d as the growth r a t e  i n c r e a s e d up t o a d i l u t i o n r a t e of 0.15 was  As shown i n F i g . 18,  hr \  at t h i s p o i n t t h e r e  a decrease i n enzyme a c t i v i t y . f.  P r o d u c t i o n o f the p r o t e a s e at d i f f e r e n t c o n c e n t r a t i o n s of hemin. I t was  found t h a t growth and p r o t e a s e p r o d u c t i o n by  2D were r o u g h l y p r o p o r t i o n a l t o hemin c o n c e n t r a t i o n i n b a t c h cultures.  To determine whether the hemin had a d i r e c t e f f e c t  the p r o t e a s e , or whether i t was  a c t i n g i n d i r e c t l y by  the growth r a t e , the organism was  on  influencing  grown i n c o n t i n u o u s c u l t u r e i n  d i f f e r e n t l e v e l s o f hemin but a t the same growth r a t e . The p r o t e a s e was  measured a t steady s t a t e s of growth  by h a r v e s t i n g the c u l t u r e and r e s u s p e n d i n g i t a f t e r washing i n PBS  t o an A,,  n  = 1.0  i n PBS.  R e s u l t s a r e shown i n T a b l e 17.  It  143  FIGURE 18.  Effect 15.  o f d i l u t i o n r a t e (D) on p r o t e a s e p r o d u c t i o n by  melaninogenicus  When a steady s t a t e had been a c h i e v e d c e l l s were h a r v e s t e d , washed and  resuspended  i n PBS  (AggQ = 1.0) and assayed  f o r p r o t e a s e by  i n c u b a t i o n f o r 1 h r a t 37°C w i t h the a z o c a s e i n s u b s t r a t e .  145  T a b l e 17.  E f f e c t o f hemin c o n c e n t r a t i o n on protease  Hemin c o n c e n t r a t i o n ug/ml medium  A  production.  660 steady r a t e a  t  f  i  n  a  l  Protease U n i t s /  - A 6 60  1  1.1  7.8  1.5  1.2  8  2  1.25  7.8  2.5  1.2  8.2  5  1.3  8.7  10  1.3  7.9  20  1.2  8.2  146  can be seen t h a t d i f f e r e n t hemin c o n c e n t r a t i o n s had no e f f e c t on the p r o t e a s e a c t i v i t y i n continuous In  s t a t e o f growth i n TYH c o n t a i n i n g 0.5%  was  the c e l l s were a t steady  medium, changing  the medium to  TYH  L - c y s t e i n e a t the same d i l u t i o n r a t e r e s u l t e d i n  decreased c e l l y i e l d s  (measured by A^Q)  p r o t e a s e a c t i v i t y per g.  culture.  the continuous c u l t u r e where the growth r a t e o f 2D  c o n t r o l l e d u s i n g the chemostat and  in  direct  and a p a r a l l e l  decrease  cell.  P r e l i m i n a r y c h a r a c t e r i z a t i o n of the c e l l u l a r  protease,  of J3. m e l a n i n o g e n i c u s . i)  E f f e c t o f r e d u c i n g agents on the c e l l u l a r  protease  The d a t a i n T a b l e 18 show the e f f e c t of d i f f e r e n t r e d u c i n g agents on the a c t i v i t y o f the p r o t e a s e in  the c e l l - e x t r a c t of 13. melaninogenicus  t r a t i o n of a t l e a s t 10 mM  r e d u c i n g agent was  maximum p r o t e o l y t i c a c t i v i t y .  c o n c e n t r a t i o n o f BME  i n only a  A p r e p a r a t i o n o f 50 or c y s t e i n e was  chosen  A  concen-  required for  I n c r e a s i n g the  t r a t i o n of r e d u c i n g agent r e s u l t e d increase i n a c t i v i t y .  2D.  concenslight  mM to be  r o u t i n e l y used i n the a s s a y s f o r p r o t e a s e . F r e s h l y prepared c u l t u r e e x t r a c t was  not  as dependent on the presence o f exogeneous r e d u c i n g agent, presumably because the e x t r a c t c o n t a i n e d endogenous r e d u c i n g systems.  A f t e r a p e r i o d of time the endogenous  s o u r c e s became o x i d i z e d and i t was  necessary to supply r e d -  u c i n g a c t i v i t y to the system. A c t i v i t y a g a i n s t a z o c o l l was  lost  147  T a b l e 18.  E f f e c t o f r e d u c i n g agents on the cellular  protease  Reducing agent  none  Protease  Units  • 6.8  cysteine  19.2  dithiothreitol  18  (3-mercaptoethanol  17.6  thioglycolate  13.4  0.5 ml samples o f c e l l e x t r a c t c o n t a i n i n g 3.2 mg p r o t e i n were assayed  i n the presence o f 50 mM  c o n c e n t r a t i o n of each of the  r e d u c i n g agents u s i n g a z o c o l l as s u b s t r a t e .  148  when the p r o t e a s e p r e p a r a t i o n 20 min,  but was  restored The  was  aerated  by a d d i t i o n of r e d u c i n g agent.  increased  a c t i v i t y of the  the presence of r e d u c i n g agents i n d i c a t e d cellular  protease i n  that  p r o t e a s e of B_. melaninogenicus c o u l d  as a s u l f h y d r y l p r o t e a s e . p r i m a r i l y by generally  for  their  contain  The  sensitivity  l a t t e r are  the be c l a s s i f i e d  characterized  to t h i o l r e a g e n t s  and  c y s t e i n e m o i e t i e s as e s s e n t i a l a c t i v e  s i t e components. ii.)  S t a b i l i t y and  t h e r m o l a b i l i t y of the  cellular  protease. The c e l l e x t r a c t was  t h e r m o l a b i l i t y of the  determined as the p e r c e n t of  l o s t a f t e r incubating temperature  protease i n  (Table  the enzyme i n PBS  19).  The  protease  a t the  was  the  activity indicated  partially  or c o m p l e t e l y i n a c t i v a t e d at temperatures of 40°C  and  above. The  p r o t e a s e a c t i v i t y was  i n the absence of 3ME  f o r 12 h r , a f t e r t h i s  g r a d u a l l o s s of a c t i v i t y . 21°C 20%  and  37°C f o r 4 hr  l o s s of a c t i v i t y .  s t a b l e at  enzyme was  resulted  more s t a b l e  in  at  i n the absence of  r e d u c i n g agents.  Freezing  and  at -70°C r e s u l t e d  i n 8-10%  l o s s of a c t i v i t y  and  a  I n c u b a t i o n of the p r o t e a s e at  room temperature than at 37°C f o r 4 hr  absence of $ME  t h e r e was  i n the presence of 3ME The  4°C  thawing of the c e l l - e x t r a c t in  14-25% l o s s of p r o t e o l y t i c  the activity  T a b l e 19.  T h e r m o l a b i l i t y of the activity  Temperature,  i n the  proteolytic  cell-extract.  Time, minutes  activity  lost  % 40  15  6  40  30  11  50  15  19  50  30  31  60  5  0  60  10  6  60  15  25  60  30  60  100  1  80  100  5  100  The p r o t e a s e was  assayed by  the a z o c a s e i n assay a t 37 C f o r 1 h r .  150  i n the presence  of 3ME.  p a r a t i o n were kept were  Therefore, protease  pre-  f r e e o f r e d u c i n g agent u n t i l  they  assayed. The  r e s u l t s suggested  t h a t the p r o t e a s e  was s u b j e c t t o a u t o d i g e s t i o n as i n a c t i v a t i o n of the enzyme was more r a p i d when r e d u c i n g agents were p r e s e n t , h.  P u r i f i c a t i o n of the c e l l u l a r p r o t e a s e of l i . melaninogenicus. i)  P r e p a r a t i o n of c e l l - e x t r a c t As the c e l l - b o u n d p r o t e a s e of 2D c o u l d be  l i b e r a t e d by d i s i n t e g r a t i o n of a suspension of b a c t e r i a l c e l l s i n b u f f e r , a s e r i e s of experiments determine  was conducted  to  the most e f f e c t i v e method f o r l i b e r a t i n g the  enzyme. There was an i n c r e a s e i n t h e t o t a l p r o t e a s e a c t i v i t y i n the broken c e l l c e l l disintegration.  suspension  cellular  after  I t i s p o s s i b l e t h a t breakage of  c e l l s might have unmasked p r o t e a s e from p r o t e i n complexes and/or s o l u b i l i z e d of  enzyme from membrane s t r u c t u r e s .  Some  the enzyme remained bound t o t h e m e c h a n i c a l l y r u p t u r e d  cell  envelopes. The  extent of c a s e i n h y d r o l y t i c  r e l e a s e d v a r i e d w i t h the t e c h n i q u e used cells  (Table 20).  Pressure c e l l  activity  t o r u p t u r e the  Breakage of the c e l l s i n the French  proved  t o be the most e f f e c t i v e method  s i n c e 80% of the c e l l u l a r p r o t e a s e was l i b e r a t e d , i n the cell-extract.  151  T a b l e 20.  Comparison o f methods f o r l i b e r a t i n g  protease  % of c e l l u l a r protease found i n the c e l l - e x t r a c t  Protease per mg/protein  1)  French P r e s s u r e c e l l  80  3.6  2)  Mini-Mill  40  1.9  3)  Sonication  25  0.8  The  c e l l s were o b t a i n e d from e a r l y  I3_. melaninogenicus i n PBS.  stationary  phase c u l t u r e s of  grown i n THY medium and were washed and suspended  152  F o l l o w i n g breakage the c e l l - e x t r a c t d i a l y z e d a g a i n s t PBS a 1.8  f o l d p u r i f i c a t i o n and  protease was  overnight  activity  at 4°C.  This resulted i n  o n l y a 6% l o s s  (Table 22).  The  was  of  the  dialyzed cell-extract  c e n t r i f u g e d at 121,000 x g f o r 1 hr t o sediment  r e s i d u a l p a r t i c u l a t e fragments.  Ninety-two percent  the p r o t e a s e remained i n the supernatant i n c r e a s e i n the s p e c i f i c a c t i v i t y .  any of  with a two-fold  Therefore,  dialysis  and u l t r a c e n t r i f u g a t i o n were used as p r e l i m i n a r y s t e p s i n the p u r i f i c a t i o n of the B_. melaninogenicus  protease  from c e l l - e x t r a c t . ii)  Ethanol The  d i a l y z e d and -10°C.  precipitation. p r o t e a s e was  precipitated  c e n t r i f u g e d c e l l - e x t r a c t by 60%  Under a p p r o p r i a t e c o n d i t i o n s , the  a c t i v i t y was  not d e s t r o y e d  almost q u a n t i t a t i v e . t h a t the p r o t e a s e  and  The  the p r e c i p i t a t i o n  C o n d i t i o n s were determined  to be separated  p r e c i p i t a t i o n was  temperature t o prevent  had  a low  h e l d at -10°C  protein denaturation.  a t every  was such  solubility high  c a r r i e d out at a  p r e c a u t i o n s were needed to i n s u r e t h a t the was  e t h a n o l at  proteolytic  when most other components of the system had ilities.  from the  solublow  Special  temperature  stage d u r i n g the p r o c e s s .  b r i e f r i s e of temperature to a few degrees above 0°C a few minutes had the sample.  A for  u n d e s i r a b l e e f f e c t s on the s t a b i l i t y  Constant  slow s t i r r i n g  was  essential in  of  153  order to prevent  any element of the p r o t e i n s o l u t i o n  from a t t a i n i n g , even t e m p o r a r i l y , an unduly h i g h  ethanol  c o n c e n t r a t i o n which might denature  The  necessary had  the p r o t e i n .  p r e c a u t i o n s were fundamentally  to be s t r i c t l y maintained  s t e p i n the  simple but  they  and not r e l a x e d i n any  process. The  e f f e c t s of d i f f e r e n t c o n c e n t r a t i o n s of  e t h a n o l on the p r e c i p i t a t i o n of p r o t e a s e at d i f f e r e n t temperatures  a r e shown i n T a b l e 21.  60% e t h a n o l at -10°C  f o r 20 min.  A c o n c e n t r a t i o n of  proved  t o be most  s a t i s f a c t o r y , g i v i n g a h i g h r e c o v e r y and increase i n s p e c i f i c a c t i v i t y .  a  significant  Other c o n d i t i o n s r e s u l t e d  i n h i g h e r r e c o v e r y of the p r o t e a s e but the enzyme had lower  specific  activity. T h i s technique  p r o v i d e d a simple  and  e f f e c t i v e s t e p f o r the p u r i f i c a t i o n of the p r o t e a s e the c e l l - e x t r a c t . then  the p e l l e t was  Sephadex G-100 The  cipitated  sample  discarded.  i n 6M  d i a l y z e d and  The  supernatant  (Table 22).  urea. centrifuged protease  from the c e l l - e x t r a c t by e t h a n o l was  column of Sephadex G-100 6M u r e a i n PBS.  The  was  and c e n t r i f u g e d a t 121,000 x g  d i a l y z e d o v e r n i g h t a g a i n s t PBS iii)  2 ml  ethanol p r e c i p i t a t e d  resuspended i n PBS  f o r 1 hr and was  The  from  (1.6 x 62 cm)  flow r a t e was  f r a c t i o n s were c o l l e c t e d .  a p p l i e d to a  equilibrated  5 ml/h  pre-  with  and  A l l of the p r o t e a s e  was  T a b l e 21.  Ethanol p r e c i p i t a t i o n from  of p r o t e a s e  cell-extract.  Protease  Activity  % Recovery Temperature °c E t h a n o l concentration  30%  Time  -5  5  -10  -20  32  10 20  — —  38 45  —  40%  5 10 20  39 46 48  60 64 66  19 53 70  50%  5 10 20  89 79 92  70 82 94  87 90 93.2  60%  5 10 20  90 98 96  90 100 100  89 102 102  70%  5 10 30  90 96 102  99 99 102  100 98 104  _  Table 22.  P u r i f i c a t i o n of protease from B. melaninogenicus - 2D °/,', Recovery  Specific Activity Units/mg P r o t e i n  Total A c t i v i t y Units  Fraction  Purification Factor4  1.  cell-extract  2080  2.8  2.  Dialysis^  1955  5.04  94  1.8  3.  Centrifugation  1799.2  8.06  86.5  2.9  4.  Ethanal precipitation  1540  19.4  74  6.9  Ultracentrif u g a t i o n of ethanol precipitate  1354.5  38.7  65.12  13.8  6.  D i a l-.y s i•s  1330  62  64  22.1  7.  Sephadex G-100 i n 6 M Urea Sepharose 2-B i n 6 M Urea  434  128  154.8  6 154  464.4 774  5.  8.  Dialysis  2  3  2662.5 A B  1302 2083.2  125.0 3203  a g a i n s t 10 volumes  of PBS  100  overnight.  C e n t r i f u g a t i o n a t 121,000 x g f o r 1 h r Dialysis  a g a i n s t 10 volumes o f PBS (pH 7.0)  Purification  overnight.  