7902088
CA5SITY, TIMOTHY RDBERT STUDIES ON THE PHYSIOLOGICAL ROLE OF THE CAPSULE PRODUCED BY BACILLUS MEGATERIUM » AND CHARACTERIZATION OF A B. MEG ATERIUM CARBOHYDRATE.
THE OHIO STATE UNIVERSITY, PH.D., 1978
University Micrdirilms International 300 n. zeeb road, ann arbor, mi 48io6 STUDIES ON THE PHYSIOLOGICAL ROIE OF THE CAPSULE
PRODUCED BY BACILLUS MEGATERIUM. AND CHARACTERIZATION
OF A B. MEGATERIUM CARBOHYDRATE
DISSERTATION
Presented, in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Timothy Robert Cassity, B.S., M.S.
* * * * *
The Ohio State University
1978
Reading Committee: Approved By
Dr. Bruno J. Kolodziej
Dr. John R. Chipley
Dr. Patrick R. Dugan ^ Adviser Department of Microbiology PLEASE NOTE: Dissertation contains glossy photographs that will not reproduce well microfilm. Filmed best way possible.
UNIVERSITY MICROFILMS ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to my advisor, Dr. Bruno J. Kolodziej, for his suggestions and assistance throughout this study. I am also indebted to Dr. John R. Chipley and Dr. Patrick R. Dugan for valuable assistance in the preparation of this manuscript, Dr. James I. Frea for use of his gas-liquid chromatograph, and Dr. Garfield P. Royer for the amino acid analysis. I would like to especially thank the two greatest people I know, my parents, whose assistance and encouragement throughout this study made possible its completion.
ii VITA
November 8 , 1951 Born, New Boston, Ohio
1973.... B. S., Ohio University, Athens, Ohio
197^-1976 Graduate Teaching Associate, Department of Micro "biology, The Ohio State University, Columbus, Ohio
1976 M. S., The Ohio State University, Columbus, Ohio
1976-1978 Graduate Teaching Associate, Department of Microbiology, The Ohio State University, Columbus, Ohio
Publications
Cassity, Timothy R., B. J. Kolodziej, and R. M. Pfister. Ultrastructure of the capsule of Bacillus megaterium ATCC 19213. Microbios in Press.
Cassity, T. R., B. J. Kolodziej, and R. M. Pfister. 1977* Ultrastruc ture of the firmly adherent capsule produced by Bacillus megaterium ATCC 19213. ASM Abstracts J 19, p. 18*K
Cassity, T. R., B. J. Kolodziej, and J. R. Chipley. 1977* Isolation, purification, and chemical composition of the firmly adherent capsule produced by Bacillus megaterium ATCC I9213. ASM Abstracts K 155> P* 212.
Cassity, T. R., and B. J. Kolodziej. 1978. Accumulation of metallic ions by normal and small capsule mutants of Bacillus megaterium. ASM Abstracts.
Uriah, N., T. R. Cassity, and J. R. Chipley. 1977- Partial characteri zation of the mode of the action of benzoic acid on aflotoxin biosyn thesis. Can. J, Microbiol. 2^: 1580-158^-,
Kolodziej, B. J., and T. R. Cassity. 1978. Isolation and characteri zation of a new Bacillus megaterium bacteriophage. ASM Abstracts.
iii TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS...... ii
V I T A . iii
LIST OF FIGUBES...... vii
LIST OF TABLES...... x
LIST OF PLATES...... xii
Chapter
INTRODUCTION ...... 1
LITERATURE REVIEW...... 3
MATERIALS AND METHODS...... 12
Microorganisms ...... 12
Culture Medium...... 12
Isolation of Small Capsule Mutants ...... 13
Preparation of Spore Stocks...... 1^
Working Spore Suspension ...... 15
Cultivation of B. megaterium...... 15
Measurement of Growth...... 16
Glassware Preparation...... 16
Isolation and Purification of B. megaterium Polysaccharide . . 16
Extraction of Polyglutamate...... 18
Column Gel Chromatography...... 18
Determination of Total Carbohydrate...... 19
Determination of Protein and Nucleic Acid...... 19
Determination of Peptides and Amino Acids...... 19
iv Chapter Page
Determination of Phosphate...... 20
Analysis of BMP by Gas-Liquid Chromatography...... 20
Polyacrylamide Gel Electrophoresis ...... 21
Concanavalin A Binding...... 21
Iodine Spectra ...... 22
Utilization of B. megaterium Polysaccharide...... 22
Endotrophic Sporulation and BMP Utilization...... 23
Bacillus megaterium BMP Hydrolase Activity ...... 23
Determination of Linkages with Hydrolytic Enzymes...... 24-
Preparation of Media and Cell Samples for Determination of Metallic Content...... 25
Atomic Absorption Spectrophotometery ...... 26
Oxygen Uptake of B. megaterium and Small Capsule Mutants in the Presence of Toxic Metals...... 27
Isolation and Purification of B. megaterium Bacteriophage. . . 28
Determination of Bacteriophage Density ...... 29
Host Range of CK-1 ...... 29
Cation Requirement of CK-1 ...... 29
pH Stability of C K - 1 ...... 3°
Thermal Stability of CK-1...... 30
Replication of CK-1 on B. megaterium of Varying Capsule S i z e ...... 30
Adsorption of Bacteriophage on Cells with Varying Size Capsules ......
Phase Contrast Microscopy...... 32
Negative Staining of Bacteriophage ...... 32
Fixation, Embedding, and Thin Sectioning ...... 32 v Chapter Page
Critical Point Drying...... 33
Electron Microscopy...... 33
Effect of Drying on B. megaterium with Varying Capsule Size ...... 33
RESULTS...... 35
Characterization of B. megaterium BLC-1 and BLC-2...... 35
Characterization of B. megaterium polysaccharide (BMP) .... 45
Extraction of Glutamyl Polypeptide ...... 72
Accumulation of Metallic ions by Normally Capsulated B. megaterium and Small Capsule Mutants...... 7^
Characterization of B. megaterium Bacteriophage CK-1 ..... $6
Replication of CK-1 in Different M e d i a ...... 108
Effect of Drying on the Viability of Normally Capsulated B. megaterium and Small Capsule Mutants ...... 114
DISCUSSION...... 120
Characterization of Small Capsule Mutants...... 120
Characterization of BMP...... 120
Extraction of Polyglutamate...... 124
Accumulation of Metallic Ions by Normally Capsulated B. megaterium and Small Capsule Mutants...... 125
Characterization of Bacteriophage CK-1 ...... 127
Replication and Adsorption of CK-1 to B. megaterium with Varying Sized Capsules...... 128
Drying of Normally Capsulated B. megaterium and BLC-1...... 129
CONCLUSIONS...... 130
APPENDIX...... 132
LITERATURE C I T E D . I63 vi LIST OF FIGURES
Figure Page
1. Growth curve of B. megaterium ATCC 19213, B. megaterium BLC-1, and BLC-2 cultured on FMS medium...... 46
2. Column gel chromatography of acid purified BMP through Bio-Gel A 1.5m...... 48
3. Amino acid analysis of the peptide-containing fractions of acid purified BMP...... 51
4. Gas-liquid chromatography analysis of the trimethylsilyl ethers of the monosaccharide components of parent strain (A) and BLC-1 (B) column purified BMP...... 55
5. Spectrophotometeic scan of BMP electrophoresed through % polyacrylamide...... 59
6 . Absorption Spectra of BMP, starch, and glycogen complexed with iodine solution...... 66
7. Concentration of BMP in cells, culture supernates, and both, during growth and sporulation of B, megaterium. .. . 68
8 . Concentration of total BMP of cells in which cells had been placed under conditions of endotrophic sporulation...... 70
9. Thin layer chromatography of hydrolyzed polyglutamate on microcrystalline cellulose ...... 75
10. Scan of a polyglutamate extract (Cu salt) from B. megaterium electrophoresed through a *(% polyacrylamide...... 77
11. Removal of sodium from FMS by normally capsulated B. megaterium (parent strain) and the small capsule mutant BLC-2...... 79
12. Removal of potassium from FMS medium by normally capsulated B. megaterium (parent strain) and the small capsule mutant BLC-2...... 82
13. Removal of magnesium from FMS medium by normally capsulated B. megaterium (parent strain) and the small capsule mutants, BI/C—1 and BLC-2...... 84
vii Figure page
14. Removal of iron from FMS medium "by normally capsulated B. megaterium (parent strain) and the small capsule mutants BLC-1 and B L C - 2...... 86
15. Removal of zinc from FMS medium by normally capsulated B. megaterium (parent strain) and the small capsulated mutants, BLC-1 and BLC-2 * ...... 88
16. Removal of calcium from FMS medium by normally capsulated B. megaterium (parent strain) and the small capsule mutants BLC-1 and B L C - 2 ...... 90
17. Removal of manganese from FMS medium by normally capsulated B. megaterium (parent strain) and the small capsule mutants, BLC-1 and BLC-2...... 93
18. Inhibition of respiration of normally capsulated B. megaterium (parent strain) and the small capsule mutant BLC-2, by 19.6 n mole of silver...... 97
19. Inhibition of respiration of normally capsulated B, megaterium (parent strain) and the small capsule mutant, BLC-2, by 115 m mole of copper...... 99
