UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

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

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1979_A1 R39.pdf [ 9.14MB ]
Metadata
JSON: 831-1.0094891.json
JSON-LD: 831-1.0094891-ld.json
RDF/XML (Pretty): 831-1.0094891-rdf.xml
RDF/JSON: 831-1.0094891-rdf.json
Turtle: 831-1.0094891-turtle.txt
N-Triples: 831-1.0094891-rdf-ntriples.txt
Original Record: 831-1.0094891-source.json
Full Text
831-1.0094891-fulltext.txt
Citation
831-1.0094891.ris

Full Text

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 present ing t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Co lumbia, I agree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re fe rence and s tudy. I f u r t h e r agree tha t permiss ion f o r ex tens i ve copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s en t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n pe rm iss i on . Department Of M i c r o b i o l o g y The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 D a t e _ J u n e _ 8 ^ 7 9 _ D E - 6 B P 7 5 - 5 1 1 E i i ABSTRACT Bacteroides melariiribgeriicus strains 2D and K110 were characterized with regard to their pathogenic, collagenolytic, proteolytic, hemagglutin-ating and metabolic a c t i v i t i e s . Both strains were members of the sub-species 13. melariinogeriicus ss. asaccharolyticus. They possessed a c e l l -bound oxygen-sensitive collagenase, a cell-bound and a soluble oxygen-sensitive hemagglutinin (HA), and a protease. Both strains produced butyric and phenylacetic acids and were infective in guinea pigs as characterized by their a b i l i t y to produce necrotic lesions and to be transferred from one animal to another. Strain 2D required hemin for growth and i t s growth rate was influenced by the addition of free amino acids to the medium. The hemagglutinating and proteolytic activities of strain 2D were investigated further to determine their relationship to infection. The soluble HA was reversibly inhibited by Hg and activity was restored in the presence of reducing agents. Iodoacetic acid caused irreversible inhibition. The HA was sensitive to heat and pronase treatment. Treatment of the red blood cells (RBC) with neuraminidase enhanced HA activity while the presence of galactose in the reaction mixture inhibited i t , suggesting the involvement of galactose residues on the RBCs in the reaction.. Adsorption of the HA to RBC followed by elution and gel f i l t r a t i o n resulted in the recovery of 50% of the HA activity and a 52-fold purification. Protease production by _B. melariiribgenicus strain 2D was dependent on the growth rate of the organism. The protease was reversibly inhibited by HgCl 0 and irreversibly inhibited by iodoacetamide and iodoacetic acid. i i i The enzyme was insensitive to serine protease inhibitors and EDTA. The pH optimum for proteolytic activity was 7.0, which correlates with the pH of i t s natural environment, the gingival crevice. It i s thus classified as a neutral sulfhydryl enzyme. A 774-fold purification of the cellular protease of 2D, with a 160% recovery of activity, was accomplished by precipitation with 60% ethanol, ultracentrifugation and gel f i l t r a t i o n through Sephadex G-100 and Sepharose 2B in the presence of urea. Electrophoretic analysis of the protease on SDS-polyacrylamide gels revealed four distinct bands, each of which was shown to be associated with carbohydrate. In the absence of SDS only one band, which did not migrate into the gel, was obtained. Any attempts to further dissociate the protease resulted in the loss of activity. The protease was active against azocoll, azocasein, casein and N,N-dimethylcasein. No glycosidase, lipase, collagenase or HA activities were detected. Protein, carbohydrate and l i p i d were detected in the preparation. The soluble protease which amounted to 20% of the cellular protease of strain 2D was subjected to gel f i l t r a t i o n on Sephadex G-100 and eluted in a single peak at the void volume. The properties of the soluble protease were identical to those of the c e l l associated enzyme, suggesting the presence of a single proteolytic enzyme which was released into the culture medium with c e l l lysis or due to shedding of outer membrane fragments. iv TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES x i ACKNOWLEDGEMENTS xiv I. INTRODUCTION A. Bacteroides melaninogenicus 1 1. Biochemical characteristics of 13. melaninogenicus 4 2. Pathogenic properties of 13. melaninogenicus 10 a. Role of B. melaninogenicus in mixed anaerobic — a 1 Q infections and periodontal diseases 1 2 b. Infectivity of 15. melaninogenicus c. Toxin production 3. Antibiotic sensitivity ^ 4. Antigenic structure and serological heterogeneity 5. Genetic properties ^ 6. Lipopolysaccharide and lipids of 15. melaninogenicus ^ B. Hemagglutinating Activity and Adherence Properties of J5. melaninogenicus  22 1. Adherence 2 S 2. Hemagglutinin C. Proteolytic Activity of 15. melaninogenicus  II. MATERIALS AND METHODS A. Organisms ^ Page II. MATERIALS AND METHODS B. Growth 4 8 1. Anaerobiosis 4 8 2. Media 4 9 3. Continuous cultures C. Protease 5 1 1. Assays for proteolytic activity ^ 2. Purification of the protease D. Hemagglutination ^ 1. Assay 6 6 2. -Preparation of red blood cells 6^ 3. Determination of the effects of various reagents on HA 6 7 4. Adhesion and elution of hemagglutinin from RBC 67 E. Infectivity 6 8 F. Metabolic End Product Analysis 69 G. Collagenase Assay 7^ H. Protein Determination 72 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 v i HI. RESULTS Page A. Characterization of IB. melaninogenicus 7o 1. Fatty acid production 76 2. Collagenase activity ^ 3. Pathogenicity 81 a. Infectivity 82 b. Vascular permeability 4. Growth of strain 2D 13. melaninogenicus ^ 84 a. Hemin requirement 87 b. Growth response to amino acids 5. Hemagglutinin and protease activity of ^ 13. melaninogenicus. 91 6. Effect of washing 2D cells on the HA and protease 7. Release of periplasmic enzymes from 2D cells 4^ B. Soluble Hemagglutinin of 13. melaninogenicus ^6 1. Adherence of 13. melaninogenicus 2D cells to formalinized human RBC 96 2. Determination of optimal conditions for the hemagglutinin assay 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. Effect of carbohydrates on HA 1 1 0 7. Stability of the soluble HA 1 1 0 8. Oxygen sensitivity of the soluble HA m 9. Effect of sulfhydryl modifiers on HA 1 1 1 10. Ultracentrifugation of soluble HA m v i i III. RESULTS Page 11. Partial purification of soluble HA H5 a. Concentration of the HA 115 b. Chromatography 115 c. Binding to Millipore f i l t e r s 121 C. 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. Effect of passaging 2D cells in guinea pigs on protease production 133 b. Effect of hemin concentration on growth and protease production 134 c. Protease production in the presence of succinate 134 d. Effect of amino acids on protease production 140 e. Growth rate and protease production 142 f. Production of the protease at different concentrations of hemin 142 g. Preliminary characterization of the cellular protease of 13. melaninogenicus  h. Purification of the cellular protease of 13. melaninogenicus 1^ 0 i . Characterization of the purified protease 184 4. Soluble protease of 13. melaninogenicus 202 a. Demonstration of an extracellular protease 202 b. Preliminary characterization of the extra-cellular protease 203 c. Partial purification of the soluble protease 208 v i i i Page IV. DISCUSSION 215 V. LITERATURE CITED 232 ix LIST OF TABLES TABLE T i t l e P a 8 e 1. Collagenase assay 71 2. Vascular permeability test 83 3. Effect 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 cells on the HA 93 6. Release of the HA from 2D cells by treatment with Polymyxin B 95 7. Influence of culture age on the adherence of 2D to FRBC 97 8. Effect of the source of red blood cells on HA activity 101 9. Effect of treatment of RBC on their a b i l i t y to hemagglutinate with soluble HA 109 10. Effect of aeration on soluble HA 112 11. Effect of sulfhydryl modifiers on HA 113 12. Ultracentrifugation of soluble HA 114 13. Characteristics of the HA eluted from RBC with urea 117 14. Analysis of the HA purification procedures 118 15. Effect of passaging 2D on protease activity 135 16. Effect of additions of amino acids toTYH medium on the proteolytic activity of B. melaninogenicus 141 17. Effect of hemin concentration on protease production 145 X TABLE T i t l e Page 18. Effect of reducing agents on the cellular protease 147 19. Thermoliability of the proteolytic activity in the cell-extract 149 20. Comparison of methods for liberating protease 151 21. Ethanol precipitation of protease from cell-extract 154 22. Purification of protease from B. melaninogenicus-2D.. 155 23. Chemical composition of the purified protease preparation 185 24. Gas-liquid chromatographic analysis of fatty acids in the purified enzyme preparation 187 25. Modification of the purified protease 192 26. Inhibition of soluble protease 207 27. Partial purification of I5_. melaninogenicus extracellular protease 209 x i LIST OF FIGURES FIGURE T i t l e Page 1. General patterns of enzyme production in continuous culture 45 2. The effect of dilution rate on the synthesis of amidase by Pseudomonas aeruginosa growing in a chemostat under steady-state conditions 45 3. Gas chromatography of volatile fatty acids produced by ]3_. melaninogenicus 78 4. Gas chromatography of non-volatile fatty acids produced by B_. melariinogenicus 80 5. Response of 13. melaninogenicus (strain 2D) to hemin 86 6. Effect of glutamic acid on growth of 13. melaninogenicus 90 7. Effect of pH on HA of B_. melaninogenicus 98 8. Optimal erythrocyte concentration for microtiter hemagglutination assay 103 9. Effects of incubation temperature on HA 105 10. Relationship of HA to culture age 108 11. Sephadex G-100 gel f i l t r a t i o n of the soluble HA 120 12. Hydrolysis of azocasein by B_. melaninogenicus protease 125 13. Effect of azocasein concentration 127 14. Effect of enzyme concentration on the azocasein assay 130 15. Relationship of protease to culture age 132 16. Effect of hemin concentration on growth and protease production of 2D 137 x i i FIGURE T i t l e Page 17. Protease production in hemin and succinate media 139 18. Effect of dilution rate (D) on protease production by I3_. melaninogenicus 144 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 thiol Sepharose-4B 165 22. Sepharose mercury chromatography 167 23. Polyacrylamide gel electrophoresis in tris-glycine-SDS buffer of the purified protease 171 24. Polyacrylamide gel electrophoresis in t r i s -glycine-SDS buffer of protease fractions obtained during various steps in the purification process 173 25. Polyacrylamide gel electrophoresis of protease fractions obtained from the different purification procedures 175 26. Polyacrylamide gel electrophoresis in t r i s -glycine buffer without SDS 178 27. Polyacrylamide electrophoresis of fraction A and B stained for li p i d s 181 28. Diagram of glycoprotein and protein bands on slab gels following electrophoresis of purified protease 183 29. Effect of pH on the purified protease activity 190 30. HgC-''2 l ^ l b l t l 0 1 1 °f t h e Purified protease .. 194 31. Inhibition of the purified protease by iodoacetamide 196 32. Effect of guanidine hydrochloride, lithium chloride and NaCl on the purified protease 199 x i i i FIGURE T i t l e 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 is gratefully acknowledged. I extend my appreciation and eternal thanks to my research advisor Dr. B.C. McBride. His invaluable academic assistance, understanding, criticism and extremely amiable attitude were necessary to complete this research. I also wish to thank Dr. Antonio Weerkamp and Mary Gisslow for their helpful comments and suggestions. Last but certainly not least, I want to record my deep feeling of gratitude to the immeasurable support and sacrifices rendered me by my immediate family, children, husband and mother, who were always there to lean on whenever necessary. This thesis is dedicated to them. 1 I. INTRODUCTION A. Bacteroides melaninogenicus The role of anaerobic bacteria in the etiology of infection and their function in 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 flora (58) and under certain conditions, particularly in compromised patients, these organisms can invade any organ of the body and cause a variety of illnesses (56) . A commonly encountered species in human infection is 13_. melanino- genicus (4,25,57,138,202,230,231) which is frequently isolated from abscesses in the upper half of the body. B_. melaninogenicus is a s t r i c t l y anaerobic, Gram-negative, non-motile, non-sporulating rod with rounded or pointed ends. The cells vary in size and shape from small coccoid forms to long filamentous organisms. 13. melaninogenicus is normally found in small numbers in the human intestine (25), ^ on male and female external genitalia (25,78), in the throat (25,163), in the oral cavity where i t is found in large numbers in the gingival crevice (25,68,69,197,204), and in supragingival human dental plaque (69,85,130,207). In the gingival crevice i t may account for as much as 5% of the total cultivable flora. It i s also present in the mouths 'of dogs but is not found in the mouths of rodents. It has been isolated in 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), bite wounds and infected surgical wounds (163,231), urine from a suspected infected kidney (163), appendicitis peritonitis (3,4,154), and the uterus and blood in patients with puerperal infections (188,212,220). The organism has been implicated in periodontal disease (137,138,208,217,230) but this relation-ship has not been proven. Characteristically, the organism is associated with abscesses populated by a mixture of non-sporulating anaerobes and/or facultative anaerobes (4,25,54,57,79,80,148,192,202,208). The occurrence of 13. melaninogenicus in c l i n i c a l specimens was described as early as 1921, but the organism is s t i l l often undetected in c l i n i c a l specimens. This may be due to improper collection of the specimens, the lengthy delay between specimen collection and culture, failure to use the proper culture medium and failure to incubate the cultures under s t r i c t l y anaerobic conditions and for sufficient 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 in growing i t in pure culture (26,54). Furthermore, isolates 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 strain 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 characteristic fermentation patterns and volatile fatty 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 differen-tiation 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 clearly show the substantial differences among the sub-species (93,189). The subspecies differ from each other in 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 identification of 13. melaninogenicus and i t provided support for the biochemical separation of this organism into the three subspecies proposed by Moore and Holdeman (155). It also provided a rapid procedure for identification of these organisms in 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 reclassified as a separate species, Bacteroides asaccharolyticus whereas the saccharolytic strains should retain 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 in the presence of excess heme. The usual procedure is to grow the organism anaerobically on blood agar medium containing laked blood. Pigmentation begins about the third day and becomes dark olive brown in a week, and f i n a l l y black in 5-14 days. Often this i s the sole criterion for identifying a member of the Bacteroides genus as I3_. melaninogenicus, as the characteristic pigmentation remains the basic criterion for differentiation from other Bacteroides species (95). The pigment of I3_. melaninogenicus was identified as extra-cellular melanin by Oliver and wherry (163), and later claimed to be intracellular colloidal 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 is dependent upon cultural conditions such as age of the culture, presence of heme or heme-contain-ing compounds in the growth medium and other factors. Therefore, the fin a l identification of B_. melaninogenicus should not be based on pigmentation characteristics 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 is a hemin derivative, and the extent of production is dependent upon the amount 5 of hemin present in the medium (187), i t i s tempting to speculate that pigment formation represents a storage mechanism for this 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 hemin-free media but f a i l to grow upon subsequent transfer (66) , supports this 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 in energy metabolism. Rizza et^ a l . (177) found that 99% of the cytochromes were located in a partially purified membrane fraction. Whether or not the electron transport system functions in the metabolism of 15. melaninogenicus remains undefined. Lev and Milford (121,122) reported that vitamin K had a specific effect on sphingolipid biosynthesis. It induced the formation of 3-keto dihydrosphingosine synthetase (123) and thus stimulated the synthesis of this novel microbial l i p i d . Sodium succinate was found to be an additional growth factor for B_. melaninogenicus in that i t could replace the required heme in the presence of vitamin K, allowing good growth of the organism. Succinate can also partially replace the required vitamin K in 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 results demonstrate a central role for succinate in the metabolism of 15. melaninogenicus and suggest that there are two pathways of succinate metabolism, mediated by heme and vitamin K, respectively. 6 The relationship between heme, vitamin K and succinate in B_. melaninogenicus is not understood. It is 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 this compound was used in energy metabolism and was not incorporated into cellular carbon (146) . This assumption was supported by the observation that only 0.5% of the succinate carbon could be found in 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 qualitatively similar but quantitatively different in cells grown in hemin free succinate medium (146). Thirty-one strains of melaninogenicus were studied by Sawyer e_t a l . (185) . A l l the strains were actively proteolytic and collagenolytic, attacking reconstituted neutral salt-extracted collagen and gelatin, 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 nitrates, none formed catalase and three distinctly different fermentative patterns were observed. A similar study done by Courant and Gibbons (35) showed the same results. Werner and Reichertz (232) characterized ten strains of Ji. melaninogenicus with regard to the inability to produce ammonia or propionate from threonine and the lack of glutamate decarboxylase activity. Burdon (25) reported on strains of B_. melaninogenicus which 7 were highly proteolytic, attacking gelatin, coagulated serum egg albumin and milk, but which did not ferment carbohydrates. Later, Finegold (59) reported that I3_. melariiribgenicus ss asacchafolyticus hydrolyzed esculin, was indole negative, clotted milk, and did not possess lipase activity. While differences have been found between strains with regards to enzyme activity, indole, esculin and starch hydrolysis, NH^ and H^ S production, fermentation patterns and end product analysis, no correlation between any of the above and pathogenicity has been established. We have found in our laboratory a very relevant classification scheme which is based on the pathogenicity of the I3_. melaninogenicus (146). In this scheme collagenase, protease, fatty acid production, hemagglutination and pathogenicity are related. After screening 200 new strains as well as strains 2D and K110, i t was concluded that irrespective of the source, the isolates could be separated into two groups. The pathogenic strains produce collagenase, high levels of protease, butyrate and phenylacetate and they agglutinate red blood c e l l s . The non-pathogenic strains produce succinate instead of phenylacetate and do not produce the other compounds. No exceptions were reported in the 200 strains isolated in the laboratory. This suggested that a study of some of these properties in greater detail, 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 active. 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 isovaleric acid. They also characterized 13. melaninogenicus in"a more recent report (232), according to the results obtained with ten strains, by the production of acetic, propionic, isobutyric, butyric and isoval-eric acids in peptone-yeast extract-glucose media. B_. melaninogenicus was separated from other Bacteroides strains which exhibit a similar pattern of acid products byr the relatively great amount of butyrate and the outcome of the glutamate decarboxylase test. 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 non-saccharolytic strains produced large amounts of propionic and butyric acids. It was found that the addition of glucose to trypticase-yeast extract medium did not enhance growth nor was the glucose metabolized to volatile acid end products (228). Studies by Finegold and Barnes (59) revealed basic differences in the acidic end products of the two fermentative groups of I3_. melaninogenicus. Isobutyric acid was produced by both groups, whereas n-butyric acid was produced only by asaccharolytic strains, and succinic acid was produced only by saccharolytic strains. Similar results were obtained by Williams and Bowden (235). Biochemical studies (35,185) have shown that most strains of 13. melaninogenicus grow well in sugar-free peptide^containing broth. 9 In addition, this growth decreased when the trypticase concentration of the medium was reduced suggesting that the fermentation of proteinaceous constituents plays an important role in the metabolism of B_. melanino- genicus and that the organisms have the potential to ferment amino acids Experiments using labelled proteins (228) indicated that strains of B_. melaninogenicus readily fermented amino acids when they were present as peptides. This suggested that peptides were more easily 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 yield of saccharolytic and asaccharolytic strains of 15. melaninogenicus. Some amino acids enhanced growth, and others inhibited i t . The significant stimulation of growth by certain amino acids in the presence of tryptic peptides in this study suggested that for some strains of 13. melaninogenicus a few amino acids are taken up as readily as peptides. The mechanism of growth inhibition by amino acids is not known; Lev and Milford (124) found that growth of I5_. melaninogenicus was inhibited by the addition of certain monosaccharides to trypticase-hemin medium. The major inhibitory effect of the sugar was to prolong the transition from the lag to logarithmic growth phase. They ascribed this to an effect on enzyme induction of which the inhibition of 3-keto dihydrosphingosine synthetase activity was one example. In actively growing cultures, addition of sugar slowed the growth rate and did not appear to be related to the activity of the synthetase enzyme. It was also possible that other enzymes were affected by the inhibitory 10 monosaccharides, contributing to a retardation of the growth rate. They also noted that sugars did not inhibit enzyme activity in vitro. 2. Pathogenic Properties of B_. melaninogenicus a. Role of B_. melaninogenicus in 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 is not unreasonable to assume that they may be involved in 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 flora not only possesses pathogenic potential, but this potential i s often realized when a suitable environment is available. B_. melaninogenicus is almost always present in the human mouth (25,68,69,197,204) and is commonly found in mixed anaerobic infections (4,25,148,192,202). Antigenic components of B^. melaninogenicus have also been demonstrated in diseased gingival tissue (34) . 13. melaninogenicus of oral origin has been reported to be involved in 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 cultivable bacteria in plaque which was removed from the base of deep gingival pockets of adults. I3_. melaninogenicus constituted almost half of 11 these Gram-negative isolates, and most of the 13_. melaninogenicus strains were non-saccharolytic and appeared to belong to the sub-species 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 this observation to show that an infection could be initiated by a combination of 13. melaninogenicus, two other bacteroides and a diphtheroid (135). The principal role played by 15. melaninogenicus in the etiology of the infection was -shown by Socransky and Gibbons (205). It was found that no infection occurred when 15. melaninogenicus was omitted from the mixture. The role of the "helper" organisms has only been partially defined (66,205,206,211). It 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 nutritional dependency (66,205,206,211). Evidence for the latter was provided in an experiment where the 15. melaninogenicus used did not require vitamin K, thus the diptheroid and one of the bacteroides were omitted without altering the pathogenicity. It was also reported that the infectivity 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 in experimental mixed infections might provide some understanding of the mechanisms involved in the pathogenicity of c l i n i c a l anaerobic infections, investigators became concerned with the production of potentially damaging metabolites, toxins, or other factors which would explain how 13. melaninogenicus and associated organisms were able to cause infections. Although Macdonald had suggested earlier that perhaps mixed infections were bacterially nonspecific but biochemically specific in 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 role of IB. melaninogenicus in the experimental system suggested that the "nonspecific" infections were, in fact, dependent on the presence of 13. melaninogenicus and that the role of other organisms was one of supporting and enhancing the in vivo growth of the primary pathogen (139,205). Evidence thus implicated I3_. melaninogenicus as the primary pathogen in mixed infections of soft tissues; consequently, 13. melaninogenicus was examined for pathogenic properties and infectivity. b. Infectivity of J3. melaninogenicus. The c r i t e r i a used for defining a typical trans-missible infection were summarized by Socransky and Gibbons (205). A successful infection occurs when: (i) inoculation results in visible necrosis, either spreading or confined to a pustular abscess; ( i i ) exudate is infective 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 infection which penetrates the peritoneal cavity and/or perforates the skin within 18 hours. The animal loses hair and necrosis of the skin occurs in the abdominal region. The fascia 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 bacteria, white blood cells 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 infection to a second animal. Successful trans-missible infections in 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 strain of 15. melaninogenicus was infective 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 strain was so harmful that rabbits frequently died during the immunization period. Therefore, the potential pathogenicity of the strain was suspected. I5_. melaninogenicus, except for two reported cases (2 1 5,1420 , does not possess any known capsular material or ahti-phagocytic surface components. However, some strains can elaborate cell-associated and extracellular enzymes which may enhance their invasive properties. These enzymes include collagenase (69), proteases and 14 hyaluronidase (1). Kestenbaum (106) demonstrated a positive correlation between collagenase activity and infectivity for four melaninogenicus strains in a guinea pig system. Collagen degradation is a feature of periodontal disease (131,206) and although ]}. melaninogenicus is 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 in oral lesions or in other mixed anaerobic infections remains unclear. Whether factors other than collagen-ase were involved in Kestenbaum's system is not known. Enhance-ment of a fusobacterial infection in rabbits by simultaneous injection of a crude cell-free preparation of B_. melaninogenicus collagenase was demonstrated by Kaufman (101). Heating eliminated both enzyme activity and the effects of the extract on infection, suggesting that collagenase may play a role in the organism's pathogenicity. This does not exclude the possibility 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 infection (134) resulting in extensive fluid accumulation. A pathogenic strain of B_. melanino- genicus ss. asaccharolyticus was reported to possess a heat 15 sensitive toxin which induced fl u i d accumulation in ligated mouse i l e a l loops (146). Vibrio cholerae toxin induces a similar response in the gut (168) . Preliminary experiments by B.C. McBride (personal communication) have indicated that culture supernatants obtained from 13. melaninogenicus ss. asaccharolyticus possess a cholera toxin-like activity as measured by the vascular permeability assay described by Craig (36). The activity i s not as strong as that of V_. cholerae and is lost when the cells are subcultured repeatedly. Toxin i s injected intracutaneously into the skin of a shaved guinea pig, and after a suitable interval, a blue dye is injected intracardially. The dye complexes with serum proteins and passes through capillary walls in areas where vascular permeability has been increased by toxin. The resulting blue area around the site of inoculation i s then measured. The ' assay is simple and sensitive. 3. Antibiotic Sensitivity A study on antibiotic susceptibility (232) showed that 13. melaninogenicus ss. asaccharolyticus was sensitive to p e n i c i l l i n , cephalosporins, bacitracin,chlortetracyclin, chloramphenicol, erythro-mycin and rifampicin and resistant to streptomycin, c o l i s t i n , polymyxin B and neomycin. Finegold (55) has found stock strains of 13. melanino- genicus to be uniquely resistant to kanamycin and vancomycin and has suggested that the isolation of B_. melaninogenicus from heavily contaminated source material would be facilitated by the incorporation of these antibiotics in the media. Loesche and Hockett (129) reported the resistance of certain strains of I3_. melaninogenicus to kanamycin 16 and that the addition of this antibiotic to a culture medium facilitated the primary isolation of the organism from source material such as dental plaque. A more recent study (189) showed that sensitivity to vancomycin and c o l i s t i n w i l l differentiate between saccharolytic and asaccharo-l y t i c strains. Most asaccharolytic strains were sensitive to vancomycin and resistant to c o l i s t i n whereas both saccharolytic groups were re s i s t -ant to vancomycin and sensitive to c o l i s t i n . 4. Antigenic Structure and Serological Heterogeneity The antigenic composition of bacteroides cells has been shown to be species-specific and potentially useful in speciation (39). However, only few studies have attempted to group strains of 13. melaninogenicus on the basis of serology. Weiss (230) extracted protein from two strains of 13. melaninogenicus which were immunologic-al l y distinct. Shevky 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 variety of serologically heterogeneous strains". The antigenicity of thirteen strains of I3_. melaninogenicus isolated from various sources was studied by Courant and Gibbons (35) who concluded that 13. melaninogenicus strains were serologically heterogeneous, and seemed to represent a spectrum of serotypes. More recently, a fluorescent antibody procedure was developed (115) which showed that human 13. melaninogenicus strains could be divided into three specific serogroups according to the bio-chemical subspecies already known. The fluorescent antibody conjugates were specific 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 cellular antibody (162) . The results indicated that I5_. melaninogenicus resident in 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 infection by the microorganism. 5. Genetic Properties Genetic studies on 13. melaninogenicus are scarce and no specific details have been published concerning the genetic properties and v a r i a b i l i t y of the organism. Colony form is usually smooth but rough variants are found (215). Pigmentation is also noticeably variable being generally black, but different shades of brown can be observed. Other properties, such as the requirement for heme and vitamin K, hemagglutinin, collagenase and protease production might also reflect some genetic variation, although this has not been proven. melaninogenicus has not been found to possess plasmids nor has i t been shown to acquire plasmids from different Gram-negative species (27). Antibiotic resistance is not related to the presence of plasmids (41). Bacteriophages capable of infecting B_. melaninogenicus have not been isolated (103). 