f a c t o r i s the f o l d i n c r e a s e i n the enzyme s p e c i f i c  activity  1  156  excluded  from t h e column and e l u t e d as a s i n g l e peak a t  the v o i d volume, i n d i c a t i n g i t had a l a r g e m o l e c u l a r weight  ( F i g . 1 9 ) . The e l u t e d p r o t e a s e r e p r e s e n t e d 220%  ,of t h e s t a r t i n g m a t e r i a l .  The i n c r e a s e i n a c t i v i t y c o u l d  be due t o removal of i n h i b i t o r s or components  t h a t were  b i n d i n g t o t h e a c t i v e s i t e s o f t h e p r o t e a s e , or t o c o n t aminating  p r o t e i n s which competed as s u b s t r a t e s .  pooled, d i a l y z e d , concentrated 7-fold  i n t h i s step.  protease present iv)  The  p r o t e a s e was p u r i f i e d  E l e c t r o p h o r e t i c a n a l y s i s of t h e  i n d i c a t e d t h a t t h e r e were a number of p r o t e i n s i n this fraction  (see S e c t i o n h - v i )  Sepharose-2B i n 6 M u r e a . The p r o t e a s e - c o n t a i n i n g e l u a t e from a  Sephadex G-100 column was d i a l y z e d a g a i n s t PBS o v e r n i g h t , c o n c e n t r a t e d by f r e e z e - d r y i n g , made 6M w i t h r e s p e c t t o u r e a and f r a c t i o n a t e d by chromatography on a column of Sepharose-2B e q u i l i b r a t e d w i t h 6 M u r e a i n PBS ( F i g . 2 0 ) . The flow r a t e was a d j u s t e d t o 6.2 ml/hr and 2.3 ml f r a c t i o n s were c o l l e c t e d .  When t h e column was e l u t e d w i t h t h e same  b u f f e r , t h e a c t i v e p r o t e a s e emerged differing i n specific activity.  i n 2 fractions  When t h e s e  fractions  (A and B) were s e p a r a t e l y chromatographed on t h e same column, each emerged  as a s i n g l e peak a t t h e o r i g i n a l  e l u t i o n volume, one i n t h e v o i d volume i n c l u d e d i n t h e column ( B ) .  (A) and t h e other  The f r a c t i o n s were pooled  s e p a r a t e l y and d i a l y z e d a g a i n s t PBS o v e r n i g h t and  FIGURE 19.  G e l f i l t r a t i o n o f the e t h a n o l p r e c i p i t a t e d  A 6 ml sample o f e t h a n o l p r e c i p i t a t e d p r o t e a s e i n g 6 M u r e a was a p p l i e d t o the Sephadex G-100 e l u t e d i n PBS-6M u r e a w i t h B l u e Dextran,  (pH 7.0).  (20.4 mg p r o t e i n ) c o n t a i n -  column and the p r o t e a s e  The v o i d volume of t h e column was  and absorbance a t 280 nm was  f r a c t i o n s were assayed  protease.  recorded.  f o r p r o t e a s e by the A z o c a s e i n  assay.  The  was  determined  158  0  20  40  60  80  F r a c t i o n Number  100  FIGURE 20.  F r a c t i o n a t i o n on Sepharose 2B  The pooled, d i a l y s e d , c o n c e n t r a t e d p r o t e a s e from Sephadex (3 ml of t o t a l 2.2 column (1.6X  28.3  mg cm)  p r o t e i n ) was  e l u t e d from a Sepharose-2B  e q u i l i b r a t e d w i t h 6M urea i n PBS  absorbance a t 280 nm was  measured and  u s i n g a z o c a s e i n as s u b s t r a t e .  G-100  (pH 7.0).  p r o t e a s e a c t i v i t y was  The  assayed  160  161  c o n c e n t r a t e d by f r e e z e - d r y i n g to t h e i r o r i g i n a l volume. At t h i s s t a g e , i t was  not p o s s i b l e t o d e c i d e whether  f r a c t i o n s A and B r e p r e s e n t e d the same o r compounds.  Both components were found  different  to c o n s i s t  of  the same p r o t e i n s when examined by SDS-polyacrylamide  gel  e l e c t r o p h o r e s i s i n t r i s - g l y c i n e - S D S b u f f e r which s e p a r a t e s p r o t e i n s u b u n i t s on the b a s i s of m o l e c u l a r weight.  Both  lacked e l e c t r o p h o r e t i c m o b i l i t y i n polyacrylamide gels without  SDS  due  t o t h e i r s i z e and not due  to  charge  (Section h - v i ) . No d i f f e r e n c e i n pH p r o f i l e and  response  to i n h i b i t o r s between f r a c t i o n s A and B were found  which  i n d i c a t e s t h a t they are the same. A summary of the enzyme p u r i f i c a t i o n scheme i s g i v e n i n T a b l e 22. i n f r a c t i o n A, and of 160%.  The  The  p r o t e a s e was  purified  464-fold  7 7 4 - f o l d i n B, w i t h a combined r e c o v e r y  enzyme p r e p a r a t i o n o b t a i n e d a f t e r  the  Sepharose-2B s t e p w i l l h e r e a f t e r be r e f e r r e d t o as purified  protease. S i n c e the p r o t e a s e was  Sepharose-2B column as two but  still  e l u t e d from  components h a v i n g  the  different,  l a r g e m o l e c u l a r weights, which d i d not  migrate  i n p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ; i t seemed p o s s i b l e t h a t the p r o t e a s e a c t i v i t y might have been a s s o c i a t e d w i t h a component of the c e l l w a l l .  T h i s would r e s u l t i n  the a s s o c i a t i o n of p r o t e o l y t i c a c t i v i t y w i t h random s i z e d  162  and charged  fragments when the c e l l s a r e d i s i n t e g r a t e d .  D i f f e r e n c e i n chromatographic A and B might have been due  m o b i l i t i e s between f r a c t i o n s  to random fragments from  w a l l b e i n g a s s o c i a t e d w i t h the p u r i f i e d  protease.  u r a t i o n of both f r a c t i o n s by b o i l i n g i n SDS t h e s e fragments r e s u l t i n g i n two  Denat-  might d i s s o c i a t e  s i m i l a r components, which  might r e p r e s e n t a p o l y m e r i c form of the enzyme. f r a c t i o n A contained very l i t t l e  Other  chromatographic Activated  Since  enzyme, d e t a i l e d  the p u r i f i e d p r o t e a s e were performed v)  s t u d i e s on  on f r a c t i o n  B.  procedures.  (SH)  t h i o l - S e p h a r o s e 4B  Sepharose-4B c o n t a i n i n g t h i o l f u n c t i o n a l groups was  used  as  i n an attempt  to  s e l e c t i v e l y bind SH-containing p r o t e i n s . t h i o l p r o t e i n s c o v a l e n t l y b i n d t o the t h i o l and  cell  The  immobilized  can be e l u t e d by r e d u c t i o n of the  bond w i t h an a p p r o p r i a t e r e d u c i n g  agent.  A 20 ml column of a c t i v a t e d Sepharose 4B was  used  S-S-  i n an attempt  the p r o t e a s e p r e s e n t i n the e t h a n o l  to  thiolimmobilize  precipitate.  P r o t e i n s not bound t o the Sepharose were e l u t e d w i t h deaerated one  PBS  c o n t a i n i n g 1 mM  Forty-  p e r c e n t of the p r o t e a s e added t o the column  d i d not b i n d ; i t had  the same s p e c i f i c  as the s t a r t i n g m a t e r i a l . the bound p r o t e a s e was HC1  EDTA.  i n PBS  (pH 7.0)  Approximately  activity 89%  of  e l u t e d w i t h 20 mM c y s t e i n e -  with a two-fold increase i n  163  specific activity  ( F i g . 21).  The  procedure  was not as e f f e c t i v e as g e l f i l t r a t i o n and t h e r e f o r e abandoned.  Both p r o t e a s e  was  fractions  behaved i n a s i m i l a r r e s p e c t w i t h r e g a r d t o inhibitors, gel electrophoresis, etc.  suggesting  t h a t t h e i r d i f f e r e n t b i n d i n g p r o p e r t i e s were due to s a t u r a t i o n of the column or masking of t h i o l group i n some of the f r a c t i o n s . Sepharose mercury (Hg) chromatography Chromatography on Sepharose c o n t a i n i n g immobilized Hg was attempted. cipitated  The e t h a n o l p r e -  sample was a p p l i e d t o a 30 ml column  of Hg-Sepharose and 44.7% of the p r o t e a s e d i d not b i n d . There was no p u r i f i c a t i o n of t h i s Eighty-two  percent of the bound p r o t e a s e  recovered with a 4-fold activity  ( F i g . 22).  p h o r e s i s i n SDS  fraction. was  increase i n s p e c i f i c  Polyacrylamide g e l e l e c t r o -  r e v e a l e d t h a t b o t h peaks c o n t a i n -  i n g p r o t e a s e were composed of s i m i l a r p r o t e i n s (Section  h-vi). The b i n d i n g of the p r o t e a s e t o both t h e  a c t i v a t e d t h i o l - S e p h a r o s e and the Sepharose mercury columns gave a d d i t i o n a l  indication  r e g a r d i n g the n a t u r e of the p r o t e a s e as a s u l f h y d r y l enzyme.  The p r o p o r t i o n of the p r o t e a s e  t h a t d i d not b i n d t o e i t h e r column might have  FIGURE 21.  Chromatography on t h i o l Sepharose-4B  5.8 ml sample o f e t h a n o l p r e c i p i t a t e (8.8 mg p r o t e i n ) was a p p l i e d to a 1.6 x 10 column.  Unbound p r o t e i n s were e l u t e d w i t h  deaerated  PBS c o n t a i n i n g 1 mM EDTA. The bound p r o t e a s e was e l u t e d w i t h c y s t e i n e - H C l i n PBS  20 mM  (pH 7.0).  F r a c t i o n s o f 1 ml were c o l l e c t e d a t a f l o w r a t e of 2.8 ml/hr. E l u t i o n o f p r o t e i n was monitored by measuring a b s o r p t i o n a t 280 nm. Enzymatic a c t i v i t y  was determined by the a z o c a s e i n  substrate.  165 Protease Units/ml  Ln  to  M  H  O  -l>-  Ui  to O  ON  CO  to Ul  U)  O  to  >  to CO o  J>-  FIGURE 22.  Sepharose mercury  The column was The e t h a n o l  chromatography  e q u i l i b r a t e d w i t h 50 mM  p r e c i p i t a t e d enzyme was  and the column was  acetate  b u f f e r pH  a p p l i e d i n 50 ml (170 mg  rinsed with acetate  buffer  ( 50 mM  f r a c t i o n s o f 2 ml were c o l l e c t e d a t a r a t e of 3 ml/hr. was  then e l u t e d w i t h 10 mM  cysteine i n acetate  buffer.  5.5. protein),  pH 5.5); Bound p r o t e i n  F r a c t i o n Number  168  been due  to o x i d a t i o n or b l o c k i n g o f t h e i r  cysteine moieties. In attempts  to s e p a r a t e c o n t a m i n a t i n g  p r o t e i n s from the p r o t e a s e complex, the procedures were a p p l i e d without  success as  judged by g e l e l e c t r o p h o r e s i s and by activity:  following  specific  G e l - f i l t r a t i o n through G-100  and  Sepharose 2B and 4B i n v a r i o u s b u f f e r s and presence of SDS  and/or u r e a at d i f f e r e n t  i n the  concent-  r a t i o n s , s e l e c t i v e heat d e n a t u r a t i o n and (NH^^SO^ p r e c i p i t a t i o n . graphy  Ion exchange chromato-  on CM-Sephadex C-50  and DEAE-Sephadex  A-50  under d i f f e r e n t c o n d i t i o n s o f b u f f e r s , pH and i n the presence of SDS  (0.1-0.2%) and/or u r e a  (2M-8M), were a l s o t r i e d  i n an e f f o r t  t o break  the p r o t e a s e complex i n the c e l l - e x t r a c t and  the  e t h a n o l p r e c i p i t a t e t o minimum f u n c t i o n a l components without s u c c e s s , s i n c e the p r o t e a s e a c t i v i t y was  p o o r l y r e c o v e r e d from a l a r g e number of  f r a c t i o n s without  significant  Hydrophobic was  purification.  i n t e r a c t i o n chromatography  used  t o b i n d the p r o t e a s e n o n - c o v a l e n t l y t o  an i n e r t  support i n phosphate b u f f e r c o n t a i n i n g  1 M NaCl.  Bound p r o t e i n s were e l u t e d  in a  20-50% g r a d i e n t o f e t h y l e n e g l y c o l i n PBS. poor r e c o v e r y r e s u l t e d significant  from t h i s column w i t h  purification.  A no  169  vi)  Gel e l e c t r o p h o r e s i s . Polyacrylamide gel e l e c t r o p h o r e s i s per- .  formed as d e s c r i b e d by Nagai et^ a l . (156) i n t r i s - g l y c i n e - S D S b u f f e r , pH 8.3,  i n a 10%  gel  demonstrated o n l y  t h r e e major bands and one minor band i n the p u r i f i e d p r o t e a s e o b t a i n e d from chromatography on Sepharose 2B (Fig.  23). Figure.24  r e p r e s e n t s the p o l y a c r y l a m i d e  gel electrophoresis i n T r i s - g l y c i n e different  SDS  b u f f e r of the  protease preparations obtained at  s t e p s of the p u r i f i c a t i o n scheme presented The  g e l r e v e a l e d the presence  bands i n the c e l l - e x t r a c t  and  different i n Table  of a t o t a l of f i f t e e n major t e n major bands i n the  c e n t r i f u g e d and d i a l y z e d e t h a n o l p r e c i p i t a t e d The  sample.  p r o t e a s e sample o b t a i n e d from g e l f i l t r a t i o n  Sephadex G-100  possessed  seven major bands w h i l e  p u r i f i e d p r o t e a s e possessed indicating  through the  o n l y f o u r bands, thereby  t h a t the p u r i f i c a t i o n procedure e l i m i n a t e d  most of the p r o t e i n f r a c t i o n s The  p r e s e n t i n the  cell-extract.  polyacrylamide gel e l e c t r o p h o r e s i s  i n T r i s - g l y c i n e - S D S b u f f e r of the crude as w e l l as partially purified different procedures  22.  enzyme p r e p a r a t i o n s throughout  chromatographic  the the  and v a r i o u s p u r i f i c a t i o n  are r e p r e s e n t e d i n F i g . 25.  o b t a i n e d from the v a r i o u s p u r i f i c a t i o n  The  samples  procedures  r e v e a l e d the removal of some major bands as compared t o  FIGURE 23.  Polyacrylamide g e l electrophoresis i n Tris-glycine-SDS b u f f e r o f the p u r i f i e d  protease.  A and B r e p r e s e n t F r a c t i o n s A and B e l u t e d from the Sepharose column w i t h 6 M u r e a .  171  A  B  FIGURE 24.  Polyacrylamide g e l electrophoresis i n b u f f e r of protease f r a c t i o n s obtained steps  i n the p u r i f i c a t i o n  Tris-glycine-SDS during  process.  