20. Inhibition of respiration of normally capsulated B. megaterium (parent strain) and the small capsule mutant, BLC-2, by 3*07 n mole of mercury...... 101
21. Lysis of normally capsulated B. megaterium by CK-1 added with the spore inoculum (0 h of incubation), at the time of germination of the spores (after 3 h of incubation), and at early log phase (after 5 h of incubation)...... 112
22. Relationship of CK-1 adsorption rate constants to size of capsule of B. megaterium ...... Il6
23. Viability of normally capsulated B. megaterium (parent strain) and the small capsule mutant BLC-1 dried in soil ...... 118
24. Standard curve for the determination of total carbohydrate by the anthrone method of Ashwell (1957)...... 133
25. Standard curve for the determination of protein by the method of Lowry et al. (1951)..... 135
26. Standard curve for the determination of protein by the method of Kalb and Bernlohr (1977)...... 137 viii Figure Page
27. Standard curve for the determination of nucleic acid by the method of Kalb and Bernlohr (1977) ...... 139
28. Standard curve for the determination of amino acids by the method of Moore and Stein (19*1-3)...... l4l
29 . Standard curve for the determination of phosphate according to the method of Ames (1966)...... 1*1-3
30. Standard curve for the determination of glucose by the glucose oxidase-peroxidase method...... 1*1-5
31. Standard curve for the determination of reducing sugars according to the method of Somoygi (1945)...... 1**7
32. Standard curve for the determination of calcium by atomic absorption spectrophotometery ...... 149
33* Standard curve for the determination of iron by atomic absorption spectrophotometery ...... 151
34. Standard curve for the determination of potassium by atomic absorption spectrophotometery...... 153
35* Standard curve for the determination of magnesium by atomic absorption spectrophotometery...... 155
36. Standard curve for the determination of manganese by atomic absorption spectrophotometery...... 157
37* Standard curve for the determination of sodium by atomic absorption spectrophotometery ...... 159
38. Standard curve for the determination of zinc by atomic absorption spectrophotometery ...... l6l
ix LIST OF TABLES
Table Page
1. Physiological characteristics and antibiotic of B. megaterium ATCC 19213. B. megaterium BLC-1, and B. megaterium BLC-2 ...... kh
2. Glucose content of B. megaterium ATCC 19213 (parent strain) and B. megaterium BLC-1 through growth and sporulation...... 53
3. Retention times of BMP components and standards...... 57
k. Hydrolysis of BMP by microorganisms which produce specific glucan hydrolases...... 6l
5 . Utilization of BMP and purified polysaccharides by an isolated Arthrobacter species and a mixed culture from s o i l ...... 63
6 . Binding of Concanavalin A by BMP, starch, and glycogen...... 64
7 . Release of reducing sugars from BMP by cell-free extracts and sedimentable cell components of B. megaterium ATCC 19213 throughout growth and forespore formation...... 73
8 . Percentage of metallic ions accumulated by small capsule mutants (BLC-1 and BLC-2) relative to the normally capsulated parent strain of B. megaterium, which was taken as 100^ ...... 95
9 . Inhibition of respiration of normally capsulated B. megaterium (parent strain) and the small capsule mutants, BLC-1 and BLC-2, by silver, copper, and mercury ...... 103
10. Host range of B. megaterium bacteriophage CK-1...... 106
11. Influence of pH on the stability of B. megaterium bacteriophage CK-1...... 107
12. Inactivation of B. megaterium bacteriophage CK-1 by h e a t • 109
x Table Page
13. Effect of metallic ions on plaque formation try C K - 1 ...... 110
14. Replication of B. megaterium bacteriophage CK-1 in media which promotes the formation of various sized capsules...... Ill
15. Adsorption of CK-1 to B. megaterium surrounded by different sized capsules...... 115
xi LIST OF PIATES
Plate Page
I. Colonial morphology of (A) B. megaterium ATCC 19213, (B) B. megaterium BLC-1, and (C) B. megaterium BLC-2 cultured on nutrient agar supplemented with 1% sucrose...... 36
12* Phase contrast photomicrographs of (A) B. megaterium ATCC 19213i (B) B. megaterium BLC-1, and (c) B. megaterium BI£-2 in india ink wet mounts...... JQ
III, Electron photomicrographs of thin sections of (A) B. megaterium ATCC 19213, (B) B. megaterium BLC-1, and (C) B. megaterium BLC-2...... 40
IV. Electron photomicrographs of (A) B. megaterium ATCC 19213, (B) B. megaterium BLC-1, and (cy B, megaterium BLC-2 which have "been critical point dried...... 42
V. Electron photomicrograph of a negatively stained preparation of B. megaterium bacteriophage C K - 1 ...... 104
xii INTRODUCTION
The theory that the bacterial capsule protects a cell (presum ably pathogenic) from phagocytosis by macrophages and polymorpho nuclear leucocytes in mammals has been well documented in the past.
However, there are a large number of bacteria which possess capsules when cultured in the laboratory, whose natural habitat is nonmammalian, and which may have never been introduced into higher organisms which possess phagocytic cells. Discounting the idea that the capsule is a
"waste product" of some other metabolic process, it appears that for saprophytic microorganisms the capsule plays a very different role in maintaining a species in its environment. It is the purpose of this study to investigate several of the roles which might be suspected.
These ares (l) the possibility that the capsule is important in binding small quantities of metallic ions, particularly toxic ones,
(2) the effectiveness of the capsule as a protective mechanism against bacteriophage infection, and (3) the effectiveness in protecting a cell from desiccation. Capsular material may also serve as a carbon and energy storage compound, since most capsules are polymers composed of energy rich compounds. At the initiation of these studies it was believed that carbon and energy storage might be the primary function of the capsule. Due to technical complications, this aspect of capsular function was not elucidated. However, a polysaccharide, most likely not of capsular origin, was characterized, and its function elucidated. Other possible capsular functions which were not investi gated include the capsule as: (l) a protective mechanism against phagocytosis by protozoans, (2) an aid in the dispersal of the micro organism, and (3) an attachment mechanism.
Bacillus megaterium was well suited for this study, since it could be cultured reproducibly in a mineral salts medium, and since, by the isolation of mutants and manipulation of culture media, it was possible to produce cells with differing quantities of capsule. LITERATURE REVIEW
The composition of the capsules produced by many microorganisms is well known, and has been reviewed elsewhere (Housewright, 19&2;
Sutherland, 1972; and Wilkinson, 1958)* Therefore it will not be mentioned in detail in this review. Several species of Bacillus have been found to produce a unique polymer, glutamyl polypeptide. Bovarnick
(19^2 ) was one of the first investigators to describe a soluble d(-) glutamic acid polypeptide produced by B. subtilis ^1259* However,
Hanby and Rydon (19^6) were the first to describe a capsular D-glutamic acid polypeptide, when they isolated this polymer from a virulent strain of B. anthracis. Later, Thome et al. (195^0 described a medium which permitted large quantities of glutamyl polypeptide to be produced by
B. subtilis ATCC 99^5A (The name of this microorganism has since been changed to B. licheniformis ATCC 99^5^, and this name, rather than
B. subtilis. will be used for this microorganism throughout the remain der of this paper). These authors showed that the glutamyl polypeptide produced by this organism contained both D and L isomers. Subsequently,
Leonard et al. (1958) showed that the D-glutamic acid to L-glutamic acid ratio produced in polymers of B. licheniformis ATCC 99^5^ was 2+ dependent upon the concentration of Mn in the medium. Thorne and
Leonard (1958) later showed that that strain produced both poly-D- glutamic acid and poly-L-glutamic acid, but little poly-D, L-glutamic acid polypeptide. Since large quantities of glutamyl polypeptide could be easily isolated in pure form from B. licheniformis ATCC 99^5^» at one time this polymer was evaluated, without success, as a clinical plasma expander. However, Bovarnick et al. (195*0 showed that it was unsuitable, since the polymer rapidly disappeared from the blood stream.