6. Outer Membrane of 13. melaninogenicus Lipopolysaccharides (LPS) are located in 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 . They represent the 0 antigen and the endotoxins of these organisms (99). Endotoxic. LPS, consisting of three major components, i.e. fatty acids, saccharides and sometimes bound amino 18 acids,, i s considered to i n c l u d e three main regions of c o n t r a s t i n g chemical 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 polysaccharide (re g i o n I ) , c a r r y i n g the 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 to the core polysaccharide (region 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 core i s l i n k e d through 2-keto-3-deoxyoctonate (KDO) to 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 aerobic Gram-negative b a c t e r i a have been the subject of d e t a i l e d i n v e s t i g a t i o n f o r many years. Considerable 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 of the chemical 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 immuno-g e n i c i t y of these outer c e l l membrane-localized antigens (17,99,133). The LPS of Salmonella i s an important v i r u l e n c e f a c t o r ; l o s s of i t s 0 - s p e c i f i c s i d e chains r e s u l t s i n l o s s of v i r u l e n c e but has no e f f e c t on endotoxic p r o p e r t i e s . Endotoxins exert 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 only 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 evident a f t e r adsorption of endotoxin onto the host c e l l u l a r membrane (17). Lipo p o l y s a c c h a r i d e (LPS) antigens of anaerobic Gram-negative b a c t e r i a have rece i v e d f a r l e s s study, because the importance of these 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 not f u l l y appreciated 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 f e a s i b l e . Some anaerobic Gram-negative b a c t e r i a c o n t a i n LPS which i s ch 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 to the endotoxins of aerobic b a c t e r i a (92). However, i t i s of i n t e r e s t that Bacteroides f r a g i l i s and JB. melaninogenicus appear to have ra t h e r unusual LPS(92,100). In s t u d i e s of these two s p e c i e s , Hofstad has noted the predominance 19 of fatty acids and neutral sugars with the absence of the sugars 2-keto-3-deoxyoctonate (KDO) and heptose, which are found uniformly in the LPS of aerobic Gram-negative bacteria. The isolation and purification 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 typical of a Gram-negative organism. With the use of gentle techniques of heat, EDTA treatment, shearing, and differential centrifugation, the outer membrane was isolated. A relatively pure preparation was suggested by the absence of nucleic acids and muramic acids, the existence of relatively 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 deoxy-cholate 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 this f i r s t component was demonstrated by agar gel diffusion. Analysis of the protein by SDS-poly-acrylamide gel electrophoresis of three strains revealed strain specificity. Further purification of this fraction showed that the polysaccharide component cross-reacted with antiserum to another strain of the same subspecies. This component probably represents the capsular antigen and may prove to be the basis of serogrouping. The second membrane fraction differed chemically from the f i r s t fraction and represents the lipopolysaccharide component of the 20 outer membrane. It consisted mainly of loosely bound l i p i d (62%), protein (5%) , and polysaccharide which was clearly distinct from that of the f i r s t fraction. Notably, this component lacked 2-keto-3-deoxyoctonate, one of the backbone components of aerobic, Gram-negative lipopolysaccharides. Purification 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 for better study of the mechanisms of immunity to infections involving this organism. Demonstration of serologic cross-reactivity 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, in the running buffer. The LPS was composed of loosely bound l i p i d (62%) and carbohydrate (32%) , with less than 5% protein. Glucose, galactose, and glucosamine were the major sugars as detected by gas-liquid chromatography (GLC). Heptose and KDO were not observed by colorimetric analysis. 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 cyclic or odd chain fatty acids, were detected. ]3_. melaninogenicus LPS preparation did not induce skin reactions in rabbits when administered in doses of up to 1 mg, compared to Salmonella typhi endotoxin which elicit e d a positive reaction in doses of 12.5 yg. L i t t l e or no endotoxic activity was demonstrated. 21 These findings are compatible with previously noted observations on LPS in anaerobic bacteria (90,100), a l l of which stand in sharp contrast to the widely known biologic activity of aerobic Gram-negative LPS. This may explain the rarity of septic shock in patients infected with anaerobic organisms. It is intriguing to speculate that the unusual pattern of fatty acids in the l i p i d A, may be responsible for the biologic impotence of the LPS of 13. melaninogenicus (145). The presence of capsular polysaccharide contaminating the phenol/water-extracted LPS was reported (142), and may explain partially the serologic heterogeneity which has been described previously in studies of the LPS of this organism (91). The factors within the LPS which determine serologic activity are extremely complex. Characterization (142) of a relatively homogeneous LPS may make further investigation of the pathogenic mechanisms and immune response to Ii. melaninogenicus somewhat more clear. The l i p i d s and related compounds in 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 are phosphosphingo-l i p i d s . The two major phosphosphingolipids have been characterized as ceramide phosphorylethanolamine (CPE) and ceramide phosphorylglycerol (CPG). The finding of phosphosphingolipids in bacteria is 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 in that part of the extractable fatty acids was present as phospholipid and that phosphatidyl ethanolamine was the predominant diacyl phospholipid. 13. melaninogenicus i s unusual in that i t contains only a small amount of non-extractable fatty acids which are usually found to be associated with the polysaccharide, and in the absence of (3-hydroxy fatty acids usually found in l i p i d A of the outer membrane of Gram-negative bacteria. The diacylphospholipids of 13. melaninogenicus consist of phos-phatidylethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid and cardiolipin. It has been reported that these bacteria do not contain glucolipids (167). Vitamin isoprenologues make up the bulk of neutral lipid s (178) which represent a small portion of the extractable fatty acids. The l i p i d s seem to be localized in the membrane fraction of I3_. melaninogenicus along with cytochrome c. The pho pholipid found in the supernatant fraction might represent very small membrane fragments (178). There was not much change in the total amount of vitamin and phospholipid in the membrane of 13. melanino- genicus grown with different levels of protoheme supplementation in the medium (178). Hemagglutinating Activity and Adherence Properties of B. melaninogenicus 1. Adherence Adherence has recently been found to be an important ecologic-a l factor in specific 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 is one requirement for colonization and in some cases f a c i l i t a t e s the invasion of that host (73). It has been recognized that Vibrio  cholerae adhere to the intestinal mucosa (61) and that the i n a b i l i t y to do so results in a reduction in virulence (63,186). Ellen and Gibbons demonstrated that virulent strains of Streptococcus pyogenes adhered well to epithelial cells whereas an avirulent strain lacked this a b i l i t y (51). Adherence is also an important ecological determinant which influences the colonization of bacteria in environments subject to the flow of liquids (73). It was shown, as early as 1954, that certain oral bacterial species preferentially colonize different sites within the mouth (112). Gibbons has shown that this selective colonization of oral bacteria is correlated with selective bacterial adherence (70). Despite the recognition of the selective adherence of oral bacteria, there i s limited information concerning the mechanism of adherence. Tentative conclusions as to the nature of the adherence of bacteria were based on either direct microscopic examination or on the results of enzymatic, physical and chemical pretreatments (125,209). However, one must be careful interpreting these data because of the gross nature of the various treatments. It was postulated that for some organisms, adherence is 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 bacterial adherence to epithelial cells with the possible involvement of neuraminic acid receptors on the host cells (209), and in other cases the possible involvement of 24 carbohydrate and teichoic acid moieties on the bacterial cells (64). Adherence can generally be classified according to: (i) attachment to a body surface, i.e. buccal epithelium or tooth ( i i ) attachment to other bacteria, i.e. plaque. Attachment involves a specific, unique receptor site on the bacterial c e l l surface and a corresponding site on the substrate to which the organism adheres. The relatively high proportions of I3_. melaninogenicus ss. asaccharolyticus found in periodontal pockets (198) and the potential of this organism to synergistically produce mixed anaerobic infections in experimental animals (72,134,139,205), suggest that i t may play an important role in the etiology and pathogenesis of perio-dontitis. L i t t l e i s known about the parameters which influence the colonization of 13. melaninogenicus in periodontal pockets or in other sites of the mouth, and l i t t l e information is available about the mechanisms of B_. melaninogenicus retention and prevalence in the oral cavity. Recently, Slots and Gibbons (199) and Edwards (50) initiated studies to determine the a b i l i t y of 13. melaninogenicus ss. asaccharo- lyticus to attach to various oral surfaces, and to evaluate the role that adherence might play in i t s oral and subgingival colonization. They found that I3_. melaninogenicus cells suspended in phosphate-buffered saline adhered well to buccal epithelial cells and to the surfaces of certain Gram-negative bacteria that are prominent in human dental plaque. They also reported that of forty eight asaccharolytic strains of B_. melaninogenicus, forty seven agglutinated human erythrocytes (199). Their data indicated that certain Gram-positive organisms found in dental plaque possess receptors for the attachment 25 of 13. melaninogenicus cells and that these receptors are different from those present on buccal epithelial cells and erythrocytes. 2. Hemagglutinin Macromolecules which react with specific components of red blood c e l l membranes leading to agglutination of these cells are not uncommon among certain bacterial species. For organizational purposes, the literature survey of bacterial hemagglutination is considered separately from studies using cells and surfaces other than erythrocytes. Although recent reviews on bacterial adherence have often overlooked the literature on bacterial hemagglutination, there is no evidence to suggest that adherence of bacteria to cells other than erythrocytes is a different phenomenon from bacterial hemagglutination (72,73). In fact, studies using hemagglutination assume that this phenomenon is an index of bacterial attachment and, at least in some cases, adherence to epithelial c e l l s has exactly the same characteristics as bacterial hemagglutination (160). Therefore, one way of studying adherence is to evaluate the hemagglutinating activity of an organism using model adherence systems of erythrocytes as relatively simple, well-characterized natural surfaces. The a b i l i t y of certain bacteria to agglutinate red blood cells was demonstrated as early as 1955 (45). In later studies, a great deal of significance was placed on the role of p i l i in hemagglutination (28,48,49,72,160,191). From studies using piliated and non-piliated enterobacteria, i t appeared that the presence of p i l i did not always seem to be necessary in order for hemagglutination to occur (46,47,224). It was concluded that there are several different characteristics 26 between a piliated and a non-piliated hemagglutination reaction. Among these there are differences in agglutination range of erythro-cyte species, elution of bacteria from erythrocytes at high temper-ature, and inhibitory effects of D-mannose (46,47,224). Although much work has been done on the adherence of oral bacteria to epithelial cells and other surfaces, l i t t l e has been done on the hemagglutination activity of the organisms. Recently, i t was reported that Streptococcus sanguis, Streptococcus mitis, and Actinomyces viscosus a l l agglutinate human red blood cells (180). Studies on the hemagglutin-ating properties of anaerobic bacteria have been minimal. Okiida and Takazoe (161) found that twenty nine of fifty-nine strains of I3_. melaninogenicus studied had hemagglutinating activity that seemed to be mediated by surface p i l i . They suggested that these structures might also mediate the attachment of B_. melaninogenicus cells to oral mucosa. Slots and Gibbons (199) reported that forty-seven of forty-eight asaccharolytic strains of 13. melaninogenicus representing fresh isolates from subgingival plaque and tonsillar swabbings agglutinated human erythrocytes, whereas none of twenty fermentative strains, 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 epithelial c e l l s . Thus, no clear relationship exists between the hemagglutinating activity of strains of 13. melaninogenicus and their a b i l i t y to attach to buccal epithelial c e l l s . The observations further suggested that several 27 types of p i l i exist on different strains and subspecies of B_. melaninogenicus (199) . C. Proteolytic Activity of B_. melaninogenicus It has been known for many years that many microorganisms produce appreciable amounts of proteolytic enzymes. The bacterial proteases are instrumental in the degradation of complex protein substrates to amino acids and peptides in 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 this may be that some proteolytic enzymes are unstable and susceptible to autodigestion; consequently, purification 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: fractional precipitation by pH changes (5), fractional denaturation by heat, fractional precipitation by salts ( i l ) , fractional precipitation with organic solvents (9), fractional adsorption (166), column chromatography (5,11) and crystallization (9). The sequence in which several or a l l of these steps are used is determined by the enzyme under investigation. There are many published reports on the purification of bacterial proteases. The following covers a few selected examples concerning the purification of cellular and extracellular proteases produced by different organisms. The extracellular protease of Pseudomonas maltophilia was partially purified by ammonium sulfate precipitation 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 was purified 80-fold by (NH^^SO^ precipitation, electrophoresis and gel f i l t r a t i o n through Sephadex G-200 (15). Once a proteolytic enzyme is discovered and partially purified, i t is interesting to investigate i t s chemical and physical properties; Inform-ation in these areas is important in order to classify the enzyme and to determine i t s function in vivo. In the past, proteolytic enzymes have been classified by several c r i t e r i a . One classification grouped proteo-l y t i c enzymes into the categories pepsin-like, trypsin-like or cathepsin-lik e . This system is based mainly on the pH optimum of the enzyme. Extracellular proteases from microorganisms have been classified 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 their action on synthetic substrates. A system such as this t e l l s the investigator something of the mode of action of a particular enzyme but this method also has i t s limitations. Enzymes of different origins may produce essentially the same action on synthetic substrates but may differ in their reactivity towards natural substrates. Another system was proposed (6) which divided the proteolytic enzymes into categories based on their behaviour toward a number of proteolytic inhibitors. Systems such as those mentioned have their inherent shortcomings. For example, i f a particular enzyme was classified as trypsin-like, one might assume that i t possessed a l l the other chemical and physical properties of trypsin whereas in reality i t did not. It is necessary therefore to characterize the proteolytic enzyme in as many ways as possible. 29 Characterization of a protease should include the determination of the site of hydrolytic attack on natural substrates, the action of the enzyme on various synthetic substrates, the nature of the reactive site of the enzyme, the response to inhibitors, the pH and the temperature st 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 specificity l i k e , or is affected by one of the inhibitors of the well-known and thoroughly studied groups of enzymes, i t is preliminary referred to as belonging to this class of enzymes. It should be noted, however, that some proteolytic enzymes do not f a l l into the major categories which are based on mechanism of action rather than origin of physiological action. These categories include four principal classes of enzymes. The f i r s t of such main groups of enzymes is the serine proteases, which are distinguished by a serine residue in the active site (86). A common test for these enzymes is the inhibition of their hydrolase activity by the reaction of this serine residue with diisopropylphosphorofluoridate (DFP). Examples include enzymes isolated from ]}_. subtilis and related strains (subtilisins) as well as proteolytic enzymes isolated from organisms such as Streptomyces griseus (227). The second group of proteolytic enzymes are dependent on sulfhydryl groups for their catalytic activity. Activation of these enzymes is usually achieved by mild reducing agents such as cysteine, sulfide and sul f i t e , which liberate a free t h i o l group on the enzyme. L i t t l e , i f any, conformational change i s associated with reduction (8). Optimum activation was found to occur upon simultaneous application of a thiol compound such as cysteine or thioglycolate and a heavy metal-binding agent like 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 in the presence of air and can be reactivated by addition of reducing agent. Heavy metal ions such as Cd^ +, Zn^ +, Fe^ +, 2+ 2+ 2+ Cu , Hg and Pb are inhibitory. The metal inactivated enzymes can be totally reactivated by addition of a reducing agent and a chelating agent. The readily reversible formation of a stable inactivate complex with mercury has been utilized as a useful step in the purification of the well known sulfhydryl enzyme, papain (21,108). A l l sulfhydryl-binding reagents can act as sulfhydryl enzyme inhibitors. Thus, p-chloromercuribenzoate forms a stable complex with the enzyme and can serve for ti 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 irreversible inactivation (109,190). Papain was found to react with the chloromethyl ketones of phenylalanine and lysine (TPCK and TLCK) with total loss of activity (233). In this case, the reagents act specifically on the active sulfhydryl group of the enzyme rather than on the imidazole group of particular h i s t i d y l residues as they do in the case of trypsin and chymotrypsin, and thus the inactivation of papain is a stoichiometric reaction. Examples of bacterial sulfhydryl enzymes are the streptococcal proteinases (53,114). Another group of proteolytic enzymes includes the acidic proteases. The presence of proteolytic enzymes with a pH optimum in the acid pH range (pH 1-5) has been reported in a variety of microorganisms where they occur both intracellularly and extracellularly. Several strains of Clostridium (C. acetobutylicum and C_. butyricum) (225) and Lactobacilli (18) have been shown to produce weak proteinase activity with an acid pH optimum. 31 There is also the group of metal proteinases which includes enzymes specific for releasing Nl^-terminal amino acids, such as aminopeptidase-P. ' This enzyme is an exopeptidase cleaving the bond between any N-terminal amino acid residue and a following proline residue (241) and is isolated from Escherichia c o l i . Thermophilic aminopeptidase-APl, produced by Bacillus stearothermophilus splits a l l amino acids from the amino end of a polypeptide; preferentially hydrolyzing peptides containing leucine, valine, and those with aromatic amino acid residues. The following are some reported examples of protease characterization. The subtilisins are alkaline proteases of broad specificity produced by strains of B_. s u b t i l i s . Three of these enzymes have been studied in considerable detail and are probably the best known of a l l microbial proteases. The subtilisins are specifically and stoichiometrically inactivated by DFP (144) , indicating that they are serine proteases. The striking feature of the subtilisins may be that their sequences bear no significant relationship to those of the pancreatic serine proteases, whereas their status as serine proteases carries the obvious implication that the active sites are in some way similar in structure to those of the pancreatic enzymes. It is nevertheless clear that there are considerable differences in the spec i f i c i t i e s of the subtilisins and the pancreatic serine proteases. Studies of the hydrolysis of ester substrates (76) suggest that, in contrast to the high specificity of chymotrypsin and trypsin, the subtilisins have rather low specificity. A number of interesting DFP-sensitive proteases from strains of Staphylococcus aureus have been studied by Drapeau and co-workers (42) and are reported to have a high specificity for glutamyl and aspartyl residues. 32 The staphylococcal enzymes thus do not resemble any of the previously known serine proteases. Streptococcal proteinase is a sulfhydryl enzyme which is elaborated by group A streptococci. It is excreted into the medium as a zymogen which is 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 readily inactivated by reagents known to react with sulfhydryl groups, such as iodoacetic acid, 2+ iodoacetamide, p-chloromercuribenzoate, Hg , and atmospheric oxygen. Streptococcal proteinase thus appears to be a classic sulfhydryl enzyme. There are comparatively few reports of extracellular 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 extracellular proteases are largely confined to the pseudomonads. This situation is also true of exoenzymes in general, and may be a reflect-ion of the difference in complexity of the c e l l envelope between Gram-negative 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 purification and properties of a protease from Pseudomonas maltophilia which is an EDTA-sensitive alkaline serine protease. Nakajima and co-workers (157) described an alkaline protease of Escherichia freundii which was sensitive to EDTA and 33 had a molecular weight of 45,000. The protease enzymes from Serratia sp. (151) resembled the subtilisins in that they have alkaline pH optima with casein as substrate; in other respects, however, they were not similar. 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 extracellular proteinases and other hydrolytic enzymes is most reasonably a nutritional one which evolved to allow the microorganism growing in i t s natural environment to u t i l i z e complex non-diffusible substrates as a source of nutrients. In addition to the nutritional role, extracellular proteinases of the genus Bacillus are thought to be required for sporulation (37) and thus clearly have an intracellular function in a specific developmental process. Limited proteolytic degradations are responsible for the induction of biological a c t i v i t i e s , as in the formation of biologically active enzymes from their inactive precursors. The i n i t i a l step in proteolytic de-gradation might be the opening of one or a few exposed peptide bonds, or the splitting of a small amount of unfolded protein in equilibrium with the native protein. An example of induction of biological activity by limited proteolysis i s the conversion of the extracellular zymogen of Group A streptococci to the active enzyme (53,127). The role of a protease 34 in natural activation of Clostridium botulinum neurotoxin has been reported by Bibhuti et^ a l . (12). The specific toxicity of the simple protein increases during incubation of the culture. Since the conversion of progenitor toxin to the more toxic form can be accomplished with trypsin, one mechanism for the natural activation of progenitor toxin would be through the action of a suitable enzyme(s) produced by the culture. One such enzyme is a protease with trypsin-like specificity which activates progenitor toxin obtained from young cultures of the same proteolytic type B strain (38). Catalytic processes involving specific enzymes in the membtane may be involved in secretion of exoenzymes. A protease located in the outer membrane of E_. c o l i was reported to cleave a protein located in the cyto-plasmic membrane, the respiratory enzyme nitrate reductase. This cleavage is accompanied by solubilization of the enzyme (140). The importance of intracellular proteolytic activity in the physiology of the bacterial c e l l has been implicated and might include roles in: protein turnover leading to continued regeneration of labile proteins, increased proteolytis during c e l l division, proteolytic maturation of proteins, and preferential breakdown of structurally altered proteins (169). It has been shown that the autolysin of Streptococcus faecalis is present in an inactive form in the c e l l wall but is activated by a neutral proteinase; and that the active form of the autolysin is 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 and for investigation of hydrolysis products which possess biological activity. This is exemplified in the case of the structure and the biochemistry of diphtheria toxin. The toxin molecule is released from the bacterial c e l l as a single polypeptide chain having two non-overlapping cystine bridges. The toxin contains a protease sensitive site which is readily hydrolyzed to yield two sulfhydryl linked polypeptides. One polypeptide is responsible for binding the toxin to i t s target c e l l ; the second hydrolysis product is responsible for inducing the biochemical lesion in the protein synthesizing system of the c e l l . A sequence containing three arginine residues is presumed to represent an exposed loop in the intact molecule since i t is abnormally sensitive to proteolytic attack. Short treatment with proteases with trypsin-like specificity yielded two large peptides, an amino-terminal fragment A and a carboxyl-terminal fragment B (74) , which facilitated further studies on the structure and characteristics of the toxin molecule. Bacteroides melaninogenicus has been shown to possess proteolytic activity but this activity has not been characterized. An organism dependent on peptides for growth (228) might be expected to be actively 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 readily dissimilate peptides (228) . Many strains of B_. melaninogenicus have been observed by Sawyer et_ a l . (185) to be proteolytic, and the organisms appear to grow well in culture media without carbohydrate supplementation, suggest-ing that the fermentation of proteinaceous constituents play an important role in the metabolism of B_. melaninogenicus. Hausman and Kaufman have found caseinolytic activity associated with a particulate fraction 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 gelatin. Hydrolysis of proteinaceous substrates by B_. melaninogenicus was also reported by Weiss (230), Schwabacher et a l . (187) and Pulverer (173). Burdon (26) reported that his asaccharolytic strains of B_. melaninogenicus were highly proteolytic, attacking gelatin, coagulated serum, egg albumin and milk. The protease found in these organisms is not l i k e l y to be collagenase since casein was attacked. Gelatin, the denatured form of collagen, is 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 is resistant to other proteolytic enzymes. I3_. melaninogenicus may elaborate more than one protease, as activity has been demonstrated in the washed cells as well as in the supernatant. The cellular and soluble. proteases of B_. melaninogenicus which hydrolyze gelatin are also active against a number of protein substrates including azocoll, casein, azocasein, and N,N-dimethylcasein. For the azocoll assay, the rate of dye released from the dye-protein conjugate reflects the proteolytic activity in the sample. This assay is usually qualitative rather than quantitative. Casein, having many different potentially .susceptible bonds, is 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 . The assay depends on the determination of the amounts of TCA soluble peptides liberated from the 37 casein substrate by the enzyme as detected by measuring absorption at 280 nm. This assay is not extremely sensitive, and is therefore not useful for measuring small amounts of proteolytic activity. Among the reasons for i t s relative insensitivity, the most important seems to be i t s high background reading. The assay also f a i l s to measure a l l bond cleavages. Hydrolysis of small numbers of bonds in such an assay would be expected to result in larger peptides, proportionally greater numbers of which would, because of their size, be precipitated by trichloroacetic acid and not be distinguished from uncleaved protein. In addition, because the assay relies upon the absorbance of soluble peptides at 280 nm and such absorbance varies from one peptide to the next, equal degrees of proteolysis by different enzymes do not result in 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 activity is followed by determining, with TNBS, the new amino groups produced after hydrolysis. The low background values obtained with N,N-dimethylcasein results in greater sensitivity and accuracy not possible with the unmodified casein. 14 The use of C-labeled N,N-dimethylcasein as a substrate for determining total proteolytic activity offers several advantages over other methods. The assay is more sensitive than spectrophotometric procedures. The labeled substrate is stable and can be stored for a long period of time; the assay is rapid and is not affected by the presence of large concent-rations of peptides or amino acids in the sample to be assayed. 38 Another assay is based on the solubilization of a covalently linked chromophore from a modified protein. An example of this type of substrate is azocasein. After incubation with the enzyme, the unhydrolyzed protein is precipitated and hydrolysis products containing coupled dye are quantitated spectrophotometrically. The important point is that the absorption maximum of the covalently linked chromophore is different than that of chromophores contaminating the enzyme preparation. In 1962, in a review that has become a landmark in the f i e l d , Pollock (170) defined an extracellular enzyme as one that "exists in the medium around the c e l l s , having originated from the c e l l without any alteration to c e l l structure greater than the maximum compatible with the cel 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 result of c e l l l y s i s , aging and leakage during division. A consideration of the possible mechanisms involved in 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 structural membrane proteins and enzymes, are inserted into the membrane basic phospholipid bilayer. May and E l l i o t t reported that a protease was secreted from I5_. subtilis cells apparently as i t was synthesized since there was no significant intracellular accumulation. They speculated that none of the enzyme molecules were ever present in a completed form inside the c e l l membrane but rather that the nascent polypeptide chain was extruded through the membrane as i t was synthesized to take up i t s tertiary structure with 39 enzyme activity only on the outside (147). A specific hypothesis to explain protein excretion, the signal hypo-thesis, has been developed. An elongation of peptide chain on membrane-bound ribosome results in discharging the nascent chain across the membrane; the signal sequence for excretion is then removed from the polypeptide chain by proteolytic cleavage, which was reported to be in the outer membrane fraction in 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 extracellular proteases may be involved in the i n i t i a l processing of proteins in the environment (82). The release of lipopolysaccharide-phospholipid-protein complexes from E_. c o l i has been observed for growing and stationary phase c e l l s . The outer membrane fragments were preferentially 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. licheniformis. After protoplast formation the enzyme i s found associated with vesicles (183,184). The outer layers of the c e l l envelope, particularly in Gram-neative bacteria, would also pose a barrier to exoprotein secretion. Certain enzymes found outside of the cytoplasmic 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 location of enzymes in bacteria has been determined by a variety 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 availability to substrates and inhibitors, elutability by nondamaging solvents, inhibition by specific antibodies and release by osmotic shock or by such compounds as polymyxin B. Preparation of protoplasts in stabilizing media with measurement of enzymes released, indicated the location of the liberated enzyme outside the permeability barrier in the intact c e l l (183). The outer membrane layer contains charged moieties but i t is 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 in association with mucopeptide (194) , with various components in the periplasmic space (30) , with lipopolysaccharide or protein of the outer membrane (96) or to be released into the menstruum (127). Clearly, the properties of the enzyme such as hydrophobicity and charge w i l l have a bearing on the location of proteins relative to the cytoplasmic membrane. Studies on the alkaline phosphatase (APase) of Pseudomonas aeruginosa by Ingram e_t a l . (96) showed that a certain percentage of the enzyme was complexed with -lipopolysaccharide which was also released during secretion. Phosphatase i s located in three areas: the culture f i l t r a t e , the outer c e l l wall surface, and the periplasmic space. The results suggest that APase may become associated with, and bound to, a c e l l wall fraction which contains LPS and liberation of the complex from the outer wall may be accomplished by mechanical shearing forces developed during growth. Cell suspensions of Micrococcus sodonensis secrete seven to ten individual proteins including an alkaline phosphatase and a protease. The appearance of enzyme acti v i t i e s in the extracellular medium was found 41 to be dependent on the co-secretion of at least one of several poly-saccharides (19). A functional membrane-bound enzyme, the galactosyl transferase system of Salmonella typhimurium, was reconstituted in vitro from purified components including lipopolysaccharide, phosphatidyl ethanolamine and enzyme protein (181). MacGregor reported a proteolytic activity which was found in extensively washed membrane preparations (140). This membrane-bound protease was found to be responsible for the cleavage and solubilization of nitrate 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 activity found in E_. c o l i i s associated with the membrane. This membrane-bound protease was found to have many characteristics in common with trypsin. As many microorganisms are known to produce extracellular proteolytic enzymes, several studies have been carried out on the regulation of the production of extracellular proteases by Gram-positive bacteria, especially Bacillus strains. However, only a few detailed reports have appeared on Gram-negative bacteria (77). Among these organisms there are marked differences in the way in which environmental factors affect enzyme production (234). In general, induction, end product inhibition and catabolic repression have been implicated in the regulation of the synthesis of these enzymes. An efficient regulatory control has been described by Tanaka and Tuchi (218) for Vibrio parahaemolyticus. In this organism the production of a protease was induced by amino acids and was subject to catabolite repression by easily metabolizable carbon sources. 