Gel^., c e l l - e x t r a c t ; G e l ^ , d i a l y s e d  and c e n t r i f u g e d  Gel-j--j-j,ethanol-precipitated sample; G e l ^ , c e n t r i f u g e d sample; G e l ^ , d i a l y s e d  after gel f i l t r a t i o n  p u r i f i e d protease preparation  Sepharose-2B.  obtained  extract;  ethanol-treated sample;  through Sephadex G-100;  a f t e r f r a c t i o n a t i o n on  Two mg p r o t e i n of samples were s o l u b i l i z e d i n a s o l u b i l i z a t i o n  mixture c o n t a i n i n g bromophenol b l u e ; water b a t h .  cell  and c e n t r i f u g e d e t h a n o l - p r e c i p i t a t e d  G e l ^ . p r o t e a s e sample o b t a i n e d Gel  various  4% SDS,  M Tris  (pH 6.8)  and  0.01%  the samples were then b o i l e d f o r 2 min i n a b o i l i n g  Ten p e r c e n t  another minute.  20% g l y c e r o l , 0.125  (3ME was added and b o i l i n g was  continued f o r  173  •  3  ][  I I I I I IV V  VI  VII  FIGURE 25.  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of p r o t e a s e o b t a i n e d from the d i f f e r e n t p u r i f i c a t i o n  procedures.  Gel^. e t h a n o l p r e c i p i t a t e d sample; G e l ^ F r a c t i o n 1 from chromatography; G e l  fractions  thiol-Sepharose  F r a c t i o n 2 from the t h i o l - S e p h a r o s e column;  G e l ^ p r o t e a s e e l u t e d from O c t y l - S e p h a r o s e from the mercury Sepharose column; Gel^  CL-4B column; Gel^  Fraction 1  F r a c t i o n 2 from the mercury  Sepharose column; G e l ^ ^ p r o t e a s e f r a c t i o n e l u t e d from Sephadex w i t h 0.1% G-100  SDS  i n PBS;  e l u t e d w i t h 0.1%  Gel SDS  e l u t e d from Sephadex G-100  G-100  p r o t e a s e f r a c t i o n o b t a i n e d from Sephadex and  4 M urea i n PBS;  with 6 M urea; Gel  Gel crude  p r o t e a s e sample cell-extract;  A  Gel  p u r i f i e d protease preparation.  F r a c t i o n 1 r e f e r s to p r o t e a s e which  d i d not b i n d to the column, F r a c t i o n 2 r e f e r s to p r o t e a s e which bound to the column and was  e l u t e d w i t h r e d u c i n g agent.  20 y l samples, each  c o n t a i n i n g 20 yg p r o t e i n , were a p p l i e d to the g e l a f t e r b o i l i n g f o r 3 min i n 4% SDS, b l u e and  20% g l y c e r o l , 0.125  10%  (BME.  M T r i s b u f f e r (pH 6.8),  0.01%  bromophenol  175  I  II  I I I IV  V  VI V I I V I I I IX X  XI  176  the c e l l - e x t r a c t ( G e l x ) , however none o f these  procedures  y i e l d e d a p u r i f i c a t i o n comparable t o t h a t o b t a i n e d (Gel  XI) by the p u r i f i c a t i o n scheme presented The  samples were e l e c t r o p h o r e s e d towards  both t h e anode and cathode denaturation.  i n T a b l e 22.  i n t h e absence of SDS w i t h o u t  The samples were d i s s o l v e d  i z a t i o n m i x t u r e without  SDS and $ME.  in a solubil-  The SDS was a l s o  omitted from t h e r u n n i n g b u f f e r , t h e s t a c k i n g and r u n n i n g gels.  Twenty Ug p r o t e i n of each sample were c a r e f u l l y  l a y e r e d through  the T r i s - g l y c i n e b u f f e r onto t h e t o p o f  the upper g e l .  I n t h e crude and p a r t i a l l y  purified  enzyme p r e p a r a t i o n s , n o t a l l p r o t e i n s migrated spots as evidenced by t h e s t a i n a t these  from t h e  locations  ( F i g . 2 6 ) . Many bands a r e separated by ever d i s t a n c e s u g g e s t i n g an e x p o n e n t i a l d e c r e a s e  increasing i n molecular  s i z e and thus a l o s s of s i m i l a r s i z e s u b u n i t s .  No  m i g r a t i o n o f samples o c c u r r e d when t h e g e l was e l e c t r o phoresed  i n the o t h e r d i r e c t i o n .  p r e p a r a t i o n r e v e a l e d the presence d i d not migrate 7.5%  The p u r i f i e d  protease  of one major band t h a t  i n the polyacrylamide g e l .  The use o f  and 5% p o l y a c r y l a m i d e g e l s d i d n o t improve t h e  e l e c t r o p h o r e t i c m o b i l i t y of the p u r i f i e d  protease.  T h e r e f o r e , i t seemed p o s s i b l e t h a t t h e p u r i f i e d  protease  might s t i l l be bound i n a l a r g e m o l e c u l a r weight complex.  FIGURE 26.  Polyacrylamide gel electrophoresis b u f f e r without  The  gel depicts  in Tris-glycine  SDS.  the e l e c t r o p h o r e t i c p r o p e r t i e s  p r e s e n t i n the d i f f e r e n t p r o t e a s e p r e p a r a t i o n s cation.  Gel^., c e l l - e x t r a c t ; Gel  of the  obtained during  precipitated f r a c t i o n ; Gel^,centrifuged centrifuged  ethanol  ethanol  ,  ethanol-  precipitated fraction;  precipitated fraction;  Gely^j., p r o t e a s e f r a c t i o n e l u t e d from Sephadex G-100 Gel  purifi-  , dialysed cell-extract;  G e l ^ ^ j , d i a l y s e d c e l l - e x t r a c t a f t e r c e n t r i f u g a t i o n ; Gel  Gel^j. d i a l y s e d and  proteins  with 6 M  , F r a c t i o n A o f the p u r i f i e d p r o t e a s e p r e p a r a t i o n ;  urea.  Gel  V J. -L J.  Fraction B X A  of the p u r i f i e d p r o t e a s e p r e p a r a t i o n ;  Gel  30 ug p r o t e i n of the F r a c t i o n B  x Gel The  XJ_  40 ug p r o t e i n o f F r a c t i o n B;  samples were s o l u b i l i z e d i n 0.01%  g l y c e r o l and  0.125  M Tris buffer  g e l w i t h o u t b o i l i n g and  (pH  Gel  , 50 ug p r o t e i n of F r a c t i o n XX X.  bromophenol b l u e c o n t a i n e d 6.8).  There were a p p l i e d  i n the absence of SDS  o m i t t e d from the r u n n i n g b u f f e r ,  the s t a c k i n g  and  BME.  SDS  was  and  running g e l s .  in  to  20%  the  also  B.  I l l  I I I IV V V I V I I V I I I IX X X I  179  Polyacrylamide gel electrophoresis i n the absence of SDS revealed  with l i p o p r o t e i n prestained  t h a t the p u r i f i e d  s t a i n i n g m a t e r i a l which was whereas a standard  protease contained  samples lipoprotein  unable to p e n e t r a t e  l i p o p r o t e i n , as r e p r e s e n t e d  the  by  gel,  rabbit  plasma,did m i g r a t e ( F i g . 27). The  purified  p r o t e a s e i s a complex of a  number of p r o t e i n s as judged by the p o l y a c r y l a m i d e  gel unless  Detection a c r y l a m i d e g e l s was c a r b o c y a n i n e dye SDS  i t s i n a b i l i t y to  i t has  been b o i l e d i n  of g l y c o p r o t e i n s  accomplished  "Stains-all"  b u f f e r system (pH 7.4).  (110)  (SA)  layered  electrophoresis.  T h i s procedure was  on each of two  One  s t a i n e d w i t h SA.  presence of f o u r  capable  at the same time,  glycoproteins.  g e l was  (3+1)  p r o t e a s e (20 u g  g e l s and  f i x e d and  p r o t e i n s w i t h 0.2% Coomassie b l u e , and and  subjected  stained  Protein s t a i n i n g revealed p r o t e i n bands.  In the  p r o t e i n s s t a i n e d b l u e , and detected  glycoproteins. atically  the p r o t e i n s  i n F i g . 28.  fixed  the  single glyco-  . r e d , the  purified  as f o u r bands which a l l s t a i n e d  These r e s u l t s are r e p r e s e n t e d  to  for  the other was  s t a i n i n g procedure w i t h " S t a i n s - a l l " , where the  p r o t e a s e was  poly-  using a t r i s - a c e t a t e -  A sample of the p u r i f i e d p r o t e i n ) was  i n SDS  SDS.  by u s i n g a c a t i o n i c  of r e l i a b l y d e t e c t i n g the p r o t e i n s and, d i f f e r e n t i a t i n g the  enter  as  diagram-  I t can be concluded t h a t a l l f o u r  180  FIGURE 27.  Polyacrylamide electrophoresis stained f o r  Gel  , F r a c t i o n A of the p u r i f i e d p r o t e a s e ; Gel.  The  and  Gel  buffered  p r e s t a i n was  the absence of  SDS.  Standard g l y c o p r o t e i n  by 0.03  1 paper.  The  Sudan b l a c k  B (pH  hour b e f o r e  samples were a p p l i e d  of 20 yg p r o t e i n each and  of  samples  M T r i s - c i t r a t e buffer  f i r s t heated a t 60°C f o r one  through Whatman No.  the g e l i n a c o n c e n t r a t i o n in  IV  samples were s o l u b i l i z e d i n 0.1%  i n ethylene g l y c o l p r e s t a i n buffered  filtration  Fraction B  II'  of r a b b i t plasma.  B  lipids.  the p u r i f i e d p r o t e a s e ; Gel. Ill  The  of f r a c t i o n s A and  to  electrophoresed  9.0).  181  I  II  III  IV  FIGURE 28.  Diagram of g l y c o p r o t e i n  and  p r o t e i n bands on  g e l s f o l l o w i n g e l e c t r o p h o r e s i s of p u r i f i e d  Comparison o f s t a i n i n g p a t t e r n s by  "Stains-all" (pH  7.4)  (SA)  Coomassie b l u e  s t a i n i n g a r e compared i n 10%  polyacrylamide  protease.  of p u r i f i e d p r o t e a s e  SDS-polyacrylamide g e l e l e c t r o p h o r e s i s .  slab  separated (CB)  and  Tris-acetate buffered  g e l e l e c t r o p h o r e s i s system c o n t a i n i n g 0.1%  SDS.  183  II  184  major bands which r e p r e s e n t the p u r i f i e d  protease  preparations are g l y c o p r o t e i n s . i.  C h a r a c t e r i z a t i o n of the p u r i f i e d i)  Chemical  protease.  composition of the p u r i f i e d  protease  T a b l e 23 i n d i c a t e s the c h e m i c a l t i o n of the p u r i f i e d p r o t e a s e .  The p r o t e i n c o n t e n t of  the p u r i f i e d p r o t e a s e p r e p a r a t i o n was Lowry method (132).  composi-  determined  D u p l i c a t e samples of the  s o l u t i o n of g l u c o s e as a s t a n d a r d . of  the p u r i f i e d p r e p a r a t i o n was  The  the  equimolar  lipid  determined  the  purified  p r o t e a s e p r e p a r a t i o n were a n a l y z e d f o r hexose by o r c i n o l - s u l f u r i c a c i d method (223), w i t h an  by  by  content the  micro-method of Pande and P a r v i n (165) which d e t e c t s l i p i d s i n the c o n c e n t r a t i o n range of 20-140 yg. f r e e and o r g a n i c phosphorous was m i c r o - d e t e r m i n a t i o n procedure  determined  by  Total the  d e s c r i b e d by Chen et^ a l .  (29). The  c h e m i c a l a n a l y s i s of the  purified  p r o t e a s e p r e p a r a t i o n demonstrated the presence carbohydrate,  lipid  No phosphate was  of  and p r o t e i n as major c o n s t i t u e n t s .  d e t e c t e d i n t o t a l sample and  i n the  l i p i d e x t r a c t from the enzyme p r e p a r a t i o n . ii)  Gas-liquid Gas  chromatography  chromatographic  a n a l y s i s (171)  n e u t r a l sugars and hexosamines p r e s e n t i n the p r o t e a s e r e v e a l e d the presence  of g l u c o s e  and  of  purified  00  Table 23.  F i n a l Protease Preparation  mg p r o t e i n mg dry weight  %  Chemical Composition of the P u r i f i e d  mg carbohydrates /mg dry weight  %  Protease Preparation  mg l i p i d s dry weight  %  Phosphates i n t o t a l Sample and l i p i d e x t r a c t s .  Fraction A  2.5  79.4  0.25  8  0.35  11  0  Fraction B  3.1  75  0.24  6  0.79  19  0  186  glucosamine i n a d d i t i o n The  to two u n i d e n t i f i e d  fatty acid  c o m p o s i t i o n of the l i p i d  e x t r a c t s o f the p u r i f i e d p r o t e a s e was a l s o The  sugars.  studied.  l i p i d s were e x t r a c t e d from the p u r i f i e d  preparation into  c h l o r o f o r m and methanol.  protease No phospho-  l i p i d s were d e t e c t e d u s i n g the t w o - d i r e c t i o n a l  thin-layer  chromatographic method d e s c r i b e d by Y a v i n (240). chromatographic a n a l y s i s  o f the n e u t r a l l i p i d s i n  f r a c t i o n A and B a r e p r e s e n t e d i n T a b l e 24.  The p r e -  dominant f a t t y a c i d s i n f r a c t i o n A were l i n o l e i c , palmitoleic  Gas  and a r a c h i d o n i c .  I n f r a c t i o n B,  s t e a r i c and o l e i c a c i d predominated.  stearic,  palmitic,  In a d d i t i o n ,  a  number of compounds which were not i d e n t i f i e d b u t which may be c y c l i c o r odd c h a i n f a t t y a c i d s  (142) were  also  found. iii)  S t a b i l i t y o f the p u r i f i e d The  stable  protease  p u r i f i e d enzyme was found to be  than the crude p r o t e a s e .  a t room temperature r e s u l t e d  E i g h t hour  less  incubation  i n about 40% l o s s o f a c t i -  v i t y , w h i l e i n c u b a t i o n a t 4°C f o r 2 days r e s u l t e d i n 70%  loss.  Boiling  loss of 100%  the enzyme f o r 1 min r e s u l t e d i n  of the p r o t e o l y t i c  activity.  Freezing  a t -20°C and thawing a t room temperature r e s u l t e d i n 24-35% l o s s o f a c t i v i t y of the p u r i f i e d p r o t e a s e p r e p a r a tion.  F i v e to ten p e r c e n t of the p r o t e o l y t i c  a c t i v i t y was  l o s t when the p r e p a r a t i o n was s t o r e d a t -70°C f o r two weeks.  187  T a b l e 24.  Gas-liquid acids  chromatographic a n a l y s i s o f f a t t y  i n the p u r i f i e d  enzyme  preparation.  Percentages o f t o t a l f a t t y a c i d measured Fatty  Acid  Fraction A  Fraction B  C-16  2  17  C-16:l  8  6  15  . 15  C-18:l  2  5  C-18:2  17  -  C-20:4  12  -  C-18  The percentage o f c o m p o s i t i o n o f f a t t y a c i d s was determined by comparing the a r e a i n mm  2  o f each peak to t h a t o f the t o t a l sample.  