Kream et al. (195*0 demonstrated that B, licheniformis glutamyl poly peptide was hydrolyzed by human red blood cells, liver kidney, spleen,' and brain cells. Tomcsik (1956), on the basis of serological reactions, showed that the capsule produced by B. megaterium was a complex struc ture. It appeared that the material which reacted with anti-poly-D- glutamic acid antiserum was distributed throughout the capsule, where as a polysaccharide was found at transverse septa and poles of the cells. This structure was not substantiated by chemical characteriza tion, however. Vennes and Gerhardt (1959) isolated a polymer by the method of Thorne _et al. (195*0» which they assumed to' be polyglutamic acid; however, these authors also did not characterize that material chemically. Torii (1959), by hydrazinolysis, showed that polyglutamic acid isolated from B. anthracis, B. licheniformis, and B, megaterium contained only y linkages.
One role of the bacterial capsule which has been well documented is that it prevents phagocytosis by leucocytes. Avery and Dubos (1931) and Sickles and Shaw (l93l) isolated microorganisms which produced enzymes which degraded Streptococcus pneumoniae capsules. When these enzymes were injected into mice previously infected with S. pneumoniae shortly before injection of the enzyme, no infection of the mice resulted, whereas mice were killed by injection of S. pneumoniae without the enzyme. Enders et al. (1936) showed that nonencapsulated' pneumoocci were readily phagocytized by leucocytes in normal serum, whereas capsulated pneumococci were not. Even though capsules block phagocytosis by leucocytes, one would expect that this is not the general primary function of a bacterial capsule, since many capsulated bacteria are free-living.
There are no documented accounts in the literature where the capsule has been shown to act as a reserve carbon and energy storage compound. Thorne et al. (195*0 demonstrated that B. licheniformis produced an enzyme which hydrolyzed the soluble glutamyl polypeptide which it produced; however, in general, capsule producers do not produce enzymes which hydrolyze their own capsules (Wilkinson, 1958)*
Conversely, intracellular polysaccharides have been shown to serve as reserve carbon and energy storage compounds in both sporeformers and non-sporeformers. Levene et al. (1953) reported the presence of glyco gen in Enterobacter aerogenes, Escherichia coli, and Salmonella montevideo. Aubert (1951) isolated two polysaccharides from B. megaterium KM. One polysaccharide, composed of glucose, galactose, and ribose, was assumed to be of capsular origin, since it was recovered from a boiling water extract. The other polysaccharide was assumed to be of intracellular origin since it was extracted with 2 N KOH, and was composed solely of glucose. The intracellular glucan gave a colored complex with iodine which had an absorption maximum between the maxima of starch and glycogen. Barry et al. (1953) further described this glucan, and showed that This enzyme produced 3 9 ”**'9^ maltose from glycogen. Later, Tinelli (1955) reported that B. megaterium contained yL% cell dry wt. poly-/3- hydroxybutyrate (PHB) and 16% cell dry wt. glucan at stationary phase. At the time of appearance of the phase bright spores, 12% cell dry wt. of the PHB and 9 cell dry wt. of the glucan remained. However, this investigator did not examine the culture medium for the presence of these constituents. About that time, Foster and Perry (195*0 reported that Bacillus cereus. subtype mycoides could sporulate endotrophically, ie. without a carbon and energy source in the medium. This led to the conclusion that many sporeformers probably derive energy for spore formation from intracellular compounds such as PHB or glucans. Several sporeformers have been shown to contain glucans, in addition to PHB. Slock and Stahly (197*+) isolated a glucan from B. cereus similar to that described by Barry et al. (1953)* The concentration of this glucan also decreased during spore formation. Goldemberg (1972) described a glycogen-like glucan from B. stearothermophilus; however, she did not examine its use as a carbon and energy reserve compound. Strasdine (1968) isolated a glucan structurally resembling amylopectin from Clostridium botulinum type E, which was further described by Whyte and Strasdine (1972). In a subsequent paper, Strasdine (1972) noted that the concentration of that glucan decreased as the cells progressed through sporulation. In the past, the linkages between glucose residues have been deduced by periodate oxidation and gas-liquid chromatography of methyl esters following exhaustive methylation and hydrolysis of the poly saccharide. Even though for most polysaccharides these methods are still preferable, in the last several years hydrolysis by specific hydrolytic enzymes has gained acceptance as a valuable tool in the elucidation of polysaccharide linkages. Tsuchiya et al. (1952) described several sources of dextranases which hydrolyzed dextran from Leuconostoc mesenteriodes. Hutson and Wiegel (1963), Zevenhuizen (1968), Sugiura et al. (1973)1 and Janson (1975) described dextranases from Peniclllium lllacinum. P. fimiculosuin, B. subtilis. and Cytophaga .johnsonii. Bacon et al. (1968), Hasegavia et al. (1969), and Zonneveld (1972) described enzymes isolated from a Streptomyces culture filtrate, Trichoderma viride QM 6A, and Aspergillus nidulans which hydrolyzed C**(l— ^3) linkages in glucans. Reese and Mandels (196*0 described an emzyme from Trichoderma viride QM 6A which hydrolyzed c<( 1— >4) linkages in glucans containing alternating o(,(l— > 3)1 c^( 1— ^ * 0 linkages. Similar enzymes were also isolated from P. melinii QM 1931 and A. luc luchensis QM 873 and described by Rosenthal and Nordin (1975)- Glucans with (l— >-2 ) linkages have been specifically hydrolyzed by enzymes produced by P. funiculosum, P. varians, P. islandicum, P. verruculosum, A. fumigatus, A. aureolus, A. auratus, A. fiseheri. and A. quadricintus (Reese et al., 1961). Several sources of ft( 1— ^6) glucanase have been described by Reese et al. (1962), Nakamura and Tanabe (1963), and Rombouts and Phaff (1976a). In addition, numerous sources of ft (l— >3 ) glucanase have been described (Reese and Mandels, 1959; Marshall, 1973; and Rombouts and Phaff, 1976b). Another possible role of the capsule could be in the binding of ions in media where few ions are present. The ion binding capacity of the cell wall has been established much more conclusively than has the ability of the capsular material, however. Ou and Marquis (1970) demonstrated that isolated cell walls of Micrococcus lysodeikticus have a greater.capacity to bind most ions than a Dowex A-l resin. 2+ Teichoic acids have been shown to have a high affinity for Mg (Lambert et al., 1975)* Marquis et al. (1976) determined the binding affinity of several cations to isolated cell walls from M. lysodeikticus ATCC 4698, Streptococcus mutans GS-5, S. faecalis ATCC 9790, B. megaterium KM, and Staphylococcus aureus strains H and 52A.5 (teichoic acid deficient). These investigators showed that the affinity series progressed from H+ > La3+> Cd2+> Sr2+> Ca2+> Mg2+> K+ > N a + > Li+t Cell walls from the teichoic acid deficient strain of S. aureus had much lower cation exchange capabilities than walls of S. aureus which contained teichoic acids. Beveridge and Murray (1976) showed that 2+ ^j* p*t* substantial amounts of Mg , Fe , Gu , Na , and K , lesser amounts of Mn2+, Zn2+, Ca2+, and small amounts of Hg2+, Sr2+, Pb2+, and Ag+ were retained by isolated cell walls from B. subtilis Marburg, These authors also showed that partial lysozyme digestion diminished the retention of Mg2+ , but did not effect the retention of Ca2+, Fe2+, 2+ or Ni , indicating that there were probably select sites for the bind ing of some cations. Rorem (1955) showed that Leuconostoc dextranicum, L. mesenteroides. and Streptococcus salivarius accumulated much more 32 86 P 0^ and Rb when cultured in media containing sucrose, which enhances dextran and levan production, compared to the same cells cultured in media without sucrose. Several years later, Friedman and Dugan (1968) reported that two sewage isolates, Zoogloea ramigera ATCC 25935 an Ogiwara and Kubota (1969) demonstrated that Fe^+ and Ge^" formed very stable