42 Repression of protease synthesis by amino acids has been widely reported as an example of end product repression in bacteria of the genera Bacillus, Serratia and Arthobacter (77), but does not appear to occur in a l l Gram-negative organisms that have been studied (218) . The production of microbial cellular and extracellular enzymes has been investigated extensively in batch cultures, but applications of continuous culture techniques in these studies have not been widespread (22,77). In the studies that have been reported, the production of constitutive and inducible enzymes followed one of two general patterns (Fig. 1), and i t is considered that the relationship between enzyme production and growth rate depends on the characteristics of the regulatory mechanism involved (22). For constitutive enzymes where the rate of enzyme production is a function of the product of c e l l concentration and growth rate, the relation-ship between the rate of enzyme production and dilution rate for such enzymes is linear (Fig. 1A). This has been observed for the penicillinases pro-duction by Bacillus licheniformis by Wouters and Buysman (239) and for several other enzymes (31,40). An example of this is seen (Fig. 2) where the content of amidase per c e l l of Pseudomonas aeruginosa rises to a peak as the growth rate (dilution rate) is increased and the content then f a l l s as the growth rate is increased beyond this point (31). A non-linear relationship between rate of enzyme production and dilution rate is sometimes found for inducible enzymes (Fig. IB). Such a behaviour is particularly apparent when organisms producing such enzymes are grown under conditions where the inducer is the growth-limiting sub-strate (40). The advantages of continuous culture are undeniable in fields of microbial biochemistry and metabolism (23,219), and chemostat experiments 43 have made a valuable contribution in 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 nutritional and other environmental conditions. The chemostat offers possibilities which are absent in any closed culture system. During growth in batch culture microbes continuously change their environment as a reuslt of consumption of nutrients and accumulation of waste products, therefore, the morphological and metabolic properties of the cells are apt to change during the growth period (226). In the chemostat cells can be grown in steady states at any of a whole range of growth rates. In addition, i t is usually possible to make any substrate growth-limiting. Thus, the nutritional 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. The culture then has become time-independent, and conditions are stable. Therefore, experiments with the chemostat are highly reproducible and are ideal for studying the properties of an organism as a function of growth rate. Growth rate of a culture can be varied by changing the dilution rate, the total c e l l mass of the culture remaining the same. However, at extremely low growth rates, a proportion of the cells may become non-viable and the growth yield, Y, which is 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 dilution rates is to be expected only when growth i s limited by the substrate whose metabolism supplies energy. FIGURE 1. General patterns of enzyme production in continuous culture. A. Constitutive enzymes whose synthesis only depends on c e l l concentration. B. Inducible enzymes. FIGURE 2. The effect of dilution rate on the synthesis of amidase by Pseudomonas aeruginosa growing in a chemostat under steady-state conditions. 46 In the case of c h e m i c a l l y complex media, where there i s an u n s a t i s f a c t o r y vagueness about the 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 that growth i s u l t i m a t e l y r e s t r i c t e d by the exhaustion of some one substance, other substances remaining 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 not to a f f e c t the growth r a t e on t h e i r own account. For example, i n the case of 13. melaninogenicus, which r e q u i r e s a complex medium f o r growth due to i t s o b l i g a t e requirement f o r hemin and peptides, hemin can thus be s i n g l e d out and c a l l e d the " l i m i t i n g s u b s t r a t e " . In continuous c u l t u r e s , the r a t e of b a c t e r i a l growth may be regulated by c o n t r o l l i n g the 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 expressed as the d i l u t i o n r a t e D, which i s the volume of n u t r i e n t added hr expressed as a f r a c t i o n of the volume of the v e s s e l . The d i l u t i o n r a t e w i l l determine the length of time that 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 the chemostat and i n the absence of b a c t e r i a l c e l l d i v i s i o n dx where x i s the number of b a c t e r i a present i n the v e s s e l . In f a c t , 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 defined by -Sr where K i s the growth constant. 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 defined by r a t e of 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 dilution rate, soon establishes steady state conditions: there i s no change in bacterial numbers. This indicates that bacterial growth exactly balances the bacteria lost by dilution: Kx = Dx or K = D When the dilution rate approaches the maximum growth rate and eventually exceeds i t , more bacteria are washed out than are produced by c e l l division and the bacterial concentration f a l l s (87). 48 II. MATERIALS AND METHODS A. Organisms B_. melaninogenicus subspecies asaccharolyticus strain K110 was obtained from Dr. P.A. Mashimo. This is a collagenolytic strain originally isolated by Macdonald and co-workers (134) from a patient with diagnosed gingivitis. melaninogenicus subspecies asaccharolyticus strain 2D was isolated in the laboratory from a gingival scraping taken from an individual with perio-dontal disease. The gingival sample was streaked on freshly poured blood agar plate and incubated at 37°C in a ^ " . ^ i C ^ (85:10:5) atmosphere. Black colonies were repeatedly subcultured on the same medium un t i l a pure culture was obtained. The isolate was characterized according to standard procedures (94) . Neither strain K110 nor 2D required vitamin K for growth. B. Growth 1. Anaerobiosis Liquid or agar cultures were usually incubated in anaerobic jars (Torball, Torsion Balance, Clifton, New Jersey) evacuated and flushed with U: CO2 (95:5) (Canadian Liquid Air, Montreal) or in an anaerobic glove box (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 levels in the chamber were 49 monitored every two days with a trace-oxygen analyzer (Lockwood and McLorie, Inc., Horsham;, Pa.). An oxygen level of two to five ppm was considered to be acceptable. In some instances, organisms were cultured in pre-reduced media in 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. The pH was adjusted to pH 7.0 with HC1 or NaOH. The hemin solution was made up by dissolving 0.05 gm of hemin in 1 ml 1 N NaOH and 100 ml d i s t i l l e d water. This solution was stored at 4°C-b. Basal medium. The basal medium consisted of trypticase (17 g/1); yeast extract (3 g/1); K2HP04 (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 in hemin-free liquid media (146). In order to obtain cells in a hemin-deficient state i t was necessary to grow them once in basal medium. Cells obtained from the basal medium would not grow when subcultured a second time in 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 in an anaerobic chamber (Coy Manufacturing, Ann Arbor, Michigan) containing an atmosphere of N2:H2:C02 (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 steel tubes (2 mm I.D.). One tube supplied gas, another growth medium, and the other carried away the effluent or overflow. The growth flask had a working volume of 100 ml. The culture was stirred vigorously by a mechanically-driven teflon-covered st i r r i n g bar. The addition of medium was controlled by a peristaltic pump equipped with variable speed control. The cultures were supposed to be in steady state after eight changes of the medium. The dilution rate, D, was determined by measuring the output of culture from the chemostat. Then D = output (hr 1) working volume 51 C. Protease 1. Assays for Proteolytic Activity a. The azocasein assay. The azocasein substrate was prepared by dissolving 2 g of azocasein in 100 ml of phosphate buffered saline (PBS) in a boiling 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 for 60 min at 37°C. The reaction was terminated and unhydrolyzed protein precipitated by the addition of 2 ml of 10% trichloroacetic acid (TCA). The acidified solution was mixed and incubated at room temperature for 20 min and filtered 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 in a l l assays. Azocasein hydrolysis was also measured on agar plates. One gram of agar (Difco) was dissolved in 50 ml boiling 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 in the agar plates and 20 y l of the enzyme solution was placed in the holes and the plates were incubated at 37°C. Enzyme activity was demonstrated by the presence of a clear zone around the holes. The logarithm of the diameter of the zones was directly proportional 52 to the actual enzyme activity. BME (final concentration 50 mM) was added to the 13. melaninogenicus culture supernatant or enzyme preparation. Samples containing known amounts of trypsin were included in 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 in the same buffer (50 mM), 0.2 ml; and supernatant from sedimented culture or enzyme preparation, 0.5 ml. The reaction components were pre-incubated at 37°C for 15 min before addition of 20 mg of azocoll. Incubation was continued at the same temperature in a shaking water bath. Two m i l l i l i t e r samples were removed at various time intervals, chilled in ice, and filtered to remove insoluble substrate. The amount of solubilized chromophore was determined by measuring the absorb ance of the f i l t r a t e at 520 nm. c. The casein assay. The substrate for the casein assay was prepared by dissolving 1 g of casein in 100 ml of 0.1 M phosphate buffer (pH 7.4), and heating for 15 min in a boiling water bath. The reaction mixture contained 2.5 ml casein, 1.0 ml BME (50 mM) in phosphate buffer, 0.5 ml of supernatant from a sedimented 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 of 5.0 ml of 10% tricholoroacetic acid. After 20 min at room temperature the contents of the tubes were filtered and the A o n „ of the f i l t r a t e s determined. 53 d. The dimethyl c a s e i n assay. The dimethyl c a s e i n substrate was prepared by the method of L i n e_t^  a l . (126), l y o p h i l i z e d and stored at -20°C. The r e a c t i o n mixture contained 0.5 ml of the enzyme and 1 ml of 0.1% dimethyl c a s e i n i n PBS pH 7.0 and was incubated f o r 1 hr at 37°C. The r e a c t i o n s were terminated by immersing t h e samples i n a b o i l i n g water bath. One m i l l i l i t e r of a s o l u t i o n of 0.1% 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 of a 4% sodium bicarbonate s o l u t i o n , pH 8.5, were added to each sample and the mixtures were incubated i n the dark f o r 30 min at 50°C. A f t e r i n c u -b a t i o n 1 ml of 10% sodium dodecyl 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 to each sample, and the absorbance at 340 nm was determined r e l a t i v e to a blank incubated w i t h a l l of the components present i n the sample except a c t i v e enzyme. e. The radiochemical assay. 14 A C-labeled N,N-dimethylcasein was used as the subst r a t e i n t h i s radiochemical assay. N,N-dimethylcasein was prepared as described by L i n et^ a l . (126) and was l a b e l e d w i t h by a d d i t i o n of 0.5 mCi [^C] formaldehyde ( s p e c i f i c a c t i v i t y 44.3 mCi/mM, I n t e r n a t i o n a l Chemical and Nuclear 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 of 0.02 yCi/mg p r o t e i n . The sub s t r a t e used i n the f o l l o w i n g procedure (43) was prepared by mixing 300 mg 14 [ C]N,N-dimethylcasein w i t h 2 gm cold Hammersten q u a l i t y c a s e i n i n 60 ml d i s t i l l e d water, and d i s s o l v i n g the suspension by r a i s i n g the s o l u t i o n pH to 12.0 w i t h 1 N NaOH. The pH of the 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 Tris buffer to 0.01 M at pH 8.0. The reaction mixture contained from 0.01 to 0.1 ml protease sample in a total 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 precipitating reagent (8% trichloroacetic acid) was added to one of two tubes containing identical amounts of enzyme immediate-ly before the addition of substrate. This tube served to determine 14 14 the background of non-precipitable 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% tri 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 precipitation 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. After removal of ether, the aqueous phase was l e f t at room temperature overnight to remove a l l traces of ether. Ether extracted f i l t r a t e 14 (0.4 ml) was assayed for C in a s c i n t i l l a t i o n counter, f. The hemoglobin assay. Hemoglobin (Hb) was denatured in alkaline urea solution by suspending 2.0 g Hb in 50 ml d i s t i l l e d water, adding 36 g urea, 8 ml 1 N NaOH and diluting 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. The substrate concentration in the assay was 6.7 mg Hb/ml reaction mixture; incubation temperature was 37°C and the substrate solution was equilibrated to 37°C before the assay. Five percent trichloroacetic acid (TCA) was added to precipitate the unhydrolyzed proteins and the pre-cipitate was filtered and A2gQ of the f i l t r a t e measured, g. Determination of esterase activity. 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). Activity against tosyl 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 prepared in 0.1 M T r i s -HC1 buffer pH 7.8 containing 0.01 M CaC^- Assays were performed in quartz cuvettes which were held at 30°C in a thermostatted compartment. The reference solution was prepared by mixing 0.5 ml of substrate solution and 0.5 ml of the same buffer in a cuvette. One half ml of substrate solution and 0.4 ml of buffer were placed in 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 in absorbance was recorded for a period of about 15 min. The rate of increase of absorbance was directly proportional to the concentration of the standard enzyme. Activity was calculated from the slope of the linear 56 portion of the reaction curve. One unit was equal to the hydro-ly s i s of 1 micromole of substrate per min. BAEE hydrolysis was indicated by an increase in absorbance at 254 nm. The reaction mixture contained 1.9 ml of enzyme preparation in 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 in absorbance at 237 nm, in a reaction mixture composed of 1.7 ml of enzyme preparation in buffer; 0.3 ml of 2M glycine-NaOH buffer (pH 9.0) and 1 ml of 0.5 mg of ATEE per ml in 0.01 M Tris-HCl (pH 8.0) 2. Purification of the Protease Unless indicated otherwise, a l l steps in the purification of the enzyme were carried out at 4°C. a. Preparation of bacterial cells for enzyme assays. Cells intended for use in 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 in the assay\ buf f er. b. Preparation of culture supernatants for enzyme assays and column chromatography. Cells were harvested from 48 hr cultures by centrifugation. The supernatant was concentrated to 1/10 the original volume by one of the following procedures: Amicon ult r a -f i l t r a t i o n using a PM-10 membrane; freeze-drying; or flash evaporation. Ammonium sulfate precipitation was also used to concentrate the protease activity of the culture supernatant. 57 c. Preparation of cell-extract for enzyme assays and protease purification. Bacterial 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 strain 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 cells were washed twice with 0.1 M PBS (pH 7.0) and resuspended in the same buffer. Cells were then broken by one of the following procedures. (i) The French pressure c e l l Harvested and washed cells from four l i t r e s of 48 hr cultures were resuspended in 80 ml PBS containing 10 yg each of protease free deoxyribonuclease and ribonuclease enzymes. The cells were then passed through the French press (American Instrument Co., Inc.) 2 three times at a pressure of 20,000 psi ( 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 Inter-national ultracentrifuge. ( i i ) The Mini-Mill Ten ml of a washed c e l l suspension ( A =5) were mixed with 15 gm of glass beads, placed in ooO the Mini-Mill (Gifford-Wood, Inc.) and stirred for 15 min at 4°C. The disintegrated cells plus liquid 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. ( i i i ) Sonication Ultrasonic disintegration was attained by the use of a Biosonik model sonicator (Bronwill Scientific, Co.) tuned to provide maximum power. The c e l l suspension was placed in an ice-cooled bath, and treated with ultrasound u n t i l microscopic examination revealed that the majority of cells were broken, d. Release of c e l l bound HA and protease from 2D c e l l s . Cell bound HA and protease were released from 15. melaninogenicus strain 2D using two methods: (i) Release of periplasmic enzymes by osmotic shock Cells were harvested and washed with 0.01 M Tris-HCl (pH 7.3)- 0.03 M NaCl. Washed cells were suspended in 0.03 M Tris-HCl (pH 7.3) at a ratio of 1 g • cells (wet weight) to 40 ml buffer. An equal volume of 1 M sucrose in 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 cells were removed by centrifugation at 0°C. The pellet of cells was resuspended in 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 fluid was assayed for HA and protease. 59 ( i i ) 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 activity. e. Ethanol precipitation. Forty ml of c e l l extract were cooled to -10°C in 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 pellet resuspended to the original volume in PBS. f. Chromatographic procedures. (i) 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. The sample volume was 2-4% of the total column volume. Columns were run at 4°C. Protease was also chromatographed on columns of Sepharose 2B and Sepharose 4B (28.3 x 1.6 cm) equilibrated with PBS or PBS containing 6 M urea and/or 0.1% SDS. 60 ( i i ) Ion-exchange chromatography Protease, concentrated from culture supernatant or precipitated with 60% ethanol from the cell-extract was applied to DEAE and CM-Sephadex ion exchange columns equilibrated in 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 linear NaCl gradient in the different buffers. In some instances, PBS containing 0.1% SDS was used to equilibrate and elute both ion exchange columns. ( i i i ) Activated thiol-Sepharose-4B Activated thiol-Sepharose-4B was swollen and additives were removed in 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 thiol 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 in PBS (pH 7.0) was added to the column and eluted with the same equilibration buffer. Low flow rates were used during sample application and elution (5 ml/hr) and 1 ml fractions were collected. The lowest possible concent-ration of reducing agent, 10 mM L-cysteine in PBS (pH 7.0), was used to elute coupled proteins. 61 (iv) Organomercurial agarose An agarose mercury column (30 ml volume) which selectively bound thiol 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 in 10 volumes of dimethyl sulfoxide (DMS0) at 0°C. Six grams of p-aminophenylmercuric acetate dissolved in 100 ml DMSO was added. After gentle stir r i n g for 20 hr at 0°C, the suspension was warmed to 35°C, filtered and washed 4 times at 37°C with 20% DMSO to remove the free mercurial compound. The agarose was resuspended in 0.1 M ethylenediamine, adjusted to pH 8.0 and gently stirred. After storage overnight at room temperature, the agarose was washed, packed into a column and reacted with 2-nitro-5-mercaptobenzoic acid in 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 in 50 mM sodium acetate pH 5.0 containing 0.1 M KC1, 0.5% butanol, 10% DMSO, 1 mM EDTA and 10 mM Na 2S0 3 (standard buffer) was passed through the column unt 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 contain-ing 1M NaCl was added to the column. The hydrophobic-a l l y bound proteins were eluted in a 20-50% gradient of ethylene glycol in PBS. g. Gel electrophoresis (i) Polyacrylamide gel (10%) electrophoresis was performed as described by Nagai et a l . (156), with 0.25 M Tris, 1.92 M glycine, 0.1% SDS buffer (pH 8.3) in a vertical gel plate apparatus. 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. The fi n a l concentration of SDS (BioRad) was 0.1% in both stacking and running gels and in the running buffer. Samples were prepared in a solubilization mixture containing 0.125 M Tris (pH 6.8), 4% SDS, 10% $ME, 20% glycerol and 0.01% bromophenol blue as a marker. The samples were denatured by heating for 2 min in a boiling water bath. The gels were stained with 0.2% Coomassie b r i l l i a n t blue in 30% methanol / 10% acetic acid for 5-12 hr and destained in 10% acetic acid. 63 ( i i ) Polyacrylamide gel electrophoresis was also performed as above without SDS in the stacking and running gels, running buffer and samples. In this system the samples were not heated prior to electrophoresis. ( i i i ) Glycoproteins were detected by staining the gels with the cationic carbocyanine dye "Stains-all" . (SA) (110). The gels were fixed and SDS removed in 25% isopropanol. A stock solution of SA 0.1% (w/v) in formamide was stored at 4°C in a brown bottle for a maximum of 6 weeks. Five m i l l i l i t e r s of the stock solution were diluted with 20 ml formamide, 100 ml iso-propanol and 275 ml of the tris-glycine buffer without SDS and the pH adjusted to 8.5 with 1 N NaOH. The gels were stained overnight in the dark and destained with 10% isopropanol for 18-36 hr at room temperature. The gel was checked carefully to ensure that the purple SA had not deteriorated due to SDS, pH or light. The glyco-proteins stained blue and the proteins red by this procedure. (iv) Lipoprotein electrophoresis Lipoproteins were prestained prior to electrophoresis. Acrylamide gels of 3%, 5% and 7% were prepared in 0.18 M Tris-citrate buffer pH 9.0 without SDS. The running buffer was 0.065 M Tri 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 dissolving 25 mg Sudan black B in 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 0 . 5 M t r i s - c i t r a t e b u f f e r (pH 9 . 0 ) . The s o l u t i o n was i n c u b a t e d a t 60°C f o r 1 h r and f i l t e r e d t h r o u g h Whatman N o . 1 p a p e r and s t o r e d a t 4°C r e f r i g e r a t o r . F i f t y u l o f r a b b i t p l a s m a i n c u b a t e d w i t h 50 u l o f t h e b u f f e r e d s t a i n a t 37°C f o r 30 m i n was u s e d a s a l i p o p r o t e i n s t a n d a r d , h . 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 c a r b o h y d r a t e s i n g l y c o p r o t e i n s . N e u t r a l s u g a r s and h e x o s a m i n e s were a n a l y z e d b y t h e c h r o m a t o g r a p h i c p r o c e d u r e d e s c r i b e d by P o r t e r ( 1 7 1 ) . N e u t r a l and amino s u g a r s we re r e l e a s e d f r o m g l y c o p r o t e i n o r g l y c o p e p t i d e s by h y d r o l y s i s w i t h Dowex 5 0 - X 2 ( H + ) r e s i n , f o l l o w e d b y n i t r o u s a c i d d e a m i n a t i o n o f t h e r e s i n bound h e x o s a m i n e t o n e u t r a l 2 , 5 - a n h y d r o -h e x o s e s . H e x o s e s and 2 , 5 - a n h y d r o h e x o s e s were t h e n r e d u c e d w i t h N a B H ^ , 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 c h r o m a t o g r a p h e d as t h e c o r r e s p o n d i n g n e u t r a l a l d i t o l a c e t a t e s . A B e n d i x - S e r i e s 2500 gas 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 p rog rammer , a h y d r o g e n f l a m e i o n i z a t i o n d e t e c t o r and a 6 f t U - s h a p e d , 1/4 i n d i a m e t e r g l a s s co l umn was u s e d . The co l umn p a c k i n g m a t e r i a l c o n s i s t e d o f 3% E C N S S - M on 1 0 0 / 1 2 0 mesh g a s - c h r o m Q ( A p p l i e d S c i e n c e L a b o r a t o r i e s , I n c . ) . The c a r r i e r gas was h e l i u m a t a f l o w r a t e o f 40 m l m i n H y d r o g e n and a i r f l o w t o e a c h d e t e c t o r we re 50 m l m i n ^ and 70 m l m i n \ r e s p e c t i v e l y . Samp les (2 u l ) were i n j e c t e d a t a co l umn t e m p e r a t u r e o f 150° and e l u t e d a s t h e 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 t o 200° a t a r a t e o f 3 ° p e r m i n . Two h u n d r e d f i f t y nanomo les o f m y o i n o s i t o l i n 50 u l was u s e d a s an i n t e r n a l s t a n d a r d . 65 i . Lipid analysis. The l i p i d s were extracted from the purified protease preparation (0.3 mg protein) by adding 3 ml of chloro-form 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 CHC13 and 3 ml of 0.74% KC1. The lower CHC13 layer was removed 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 2, resuspended in 0.5 ml acetone and evaporated to dryness, the procedure was repeated and the residue dissolved in CHC13 and stored at -20°C. Phospholipids were analyzed by the thin-layer chromato-graphic method described by Yavin (240) using the li p i d s extracted from the purified 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 fatty acid analysis, the extracted l i p i d was fractionated by thin-layer chromatography to remove the free fatty-acids (20), and the neutral l i p i d s were eluted from the plate and saponified with 15% K0H in methanol at 70°C for 1 hr. The solution was acidified 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 fatty 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 fatty acid methyl esters were identified by comparison of their retention times with those of known standards. D. Hemagglut inat ion 1. Assay Cells from a 48 hr culture were harvested by centrifugation, washed twice in PBS and resuspended in PBS to give an absorbance at 660 nm of 1.0. Growth liquor obtained from the centrifugation of a 48 hr culture was also assayed. Hemagglutination was measured in microtiter plates by adding 0.025 ml of a 2.5% suspension of formalinized human red blood cells (FRBC) in PBS to 0.025 ml of a 2-fold serial dilution of the sample. The samples were diluted in PBS containing 25 mM 3ME. Results were recorded after 30 min incubation at 37°C. The HA activity was recorded as the reciprocal of the highest dilution showing complete hemagglutination (no erythrocyte pellet formation) and was considered as the HA t i t e r . In some instances, HA was measured in 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 bacterial c e l l suspension. The mixture was agitated gently for 10 min at room temperature. Samples possessing hemagglutinating activity usually clumped the RBC within 10 min. The extent of clumping was assessed visually and scored on a 0 to 44 basis. 2. Preparation of Red Blood Cells Hemagglutination was assayed with formalinized and non-formalinized human red blood cells (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 in the same buffer. 67 Formalinized human RBC were prepared by resuspending 25 ml of washed packed cells in 200 ml PBS pH 7.2. Fifty ml of formalin was placed in a dialysis tube and this was submerged in the c e l l suspension, and the mixture was gently agitated at 20°C. After 4 hr, the remaining formalin was transferred from the dialysis sac to the c e l l suspension and this mixture was stirred slowly for 18 hr. Cells were washed free of formalin with 0.9% NaCl. The ce l l s were stored at 4°C as a 25 percent suspension in PBS containing 0.02% sodium azide. 3. Determination of the Effects 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 activity by the microtiter method. A control using PBS instead of the culture supernatant was always assayed in parallel with the samples. In some instances, the effect of a reagent on HA was measured by serially diluting the sample in PBS containing an appropriate concent-ration of the reagent. Treatment of RBC with different 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 cel l s were washed three times and resuspended to 2.5% in PBS. 4. 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 for the presence of unadsorbed hemagglutinin. The RBCs with the adsorbed HA 68 were resuspended in 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 dialysis against PBS. E. Infectivity Cells used for inoculation of animals were harvested from blood agar 9 plates or from broth cultures and resuspended to 10 cells/ml in phosphate-buffered saline, pH 7.0. Guinea pigs weighing 150 to 200 g were shaved on the abdomen and injected subcutaneously with 0.5 ml of either in vitro cultured cells or exudate aspirated from an infected guinea pig. The animals were observed for up to four weeks. The c r i t e r i a for evaluating a positive infection were: (i) the presence of an abscess (pustular or necrotic); ( i i ) the transmissibility of the disease. The latter was demonstrated by injecting material aspirated from a lesion into a second animal to produce a similar pathology. Exudate was aspirated from infected guinea pigs using a sterile disposable syringe while the animal was under light 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. Purified cholera toxin (10 ug/ml) served as a test control. 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 in duplicate. Six to nine injections were made on each guinea pig. After 20-24 hr, filtered Evans Blue dye (5% in saline) was injected intracardially, (0.1 ml/100 g body weight). The diameter of the resulting blue area around the site of inoculation was then 69 measured. The control was sterile medium concentrated 10 times in a Diaflo u l t r a f i l t r a t i o n apparatus. F. Metabolic End Product Analysis 1. Preparation of Samples Volatile and non-volatile fatty acids were analyzed by gas liquid chromatography (GLC) as described by Holdeman and Moore (94).. For the analysis of volatile fatty acids, culture supernatants were acidified to pH 2 or below with 50% aqueous IL^SO^, and the vo l a t i l e fatty 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 fatty acids, 1.5 ml of the culture supernatant acidified as described above was methylated by addition of either 2 ml of methanol or 1 ml of borontrifluoride-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 volatile and methylated fatty acids were prepared with each set of samples. 2. Operating conditions of the gas chromatograph Samples were analyzed in a Bendix model 2500 gas chromatograph (Canadian Dynamics, Vancouver, B.C.) equipped with a hydrogen flame ionization detector. The carrier gas flow was set at 90 ml min and the oven temperature at 120°C for volatile acids and 125°C for non-volatile acids. 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 in 20 ml of chloroform. The mixture was 70 m i x e d g e n t l y u n t i l t h e CHCl- j had 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 a 6 ' x 1 / 4 " U - s h a p e d t u b e . G . C o l l a g e n a s e A s s a y C o l l a g e n a s e was measured as d e s c r i b e d by G i s s l o w and M c B r i d e ( 7 5 ) . 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 f r o m f r e s h f e t a l c a l f s k i n as d e s c r i b e d by G a l l o p and S e i f e r (65) , e x c e p t t h a t p a r t i c u l a t e m a t t e r was removed by f i l t r a t i o n . 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 - 2 0 ° C . 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 .01% c o l d a c e t i c a c i d a t a c o n c e n t r a t i o n o f 2 m g / m l , and t h e pH a d j u s t e d t o 8 . 5 by t h e a d d i t i o n o f 1 M K^HPO^ and was 14 t h e n a c e t y l a t e d w i t h a c e t i c - C - a n h y d r i d e i n b e n z e n e . The l a b e l e d m i x t u r e 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 c o l l a g e n 14 14 d i a l y z e d a g a i n s t c o l d d i s t i l l e d w a t e r t o remove C - a c e t i c a c i d . The C -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 - 20°C u n t i l n e e d e d . The s u b s t r a t e 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 .01% a c e t i c a c i d a t a c o n c e n t r a t i o n o f 1 mg/ml by s t i r r i n g o v e r n i g h t a t 4 ° C . A t y p i c a l r e a c t i o n m i x t u r e f o r C_. h i s t o l y t i c u m c o l l a g e n a s e and I3_. m e l a n i n o - g e n i c u s c o l l a g e n a s e i s shown i n T a b l e 1 . Enzyme , b u f f e r and c y s t e i n e we re i n c u b a t e d f o r 15 m i n a t 20°C b e f o r e a d d i t i o n o f t h e s u b s t r a t e . A t t h e a p p r o p r i a t e t i m e 0 . 1 m l o f t h e r e a c t i o n m i x t u r e was added t o a " m i c r o f u g e " t u b e (Beckman I n s t r u m e n t s , I n c . ) , c o n t a i n i n g 50 y l o f 0 . 04 N p h o s p h o -t u n g s t i c a c i d and 50 y l o f 2 N H C 1 . The s a m p l e s we re l e f t a t room t e m p e r -a t u r e f o r 10 m i n and t h e n c e n t r i f u g e d f o r 5 m i n i n a B e c k m a n - S p i n c o m i c r o f u g e (model 1 5 2 , Beckman I n s t r u m e n t s , I n c . ) . One h u n d r e d y l o f t h e 14 s u p e r n a t a n t was a n a l y z e d f o r C . The c o n t r o l samp le f o r e a c h a s s a y c o n t a i n e d b u f f e 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 c e l l s . 71 TABLE I Collagenase Assay Components 14 C-collagen (0.1% in 0.01% acetic acid) Tris-HCl Buffer (0.05 M, pH 7.2) )5 .1 with CaCl 2 (0.00 M) Cysteine (0.05 M)' Collagenase (30 units/ml) 2 Cell suspension Volume (ml) B. melaninogenicus C_. histolyticum Control 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.2 0.2 0.1 "''Cysteine hydrochloride was made up to 0.05 M concentration in 0.05 M Tris-HCl buffer and neutralized by adding 5 N NaOH. Cells were resuspended in 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 Lipids 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 l i p i d An aliquot of the l i p i d solution to be analyzed was oxidized with acid dichromate. The reaction was followed by direct colorimetry (micro method), and by an iodometric colorimetry (ultramicro method). In the ultramicro method, 1.0 ml of 0.034% (w/v) potassium dichromate in 97% sulfuric acid was added to 2-12 Vg dried l i p i d sample. A control tube was also included which did not contain any l i p i d . The tubes were placed in a boiling water bath for 15 min and then cooled; 9.0 ml water was added to a l l the tubes, contents were mixed well, and 0.5 ml of these solutions was added to 4.5 ml Cd^-starch reagent. The reagent blank was prepared by adding 0.5 ml 3.6 N sulfuric acid to 4.5 ml Cdl^-starch reagent. The color intensities were read against the reagent blank at 575 nm. In the micro method, 2.0 ml 0.15% potassium dichromate in 96% (w/v) sulfuric 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 (w/v) was added to reduce the dichromate in one of the blanks. A l l tubes were read against the reduced blank at 440 nm. 