188  Longer s t o r a g e p e r i o d s r e s u l t e d  i n progressively greater  losses. iv)  pH optimum o f the p u r i f i e d The  protease  e f f e c t of pH on the a c t i v i t y of the  p u r i f i e d p r o t e a s e was determined  by t h e a z o c a s e i n method  i n v a r i o u s b u f f e r s o f 0.1 M i o n i c s t r e n g t h . F i g . 29, the p u r i f i e d  As shown i n  p r o t e a s e was a c t i v e over a wide pH  range w i t h a pH optimum of 7.0. The e f f e c t of d i f f e r e n t b u f f e r s on t h e p r o t e o l y s i s as measured i n the a z o c a s e i n assay t h a t a c t i v i t y i n T r i s b u f f e r was reduced PBS, HEPES  indicated  25% compared to  (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic  a c i d ) , EPPS ( N - 2 - h y d r o x y e t h y l p i p e r a z i n e propane s u l f o n i c a c i d ) , MOPS (morpholinopropane buffer.  T h e r e f o r e , i n cases where the phosphate i o n s had  t o be excluded  from the p u r i f i e d  EPPS b u f f e r was used v)  protease preparation,  i n s t e a d of PBS.  M o d i f i c a t i o n of protease The  were undertaken  activity  r e s u l t s of a number of experiments  which  t o show the e f f e c t s o f v a r i o u s r e a g e n t s  on the a c t i v i t y o f p u r i f i e d The  s u l f o n i c a c i d ) and g l y c i n e  p r o t e a s e a r e shown i n T a b l e 25.  s u b s t r a t e f o r a l l the r e a c t i o n s was  type of i n h i b i t o r  azocasein.  f r e q u e n t l y p r o v i d e s an i n s i g h t  a c t i v e f u n c t i o n a l group of the enzyme. p r o t e a s e was i n h i b i t e d by H g C ^  The  i n t o the  The p u r i f i e d  and a l k y l a t i n g agents  as i o d o a c e t i c a c i d and iodoacetamide  ( F i g . 30,31).  such  The  FIGURE 29.  E f f e c t of pH on  the p u r i f i e d p r o t e a s e  Symbols: ( t ) 0.1  M phosphate b u f f e r , ( A )  ( 0 ) 0.1  M g l y c i n e - NaOH b u f f e r  P u r i f i e d p r o t e a s e p r e p a r a t i o n (0.09 0.75  activity.  mg  p r o t e i n ) was  ml of the a p p r o p r i a t e b u f f e r c o n t a i n i n g 50 mM  0.1  M Tris-HCl buff  incubated  (3ME f o r 15 min  37°C; 1 ml o f a z o c a s e i n  (2%) was  i n c u b a t i o n , 2 ml of 10%  TCA were added and p r e c i p i t a t e d a z o c a s e i n  f i l t e r e d and mixtures  was  added to each tube, and  of the f i l t r a t e was checked and  found  measured.  The  to be as i n d i c a t e d .  a f t e r one  with at hr was  pH of the r e a c t i o n  190  191  T a b l e 25.  M o d i f i c a t i o n o f the p u r i f i e d  protease  P u r i f i e d enzyme (0.09 mg p r o t e i n / m l ) c o n c e n t r a t e d and d i a l y s e d o v e r n i g h t a g a i n s t PBS  (pH 7.2) was i n c u b a t e d a t room temperature  the i n h i b i t o r f o r 30 min. and c o n t r o l s  containing  a c i d and iodoacetamide  with  PMSF and TPCK were d i s s o l v e d i n 1-propanol  o n l y 1-propanol  were r u n .  CaC^,  iodoacetic  were d i s s o l v e d i n T r i s - H C l b u f f e r pH 7.2.  HgC^,  EDTA, urea, SDS, guanidine HC1, l i t h i u m c h l o r i d e and NaCl were d i s s o l v e d i n phosphate b u f f e r pH 7.0.  I n h i b i t i o n of a c t i v i t y was expressed  to the c o n t r o l i n c u b a t e d i n the same b u f f e r w i t h o u t  reagent.  relative  T a b l e 25.  Reagent  Mercuric chloride  M o d i f i c a t i o n o f the p u r i f i e d p r o t e a s e  Concentration  0.01 mM 3 mM  % I n h i b i t i o n of Protease A c t i v i t y  20 100  Iodoacetic acid  10 50  mM mM  28 55  Iodoacetamide  10  mM  AO  50  mM  82  PMSF  10  mM  0  TPCK  10  mM  0  EDTA  10  mM  0  Urea  8  mM  0  SDS  0.1% 0.3%  0 65  Guanidine HC1  0.5 M 4 M  20.5 66.7  Lithium chloride  0.5 M  38.5  3  74.4  M  Acetone  100%  80  Butanol  100%  100  Phenol  100%  100  Chloroform  100%  100  T r i t o n X-100  0.5%  100  20% 50% 5% 10%  0 15 0 5  Ethylene g l y c o l DMSO Sodium c h l o r i d e  1 3  M M  Calcium c h l o r i d e  0.1 M 0.5 M  5 50 0 0  193  FIGURE 30.  HgCl  2  i n h i b i t i o n o f the p u r i f i e d  protease  Enzyme (0.09 mg p r o t e i n ) was i n c u b a t e d a t 37°C i n the a n a e r o b i c chamber w i t h i n d i c a t e d c o n c e n t r a t i o n s of HgCL^ i n PBS pH 7.0 f o r 30 min and then assayed w i t h a z o c a s e i n as s u b s t r a t e .  Percent  ho o  o  Inhibition  o  oo o  o o  FIGURE 31.  I n h i b i t i o n o f the p u r i f i e d p r o t e a s e by  iodoacetamide.  Enzyme p r e p a r a t i o n (0.09 mg p r o t e i n ) was i n c u b a t e d  f o r 30 min a t  room temperature w i t h T r i s - H C l b u f f e r (pH 8.2) c o n t a i n i n g the a p p r o p r i a t e c o n c e n t r a t i o n o f the reagent w i t h 50 mM BME.  One ml o f  2% a z o c a s e i n was used as a s u b s t r a t e and the r e a c t i o n m i x t u r e incubated  f o r 1 h r a t 37°C.  was  P e r c e n t Remaining A c t i v i t y  I—  NJ  CO  J>-  Ln  o  o  o  o  o  1  o  ON  o  VO ON  197  metal c h e l a t i n g agent EDTA and had  no  the  serine  inhibitors  i n h i b i t o r y e f f e c t on the p r o t e a s e .  of these f i n d i n g s , the p r o t e a s e c o u l d be c l a s s i f i e d  On  the  of B_. m e l a n i n o g e n i c u s  as a s u l f h y d r y l enzyme.  Guanidine  HC1,which g e n e r a l l y uncouples p o l a r bonds and p r o t e i n s caused up activity.  The  to 66.7%  denatures  i n h i b i t i o n of the p r o t e o l y t i c  i n h i b i t o r y e f f e c t might a l s o . b e  i n f l u e n c e of i o n i c s t r e n g t h on the enzymatic L i t h i u m c h l o r i d e at 3 M c o n c e n t r a t i o n of the p r o t e o l y t i c a c t i v i t y  basis  due  activity.  inhibited  ( F i g . 32).  to  74.4%  Phenol and  chloro-  form e x t r a c t i o n as w e l l as treatment w i t h b u t a n o l , T r i t o n X-100  and  protease  acetone caused almost complete i n h i b i t i o n of but  t h i s was  probably  However, the p o l a r o r g a n i c (DMSO) and  ethylene  due  to p r o t e i n  solvents dimethyl  g l y c o l had  little  Urea (8M)  not  but 8 M u r e a had  i n h i b i t the p r o t e a s e ;  effect.  The  denaturation  and  reason f o r t h i s c o u l d be  denaturation. sulfoxide  inhibitory  on the enzyme a c t i v i t y .  0.1% a  the  SDS  effect did  stimulatory  t h a t u r e a caused  of the p r o t e i n complexes t h a t c o u l d mask  some of the a c t i v e s i t e s of the enzyme.  It i s also  p o s s i b l e t h a t h i g h c o n c e n t r a t i o n s of u r e a might modify s u b s t r a t e by making i t more a c c e s s i b l e to the  the  protease  enzyme. The enzymatic a c t i v i t y  i n f l u e n c e of i o n i c s t r e n g t h on is illustrated  presence of NaCl at c o n c e n t r a t i o n s  i n F i g . 32. above 0.5  the  The M decreased  FIGURE 32.  E f f e c t o f g u a n i d i n e h y d r o c h l o r i d e , l i t h i u m c h l o r i d e and NaCl on the p u r i f i e d  protease.  Enzyme p r e p a r a t i o n (0.09 mg p r o t e i n ) was i n c u b a t e d w i t h 0.75 ml of the a p p r o p r i a t e c o n c e n t r a t i o n o f the reagent i n phosphate b u f f e r (pH 7.0) c o n t a i n i n g 50 mM  BME f o r 30 min a t 37°C; 1 ml a z o c a s e i n (2%)  was added and the r e a c t i o n m i x t u r e was i n c u b a t e d  f o r 1 hr.  199  200  the enzyme a c t i v i t y . The to  p u r i f i e d p r o t e a s e was v e r y  HgCl-2 s i n c e low c o n c e n t r a t i o n of the i n h i b i t o r  complete l o s s o f a c t i v i t y .  percent  caused  The i n h i b i t i o n c o u l d be  r e v e r s e d by r e d u c i n g agents.  al  sensitive  As shown i n F i g . 30, the  i n h i b i t i o n o f p r o t e o l y t i c a c t i v i t y was p r o p o r t i o n -  t o t h e c o n c e n t r a t i o n o f t h e HgCl^. The  f u n c t i o n of the carbohydrate  components i n t h e p u r i f i e d  and l i p i d  p r o t e a s e was i n v e s t i g a t e d by  t e s t i n g t h e e f f e c t o f mixed g l y c o s i d a s e s on t h e a c t i v i t y of  the p u r i f i e d  p r o t e a s e , and by e x t r a c t i n g t h e l i p i d  the p u r i f i e d p r o t e a s e or  p r e p a r a t i o n w i t h phenol,  from  chloroform  butanol. E x t r a c t i o n of t h e l i p i d s  from t h e p u r i f i e d  p r o t e a s e r e s u l t e d i n complete l o s s of a c t i v i t y , but t h i s e f f e c t might have been due t o d e n a t u r a t i o n of the p r o t e i n by t h e o r g a n i c s o l v e n t s used i n t h e e x t r a c t i o n .  Restor-  a t i o n of the p r o t e o l y t i c a c t i v i t y with the d i a l y z e d e x t r a c t e d l i p i d d i a l y z e d a g a i n s t PBS was u n s u c c e s s f u l . Incubation  of the p u r i f i e d  protease  with  mixed g l y c o s i d a s e s f o r 10 h r a t 37°C caused 43% i n h i b i t i o n of  the p r o t e a s e  activity,  suggesting  that the carbohydrate  p a r t o f the p r o t e a s e might be e s s e n t i a l or r e q u i r e d f o r activity.  201  vi)  S u b s t r a t e s p e c i f i c i t y of the p u r i f i e d The  purified  protease  p r o t e a s e was a c t i v e a g a i n s t  a z o c a s e i n , c a s e i n , N , N - d i m e t h y l c a s e i n and a z o c o l l as substrates.  No a c t i v i t y c o u l d be demonstrated  hemoglobin, bovine  against  serum albumin, or C - c o l l a g e n .  The  1 4  p u r i f i e d protease d i d not hydrolyze the s y n t h e t i c s u b s t r a t e s TAME, t o s y l a r g i n i n e methyl e s t e r ; BAEE, benzoylarginine ethyl ester  o r ATEE, a c e t y l t y r o s i n e e t h y l  ester. An  i d e n t i c a l s p e c i f i c i t y was e x h i b i t e d by  the enzyme from t h e c e l l - e x t r a c t and the p r e p a r a t i o n obtained  a f t e r e t h a n o l p r e c i p i t a t i o n as w e l l as o t h e r  preparationsobtained suggesting  through the p u r i f i c a t i o n  procedure,  t h a t a s i n g l e p r o t e o l y t i c enzyme was produced  by t h e organism. The  purified  p r e p a r a t i o n had no l i p a s e ,  a o r B - g l y c o s i d a s e , c o l l a g e n a s e or a c t i v i t y when assayed vii)  hemagglutinating  as d e s c r i b e d i n Methods.  P a t h o l o g i c a l a c t i v i t y of t h e p u r i f i e d A concentrated  purified  protease  preparation of the  p r o t e a s e c o n t a i n i n g 2 mg p r o t e i n / m l was d i a l y z e d a g a i n s t PBS and i n j e c t e d guinea to  subcutaneously  p i g u s i n g 0.5 ml.  i n t o the g r o i n of a 200 g  The animal was observed  4 weeks f o r t h e presence o f a b s c e s s .  The p u r i f i e d  p r o t e a s e had no b i o l o g i c a l a c t i v i t y by i t s e l f . protease  preparation to a c e l l  suspension  f o r up  Adding t h e  o f 2D t h a t caused  202  a t y p i c a l i n f e c t i o n i n the guinea p i g model system resulted  in a slight  i n c r e a s e i n the n e c r o s i s of  the  l e s i o n when compared to the n e c r o t i c l e s i o n s caused 2D  c e l l s without a d d i t i o n of the p r o t e a s e  Whether the p u r i f i e d for  protease  was  by  preparation.  the f a c t o r r e s p o n s i b l e  t h i s more pronounced i n f e c t i o n , or  experimental  v a r i a t i o n s which are common i n these k i n d of experiments, c o u l d not be  verified. When the p u r i f i e d  was  tested for  vascular permeability  f o l l o w i n g the method of C r a i g  no b l u e i n g e f f e c t was  observed.  did  The  purified  c u l t u r e supernatant of  S o l u b l e P r o t e a s e of 15. melaninogenicus  cell-bound  protease(s)  are  the same o r d i f f e r e n t  cell-  entities  by comparison of t h e i r pH optima, e f f e c t s of i n h i b i t o r s and during  con-  2D.  S t u d i e s were c a r r i e d out to i n v e s t i g a t e whether the f r e e and  (36),  protease  not enhance the b l u e i n g e f f e c t produced by the  centrated '4.  protease  properties  chromatography. a.  Demonstration of an e x t r a c e l l u l a r p r o t e a s e . P r o t e o l y t i c a c t i v i t y against a z o c o l l i n c u l t u r e  s u p e r n a t a n t s of Ii. melaninogenicus s t r a i n 2D was i n the e x p o n e n t i a l  growth phase ( F i g . 15), and  increase i n proportion with  detected  was  found to  i n c r e a s i n g c e l l numbers u n t i l mid-  l a t e s t a t i o n a r y phase when a f u r t h e r i n c r e a s e i n a c t i v i t y noted which continued  early  as the c e l l s began to l y s e .  was  to  203  Preliminary c h a r a c t e r i z a t i o n of the e x t r a c e l l u l a r protease. i)  Oxygen  sensitivity The  supernatant  s o l u b l e protease i n the c u l t u r e  was i n a c t i v a t e d by oxygen.  