complexes with cellulosic fibrils. Other ions were also bound, but did not form such stable complexes. Engel and Owen (1969) reported that several capsulated microorganisms associated with hydrocarbon oxidation accumulated metal ions. In a series of studies, Dugan and his associates showed that ions from acid mine drainage, as well as several other chemicals, were removed by bacteria which formed fibrillar floes, particularly Zoogloea species (Dugan, 1970; Dugan et al. 1971; and Dugan, 1971). There have been no studies in the literature in which the binding of ions by polyglutamate, or bacteria which produce polyglutamate capsules, has been reported. Kreuger (1972) showed that B. megaterium cultured on fructose mineral salts medium had smaller, less dense, capsules than B. megaterium cultured on sucrose mineral 2+ 2+ salts medium. The fructose-grown cells removed less Ga , Mg , and 2+ 2+ Mn than cells cultured on sucrose, while Gu uptake was similar for both cell types. However, it was not established that both cell types possessed the same type of capsular material. For some time it had been suspected that the bacterial capsule may protect a bacterial cell from attack by bacteriophages. McCloy (1951) isolated a bacteriophage specific for B. anthracis; however, when B. anthracis was cultured on 2Qffo serum agar in an atmo sphere with a high CO^ concentration, the cells became mucoid, and were no longer infected by the bacteriophage.- Maxted (1952) reported that Group A Streptococcus strain A6, which forms a hyaluronic acid capsule, was not infected by Streptococcus bacteriophage A6. However, the bacterial strain was infected by the bacteriophage when hyaluronidase was added to the medium. In a more recent article, Bernheimer and Tiraby (197&) showed that three different bacteriophages (W2, W3 , and W8) lysed nine nonencapsulated strains and two C capsulated strains s of S. pneumoniae, while forty-one encapsulated strains were not lysed, Capsulated S. pneumoniae cells in which the capsules had been enzymatically removed were infected with phage W3» even though the capsule had been resynthesized by the time of release of mature phage from the host cells. This showed that the capsule blocked the phage receptor site on the S. pneumoniae cell wall, Burt et al. (1978) Isolated a Bacteriodes species (B. thetaiotaomicron) which gave rough colonies composed of nonencapsulated cells and mucoid colonies, composed of capsulated cells. They noted that the encapsulated variant was not infected by a bacteriophage specific for this organism. These studies showed that the capsules produced by some microorganisms do indeed inhibit bacteriophage infection. However, bacteriophages have been described which infect heavily encapsulated bacteria. Adams and Park (1958), and Park (1958), demonstrated that encapsulated Klebsiella pneumoniae type 2 could be infected with bacteriophage Kp, but that the bacteriophage produced an enzyme which depolymerized the hosts capsule. The genetic information for the production of the enzyme was contained in the phage genome. Later, Thurow et al. (1974) isolated a particulate fraction from two bacteriophages which exhibited glucosidase activity on Klebsiella strain K 20 and galactosidase activity on Klebsiella strain K 24, causing extensive depolymerization of the capsular material of each strain. Eklund and Wyss (1962) reported that Azotobacter vinelandii strain 0 , when infected with certain bacteriophages, formed a capsule degrading enzyme early during replication of the phage. When cells were finally lysed, much of the enzyme was released into the medium, but some was found to be carried with the bacteriophage. Sutherland and Wilkinson (1965) described several bacteriophages which contained genetic information for the production of an exopolysaccharide depolymerase for the exopolysaccharide produced by E. coli K 12 and other microorganisms with exopolysaccharides similar to that of E. coli K 12. Similarly, Bartell et al. (1966) described a Pseudomonas 11 aeruginosa bacteriophage (Phage 2) which specifically depolymerized P. aeruginosa exopolysaccharide. Again the genetic information for the production of the enzyme was found to be within the phage genome. Yurewicz et al. (1971) examined, in detail, the properties of an enzyme produced by a virulent Enterobacter aerogenes bacteriophage isolated from sewage. The enzyme specifically attacked galactosyl- O H — galactose linkages. These studies demonstrate that unless a bacteriophage can hydrolyze capsular material, they most likely will not infect a capsulated cell if the receptor cite for the phage is on the cell wall- Several bacteriophages have been described which will infect B. megaterium (Friedman and Cowles, 1953 > Ehrlich and Pfau, 1957? Murphy, 1957; Cooney et al.. 1975; and Carvalho and Vary, 1977); however, no studies have appeared in the literature in which the capsule has been considered to play any role in phage infection. It had been speculated for some time that the bacterial capsule may protect cells from desiccation. Morgan and Beckwith (1939) reported that mucoid species of various Enterobacteriaceae dried on coverslips and stored at room temperature survived for longer periods of time than nonmucoid species which had been similarly treated. However, Bitton et al. (1976) showed that nonencapsulated Klebsiella aerogenes survived as well as encapsulated K. aerogenes when both types were dried in different types of soil. MATERIALS AND METHODS Microorganisms The primary microorganism used throughout this study was Bacillus megaterium ATCG 19213 (OSU 125) a^d small capsule mutants derived from this strain. A description of the B. megaterium mutants will be given later, and the use of numerous other microorganisms is described with the methodology of the experiment in which they were used. Culture Medium The primary culture medium employed for the growth of B. megaterium and its mutants throughout this study was a fructose mineral salts medium (fflS), This medium was modified from that origi nally described by Slepecky and Foster (1959)* and is composed of (on a w/v basis)i fructose, 0.1$; KNO^, 0.1$; NaCl, 0,1$; MgSO^'TKgO* 0.02$; FeS0^’7H20, 0.001$; ZnSO^THgO, 0.001$; MnSO^'HgO, 0.000?$; CaCl2 *2H20 , 0 .0005$, CuSO^T^O, 0 .00005$; (NH^HPO^, 0 .55$; and KHgPO^, 0.12$. All of the chemical constituents of this medium were of reagent grade quality. Dibasic ammonium phosphate was purchased from Mallincrodt Chemical Co. (St. Louis, Mo.), since this chemical as supplied by other manufacturers contained substances toxic to B. megaterium. For mineral accumulation experiments, trace salts (FeS0^ ‘7H20 » ZnS0^*7H20 , MnSO^’^O , CaCl2 -2H20 , and C u S O ^ ^ O ) were 12 13 made 100 times concentrated, filter sterilized, and stored at ^ C, and added to the other medium constituents after they had been autoclaved and cooled. In media used for the growth of cells for the preparation of B. megaterium polysaccharide or polyglutamate, 100 times concen trated mineral salts and 100 times concentrated 0.005% (w/v) disodium dihydrogen ethylenediaminetetraacetate (EDTA) were added to the other media constituents prior to autoclaving. In all MS, phosphate compon ents were autoclaved separately as 20 times concentrated solutions, and added to the rest of the medium just prior to inoculation. The final pH of the medium was 7-2 - 0.05 without adjustment. Isolation of Small Capsule Mutants Cells from 100 ml of three-quarter logarithmic M S culture of B. megaterium ATCC 19213 were pelleted at 6,000 X g for 10 min at 25 C, resuspended in 25 ml of fresh MS, and 2.5 ml of a 500 }ig/ml solution of N-methyl-N'-nitro-N-nitrosoguanidine added. This culture was incubated at 30 C for 60 min. The cells were then pelleted, washed twice with 0.85$> saline, and resuspended in 25 ml of MS. This culture was incubated for 90 min at 30 C with shaking at 200 rpm. The cells were diluted 100-fold in 0.05 M phosphate buffer (pH 7*2) and plated on trypticase soy agar (BBL) supplemented with 1 % (w/v) sucrose. Following incubation at 30 C for 20 h, less mucoid colonies were picked and observed in india ink wet mounts by phase contrast microscopy for the presence of capsule. Those colonies which were composed of cells with small capsules were propagated in 10 ml of