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 in acid-alkali cleaned thick 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 trioxide appeared. Two drops of perchloric acid (72%) were then added and the samples heated u n t i l liquids became clear, and then cooled and volumes were adjusted to 25 ml in 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 activities were determined with p-nitrophenyl a-D-glucoside (a-PNPG) and p-nitrophenyl 3-D-glucoside (B-PNPG) (Calbiochem) as substrates, respectively. The enzyme activity was assayed in the purified 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 initiated by the addition of 1.0 ml of 10 mM prewarmed solution of the appropriate substrate in PBS (pH 7.0). The assay was terminated by placing the tubes in an ice bath and 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 in a Perkin Elmer-Hitachi spectrophotometer model 124. M. Lipase Assay The lipase activity in the purified protease preparation was determined by a modification of the method of Huggins, Charles and Lapides as described by Winters (237), which is 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 M sub-strate in the same buffer. 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: trypsin, papain, neuraminidase Type VI, C_. histolyticum collagen-ase, wheat germ lipase, pronase, BSA, dithiothreitol, trinitrobenzene-sulfonic 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 fluoride (PMSF), azocoll 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 t h i o l -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 Scientific Company). The results of the present investigation may be divided into three sections. In the f i r s t , the preliminary identification of 1J. melaninogenicus strains i s reported. The second section deals with the partial character-ization and purification of the soluble hemagglutinin. The last section presents the techniques used in attempt to purify and characterize the soluble and cell-bound protease(s) of I3_. me 1 aninogenicus. 76 III. RESULTS A. Characterization of ]3. melaninogenicus The purpose of this part of the thesis is to describe experiments which were carried out to characterize B_. melaninogenicus strains 2D and K110 and further to investigate strain 2D which was used in this study. 1. Fatty Acid Production The f i r s t step in characterizing the organisms'was . to subdivide them on the basis of the acidic end products produced during growth. Organisms were cultured for 48 hr in TYH medium and the supernatants analyzed for volatile and non-volatile fatty acids by gas chromatography (Fig. 3 and 4). The peaks were identified by comparing their retention times with those of standards. Both K110 and 2D strains produced acetic, propionic, isovaleric, isobutyric, and butyric acids in addition to an unknown compound which was shown to be phenylacetic acid by mass spectroscopy and gas chromatography (Susan Jensen, personal communication). The results indicated that both strains correspond to the subspecies known as melaninogenicus ss. asaccharolyticus. The presence of phenylacetic acid in cultures of 15_. melaninogenicus ss. asaccharolyticus had not been noted previously and should provide a useful tool in identifying this organism in mixed infection. FIGURE 3. Gas chromatography of volatile fatty acids produced by ]}. melaninogenicus Volatile fatty acids were extracted from acidified culture supernatant. a = acetic acid; p = propionic acid; iso b = isobutyric acid; b = butyric acid; iso v = isovaleric acid 78 Retention Time (min) FIGURE 4. Gas chromatography of non-volatile fatty acids produced by 15. melaningenicus. Methylated fatty acids were extracted from acidified culture supernatant. £ = la c t i c acid pa = phenylacetic acid Retention Time (min) 81 2. Collagenase Activity J3. melaninogenicus has been reported to possess a c e l l bound collagenase which is believed to be associated with pathogenicity (67,83,84). In order to determine i f collagenase was associated with K110 and 2D strains, 48 hr cultures were harvested and cells assayed 14 for collagenase by incubating them with C-collagen. Both K110 and 2D strains possess a c e l l bound collagenase. Unlike other microbial c o l l -agenases, the 13. melaninogenicus enzyme is oxygen sensitive and is stimulated by reducing agents. No soluble collagenase was detected in the culture supernatant of either organism. The specific activity of the c e l l bound collagenase was expressed as the ug of collagen solubilized per hr by 1 ml of cells (Absorbance^r. = 1.0). B. melaninogenicus strain K110 had a specific 660 — ° activity of 122 pg/hr/O.D. Strain 2D 13. melaninogenicus had a specific activity of 140 ug/hr/O.D. 3. Pathogenicity a. Infectivity. _B. melaninogenicus strain 2D was tested for i t s ab i l i t y to produce an infection in the guinea pig model system. Cells from a 48 hr liquid culture were washed, resuspended i n ster i l e PBS and injected into the groin of a 200 g guinea pig. Within 18 hr the animal developed symptoms of a rapidly spreading infection: darkening of skin and loss of hair in the thoracic area and accumulation of a large volume of f l u i d . Material aspirated from the animal was dark in 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 ce l l s indicating that the organism had invaded the circulatory system. Culture of the exudate on blood agar plates confirmed that i t contained a pure culture of melanino- genicus . Fatty acid analysis revealed that acetic, isobutyric, butyric and phenylacetic acids were present. These acids are produced by I5_. melaninogenicus in in vitro culture. The trans-missible nature of the infection was proven by using the exudate to infect another guinea pig. Sterilizing the exudate by auto-claving resulted in a loss of infectivity. Strain 2D was thus one of the few I3_. melaninogenicus strains capable of producing an infection without the support of other organisms (139,215). The infection produced symptoms similar to those described in the literature for infections produced by CR2A (139). Strain K110 failed to produce infection when pure cultures were injected into guinea pigs but was infective in mixed culture (146). Generally, successful infections resulted when 200-250 g guinea pigs were infected instead of larger guinea pigs and when 48 hr cells were used instead of 24 hr c e l l s . b. Vascular permeability. Filtrates from stationary phase cultures of IJ. melaninogenicus strains 2D and K110 were sterilized by f i l t r a t i o n through a 0.45 u millipore f i l t e r and concentrated 10 times by ul 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 injecting 0.2 ml intracutaneously 83 Table 2. Vascular Permeability Test Inoculum Blueing diameter (mm) Cholera toxin 10 2D 7 K110 4 Control 0 The test samples were injected intracutaneously, in a random sequence, into the shaved backs of three guinea pigs in duplicates, and six injections were made on each guinea pig. Evans Blue (5% in saline) was injected intracardially, (0.1 ml/100 g body weight), about 20-24 hr after injection of samples. The dye was disseminated throughout the vascular system almost immediately, and the guinea pigs were sacrificed after 10 min. 84 into each of two guinea pigs. The co n t r o l was s t e r i l e medium concentrated 10 times i n the same way as the culture supernatant. Aft e r 24 hr, a blue dye was injected into the t e s t guinea pigs by cardiac puncture. The diameter of the blue areas around the s i t e of inoculation of the d i f f e r e n t samples represented the e f f e c t of these samples on vascular permeability i n the test animals (Table 2 ) . It was found that B_. melaninogenicus produced a factor which was released from the c e l l s and which exhibited b i o l o g i c a l properties s i m i l a r to V i b r i o cholerae enterotoxin. B o i l i n g of the concentrated culture supernatant destroyed the blueing factor suggesting that i t was a heat s e n s i t i v e protein. 4. Growth of S t r a i n 2D 15. melaninogenicus a. Hemin requirement. Str a i n 2D of I5_. melaninogenicus has an obligate requirement for hemin. The response of 2D to varying amounts of hemin was followed. The r e s u l t s are shown i n F i g . 5. It may be seen that growth of the bacterium was roughly proportional to the hemin concentration over a range of 0.25 to 2.5 yg hemin per ml. The organism grew i n a medium free of hemin for one generation but when transferred to fresh medium lacking hemin, l i t t l e i f any growth occurred whereas subculture to hemin containing media always resulted i n good growth. The organism grew i n the presence of a concentration of hemin as low as 0.2 yg/ml medium; concentrations of hemin higher than 20 yg/ml had no further enhancing e f f e c t on the growth of the organism. Addition of glucose to the TYH medium i n h i b i t e d the growth of 2D. 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 different concentrations of hemin. Growth was measured (A,,,.) after 24 hr incubation anaerobically ooU at 37°C. yg Hemin/ml medium 87 b. Growth response to amino acids. The a d d i t i o n of glutamic acid to cultures of 2D i n TYH medium resulted i n a marked increased i n growth rate and t o t a l c e l l y i e l d when compared to unsupplemented medium (Table 3) . The growth response of 2D was dependent on the concentration of glutamic acid over the range of 0.2 to 4 mg/ml (Fig. 6). Enhancement of growth also occurred when L-serine, L-asparagine, L-methionine and L-proline were added to TYH medium. Addition of L-cysteine, DL-valine, L - h i s t i d i n e , L-tryptophan, glycine or L-arginine to TYH medium produced marked i n h i b i t i o n of the growth of s t r a i n 2D. L-leucine caused 66% i n h i b i t i o n of growth. Growth was not affected by the addi t i o n of L- l y s i n e or L-phenylalanine to 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 acids when comparing 24 and 40 hr cultures ( T a b l e 3) could be due to the d i f f e r e n t times at which the cultures reach the stationary phase of growth. Addition of L-cysteine, adjusted to pH 7.0 by NaOH, to a growing culture of 2D i n TYH medium (28 hr) caused i n h i b i t i o n of growth (no further increase i n A,,, was detected). 660 Although glutamic acid or asparagine caused enhancement of growth of 2D i n TYH medium they were unable to replace hemin i n t r y p t i c a s e yeast-extract medium. 5. Hemagglutinin and Protease A c t i v i t y of 13. melaninogenicus ]3. melaninogenicus s t r a i n s K110 and 2D produce both a soluble and a c e l l bound hemagglutinin (HA). They agglutinate- r a p i d l y with 88 Table 3. E f f e c t of the addition of amino acids on Growth of I5_. melaninogenicus ss. asaccharolyticus 2D i n TYH medium Amino acid added Concentration A, (mM) 6 6 0 24 h 40 h None - 0.38 0.98 Glutamic acid 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 - h i s t i d i n e 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- l y s i n e 34.2 0.4 0.98 L-phenylalanine 30.3 0.36 1.0 FIGURE 6 E f f e c t of glutamic acid on growth of 13. melaninogenicus. TYH media containing increasing concentrations of glutamic acid were inoculated with 2D and growth response was recorded at 24 hr. Percent Increase i n A 91 human red blood c e l l s . Both s t r a i n s produce soluble and c e l l u l a r protease(s) which are a c t i v e against a number of substrates including a z o c o l l , casein and azocasein. Table 4 presents the soluble and c e l l u l a r HA and protease a c t i v i t i e s of both 2D and K110. Due to greater pathogenicity of the 2D s t r a i n of 1$. melanino- genicus and i t s production of greater amounts of HA and protease, t h i s organism was chosen f o r d e t a i l e d studies of the soluble hemagglutinin and soluble and c e l l u l a r proteases. 6. E f f e c t of Washing 2D C e l l s on the HA and Protease C e l l s from a 4 day old culture were harvested by c e n t r i f u -gation and resuspended i n 1/10 of the o r i g i n a l volume of PBS-8ME (50 mM). The c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n and the process repeated s i x times. C e l l s and supernatant from each washing were assayed for hema-gg l u t i n i n and protease. HA can be sequentially eluted from the b a c t e r i a l c e l l s by washing with phosphate buffered s a l i n e i n the presence of a reducing agent (Table 5). In the meantime the HA t i t e r of the c e l l s remained the same. HA could not be eluted from c e l l s harvested from a 24 hr culture. T r i s buffer containing 25 mM BME or cysteine could not replace the PBS i n r e l e a s i n g the cell-bound HA. The presence of reducing agent i n the washing bu f f e r resulted i n better e l u t i o n of the HA from 2D c e l l s than using only b u f f e r . Anaerobic e l u t i o n of 2D HA using PBS i n the anaerobic chamber gave the same r e s u l t s . When the HA e l u t e d f r o m 2D c e l l s was compared to the soluble HA i n culture supernatants, no differences were noticed 92 Table 4. Hemagglutinin and Protease of 2D and K110 S t r a i n Hemagglutinin Protease Soluble C e l l bound Soluble C e l l bound Titer/ml Titer/ml/1 A 660 Units/ml Units/ml/1 A 660 2D 1280 20480 7.2 28.4 K110 640 5120 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 using washed c e l l s resuspended i n PBS (A.,- = 1.0) and the supernatant from a 48 hr cul t u r e of 660 2D and K110. Formalinized human RBC were used f o r the HA assay. The protease was assayed i n both c e l l s and culture supernatant using casein as substrate. Table 5. E f f e c t of washing 2D c e l l s on the HA Sample HA t i t e r of Supernatant HA t i t e r of P e l l e t Growth l i q u i d 64 256 -F i r s t wash 64 256 Second wash 64 256 Third wash 32 256 Fourth wash 32 256 F i f t h wash 4 256 Sixth wash 16 256 A ten-times-concentrated suspension of four days o ld 2D cul t u r e was washed s i x times with 25 mM $ME i n PBS, and the washing and the p e l l e t were assayed f o r HA by the m i c r o t i t e r p l a t e . 94 between the two i n respect to optimum pH, s t a b i l i t y and the e f f e c t of i n h i b i t o r s on the hemagglutinin. Centrifugation of the eluted HA at 100,000 x g resulted i n the recovery of most of the HA i n the pellet;, which implies that the release 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 of the outer membrane of the 2D c e l l s . Protease was not eluted from 2D c e l l s by either procedure suggesting that the protease enzyme may be more t i g h t l y bound to the c e l l s than i s the c e l l u l a r HA. 7. Release of Periplasmic Enzymes from 2D C e l l s Since the c e l l u l a r HA and protease of B_. melaninogenicus are accessible to RBC and high molecular weight substrates r e s p e c t i v e l y , i t i s probable that both are l o c a l i z e d external to the cytoplasmic membrane. An attempt was made to determine i f the HA and protease were located i n the periplasmic space, by osmotic shock treatment, and by treatment with 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 protease was released by either procedure , therefore the enzyme cannot be considered as periplasmic. The HA seemed to be l e s s t i g h t l y bound to the 2D c e l l s than the protease, c o r r e l a t i n g with the r e s u l t s obtained from e l u t i o n of the HA and protease by successive washings of the c e l l s . The release of the HA from the c e l l s by polymyxin B treatment was rel a t e d to the time of exposure to the a n t i b i o t i c (Table 6). Most of the c e l l s treated with 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 occurred as judged by microscopic examination. There was a rough c o r r e l a t i o n between the appearance of soluble HA and loss of 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 assay. Table 6. Release of the HA from 2D c e l l s by treatment with Polymyxin B Time of exposure min HA t i t e r of p e l l e t HA t i t e r of supernatant 0 512 -1 512 0 5 256 4 15 128 8 30 64 16 60 64 64 A 400 ml culture of 2D I5_. melaninogenicus was harvested, washed and resuspended i n 20 ml PBS with 25 mM (3ME. 5 ml of c e l l suspension was mixed with 5 ml of 0.4 mg/ml Polymyxin B and incubated at 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 and supernatant were assayed f o r HA by the m i c r o t i t e r method. 96 B. Soluble Hemagglutinin of 13. melaninogenicus 13. melaninogenicus possesses a cell-bound and a soluble hemagglutinin(s) which i s thought to be c e l l associated hemagglutinin which has been released from the c e l l surface. Studies were c a r r i e d out to provide evidence to support t h i s assumption and at the same time to define the adherence properties of ]3_. melaninogenicus which might contribute to the establishment of the organism i n the g i n g i v a l crevice. 1. Adherence of 13. melaninogenicus 2D C e l l s to Formalinized Human RBC Cultures of 2D c e l l s were tested f or adherence to RBC by harvesting the c e l l s , resuspending them i n PBS (A,,.. = 1.0) and mixing with packed FRBC f o r 30 min at room temperature with shaking. Hemagglutination was determined by the standard test tube assay and the samples were observed microscopically f or J3. melaninogenicus bound to RBC. By following FRBC adherence versus culture age of J3. melaninogenicus 2D, i t was found that the adhesion to RBC occurred at 24 hr (Table 7). Wash-ing the bacteria-RBC aggregates 4 times with PBS did not dislodge the microorganisms. 2. Determination of Optimal Conditions for the Hemagglutinin  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. Optimum a c t i v i t y occurred over a pH range of 7.0 to 7.5, but good a c t i v i t y could be demonstrated i n the range of pH 6.0 to 8.0. Hemagglutination a c t i v i t y decreased r a p i d l y below pH 6.0 and above pH 8.0. Red blood c e l l s from a number of animals and from humans were Table 7. Influence of culture age on the adherence of 2D to FRBC. Culture age hrs. Adherence with checked by the tube method FRBC test % of 2D c e l l s not adhering to FRBC 10 0 100 16 0 100 24 +++ 50 36 +++ 40 48 IIII 20 60 ++++ 30 The extent of adherence was assessed v i s u a l l y by the test-tube HA assay, and expressed as the percent of non-adhering c e l l s to FRBC a f t e r microscopic examination. FIGURE 7. E f f e c t of pH on HA of 15. melaninogenicus Supernatant of 48 hr culture was tested f o r HA by the m i c r o t i t e r plate using 0.2 M each of Na acetate buffer at pH 5.0-6.0; phosphate buffer at pH 6.0-7.5 and t r i s buffer at pH 7.5-9.0. 99 pH 100 tested in the HA assay using the microtiter plate. The results are shown in Table 8. Rabbit and human RBC showed the strongest activity 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 in the Materials and Methods, were as effective in the HA assay as were fresh RBC. The formalinized RBC were more stable than were non-formalinized RBC. Furthermore, the cells retained the microscopic appearance and shape of fresh normal RBC. For the quantitative studies of the adsorption or elution of soluble hemagglutinin, formalinized cells can be used advantageously in place of normal c e l l s . Formalinized cells were found to pack more quickly and firmly than normal cells upon centrifugation so that greater accuracy was obtained in preparing suspensions. A stock suspension of the modified cells may be prepared and used over a period of months with assured constancy of concentration and reactivity. The microtiter assay was also performed using a range of RBC concentrations as shown i n Fig. 8. It was determined that the optimal RBC concentration necessary to observe HA was in the range of 2.5 to .1.25%. The effect of incubation temperature on the hemagglutination assay is shown in Fig. 9. From these results, 37°C was chosen as the most satisfactory incubation temperature for the HA assay using an incubation period of 30 min. Hemagglutination occurred at 4°C, but was very slow. 101 Table 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 . Source of RBC HA t i t e r Guinea pig 8 Rabbit 32 Frog 16 Human 64 Formalized human 64 Formalized rabbit 64 The m i c r o t i t e r plate method was used to assay the HA i n a 72 hr culture supernatant with 2.5% washed RBC of d i f f e r e n t sources. 102 FIGURE 8 Optimal erythrocyte concentrations for m i c r o t i t e r hemagglutination assay. . Assay conditions: RBC suspended i n PBS (pH 7.0) containing 50 mM $ME, culture supernatant from a 48 hr cul t u r e . The m i c r o t i t e r plates were incubated f o r 30 min at 37°C. The concentrations of RBCs were expressed as percentage of packed c e l l s . 103 Percent RBC FIGURE 9. E f f e c t s of incubation temperature on HA. Assay conditions: 1.25% (vol/vol) RBC suspended i n PBS (pH 7.0) containing 50 mM 3ME, culture supernatant from a 48 hr cul t u r e . The m i c r o t i t e r plates were incubated f or 30 min at various temperatures. 106 3. Relationship of HA to Culture Age The appearance of cellular and soluble hemagglutinins was followed as a function of the age of the culture. The organisms were grown at 37°C under anaerobic conditions. As shown in Fig. 10, the production of cell-bound HA paralleled 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 infectivity of the organism. It also continued to increase after growth had ceased, presumably due to l i b -eration of cellular HA as a result of c e l l l y s i s . 4. Effects of RBC Modification on HA The results of the treatment of RBC with various enzymes are presented in Table 9. Treatment with trypsin and a-chymotrypsin appears to have some inhibitory effect. However, since the inhibitory effects represent only a two-fold dilution, this i s probably not significant. Treatment of the RBC with neuraminidase caused enhancement of the HA activity, which may have been due to the unmasking of the active receptor or binding site for the HA on the RBC. Pronase, s u b t i l i s i n and galactosidase treatments of RBC caused inhibition of the HA activity which may have been due to alteration of the receptor structure for the HA on the RBC. 5. Modification of the HA In order to obtain information concerning the nature of the component(s) necessary for HA, the culture supernatant was treated with several reagents and salts prior to the HA assay. A number of salts had no effect on the HA, which might suggest FIGURE 10. Relationship of HA to culture age. C e l l s grown on TYH medium were harvested, washed and resuspended i n PBS (A.,,,, = 1.0). Culture supernatant was assayed without boo concentration. 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 of wells of p o s i t i v e HA. Time - hr 109 Table 9. E f f e c t of treatment of RBC on t h e i r a b i l i t y to hemagglutinate with soluble HA. Reagent Concentration Units/ HA t i t e r mg Trypsin 1 mg/ml 65 16 Control 32 a~ chymo tryp s i n 1 mg/ml 65 16 Control 32 Pronase 1 mg/ml 49 4 Control 32 S u b t i l i s i n 1 mg/ml 10 8 Control 32 DNase 1 mg/ml 1200 32 Control 32 Neuraminidase 250 yg/ml 1.0 64 Control 16 3-galactosidase 250 yg/ml 4.0 4 Control 16 Dextranase 250 yg/ml 4.0 32 Control 32 Red blood c e l l s were exposed to the d i f f e r e n t enzymes for 30 min at 37°C i n 0.1 M acetate buffer (pH 4.0) i n case of neuraminidase, 3-galactosidase and dextranase, and i n PBS (pH 7.0) for the other reagents i n the presence of 25 mM using 48 hr culture supernatant of 2D I5_. melaninogenicus. 110 that agglutination of the HA and RBC i s not an i o n i c i n t e r -action. EDTA had no e f f e c t on the HA a c t i v i t y . Pretreatments with reagents which normally i n t e r a c t with proteins ( t r y p s i n and formalin) i n h i b i t e d the HA reaction. Treatment with sodium periodate also i n h i b i t e d the HA reaction. These r e s u l t s give some i n d i c a t i o n that protein and/or carbohydrate moieties as w e l l as d i s u l f i d e : bonds may be necessary for HA to occur. However, these conclusions are very tentative because of the crude and r e l a t i v e l y non-s p e c i f i c nature of these treatments. 6. E f f e c t of 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 HA reactions are i n h i b i t e d by incubation with s p e c i f i c carbohydrates, suggesting that these compounds may be receptors. The r o l e of carbo-hydrates i n the Ji. melaninogenicus HA r e a c t i o n was assessed by pre-incubating the culture supernatant for 30 min with various carbohydrates. Glucose, lactose, fructose, rhamnose, ribose, c e l l o b i o s e , fucose and sucrose at a concentration of 5% had no e f f e c t on the HA r e a c t i o n . Galactose i n h i b i t e d the HA t i t e r by 75%. 7. S t a b i l i t y of the Soluble HA The HA a c t i v i t y was stable at 4°C for 3 days i n the absence of a reducing agent. At 37°C and 20°C, the a c t i v i t y was stable for 48 hr i n the absence of a reducing agent. Heating at 57°C for 30 min did not i n h i b i t the HA a c t i v i t y , but heating at 70°-80°C for 10 min or 100°C for 5 min completely destroyed hemagglutination. No change i n a c t i v i t y occurred a f t e r f r e e z i n g and thawing i n the absence of BME. I l l •8. Oxygen S e n s i t i v i t y of the Soluble HA The e f f e c t of oxygen on HA was demonstrated i n an experiment i n which the supernatant from a 48 hr culture of 2D was subjected to vigorous aeration for 30 min p r i o r to the assay (Table 10). The HA a c t i v i t y was constantly i n h i b i t e d by 2 t i t e r s upon aeration, but a c t i v i t y could be r e -stored by the a d d i t i o n of 3-mercaptoethanol (25 mM). A d e f i n i t e increase i n the HA t i t e r occurred when a reducing agent such as (3ME "or cysteine was included i n the HA assay, although the e f f e c t i n t h i s case was not as pronounced because the supernatant contained endogenous reducing a c t i v i t y . When the culture supernatant was c o l l e c t e d and assayed i n the anaerobic chamber, reducing agents had no b e n e f i c i a l e f f e c t on the HA a c t i v i t y . 9. E f f e c t s of Sulfhydryl Modifiers on HA Since reducing conditions are required for the HA a c t i v i t y , reagents that bind to s u l f h y d r y l compounds were tested for i n h i b i t i o n of the HA (Table 11) . HgC^ i n h i b i t e d HA and i n h i b i t i o n could be reversed by addingBME. Iodoacetic acid and iodoacetamide, which 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 the soluble HA. 10. U l t r a c e n t r i f u g a t i o n of Soluble HA U l t r a c e n t r i f u g a t i o n of the supernatant at 141,000 x g for 1 hr resulted i n the sedimentation of 83% of the HA a c t i v i t y . A small amount remained i n the supernatant (Table 12). The majority of the HA i n the culture supernatant seemed to be of high molecular weight and might be particle-bound; i t i s not known whether the non sedimenting HA represented a d i f f e r e n t HA or the HA i n a d i f f e r e n t form. 112 Table 10. E f f e c t of aeration on soluble HA Aeration 3ME* HA t i t e r - - 16 - + 64 + + 64 + - 8 (3ME was added immediately before the assay. 113 Table 11. E f f e c t of s u l f h y d r y l modifiers on HA * I n h i b i t o r s Concentration mM HA t i t e r Control 0 16 Control + BME 0 64 HgCl 2 5 2 HgCl2+BME 5 32 Iodoacetic acid + BME 25 2 Iodoacetamide + BME 25 2 *Forty-eight hr culture supernatant was incubated for 30 min at room temperature with the reagent i n Tris-HCl buffer, pH 8.2 before assaying i t for HA. BME was used at a concentration of 25 mM. Table 12. U l t r a c e n t r i f u g a t i o n of soluble HA HA HA t i t e r % of t o t a l HA A c t i v i t y p r i o r to cen t r i f u g a t i o n 2448 100 P e l l e t 2034 83.0 Supernatant 388 15.8 The HA was assayed by the m i c r o t i t e r plate method and was expressed as HA t i t e r per t o t a l volume of the sample. 115 P a r t i a l P u r i f i c a t i o n of soluble HA a. Concentration of the HA. Because the HA i s e x t r a c e l l u l a r and therefore d i l u t e , i t was necessary to concentrate i t as a f i r s t step i n p u r i f i c a t i o n procedures. A number of procedures were tested to determine the most e f f e c t i v e method. The HA i n the supernatant could be concentrated by freeze-drying, by f l a s h evaporation and by (NH^^SO^ p r e c i p i t a t i o n . It could not be concentrated by the use of 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 or XM-50 membranes due to adsorption of the HA to the d i a l y s i s tubing and to the 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 binds to RBCs. I t seemed therefore possible to devise an a f f i n i t y chromatography procedure which would take advantage of t h i s character-i s t i c . Formalinized RBCs were mixed for 30 min at 4°C with supernatant containing HA, and washed as described i n Materials and Methods. Hemagglutinin was eluted most s a t i s f a c t o r -i l y with 8 M urea containing 25 mM |3ME. A number of s a l t s i ncluding NaCl, NH4C1, MgCl 2 > MgSO^, CaCl 2,'KCl and L i C l were not e f f e c t i v e i n re l e a s i n g HA. A v a r i e t y of s a l t concentrations (up to 6 M) and pH conditions were tested but none proved to be e f f e c t i v e . Galactose, glucose and ce l l u b i o s e did not elute the HA. Acetaldehyde, t r i t o n X-100, H o0-saturated 116 butanol as well as guanidine HC1 were not e f f e c t i v e i n e l u t i n g the HA from RBCs. The c h a r a c t e r i s t i c s of the urea eluate are shown i n Table 13. Some b a c t e r i a have been shown to bind to neuraminic acid residues on RBC (209). This does not appear to be the case with the B. melaninogenicus HA, as the removal of neuraminic acid residues with neuraminidase increased the a b i l i t y of RBC to bind HA, and e l u t i o n of HA adsorbed to neuraminidase-treated RBCs with 8 M urea was found l e s s e f f e c t i v e than e l u t i o n of adsorbed HA from untreated RBCs which might be due to a t i g h t e r binding of soluble HA to neuraminidase-treated RBC. This 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% recovery of the soluble HA of the supernatant (Table 14). A second treatment of the RBC with 8 M urea yielded more of the HA, but there was no increase i n the s p e c i f i c a c t i v i t y of the eluted HA. This might be due to e l u t i o n of protein constituents from the RBC together with the HA. A c o n t r o l of RBC treated with PBS instead of culture supernatant was also eluted with 8 M urea. i i ) Gel f i l t r a t i o n on Sephadex G-100 The HA eluted from RBC was l y o p h i l i z e d , resuspended i n PBS and applied to a Sephadex G-100 column and eluted with PBS. The r e s u l t s are shown i n F i g . 11 and Table 14. Even though the HA was excluded from the beads Table 13. Characteristics of the HA eluted from RBC with Urea HA i n the supernatant Adsorbed HA % to FRBC Urea % eluted HA Protein content ug/ml Carbohydrate content ug/ml U l t r a c e n t r i f u g a t i o n of Urea eluted HA P e l l e t % Supernatant % 81920 80600 98.4 40960 50.8 5600 800 31949 78 9011 22 The HA eluted from RBC with urea was centrifuged at 141,000xg for 1 hr. The HA was assayed by the m i c r o t i t e r plate method and i s expressed as the t i t e r produced i n t o t a l volume of samples. TABLE 14. Analysis of the HA P u r i f i c a t i o n Procedures Methods of Total HA To t a l Protein HA titer/mg P u r i f i c a t i o n '% P u r i f i c a t i o n t i t e r mg protein factor Recovery 1. Culture f i l t r a t e 1024 6.8 150 2. E l u t i o n from RBC 512 0.31 1652 11 50 3. Sephadex G-100 512 0.06 - 8533 57.2 50 The HA was determined by the m i c r o t i t e r plate and the protein was measured by the Lowry method (132). FIGURE 11. Sephadex G-100 g e l f i l t r a t i o n of the soluble HA. A 59 X 1.6 cm column was used at 5.6 ml/hr flow rate. The void volume of the column was determined with Blue Dextran 2000, and a sample of 0.06 mg protein of HA eluted from RBC was applied to the column and 3.2 ml factions were c o l l e c t e d . The o p t i c a l density was followed at 280 nm f o r protein determination and the HA was assayed by the m i c r o t i t e r method. 120 Fr a c t i o n Number 121 and appeared i n the void volume of the column, the recovery was s a t i s f a c t o r y and good p u r i f i c a t i o n was achieved. The adsorption and e l u t i o n of the soluble HA from RBC followed by gel f i l t r a t i o n through Sephadex G-100 resulted i n a 57.2 f o l d - p u r i f i c a t i o n and' 50% recovery. The p a r t i a l l y p u r i f i e d HA had the same c h a r a c t e r i s t i c s as the HA of the culture supernatant with regard to pH optima, s t a b i l i t y and i n h i b i t i o n by HgC^-The presence of reducing agents enhanced the HA t i t e r of the p a r t i a l l y p u r i f i e d preparation, c. Binding to M i l l i p o r e f i l t e r s I t was noted that HA a c t i v i t y was l o s t when the culture supernatant was passed through a M i l l i p o r e f i l t e r ; i t seemed possible that some type of binding was occurring and t h i s could 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. A l l the reagents used i n the attempts to elute the HA from RBCs were tested i n the M i l l i p o r e f i l t e r system and were found to have no e f f e c t . It was also found that the M i l l i p o r e f i l t e r had a f i n i t e capacity for binding HA, so i t was l i k e l y a binding i n t e r a c t i o n rather than just adsorption or pore-size binding. The speculations that the binding phenomena of HA to M i l l i p o r e f i l t e r would be advantageous i n the p u r i f i c a t i o n of soluble HA were corre c t ; 30.4 f o l d p u r i f i c a t i o n was accomplished, but the recovery of the HA was r e l a t i v e l y low. 122 C. Protease of B_. melaninogenicus A c t i v e l y growing cultures of B_. melaninogenicus-2D produce protease(s) which are bound to the c e l l as well as being free i n the growth media. When measured by the a b i l i t y to hydrolyze casein, 80% of the p r o t e o l y t i c a c t i v i t y was found to be c e l l associated. It i s not known whether the c e l l - f r e e and cell-bound enzymes are the same or whether they are d i f f e r e n t gene products. 