A definite i n -  c r e a s e i n the r a t e o f dye r e l e a s e d from a z o c o l l  occurred  when 0.01 M c y s t e i n e o r 25 mM gME was i n c l u d e d i n t h e r e a c t i o n mixture. the supernatant with  A c t i v i t y a g a i n s t a z o c o l l was l o s t when  was a e r a t e d f o r 20 min, b u t was r e s t o r e d  the r e d u c i n g agent. ii.  S t a b i l i t y o f the p r o t e a s e The  s t a b i l i t y of t h e p r o t e a s e  rated c u l t u r e supernatants  i n concent-  was examined by s t o r i n g t h e  enzyme a t -70°C, -20°C, 4°C, 20°C and 37°C w i t h and without  r e d u c i n g agent and a s s a y i n g p e r i o d i c a l l y f o r  protease a c t i v i t y .  S o l u b l e p r o t e a s e was found  t o be more  s t a b l e i n t h e absence o f the r e d u c i n g agent when s t o r e d o r incubated a t any of the above s t a t e d  temperatures.  T h i s may be due t o i n c r e a s e d r a t e s of a u t o d i g e s t i o n i n the presence activity.  o f t h e r e d u c i n g agents needed f o r p r o t e o l y t i c  Storage  o f t h e p r o t e a s e a t -70°C was found t o  be more e f f e c t i v e than a t -20°C where some of t h e a c t i v i t y was l o s t even i n t h e absence of BME. The  p r o t e a s e was s t a b l e when i n c u b a t e d a t  room temperature o n l y f o r a p e r i o d of 3 t o 4 h r . I n c u b a t i o n a t 37°C r e s u l t e d  i n a greater l o s s of the  204  p r o t e a s e a c t i v i t y , e s p e c i a l l y i n the presence I t was  found  t o be s t a b l e f o r 8 h r a t 4°C.  c u l t u r e supernatant  for 5  min r e s u l t e d  of  3ME.  Boiling  the  i n complete l o s s  j  of protease iii.  activity. E f f e c t of pH on p r o t e a s e P r o t e a s e a c t i v i t y was  range of 4.0 4.0  t o 5.5,  t o 9.0.  activity measured over a  A c e t a t e b u f f e r s were used  phosphate b u f f e r s from pH 6.0  T r i s - H C l b u f f e r s from pH 7.0  t o 9.0  from  t o 7.5  pH pH  and  ( F i g . 33).  The  optimum pH f o r p r o t e a s e a c t i v i t y a g a i n s t a z o c a s e i n was  between 7.0  a slight  and 7.5.  T r i s - H C l b u f f e r caused  i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y  when used  i n the assay m i x t u r e  (15-20%)  as compared to the  phosphate b u f f e r . iv)  I n h i b i t i o n of s o l u b l e p r o t e a s e by v a r i o u s reagents T a b l e 26 summarizes the e f f e c t of a number  of i n h i b i t o r s of p r o t e o l y t i c enzymes on the s o l u b l e p r o t e a s e o f I5_. m e l a n i n o g e n i c u s . completely  The  s o l u b l e protease  i n h i b i t e d by m e r c u r i c c h l o r i d e , the  was  inhibition  c o u l d be r e v e r s e d by a d d i t i o n of the Hg b i n d i n g reagent BME.  The  acetamide.  s u l f h y d r y l reagents  i o d o a c e t i c a c i d and  i n h i b i t e d the p r o t e a s e a c t i v i t y .  Sulfhydryl  groups a r e a p p a r e n t l y r e q u i r e d f o r p r o t e o l y t i c Phenyl methyl s u l f o n y l f l u o r i d e  iodo-  activity.  (PMSF) and T o s y l - p h e n y l -  e t h y l - c h l o r o m e t h y l ketone (TPCK), which  inhibit  FIGURE 33.  The by  pH optimum of the s o l u b l e  e f f e c t of pH on the s o l u b l e p r o t e a s e a c t i v i t y was determined  the a z o c a s e i n  ( • ) acetate Dialyzed  protease.  method i n v a r i o u s  buffer,  (A)  c u l t u r e supernatant  f o r 1 h r a t 37°C.  b u f f e r s of 0.1 M i o n i c  phosphate b u f f e r ,  ( 0 ) Tris  strength. buffer.  (1.5 mg) was i n c u b a t e d w i t h the s u b s t r a t e  Absorbance (370 nm)  207  T a b l e 26.  I n h i b i t i o n of soluble  protease  P r o t e a s e was i n c u b a t e d a t 37°C f o r 30 min w i t h each of the compounds b e f o r e measuring p r o t e o l y t i c a c t i v i t y a g a i n s t p r o t e a s e a c t i v i t y o f unmodified presence  casein.  c u l t u r e supernatant was assayed  i n the  and absence o f BME i n same b u f f e r s and the r e s u l t s a r e  expressed as % i n h i b i t i o n o f p r o t e a s e a c t i v i t y of the c o n t r o l PMSF and TPCK were d i s s o l v e d  i n 1-propanol;  PBS (pH 7.0) and EDTA and a l k y l a t i n g agents  Inhibitors  HgCl  The  Concentration (nM)  reactions.  HgC^was d i s s o l v e d i n i n 0.1 M T r i s - H C l  (pH 8.0).  % Inhibition  2 5  100 100  2 5  10 75  PMSF  1 10  0 0  TPCK  1 10  2 4  EDTA  1 10 50  5 15 60  1 16  24.2 40.5  1 16  60 80.7  2  HgCl +BME 2  Iodoacetic  acid  Iodoacetamide  208  p r o t e a s e s r e q u i r i n g s e r i n e a t t h e a c t i v e s i t e , had no e f f e c t on t h e s o l u b l e p r o t e a s e .  Incubation with the  m e t a l c h e l a t o r EDTA i n h i b i t e d t h e e x t r a c e l l u l a r by c.  protease  5-60%.  P a r t i a l p u r i f i c a t i o n o f the s o l u b l e p r o t e a s e . i)  D i a l y s i s of culture  supernatant  The d i a l y s i s o f c u l t u r e o v e r n i g h t a t 4°C a g a i n s t PBS r e s u l t e d  supernatant i n 2.1 f o l d -  p u r i f i c a t i o n most p r o b a b l y due t o l o s s o f d i a l y z a b l e p e p t i d e s ; and 6% l o s s o f p r o t e o l y t i c a c t i v i t y o c c u r r e d (Table 27). ii)  Concentration of c u l t u r e  supernatant  Due t o t h e o b s e r v a t i o n t h a t t h e s o l u b l e p r o t e a s e was u n s t a b l e , a v a r i e t y o f methods f o r c o n c e n t r a t i n g t h e p r o t e a s e were t r i e d  i n order t o f i n d t h e  method which would l e a d t o t h e l e a s t  inactivation.  F r e e z e - d r y i n g was found t o be t h e most e f f e c t i v e c e n t r a t i n g procedure  p r o v i d e d t h a t t h e glassware was  coated w i t h S i l a n e (Bio-Rad). procedure  t h a t proved  Another c o n c e n t r a t i o n  e f f e c t i v e was t o remove water by  blowing a i r over t h e supernatant tubing.  con-  contained i n d i a l y s i s  E v a p o r a t i o n phenomena kept t h e temperature o f  the enzyme s o l u t i o n low and r e s u l t e d of t h e p r o t e a s e . ultrafiltration  i n less  inactivation  Ammonium s u l f a t e p r e c i p i t a t i o n and (Amicon PM-30 and XM-50 membranes)  not e f f e c t i v e procedures  were  f o r c o n c e n t r a t i n g t h e enzyme.  209  T a b l e 27.  Partial purification extracellular  Procedure  of JB_. melaninogenicus  protease  T o t a l Protease Units  Protease s p e c i f i c activity units/mg p r o t e i n  Yield  Purification Factor  Culture f i l t r a t e  72  2.8  100  Dialysis  67.7  5.8  94  2.1  Freeze-drying  51.0  3.4  70.84  1.2  Sephadex G-100 gel f i l t r a t i o n  32.8  35.4  1  D i a l y s i s was a g a i n s t 10 volumes o f PBS f o r o v e r n i g h t .  45.6  12.6  210  iii)  Purification  procedures  C u l t u r e supernatant of 2D was a DEAE-Sephadex A-50 0.05  M NaCl  column packed  i n phosphate b u f f e r .  applied to  and washed w i t h The  p r o t e a s e was  to the DEAE-Sephadex and o n l y 20% of the a c t i v i t y recovered. gradient. the s a l t  T h i s ; was  e l u t e d w i t h a 0.05-2 N  teolytic activity. achieved.  No  significant  pro-  was  at f r a c t i o n a t i o n of the  c o n c e n t r a t e d supernatant r e s u l t e d  i n v e r y low r e c o v e r y .  When a sample of c o n c e n t r a t e d 48 hr c u l t u r e was  with  substantial purification  F u r t h e r attempts  was  NaCl  S e v e r a l peaks of p r o t e i n were e l u t e d g r a d i e n t , but none possessed  bound  chromatographed on a CM-Sephadex C-50  supernatant  and  G-200 columns, a c t i v i t y a g a i n s t a z o c o l l was many f r a c t i o n s , s u g g e s t i n g t h a t the p r o t e a s e  Sephadex  detected i n activity  might be a s s o c i a t e d w i t h another c o n s t i t u e n t of the supernatant. to be  binding  G e n e r a l l y , the p r o t e a s e a c t i v i t y  appeared  n o n s p e c i f i c a l l y t o the columns w i t h  s i d e r a b l e v a r i a t i o n among the e l u t i o n p a t t e r n s of ent samples. i t may  condiffer-  The reason f o r t h i s i s not known a l t h o u g h  have been due t o p r o t e o l y t i c a c t i o n on some o f  the p r o t e i n components of the s u p e r n a t a n t . p o s s i b l e t h a t t h e p r o t e a s e may  It i s also  have been a s s o c i a t e d w i t h  a component of the c e l l w a l l , which would r e s u l t a s s o c i a t i o n of the enzyme w i t h random s i z e d and fragments.  i n the charged  211  iv.)  G e l f i l t r a t i o n on Sephadex A Sephadex G-100  i b r a t e d and  e l u t e d w i t h PBS  soluble protease. supernatant was  The  was  G-100  column packed, used  to f r a c t i o n a t e  as a s i n g l e peak immediately  column  a f t e r the v o i d volume, as  T h i s i n d i c a t e d an approximate m o l e c u l a r  weight of 100,000 or more.  64.4%  of the  a c t i v i t y a p p l i e d t o the column was  proteolytic  recovered,- s u g g e s t i n g  the p o s s i b i l i t y t h a t some of the p r o t e a s e was i n a c t i v a t e d or s t i l l steps r e s u l t e d  the  p r o t e a s e a c t i v i t y i n the c u l t u r e  e l u t e d from the Sephadex G-100  shown i n F i g . 34.  equil-  on the column.  i n an a p p r o x i m a t e l y  The 12.6  either  purification fold  increase i n  s p e c i f i c a c t i v i t y over the s t a r t i n g m a t e r i a l w i t h a t o t a l 46% r e c o v e r y ( T a b l e 27). G e l e l e c t r o p h o r e s i s of the  partially  purified  p r o t e a s e r e v e a l e d many p r o t e i n s and  attempts  t o p u r i f y the p r o t e a s e were u n s u c c e s s f u l .  partially purified  p r o t e a s e was  shown t o be a  enzyme h a v i n g the same c h a r a c t e r i s t i c s as the p r o t e a s e i n the c u l t u r e I t was p r o t e a s e was  further The  sulfhydryl crude  supernatant.  determined  t h a t the  extracellular  l a r g e , p o s s i b l y membrane or p a r t i c l e bound.  T h e r e f o r e , t h e r e would be no advantage i n a t t e m p t i n g  to  p u r i f y i t any f u r t h e r , as s i m i l a r m a t e r i a l c o u l d more c o n v e n i e n t l y be prepared  from e x p o n e n t i a l phase c e l l s .  212  FIGURE 34.  Sephadex G-100  g e l f i l t r a t i o n of the s o l u b l e  A 59 x 1.6 cm column was used a t 5.5 ml/hr f l o w r a t e . v o i d volume of the column was determined  w i t h Blue Dextran  protease.  The 2000,  and a sample of 18.2 mg p r o t e i n of d i a l y z e d c u l t u r e supernatant applied  to the column and 1.8 ml f r a c t i o n s were c o l l e c t e d .  d e n s i t y was  f o l l o w e d a t 280 nm f o r p r o t e i n d e t e r m i n a t i o n .  was assayed by the a z o c o l l a s s a y .  was  The o p t i c a l The p r o t e a s e  213 Protease units/ml  P>  n  rt H-  O 3  a i fD  A  280  214  The r e s u l t s i n d i c a t e t h a t the s o l u b l e p r o t e a s e was  s i m i l a r t o the c e l l - b o u n d enzyme w i t h  r e s p e c t t o pH optima, s p e c i f i c i t y and  response  p r o p e r t i e s d u r i n g chromatography.  f o r e , i t can be c o n c l u d e d " t h a t produced  from the  cells.  There-  o n l y one major p r o t e a s e  by the s t r a i n 2D of 15. melaninogenicus  the e x t r a c e l l u l a r p r o t e a s e was leakage  to i n h i b i t o r s , substrate  p r o b a b l y due  and  was  that  to l y s i s or  215  IV.  DISCUSSION  melaninogenicus has been i m p l i c a t e d a n a e r o b i c i n f e c t i o n s i n man  i n the p a t h o g e n e s i s of mixed  (4,25,57,231) and i n the development  experimental anaerobic i n f e c t i o n s  (137,205).  of  I t i s known t o p o s s e s s  a t t r i b u t e s which would i m p l i c a t e i t i n human p e r i o d o n t a l d i s e a s e  (138,139).  T h e r e f o r e , the organism has a t t r a c t e d the i n t e r e s t of a number of i n v e s t i gators  throughout the y e a r s .  However, d e t a i l e d  investigations concerning  the b i o c h e m i c a l and pathogenic c a p a c i t i e s of the organism have been hampered by d i f f i c u l t i e s  i n growing many s t r a i n s i n pure c u l t u r e and by  inadequate taxonomy (26,54).  These  f a c t s have been r e s p o n s i b l e , a t l e a s t  i n p a r t , f o r the c o n f l i c t i n g r e s u l t s i n the  literature.  A study o f the p r o t e o l y t i c and h e m a g g l u t i n a t i n g a c t i v i t y o f t h e organisms might p r o v i d e i n s i g h t i n t o the n a t u r e of t h e i r p a t h o g e n i c i t y and i n t o t h e i r f u n c t i o n and behaviour as members of the i n d i g e n o u s human g i n g i v a l c r e v i c e .  f l o r a of the  S t u d i e s i n our l a b o r a t o r y tend t o v a l i d a t e the  r e c e n t p r o p o s a l (98) t h a t e x i s t i n g s u b s p e c i e s of B_. m e l a n i n o g e n i c u s can be classified  i n t o a t l e a s t two  number of new criteria,  taxonomic  species.  Our c l a s s i f i c a t i o n  c r i t e r i a , which taken t o g e t h e r w i t h the s t a n d a r d  emphasize the unique c h a r a c t e r i z a t i o n of B_. m e l a n i n o g e n i c u s  ss. asaccharolyticus.  