MS. Following incubation, this culture was layered on a 70^, 60fo, 50fo, yrfo, 20% (v/v) discontinuous RENOgraphin-76 gradient and centrifuged ]A at 20,000 X £ for 60 min at 4 G in a swinging “bucket rotor. The cells at the 60-70^ interface were removed and propagated on FMS. The mutants derived in this manner were prototrophic, and otherwise very similar to the parent strain with respect to physiological charac teristics. Two of the mutants isolated in this manner were used exten sively throughout this study. These were named BLC-1 and BLC-2. Preparation of Spore Stocks In order to limit the number of transfers of B. megaterium and its mutants, and insure the preservation of their characteristics, spore stocks were prepared in the following manner. Bacillus megaterium was received from the OSU culture collection on nutrient agar slants. The mutants were streaked onto nutrient agar slants and incubated for 20 h. The growth from one such slant was removed with 20 ml of sterile double distilled water (SDDW), and the cells pelleted and washed twice by centrifugation at ^,000 X £ for 10 min at ambient temperature. The final pellet was resuspended in 10 ml of SDDW and used to inoculate 200 ml of FMS in a 1 liter Erlenmeyer flask. Following 12 h of incubation at 30 G in a Psychotherm Incubator Shaker (New Brunswick Scientific) with agitation at 200 rpm, the culture was used as an inoculum for 8 liters of FMS in a 1^ liter Microferm fermenter jar (New Brunswick Scientific). The fermenter was sparged with 6 liter/min of filtered air and mechanically agitated at 200 rpm. Following 36 h of incubation, the spores were aseptically harvested by centrifugation at 6,000 X £ for 10 min at 0 C. The upper slimy layer of the pellet, which contained primarily dead cells and sporangia, was discarded, and the spores washed 5 times with SDDW. The final 1 5 spore pellet was resuspended in 25 ml of SDDW and dispensed in 1 ml aliquots into sterile 5 ml lyophilization ampules. These were frozen in a d:?y ice-acetone bath and dried in vacuo. The lyophilized spores were stored at ambient temp, until used. Working Spore Suspension To prepare a working spore suspension, which was used as inoculum for vegetative cultures, the contents of an ampule of lyophilized spores was suspended in 20 ml of SDDW, heat shocked for 60 min at 60 G, and used to inoculate 400 ml of M S contained in a 2 liter Erlenmeyer flask. This culture was incubated for 48 h at 30 C in a Psychotherm Shaker Incubator with agitation at 200 rpm. The working spore suspension was prepared monthly. Cultivation of B. megaterium Bacillus megaterium and its mutants were routinely cultured in the following manner. Twenty milliliters of working spore suspension were centrifuged at 3,000 X £ for 10 min. The pellet was washed once with SDDW, resuspended in 10 ml of SDDW, and heat shocked at 60 G for 60 min. These spores were used to inoculate 200 ml of M S in a 1 liter Erlenmeyer flask, which was incubated for 14 h at JO C with agitation of 200 rpm. This entire culture was added to 8 liters of M S in a 14 liter Microferm fermenter jar prewarmed to 30 G and sparged with 6 liter/min of filtered air. When the culture was to be used for the preparation of polysaccharide or polyglutamate, the cells were harvested at three-quarters logarithmic growth (approximately 7 h of incubation) by centrifigation at 6,000 X g for 10 min at 0 G in a Sorvall RG2-B refrigerated centrifuge. Cells cultured in this manner were either 16 used immediately or frozen at -70 C. When smaller cultures were desired, the amount of medium and inoculum was scaled down propor tionately. Measurement of Growth Growth was followed "by measuring culture turbidity (O.D.) at 5^0 nm (filter no. 5*0 using a Klett-Summerson photoelectric colorimeter. Dry weight determinations were used to quantitate large numbers of cells. Glassware Preparation All glassware and polyethylene items used for mineral accumu lation experiments, and glassware used in carbohydrate and phosphorus analysis, phage binding, and mutant isolation procedures, were cleaned by soaking in dichromate cleaning solution for at least 12 h. This glassware was rinsed at least 10 times with tap water and 7 times with distilled water. Fourteen .liter fermenter jars were scrubbed with Haemo-Sol cleanser and rinsed well with generous quantities of tap and distilled water. Glassware used in collecting fractions or quantitating peptides was soaked in saturated KOH solution at 90-100 G for 2 h. All traces of the KOH were removed by rinsing 10 times with tap water and 7 times with distilled water. Isolation and Purification of B. megaterium Polysaccharide Bacillus megaterium polysaccharide (BMP) was extracted from three-quarter logarithmic cells by incubating a thick cell slurry in 0.2M sucrose-0.05M phosphate buffer (pH 7*2) containing 300 pig/ml of lysozyme (3 X crystaliz-ed, Sigma Chemical Go.) at 30 C for 2 h. Following incubation, protoplasts and dense cell debris were removed by centrifugation at 20,000 X £ for 10 min at 0 C. The sucrose was replaced with 0.05 M phosphate buffer (pH 7.2) by ultrafiltration through an XM 100A membrane in an Amicon membrane ultrafiltration apparatus (Amicon Corp., Lexington, Mass.) The retentate was precipitated by the addition of 3 vol of -20 C 95% ethanol (EtOH). The precipitate was collected by centrifugation and lyophilized. In an alternative procedure, cells were lysed by two passages through a French pressure cell at a pressure greater than 20,000 psi. The large particulate matter was removed by centrifugation at 5>000 X £ for 10 min at 0 C, and the supernate extracted with an equal volume of 90% (v/v) aqueous phenol at 60 C as described by Wicken et al. (1973)* Carbohydrate was precipitated from the aqueous layer by addition of 3 vol of -20 C 95% EtOH. The ppt. was collected by centrifugation at 20,000 X £ for 10 min, and lyophilized. Nucleic acids were removed from these preparations by dissolving 0.5 g of lyophilized ppt. in 100 ml of DDW at 4 C. The pH of this solution was lowered to 3*5 by the dropwise addition of glacial acetic acid, and 0.35 vol of $5% EtOH (4 C) added. The resulting ppt. (primarily nucleic acids and protein) was removed by centrifugation at 20,000 X £ for 10 min at 0 C. The soluble polysaccharide remaining in the supernate was precipitated by the addition of 3 vol of -20 C 95^ EtOH and 0.01% NaCl. The resulting ppt. was collected by centrifugation, washed with 95% EtOH, and lyophilized. This material was labeled acid purified BMP. « 18 Extraction of Poly-glutamate Poly-glutamic acid was extracted from the cells by a modifica tion of the method used by Bovarnick (19^2) to prepare soluble poly glutamate from B. subtilis. A thick suspension of B. megaterium cells was autoclaved for ^5 min, and the cells removed by centrifugation (20,000 X g for 10 min at 0 C). Saturated CuSO^. 5*^0 was added to the supernate until a heavy ppt. formed. The ppt. was collected by centrifugation and dissolved in 2^ ml of 1.5 N HG1. Polyglutamate was precipitated by raising the pH to 10.0, and the ppt. was collected by centrifugation at 20,000 X g for 10 min at 0 G. This method was found to be superior to the methods of Hanby and Rydon (19^+6), Thorne et al. (195^)» and Thorne and Leonard (1958). Column Gel Chromatography Samples were chromatographed on a 1,5 X 90 cm column packed with Bio-Gel A 1.5m (Bio’Rad Laboratories, Richmond, Cal). This gel 6 4 had an operating range of 1.5 X 10 -1.0 X 10 . In a typical run, 0.03 g of sample dissolved in 2 ml of 0.05 M phosphate buffer (pH 7*2) was applied to the column and eluted with 0.05 M phosphate buffer (pH 7.2) at ambient temp with a flow rate of 0.125 ml/min. Five milliliter fractions were collected on an Isco model 270 fraction collector equipped with a 5 ml siphon. The void volume of the column was determined by chromatographing a solution of blue dextran 2,000 (Pharmacia, mol. wt. 2 X 10^) through the gel. Determination of Total Carbohydrate The presence of carbohydrate was detected and quantitated by the anthrone method of Ashwell (1957)* Absorbance was measured at 625 m on a Bausch and Lomb Spectronic 20 spectrophotometer. Glucose was used as standard, and the standard curve is shown in Fig. 24 (Appendix). Determination of Protein and Nucleic Acid Protein