1. Protease Assays The c e l l u l a r and soluble protease(s) of 15. melaninogenicus are a c t i v e against a number of protein substrates including a z o c o l l , casein, azocasein and N,N-dimethylcasein. For the a z o c o l l assay, the amount of dye released, 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 at 520 nm. This assay i s usually q u a l i t a t i v e rather than quantitative. The casein assay depends on the determination of the amounts of TCA soluble peptides l i b e r a t e d from the casein substrate by the enzyme. The assay depends on measuring the absorbance at 0^280' a n t^ i n most cases, 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 i n t h i s study contained large amounts of materials that absorb at 280 nm. The assay was, therefore, affected by the presence of large concentrations of peptides or amino acids i n the sample to be assayed. The dimethylcasein assay was also used. The p r o t e o l y t i c a c t i v i t y was followed by determining with TNBS the production of new amino groups upon h y d r o l y s i s . Low blank values were obtained with t h i s assay, however the preparation of the substrate and the assay procedure i t s e l f were time consuming and not reproducible. 123 C-labeled N,N-dimethylcasein was also used as a substrate for determining 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 the assay was more se n s i t i v e than the spectrophotometric procedures. Since the azocasein assay also gave r e l i a b l e r e s u l t s but was l e s s expensive, t h i s assay' was used throughout the experimental study. The azocasein 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 protein. The absorption maximum of t h i s substrate was 370 nm. The maximum absorbance of the cult u r e supernatant and c e l l - e x t r a c t was found to occur at a range of wavelength between 220-320 nm and l i t t l e or no absorbance occurred at 370 nm. Therefore, there was no background, the assay was reproducible, quick and r e l i a b l e ; and the substrate was easy to prepare. The azocasein assay proved to be the easiest to perfom. Control values were obtained by assay without enzyme and were subtracted from experimental values. The absorbance of the cont r o l never exceeded 0.1. The enzyme preparations had no absorbance at t h i s wavelength. Azocasein units were defined as the change i n absorbance at 370 nm per 60 min per ml at 37°C i n the presence of PBS (pH 7.0). The time course of release of azoMye from the azocasein by IS_. melaninogenicus i n t r a c e l l u l a r protease i s shown i n F i g . 12. The rate of release of the chromophore was l i n e a r during the early part of the reaction . The e f f e c t of substrate concentration on the release of dye from azocasein was also followed ( F i g . 13). A 2% so l u t i o n of azocasein was adequate to 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 concentration and hydro l y s i s FIGURE 12. Hydrolysis of azocasein by J3. melaninogenicus protease Each reaction mixture contained: c e l l - e x t r a c t , 2 mg protein/0.25 ml; 50 mM BME i n PBS, 0.75 ml: 2% azocasein, 1 ml. Incubating temperature was 37°C. 125 0 20 40 60 80 100 Time - Minute FIGURE 13. E f f e c t of azocasein concentration. Each reaction mixture contained: c e l l - e x t r a c t , 2 mg protein; 50 mM (3ME i n PBS, 0.75 ml: concentrations of azocasein i n 1 ml PBS as indicated. Reaction mixtures were incubated at 37°C for 60 min. 127 r Azocasein (mg) 128 of the substrate i s described i n F i g . 14. As can be seen, there i s a l i n e a r r e l a t i o n s h i p between the enzyme concentration and p r o t e o l y t i c a c t i v i t y . The assay i s s e n s i t i v e , and by increasing incubation times, small amounts of enzyme could be measured. Azocasein was incorporated into agar to assay various preparations of protease, as mentioned i n the Materials and Methods. The azocasein agar assay was u s e f u l for assaying protease i n electrophoretic polyacrylamide gels. The c e l l u l a r and e x t r a c e l l u l a r proteases of B_. melaninogenicus were unable to hydrolyze hemoglobin and bovine serum albumin (BSA) at l e a s t to the extent that i t was possible to detect hydrolysis products spectrophotometrically (1-3.2 mg of c e l l - e x t r a c t and c u l t u r e supernatant were incubated for 1 hr with these substrates at 37°C). 2. Relationship of Protease to Culture Age The appearance of c e l l u l a r and soluble proteases was followed as a function of the age of the cu l t u r e . The organisms were grown at 37°C under anaerobic conditions. As shown in F i g . 15, the production of the c e l l u l a r and soluble proteases p a r a l l e l e d the growth curve of the organism u n t i l 48 hr when a further increase of the soluble protease was noted and continued as the c e l l s began to l y s e . 3. Cell-bound Protease of IS. melaninogenicus The 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 culture super-natant and f a i l u r e to concentrate the soluble protease without decrease i n a c t i v i t y indicated that i t would be wise to proceed with studies of the cell-bound protease. The c e l l u l a r protease of 15. melaninogenicus i s l o c a l i z e d 129 FIGURE 14. E f f e c t of enzyme concentration on the azocasein assay. Each reaction mixture contained: c e l l - e x t r a c t , as indicated and 50 mM BME i n PBS up to 1 ml; 2% azocasein, 1 ml. Tubes were incubated for 1 hour at 37°C. 130 FIGURE 15» Relationship of protease to culture age. C e l l s grown on TYH medium were harvested, washed and resuspended i n PBS (A.£(_ = 1.0). Culture supernatant was assayed without 660 concentration. Casein was used as the protease substrate. 132 133 i n the c e l l envelope, probably near the c e l l surface since i t i s accessible to high molecular weight substrates. Cell-bound protease could 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 er. In t h i s study, attempts were made to separate the cell-bound p r o t e o l y t i c a c t i v i t y from contaminating c e l l u l a r material and to study the r o l e of the protease i n i n f e c t i o n s by B_. melaninogenicus. a. E f f e c t of passaging 2D c e l l s i n guinea pigs on protease production. A 10 ml sample of a 48 hr culture of 2D was harvested and resuspended a s e p t i c a l l y i n 1 ml of s t e r i l e PBS. A 200 g guinea p i g was injected i n the groin with 0.5 ml of c e l l suspen-sion. A f t e r 24 hr the guinea pig had a very pronounced i n f e c t i o n . A large amount of f l u i d had c o l l e c t e d i n the thoracic area. The animal was anaesthetized and exudate withdrawn a s e p t i c a l l y and placed i n a s t e r i l e tube. A 0.5 ml sample of the exudate was transferred to a second guinea pig, and 0.2 ml samples of exudate were used to inoculate two tubes of TYH medium. P u r i t y of the exudate was checked on blood agar. A 24 hr old culture of 2D i n TYH medium inoculated with the laboratory non-passaged s t r a i n of 2D, as well as one tube of TYH medium inoculated with the exudate a f t e r passage i n the guinea pig was harvested, washed i n PBS, resuspended i n PBS containing $ME 50 mM (A,,- = 1.0) and assayed OOU for protease a c t i v i t y using azocasein as substrate. 134 A f t e r 24 hr, the second guinea pig was also infected and 0.5 ml of exudate was transferred to another guinea pig. The exudate appeared to be more i n f e c t i v e a f t e r passage. The procedure was then repeated as mentioned above. An increase i n protease production was associated with animal passage of the 2D containing exudate when assayed jLn v i t r o by the azocasein substrate (Table 15). In order to investigate the r e l a t i o n s h i p of the cell-bound protease to the growth of 13. melaninogenicus, protease production was followed during growth of the organism i n d i f f e r -ent media and under d i f f e r e n t growth conditions. b. E f f e c t of hemin concentration on growth and protease production. J3. melaninogenicus has an obligate requirement for hemin and the growth rate i s dependent on the amount of hemin present 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-tions of hemin on the growth and protease production of 2D was determined ( F i g . 16). Growth and protease production were r e l a t e d to the hemin concentration up to a l e v e l of 2.5 yg hemin/ml medium; increasing the concentration of hemin beyond 2.5 yg/ml had 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 a c t i v i t y . c. Protease production i n the presence of succinate. Protease a c t i v i t y was followed during growth of 2D i n 0.1% succinate-trypticase medium and was compared to the protease produced by 2D grown on TYH medium. Figure 17 represents the growth curve of 2D c e l l s on hemin and on succinate medium, as well as the respective protease a c t i v i t i e s at d i f f e r e n t culture ages. 135 Table 15. E f f e c t of Passaging 2D on Protease A c t i v i t y Exudates from the passage of the organism i n the f i r s t , second and t h i r d were inoculated into TYH medium and cultures were incubated anaerobically f o r 24 hr, checked f or p u r i t y , harvested, washed, resuspended i n PBS (A^^Q = 1.0), and assayed f o r protease by the azocasein assay. A 24 hr culture of 2D was used as a control i n each case. Sample Protease Units/ml/1 A,, n oou % increase i n Protease a c t i v i t y 1) 2 D c e l l s 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 r e s p e c t i v e l y . ' 1 FIGURE 16. E f f e c t of hemin concentration on growth and protease production of 2D. The A, r r. of 24 hr cultures of 2D i n d i f f e r e n t concentrations of 660 hemin i n the medium was measured and then c e l l s were harvested, washed and resuspended to A^ ^Q = 1*0 i n PBS and assayed f o r protease by the azocasein assay. Absorbance (660 nm) Protease Units/A 138 FIGURE 17. Protease production i n hemin and succinate media. Media: Trypticase-yeast medium containing 0.1% succinate as w e l l as TYH medium (as i n Materials and Methods) were inoculated with a hemin depleted 48 hr culture, A^Q = 0.8 (inoculum was 1% of the t o t a l media volume). At d i f f e r e n t culture ages, the of the culture was measured, and the c e l l s were harvested, washed and resuspended i n PBS (A,,_ = 1.0) and assayed for protease a c t i v i t y using azocasein as 660 substrate. A ^ , „ : # , Hemin; O , 0.1% succinate. 660 Protease: B , Hemin;• , 0.1% succinate. Absorbance (660 nm) 140 There was 60% greater protease a c t i v i t y i n hemin medium than i n succinate medium where the growth rate was slower. When 2D was inoculated into t r y p t i c a s e medium containing hemin (10 ug/ml) and succinate (0.1%), the growth of the organism and i t s protease production were the same as when the organism was grown on hemin medium (10 ug/ml) without succinate. Therefore, succinate had no e f f e c t on the growth rate or the protease production of 2D c e l l s when hemin was present i n the med ium. d. E f f e c t of amino acids on protease production. The addition of amino acids to TYH medium affected the growth rate and f i n a l y i e l d s of 2D 13. melaninogenicus as well as protease production (Table 16). The amino acids were added to the TYH medium ( f i n a l concentration 0.5%) and growth of 2D was followed. Cultures were harvested during the period of most rapid growth, washed and resuspended to A = 1.0 i n PBS and protease a c t i v i t y assayed using the azocasein substrate. Amino acids d i d not have any e f f e c t on the protease assay. A 31-75% increase i n protease production per c e l l occurred when the amino acids L-asparagine, glutamic a c i d , L-serine, L-proline and L-methionine were included i n the growth medium. This c o r r e l a t e s with the growth enhancing properties of these compounds (Table 3). The amino acids L - l y s i n e and L-phenylalanine, which had no e f f e c t on the growth of the organism, did not a f f e c t the protease production. Table 16. E f f e c t of additions of amino acids to TYH medium on the p r o t e o l y t i c a c t i v i t y of I3_. melaninogenicus 141 Amino a c i d added Concentration mM 660 Protease Units/ 1 A660 None Glutamic acid L-asparagine L-serine L-proline L-methionine L - l y s i n e L-phenylalanine L-leucine L-cysteine L - h i s t i d i n e 0.38 16 34 0.75 28 37.8 0.62 24 47.6 0.58 22.5 43.4 0.5 24 33.5 0.54 21 34.2 0.36 17 30.3 0.34 14 38.1 0.12 5.8 41.3 0.04 2.6 32.2 0.08 3.7 142 The f i n d i n g obtained i n batch cultures that under d i f f e r e n t n u t r i t i o n a l conditions, the organism d i f f e r e d not only i n i t s growth rate but also i n protease production, suggested that i t might be valuable to explore the r e l a t i o n s h i p between growth rate and protease a c t i v i t y . This was accomplished by growing 13. melaninogenicus i n continuous culture at d i f f e r e n t d i l u t i o n r ates. e. Growth rate and protease production. A chemostat was designed for use i n the anaerobic chamber (see Materials and Methods) for the continuous cu l t u r e of 2D, and protease 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 rates under steady state conditions of growth. As shown i n F i g . 18, the amount of protease per c e l l increased as the growth rate increased up to a d i l u t i o n rate of 0.15 hr \ at t h i s point there was a decrease i n enzyme a c t i v i t y . f. Production of the protease at d i f f e r e n t concentrations of hemin. It was found that growth and protease production by 2D were roughly proportional to hemin concentration i n batch cu l t u r e s . To determine whether the hemin had a d i r e c t e f f e c t on the protease, or whether i t was acting i n d i r e c t l y by i n f l u e n c i n g the growth rate, the organism was grown i n continuous culture i n d i f f e r e n t l e v e l s of hemin but at the same growth rate. The protease was measured at steady states of growth by harvesting the culture and resuspending i t a f t e r washing i n PBS to an A,, n = 1.0 i n PBS. Results are shown i n Table 17. It 143 FIGURE 18. E f f e c t of d i l u t i o n rate (D) on protease production by 15. melaninogenicus When a steady state had been achieved c e l l s were harvested, washed and resuspended i n PBS (AggQ = 1.0) and assayed f o r protease by incubation f o r 1 hr at 37°C with the azocasein substrate. Table 17. E f f e c t of hemin concentration on protease production. 145 Hemin concentration ug/ml medium A 660 a t f i n a l steady rate Protease Units/ - A 6 60 1 1.5 2 2.5 5 10 20 1.1 1.2 1.25 1.2 1.3 1.3 1.2 7.8 8 7.8 8.2 8.7 7.9 8.2 146 can be seen that d i f f e r e n t hemin concentrations had no d i r e c t e f f e c t on the protease a c t i v i t y i n continuous culture. In the continuous culture where the growth rate of 2D was controlled using the chemostat and the c e l l s were at steady state of growth i n TYH medium, changing the medium to TYH containing 0.5% L-cysteine at the same d i l u t i o n rate 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) and a p a r a l l e l decrease i n protease a c t i v i t y per c e l l . g. Preliminary 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. melaninogenicus. i ) E f f e c t of reducing agents on the c e l l u l a r protease The data i n Table 18 show the e f f e c t of d i f f e r e n t reducing agents on the a c t i v i t y of the protease i n the c e l l - e x t r a c t of 13. melaninogenicus 2D. A concen-t r a t i o n of at l e a s t 10 mM reducing agent was required f o r maximum p r o t e o l y t i c a c t i v i t y . Increasing the concen-t r a t i o n of reducing agent resulted i n only a s l i g h t increase i n a c t i v i t y . A preparation of 50 mM concentration of BME or cysteine was chosen to be r o u t i n e l y used i n the assays for protease. Freshly prepared culture extract was not as dependent on the presence of exogeneous reducing agent, presumably because the extract contained endogenous reducing systems. A f t e r a period of time the endogenous sources became oxidized and i t was necessary to supply red-ucing a c t i v i t y to the system. A c t i v i t y against a z o c o l l was l o s t 147 Table 18. E f f e c t of reducing agents on the c e l l u l a r protease Reducing agent Protease Units none • 6.8 cysteine 19.2 d i t h i o t h r e i t o l 18 (3-mercaptoethanol 17.6 thio g l y c o l a t e 13.4 0.5 ml samples of c e l l extract containing 3.2 mg protein were assayed i n the presence of 50 mM concentration of each of the reducing agents using a z o c o l l as substrate. 148 when the protease preparation was a e r a t e d for 20 min, but was restored by addition of reducing agent. The increased a c t i v i t y of the protease i n the presence of reducing agents indicated that the c e l l u l a r protease of B_. melaninogenicus could be c l a s s i f i e d as a s u l f h y d r y l protease. The l a t t e r are characterized p r i m a r i l y by t h e i r s e n s i t i v i t y to t h i o l reagents and generally contain cysteine moieties 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 c e l l u l a r protease. The t h e r m o l a b i l i t y of the protease i n the c e l l extract was determined as the percent of a c t i v i t y l o s t a f t e r incubating the enzyme i n PBS at the indicated temperature (Table 19). The protease was p a r t i a l l y or completely inactivated at temperatures of 40°C and above. The protease a c t i v i t y was stable at 4°C i n the absence of 3ME for 12 hr, a f t e r t h i s there was a gradual l o s s of a c t i v i t y . Incubation of the protease at 21°C and 37°C for 4 hr i n the presence of 3ME resulted i n 20% l o s s of a c t i v i t y . The enzyme was more stable at room temperature than at 37°C for 4 hr i n the absence of reducing agents. Freezing and thawing of the c e l l - e x t r a c t at -70°C resulted i n 8-10% l o s s of a c t i v i t y i n the absence of $ME and 14-25% l o s s of p r o t e o l y t i c a c t i v i t y Table 19. Thermolability of the p r o t e o l y t i c a c t i v i t y i n the c e l l - e x t r a c t . Temperature, Time, minutes a c t i v i t y l o s t % 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 protease was assayed by the azocasein assay at 37 C f o r 1 hr. 150 i n the presence of 3ME. Therefore, protease pre-paration were kept free of reducing agent u n t i l they were assayed. The r e s u l t s suggested that the protease was subject to autodigestion as i n a c t i v a t i o n of the enzyme was more rapid when reducing agents were present, 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 l i . melaninogenicus. i ) Preparation of c e l l - e x t r a c t As the cell-bound protease of 2D could 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 buffer, a serie s of experiments was conducted to determine the most e f f e c t i v e method for l i b e r a t i n g the enzyme. There was an increase i n the t o t a l c e l l u l a r protease a c t i v i t y i n the broken c e l l suspension a f t e r c e l l d i s i n t e g r a t i o n . I t i s possible that breakage of c e l l s might have unmasked protease from protein complexes and/or s o l u b i l i z e d enzyme from membrane structures. Some of the enzyme remained bound to the mechanically ruptured c e l l envelopes. The extent of casein h y d r o l y t i c a c t i v i t y released varied with the technique used to rupture the c e l l s (Table 20). Breakage of the c e l l s i n the French Pressure c e l l proved to be the most e f f e c t i v e method since 80% of the c e l l u l a r protease was liberated, i n the c e l l - e x t r a c t . 151 Table 20. Comparison of methods for l i b e r a t i n g protease % of c e l l u l a r protease Protease found i n the c e l l - e x t r a c t per mg/protein 1) French Pressure c e l l 80 3.6 2) M i n i - M i l l 40 1.9 3) Sonication 25 0.8 The c e l l s were obtained from early stationary phase cultures of I3_. melaninogenicus grown i n THY medium and were washed and suspended i n PBS. 152 Following breakage the c e l l - e x t r a c t was dialyzed against PBS overnight at 4°C. This resulted i n a 1.8 f o l d p u r i f i c a t i o n and only a 6% l o s s of the protease a c t i v i t y (Table 22). The dialyzed c e l l - e x t r a c t was centrifuged at 121,000 x g for 1 hr to sediment any r e s i d u a l p a r t i c u l a t e fragments. Ninety-two percent of the protease remained i n the supernatant with a two-fold increase i n the s p e c i f i c a c t i v i t y . Therefore, d i a l y s i s and u l t r a c e n t r i f u g a t i o n were used as preliminary steps 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 . i i ) Ethanol p r e c i p i t a t i o n . The protease was p r e c i p i t a t e d from the dialyzed and centrifuged c e l l - e x t r a c t by 60% ethanol at -10°C. Under appropriate conditions, the p r o t e o l y t i c a c t i v i t y was not destroyed and the p r e c i p i t a t i o n was almost quantitative. Conditions were determined such that the protease to be separated had a low s o l u b i l i t y when most other components of the system had high solub-i l i t i e s . The p r e c i p i t a t i o n was c a r r i e d out at a low temperature to prevent protein denaturation. Special precautions were needed to insure that the temperature was held at -10°C at every stage during the process. A b r i e f r i s e of temperature to a few degrees above 0°C for a few minutes had undesirable e f f e c t s on the s t a b i l i t y of the sample. Constant slow s t i r r i n g was e s s e n t i a l i n 153 order to prevent any element of the protein s o l u t i o n from a t t a i n i n g , even temporarily, an unduly high ethanol concentration which might denature the protein. The necessary precautions were fundamentally simple but they had to be s t r i c t l y maintained and not relaxed i n any step i n the process. The e f f e c t s of d i f f e r e n t concentrations of ethanol on the p r e c i p i t a t i o n of protease at d i f f e r e n t temperatures are shown i n Table 21. A concentration of 60% ethanol at -10°C for 20 min. proved to be most s a t i s f a c t o r y , giving a high recovery and a s i g n i f i c a n t increase i n s p e c i f i c a c t i v i t y . Other conditions resulted i n higher recovery of the protease but the enzyme had lower s p e c i f i c a c t i v i t y . This technique provided a simple and e f f e c t i v e step for the p u r i f i c a t i o n of the protease from the c e l l - e x t r a c t . The ethanol p r e c i p i t a t e d sample was then resuspended i n PBS and centrifuged at 121,000 x g for 1 hr and the p e l l e t was discarded. The supernatant was dialyzed overnight against PBS (Table 22). i i i ) Sephadex G-100 i n 6M urea. The dialyzed and centrifuged protease pre-c i p i t a t e d from the c e l l - e x t r a c t by ethanol was applied to a column of Sephadex G-100 (1.6 x 62 cm) e q u i l i b r a t e d with 6M urea i n PBS. The flow rate was 5 ml/h and 2 ml f r a c t i o n s were c o l l e c t e d . A l l of the protease was Table 21. Ethanol p r e c i p i t a t i o n of protease from c e l l - e x t r a c t . Protease A c t i v i t y % Recovery Temperature °c Ethanol con- Time -5 -10 -20 centration 30% 5 32 10 — 38 _ 20 — 45 — 40% 5 39 60 19 10 46 64 53 20 48 66 70 50% 5 89 70 87 10 79 82 90 20 92 94 93.2 60% 5 90 90 89 10 98 100 102 20 96 100 102 70% 5 90 99 100 10 96 99 98 30 102 102 104 Table 22. P u r i f i c a t i o n of protease from B. melaninogenicus - 2D Fra c t i o n Total A c t i v i t y Units S p e c i f i c A c t i v i t y °/, Units/mg Protein ', Recovery P u r i f i c a t i o n Factor4 1. c e l l - e x t r a c t 2080 2.8 100 1 2. D i a l y s i s ^ 1955 5.04 94 1.8 3. 2 Centrifugation 1799.2 8.06 86.5 2.9 4. Ethanal p r e c i p i t a t i o n 1540 19.4 74 6.9 5. U l t r a c e n t r i -fugation of ethanol p r e c i p i t a t e 1354.5 38.7 65.12 13.8 6. -. • 3 D i a l y s i s 1330 62 64 22.1 7. 8. Sephadex G-100 i n 6 M Urea Sepharose 2-B i n 6 M Urea 2662.5 A 125.0 B 3203 434 1302 2083.2 128 6 154 154.8 464.4 774 D i a l y s i s against 10 volumes of PBS overnight. Centrifugation at 121,000 x g for 1 hr D i a l y s i s against 10 volumes of PBS (pH 7.0) overnight. P u r i f i c a t i o n factor i s the f o l d increase i n the enzyme s p e c i f i c a c t i v i t y 156 excluded from the column and eluted as a sing l e peak at the void volume, i n d i c a t i n g i t had a large molecular weight (Fig. 19). The eluted protease represented 220% ,of the s t a r t i n g material. The increase i n a c t i v i t y could be due to removal of i n h i b i t o r s or components that were binding to the act i v e s i t e s of the protease, or to cont-aminating proteins which competed as substrates. The pooled, dialyzed, concentrated protease was p u r i f i e d 7-fold i n t h i s step. Electrophoretic analysis of the protease indicated that there were a number of proteins present i n t h i s f r a c t i o n (see Section h-vi) i v ) Sepharose-2B i n 6 M urea. The protease- containing eluate from a Sephadex G-100 column was dialyzed against PBS overnight, concentrated by freeze-drying, made 6M with respect to urea and fractionated by chromatography on a column of Sepharose-2B e q u i l i b r a t e d with 6 M urea i n PBS (Fig. 20). The flow rate was adjusted to 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 the column was eluted with the same bu f f e r , the active protease emerged i n 2 f r a c t i o n s d i f f e r i n g i n s p e c i f i c a c t i v i t y . When these f r a c t i o n s (A and B) were separately chromatographed on the same column, each emerged as a sing l e peak at the o r i g i n a l e l u t i o n volume, one i n the void volume (A) and the other included i n the column (B). The f r a c t i o n s were pooled separately and dialyzed against PBS overnight and FIGURE 19. Gel f i l t r a t i o n of the ethanol p r e c i p i t a t e d protease. A 6 ml sample of ethanol p r e c i p i t a t e d protease (20.4 mg protein) contain-ing 6 M urea was applied to the Sephadex G-100 column and the protease was eluted i n PBS-6M urea (pH 7.0). The void volume of the column was determined with Blue Dextran, and absorbance at 280 nm was recorded. The fr a c t i o n s were assayed for protease by the Azocasein assay. 158 0 20 40 60 80 100 Fra c t i o n Number FIGURE 20. Fr a c t i o n a t i o n on Sepharose 2B The pooled, dialysed, concentrated protease from Sephadex G-100 (3 ml of t o t a l 2.2 mg protein) was eluted from a Sepharose-2B column (1.6X 28.3 cm) e q u i l i b r a t e d with 6M urea i n PBS (pH 7.0). The absorbance at 280 nm was measured and protease a c t i v i t y was assayed using azocasein as substrate. 160 161 concentrated by freeze-drying to t h e i r o r i g i n a l volume. At t h i s stage, i t was not possible to decide whether f r a c t i o n s A and B represented the same or d i f f e r e n t compounds. Both components were found to consist of the same proteins when examined by SDS-polyacrylamide gel electrophoresis i n tris-glycine-SDS buffer which separates protein subunits on the basis of molecular weight. Both lacked electrophoretic m o b i l i t y i n polyacrylamide gels without SDS due to 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 indicates that they are the same. A summary of the enzyme p u r i f i c a t i o n scheme i s given i n Table 22. The protease was p u r i f i e d 464-fold i n f r a c t i o n A, and 774-fold i n B, with a combined recovery of 160%. The enzyme preparation obtained a f t e r the Sepharose-2B step w i l l hereafter be referred to as p u r i f i e d protease. Since the protease was eluted from the Sepharose-2B column as two components having d i f f e r e n t , but s t i l l large molecular weights, which did not migrate i n polyacrylamide gel electrophoresis; i t seemed possible that the protease a c t i v i t y might have been associated with a component of the c e l l w a l l . This would r e s u l t i n the as 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 with random sized 162 and charged fragments when the c e l l s are d i s i n t e g r a t e d . Difference i n chromatographic m o b i l i t i e s between f r a c t i o n s A and B might have been due to random fragments from c e l l w a ll being associated with the p u r i f i e d protease. Denat-uration of both f r a c t i o n s by b o i l i n g i n SDS might d i s s o c i a t e these fragments r e s u l t i n g i n two s i m i l a r components, which might represent a polymeric form of the enzyme. Since f r a c t i o n A contained very l i t t l e enzyme, d e t a i l e d studies on the p u r i f i e d protease were performed on f r a c t i o n B. v) Other chromatographic procedures. Activated (SH) thiol-Sepharose 4B Sepharose-4B containing t h i o l as f u n c t i o n a l groups was used i n an attempt to s e l e c t i v e l y bind SH-containing proteins. The t h i o l proteins covalently bind to the immobilized t h i o l and can be eluted by reduction of the S-S-bond with an appropriate reducing agent. A 20 ml column of activated t h i o l -Sepharose 4B was used i n an attempt to immobilize the protease present i n the ethanol p r e c i p i t a t e . Proteins not bound to the Sepharose were eluted with deaerated PBS containing 1 mM EDTA. Forty-one percent of the protease added to the column did not bind; i t had the same s p e c i f i c a c t i v i t y as the s t a r t i n g material. Approximately 89% of the bound protease was eluted with 20 mM cysteine-HC1 i n PBS (pH 7.0) with a two-fold increase i n 163 s p e c i f i c a c t i v i t y (Fig. 21). The procedure was not as e f f e c t i v e as gel f i l t r a t i o n and was therefore abandoned. Both protease f r a c t i o n s behaved i n a s i m i l a r respect with regard to i n h i b i t o r s , gel electrophoresis, etc. suggesting that t h e i r d i f f e r e n t binding properties were due to saturation 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 containing immobilized Hg was attempted. The ethanol pre-c i p i t a t e d sample was applied to a 30 ml column of Hg-Sepharose and 44.7% of the protease did not bind. There was no p u r i f i c a t i o n of t h i s f r a c t i o n . Eighty-two percent of the bound protease was recovered with a 4-fold increase i n s p e c i f i c a c t i v i t y ( F ig. 22). Polyacrylamide gel e l e c t r o -phoresis i n SDS revealed that both peaks contain-ing protease were composed of s i m i l a r proteins (Section h - v i ) . The binding of the protease to both the activated thiol-Sepharose and the Sepharose mercury columns gave a d d i t i o n a l i n d i c a t i o n regarding the nature of the protease as a s u l f -hydryl enzyme. The proportion of the protease that did not bind to either column might have FIGURE 21. Chromatography on t h i o l Sepharose-4B 5.8 ml sample of ethanol p r e c i p i t a t e (8.8 mg protein) was applied to a 1.6 x 10 column. Unbound proteins were eluted with deaerated PBS containing 1 mM EDTA. The bound protease was eluted with 20 mM cysteine-HCl i n PBS (pH 7.0). Fractions of 1 ml were c o l l e c t e d at a flow rate of 2.8 ml/hr. E l u t i o n of protein was monitored by measuring absorption at 280 nm. Enzymatic a c t i v i t y was determined by the azocasein substrate. 165 Protease Units/ml M H to to U ) Ln O Ui O Ul O to -l>- O N CO to J>-> to CO o FIGURE 22. Sepharose mercury chromatography The column was e q u i l i b r a t e d with 50 mM acetate buffer pH 5.5. The ethanol p r e c i p i t a t e d enzyme was applied i n 50 ml (170 mg p r o t e i n ) , and the column was rinsed with acetate buffer ( 50 mM pH 5.5); f r a c t i o n s of 2 ml were c o l l e c t e d at a rate of 3 ml/hr. Bound protein was then eluted with 10 mM cysteine i n acetate buffer. Fraction Number 168 been due to oxidation or blocking of t h e i r cysteine moieties. In attempts to separate contaminating proteins from the protease complex, the following procedures were applied without success as judged by gel electrophoresis and by s p e c i f i c a c t i v i t y : G e l - f i l t r a t i o n through G-100 and Sepharose 2B and 4B i n various buffers and i n the presence of SDS and/or urea at d i f f e r e n t concent-r a t i o n s , s e l e c t i v e heat denaturation and (NH^^SO^ p r e c i p i t a t i o n . Ion exchange chromato-graphy on CM-Sephadex C-50 and DEAE-Sephadex A-50 under d i f f e r e n t conditions of b u f f e r s , pH and i n the presence of SDS (0.1-0.2%) and/or urea (2M-8M), were also t r i e d i n an e f f o r t to break the protease complex i n the c e l l - e x t r a c t and the ethanol p r e c i p i t a t e to minimum fun c t i o n a l compo-nents without success, since the protease a c t i v i t y was poorly recovered from a large number of f r a c t i o n s without s i g n i f i c a n t p u r i f i c a t i o n . Hydrophobic i n t e r a c t i o n chromatography was used to bind the protease non-covalently to an i n e r t support i n phosphate buffer containing 1 M NaCl. Bound proteins were eluted i n a 20-50% gradient of ethylene g l y c o l i n PBS. A poor recovery resulted from t h i s column with no s i g n i f i c a n t p u r i f i c a t i o n . 169 v i ) Gel electrophoresis. Polyacrylamide gel electrophoresis per- . formed as described by Nagai et^ a l . (156) i n a 10% gel i n tris-glycine-SDS buffer, pH 8.3, demonstrated only three major bands and one minor band i n the p u r i f i e d protease obtained from chromatography on Sepharose 2B (Fig. 23). Figure.24 represents the polyacrylamide gel electrophoresis i n T r i s - g l y c i n e SDS buffer of the d i f f e r e n t protease preparations obtained at d i f f e r e n t steps of the p u r i f i c a t i o n scheme presented i n Table 22. The gel revealed the presence of a t o t a l of f i f t e e n major bands i n the c e l l - e x t r a c t and ten major bands i n the centrifuged and dialyzed ethanol p r e c i p i t a t e d sample. The protease sample obtained from gel f i l t r a t i o n through Sephadex G-100 possessed seven major bands while the p u r i f i e d protease possessed only four bands, thereby i n d i c a t i n g that the p u r i f i c a t i o n procedure eliminated most of the protein f r a c t i o n s present i n the c e l l - e x t r a c t . The polyacrylamide gel electrophoresis i n Tris-glycine-SDS buffer of the crude as well as the p a r t i a l l y p u r i f i e d enzyme preparations throughout the d i f f e r e n t chromatographic and various p u r i f i c a t i o n procedures are represented i n F i g . 25. The samples obtained from the various p u r i f i c a t i o n procedures revealed the removal of some major bands as compared to FIGURE 23. Polyacrylamide g e l electrophoresis i n Tris-glycine-SDS bu f f e r of the p u r i f i e d protease. A and B represent Fractions A and B eluted from the Sepharose column with 6 M urea. 171 A B FIGURE 24. Polyacrylamide gel electrophoresis i n Tris-glycine-SDS buffer of protease f r a c t i o n s obtained during various steps i n the p u r i f i c a t i o n 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 centrifuged c e l l extract; Gel-j--j-j,ethanol-precipitated sample; G e l ^ , c e n t r i f u g e d ethanol-treated sample; Gel^,dialysed and centrifuged ethanol-precipitated sample; Gel^. protease sample obtained a f t e r gel f i l t r a t i o n through Sephadex G-100; Gel p u r i f i e d protease preparation obtained a f t e r f r a c t i o n a t i o n on Sepharose-2B. Two mg protein 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 containing 4% SDS, 20% g l y c e r o l , 0.125 M T r i s (pH 6.8) and 0.01% bromophenol blue; 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 water bath. Ten percent (3ME was added and b o i l i n g was continued f o r another minute. 