These c r i t e r i a a r e p a t h o g e n i c i t y , HA  c o l l a g e n a s e and the p r o d u c t i o n o f p h e n y l a c e t i c a c i d . may  have some importance  i n exudate from animals organisms  i s based on a  characterized  activity,  P h e n y l a c e t i c acid,  i n c l i n i c a l d i a g n o s i s s i n c e i t has been found . (S. Jensen, p e r s o n a l communication).  Both  f o r t h i s s t u d y , produced b u t y r i c a c i d ,  phenylacetic  216  a c i d , hemagglutinin, high l e v e l of protease collagenase Therefore, As  they can be c l a s s i f i e d  as Ji_. m e l a n i n o g e n i c u s s s .  expected, s t r a i n 2D r e q u i r e d hemin f o r growth.  grow on as low c o n c e n t r a t i o n growth i s r o u g h l y  and were i n f e c t i v e . asaccharolyticus.  The organism can  o f hemin as 0.25 ug hemin/ml medium,and t h e  p r o p o r t i o n a l t o t h e hemin c o n c e n t r a t i o n  r a t i o n of 2.5 ug hemin/ml medium.  Higher ..concentrations  up t o a concent-  1  up t o 20 ug/ml  medium had n e i t h e r an i n h i b i t o r y n o r an enhancing e f f e c t on growth. The glutamic (Table  growth r a t e o f 2D was i n f l u e n c e d by f r e e amino a c i d s .  a c i d , L-methionine, L - p r o l i n e and L - a s p a r a g i n e enhanced growth  3).  G l u t a m i c a c i d had t h e most pronounced e f f e c t ,  a t 24 h r by 45%. DL-valine,  i n c r e a s i n g growth  Growth was i n h i b i t e d almost c o m p l e t e l y by L - c y s t e i n e ,  L - h i s t i d i n e , L-tryptophan, g l y c i n e or L-arginine.  i n h i b i t e d growth by 66% w h i l e L - l y s i n e and L - p h e n y l a l a n i n e Our  L-serine,  L-leucine  had no e f f e c t .  r e s u l t s c o r r e l a t e w i t h t h e work done by M i l e s and Wong (149) on  the i n h i b i t i o n o f growth o f an a s a c c h a r o l y t i c s t r a i n o f J3. m e l a n i n o g e n i c u s by v a l i n e , t r y p t o p h a n , g l y c i n e , h i s t i d i n e and a r g i n i n e as w e l l as t h e enhancement o f growth by a s p a r a g i n e . L-phenylalanine  These workers a l s o r e p o r t e d  that  and L - l y s i n e had no e f f e c t on t h e growth r a t e , a c o n c l u s i o n  which was a l s o confirmed by our s t u d i e s . which were r e p o r t e d  to i n h i b i t  However, s e r i n e and m e t h i o n i n e ,  t h e growth o f the a s a c c h a r o l y t i c  Ii. m e l a n i n o g e n i c u s , were found t o enhance the growth o f s t r a i n 2D o f B.  melaninogenicus.  Glutamic a c i d which was found to cause the maximum  growth enhancement o f 2D was r e p o r t e d  by M i l e s and Wong as having no e f f e c t ;  a l s o , the amino a c i d s L - s e r i n e , L - c y s t e i n e  and L - l e u c i n e were found t o  i n h i b i t growth of 2D i n our s t u d i e s , w h i l e having no e f f e c t on the growth of the a s a c c h a r o l y t i c s t r a i n s t u d i e d by M i l e s  (149).  217  The  a b i l i t y of amino a c i d s t o i n f l u e n c e growth i n d i c a t e s t h a t  were p r o b a b l y a s s i m i l a t e d by the microorganism. conclusions  of Wahren et^ a l . (228)  who  reported  unable to a s s i m i l a t e these compounds. a c t u a l l y consumed i s l a c k i n g and  This contradicts  they the  t h a t the organisms were  Proof t h a t the amino a c i d s were  should  be  i n v e s t i g a t e d by  i n c o r p o r a t i o n or metabolism of l a b e l e d amino a c i d s .  following  Glutamic a c i d  was  u n a b l e t o r e p l a c e hemin as a growth f a c t o r , a l t h o u g h i t seems p o s s i b l e t h a t glutamic  a c i d c o u l d be deaminated and  s u c c i n i c a c i d which was The known.  reasons why  reported  decarboxylated  to form  as a p o s s i b l e replacement f o r hemin (146).  some of the amino a c i d s i n f l u e n c e growth i s not  I n t e r e s t i n g l y , the amino a c i d s t h a t i n h i b i t e d growth of  melaninogenicus i n t h i s study are of d i f f e r e n t c h e m i c a l groups. example, i n h i b i t i o n was and  L - t r y p t o p h a n , by  by L - c y s t e i n e ,  caused by the h e t e r o c y c l i c amino a c i d s  the a l i p h a t i c amino a c i d s  a s u l f u r - c o n t a i n i n g compound.  ory mechanism i s u n l i k e l y . i o n of uptake of r e q u i r e d  g l y c i n e and Therefore,  acids. and by  t r a n s p o r t may  amino a c i d s i s o c c u r r i n g , because the few  and  inhibitinhibit-  growth f r e e amino  i n h i b i t i o n between amino a c i d s  e x i s t , s i n c e i t i s known t h a t p e p t i d e s  are used  m e l a n i n o g e n i c u s (228) . ]3_. melaninogenicus s t r a i n 2D was  lytic  valine,  A l s o , i t i s u n l i k e l y that c o m p e t i t i v e  I t i s p o s s i b l e that competitive  peptide  L-histidine  a common  s t u d i e s were done i n the presence of t r y p t i c a s e , which has  For  shown to be one  i s o l a t e s which i s i n f e c t i v e as a monoculture and  more v i r u l e n t s t r a i n s of t h i s s p e c i e s . r a p i d l y and  The  l e a d s to death w i t h i n 48 h r s .  shock r e s u l t i n g from massive b a c t e r e m i a and  of the few  asaccharo-  i s thus one  i n f e c t i o n i s severe, Death i s p r o b a b l y due i n f e c t i o n of the  of  the  develops to s e p t i c  peritoneal  218  cavity.  The  i n f e c t i o n can be  another p r o v i d i n g hypersensitive  The  directly  not  a n i m a l to  i n d u c t i o n of  of 15. melaninogenicus can be r e l a t e d to a number of  r e s u l t s reported  here have shown t h a t s t r a i n 2D possesses a the v a s c u l a r p e r m e a b i l i t y  seems h i g h l y u n l i k e l y t h a t the t o x i c m a t e r i a l i s a l i k e endotoxin s i n c e heating i n f e c t i o n might a l s o be due  destroyed  of whether c o l l a g e n a s e  infective strain differing or p r o t e a s e  The  rapidly  or p r o t e a s e a c t i v i t y  i s essential for an  activity.  to cause clumping of RBC  Culture  supernatant c o n t a i n s  HA  and  to b i n d  a c t i v i t y as evidenced by i t s  s t r o n g l y to RBC  surfaces.  a c t i v i t y which i s thought to be due  been r e l e a s e d  to support t h i s assumption i s as  from the c e l l s u r f a c e .  The  can be r e s t o r e d by  and  (Table  11).  (c)  Both HA  are i n s e n s i t i v e to the same s a l t s , EDTA and  however, both were s e n s i t i v e to  HA  are i n a c t i v a t e d by  can be  and (Table  a r e s e n s i t i v e to Hg  (e)  evidence  agents  Both HA  Both HA  cell  a d d i t i o n of r e d u c i n g  (b)  (d)  to  follows:  Both h e m a g g l u t i n i n a c t i v i t i e s are s e n s i t i v e to o x i d a t i o n activity  The  from the p a r e n t s t r a i n o n l y i n the absence of  ability  (a)  spreading  and/or p r o t e a s e a c t i v i t y .  15. m e l a n i n o g e n i c u s s t r a i n 2D p o s s e s s e s HA  a s s o c i a t e d HA which has  It  lipopolysaccharide-  i t s activity.  to c o l l a g e n a s e  assay.  a s u c c e s s f u l i n f e c t i o n w i l l r e q u i r e the i s o l a t i o n of a mutant of  collagenase  a  response.  c h o l e r a - l i k e t o x i n when t e s t e d by  determination  from one  t h a t i t i s t r u l y an i n f e c t i o n and  Pathogenicity factors.  transmitted  iodoacetic acid  carbohydrates,  galactose.  heating.  removed from c e l l s by m i l d procedures such as washing  with buffer  (Table  5).  10).  219  (f)  E l e c t r o n m i c r o s c o p i c s t u d i e s have shown t h a t the s o l u b l e HA i s p r e s e n t i n a membranous s t r u c t u r e  (Thanks t o Dr. P a u l Osmanski  and Susan J e n s e n ) . (g)  S o l u b l e HA i s a l a r g e m o l e c u l e as evidenced by g e l f i l t r a t i o n and s e d i m e n t a t i o n a t 100,000 x g ( F i g . 11 and T a b l e 12).  (h)  An i n c r e a s e i n s o l u b l e HA i s found i n o l d c u l t u r e s  ( F i g . 10).  ( A l l i n f o r m a t i o n on the c e l l - b o u n d HA was o b t a i n e d from S. Jensen). Hemagglutinin in  a c t i v i t y was found a s s o c i a t e d w i t h the c e l l s as w e l l as  the c u l t u r e s u p e r n a t a n t s o f s t r a i n 2D.  study  The s o l u b l e HA was chosen f o r  s i n c e i t i s f r e e o f c e l l s and t h e r e f o r e , l i k e l y  to be p u r i f i e d  more e a s i l y , and a l s o t o p r o v i d e e v i d e n c e t o support t h e assumption  that  the s o l u b l e HA i s the same as t h a t which i s c e l l a s s o c i a t e d . The requirement  o f r e d u c i n g substances  c o r r e l a t e s w e l l w i t h the h i g h l y reduced of  the organism.  weight  f o r hemagglutination  s t a t e o f the n a t u r a l  activity  environment  As p o i n t e d o u t , the c e l l - f r e e HA was o f h i g h m o l e c u l a r  ( F i g . 1 and T a b l e 12) which i s p r o b a b l y due t o the a s s o c i a t i o n o f HA  w i t h fragments  o f the o u t e r membrane.  T h e r e f o r e , the observed  e f f e c t s of  i n h i b i t o r s and enzymes on h e m a g g l u t i n a t i n g a c t i v i t y might be e i t h e r the r e s u l t o f changing  the p r o p e r t i e s o f the a s s o c i a t e d membrane p a r t i c l e , o r  a d i r e c t e f f e c t on the HA  itself.  The s o l u b l e HA o f 15. melaninogenicus w i t h the e x c e p t i o n o f g a l a c t o s e .  was i n s e n s i t i v e t o c a r b o h y d r a t e s  G a l a c t o s e was a l s o found t o i n h i b i t t h e  h e m a g g l u t i n i n of the o r a l s t r a i n s of Fusobacterium T h e r e f o r e , i t appears HA moiety  nucleatum  (153).  t h a t the c e l l r e c e p t o r f o r the 15. melaninogenicus  contains D-galactose.  h e m a g g l u t i n a t i o n of E_. c o l i  D-mannose has been shown t o i n h i b i t  (160), which was a t t r i b u t e d to t h e p r e s e n c e  220  of a m a n n o s e - s p e c i f i c l e c t i n - l i k e p r o t e i n on the E_. c o l i c e l l s u r f a c e .  The  s o l u b l e HA o f 2D was s e n s i t i v e t o heat and to treatment w i t h pronase indicating  that p r o t e i n a c e o u s substances a r e i n v o l v e d .  s o l u b l e HA to d i f f e r e n t s a l t s might  I n s e n s i t i v i t y of the  suggest t h a t a g g l u t i n a t i o n o f the HA  and RBC does not occur through an i o n i c  interaction.  In o r d e r t o o b t a i n some i n f o r m a t i o n c o n c e r n i n g the n a t u r e o f the . component(s) n e c e s s a r y f o r HA, the e f f e c t o f treatment o f RBC on t h e i r ability  t o hemagglutinate w i t h s o l u b l e HA was determined  (Table 9).  Pronase  and g a l a c t o s i d a s e treatments o f RBC cause i n h i b i t i o n o f the HA a c t i v i t y which might  i n d i c a t e t h a t p r o t e i n and/or  n e c e s s a r y f o r HA to o c c u r .  Treatment  enhancement o f t h e HA a c t i v i t y .  carbohydrate m o i e t i e s are  of t h e RBC w i t h neuraminidase  caused  E l u t i o n o f HA adsorbed t o n e u r a m i n i d a s e -  t r e a t e d RBCs w i t h 8 M u r e a was l e s s e f f e c t i v e than e l u t i n g adsorbed HA from u n t r e a t e d RBCs.  T h i s might be due to a t i g h t e r b i n d i n g of s o l u b l e HA to  n e u r a m i n i d a s e - t r e a t e d RBC. galactose  The f a c t t h a t n e u r a m i n i c a c i d i s o f t e n l i n k e d to  (238)* suggest t h a t removal of neuraminic a c i d s may unmask  and then c r e a t e many new r e c e p t o r s o r b i n d i n g s i t e s f o r the HA on t h e RBC. P r e t r e a t m e n t o f RBC w i t h neuraminidase was r e p o r t e d t o r e s u l t i n an i n c r e a s e d b i n d i n g o f the HA p r e p a r a t i o n s of the o r a l b a c t e r i u m F. nucleatum  t o the RBC  (153).  An a f f i n i t y a d s o r p t i o n system u s i n g f o r m a l i n i z e d RBC was developed and used t o a c c o m p l i s h some p u r i f i c a t i o n of the s o l u b l e HA i n c u l t u r e s u p e r n a t a n t , f o l l o w e d by g e l f i l t r a t i o n on Sephadex G-100. 50% o f the HA was accomplished w i t h a 5 2 - f o l d p u r i f i c a t i o n .  Recovery o f However,  p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f the p a r t i a l l y p u r i f i e d HA r e v e a l e d a heterogenous  preparation.  I f the HA i s a s s o c i a t e d w i t h the o u t e r  221  membrane, i t may many p r o t e i n s .  e x i s t i n a l a r g e complex, and F u r t h e r attempts  the p a r t i c u l a t e fragments it  f i e d HA.  c o n d i t i o n s were  from  o n l y be a c t i v e i n t h i s complex form.  r e q u i r e d f o r a c t i v i t y of the p a r t i a l l y  Reagents known to r e a c t w i t h s u l f h y d r y l groups such as  a c i d , iodoacetamide  and H g C ^  p a r t i a l l y p u r i f i e d HA concluded  to d i s s o c i a t e the HA a c t i v i t y  r e s u l t e d i n g r e a t l o s s of a c t i v i t y ; t h e r e f o r e ,  i s p o s s i b l e t h a t the HA may  Reducing  thus be a s s o c i a t e d w i t h  partly inhibited  i n c u l t u r e supernatant.  