concentration was determined colorimetrically by the method of Lowry et al. (l95l)» Bovine serum albumin was used as standard. Absorbance was measured at 540 nm in a Bausch and Lomb Spectronic 20 Spectrophotometer. The standard curve is shown in Fig, 25 (Appendix). Alternately, protein and nucleic acid concentra tions were determined by the 230/260 method of Kalb and Bernlohr (1976) using a Gilford model 2400 spectrophotometer. Standard curves using Bovine serum, albumin and sperm DNA for protein and nucleic acid, respectively, are shown in Figs. 26 and 27 (Appendix). This method proved to be superior to the Lowry et al. (l95l) method for most samples. Determination of Peptides and Amino Acids For quantitative analyses of peptides, the peptide was first hydrolyzed by the alkaline hydrolysis method of Hirs (1967). After adjustment of the pH to 5*0 with glacial acetic acid, the amino acids were quantitated by the ninhydrin method of Moore and Stein (1948). L-glutamic acid was used as standard. Absorbance was measured at 570 nm with a Bausch and Lomb Spectronic 20 spectrophotometer. The 20 standard curve is shown in Fig, 28 (Appendix), When both qualitative and quantitative determinations of amino acids were desired, the sample was hydrolyzed with 6 N mineral-free HG1 for 24 h at 110 C in vacuo. The HG1 was removed over KOH in vacuo. Amino acids were separated and quantitated with a Beckman model 116 amino acid analyzer equipped with a 0,9 x 30 ci column packed with Durrum DC 6A resin, or by thin layer chromatography on SigmaCell Type 20 microcrystalline cellulose (Sigma Chemical Co.). Plates were developed in a solution of n-butanol, pyridine, and water in a ratio of 3*2:1.5 i respectively. Amino acids were detected by the ninhydrin solution of Salander et al. (1953). Determination of Phosphate Organic and inorganic phosphate was determined by the method of Ames (1966). Absorbance was measured at 625 nm in a Bausch and Lomb Spectronic 5®5 recording spectrophotometer. The standard curve is shown in Fig. 29 (Appendix). Analysis of BMP by Gas-Liquid Chromatography The monosaccharide constituents of the BMP was determined in the following manner. Ten milligrams of column purified BMP, or carbohydrate standard, was hydrolyzed with 1 ml of 4 N HC1 at 100 C for 3 h in vacuo. The HC1 was removed over KOH in vacuo and the monosaccharides silanized as described by Sweeley et al. (1966). A l/8" x 6 ' stainless steel column packed with 8% Dow Silicone SE-30 on Gas Chrom Q, was used throughout this study. The column and glass wool plugs were silanized before packing the column. Samples (1-2 pi) were run on a Varian Aerograph Series 2700 gas-liquid chromatograph at 155 C with a flow rate of 30 nil of gas per nin. Monosaccharides were detected by flame ionization. Polyacrylamide Gel Electrophoresis Bacillus megaterium polysaccharide preparations (BMP) were electrophoresed on 5% polyacrylamide gels (7 x 125 mm) prepared using an Eastman Standard Acrylamide Electrophoresis Reagents Kit, Electrophoresis was carried out at 10 C for 14 h using a current of 2 mA per tube supplied by a Heath-Shumberger model SP-17A Regulated HV controlled power supply, A 0.1 M Tris-glycine buffer (pH 8.4) was used as running buffer. Following removal of the gels from their tubes, the gels were stained and destained by the method of Zacharias and Zell (1969) using the fuchsin-sulfite staining reagent of McGuckin and McKenzee (1958) and scanned at 430 nm on a Gilford model 2400 spectrophotometer equipped with a model 2410 linear transport apparatus. Polyglutamate was electrophoresed similar to HIP, except that 7% gels were used, electrophoresis was allowed to proceed only 3 h, and the gels were stained with Coomasie Brilliant Blue R-250 and destained with 12.5% (w/v) trichloroacetic acid. Gels were scanned at 540 nm. Goncanavalin A Binding One milliliter of a 5 mg/ml solution of HIP in 0.05 M phosphate buffer (pH 7*2) was mixed with a 1 ml of a 5 mg/ml solution of the lectin Concanavalin A (Con A) and incubated at ambient temp for 1 h, then at 4 G for 24 h. Following incubation, the ppt. was removed by centrifugation, and the concentration of unprecipitated Con A in the supernates determined by protein analysis. 22 Iodine Spectra A 1 mg/ml solution of BMP or standard carbohydrate was mixed with an equal volume of a 0.05 M I^-O.IO M KI solution. Spectra were recorded from nm to 600 nm using a Bausch and Lomb 505 recording spectrophotometer. Utilization of B. megaterium Polysaccharide In order to determine if BMP was utilized by B. megaterium, eight liters of B. megaterium were cultured in a 14- liter fermenter jar in FMS at 30 C sparged with 6 liter/min of filtered air and mechan ically agitated at 200 rpm. At specific times throughout growth and sporulation, ^00-700 ml of culture were removed and the cells separated from the medium by centrifugation at 6,000 X £ for 10 min at 0 G. Cell-free media samples (10 ml) were placed in dialysis bags, which had been prepared by boiling in distilled water 3 times, and dialyzed for 3 days against 5 changes of 10 liters each of DDW at k G. Cells were washed with DDW once and the volume of the final pellet recorded. Aliquots of cells were removed for dry weight determinations, and the remaining cell slurry either analyzed by the anthrone method, or 1.8 ml of cell slurry mixed with 0.2 ml of 20 N HgSO^ and hydrolyzed at 100 G for 3 h. The pH of the suspensions that were hydrolyzed with HJ30. 2 Hr were adjusted to 6.5 by the addition of 4N NaOH and 2 M Tris buffer, and analyzed by the glucose oxidase-peroxidase method as described in the Sigma Technical Bulletin No. 510 using a commercial kit available from Sigma Chemical Co. (St. Louis, Mo.). This method was essentially that of Raabo and Terkildsen (i960). Absorbance was measured at ^50 11111 with a Bausch and Lomb Spectronic 20 spectrophotometer. A standard curve 23 is shown in Fig. JO (Appendix). Following dialysis, the carbohydrate concentrations of the supernates were determined by the anthrone method. Endotrophic Sporulation and BMP Utilization A /J-00 ml culture of B. megaterium was grown on M S at 30 C and shaken at 200 rpm. After 3 h, 6 h, and 8 h of incubation, 100 ml of cells were aseptically removed, pelleted by centrifugation at 6,000 X g for 10 min at 28 G, and washed twice with sterile 0.05 M phosphate buffer (pH 7.2) supplemented with filter-sterilized M S trace salts. The final pellet of the 3 h sample was resuspended in 50 ml of 0.05 M phosphate buffer (pH 7*2) containing filter-sterilized trace salts, and the final pellets of the 6 h and 8 h samples resuspended in 100 ml of sterile 0.05 M phosphate buffer containing filter- sterilized FMS trace salts. The samples were then incubated at JO C with shaking at 200 rpm. Periodically, samples were taken for total carbohydrate analysis, and wet mounts were observed by phase contrast microscopy. Bacillus megaterium BMP Hydrolase Activity Eight liters of B. megaterium was cultured in a 14 liter fermenter jar at JO G, sparged with filtered air, and mechanically agitated at 200 rpm. After 5 h t 7 h, 11 h, and 15 h of incubation, 1 liter was removed, and the cells pelleted by centrifugation at 6,000 X g for 10 min at 0 C and washed twice with DDW. The cells were lysed by passing them through a French pressure cell at a pressure greater than 20,000 psi. Sedimentable cell components were separated from the cell-free extract by centrifugation at 22,000 X g for JO min at 0 C. 24 Following centrifugation, the supernate was collected and the sedimen table cell components resuspended in 5 ml of DDW. Bacillus megaterium BMP hydrolase was assayed hy adding 0.8 ml of cell-free extract or sedimentable cell components suspension to 0.2 ml of a 10 mg/ml solution of acid purified BMP in 0.2 M phosphate buffer (pH 7.2) or 0.2 ml of buffer alone, and incubating at 30 G for 12 h. Following incubation, any insoluble material was removed by centrifugation and 0.2 ml of supernate was analyzed for the presence of reducing sugars by the arsenomolybdate method of Somoygi (1945)* Absorbance was measured at 5^0 nm using a Bausch and Lomb Spectronic 20 spectro photometer. Glucose was used as standard, and the standard curve is shown in Fig. 31 (Appendix), Determination of Linkages with Hydrolytic Enzymes In order to