173 • 3 ] [ II III IV V VI VII FIGURE 25. Polyacrylamide g e l electrophoresis of protease f r a c t i o n s 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. Gel^. ethanol 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 thiol-Sepharose chromatography; Gel F r a c t i o n 2 from the thiol-Sepharose column; G e l ^ protease eluted from Octyl-Sepharose CL-4B column; Gel^ F r a c t i o n 1 from the mercury Sepharose column; Gel^ F r a c t i o n 2 from the mercury Sepharose column; G e l ^ ^ protease f r a c t i o n eluted from Sephadex G-100 with 0.1% SDS i n PBS; Gel protease f r a c t i o n obtained from Sephadex G-100 eluted with 0.1% SDS and 4 M urea i n PBS; Gel protease sample eluted from Sephadex G-100 with 6 M urea; Gel crude c e l l - e x t r a c t ; A Gel p u r i f i e d protease preparation. Fraction 1 re f e r s to protease which did not bind to the column, F r a c t i o n 2 r e f e r s to protease which bound to the column and was eluted with reducing agent. 20 y l samples, each containing 20 yg protein, were applied to the gel a f t e r b o i l i n g f o r 3 min i n 4% SDS, 20% g l y c e r o l , 0.125 M T r i s buffer (pH 6.8), 0.01% bromophenol blue and 10% (BME. 175 I II I I I IV V VI VII VIII IX X XI 176 the c e l l - e x t r a c t ( G e l x ) , however none of these procedures yielded a p u r i f i c a t i o n comparable to that obtained (Gel XI) by the p u r i f i c a t i o n scheme presented i n Table 22. The samples were electrophoresed towards both the anode and cathode i n the absence of SDS without denaturation. The samples were dissolved i n a s o l u b i l -i z a t i o n mixture without SDS and $ME. The SDS was also omitted from the running buffer, the stacking and running gels. Twenty Ug protein of each sample were c a r e f u l l y layered through the T r i s - g l y c i n e buffer onto the top of the upper g e l . In the crude and p a r t i a l l y p u r i f i e d enzyme preparations, not a l l proteins migrated from the spots as evidenced by the s t a i n at these l o c a t i o n s (Fig. 26). Many bands are separated by ever increasing distance suggesting an exponential decrease 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 subunits. No migration of samples occurred when the gel was e l e c t r o -phoresed i n the other d i r e c t i o n . The p u r i f i e d protease preparation revealed the presence of one major band that did not migrate i n the polyacrylamide gel. The use of 7.5% and 5% polyacrylamide gels did not improve the electrophoretic m o b i l i t y of the p u r i f i e d protease. Therefore, i t seemed possible that the p u r i f i e d protease might s t i l l be bound i n a large molecular weight complex. FIGURE 26. Polyacrylamide gel electrophoresis i n T r i s - g l y c i n e b u f f e r without SDS. The gel depicts the electrophoretic properties of the proteins present i n the d i f f e r e n t protease preparations obtained during p u r i f i -cation. Gel^., c e l l - e x t r a c t ; Gel , dialysed c e l l - e x t r a c t ; G e l ^ ^ j , dialysed 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 , ethanol-p r e c i p i t a t e d f r a c t i o n ; Gel^,centrifuged ethanol p r e c i p i t a t e d f r a c t i o n ; Gel^j. dialysed and centrifuged ethanol p r e c i p i t a t e d f r a c t i o n ; Gely^j., protease f r a c t i o n eluted from Sephadex G-100 with 6 M urea. Gel , Fraction A of the p u r i f i e d protease preparation; Gel F r a c t i o n B V J. -L J. X A of the p u r i f i e d protease preparation; Gel 30 ug protein of the F r a c t i o n B x Gel 40 ug protein of F r a c t i o n B; Gel , 50 ug protein of F r a c t i o n B. XJ_ XX X. The samples were s o l u b i l i z e d i n 0.01% bromophenol blue contained i n 20% g l y c e r o l and 0.125 M T r i s b u f f e r (pH 6.8). There were applied to the gel without b o i l i n g and i n the absence of SDS and BME. SDS was also omitted from the running buffer, the stacking and running gels. I l l I I I IV VVI VII VIII IX XXI 179 Polyacrylamide gel electrophoresis i n the absence of SDS with l i p o p r o t e i n prestained samples revealed that the p u r i f i e d protease contained l i p o p r o t e i n s t a i n i n g material which was unable to penetrate the g e l , whereas a standard l i p o p r o t e i n , as represented by rabbit plasma,did migrate (Fig. 27). The p u r i f i e d protease i s a complex of a number of proteins as judged by i t s i n a b i l i t y to enter the polyacrylamide gel unless i t has been boiled i n SDS. Detection of glycoproteins i n SDS poly-acrylamide gels was accomplished (110) by using a c a t i o n i c carbocyanine dye " S t a i n s - a l l " (SA) using a t r i s - a c e t a t e -SDS buffer system (pH 7.4). This procedure was capable of r e l i a b l y detecting the proteins and, at the same time, d i f f e r e n t i a t i n g the glycoproteins. A sample of the p u r i f i e d protease (20 u g protein) was layered on each of two gels and subjected to electrophoresis. One gel was fixed and stained for proteins with 0.2% Coomassie blue, and the other was fixed and stained with SA. Protein s t a i n i n g revealed the presence of four (3+1) protein bands. In the s i n g l e s t a i n i n g procedure with " S t a i n s - a l l " , where the glyco-proteins stained blue, and the proteins .red, the p u r i f i e d protease was detected as four bands which a l l stained as glycoproteins. These r e s u l t s are represented diagram-a t i c a l l y i n F i g . 28. I t can be concluded that a l l four 180 FIGURE 27. Polyacrylamide electrophoresis of f r a c t i o n s A and B stained f o r l i p i d s . Gel , F r a c t i o n A of the p u r i f i e d protease; Gel. I I ' F r a c t i o n B of the p u r i f i e d protease; Gel. I l l and Gel IV Standard glycoprotein samples of rabbit plasma. The samples were s o l u b i l i z e d i n 0.1% Sudan black B i n ethylene g l y c o l prestain buffered by 0.03 M T r i s - c i t r a t e buffer (pH 9.0). The buffered p r e s t a i n was f i r s t heated at 60°C for one hour before f i l t r a t i o n through Whatman No. 1 paper. The samples were applied to the gel i n a concentration of 20 yg protein each and electrophoresed i n the absence of SDS. 181 I II III IV FIGURE 28. Diagram of glycoprotein and protein bands on slab gels following electrophoresis of p u r i f i e d protease. Comparison of s t a i n i n g patterns of p u r i f i e d protease separated by SDS-polyacrylamide gel electrophoresis. Coomassie blue (CB) and " S t a i n s - a l l " (SA) s t a i n i n g are compared i n 10% Tris-acetate buffered (pH 7.4) polyacrylamide gel electrophoresis system containing 0.1% SDS. 183 II 184 major bands which represent the p u r i f i e d protease preparations are glycoproteins. i . C haracterization of the p u r i f i e d protease. i ) Chemical composition of the p u r i f i e d protease Table 23 i n d i c a t e s the chemical composi-t i o n of the p u r i f i e d protease. The protein content of the p u r i f i e d protease preparation was determined by the Lowry method (132). Duplicate samples of the p u r i f i e d protease preparation were analyzed f o r hexose by the o r c i n o l - s u l f u r i c a c i d method (223), with an equimolar s o l u t i o n of glucose as a standard. The l i p i d content of the p u r i f i e d preparation was determined by the micro-method of Pande and Parvin (165) which detects l i p i d s i n the concentration range of 20-140 yg. Total free and organic phosphorous was determined by the micro-determination procedure described by Chen et^ a l . (29). The chemical analysis of the p u r i f i e d protease preparation demonstrated the presence of carbohydrate, l i p i d and protein as major constituents. No phosphate was detected i n t o t a l sample and i n the l i p i d extract from the enzyme preparation. i i ) Gas-liquid chromatography Gas chromatographic analysis (171) of neutral sugars and hexosamines present i n the p u r i f i e d protease revealed the presence of glucose and 00 Table 23. Chemical Composition of the P u r i f i e d Protease Preparation F i n a l Protease Preparation mg protein % mg dry weight mg carbohydrates % /mg dry weight mg l i p i d s % dry weight Phosphates i n t o t a l Sample and l i p i d extracts. F r a c t i o n A 2.5 79.4 0.25 8 0.35 11 0 Fract i o n B 3.1 75 0.24 6 0.79 19 0 186 glucosamine i n addition to two u n i d e n t i f i e d sugars. The f a t t y acid composition of the l i p i d extracts of the p u r i f i e d protease was also studied. The l i p i d s were extracted from the p u r i f i e d protease preparation into chloroform and methanol. No phospho-l i p i d s were detected using the two-directional t h i n - l a y e r chromatographic method described by Yavin (240). Gas chromatographic analysis of the neutral l i p i d s i n f r a c t i o n A and B are presented i n Table 24. The pre-dominant f a t t y acids i n f r a c t i o n A were l i n o l e i c , s t e a r i c , p a l m i t o l e i c and arachidonic. In f r a c t i o n B, p a l m i t i c , s t e a r i c and o l e i c a c i d predominated. In add i t i o n , a number of compounds which were not i d e n t i f i e d but which may be c y c l i c or odd chain f a t t y acids (142) were also found. i i i ) S t a b i l i t y of the p u r i f i e d protease The p u r i f i e d enzyme was found to be les s stable than the crude protease. Eight hour incubation at room temperature resulted i n about 40% loss of a c t i -v i t y , while incubation at 4°C f o r 2 days resulted i n 70% l o s s . B o i l i n g the enzyme for 1 min resulted i n loss of 100% of the p r o t e o l y t i c a c t i v i t y . Freezing at -20°C and thawing at room temperature resulted i n 24-35% loss of a c t i v i t y of the p u r i f i e d protease prepara-t i o n . Five to ten percent 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 preparation was stored at -70°C f o r two weeks. 187 Table 24. Gas-liquid chromatographic analysis of f a t t y acids i n the p u r i f i e d enzyme preparation. Fatty Acid Percentages of t o t a l f a t t y a c i d measured Fract i o n A Fra c t i o n B C-16 2 17 C-16:l 8 6 C-18 15 . 15 C-18:l 2 5 C-18:2 17 -C-20:4 12 -The percentage of composition of f a t t y acids was determined by 2 comparing the area i n mm of each peak to that of the t o t a l sample. 188 Longer storage periods resulted i n progressively greater losses. i v ) pH optimum of the p u r i f i e d protease The 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 protease was determined by the azocasein method i n various buffers of 0.1 M i o n i c strength. As shown i n Fig . 29, the p u r i f i e d protease was ac t i v e over a wide pH range with a pH optimum of 7.0. The e f f e c t of d i f f e r e n t b uffers on the pr o t e o l y s i s as measured i n the azocasein assay indicated that a c t i v i t y i n T r i s buffer was reduced 25% compared to PBS, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic a c i d ) , EPPS (N-2-hydroxyethylpiperazine propane s u l f o n i c a c i d ) , MOPS (morpholinopropane s u l f o n i c acid) and glycine buffer. Therefore, i n cases where the phosphate ions had to be excluded from the p u r i f i e d protease preparation, EPPS buffer was used instead of PBS. v) M o d i f i c a t i o n of protease a c t i v i t y The r e s u l t s of a number of experiments which were undertaken to show the e f f e c t s of various reagents on the a c t i v i t y of p u r i f i e d protease are shown i n Table 25. The substrate f o r a l l the reactions was azocasein. The type of i n h i b i t o r frequently provides an insi g h t into the act i v e f u n c t i o n a l group of the enzyme. The p u r i f i e d protease was i n h i b i t e d by HgC^ and a l k y l a t i n g agents such as iodoacetic acid and iodoacetamide (Fig. 30,31). The FIGURE 29. E f f e c t of pH on the p u r i f i e d protease a c t i v i t y . Symbols: ( t ) 0.1 M phosphate buffer, (A) 0.1 M Tris-HCl buff ( 0 ) 0.1 M g l y c i n e - NaOH buffer P u r i f i e d protease preparation (0.09 mg protein) was incubated with 0.75 ml of the appropriate buffer containing 50 mM (3ME f o r 15 min at 37°C; 1 ml of azocasein (2%) was added to each tube, and a f t e r one hr incubation, 2 ml of 10% TCA were added and p r e c i p i t a t e d azocasein was f i l t e r e d and of the f i l t r a t e was measured. The pH of the reaction mixtures was checked and found to be as indicated. 190 191 Table 25. Modi f i c a t i o n of the p u r i f i e d protease P u r i f i e d enzyme (0.09 mg protein/ml) concentrated and dialysed overnight against PBS (pH 7.2) was incubated at room temperature with the i n h i b i t o r f o r 30 min. PMSF and TPCK were dissolved i n 1-propanol and controls containing only 1-propanol were run. CaC^, iodoacetic acid and iodoacetamide were dissolved i n Tris-HCl buffer pH 7.2. HgC^, EDTA, urea, SDS, guanidine HC1, l i t h i u m chloride and NaCl were dissolved i n phosphate buffer pH 7.0. I n h i b i t i o n of a c t i v i t y was expressed r e l a t i v e to the control incubated i n the same buffer without reagent. Table 25. Modif i c a t i o n of the p u r i f i e d protease Reagent Concentration % I n h i b i t i o n of Protease A c t i v i t y Mercuric chloride 0.01 mM 20 3 mM 100 Iodoacetic acid 10 mM 28 50 mM 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 0.3% 65 Guanidine HC1 0.5 M 20.5 4 M 66.7 Lithium c h l o r i d e 0.5 M 38.5 3 M 74.4 Acetone 100% 80 Butanol 100% 100 Phenol 100% 100 Chloroform 100% 100 Tr i t o n X-100 0.5% 100 Ethylene g l y c o l 20% 0 50% 15 DMSO 5% 0 10% 5 Sodium chl o r i d e 1 M 5 3 M 50 Calcium chloride 0.1 M 0 0.5 M 0 193 FIGURE 30. HgCl 2 i n h i b i t i o n of the p u r i f i e d protease Enzyme (0.09 mg protein) was incubated at 37°C i n the anaerobic chamber with indicated concentrations of HgCL^ i n PBS pH 7.0 f o r 30 min and then assayed with azocasein as substrate. Percent I n h i b i t i o n ho o o o oo o o o FIGURE 31. I n h i b i t i o n of the p u r i f i e d protease by iodoacetamide. Enzyme preparation (0.09 mg protein) was incubated f o r 30 min at room temperature with Tris-HCl b u f f e r (pH 8.2) containing the appropriate concentration of the reagent with 50 mM BME. One ml of 2% azocasein was used as a substrate and the reaction mixture was incubated f o r 1 hr at 37°C. Percent Remaining A c t i v i t y I—1 NJ CO J>- Ln ON o o o o o o o V O O N 197 metal chelating agent EDTA and the serine i n h i b i t o r s had no i n h i b i t o r y e f f e c t on the protease. On the basis of these fi n d i n g s , the protease of B_. melaninogenicus could be c l a s s i f i e d as a s u l f h y d r y l enzyme. Guanidine HC1,which generally uncouples polar bonds and denatures proteins caused up to 66.7% 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 . The i n h i b i t o r y e f f e c t might also.be due to influence of i o n i c strength on the enzymatic a c t i v i t y . Lithium c h l o r i d e at 3 M concentration i n h i b i t e d 74.4% of the p r o t e o l y t i c a c t i v i t y ( F i g . 32). Phenol and chloro-form extraction as well as treatment with butanol, T r i t o n X-100 and acetone caused almost complete i n h i b i t i o n of the protease but t h i s was probably due to protein denaturation. However, the polar organic solvents dimethyl sulfoxide (DMSO) and ethylene g l y c o l had l i t t l e i n h i b i t o r y e f f e c t on the enzyme a c t i v i t y . Urea (8M) and 0.1% SDS did not i n h i b i t the protease; but 8 M urea had a stimulatory e f f e c t . The reason for t h i s could be that urea caused denaturation of the protein complexes that could mask some of the a c t i v e s i t e s of the enzyme. It i s also possible that high concentrations of urea might modify the substrate by making i t more accessible to the protease enzyme. The influence of i o n i c strength on the enzymatic a c t i v i t y i s i l l u s t r a t e d i n F i g . 32. The presence of NaCl at concentrations above 0.5 M decreased FIGURE 32. E f f e c t of guanidine hydrochloride, l i t h i u m chloride and NaCl on the p u r i f i e d protease. Enzyme preparation (0.09 mg protein) was incubated with 0.75 ml of the appropriate concentration of the reagent i n phosphate buffer (pH 7.0) containing 50 mM BME for 30 min at 37°C; 1 ml azocasein (2%) was added and the reaction mixture was incubated f o r 1 hr. 199 200 the enzyme a c t i v i t y . The p u r i f i e d protease was very s e n s i t i v e to HgCl-2 since low concentration of the i n h i b i t o r caused complete l o s s of a c t i v i t y . The i n h i b i t i o n could be reversed by reducing agents. As shown i n F i g . 30, the percent i n h i b i t i o n of p r o t e o l y t i c a c t i v i t y was proportion-a l to the concentration of the HgCl^. The function of the carbohydrate and l i p i d components i n the p u r i f i e d protease was investigated by t e s t i n g the e f f e c t of mixed glycosidases on the a c t i v i t y of the p u r i f i e d protease, and by extracting the l i p i d from the p u r i f i e d protease preparation with phenol, chloroform or butanol. Extraction of the l i p i d s from the p u r i f i e d protease resulted 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 to denaturation of the protein by the organic solvents used i n the extr a c t i o n . Restor-ation of the p r o t e o l y t i c a c t i v i t y with the dialyzed extracted l i p i d dialyzed against PBS was unsuccessful. Incubation of the p u r i f i e d protease with mixed glycosidases for 10 hr at 37°C caused 43% i n h i b i t i o n of the protease a c t i v i t y , suggesting that the carbohydrate part of the protease might be e s s e n t i a l or required for a c t i v i t y . 201 v i ) Substrate s p e c i f i c i t y of the p u r i f i e d protease The p u r i f i e d protease was ac t i v e against azocasein, casein, N,N-dimethylcasein and a z o c o l l as substrates. No a c t i v i t y could be demonstrated against hemoglobin, bovine serum albumin, or 1 4 C - c o l l a g e n . The p u r i f i e d protease d i d not hydrolyze the synthetic substrates TAME, t o s y l arginine methyl ester; BAEE, benzoy-l a r g i n i n e ethyl ester or ATEE, ac e t y l tyrosine 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 exhibited by the enzyme from the c e l l - e x t r a c t and the preparation obtained a f t e r ethanol p r e c i p i t a t i o n as w e l l as other preparationsobtained through the p u r i f i c a t i o n procedure, suggesting that a single p r o t e o l y t i c enzyme was produced by the organism. The p u r i f i e d preparation had no l i p a s e , a or B-glycosidase, collagenase or hemagglutinating a c t i v i t y when assayed as described i n Methods. v i i ) Pathological a c t i v i t y of the p u r i f i e d protease A concentrated p u r i f i e d preparation of the protease containing 2 mg protein/ml was dialyzed against PBS and injected subcutaneously into the groin of a 200 g guinea pig using 0.5 ml. The animal was observed for up to 4 weeks for the presence of abscess. The p u r i f i e d protease 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 . Adding the protease preparation to a c e l l suspension of 2D that caused 202 a t y p i c a l i n f e c t i o n i n the guinea pig model system resulted i n a s l i g h t increase i n the necrosis of the l e s i o n when compared to the necrotic l e s i o n s caused by 2D c e l l s without addition of the protease preparation. Whether the p u r i f i e d protease was the factor responsible for 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 kind of experiments, could not be v e r i f i e d . When the p u r i f i e d protease was tested for vascular permeability following the method of Craig (36), no blueing e f f e c t was observed. The p u r i f i e d protease did not enhance the blueing e f f e c t produced by the con-centrated culture supernatant of 2D. '4. Soluble Protease of 15. melaninogenicus Studies were c a r r i e d out to investigate whether the c e l l -free and cell-bound protease(s) are the same or d i f f e r e n t e n t i t i e s 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 properties during chromatography. a. Demonstration of an e x t r a c e l l u l a r protease. 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 culture supernatants of Ii. melaninogenicus s t r a i n 2D was detected e a r l y i n the exponential growth phase ( F i g . 15), and was found to increase i n proportion with increasing c e l l numbers u n t i l mid- to l a t e stationary phase when a further increase i n a c t i v i t y was noted which continued as the c e l l s began to l y s e . 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 s e n s i t i v i t y The soluble protease i n the culture supernatant was inactivated by oxygen. A d e f i n i t e i n -crease i n the rate of dye released from a z o c o l l occurred when 0.01 M cysteine or 25 mM gME was included i n the reac t i o n mixture. A c t i v i t y against a z o c o l l was l o s t when the supernatant was aerated f o r 20 min, but was restored with the reducing agent. i i . S t a b i l i t y of the protease The s t a b i l i t y of the protease i n concent-rated culture supernatants was examined by s t o r i n g the enzyme at -70°C, -20°C, 4°C, 20°C and 37°C with and without reducing agent and assaying p e r i o d i c a l l y for protease a c t i v i t y . Soluble protease was found to be more stable i n the absence of the reducing agent when stored or incubated at any of the above stated temperatures. This may be due to increased rates of autodigestion i n the presence of the reducing agents needed for p r o t e o l y t i c a c t i v i t y . Storage of the protease at -70°C was found to be more e f f e c t i v e than at -20°C where some of the a c t i v i t y was l o s t even i n the absence of BME. The protease was stable when incubated at room temperature only for a period of 3 to 4 hr. Incubation at 37°C resulted i n a greater l o s s of the 204 protease a c t i v i t y , e s p e c i a l l y i n the presence of 3ME. It was found to be stable f or 8 hr at 4°C. B o i l i n g the culture supernatant for 5 min resulted i n complete l o s s j of protease a c t i v i t y . i i i . E f f e c t of pH on protease a c t i v i t y Protease a c t i v i t y was measured over a pH range of 4.0 to 9.0. Acetate buffers were used from pH 4.0 to 5.5, phosphate buffers from pH 6.0 to 7.5 and Tri s - H C l buffers from pH 7.0 to 9.0 (Fig. 33). The optimum pH for protease a c t i v i t y against azocasein was between 7.0 and 7.5. Tr i s - H C l buffer caused a s l i g h t 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 (15-20%) when used i n the assay mixture as compared to the phosphate buffer. i v ) I n h i b i t i o n of soluble protease by various reagents Table 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 soluble protease of I5_. melaninogenicus. The soluble protease was completely i n h i b i t e d by mercuric c h l o r i d e , the i n h i b i t i o n could be reversed by addition of the Hg binding reagent BME. The s u l f h y d r y l reagents iodoacetic acid and iodo-acetamide. i n h i b i t e d the protease a c t i v i t y . Sulfhydryl groups are apparently required for p r o t e o l y t i c a c t i v i t y . Phenyl methyl s u l f o n y l f l u o r i d e (PMSF) and Tosyl-phenyl-ethyl-chloromethyl ketone (TPCK), which i n h i b i t FIGURE 33. pH optimum of the soluble protease. The e f f e c t of pH on the soluble protease a c t i v i t y was determined by the azocasein method i n various buffers of 0.1 M i o n i c strength. ( • ) acetate buffer, (A) phosphate buffer, ( 0 ) T r i s b u f f e r . Dialyzed culture supernatant (1.5 mg) was incubated with the substrate for 1 hr at 37°C. Absorbance (370 nm) 207 Table 26. I n h i b i t i o n of soluble protease Protease was incubated at 37°C f o r 30 min with each of the compounds before measuring p r o t e o l y t i c a c t i v i t y against casein. The protease a c t i v i t y of unmodified culture supernatant was assayed i n the presence and absence of BME i n same buffers and the r e s u l t s are expressed as % i n h i b i t i o n of protease a c t i v i t y of the control reactions. PMSF and TPCK were dissolved i n 1-propanol; HgC^was dissolved i n PBS (pH 7.0) and EDTA and a l k y l a t i n g agents i n 0.1 M Tris-HCl (pH 8.0). In h i b i t o r s Concentration (nM) % I n h i b i t i o n HgCl 2 2 100 5 100 HgCl2+BME 2 10 5 75 PMSF 1 0 10 0 TPCK 1 2 10 4 EDTA 1 5 10 15 50 60 Iodoacetic a c i d 1 24.2 16 40.5 Iodoacetamide 1 60 16 80.7 208 proteases r e q u i r i n g serine at the act i v e s i t e , had no ef f e c t on the soluble protease. Incubation with the metal chelator EDTA i n h i b i t e d the e x t r a c e l l u l a r protease by 5-60%. c. P a r t i a l p u r i f i c a t i o n of the soluble protease. i ) D i a l y s i s of cult u r e supernatant The d i a l y s i s of culture supernatant overnight at 4°C against PBS resulted i n 2.1 f o l d -p u r i f i c a t i o n most probably due to loss of d i a l y z a b l e peptides; and 6% lo s s of p r o t e o l y t i c a c t i v i t y occurred (Table 27). i i ) Concentration of culture supernatant Due to the observation that the soluble protease was unstable, a v a r i e t y of methods for con-centrating the protease were t r i e d i n order to find the method which would lead to the l e a s t i n a c t i v a t i o n . Freeze-drying was found to be the most e f f e c t i v e con-centrating procedure provided that the glassware was coated with Silane (Bio-Rad). Another concentration procedure that proved e f f e c t i v e was to remove water by blowing a i r over the supernatant contained i n d i a l y s i s tubing. Evaporation phenomena kept the temperature of the enzyme so l u t i o n low and resulted i n l e s s i n a c t i v a t i o n of the protease. Ammonium s u l f a t e p r e c i p i t a t i o n and u l t r a f i l t r a t i o n (Amicon PM-30 and XM-50 membranes) were not e f f e c t i v e procedures for concentrating the enzyme. 209 Table 27. P a r t i a l p u r i f i c a t i o n of JB_. melaninogenicus e x t r a c e l l u l a r protease Procedure T o t a l Protease Protease s p e c i f i c Y i e l d P u r i f i c a t i o n Units a c t i v i t y Factor units/mg protein Culture f i l t r a t e 72 2.8 100 D i a l y s i s 1 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 45.6 12.6 D i a l y s i s was against 10 volumes of PBS for overnight. 210 i i i ) P u r i f i c a t i o n procedures Culture supernatant of 2D was applied to a DEAE-Sephadex A-50 column packed and washed with 0.05 M NaCl i n phosphate buffer. The protease was bound to the DEAE-Sephadex and only 20% of the a c t i v i t y was recovered. This; was eluted with a 0.05-2 N NaCl gradient. Several peaks of protein were eluted with the s a l t gradient, but none possessed s i g n i f i c a n t pro-t e o l y t i c a c t i v i t y . No substantial p u r i f i c a t i o n was achieved. Further attempts at f r a c t i o n a t i o n of the concentrated supernatant resulted i n very low recovery. When a sample of concentrated 48 hr culture supernatant was chromatographed on a CM-Sephadex C-50 and Sephadex G-200 columns, a c t i v i t y against a z o c o l l was detected i n many f r a c t i o n s , suggesting that the protease a c t i v i t y might be associated with another constituent of the supernatant. Generally, the protease a c t i v i t y appeared to be binding n o n s p e c i f i c a l l y to the columns with con-siderable v a r i a t i o n among the e l u t i o n patterns of d i f f e r -ent samples. The reason for t h i s i s not known although i t may have been due to p r o t e o l y t i c a c t i o n on some of the protein components of the supernatant. I t i s also possible that the protease may have been associated with a component of the c e l l w a l l , which would r e s u l t i n the association of the enzyme with random sized and charged fragments. 211 iv.) Gel f i l t r a t i o n on Sephadex G-100 A Sephadex G-100 column packed, e q u i l -ibrated and eluted with PBS was used to f r a c t i o n a t e the soluble protease. The protease a c t i v i t y i n the c u l t u r e supernatant was eluted from the Sephadex G-100 column as a si n g l e peak immediately a f t e r the void volume, as shown i n F i g . 34. This indicated an approximate molecular weight of 100,000 or more. 64.4% of the p r o t e o l y t i c a c t i v i t y applied to the column was recovered,- suggesting the p o s s i b i l i t y that some of the protease was e i t h e r inactivated or s t i l l on the column. The p u r i f i c a t i o n steps resulted i n an approximately 12.6 f o l d 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 material with a t o t a l 46% recovery (Table 27). Gel electrophoresis of the p a r t i a l l y p u r i f i e d protease revealed many proteins and further attempts to p u r i f y the protease were unsuccessful. The p a r t i a l l y p u r i f i e d protease was shown to be a s u l f h y d r y l enzyme having the same c h a r a c t e r i s t i c s as the crude protease i n the c u l t u r e supernatant. It was determined that the e x t r a c e l l u l a r protease was large, possibly membrane or p a r t i c l e bound. Therefore, there would be no advantage i n attempting to p u r i f y i t any further, as s i m i l a r material could more conveniently be prepared from exponential phase c e l l s . 212 FIGURE 34. Sephadex G-100 gel f i l t r a t i o n of the soluble protease. A 59 x 1.6 cm column was used at 5.5 ml/hr flow rate. The void volume of the column was determined with Blue Dextran 2000, and a sample of 18.2 mg protein of dialyzed culture supernatant was 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 . The o p t i c a l density was followed at 280 nm for protein determination. The protease was assayed by the a z o c o l l assay. 213 Protease units/ml P> n r t H-O 3 a i fD A280 214 The r e s u l t s indicate that the soluble protease was s i m i l a r to the cell-bound enzyme with respect to pH optima, response to i n h i b i t o r s , substrate s p e c i f i c i t y and properties during chromatography. There-fore, i t can be concluded"that only one major protease was produced by the s t r a i n 2D of 15. melaninogenicus and that the e x t r a c e l l u l a r protease was probably due to l y s i s or leakage from the c e l l s . 215 IV. DISCUSSION melaninogenicus has been implicated i n the pathogenesis of mixed anaerobic i n f e c t i o n s i n man (4,25,57,231) and i n the development of experimental anaerobic i n f e c t i o n s (137,205). I t i s known to possess a t t r i b u t e s which would implicate i t i n human periodontal disease (138,139). Therefore, the organism has attracted the i n t e r e s t of a number of i n v e s t i -gators throughout the years. However, d e t a i l e d i n v e s t i g a t i o n s concerning the biochemical 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 cu l t u r e and by inadequate taxonomy (26,54). These fac t s have been responsible, at l e a s t i n part, for the c o n f l i c t i n g r e s u l t s i n the l i t e r a t u r e . A study of the p r o t e o l y t i c and hemagglutinating a c t i v i t y of the organisms might provide ins i g h t into the nature of t h e i r pathogenicity and into t h e i r function and behaviour as members of the indigenous f l o r a of the human g i n g i v a l c r e v i c e . Studies i n our laboratory tend to v a l i d a t e the recent proposal (98) that e x i s t i n g subspecies of B_. melaninogenicus can be c l a s s i f i e d into at l e a s t two species. Our c l a s s i f i c a t i o n i s based on a number of new taxonomic c r i t e r i a , which taken together with the standard c r i t e r i a , emphasize the unique c h a r a c t e r i z a t i o n of B_. melaninogenicus ss. asaccharolyticus. These c r i t e r i a are pathogenicity, HA a c t i v i t y , collagenase and the production of phenylacetic a c i d . Phenylacetic acid, may have some importance i n c l i n i c a l diagnosis since i t has been found . i n exudate from animals (S. Jensen, personal communication). Both organisms characterized for t h i s study, produced butyric a c i d , phenylacetic 216 a c i d , hemagglutinin, high l e v e l of protease collagenase and were i n f e c t i v e . Therefore, they can be c l a s s i f i e d as Ji_. melaninogenicus ss. asaccharolyticus. As expected, s t r a i n 2D required hemin for growth. The organism can grow on as low concentration of hemin as 0.25 ug hemin/ml medium,and the growth i s roughly proportional to the hemin concentration up to a concent- 1 r a t i o n of 2.5 ug hemin/ml medium. Higher ..concentrations up to 20 ug/ml medium had neither an i n h i b i t o r y nor an enhancing e f f e c t on growth. The growth rate of 2D was influenced by free amino acids. L-serine, glutamic a c i d , L-methionine, L-proline and L-asparagine enhanced growth (Table 3). Glutamic acid had the most pronounced e f f e c t , increasing growth at 24 hr by 45%. Growth was i n h i b i t e d almost completely by L-cysteine, DL-valine, L - h i s t i d i n e , L-tryptophan, glycine or L-arginine. L-leucine i n h i b i t e d growth by 66% while L - l y s i n e and L-phenylalanine had no e f f e c t . Our r e s u l t s c o r r e l a t e with the work done by Miles and Wong (149) on the i n h i b i t i o n of growth of an asaccharolytic s t r a i n of J3. melaninogenicus by v a l i n e , tryptophan, g l y c i n e , h i s t i d i n e and arginine as we l l as the enhancement of growth by asparagine. These workers also reported that L-phenylalanine and L - l y s i n e had no e f f e c t on the growth rate, a conclusion which was also confirmed by our studies. However, serine and methionine, which were reported to i n h i b i t the growth of the asaccharolytic Ii. melaninogenicus, were found to enhance the growth of s t r a i n 2D of B. melaninogenicus. Glutamic acid which was found to cause the maximum growth enhancement of 2D was reported by Miles and Wong as having no e f f e c t ; also, the amino acids L-serine, L-cysteine and L-leucine were found to i n h i b i t growth of 2D i n our studies, while having no e f f e c t on the growth of the asaccharolytic s t r a i n studied by Miles (149). 