puriiodoacetic  the crude HA as w e l l as T h e r e f o r e , i t can  the  be  t h a t s u l f h y d r y l groups are an i n t e g r a l p a r t of the h e m a g g l u t i n a t i o n  activity. J3. melaninogenicus  produced  both c e l l - b o u n d and c e l l - f r e e  a c t i v i t y which amounted to a p p r o x i m a t e l y 80% and t o t a l a c t i v i t y d u r i n g e x p o n e n t i a l growth. activity  of J3. melaninogenicus  resembles  The  cell-free  cell-bound protease  the p r o t e i n a s e s i n S t r e p t o c o c c u s  (221) and i n B a c t e r o i d e s amylophilus H18 localized  20% r e s p e c t i v e l y o f the  The p r o p o r t i o n of  i n c r e a s e d i n s t a t i o n a r y phase c u l t u r e s .  proteolytic  (15).  lactis  A l l three proteases are  i n the c e l l envelope, p r o b a b l y near the c e l l w a l l s u r f a c e s i n c e  they a r e a c c e s s i b l e to h i g h m o l e c u l a r weight s u b s t r a t e s . Both c e l l u l a r and s o l u b l e p r o t e a s e s of 15. melaninogenicus  were a c t i v e  a g a i n s t a number of p r o t e i n s u b s t r a t e s i n c l u d i n g a z o c o l l , c a s e i n , a z o c a s e i n and N,N-dimethylcasein. ory  The a z o c a s e i n assay was  m a i n l y because of low background.  found  The assay was  to be most  most  satisfact-  effective,  r e p r o d u c i b l e and e a s i e s t t o perform. P r o t e a s e p r o d u c t i o n by Gram-negative a n a e r o b i c b a c t e r i a has been i n v e s t i g a t e d ; the study o f the p r o t e o l y t i c a c t i v i t y of 15. thus seemed to be of g e n e r a l i n t e r e s t .  little  melaninogenicus  222  In o r d e r to i n v e s t i g a t e the r e l a t i o n s h i p o f the c e l l - b o u n d p r o t e a s e to the growth of 13. m e l a n i n o g e n i c u s , p r o t e a s e growth of the organism I t was of the  p r o d u c t i o n was  followed during  i n d i f f e r e n t media and under d i f f e r e n t  conditions.  found t h a t the p r o t e a s e p r o d u c t i o n c o r r e l a t e d w i t h the growth r a t e organism.  The evidence s u p p o r t i n g t h i s assumption (a)  (b)  was  as  follows:  I n c r e a s i n g the growth r a t e o f a continuous c u l t u r e o f the organism  r e s u l t e d i n an i n c r e a s e i n p r o t e a s e p r o d u c t i o n by  organism  ( F i g . 18).  Amino a c i d s t h a t s t i m u l a t e d growth i o n and  t h i s was  the  stimulated protease product-  not r e l a t e d to a d i r e c t e f f e c t on the a c t i v i t y  of the enzyme ( T a b l e 16). (c)  When the growth r a t e was s y n t h e s i s was was  slowed  l i m i t e d by hemin c o n c e n t r a t i o n p r o t e a s e  ( F i g . 16).  Under c o n d i t i o n s where hemin  not growth l i m i t i n g , f u r t h e r i n c r e a s i n g the hemin  concent-  r a t i o n d i d not have an e f f e c t on p r o t e a s e p r o d u c t i o n (Table 17). (d)  There was  a 60% decrease i n p r o t e a s e a c t i v i t y i n s u c c i n a t e medium  compared to TYH medium w i t h a simultaneous r a t e due  to the replacement  decrease i n the growth  of hemin by s u c c i n a t e ( F i g . 17).  s u c c i n a t e had no d i r e c t e f f e c t on the p r o t e a s e The r e a s o n why  The  activity.  p r o t e a s e s y n t h e s i s would be dependent upon the growth  r a t e i s not known, but these f i n d i n g s c o r r e l a t e w i t h f i n d i n g s r e p o r t e d f o r enzyme p r o d u c t i o n i n Pseudomonas a e r u g i n o s a the former amidase s y n t h e s i s and  (31) and V i b r i o SA1  i n the l a t t e r e x t r a c e l l u l a r  p r o d u c t i o n were r e l a t e d to the growth r a t e of the organisms studies.  (234).  In  protease i n chemostat  I t i s most p r o b a b l e t h a t t h i s r e f l e c t s a complex c o n t r o l  system.  223  S t u d i e s w i t h m a t e r i a l a s p i r a t e d from guinea p i g i n f e c t i o n s have shown t h a t 2D e l a b o r a t e s the s u l f h y d r y l p r o t e a s e i n the i n f e c t i o n as w e l l as in vitro.  An  i n c r e a s e i n p r o t e a s e p r o d u c t i o n was  of the 2D c o n t a i n i n g exudate when assayed  a s s o c i a t e d w i t h animal  in vitro  i n d i c a t e a r o l e o f the p r o t e a s e i n the i n f e c t i v e An e x t e n s i v e study was bound p r o t e a s e , i s o l a t e d  ( T a b l e 1 5 ) , which might  process.  made of the b i o c h e m i c a l p r o p e r t i e s of the  from m e c h a n i c a l l y d i s r u p t e d c e l l s .  p r o t e a s e s a r e o f t e n c l a s s i f i e d by pH optimum and  cell-  Microbial  inhibitor s e n s i t i v i t y rather  than by the most r e a d i l y h y d r o l y z e d s u b s t r a t e (102).  In a d d i t i o n ,  specific  i n h i b i t o r s of p r o t e o l y t i c a c t i v i t y f r e q u e n t l y p r o v i d e i n s i g h t i n t o the of the f u n c t i o n a l groups on the enzyme. I3_. melaninogenicus  appeared  t o be due  The  passage  nature  p r o t e o l y t i c a c t i v i t y of  to s u l f h y d r y l p r o t e a s e ( s ) , s i n c e t h e 2+  c e l l u l a r a c t i v i t y was  completely  i n h i b i t e d by Hg  r e s t o r a t i o n of a c t i o n w i t h r e d u c i n g agents a l k y l a t i n g agents  i o d o a c e t i c a c i d and  The metal c h e l a t i n g agent  the i n h i b i t o r y e f f e c t of the  iodoacetamide  supported  this  assumption.  not i n h i b i t e d by the s e r i n e i n h i b i t o r s ,  i t a c t i v a t e d by d i v a l e n t i o n s , and was  suggests  S e n s i t i v i t y to o x i d a t i o n ,  EDTA had no e f f e c t on the p r o t e o l y t i c a c t i v i t y .  o b s e r v a t i o n t h a t the p r o t e a s e was was  and  .  The nor  a c t i v e a t n e u t r a l pH, s t r o n g l y  t h a t i t i s not an a c i d i c , s e r i n e or metalloenzyme type of p r o t e a s e .  On the b a s i s of these f i n d i n g s t h e c e l l u l a r p r o t e a s e of B_. should be c l a s s i f i e d  melaninogenicus  as a s u l f h y d r y l enzyme.  G e n e r a l l y , the e x i s t e n c e o f an a c t i v e t h i o l group i s p r i m a r i l y a c h a r a c t e r i s t i c of the n e u t r a l p r o t e a s e s .  The  c e l l u l a r enzyme of 2D had  broad  and  10.5,  spectrum  of a c t i v i t y between pH 5.5  c a s e i n h y d r o l y t i c a c t i v i t y a t pH 7.0 between pH 8.0  and  9.0.  The  and  a second  a  w i t h a sharp peak of  p l a t e a u of  activity  pH optima of the c e l l u l a r p r o t e a s e  correlate  224  with  the n e u t r a l o r s l i g h t l y a l k a l i n e pH o f i t s n a t u r a l environment i n the  gingival crevice. The  c e l l u l a r protease  autodigestion, p a r t i c u l a r l y  o f 15. m e l a n i n o g e n i c u s was found to be s u b j e c t t o i n the p r e s e n c e of r e d u c i n g  a d d i t i o n , i t was not g e n e r a l l y v e r y  Among these p r e c a u t i o n s  In  s t a b l e which hampered i t s p u r i f i c a t i o n .  However, the f r a c t i o n a t i o n scheme p r e s e n t e d and was e a s i l y r e p r o d u c i b l e p r o v i d e d  agents.  (Table 22) gave a good  recovery  that c e r t a i n p r e c a u t i o n s were f o l l o w e d .  a r e the e x c l u s i o n o f r e d u c i n g agent d u r i n g a l l  steps i n the p u r i f i c a t i o n ,  the u l t i m a t e c a r e i n keeping the temperature  below 4°C and a p p r o p r i a t e c o n d i t i o n s of c o o l i n g and s t i r r i n g d u r i n g the p r e c i p i t a t i o n of the p r o t e o l y t i c a c t i v i t y by e t h a n o l . The  p u r i f i c a t i o n o f the c e l l u l a r p r o t e a s e was accomplished by d i a l y s i s  of the, c e l l - e x t r a c t , u l t r a c e n t r i f u g a t i o n a t 121,000 x g f o r 1 h r , p r e c i p i t a t i o n with  60% e t h a n o l a t -10°C f o l l o w e d by u l t r a c e n t r i f u g a t i o n a t  100,000 x g and d i a l y s i s ; g e l f i l t r a t i o n urea; The  and g e l f i l t r a t i o n  through Sephadex G-100 i n 6 M  through Sepharose-2B i n PBS c o n t a i n i n g 6 M  s p e c i f i c a c t i v i t y of the p u r i f i e d p r e p a r a t i o n was i n c r e a s e d  urea.  774 times  over t h a t of the crude p r e p a r a t i o n , and a 160% f i n a l r e c o v e r y was  obtained  (Table 22). The  f a c t t h a t an i n c r e a s e i n the r e c o v e r y  o f the p r o t e o l y t i c  activity  was found a f t e r chromatography o f the e t h a n o l p r e c i p i t a t e d p r o t e a s e suggested t h a t the p u r i f i c a t i o n s t e p s might have unmasked the p r o t e a s e  from  p r o t e i n complexes, removed some i n h i b i t o r s or components t h a t were b i n d i n g to a c t i v e s i t e s o r removed endogenous s u b s t r a t e . Polyacrylamide  g e l e l e c t r o p h o r e s i s showed t h e p u r i f i e d p r o t e a s e  to consist  of f o u r e l e c t r o p h o r e t i c a l l y d i s t i n c t bands as compared t o 15 major bands  225  i n the crude enzyme i n c e l l - e x t r a c t ( F i g . 23,24). was  Each of the f o u r bands  f i r m l y bound to c a r b o h y d r a t e moiety as i n d i c a t e d by  stain.  When the p u r i f i e d p r o t e a s e p r e p a r a t i o n was s u b j e c t e d  acrylamide g e l e l e c t r o p h o r e s i s without denaturation SDS,  the  i t revealed  the p r e s e n c e of o n l y one  m i g r a t e i n t o the g e l ( F i g . 26).  band and  Therefore,  and  glycoprotein to  poly-  i n the absence of  t h a t band d i d  i t seems l i k e l y  not  t h a t the p u r i -  f i e d p r o t e a s e s t i l l might have been bound i n a l a r g e m o l e c u l a r weight complex, as was urea.  a l s o i n d i c a t e d by  i t s e x c l u s i o n from G-100  It i s s i m i l a r , i n this respect,  Bacteroides penetrate  to the c e l l u l a r p r o t e a s e of  a m y l o p h i l u s r e l e a s e d by c e l l  polyacrylamide  g e l s . (14).  Sephadex i n 6 M  d i s i n t e g r a t i o n which a l s o d i d  I t a l s o resembles the p e n i c i l l i n a s e  B a c i l l u s l i c h e n i f o r m i s which on s o l u b i l i z a t i o n by d e o x y c h o l a t e and was  p a r t i c u l a t e as judged by  phoresis The  urea  (116). p u r i f i e d protease could represent The  a p o l y m e r i c form of enzyme f a c t t h a t the  sub-  purification  procedure used i n t h i s study always r e s u l t e d i n f r a c t i o n a t i o n of the  crude  p r o t e a s e i n t o f o u r major e l e c t r o p h o r e t i c a l l y d i s t i n c t bands, suggests the components of the complex p u r i f i e d p r o t e a s e were f i r m l y bound not  coincidentally associated  also implies  the p o l y a c r y l a m i d e  that  together  through the p u r i f i c a t i o n s t e p s .  It  t h a t the p u r i f i e d p r o t e a s e i s the "minimum b i o l o g i c a l l y  u n i t " , which i s p r e s e n t enter  of  i t s poor m o b i l i t y i n s t a r c h g e l e l e c t r o -  u n i t s bound to a c e l l w a l l component.  and  not  active  as a complex of a number of p r o t e i n s unable to g e l unless  i t has  been denatured i n SDS.  Any  further  attempts to break down the p u r i f i e d p r o t e a s e i n t o a l e s s complex u n i t r e s u l t e d i n l o s s of the p r o t e o l y t i c a c t i v i t y .  I t was  reported  t h a t some b a s i c  p r o p e r t i e s of c e r t a i n enzymes such as the d i m e r i z a t i o n of s u b u n i t s  might  dependent on the r e t e n t i o n of the enzyme i n a s s o c i a t i o n w i t h the c e l l w a l l  be (30) .  226  More d e f i n i t e c o n c l u s i o n s  c o u l d be drawn i f one was a b l e  which o f the f o u r bands found upon g e l - e l e c t r o p h o r e s i s proteolytic activity. use  to d e f i n e  c o n t a i n ( s ) the  F e a s i b l e approaches to t h i s problem c o u l d be the  of mutants d e f i c i e n t i n p r o t e o l y t i c a c t i v i t y , or c h e m i c a l l y  linking a  radioactively labeled  substrate  cross-  t o the enzyme.  In the f i n a l step o f p u r i f i c a t i o n , the a c t i v e p r o t e a s e emerged in.two f r a c t i o n s d i f f e r i n g i n both s p e c i f i c a c t i v i t y and chromatographic m o b i l i t y . E v i d e n c e was o b t a i n e d t h a t both f r a c t i o n s r e p r e s e n t  the same p r o t e a s e .  i n f l u e n c e of pH on p r o t e o l y t i c a c t i v i t y was i d e n t i c a l .  The  No d i f f e r e n c e was  found i n the response t o i n h i b i t o r s between the two f r a c t i o n s .  