determine the possible linkages of the BMP by enzymatic methods, two approaches were taken. First, microorganisms known to produce polysaccharide hydrolases specific for individual linkages were incubated in 1 ml of BMP mineral salts medium, (BMEMS), which was identical to IMS, with the exception that 0,1$ fructose was replaced with 0.3$ BMP. Utilization, and thus hydrolysis, of the BMP was determined by analyzing the carbohydrate content of such cultures. The microorganisms used in this study included Aspergillus fumigatus, Sporotrichum pruinosum ATCG 24782, B. subtilis Marburg, Trichoderma viride ATGG 13631, Penicillium brefeldianum ATCC 10417, and Aspergillus niger. The second approach invo3.ved isolating microorganisms from the soil which would degrade BMP. The linkages in the BMP might then be deduced by testing such isolates for hydrolytic activity on carbohy drates tilth known linkages. This was performed in the following manner. Samples of soil or decaying vegetative matter were collected over an area of approximately ^0 sq.. mi. of forest. Approximately 5 S of sample was added to 5 ml of SDDW and shaken well on a vortex mixer. The heavy particulate matter was permitted to settle, and 1 ml used to inoculate 10 ml of BMPMS medium contained in a 50 ml Erlenmeyer flask. Such enrichment cultures were incubated at 30 C for 3 weeks with shaking at 200 rpm. Samples were taken periodically and assayed for total carbohydrate content. When a sizable decrease in polysaccharide was noted, 0.01 ml of sample was streaked on BMPMS solidified with 0.8/6 agarose and incubated until growth was noted. Isolated colonies were removed and restreaked on BMPMS agarose medium. It was often noted that the carbohydrate content of the enrichment flasks would decrease without concominant growth on streak plates. Isolates were tested for the degradation of dextrin, cellobiose, starch, glycogen, lichenan, gentiobiose, trehalose, and BMP in 1 ml cultures. To determine if additional nutrients (ie. yeast extract, peptone, etc.) would enhance degradation of BMP, 10 ml cultures were used. Preparation of Media and Cell Samples for Determination of Metallic Content For analysis of the metallic ion content of the medium, a 20 ml sample of culture was removed periodically (usually hourly) and the cells removed by centrifugation at 2^,000 X g for 20 min at 0 C. The supernates were collected and refrigerated until analyzed. For analysis of cell samples, 1 liter of culture was removed, the cells pelleted by centrifugation at 6,000 X g for 10 min at 0 C, and washed 26 twice with double distilled demineralized water (DDDW). The final pellet was lyophilized and further dried in an oven at 80 C for 3 h. The cell samples were then weighed and digested with 3 ml of a 2:1 mixture of HNO^ and H2S0^, respectively, by heating at 150 G for 4-6 h. The samples were then diluted by the addition of 17 ml of DDDW. Atomic Absorption Spectrophotometery Media and cell samples were analyzed by atomic absorption spectrophotometery using a Perkin-Eliner model 403 atomic absorption spectrophotometer. Hollow cathode lamps were used, and the absorbance recorded at the wavelength specified for each element. Samples in which the concentration of a metal was too high to be accurately deter mined were diluted with DDDW. For the analysis of Ca.and Mg, 1 ml of a 1($ solution of lanthanum oxide was added to 4 ml of sample in order to determine reduce interference from sulfates and phosphates. The 10% lanthanum oxide solution was prepared by dissolving 29;325 g of La.0„ in 62.5 nil of concentrated HG1 and diluting to a final volume of 250 ml. Standard curves were prepared by diluting standard stock solutions (ARRO Scientific Laboratories, Inc.) containing 1000 pg of metal/ml solution to the appropriate concentrations to fall within the linear range of the instrument. Standard curves for the analysis of Ca, Fe, K, Mg, Mn, Na, and Zn are shown in Figs, 32, 33, 34, 35 , 36, 37, and 38 respectively, (Appendix). 27 Oxygen Uptake of B. megaterium and Small Capsule Mutants in the Presence of Toxic Metals One hundred milliliters of mid-log M S culture containing B. megaterium or BLC-2 cells were removed and centrifuged at 6,000 X g for 10 min at 28 C, resuspended in 50 ml of fresh MS, and the O.D. of each culture standardized. Each culture was maintained at 30 C, and sparged with air supplied from a laboratory air line. One milliliter of the culture was aided to the chamber of a Yellow Springs Oxygen Polarograph (l.7 ml chamber) maintained at 30 C» and the rate of 0^ consumption measured. After the culture had removed approximately 50?S of the 0g from the medium, 0.5 ml of (^-saturated AgNO^ (0.196-0.392 mM final concentration), CuS0^ ’5H20 (0.115-0.0575 mM final concentration), or HgClg (5 *0-2.3 nM final concentration) was added to the chamber of the oxygen polaragraph, and the rate of 0^ consumption again measured. As a measure of the effectiveness of the metal to inhibit respiration of the cells, the time required to completely inhibit respiration was recorded, and a coefficient of inhibition (C) calculated by the equation: 0- ^inh , H o where is "the time required to completely inhibit respiration (in min), and Rq is the rate of 0^ consumption (in % saturation/min) before addition of the toxic metal. 28 Isolation and Purification of B. megaterium Bacteriophage Approximately 500 g of moist fertile soil was mixed with 1000 ml of distilled water in a Waring "blender for 1 min. Soil particles were removed by centrifugation at 3*000 X g for 5 rain and the supernate filtered through a 0.22 ;jm Millipore membrane filter. The filtrate was concentrated to approximately 50 ml in an Amicon ultrafiltration apparatus using an XM 300 membrane. Bacteria were removed from the retentate by membrane filtration, and 1 ml aliqouts of the filtrate plated onto nutrient agar (Difco) supplemented with 12 mM MgCl^*6H2O by the agar overlay method of Adams (1959 a). Bacillus megaterium spores were used as a source of host bacterium. Following incubation at 30 C, plates were observed for plaques. Several plaques were apparent, and these were removed, mixed with 5 ral of nut rient broth, and replated on the same medium. One phage was isolated and named CK-1. An 8 liter batch of CK-1 was prepared in a 14 liter Microferm fermenter jar in the PA medium of Thorne (1962) without agar. Bacillus megaterium spores were used as source of host organism, and the phage was permitted to lyse the cells for 12 h at 30 G. The resulting phage lysate was concentrated and purified with polyethylene glycol 6,000 by the method of Yamamoto et al. (1970). CK-1 was further purified by dissolving 13*472 g of GsGl in 20 ml of 0,02 Tris buffer (pH 7.2) supplemented with 6 mM MgCl^'and 0.01% gelatin (phage buffer) and centrifuging at 120,000 X g in an AH-65 rotor in a Beckman L3-50 ultracentrifuge at 5 C for 42 h. The resulting phage band was collected and the GsGl removed by dialysis against 3 changes 29 of phage "buffer. The phage suspension was then filtered through a 0.22 pm Millipore filter and stored at 4 C until used. Determination of Bacteriophage Density The density of CK-1 was determined by isopycnic ultracentri fugation in CsCl gradients in a Beckman L3-50 ultracentrifuge at 120,000 X g for 42 h at 5 C* The refractive index of the phage band was determined using a Bausch and Lomb ABBE-31 refractometer, and the , 25, density calculated using the equation /?