217 The a b i l i t y of amino acids to influence growth indicates that they were probably assimilated by the microorganism. This contradicts the conclusions of Wahren et^ a l . (228) who reported that the organisms were unable to assimilate these compounds. Proof that the amino acids were a c t u a l l y consumed i s l a c k i n g and should be investigated by following incorporation or metabolism of labeled amino acids. Glutamic acid was unable to replace hemin as a growth f a c t o r , although i t seems possible that glutamic acid could be deaminated and decarboxylated to form su c c i n i c acid which was reported as a possible replacement for hemin (146). The reasons why some of the amino acids influence growth i s not known. I n t e r e s t i n g l y , the amino acids that 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 chemical groups. For example, i n h i b i t i o n was caused by the h e t e r o c y c l i c amino acids L - h i s t i d i n e and L-tryptophan, by the a l i p h a t i c amino acids glycine and v a l i n e , and by L-cysteine, a sulfur-containing compound. Therefore, a common i n h i b i t -ory mechanism i s u n l i k e l y . Also, i t i s u n l i k e l y that competitive i n h i b i t -ion of uptake of required amino acids i s occurring, because the growth studies were done i n the presence of t r y p t i c a s e , which has few free amino acids. I t i s possible that competitive i n h i b i t i o n between amino acids and peptide transport may e x i s t , since i t i s known that peptides are used by melaninogenicus (228) . ]3_. melaninogenicus s t r a i n 2D was shown to be one of the few asaccharo-l y t i c i s o l a t e s which i s i n f e c t i v e as a monoculture and i s thus one of the more v i r u l e n t s t r a i n s of t h i s species. The i n f e c t i o n i s severe, develops r a p i d l y and leads to death within 48 hrs. Death i s probably due to s e p t i c shock r e s u l t i n g from massive bacteremia and i n f e c t i o n of the peritoneal 218 cavity. The i n f e c t i o n can be transmitted d i r e c t l y from one animal to another providing that i t i s t r u l y an i n f e c t i o n and not induction of a hypersensitive response. Pathogenicity of 15. melaninogenicus can be r e l a t e d to a number of f a c t o r s . The r e s u l t s reported here have shown that s t r a i n 2D possesses a c h o l e r a - l i k e toxin when tested by the vascular permeability assay. I t seems highly u n l i k e l y that the toxic material i s a lipopolysaccharide-l i k e endotoxin since heating destroyed i t s a c t i v i t y . The r a p i d l y spreading i n f e c t i o n might also be due to collagenase and/or protease a c t i v i t y . The determination of whether collagenase or protease a c t i v i t y i s e s s e n t i a l for a successful i n f e c t i o n w i l l require the i s o l a t i o n of a mutant of an i n f e c t i v e s t r a i n d i f f e r i n g from the parent s t r a i n only i n the absence of collagenase or protease a c t i v i t y . 15. melaninogenicus s t r a i n 2D possesses HA a c t i v i t y as evidenced by i t s a b i l i t y to cause clumping of RBC and to bind strongly to RBC surfaces. Culture supernatant contains HA a c t i v i t y which i s thought to be due to c e l l associated HA which has been released from the c e l l surface. The evidence to support t h i s assumption i s as follows: (a) Both hemagglutinin a c t i v i t i e s are s e n s i t i v e to oxidation and a c t i v i t y can be restored by addition of reducing agents (Table 10). (b) Both HA are s e n s i t i v e to Hg and iodoacetic acid (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 carbohydrates, however, both were s e n s i t i v e to galactose. (d) Both HA are i n a c t i v a t e d by heating. (e) HA can be removed from c e l l s by mild procedures such as washing with buffer (Table 5). 219 (f) E l e c t r o n microscopic studies have shown that the soluble HA i s present i n a membranous structure (Thanks to Dr. Paul Osmanski and Susan Jensen). (g) Soluble HA i s a large molecule as evidenced by gel f i l t r a t i o n and sedimentation at 100,000 x g (Fig. 11 and Table 12). (h) An increase i n soluble HA i s found i n old cultures (Fig. 10). ( A l l information on the cell-bound HA was obtained from S. Jensen). Hemagglutinin a c t i v i t y was found associated with the c e l l s as well as i n the culture supernatants of s t r a i n 2D. The soluble HA was chosen f o r study since i t i s free of c e l l s and therefore, l i k e l y to be p u r i f i e d more e a s i l y , and also to provide evidence to support the assumption that the soluble HA i s the same as that which i s c e l l associated. The requirement of reducing substances for hemagglutination a c t i v i t y correlates w e l l with the highly reduced state of the natural environment of the organism. As pointed out, the c e l l - f r e e HA was of high molecular weight (Fig. 1 and Table 12) which i s probably due to the as s o c i a t i o n of HA with fragments of the outer membrane. Therefore, the observed e f f e c t s of i n h i b i t o r s and enzymes on hemagglutinating a c t i v i t y might be either the r e s u l t of changing the properties of the associated membrane p a r t i c l e , or a d i r e c t e f f e c t on the HA i t s e l f . The soluble HA of 15. melaninogenicus was i n s e n s i t i v e to carbohydrates with the exception of galactose. Galactose was also found to i n h i b i t the hemagglutinin of the o r a l s t r a i n s of Fusobacterium nucleatum (153). Therefore, i t appears that the c e l l receptor f o r the 15. melaninogenicus HA moiety contains D-galactose. D-mannose has been shown to i n h i b i t hemagglutination of E_. c o l i (160), which was at t r i b u t e d to the presence 220 of a mannose-specific 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 surface. The soluble HA of 2D was s e n s i t i v e to heat and to treatment with pronase i n d i c a t i n g that proteinaceous substances are involved. I n s e n s i t i v i t y of the soluble HA to d i f f e r e n t s a l t s might suggest that agglutination of the HA and RBC does not occur through an i o n i c i n t e r a c t i o n . In order to obtain some information concerning the nature of the . component(s) necessary for HA, the e f f e c t of treatment of RBC on t h e i r a b i l i t y to hemagglutinate with soluble HA was determined (Table 9). Pronase and galactosidase treatments of RBC cause i n h i b i t i o n of the HA a c t i v i t y which might i n d i c a t e that protein and/or carbohydrate moieties are necessary f o r HA to occur. Treatment of the RBC with neuraminidase caused enhancement of the HA a c t i v i t y . E l u t i o n of HA adsorbed to neuraminidase-treated RBCs with 8 M urea was les s e f f e c t i v e than e l u t i n g adsorbed HA from untreated RBCs. This might be due to a ti g h t e r binding of soluble HA to neuraminidase-treated RBC. The f a c t that neuraminic acid i s often linked to galactose (238)* suggest that removal of neuraminic acids may unmask and then create many new receptors or binding s i t e s f o r the HA on the RBC. Pretreatment of RBC with neuraminidase was reported to r e s u l t i n an increased binding of the HA preparations of the o r a l bacterium F. nucleatum to the RBC (153). An a f f i n i t y adsorption system using formalinized RBC was developed and used to accomplish some p u r i f i c a t i o n of the soluble HA i n culture supernatant, followed by g e l f i l t r a t i o n on Sephadex G-100. Recovery of 50% of the HA was accomplished with a 52-fold p u r i f i c a t i o n . However, polyacrylamide g e l electrophoresis of the p a r t i a l l y p u r i f i e d HA revealed a heterogenous preparation. If the HA i s associated with the outer 221 membrane, i t may e x i s t i n a large complex, and thus be associated with many proteins. Further attempts to d i s s o c i a t e the HA a c t i v i t y from the p a r t i c u l a t e fragments resulted i n great los s of a c t i v i t y ; therefore, i t i s possible that the HA may only be a c t i v e i n t h i s complex form. Reducing conditions were required for a c t i v i t y of the p a r t i a l l y p u r i -f i e d HA. Reagents known to react with s u l f h y d r y l groups such as iodoacetic a c i d , iodoacetamide and HgC^ p a r t l y i n h i b i t e d the crude HA as well as the p a r t i a l l y p u r i f i e d HA i n culture supernatant. Therefore, i t can be concluded that s u l f h y d r y l groups are an i n t e g r a l part of the hemagglutination a c t i v i t y . J3. melaninogenicus produced both cell-bound and c e l l - f r e e p r o t e o l y t i c a c t i v i t y which amounted to approximately 80% and 20% r e s p e c t i v e l y of the t o t a l a c t i v i t y during exponential growth. The proportion of c e l l - f r e e a c t i v i t y increased i n stationary phase cultures. The cell-bound protease of J3. melaninogenicus resembles the proteinases i n Streptococcus l a c t i s (221) and i n Bacteroides amylophilus H18 (15). A l l three proteases are l o c a l i z e d i n the c e l l envelope, probably near the c e l l w a l l surface since they are accessible to high molecular weight substrates. Both c e l l u l a r and soluble proteases of 15. melaninogenicus were a c t i v e against a number of protein substrates including a z o c o l l , casein, azocasein and N,N-dimethylcasein. The azocasein assay was found to be most s a t i s f a c t -ory mainly because of low background. The assay was most e f f e c t i v e , reproducible and easiest to perform. Protease production by Gram-negative anaerobic b a c t e r i a has been l i t t l e i nvestigated; the study of the p r o t e o l y t i c a c t i v i t y of 15. melaninogenicus thus seemed to be of general i n t e r e s t . 222 In order to inv e s t i g a t e the r e l a t i o n s h i p of the cell-bound protease to the growth of 13. melaninogenicus,protease production was followed during growth of the organism i n d i f f e r e n t media and under d i f f e r e n t conditions. It was found that the protease production correlated with the growth rate of the organism. The evidence supporting t h i s assumption was as follows: (a) Increasing the growth rate of a continuous culture of the organism resulted i n an increase i n protease production by the organism (Fig. 18). (b) Amino acids that stimulated growth stimulated protease product-ion and t h i s was 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 (Table 16). (c) When the growth rate was l i m i t e d by hemin concentration protease synthesis was slowed (Fig. 16). Under conditions where hemin was not growth l i m i t i n g , further increasing the hemin concent-r a t i o n did not have an e f f e c t on protease production (Table 17). (d) There was a 60% decrease i n protease a c t i v i t y i n succinate medium compared to TYH medium with a simultaneous decrease i n the growth rate due to the replacement of hemin by succinate (Fig. 17). The succinate had no d i r e c t e f f e c t on the protease a c t i v i t y . The reason why protease synthesis would be dependent upon the growth rate i s not known, but these findings c o r r e l a t e with findings reported for enzyme production i n Pseudomonas aeruginosa (31) and V i b r i o SA1 (234). In the former amidase synthesis and i n the l a t t e r e x t r a c e l l u l a r protease production were r e l a t e d to the growth rate of the organisms i n chemostat studies. I t i s most probable that t h i s r e f l e c t s a complex c o n t r o l system. 223 Studies with material aspirated from guinea p i g i n f e c t i o n s have shown that 2D elaborates the s u l f h y d r y l protease i n the i n f e c t i o n as well as i n v i t r o . An increase i n protease production was associated with animal passage of the 2D containing exudate when assayed i n v i t r o (Table 15), which might in d i c a t e a r o l e of the protease i n the i n f e c t i v e process. An extensive study was made of the biochemical properties of the c e l l -bound protease, i s o l a t e d from mechanically disrupted c e l l s . M i c r o b i a l proteases are often c l a s s i f i e d by pH optimum and i n h i b i t o r s e n s i t i v i t y rather than by the most r e a d i l y hydrolyzed substrate (102). In a d d i t i o n , s p e c i f i c 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 frequently provide ins i g h t into the nature of the functional groups on the enzyme. The p r o t e o l y t i c a c t i v i t y of I3_. melaninogenicus appeared to be due to s u l f h y d r y l protease(s) , since the 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 . S e n s i t i v i t y to oxidation, r e s t o r a t i o n of action with reducing agents and the i n h i b i t o r y e f f e c t of the a l k y l a t i n g agents iodoacetic acid and iodoacetamide supported t h i s assumption. The metal chelating agent 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 . The observation that the protease was not i n h i b i t e d by the serine i n h i b i t o r s , nor was i t activated by divalent ions, and was active at neutral pH, strongly suggests that i t i s not an a c i d i c , serine or metalloenzyme type of protease. On the basis of these findings the c e l l u l a r protease of B_. melaninogenicus should be c l a s s i f i e d as a s u l f h y d r y l enzyme. Generally, the existence of 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 neutral proteases. The c e l l u l a r enzyme of 2D had a broad spectrum of a c t i v i t y between pH 5.5 and 10.5, with a sharp peak of casein h y d r o l y t i c a c t i v i t y at pH 7.0 and a second plateau of a c t i v i t y between pH 8.0 and 9.0. The pH optima of the c e l l u l a r protease c o r r e l a t e 224 with the ne u t r a l or s l i g h t l y a l k a l i n e pH of i t s natural environment i n the g i n g i v a l c r e v i c e . The c e l l u l a r protease of 15. melaninogenicus was found to be subject to autodigestion, p a r t i c u l a r l y i n the presence of reducing agents. In addi t i o n , i t was not generally very stable 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 presented (Table 22) gave a good recovery and was e a s i l y reproducible provided that c e r t a i n precautions were followed. Among these precautions are the exclusion of reducing agent during a l l steps i n the p u r i f i c a t i o n , the ultimate care i n keeping the temperature below 4°C and appropriate conditions of cooling and s t i r r i n g during 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 ethanol. The p u r i f i c a t i o n of the c e l l u l a r protease 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 at 121,000 x g for 1 hr, pre-c i p i t a t i o n with 60% ethanol at -10°C followed by u l t r a c e n t r i f u g a t i o n at 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 through Sephadex G-100 i n 6 M urea; and g e l f i l t r a t i o n through Sepharose-2B i n PBS containing 6 M urea. The 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 preparation was increased 774 times over that of the crude preparation, and a 160% f i n a l recovery was obtained (Table 22). The fa c t that an increase i n the recovery of the p r o t e o l y t i c a c t i v i t y was found a f t e r chromatography of the ethanol p r e c i p i t a t e d protease suggested that the p u r i f i c a t i o n steps might have unmasked the protease from pr o t e i n complexes, removed some i n h i b i t o r s or components that were binding to a c t i v e s i t e s or removed endogenous substrate. Polyacrylamide g e l electrophoresis showed the p u r i f i e d protease to consist of four 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 to 15 major bands 225 i n the crude enzyme i n c e l l - e x t r a c t (Fig. 23,24). Each of the four bands was f i r m l y bound to carbohydrate moiety as indicated by the glycoprotein s t a i n . When the p u r i f i e d protease preparation was subjected to poly-acrylamide g e l electrophoresis without denaturation and i n the absence of SDS, i t revealed the presence of only one band and that band did not migrate into the g e l (Fig. 26). Therefore, i t seems l i k e l y that the p u r i -f i e d protease s t i l l might have been bound i n a large molecular weight complex, as was also indicated by i t s exclusion from G-100 Sephadex i n 6 M urea. I t i s s i m i l a r , i n t h i s respect, to the c e l l u l a r protease of Bacteroides amylophilus released by c e l l d i s i n t e g r a t i o n which also did not penetrate polyacrylamide gels. (14). It also resembles the p e n i c i l l i n a s e of 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 deoxycholate and urea was p a r t i c u l a t e as judged by i t s poor mobility i n starch g e l e l e c t r o -phoresis (116). The p u r i f i e d protease could represent a polymeric form of enzyme sub-units bound to a c e l l w a l l component. The f a c t that the p u r i f i c a t i o n procedure used i n t h i s study always resulted i n f r a c t i o n a t i o n of the crude protease into four 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 that the components of the complex p u r i f i e d protease were f i r m l y bound together and not c o i n c i d e n t a l l y associated through the p u r i f i c a t i o n steps. I t also implies that the p u r i f i e d protease i s the "minimum b i o l o g i c a l l y a c t i v e u n i t " , which i s present as a complex of a number of proteins unable to enter the polyacrylamide 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 protease into a l e s s complex unit resulted i n loss of the p r o t e o l y t i c a c t i v i t y . It was reported that some basic properties of c e r t a i n enzymes such as the dimerization of subunits might be dependent on the retention of the enzyme i n association with the c e l l wall (30) . 226 More d e f i n i t e conclusions could be drawn i f one was able to define which of the four bands found upon gel-electrophoresis contain(s) the p r o t e o l y t i c a c t i v i t y . Feasible approaches to t h i s problem could be the use 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 chemically cross-l i n k i n g a r a d i o a c t i v e l y labeled substrate to the enzyme. In the f i n a l step of p u r i f i c a t i o n , the a c t i v e protease 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 mob i l i t y . Evidence was obtained that both f r a c t i o n s represent the same protease. The influence 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 . No differ e n c e was found i n the response to i n h i b i t o r s between the two f r a c t i o n s . Both were stained f o r l i p i d s , had 4 d i s t i n c t e l e c trophoretic bands of glycoproteins and lacked electrophoretic mobili ty i n the absence of SDS. The dif f e r e n c e 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 23) which could be a reason behind the dif f e r e n c e i n t h e i r chromatographic m o b i l i t y . It can be deduced that the c e l l u l a r protease released 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 could not be completely l i b e r a t e d . In t h i s respect, i t resembled the cell-bound protease of Bacteroides amylophilus H18 which, when l i b e r a t e d by sonic d isruption of c e l l s harvested during exponential phase, was particle-bound and could not be e a s i l y p u r i f i e d (14). Also, the p e n i c i l l i n -ase of 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 (116). As i t was assumed that the protease a c t i v i t y might have been associated with 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 as 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 with random sized and charged fragments when the c e l l s were dis i n t e g r a t e d . This i s supported by the r e s u l t s obtained from 227 ion exchange chromatography of the crude protease under d i f f e r e n t con-d i t i o n s of buf f e r s , pH and i n the presence of denaturing agents which resulted i n poor recovery of the protease from a large number of f r a c t i o n s . This assumption can also account f o r the v a r i a t i o n s i n protease l i b e r a t e d into the c e l l - e x t r a c t that was found between batches of 48 hr cultures of ba c t e r i a disintegrated at d i f f e r e n t times without major changes'-i n the proportion of unbroken c e l l s . The p u r i f i e d protease of ]5. melaninogenicus was ac t i v e against a number of substrates in c l u d i n g a z o c o l l , azocasein, casein and N,-N-dimethyl casein, and had no glycosidase, l i p a s e , collagenase or hemagglutinating a c t i v i t i e s . The p u r i f i e d protease was proven to be s i m i l a r to the crude enzyme i n c e l l - e x t r a c t with respect to 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. EDTA and cations (Ca ) had no e f f e c t on the p u r i f i e d protease. The pH optimum was found to be at pH 7.0 with higher a c t i v i t y at a l k a l i n e pH values than at acid pH. The serine i n h i b i t o r s PMSF and TPCK had no e f f e c t on the p u r i f i e d protease, therefore, the c e l l -u l a r protease of s t r a i n 2D of J3. melaninogenicus appears to be a s u l f h y d r y l enzyme. The protease had c e r t a i n s t a b i l i t y aspects i n common with papain enzyme, which i s a t y p i c a l s u l f h y d r y l enzyme that has been extensively studied. Resistance to the organic solvent dimethylsulfoxide (DMSO) and to 8 M urea, and s e n s i t i v i t y to guanidine hydrochloride are s i m i l a r i n both enzymes (88,190). The increase i n p r o t e o l y t i c a c t i v i t y caused by high concentrations of urea i n the assay mixture could be explained by the p o s s i b i l i t y of that urea modifies the substrate or makes i t more accessible to the enzyme. I t might also be due to the unmasking of ac t i v e s i t e s which 228 were blocked by other components of the enzyme complex. The r e s u l t s have shown that the c h a r a c t e r i s t i c s exhibited by the crude 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 with those of the p u r i f i e d enzyme which strongly suggests that one c e l l u l a r protease was produced by Ii. melaninogenicus which was 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 analysis of the p u r i f i e d protease preparation demonstrated the presence of carbohydrate, l i p i d and protein. The function of the carbohydrate and l i p i d i s not known. The c h a r a c t e r i z a t i o n of the carbo-hydrate and l i p i d moieties of the p u r i f i e d protease revealed the presence of components which were previously reported to be found i n i s o l a t e d lipopolysaccharide from the outer membrane complex of 13. melaninogenicus ss. asaccharolyticus (142). Glucose, galactose and glucosamine were reported as the predominant sugars i n the LPS preparations. The pre-dominance of p a l m i t i c , p a l m i t o l e i c and s t e a r i c acid was also reported as w e l l as the presence of two unknown f a t t y acids that were assumed to be c y c l i c or odd chain f a t t y acids (142). Attempts were made to determine i f the protease of 15. melaninogenicus was located i n the periplasmic space by osmotic shock and treatment with polymyxin B. Only 10-12% of the protease a c t i v i t y was l i b e r a t e d , and the protease could not, therefore, be c l a s s i f i e d as periplasmic. Ingram et_ a l . found that some of the a l k a l i n e phosphatase of Pseudomonas aeruginosa was located e x t e r i o r to the outer t r i p a r t i t e layer and was complexed with lipopolysaccharide which was also released during secretion (96). They hypothesized that mechanical shearing forces associated with growth lead to a s t r i p p i n g of LPS-alkaline phosphatase aggregates on the external wall surface. 229 Various enzymes found outside of the cytoplasmic membrane were not released into the medium, but were bound to the outer membrane of the c e l l envelope which contains charged moieties. A molecule might remain bound to the c e l l either i n a s s o c i a t i o n with mucopeptide (194) , with various components of the periplasmic space (30) or with lipopolysaccharide of the outer membrane (96), depending on the nature of the enzyme such as the amount of hydrophobocity and number of charged groups. The secretion of a protease enzyme by Micrococcus sodonensis was found to be dependent on the co-secretion of at l e a s t one of several poly-saccharides, also elaborated by these c e l l s (19). Regnier and Thang (174) reported that at l e a s t 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. There are very few reports on e x t r a c e l l u l a r enzymes of Gram-negative anaerobic organisms. Blackburn reported that a protease was l i b e r a t e d into the growth medium by exponentially growing cultures of Bacteroides amylo- philus s t r a i n H18 (13). In t h i s study, evidence was obtained suggesting that the e x t r a c e l l u l a r protease of IS. melaninogenicus i s the cell-bound protease which was probably l i b e r a t e d from the c e l l s by release of outer membrane during c e l l growth or c e l l l y s i s . The optimum pH for the e x t r a c e l l u l a r protease of 2D with azo-casein as substrate was found to be between 7.0 and 7.5 which i s s i m i l a r to that of the c e l l associated protease. The e x t r a c e l l u l a r protease of IS. melaninogenicus was also c l a s s i f e d as a s u l f h y d r y l enzyme due to i t s dependence on reducing agents and s e n s i t i v i t y to SH-inactivating agents. It was also found that the e x t r a c e l l u l a r protease of 2D was unstable and subject to audodigestion. From the studies 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, i t was concluded that the e x t r a c e l l u l a r protease of B_. melaninogenicus was of high molecular weight and presumably membrane or p a r t i c l e bound. The metal chelating agent EDTA caused 60% i n h i b i t i o n of the extra-c e l l u l a r protease of 13. melaninogenicus but did not a f f e c t the cell-bound protease. The d i f f e r e n c e might be due to differences i n the complex i n which the enzymes are bound. Generally, cations can be required for a c t i v i t y or for protection of the enzyme against autodigestion. A proteo-l y t i c enzyme i s o l a t e d from Clostridium botu'linum type B which was a c t i v e only when i n the reduced state was reported to be i n a c t i v a t e d by EDTA (11). The small proportion of protease located i n the culture supernatant during logarithmic growth and the increase i n proportion only during stationary phase made i t l i k e l y that the e x t r a c e l l u l a r enzyme was l i b e r a t e d because of release of outer membrane during c e l l growth or a u t o l y s i s of only a few b a c t e r i a l c e l l s . Generally, 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 involves some s p e c i a l problems. The enzyme concentration i n the growth medium i s usually low and large quantities of s a l t s and extraneous compounds must be removed. Several b a c t e r i a l proteinases have been p a r t l y p u r i f i e d but only a few of these have been i s o l a t e d i n a pure state and characterized i n some d e t a i l . The protease of the dialyzed and concentrated culture supernatant df 2D was p a r t i a l l y p u r i f i e d by gel f i l t r a t i o n through Sephadex G-100. The protease was eluted as a s i n g l e peak at the void volume of the column with a f i n a l recovery of only 46%. Gel electrophoresis of the p a r t i a l l y p u r i -f i e d protease revealed many protein bands; and further attempts to 231 disaggregate and separate the protease i n a p u r i f i e d form were unsuccessful. The possible 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 been suggested by various studies. Kogoma and N i s h i (111) had found an increase at d i v i s i o n followed by a decrease of an i n t r a c e l l u l a r proteinase i n synchronously d i v i d i n g c e l l s of Escherichia c o l i . Bufdett and Murray (24) presented electron microscopic 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 at the s i t e of septum formation i n E. c o l i . I t has been shown that the auto-l y s i n of Streptococcus f a e c a l i s i s present i n an i n a c t i v e form i n the c e l l w a l l but i s activated by a neutral proteinase; and that the active form of the a u t o l y s i n i s associated with recently synthesized w a l l (193). The major function of e x t r a c e l l u l a r proteinases and other h y d r o l y t i c 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 substrates as sources of nutri e n t s . The r e s u l t s presented i n t h i s study suggest that the protease of s t r a i n 2D of 15. melaninogenicus can be c l a s s i f i e d as a s u l f h y d r y l enzyme. The observation that the s p e c i f i c i t y exhibited by the crude enzyme i n the c e l l -extract was i d e n t i c a l with that of the soluble enzyme strongly suggests that one c e l l u l a r protease was produced by 15. melaninogenicus which was l i b e r a t e d from the c e l l s by release of outer membrane during c e l l growth. 232 V. LITERATURE CITED 1. Aisenberg, M.S. and A.D. Aisenberg. 1951. Hyaluronidase i n p e r i o -dontal disease. Oral Surg. Oral Med. Oral Pathol. _4:317. 2. A l l i s o n , F. J r . 1975. Anaerobic b a c t e r i a l i n f e c t i o n i n man. South Med. J . 68:1088. 3. Altemeier, W.A. 1938. The b a c t e r i a l f l o r a of acute perforated appendicitis with p e r i t o n i t i s . Ann. Surg. 107:517. 4. Altemeier, W.A. 1942. The pathogenicity of appendicitis p e r i t o n i t i s . Surgery 11:374. 5. Arvidson, S., T. Holme and B. Lindholm. 1973. Studies on 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 from Staphylococcus aureus. I. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of one ne u t r a l and one a l k a l i n e protease. Biochim. Biophys. Acta.307:135. 6. Astrup, T. and N. A l k j a e r s i g . 1952. C l a s s i f i c a t i o n of p r o t e o l y t i c enzymes by means of t h e i r i n h i b i t o r s . Nature 169:214. 7. Bamirez-Ronda, C H . , R.K. Holmes and J.P. Sanford. 1975. Adherence of b a c t e r i a to .heart valves i n v i t r o . J . C l i n . Invest. 56:1364. 8. Barel, A.O. and A.N. Glazer. 1969. Spectroscopic studies on papain and some i n a c t i v e d e r i v a t i v e s . J . B i o l . Chem. 244:268. 9. Bengt, V.H., H.V. Kley and D. Eaker. 1965. An e x t r a c e l l u l a r proteo-l y t i c enzyme from a s t r a i n of arthobacter. I I . P u r i f i c a t i o n and chemical properties of the enzyme. Biochim. Biophys. Acta. 110:585. 10. Bergmann, M. 1942. A c l a s s i f i c a t i o n of p r o t e o l y t i c enzymes. Adv. Enz. 2^ :49. 11. Bibhuti, R. and H. Sugiyama. 1972. I s o l a t i o n and ch a r a c t e r i z a t i o n of a protease from Clostridium botulinum type B. Biochim. Biophys. Acta. 268:719. 12. Bibhuti, R., B.R. Das Gupta and H. Sugiyama. 1972. Role of a protease i n natural a c t i v a t i o n of Clostridium botulinum neurotoxin. Infect. Immununity 6:587. 13. Blackburn, T.H. 1968. Protease production by Bacteroides amylophilus s t r a i n H18. J . Gen. M i c r o b i o l . 53:27. 233 14. Blackburn, T.H. 1968. The protease l i b e r a t e d from Bacteroides amylophilus s t r a i n H18 by mechanical d i s i n t e g r a t i o n . J . Gen. M i c r o b i o l . 53:37. 15. Blackburn, T.H. and W.A. Hullah. 1974. The cell-bound protease of Bacteroides amylophilus H18. Can. J . M i c r o b i o l . 20:435. 16. Boethling, R.S. 1975. P u r i f i c a t i o n and properties of a serine protease from Pseudomonas ma l t o p h i l i a. J . B a c t e r i o l . 121:933. 17. Bona, C.A. 1973. B a c t e r i a l Lipopolysaccharides. The chemistry, biology, and c l i n i c a l s i g n i f i c a n c e of endotoxins, p. 66. In E.H. Kass and S.M. Wolff (eds.) University of Chicago Press. 18. B o t t a z z i , V. 1962. P r o t e o l y t i c a c t i v i t y of some s t r a i n s of thermo-p h i l i c L a c t o b a c i l l i . Intern. Dairy. Cong. Proc. 16th, Copenhagen _2:522. 19. Braatz, A.J. and E.C. Heath. 1974. The r o l e of polysaccharide i n the secretion of p r o t e i n by Micrococcus sodonensis. J . B i o l . Chem. 249:2536. 20. Brown, J.L. and J.M. Johnson. 1962. Radioassay of l i p i d components separated by t h i n layer chromatography. J . L i p i d Res. 3^:480. 21. Brubacher, L.J. and M.L. Bender. 1966. The preparation and properties of trans-cinnamoyl-papain. J . Am. Chem. Soc. 88:5871. 22. B u l l , A.T. 1972. Environmental factors i n f l u e n c i n g the synthesis and excretion of e x t r a c e l l u l a r macromolecules. J . Appl. Chem. Biotechnol. 22:261. 23. B u l l , A.T. 1974. In : Companion to biochemistry, p. 415. A.T. B u l l , J.R. Lagnado, K.F. Tipton and J.O. Thomas, (eds.) Longman, London. 24. Burdett, I. and R.G.E. Murray. 1974. Septum formation i n Esch e r i c h i a c o l i : Characterization of septal structure and the e f f e c t s of a n t i b i o t i c s on c e l l d i v i s i o n . J. B a c t e r i o l . 119:303. 25. Burdon, K.L. 1928. Bacterium melaninogenicum from normal "and pathogenic tissu e s . J . Infect. Dis. 42:161. 26. Burdon, K.L. 1932. I s o l a t i o n and c u l t i v a t i o n of Bacterium melaninogenicum. Proc. Soc. Exp. B i o l . Med. 29:1144. 27. Burt, S.J. and D.R. Woods. 1976. R factor transfer to obligate anaerobe from Escherichia c o l i . J . Gen. M i c r o b i o l . 93:405. 28. Chan, K. and G.M. Wiseman. 1975. A new c o l o n i a l type of N e i s s e r i a gonorrhoeae. Br. J. Vener. Dis. 51:251. 234 29. Chen, P.S., T.Y. Toribara and H. Warner. 1956. Microdetermination of phosphorous. Anal. Chem. 28:1756. 30. Cheng, K.-J., J.M. Ingram, and J.W. Costerton. 1971. Interaction of a l k a l i n e phosphatase and the c e l l w a l l of Pseudomonas aeruginosa. J. B a c t e r i o l . 107:325. 31. Clarke, P. 1968. Catabolite repression and the induction of amidase synthesis by Pseudomonas aeruginosa 8602 i n continuous cu l t u r e . J . Gen. M i c r o b i o l . 51:225. 32. Cohen, J . 1932. The bacteriology of abscess of the lung, and methods for i t s study. Arch. Surg. 24:171. 33. Costerton, J.W., J.M. Ingram, and K.-J. Cheng. 1974. Structure and function of the c e l l envelope of Gram-negative b a c t e r i a . B a c t e r i o l . Rev. 38:87. 34. Courant, P.R. and H. Bader. 1966. Bacteroides melaninogenicus and i t s products i n the gingiva of man. Periodontics _4:131.-35. Courant, P.R. and R.J. Gibbons. 1967. Biochemical and immunological heterogeneity of Bacteroides melaninogenicus. Arch. Oral B i o l . 12:1605. 36. Craig, J.P. 1965. A permeability f a c t o r (toxin) found i n cholera stools and culture f i l t r a t e s and i t s n e u t r a l i z a t i o n by con-valescent cholera sera. Nature 207:614. 37. Dancer, B.N. and J . Mandelstam. 1975. Production and possible . function of serine protease during sporulation of B a c i l l u s s u b t i l i s . J . B a c t e r i o l . 121:406. 38. DasGupta, B.R. 1971. A c t i v a t i o n of Clostridium botulinum type B toxin by an endogenous enzyme. J . B a c t e r i o l . 108:1051. 39. De l a Cruz, B. and C. Cuadra. 1969. Antigenic c h a r a c t e r i z a t i o n of f i v e species of human Bacteroides. J . B a c t e r i o l . 100:1116. 