Both were  s t a i n e d f o r l i p i d s , had 4 d i s t i n c t e l e c t r o p h o r e t i c bands o f g l y c o p r o t e i n s and  lacked  e l e c t r o p h o r e t i c m o b i l i t y i n the absence o f SDS.  between the 2 f r a c t i o n s i s i n t h e i r l i p i d  content  (Table  The d i f f e r e n c e  23) which c o u l d be  a r e a s o n b e h i n d the d i f f e r e n c e i n t h e i r chromatographic m o b i l i t y . I t can be deduced t h a t the c e l l u l a r p r o t e a s e r e l e a s e d  by d i s i n t e g r a t i o n  of the c e l l s remained f i r m l y bound to c e l l u l a r components from which i t c o u l d n o t be c o m p l e t e l y l i b e r a t e d . cell-bound by  p r o t e a s e of B a c t e r o i d e s  i t resembled the  a m y l o p h i l u s H18 which, when l i b e r a t e d  sonic d i s r u p t i o n of c e l l s harvested during  particle-bound ase  In t h i s r e s p e c t ,  exponential  and c o u l d n o t be e a s i l y p u r i f i e d (14).  phase, was  Also,  the p e n i c i l l i n -  o f B a c i l l u s l i c h e n i f o r m i s , when l i b e r a t e d by lysozyme treatment,  appeared to be bound to membrane fragments As  (116).  i t was assumed t h a t the p r o t e a s e a c t i v i t y might have been  associated  w i t h a component o f the c e l l w a l l , t h i s would r e s u l t i n the a s s o c i a t i o n o f p r o t e o l y t i c a c t i v i t y w i t h random s i z e d and charged fragments when the c e l l s were d i s i n t e g r a t e d .  T h i s i s supported by the r e s u l t s o b t a i n e d  from  227  i o n exchange chromatography o f t h e crude p r o t e a s e under d i f f e r e n t  con-  d i t i o n s o f b u f f e r s , pH and i n t h e presence o f d e n a t u r i n g agents which r e s u l t e d i n poor r e c o v e r y o f the p r o t e a s e from a l a r g e number o f f r a c t i o n s . T h i s assumption  can a l s o account f o r t h e v a r i a t i o n s i n p r o t e a s e  l i b e r a t e d i n t o the c e l l - e x t r a c t t h a t was found between batches o f 48 h r cultures of bacteria disintegrated at different i n t h e p r o p o r t i o n o f unbroken  times w i t h o u t major changes'-  cells.  The p u r i f i e d p r o t e a s e o f ]5. melaninogenicus  was a c t i v e a g a i n s t a number  o f s u b s t r a t e s i n c l u d i n g a z o c o l l , a z o c a s e i n , c a s e i n and N,-N-dimethyl c a s e i n , and had no g l y c o s i d a s e , l i p a s e , c o l l a g e n a s e o r h e m a g g l u t i n a t i n g The p u r i f i e d p r o t e a s e was proven  activities.  t o be s i m i l a r to t h e crude enzyme i n  c e l l - e x t r a c t w i t h r e s p e c t t o oxygen s e n s i t i v i t y , r e v e r s i b l e i n h i b i t i o n by HgCl  and i r r e v e r s i b l e i n a c t i v a t i o n by the a l k y l a t i n g agents  iodoacetamide  2+ and  iodoacetic acids.  p u r i f i e d protease.  EDTA and c a t i o n s (Ca  ) had no e f f e c t on t h e  The pH optimum was found t o be a t pH 7.0 w i t h h i g h e r  a c t i v i t y a t a l k a l i n e pH v a l u e s than a t a c i d pH.  The s e r i n e  inhibitors  PMSF and TPCK had no e f f e c t on the p u r i f i e d p r o t e a s e , t h e r e f o r e , t h e c e l l u l a r p r o t e a s e o f s t r a i n 2D of J3. melaninogenicus enzyme.  appears  t o be a s u l f h y d r y l  The p r o t e a s e had c e r t a i n s t a b i l i t y a s p e c t s i n common w i t h  p a p a i n enzyme, which i s a t y p i c a l s u l f h y d r y l enzyme t h a t has been e x t e n s i v e l y studied.  R e s i s t a n c e t o the o r g a n i c s o l v e n t d i m e t h y l s u l f o x i d e (DMSO) and t o  8 M urea, and s e n s i t i v i t y enzymes (88,190).  t o g u a n i d i n e h y d r o c h l o r i d e a r e s i m i l a r i n both  The i n c r e a s e i n p r o t e o l y t i c a c t i v i t y caused by h i g h  c o n c e n t r a t i o n s o f u r e a i n the assay mixture c o u l d be e x p l a i n e d by the p o s s i b i l i t y o f t h a t urea m o d i f i e s the s u b s t r a t e or makes i t more a c c e s s i b l e to t h e enzyme.  I t might a l s o be due t o the unmasking o f a c t i v e s i t e s which  228  were b l o c k e d The  by other  components of the enzyme complex.  r e s u l t s have shown t h a t the c h a r a c t e r i s t i c s e x h i b i t e d by  the  enzyme i n the c e l l - e x t r a c t were i d e n t i c a l w i t h those of the p u r i f i e d which s t r o n g l y suggests t h a t one Ii. melaninogenicus which was  c e l l u l a r p r o t e a s e was  produced  the presence of c a r b o h y d r a t e , l i p i d  h y d r a t e and  lipid  and  i s not known.  l i p i d moieties  protein.  The  ss.  asaccharolyticus  reported  (142).  outer  f u n c t i o n of  the  to be  the  carbopresence  found i n i s o l a t e d  membrane complex of 13. m e l a n i n o g e n i c u s  Glucose, galactose  dominance of p a l m i t i c , p a l m i t o l e i c and  c y c l i c or odd  The  demonstrated  of the p u r i f i e d p r o t e a s e r e v e a l e d  as the predominant sugars i n the LPS  w e l l as the p r e s e n c e of two  by  c h a r a c t e r i z a t i o n of the  of components which were p r e v i o u s l y r e p o r t e d l i p o p o l y s a c c h a r i d e from the  enzyme  l i b e r a t e d from the c e l l s by d i s i n t e g r a t i o n .  Chemical a n a l y s i s of the p u r i f i e d p r o t e a s e p r e p a r a t i o n  c a r b o h y d r a t e and  crude  and  glucosamine were  preparations.  s t e a r i c a c i d was  The  pre-  also reported  unknown f a t t y a c i d s t h a t were assumed t o  chain f a t t y acids  as be  (142).  Attempts were made to determine i f the p r o t e a s e of 15. m e l a n i n o g e n i c u s was  l o c a t e d i n the p e r i p l a s m i c  polymyxin B.  space by osmotic shock and  Only 10-12% of the p r o t e a s e a c t i v i t y was  p r o t e a s e c o u l d not,  t h e r e f o r e , be c l a s s i f i e d as  treatment w i t h  l i b e r a t e d , and  periplasmic.  Ingram et_ a l . found t h a t some of the a l k a l i n e phosphatase Pseudomonas a e r u g i n o s a was and  was  l o c a t e d e x t e r i o r to the outer  complexed w i t h l i p o p o l y s a c c h a r i d e which was  secretion  (96).  also released  a s s o c i a t e d w i t h growth l e a d to a s t r i p p i n g of L P S - a l k a l i n e surface.  of  tripartite  They h y p o t h e s i z e d t h a t m e c h a n i c a l s h e a r i n g  aggregates on the e x t e r n a l w a l l  the  layer  during  forces phosphatase  229  V a r i o u s enzymes found o u t s i d e of the c y t o p l a s m i c membrane were not r e l e a s e d i n t o the medium, but were bound to the outer membrane of the c e l l envelope which c o n t a i n s charged m o i e t i e s . bound t o the c e l l  either  A molecule might remain  i n a s s o c i a t i o n w i t h mucopeptide (194) , w i t h v a r i o u s  components of the p e r i p l a s m i c space  (30) or w i t h l i p o p o l y s a c c h a r i d e of the  o u t e r membrane ( 9 6 ) , depending on the n a t u r e of the enzyme such as amount of h y d r o p h o b o c i t y The to  and number of charged  the  groups.  s e c r e t i o n of a p r o t e a s e enzyme by M i c r o c o c c u s  sodonensis was  found  be dependent on the c o - s e c r e t i o n of a t l e a s t one of s e v e r a l p o l y -  s a c c h a r i d e s , a l s o e l a b o r a t e d by t h e s e c e l l s  (19).  r e p o r t e d t h a t a t l e a s t 50% of the p r o t e a s e a c t i v i t y  Regnier and Thang found  (174)  i n E_. c o l i i s  a s s o c i a t e d w i t h the membrane. There a r e v e r y few r e p o r t s on e x t r a c e l l u l a r enzymes of Gram-negative a n a e r o b i c organisms.  B l a c k b u r n r e p o r t e d t h a t a p r o t e a s e was  liberated  into  the growth medium by e x p o n e n t i a l l y growing c u l t u r e s of B a c t e r o i d e s amylop h i l u s s t r a i n H18 In  (13).  t h i s study, evidence was  p r o t e a s e of IS. melaninogenicus  o b t a i n e d s u g g e s t i n g t h a t the  extracellular  i s the c e l l - b o u n d p r o t e a s e which was  l i b e r a t e d from t h e c e l l s by r e l e a s e of o u t e r membrane d u r i n g c e l l cell lysis.  The  found  to be between 7.0  associated protease.  IS. melaninogenicus  was  and  a l s o found  The  and 7.5  e x t r a c e l l u l a r p r o t e a s e of to i t s  s e n s i t i v i t y to S H - i n a c t i v a t i n g agents.  t h a t the e x t r a c e l l u l a r p r o t e a s e of 2D was  subject to audodigestion.  azo-  which i s s i m i l a r to  a l s o c l a s s i f e d as a s u l f h y d r y l enzyme due  dependence on r e d u c i n g agents and I t was  growth o r  optimum pH f o r the e x t r a c e l l u l a r p r o t e a s e of 2D w i t h  c a s e i n as s u b s t r a t e was t h a t of the c e l l  probably  unstable  From the s t u d i e s on the p u r i f i c a t i o n of  the  230  c e l l - f r e e protease  of 2D,  of B_. melaninogenicus was  i t was  concluded  of h i g h m o l e c u l a r  that the e x t r a c e l l u l a r weight and  protease  presumably membrane  or p a r t i c l e bound. The m e t a l c h e l a t i n g agent EDTA caused 60% c e l l u l a r protease protease.  The  inhibition  of the e x t r a -  of 13. melaninogenicus but d i d not a f f e c t the  d i f f e r e n c e might be due  which the enzymes a r e bound.  cell-bound  to d i f f e r e n c e s i n the complex i n  G e n e r a l l y , c a t i o n s can be r e q u i r e d f o r  a c t i v i t y or f o r p r o t e c t i o n of the enzyme a g a i n s t a u t o d i g e s t i o n .  A  l y t i c enzyme i s o l a t e d from C l o s t r i d i u m botu'linum type B which was o n l y when i n the reduced s t a t e was The  l o c a t e d i n the c u l t u r e  the i n c r e a s e i n p r o p o r t i o n o n l y  s t a t i o n a r y phase made i t l i k e l y  active  r e p o r t e d to be i n a c t i v a t e d by EDTA  s m a l l p r o p o r t i o n of p r o t e a s e  d u r i n g l o g a r i t h m i c growth and  proteo-  (11).  supernatant during  t h a t the e x t r a c e l l u l a r enzyme was  liberated  because of r e l e a s e of outer membrane d u r i n g c e l l growth or a u t o l y s i s of o n l y a few b a c t e r i a l  cells.  G e n e r a l l y , the p u r i f i c a t i o n of e x t r a c e l l u l a r enzymes from b a c t e r i a i n v o l v e s some s p e c i a l problems. medium i s u s u a l l y low and compounds must be removed. p u r i f i e d but  o n l y a few  The  enzyme c o n c e n t r a t i o n i n the growth  l a r g e q u a n t i t i e s of s a l t s and  extraneous  S e v e r a l b a c t e r i a l p r o t e i n a s e s have been p a r t l y  of these have been i s o l a t e d i n a pure s t a t e and  c h a r a c t e r i z e d i n some d e t a i l . The was  protease  of the d i a l y z e d and  concentrated  p a r t i a l l y p u r i f i e d by g e l f i l t r a t i o n  p r o t e a s e was  through Sephadex G-100.  The  e l u t e d as a s i n g l e peak at the v o i d volume of the column w i t h  a f i n a l recovery f i e d protease  c u l t u r e supernatant df  of o n l y 46%.  G e l e l e c t r o p h o r e s i s of the p a r t i a l l y  r e v e a l e d many p r o t e i n bands; and  f u r t h e r attempts to  puri-  2D  231  d i s a g g r e g a t e and  s e p a r a t e the p r o t e a s e i n a p u r i f i e d form were  unsuccessful. The p o s s i b l e r o l e of p r o t e o l y t i c a c t i v i t y i n c e l l d i v i s i o n has suggested at  by v a r i o u s s t u d i e s .  Kogoma and N i s h i  (111) had  been  found an i n c r e a s e  d i v i s i o n f o l l o w e d by a decrease of an i n t r a c e l l u l a r p r o t e i n a s e i n  synchronously  d i v i d i n g c e l l s of E s c h e r i c h i a c o l i .  B u f d e t t and Murray  (24)  p r e s e n t e d e l e c t r o n m i c r o s c o p i c evidence of l o c a l i z e d h y d r o l y t i c a c t i v i t y the s i t e of septum f o r m a t i o n i n E.  coli.  I t has been shown t h a t the  auto-  l y s i n o f S t r e p t o c o c c u s f a e c a l i s i s p r e s e n t i n an i n a c t i v e form i n the w a l l but i s a c t i v a t e d by a n e u t r a l p r o t e i n a s e ; and  at  cell  t h a t the a c t i v e form of  the a u t o l y s i n i s a s s o c i a t e d w i t h r e c e n t l y s y n t h e s i z e d w a l l The major f u n c t i o n of e x t r a c e l l u l a r p r o t e i n a s e s and  (193).  other h y d r o l y t i c  enzymes, i s most r e a s o n a b l y a n u t r i t i o n a l one which e v o l v e d to a l l o w the microorganism  growing i n i t s n a t u r a l environment to u t i l i z e  complex  s u b s t r a t e s as sources of n u t r i e n t s . The r e s u l t s p r e s e n t e d i n t h i s study suggest 2D of 15. melaninogenicus  can be c l a s s i f i e d  t h a t the p r o t e a s e of  as a s u l f h y d r y l enzyme.  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