40. Dean, A.C.R. 1972. Influence of environment on the co n t r o l of enzyme synthesis. J . Appl. Chem. Biotechnol. 22:245. 41. Del Bene, V.E., A. Rogers and W.E. Farrar, J r . 1976. Attempted transfer of a n t i b i o t i c resistance between Bacteroides and Escherichia c o l i . J. Gen. M i c r o b i o l . 92:384. 42. Drapeau, G.R., Y. Bo i l y and J . Houmard. 1972. P u r i f i c a t i o n and properties of an e x t r a c e l l u l a r protease of Staphylococcus aureus. J. B i o l . Chem. 247:6720. 235 43. Drucker, H. 1972. Sensitive radiochemical assay for p r o t e o l y t i c a c t i v i t i e s . Anal. Biochem. 46:598. 44. Duerden, B.I. 1975. Pigment production by Bacteroides species with reference to s u b - c l a s s i f i c a t i o n . J. Med. M i c r o b i o l . J5_:113. 45. Duguid, J.P., I.W. Smith, G. Dempster and P.N. Edmunds. 1955. Non-f l a g e l l a r filamentous appendages (Fimbriae) and hemagglutinin-ati o n a c t i v i t y i n E_. c o l i . J. Path. B a c t e r i o l . 70:335. 46. Duguid, J.P. and R.R. G i l l i e s . 1957. Fimbriae and adhesive properties i n dysentery b a c i l l i . J. Path. B a c t e r i o l . 74:397. 47. Duguid, J.P., E.S. Anderson and I. Campbell. 1966. Fimbriae and adhesive properties i n Salmonellae. J . Path. B a c t e r i o l . 92:109. 48. Duguid, J.P. 1966. Notes on the terms "Fimbriae" and " P i l i " . J. Path. B a c t e r i o l . 92:137. 49. Duguid, J.P. 1968. The function of b a c t e r i a l fimbriae. Arch. Immunol. Ther. Exp. 16:173. 50. Edwards, T., J.J.R. Campbell and B.C. McBride. 1975. Hemagglutinating a c t i v i t y of pathogenic s t r a i n s of Bacteroides melaninogenicus. (Abst.), Amer. Soc. M i c r o b i o l . 75th meeting. 51. E l l e n , R.P. and J . Gibbons. 1972. M-protein associated adherence of Streptococcus pyogenes to e p i t h e l i a l surfaces : Pr e r e q u i s i t e for vir u l e n c e . Infect. Immunity _5:826. 52. E l l e n , R.P. and R.J. Gibbons. 1974. Parameters a f f e c t i n g the adherence and t i s s u e tropisms of Streptococcus pyogenes. Infect. Immunity 9^ :85. 53. E l l i o t t , S.D. and V.P. Dole. 1947. An i n a c t i v e precursor of streptococcal proteinase. J. Expt. Med. 85:305. 54. Felner, J.M. and V.R. Dowell, J r . 1971. "Bacteroides" bacteremia. Amer. J. Med. 50:787. 55. Finegold, S.M., A.B. M i l l e r and D.L. Posnick. 1965. Further studies' on s e l e c t i v e media for Bacteroides and other anaerobes. Ernaehrungsf. 10:517. 56. Finegold, S.M., V.H. Marsh and J.G. B a r t l e t t . 1970. Anaerobic i n f e c t i o n s i n the compromised host. Proc. Internat. Conf. on Ho s p i t a l I n f e c t i o n s , Atlanta. 1971. Amer. Hosp. Assoc. Chicago. 236 57. Finegold, S.M. 1974. Infections due to anaerobic organisms other than C l o s t r i d i a . In P r a c t i c e of Medicine, Vol. I l l , Chapter 27. Harper and Row. 58. Finegold, S.M. 1977. Anaerobic b a c t e r i a in human disease. Academic Press, New York. 59. Finegold, S.M. and E.M. Barnes. 1977. Report of the ICSB taxonomic subcommittee on Gram-negative anaerobic rods. Int. J. Syst. B a c t e r i o l . 27;388. 60. F i n k l e , B.J. and E.L. Smith. 1958. C r y s t a l l i n e papain: Number and r e a c t i v i t y of t h i o l groups; chromatographic behavior. J. B i o l . Chem. 230:669. 61. Freter, R. 1969. Studies of the mechanism of action of i n t e s t i n a l antibody i n experimental cholera. Tex. Rep. B i o l . Med. 27:299. 62. Frost, A.J. 1975. Selective adhesion of microorganisms to the ductular epithelium of the bovine mammary gland. Infect. Immunity 12:1154. 63. Fubara, E.S. and R. Freter. 1973. Protection against enteric b a c t e r i a l i n f e c t i o n by secretory IgA antibodies. J. Immunol. I l l : 3 9 5 . 64. F u l l e r , R. 1975. Nature of the determinant responsible for the adherence of L a c t o b a c i l l i to chicken crop e p i t h e l i a l c e l l s . J. Gen. M i c r o b i o l . 87:245. 65. Gallop, P.M. and S. S e i f e r t . 1963. Soluble collagens, p. 635; In S.P. Colowick and N.D. Kaplan (eds.) Methods i n enzymology, Vol. 6. Academic Press, Inc., New York. 66. Gibbons, R.J. and J.B. Macdonald. 1960. Hemin and vitamin K compounds as required factors for the c u l t i v a t i o n of c e r t a i n s t r a i n s of Bacteroides melaninogenicus. J. B a c t e r i o l . 80:164. 67. Gibbons, R.J. and J.B. Macdonald. 1961. Degradation of collagenase substrates by Bacteroides melaninogenicus. J. B a c t e r i o l . 81:614. 68. Gibbons, R.J., S.S. Socransky, S. Sawyer, B. Kapsimalis and J.B. Macdonald. 1963. The microbiota of the g i n g i v a l c r e v i c e area of man. I I . The predominant c u l t i v a b l e organisms. Arch. Oral B i o l . 8_:28l. 69. Gibbons, R.J., B. Kapsimalis and S.S. Socransky. 1964. The source of s a l i v a r y b a c t e r i a . Arch. Oral B i o l . 9_:101. 70. Gibbons, R.J. and J . Van Houte. 1971. Selective b a c t e r i a l adherence to o r a l e p i t h e l i a l surfaces and i t s r o l e as an e c o l o g i c a l determinant. Infect. Immunity 3_:567 . 237 71. Gibbons, R.J., J. Van Houte and W.F. Liljemark. 1972. Parameters that a f f e c t the adherence of J3. s a l i v a r i u s to o r a l e p i t h e l i a l surface. J. Dent. Res. 51:424. 72. Gibbons, R.J. 1974. Aspects of the pathogenicity and ecology of the indigenous o r a l f l o r a of man. In_ A. Ballows, R.M. Dehaan, V.R. Dowell, and L.B. Cruze (eds.) Anaerobic b a c t e r i a : r o l e i n disease. Charles C. Thomas, Publisher, S p r i n g f i e l d , 111. 73. Gibbons, R.J. and J. Van Houte. 1975. B a c t e r i a l adherence i n o r a l microbial ecology. Ann. Rev. M i c r o b i o l . 29:19. 74. G i l l , D.M. and L.L. Dinius. 1971. Observations on the structure of di p h t h e r i a t o x i n . J . B i o l . Chem. 246:1485. 75. Gisslow, M.T. and B.C. McBride. 1975. A rapid s e n s i t i v e collagenase assay. Anal. Biochem. 68:70. 76. Glazer, A.N. 1967. E s t e r a t i c reactions catalyzed by s u b t i l i s i n s . . J. B i o l . Chem. 242:433. 77. Glenn, A.R. 1976. Production of e x t r a c e l l u l a r proteins by b a c t e r i a . Ann. Rev. M i c r o b i o l . 30:41. 78. Gorbach, S.L., K.B. Menda, H. Thadepalli and L. Keith. 1973. Anaerobic m i c r o f l o r a of the cervix i n healthy women. Am. J. Obstet. Gynecol. 117:1053. 79. Gorbach, S.L. and J.G. B a r l e t t . 1974. Anaerobic i n f e c t i o n s : old myths and new r e a l i t i e s . J. Infect. Dis. 130:307. 80. Gorbach, S.L. and J.G. B a r l e t t . 1974. Anaerobic i n f e c t i o n s . New England J. Med. 290:1177 Part I, 1237 Part I I , 1289 Part I I I . 81. Hagihara, B. 1958. The enzymes, Vol. 4, p. 193, P.D. Boyer, H. Lardy and K. Myrback (eds.). Academic Press, Inc., New York. 82. Hancock, R.E.W. and H. Nikaido. 1978. Outer membrane of Gram-negative b a c t e r i a . XIX. I s o l a t i o n from Pseudomonas aeruginosa PA01 and use i n r e c o n s t i t u t i o n and d e f i n i t i o n of the permeability b a r r i e r . J. B a c t e r i o l . 136 :381. 83. Hausmann, E., P. Courant and D.S. Arnold. 1967. Conditions for the demonstration of c o l l a g e n o l y t i c a c t i v i t y i n Bacteroides  melaninogenicus. Arch. Oral B i o l . 12:317» -84. Hausmann, E. and E. Kaufman. 1969. Collagenase a c t i v i t y i n a p a r t i c u l a t e f r a c t i o n from Bacteroides melaninogenicus. Biochim. Biophys. Acta. 194:612. 238 85. Hardie, J.M. and G.H. Bowden. 1975. B a c t e r i a l f l o r a of dental plaque. Br. Med. B u l l . 31:131. 86. Hartley, B.S. 1960. P r o t e o l y t i c enzymes. Ann. Rev. Biochem. 29:45. 87. Herbert, D. 1956. The continuous culture of b a c t e r i a ; a t h e o r e t i c a l and experimental study. J. Gen. Mi c r o b i o l . 14:601. 88. H i l l , R.L., H.C. Schwartz and E.L. Smith. 1959. The e f f e c t of urea and guanidine hydrochloride on a c t i v i t y and o p t i c a l r o a t i o n of c r y s t a l l i n e papain. J. B i o l . Chem. 234:572. 89. Hite, K.E., M. Loclae and H.C. Hesseltine. 1949. Synergism i n experimental i n f e c t i o n s with nonsporulating anaerobic b a c t e r i a . J . Infect. Dis. 84:1 90. Hofstad, T. 1970. B i o l o g i c a l a c t i v i t i e s of endotoxin from Bacteroides melaninogenicus. Arch. Oral B i o l . 15:343. 91 Hofstad, T. and T. K r i s t o f f e r s e n . 1971. Lipopolysaccharide from Bacteroides melaninogenicus i s o l a t e d from the supernatant f l u i d a f t e r c e n t r i f u g a t i o n of the water phase following phenol-water extraction. Acta. Path. M i c r o b i o l . Scand. 79:12. 92. Hofstad, T. 1974. Endotoxins of anaerobic Gram-negative micro-organisms (Chapt. XXIII). In Anaerobic Bacteria. A. Balows, R.M. Dehaan, V.R. Dowell, and L.B. Guze (eds.). Charles C. Thomas, S p r i n g f i e l d , 111. 93. Holbrook, W.P., B.I. Duerden and A.G. Deacon. 1977. The c l a s s i f i c a t i o n of Bacteroides melaninogenicus and related species. J. Appl. B a c t e r i o l . 42:259. 94. Holdeman, L.V. and W.E.C. Moore. 1972. Bacteroides. In Anaerobe Laboratory Manual. Holdeman, L.V. and W.E.C. Moore (eds.) VPI Anaerobe Laboratory, Blacksburg, V i r g i n i a . 95. Holdeman, L.V. and W.E.C. Moore. 1974. Gram-negative anaerobic b a c t e r i a . In Bergey's Manual of Determinative Bacteriology. Buchanan, R.E. and Gibbons, N.E. (eds.), 8th Ed., Baltimore: The Williams and Wilkins Co. 96. Ingram, J.M., K.-J. Cheng and J.W. Costerton. 1973. A l k a l i n e phos-phatase of Pseudomonas aeruginosa: the mechanism of secretion and release of the enzyme from whole c e l l s . Can. J. M i c r o b i o l . 19:1407. 97. Inouye, H. and J. Beckwith. 1977. Synthesis and processing of an Esch e r i c h i a c o l i a l a k l i n e phosphatase precursor iiv v i t r o . Proc. N a t l . Acad. S c i . 74:1440. 239 98. International Committee on Systematic Bacteriology. 1977. Sub-committee on Gram-negative anaerobic rods. Minutes of the Meeting, 29 and 30 May 1976. Int. J. Syst. B a c t e r i o l . 27:61 99. Kadis, S., G. Weinbaum and S.J. A j l (eds.). 1971. M i c r o b i a l toxins. V o l . 5. p. 507. Academic Press, New York. 100. Kasper, D.L. 1976. Chemical and b i o l o g i c a l c h a r a c t e r i z a t i o n of the lipopolysaccharide of Bacteroides f r a g i l i s subspecies f r a g i l i s . J. Infect. Dis. 134:59. 101. Kaufman, E.J., P.A. Mashimo, E. Hausmann, C.T. Hanks and S.A. E l l i s o n . 1972. Fusobacterial i n f e c t i o n : enhancement by c e l l - f r e e extracts of Bacteroides melaninogenicus possessing c o l l a g e n o l y t i c a c t i v i t y . Arch. Oral B i o l . 17:577. 102. Keay, L. 1971. M i c r o b i a l proteases. Process Biochem. 6^ :17. 103. Keller,. R. and N. Traub. 1974. The c h a r a c t e r i z a t i o n of B_. f r a g i l i s bacteriophage recovered from animal sera: observations on the nature of bacteroides phage c a r r i e r c u ltures. J. Gen. V i r o l . 24:179. 104. Kerebel, B. and A. Seda l l i a n . 1972. Action de Bacteroides melanino-genicus sur l a dentine i n v i t r o . Schweiz. Monatsschr. Zahnheilk 82:731. 105. Kestenbaum, R.C. and S. Weiss. 1962. The production of experimental i n f e c t i o n s with combinations of microorganisms of human o r i g i n . (Abst.), IADR 40th meeting. 106. Kestenbaum, R.C, J . Massing and S. Weiss. 1964. The r o l e of collagenase i n mixed i n f e c t i o n s containing Bacteroides melanino- genicus . (Abst.), IADR 42nd meeting. 107. Kettner, C , J. Rodriguez-Absi, G.I. Glover and J.M. Presscott. 1974. The p u r i f i c a t i o n of Aeromonas aminopeptidase by a f f i n i t y chromato-graphy. Arch. Biochem. Biophys. 162:56. 108. Kimmel, J.R. and E.L. Smith. 1954. C r y s t a l l i n e papain: I. Preparation, s p e c i f i c i t y and a c t i v a t i o n . J . B i o l . Chem. 207:515. 109. Kimmel, J.R., H.J. Rogers and E.L. Smith. 1965. T r y p t i c digest of papain: I I . I s o l a t i o n and sequences of peptides from the carboxy-methylated protein. J . B i o l . Chem. 240:266. 110. King, L.E. and M. Morrison. 1976. The v i s u a l i z a t i o n of human erythrocyte membrane proteins and glycoproteins i n SDS poly-acrylamide gels employing a s i n g l e s t a i n i n g procedure. Anal. Biochem. 71:223. 240 111. Kogoma, T. and A. N i s h i . 1965. Rhythmic v a r i a t i o n s i n p r o t e o l y t i c a c t i v i t i e s during the c e l l c y c l e of E s c h e r i c h i a c o l i . J. Gen. Appl. M i c r o b i o l . 11:321. 112. Krasse, B. 1954. The proportional d i s t r i b u t i o n of S^. s a l i v a r i u s and other s t r e p t o c o c c i i n various parts of the mouth. Odontol. Revy 5/.203. 113. Kunsman, J.E. J r . , I. Katz and M. Keeny. 1966. L i p i d content of a rumen microorganism L a c t o b a c i l l i GD31. (Abst.), Amer. Chem. Soc. 152nd meeting, New York. 114. Lamanna, C , M.F. Malette and L. Zimmermann. 1973. Basic bacteriology. Williams and Wilkins, Baltimore. 115. Lambe, D.W. J r . 1974. Determination of Bacteroides melaninogenicus serogroups by fluorescent antibody s t a i n i n g . Appl. M i c r o b i o l . 28_:561. 116. Lampen, J.O. 1967. C e l l bound p e n i c i l l i n a s e of B a c i l l u s l i c h i n e f o r m i s . Properties and p u r i f i c a t i o n . J. Gen. M i c r o b i o l . 48:249. 117. Ledger, W.J. 1975. Anaerobic i n f e c t i o n s . Am. J. Obstet. Gynecol. 123:111. 118. Lennette, E.H., E.H. Spaulding and J.P. Truant (eds.). 1974. Manual of C l i n i c a l Microbiology, p. 363. Washington,: American Society for Microbiology. 119. Lev, M. 1968. Vitamin K d e f i c i e n c y i n Fusiformis nigrescens. I. Influence on whole c e l l s and c e l l envelope c h a r a c t e r i s t i c s . J. B a c t e r i o l . 95:2317. 120. Lev, M., K.C. Keudell and A.F. M i l f o r d . 1971. Succinate as a growth factor for Bacteroides melaninogenicus. J. B a c t e r i o l . 108:175. 121. Lev, M. and A.F. M i l f o r d . 1971. Vitamin K stimulation of sphingo-l i p i d synthesis. Biochem. Biophys. Res. Commun. 45:358. 122. Lev, M. and A.F. M i l f o r d . 1972. E f f e c t of vitamin K depletion and r e s t o r a t i o n on sphingolipid metabolism i n Bacteroides melanino- genicus . J. L i p i d Res. 13:364. 123. Lev, M. and A.F. M i l f o r d . 1973. The 3-keto-dihydrosphingosine synthetase of Bacteroides melaninogenicus: Induction by vitamin K. Arch. Biochem. Biophys. 157:500. 124. Lev, M. and A.F. M i l f o r d . 1975. S e n s i t i v i t y of a Bacteroides melaninogenicus s t r a i n to monosaccharides: E f f e c t on enzyme induction. J. B a c t e r i o l . 121:152. 241 125. Liljemark, w.F. and R.J. Gibbons. 1972. The proportional d i s t r i b u t i o n and r e l a t i v e adherence of S_. m i t i s i n the human o r a l c a v i t y . Infect. Immunity 6/.852. 126. L i n , Y., G.E. Means and R.E. Feeney. 1969. The action of p r o t e o l y t i c . enzymes on N,N-dimethyl proteins. J. B i o l . Chem. 244:789. 127. Lindsay, S.S., B. Wheeler, K.E. Sanderson and J.W. Costerton. 1973. The release of a l k a l i n e phosphatase and of lipopolysaccharide during the growth of rough and smooth s t r a i n s of Salmonella typhimurium. Can. J. M i c r o b i o l . 19^:335. 128. Loesche, W.J. and R.J. Gibbons. 1965. A p r a c t i c a l scheme for i d e n t i f i c a t i o n of the most numerous o r a l Gram-negative anaerobic rods. Arch. Oral B i o l . 10:723. 129. Loesche, W.J., R. Hockett and S.A. Syed. 1971. Evaluation of kanamycin as an aid i n the i s o l a t i o n of Bacteroides melaninogenicus from dental plaque. Arch. Oral B i o l . 16:813. 130. Loesche, W.J., R.N. Hockett and S.A. Syed. 1972. The predominant c u l t i v a b l e f l o r a of tooth surface plaque removed from i n s t i t u t i o n -a l i z e d subjects. Arch. Oral B i o l . 17:1311. 131. Loesche. W.J., K.U. Paunio, M.P. Wooldolk and R.N. Hockett. 1974. C o l l a g e n o l y t i c a c t i v i t y of dental plaque associated with periodontal pathology. Infect. Immunity 9^:329. 132. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. Protein measurement with the F o l i n phenol reagent. J. B i o l . Chem. 193:265. 133. L i i d e r i t z , 0., 0. Westphal, A.M. Staub, and H. Nikaido. 1971. I s o l a t i o n and chemical and immunological c h a r a c t e r i z a t i o n of b a c t e r i a l l i p o -polysaccharides. p. 145. In_ C. Weinbaum, S. Kadis, S.J. A j l (eds.) M i c r o b i a l toxins. V o l. 4. Academic Press, New York. 134. Macdonald, J.B., R.M. Sutton and M.L. K n o l l . 1954. The production of fusospirocheatal i n f e c t i o n s i n guinea pigs with recombined pure cultures. J. Infect. Dis. 95:275. 135. Macdonald, J.B., R.M. Sutton, M.L. K n o l l , E.U. Madlener and R.M. Grainger. 1956. The pathogenic components of an experimental fusosphirocheatal i n f e c t i o n . J. Infect. Dis. 98_:15. 136. Macdonald, J.B., R.J. Gibbons and S.S. Socransky. 1960. B a c t e r i a l mechanisms i n periodontal disease. Ann. N.Y. Acad. S c i . 85:467. 137. Macdonald, J.B. 1962. On the pathogenesis of mixed anaerobic i n f e c t i o n s of mucous membranes. Ann. Roy. C o l l . Surg. Engl. 31:361. 242 138. Macdonald, J.B. and R.J. Gibbons. 1962. The r e l a t i o n s h i p of indigenous b a c t e r i a to periodontal disease. J. Dent. Res. 41:320. 139. Macdonald, J.B., S.S. Socranosky and R.J. Gibbons. 1963. Aspects of the pathogenesis of mixed anaerobic i n f e c t i o n s of mucous membranes. J. Dent. Res. 42:529. 140. MacGregor, C.H., C.W. Bishop and J.E. Blech. 1979. L o c a l i z 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 i n the outer membrane of Escherichia c o l i . J. B a c t e r i o l . 137:574. 141. Mansheim, J.B. and D.L. Kasper. 1977. P u r i f i c a t i o n and immunochemical cha r a c t e r i z a t i o n of the outer membrane complex of Bacteroides  melaninogenicus subspecies asaccharolyticus. J. Infect. Dis. 135:787. 142. Mansheim, J.B., A.B. Onderdonk and D.L. Kasper. 1978. Immunochemical and b i o l o g i c studies of the lipopolysaccharide of Bacteroides  melaninogenicus subspecies asaccharolyticus. J . Immunol. 120:72. 143. Mardb, P.-A. and L. Westrom. 1976. Adherence of b a c t e r i a to vaginal e p i t h e l i a l c e l l s . Infect. Immunity 13:661. 144. Matsubara, H. and S. Nishimura. 1958. C r y s t a l l i n e b a c t e r i a l proteinase. V. On the r e a c t i o n with di-isopropylfluorophosphate. J . Biochem. (Tokyo) 45_:503. 145. Mattsby-Baltzer, I. and B. K a i j s e r . 1979. L i p i d A and a n t i - l i p i d A. Infect. Immunity 23:758. 146. Mayrand, D. 1978. Characterization of Bacteroides melaninogenicus and i t s r o l e i n mixed anaerobic i n f e c t i o n s . Ph.D. t h e s i s , B r i t i s h Columbia U n i v e r s i t y , B.C. 147. May, B.K. and W.H. E l l i o t t . 1968. C h a r a c t e r i s t i c s of e x t r a c e l l u l a r protease formation by B a c i l l u s s u b t i l i s and i t s c o n t r o l by amino acid repression. Biochim. Biophys. Acta 157 :607. 148. Meleney, P.L. 1931. B a c t e r i a l synergism i n disease process with a confirmation of the s y n e r g i s t i c b a c t e r i a l e t i o l o g y of a c e r t a i n type of progressive gangrene of the stomach w a l l . Ann. Surg. 94:961. 149. Miles, D.O., J.K. Dyer and J.C. Wong. 1976. Influence of amino acids on the growth of Bacteroides melaninogenicus. J. B a c t e r i o l . 127:899. 150. M i r a g l i a , G.J. 1974. Pathogenic anaerobic b a c t e r i a . CRC C r i t i c a l Reviews i n M i c r o b i o l . 3:161. 243 151. Miyata, K., K. Tomoda and M. Isono. 1970. S e r r a t i a protease. I. P u r i f i c a t i o n and general properties of the enzyme. Agr i c . B i o l . Chem. 34;310. 152. Miyata, K., K. Tomoda and M. Isono. 1971. S e r r a t i a protease. I I I . C h a r a c t e r i s t i c s of the enzyme as metalloenzyme. Ag r i c . B i o l . Chem. 35:460. 153. Mongiello, J.R. 1978. Sugar i n h i b i t i o n of o r a l Fusobacterium nucleatum hemagglutination. J. Dent. Res. 5_7_ (Special Issue A, Abst.) p. 77. 154. Moore, W.E.C., E.P. Cato and L.V. Holdeman. 1969. Anaerobic b a c t e r i a of the g a s t r o i n t e s t i n a l f l o r a and t h e i r occurrence i n c l i n i c a l i n f e c t i o n . J . Infect. Dis. 119:641. 155. Moore, W.E.C. and L.V. Holdeman. 1973. New names and combinations i n the genera Bacteroides C a s t e l l a n i and Chalmers, Fusobacterium  Rnorr, Eubacterium Prevot, Propionibacterium Delwich, and La c t o b a c i l l u s Orla-Jensen. Int. J. Syst. B a c t e r i o l . 23:69. 156. Nagai, Y., J . Gross and K.A. Piez. 1964. Disc, electrophoresis of collagen components. Ann. N.Y. Acad. S c i . 121:494. 157. Nakajima, M., K. Mizusawa and F. Yoshida. 1974. P u r i f i c a t i o n and properties of an e x t r a c e l l u l a r proteinase of psychrophilic E s c h e r i c h i a f r e u n d i i . Eur. J. Biochem. 44:87. 158. Nobles, E.R. 1973. Bacteroides i n f e c t i o n s . Ann. Surg. 177 -.601. 159. Ofek, I., E.H. Beachey, W. Jefferson and G.L. Campbell. 1975. C e l l membrane binding properties of group-A streptococcal l i p o t e i c h o i c acid. J . Exp. Med. 141:990. 160. Ofek, I., D. Mirelman and N. Sharon. 1977. Adherence of E. c o l i to human mucosal c e l l s mediated by mannose receptors. Nature 265:623. 161. Okuda, K. and I. Takazoe. 1974. Haemagglutinating a c t i v i t y of Bacteroides melaninogenicus. Arch. Oral B i o l . 19:415. 162. Okuda, K. and I. Takazoe. 1974. A delayed h y p e r s e n s i t i v i t y i n animals immunized with Bacteroides melaninogenicus. B u l l . Tokyo Dent. C o l l . 15_:43. 163. O l i v e r , W.W. and W.B. Wherry. 1921. Notes on some b a c t e r i a l parasites of the human mucous membranes. J . Infect. Dis. 28:341. 244 164. Opstelten, M.D. and B. Withold. 1978. P r e f e r e n t i a l release of new outer membrane fragments by exponentially growing E s c h e r i c h i a  c o l i . Biochim. Biophys. Acta. 508:287. 165. Pande, S.V., R. Parvin Khan and T.A. Venkitasubramanian. 1962. Microdetermination of l i p i d s and serum t o t a l f a t t y acids. Anal. Biochem. 6^:415. 166. Pangburn, M.K. , Y. Burstein, P.H. Morgan, K.A. Walsh and H. Neurath. 1973. A f f i n i t y chromatography of thermolysin and of neutral proteases from B_. s u b t i l i s . Biochem. Biophys. Res. Commun. 54:371. 167. Parker, L.J. and D.C. White. 1969. I d e n t i f i c a t i o n of ceramide phosphorylethanolamine and ceramide phosphorylglycerol i n the l i p i d s of an anaerobic bacterium. J. L i p i d Res. 10:528. 168. Pierce, N.F., W.B. Greenough and C.C.J. Carpenter,„Jr. 1971. V i b r i o cholera enterotoxin and i t s mode of action. Bact. Reviews 35:1. 169. Pine, M.J. 1972. Turnover of i n t r a c e l l u l a r proteins. Ann. Rev. Mic r o b i o l . 26:103. 170. Pollock, M.R. 1963. Exoenzymes, p. 121. In_ I.C. Gunsalus and R.Y. Stanier (eds.), The Bacteria, V o l . 4, Academic Press Inc., New York. 171. Porter, H.W. 1975. Appl i c a t i o n of nitrous acid deamination of hexosamines to the simultaneous GLC determination of neutral and amino sugars i n glycoproteins. Anal. Biochem. 63:27. 172. Prestidge, L., V. Gage and J. Spizezen. 1971. Protease a c t i v i t i e s during the course of sporulation i n B a c i l l u s s u b t i l i s . J . B a c t e r i o l . 107:815. 173. Pulverer, G. 1958. Zur. morphologie, biochemie, and serologie des Bacteroides melaninogenicus. Z. Hyg. Infektkrankh. 145 :293. 174. Regnier, Ph. and N.M. Thang. 1973. Membrane associated proteases i n E_. c o l i . FEBS Let t e r s 36_:31. * 175. Reichertz, C. 1971. ButterSaurebildende Bacteroides-Kulturen. Z b l . Bakt., I Abt. 217:206. 176. Reysset, G. and J. M i l l e t . 1972. Characterization of an i n t r a -c e l l u l a r protease i n ]}. s u b t i l i s during sporulation. Biochem. Biophys. Res. Commun. 49:328. 245 177. Rizza, V., P.R. S i n c l a i r , D.C. White and P.R. Courant. 1968. Electron transport system of the protoheme-requiring anaerobe Bacteroides  melaninogenicus. J. B a c t e r i o l . 96:665. 178. Rizza, V., A.N. Tucker and D.C. White. 1970. L i p i d s of Bacteroides melaninogenicus. J . B a c t e r i o l . 101:84. 179. Robins, D.J., R.B. Yee and R. Bentley. 1973. Biosynthetic precursors of vitamin K as growth promoters for Bacteroides melaninogenicus. J. B a c t e r i o l . 116:965. 180. R o l l a , G. and M. K i l i a n . 1977. Haemagglutination a c t i v i t y of plaque-forming b a c t e r i a . Caries Res. 11:85. 181. Romeo, D., A. Girard and L. R o t h f i e l d . 1970. Reconstitution of a functional membrane enzyme system i n a monomolecular f i l m . 1. Formation of a mixed monolayer of lipopolysaccharide and phospholipid. J. Mol. B i o l . 53 :475. 182. Sabbaj, J . , V.L. Sutter and S.M. Finegold. 1972. Anaerobic pyogenic l i v e r abscess. Ann. Intern. Med. 77:629. 183. Sargent, M.G., B.K. Ghosh and J.O. Lampen. 1969. C h a r a c t e r i s t i c s of p e n i c i l l i n a s e secretion by growing c e l l s and protoplasts of B a c i l l u s l i c h e n i f o r m i s . J . B a c t e r i o l . 97:820. 184. Sargent, M.G. and J.O. Lampen. 1970. A mechanism for p e n i c i l l i n a s e secretion i n B a c i l l u s l i c h e n i f o r m i s . Proc. Nat. Acad. S c i . 65:962. 185. Sawyer, S.J., J.B. Macdonald and R.J. Gibbons. 1962. Biochemical c h a r a c t e r i s t i c s of Bacteroides melaninogenicus. Arch. Oral B i o l . 1:685. 186. Schrank, G.O. and W.F. Verwey. 1976. D i s t r i b u t i o n of cholera organisms i n V i b r i o cholerae i n f e c t i o n s ; proposed mechanisms of pathogenesis and a n t i b a c t e r i a l immunity. Infect. Immunity 13:195. 187. Schwabacher, H., D.R. Lucas and C. Rimington. 1947. Bacterium melaninogenicum - a misnomer. J. Gen. M i c r o b i o l . 1^:109. 188. Schwarz, O.H. and W.J. Dierkmann. 1927. Puerpural i n f e c t i o n due to anaerobic s t r e p t o c o c c i . Amer. J. Obstet. Gynecol. 13:467. 189. Shah, H.N., R.A.D. Williams, G.H. Bowden and J.M. Hardie. 1976. Comparison of the biochemical properties of Bacteroides melanino- genicus from human dental plaque and other s i t e s . J . Appl. B a c t e r i o l . 41:473. 246 190. Shapira, E. and R. Amon. 1969. Cleavage of one s p e c i f i c d i s u l f i d e bond i n papain. J. B i o l . Chem. 244;1026. 191. Shedden, W.I.H. 1962. Fimbriae and hemagglutinating a c t i v i t y i n s t r a i n s of Proteus bauseri. J. Gen. M i c r o b i o l . 28;1 192. Shevky, M., C. Kohl and M.S. Marshall. 1934. Bacterium melaninogenicum. J. Lab. C l i n . Med. 19_:689. 193. Shockman, G.D. , H.M. Pooley and J.S. Thompson. 1967. A u t o l y t i c enzyme system of Streptococcus f aecalis. I I I . L o c a l i z a t i o n of the a u t o l y s i n at the s i t e s of c e l l w all synthesis. J. B a c t e r i o l . 94:1525. 194. Schockman, G.D. and M.C. Cheney. 1969. A u t o l y t i c enzyme system of Streptococcus f a e c a l i s . V. Nature of the a u t o l y s i n - c e l l wall complex and i t s r e l a t i o n s h i p to properties of the a u t o l y t i c enzyme of Streptococcus f a e c a l i s . j . B a c t e r i o l . 98:1199. 195. Sikyta, B. and Z. Fencl. 1976. Continuous production of enzymes. In: Continuous Culture 6 : Applications and New F i e l d s , p. 158. A.C.R. Dean, D.C.Ellwood, C.G.T. Evans and J . Melling (eds.). Chichester, England. 196. S l i f k i n , M. and H.J. Hercher. 1974. Paper chromatography as an adjunct i n the i d e n t i f i c a t i o n of anaerobic b a c t e r i a . Appl. M i c r o b i o l . 27_:500. 197. S l o t s , J. 1976. The predominant c u l t i v a b l e organisms i n j u v e n i l e p e r i o d o n t i t i s . Scand. J. Dent. Res. 84:1. 198. S l o t s , J . 1977. The predominant c u l t i v a b l e m i c r o f l o r a of advanced p e r i o d o n t i t i s . Scand. J. Dent. Res. 85:114. 199. S l o t s , J. and R.J. Gibbons. 1978. Attachment of Bacteroides melanino-genicus subsp. asaccharolyticus to o r a l surfaces and i t s possible r o l e i n c o l o n i z a t i o n of the mouth and of periodontal pockets. Infect. Immunity 19 :254. 200. Sluyterman, L.A.E. and J. Wijdenes. 1970. An agarose mercurial column for the separation of mercatopapain and nonmercaptopapain. Biochim. Biophys. Acta. 200:593. 201. Smith, D.T. 1930. Fusospirocheatal disease of the lungs produced with cultures from Vincent's angina. J. Infect. Dis. 46:303 202. Smith, L.D.S. and L.V. Holdeman. 1968. The pathogenic anaerobic b a c t e r i a , p. 118. In Charles C. Thomas, Publisher , S p r i n g f i e l d , 111. 203. Smith, L.D.S. 1975. The pathogenic anaerobic b a c t e r i a . Charles C. Thomas, Publisher, S p r i n g f i e l d , 111. 247 204. Socransky, S.S., R.J. Gibbons, H.C. Dale, L. Bortnick, E. Rosenthal and J.B. Macdonald. 1963. The microbiota of the g i n g i v a l crevice area of man. I. T o t a l microscopic and v i a b l e counts and counts of s p e c i f i c organisms. Arch. Oral B i o l . 8_:275. 205. Socransky, S.S. and R.J. Gibbons. 1965. Required r o l e of Bacteroides melaninogenicus i n mixed anaerobic i n f e c t i o n s . J. Infect. Dis. 115:247. 206. Socransky, S.S. 1970. Relationship of b a c t e r i a to the et i o l o g y of periodontal disease. J. Dent. Res. 49:203. 207. Socransky, S.S. and S.D. Manganiello. 1970. The o r a l microbiota of man from b i r t h to s e n i l i t y . J. Periodont. 42:485. 208. Socransky, S.S. 1977. Microbiology of periodontal disease. Present status and future considerations. J. Periodont. 48:497. 209. Solesslavsky, 0., B. Prescott and R.M. Chahoch. 1968. Adsorption of Mycoplasma pneumoniae to neuraminic acid receptors of various c e l l s and possible r o l e i n vi r u l e n c e . J . B a c t e r i o l . 96:695. 210. Stockel, A. and E.L. Smith. 1967. K i n e t i c s of papain a c t i o n : I. Hydro-l y s i s of benzoyl-L-argininamide. J. B i o l . Chem. 227:1. 211. Stone, J . J . and J.D. Martin, J r . 1972. Syngergistic n e c r o t i s i n g c e l l u l i t i s . Ann. Surg. 175:702. 212. Swenson, R.M., T.C. Michaelson, M.J. Daly and E.H. Spaulding. 1973. Anaerobic b a c t e r i a l i n f e c t i o n s of the female g e n i t a l t r a c t . Obstet. Gynecol. 42:538. 213. Sutter, V.L., V.L. Vargo and S.M. Finegold. 1975. Wadsworth Anaerobic Bacteriology Manual, 2nd E d i t i o n . Department of Continuing Education i n Health Sciences U n i v e r s i t y Extension, and the School of Medicine, UCLA. 214. Swindlehurst, C.A., H.A. Shah, C.W. Parr and R.A.D. Williams. 1977. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of peptides from Bacteroides melaninogenicus. J. Appl. B a c t e r i o l . 42:319. 215. Takazoe, I., M. Tanaka and T. Homma. 1971. A pathogenic s t r a i n of Bacteroides melaninogenicus. Arch. Oral B i o l . 16:817. 216. Takazoe, I. and T. Nakamiira. 1971. Experimental mixed i n f e c t i o n by human g i n g i v a l c r e v i c e material. B u l l . Tokyo Dent. C o l l . 12:85 248 217. Takeuchi, H., M. Sumitani, K. Tsubakinoto and M. Tsutsui. 1974. Oral microorganisms i n the gingiva of i n d i v i d u a l s with periodontal disease. J. Dent. Res. 53:136. 218. Tanaka, S. and S. Iuchi. 1971. Induction and repression of an extra-c e l l u l a r proteinase i n V i b r i o parahaemolyticus. Biken J . 14:81. 219. Tempest, D.W. 1970. The place of continuous culture i n m i c r o b i o l o g i c a l research. Adv. M i c r o b i o l . P h y s i o l . 4_:223. 220. Thadepalli, H., S.L. Gorbach and L. Keith. 1973. Anaerobic i n f e c t i o n s of the female g e n i t a l t r a c t ; B a c t e r i o l o g i c and therapeutic aspects. Amer. J. Obstet. Gynecol. 117:1034. 221. Thomas, T.D., B.D. J a r v i s and N.A. Skipper. 1974. L o c a l i z a t i o n of proteinase(s) near the c e l l surface of Streptococcus l a c t i s . J. B a c t e r i o l . 118:329. 222. Tracy, 0. 1969. Pigment production i n Bacteroides. J. Med. M i c r o b i o l . 2_:309. 223. Trevelyan, W.E. and J.S. Harrison. 1952. Studies on yeast metabolism. I. F r actionation and microdetermination of c e l l carbohydrates. Biochem. J . 50:298. 224. Tweedy, J.N., R.W.A. Park and W. Hodgkiss. 1968. Evidence for the presence of Fimbriae ( P i l i ) on V i b r i o species. J. Gen. M i c r o b i o l . 51:235. 225. Uchino, F., K. Miura and S. Doi. 1968. P r o t e o l y t i c a c t i v i t i e s of butyric acid b a c t e r i a and "Butyl organisms". J . Ferment. Technol. 46:188. 226. Veldkamp, H. 1976. Continuous culture i n microbial physiology and ecology. p. 1. J. Gordon Cook (ed.). Bushey, England. 227. Wahlby, S. 1969. Studies on Streptococcus griseus protease. Biochim. Biophys. Acta. 185:178. 228. Wahren, A. and R.J. Gibbons. 1970. Amino acid fermentation by Bacteroides melaninogenicus. Antonie van Leeuwenhoek. 36:149. 229. Walsh, K.A. 1970. Trypsinogen and trypsins of various species. In: G.E. Perlmann and L. Lorand (eds.), Methods i n enzymology, V o l . XIX, Academic Press, Inc., New York. 230. Weiss, C. 1937. Observations on Bacterium melaninogenicum: demonstration of f i b r i n o l y s i n , pathogenicity and s e r o l o g i c a l types. Proc. Soc. Expt. B i o l . Med. 37:473. 249 231. Weiss, C. 1943. The pathogenicity of Bacteroides melaninogenicus and i t s importance i n s u r g i c a l i n f e c t i o n s . Surgery 13:638. 232. Werner, H., G. Pulverer and C. Reichertz. 1971. The biochemical properties and a n t i b i t o i c s u s c e p t i b i l i t y of Bacteroides melanino-;  genicus. Med. M i c r o b i o l . Immunol. 157:3. 233. Whitaker, J.R. and J. Perez-Villaseiior. 1968. Chemical modification of papain: I. Reaction with the chloromethyl ketones of phenylalanine and l y s i n e and with phenyl-methyl-sulfonyl f l u o r i d e . Arch. Biochem^ Biophys. 124:70. 234. Wiersma, M., T.A. Hansen and W. Harder. 1978. E f f e c t of environmental conditions on the production of two 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 by V i b r i o SA1. Antonie van Leeuwenhoek. 44:129. 235. Williams, R.A.D., G.H. Bowden, J.M. Hardie and H. Shah. 1975. Biochemical properties of Bacteroides melaninogenicus subspecies. Int. J. Syst. B a c t e r i o l . 25:298. 236. Wilson, M.R. and A.W. Hohmann. 1974. Immunity to Escherichia c o l i i n pigs: Adhesion of enteropathogenic Escherichia c o l i to i s o l a t e d i n t e s t i n a l e p i t h e l i a l c e l l s . Infect. Immunity. 10:776. 237. Winters, H. 1970. E x t r a c e l l u l a r enzymes produced by a s t r a i n of Pseudomonas fluoroscens. Ph.D. t h e s i s , Columbia Un i v e r s i t y , New York. 238. Winzler, R.J. 1972. Glycoproteins of plasma membranes. Chemistry and function, p. 1268. In: Glycoproteins. A. Gottschalk (ed.). 239. Wouters, J.T.M. and P.J. Buyman. 1977. Production of some ex o c e l l u l a r enzymes by B a c i l l u s l i c h e n i f o r m i s 749/c i n chemostat cultures. FEMS L e t t e r s 1:109. 32 240. Yavin, E. and A. Zutra. 1977. Separation and analysis of P-labeled phospholipids by a simple and rapid thin-layer chromatographic procedure and i t s a p p l i c a t i o n to cultured Neuroblastoma c e l l s . Anal. Biochem. 80:430. 241. Yaron, A. and D. Mlynar. 1968. Aminopeptidase-P. Biochem. Biophys. Res. Commun. 32:658. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0094891/manifest

Comment

Related Items