=a(YyD )-b, where is the density of the CsCl-phage suspension (in g/crrP), a and b are constants, andy^5 is the refractive index of the CsCl at 25 C. Host Range of CK-1 Bacteriophage CK-1 was plated with B. megaterium ATCC 19213» B. megaterium 0SU 223. B» megaterium NRRL-B-3694, B. megaterium NRRL-B-3695, B. megaterium BLC-1, B. megaterium BLC-2, B. megaterium KM, B. megaterium QM, B. megaterium V, Bacillus M, B. subtilis Marburg, B. globigii OSU 55&, B. cereus 0SU 123> B. brevis 0SU 197, Pseudomonas fluorescens OSU 197, and Micrococcus luteus OSU 459 on nutrient agar supplemented with 12 mM MgCl^^H^O using the agar overlay method of Adams (l959 a). Spores were used as the source of inoculum of the host organisms for all Bacillus species. A strain was regarded as sensative if plaques were apparent after 16 h of incubation at 30 0 . Cation Requirement of CK-1 The cation requirement of CK-1 was determined by plating the phage on media composed of nutrient broth solidified with 0 .8% agarose which contained 0 mM, 0.3125 mM, 0.625 mM, 1.25 mM, 2.5 mM, 5*0 mM, 30 and 10 mM of CaCl *6K_0, MnSO./H-O, MgCl *6Ho0, and NaGl by the agar £ nr £ £ £ overlay method of Adams (1959 a). Following incubation at 30 C for 16 h, the plaques were counted. pH Stability of CK-1 The pH of solutions of 0.8$ peptone in screw cap tubes were adjusted from ^.0-10.0 with HC1 or NaOH, and the volumes adjusted to 9.0 ml. One milliliter of the bacteriophage (6.7 x 10^ PFU/ml) was added to each peptone tube, and the suspension incubated at 37 0 for 1 h. Following incubation, the suspensions were diluted 1:100 in cold 0.8$ peptone (pH 7.0) and plated immediately on the PA agar of Thorne (1962) by the agar overlay method of Adams (1959 a). The plates were incubated at 30 C for 16 h and the resulting plaques counted. Thermal Stability of CK-1 Suspensions of CK-1 in 0.8$ peptone (pH 7*0) were placed in water baths at 60 C and 70 G. Samples from the phage suspensions were diluted in cold 0.8$ peptone and titered'on the PA agar of Thorne (1962) after 0 min, 5 min, and ^5 min of incubation. Plaques were counted following 16 h of incubation at 30 C. Replication of CK-1 on B. megaterium of Varying Capsule Size Bacillus megaterium ATCC 19213 was cultured on 10 ml of FMS, nutrient broth supplemented with FMS trace salts, nutrient broth supplemented with 0.2$ sucrose and trace salts from PA medium, nutrient broth supplemented with trace salts and phosphate components from FMS, and nutrient broth supplemented with PA trace salts at 30 G with shaking at 200 rpm. Bacillus megaterium ELC-1 was cultured on nutrient Broth supplemented with M S trace salts. Each flask was inoculated with approximately 8 x 10^ heat-shocked spores. Bacteriophage was added either at the time of inoculation of the spores, at the time of germina tion of the spores, or at early log phase, at a multiplicity of infection of approximately 7*5 PFU/CFU. Samples were removed and diluted in 0 .8^ peptone at the time of addition of the phage and at 7 h post infection and titered on the PA agar of Thorne (1962) by the overlay method of Adams (1959 a )■ Plaques were counted after 16 h of incubation at 30 C. Adsorption of Bacterionhage on Cells with Varying Size Capsules Measurement of the adsorption rate of CK-1 to B. megaterium ATCC 19213 cultured on liquid PA medium, and liquid PA medium supple mented with 0.2?$ sucrose, and to B. megaterium BLC-1 cultured on liquid PA medium was carried out by a modification of the method of Adams (1959 b) as follows. All cells were pelleted by centrifugation at 6,000 X £ for 10 min at 0 C and washed twice with DDW. The final pellet was resuspended in 10 ml of liquid PA medium and equilibrated at 30 C. A cell sample was removed and titered on nutrient agar. Bacteriophage was then added at a multiplicity of infection of approxi mately 0.01 PFU/CFU, mixed well, and an initial sample immediately removed and diluted Is100 in cold 0.8^ peptone. Adsorption mixtures were incubated at 3 C with shaking at 200 rpm. Periodically, samples were removed, diluted Is100 at 4 C peptone, and centrifuged at 6,000 x £ for 10 min to remove bacteria and adsorbed phage. Chloroform could not be used, since CK-1 was sensative to chloroform. Free phage was diluted in cold 0.8^ peptone and titered on the PA agar of Thorne (1969) by the agar overlay method of Adams (1959 a-). Adsorption rate constants were calculated as suggested by Adams (1959 b). Phase Contrast Microscopy Wet mounts and'india ink wet mounts were routinely observed in a Nikon phase contrast microscope. Photographs were taken on a Carl Zeiss Photomicroscope equipped with phase contrast optics using Panatomic X film (Kodak). Negative Staining of Bacteriophage A formvar-carbon coated standard 3 mm 200 mesh copper grid was floated on a drop of bacteriophage suspension (approximately 10^ PFU/ml) for 5 min, then on a drop of 1$ uranyl acetate for 1 min. Excess stain was removed by capillary action with bibulous paper, and the grids dried in a dust-free atmosphere. Fixation. Embedding, and Thin Sectioning Bacillus megaterium ATCC 19213» B, megaterium BLC-1, and B. megaterium BLC-2 cultured on M S medium were fixed by the method of Kellenberger et al. (1958)» dehydrated in a graded alcohol series and propylene oxide, and embedded in Epon 812 complete monomer. Thin sections were cut from trimmed blocks on a Porter-Blum MT-1 ultramicro tome using glass knives broken on an 3KB glass knife maker. Sections were collected on uncoated J00 mesh copper grids and stained for 5 rain with 1% uranyl acetate and for 20 min with Reynold's lead citrate. 33 Critical Point'Drying Bacillus megaterium ATCC 19213, B. megaterium BLC-1, and B. megaterium BLC-2. were critical point dried by a modification of the method of Cagle (1975)* A formvar-carbon coated 200 mesh copper grid was floated on a drop of heavy cell suspension for 5 min, then on a drop of 3% (v/v) glutaraldehyde for 5 min. Grids were then transferred to a BRC grid holder (Biodynamics Research Corporation), and the cells dehydrated by passage through a graded alcohol series. The grids were then transferred to amyl acetate for 5 min before being critical point dried in a Samdri PVT-3 critical point drying apparatus. Specimens were rotary coated with 100 A of carbon and stored over phosphorus pentoxide until observed. Electron Microscopy Specimens were observed and photographed on a Carl Zeiss EM-9S electron microscope with an accelerating voltage of 50 Kv. Effect of Drying on B. megaterium with Varying Capsule Size The effect of capsule on the survival of B. megaterium when dried was accessed as follows. One gram of sterile garden soil, as a matrix for desiccation, was placed in a small screw capped tube for 1 h. Mid-log B. megaterium ATCC 19213 and B. megaterium BLC-1 cultured on FMS medium were r e m o v e d from their medium by centrifugation at 6,000 X £ for lu min and washed twice with DDW. The final pellet was resuspended in a small amount of DDW, and 0.1 ml added to the dried soil. The soil was quickly redried by passing filtered air through the sample. The samples were stored over silica gel. Periodically, a tube was removed from the dessicator, 9 ml of 0 .8^ peptone added, and the soil suspension rapidly mixed on a vortex mixer for JO sec. Particulate matter was permitted to settle for 20 min, and the bacteria in the suspension 1 cm below the surface titered on nutrient agar. In order to determine the number of spores present, the soil suspension was heated at 80 G for JO min and again titered. The number of spores was subtracted from the total cell count to yield the number of vegetative cells. RESULTS Characterization of B. megaterium BLC-1 and BLC-2 The colonial morphology of B. megaterium ATCC 19213 (Parent strain), BLC-1, and BLC-2 is shown in Plate X. It was noted that the capsulated strain was much more mucoid than either of the small capsule mutants. The cellular morphology of the parent strain and small capsule mutants are shown in Plates31,115, and ly. Small capsules were seen surrounding BLC-1 and BLC-2, as compared to the parent strain, when viewed in wet mounts prepared in India ink (Plate n) . Electron photomicrographs of thin sections of the mutants BLC-1 and BLC-2 revealed that there was a much smaller region of fibrillar matrix surrounding the cells, even though the same intracellular structures were present (Plate 35). Electron photomicrographs of critical point dried preparations substantiated these previous observations. Again very little fibrillar matrix was noted surrounding the mutant cells as compared to normally capsulated parent strain (Plate Xv), The results of physiological and antibiotic sensitity tests of the parent strain, BLC-1, and BLC-2 are shown in Table 1. It was noted that the mutant strains were identical to the normally capsulated parent strain, indicating that the mutants were indeed B. megaterium, and not contaminants that were picked up during the isolation procedure. 35 .36 Plate I. Macroscopic morphology of (A) IL_ megaterium ATCC 19213» (B) B^_ megaterium BLC-1, and (c) B^ megaterium BLC-2 cultured on nutrient agar supplemented with 1% sucrose. 37 Plate I . 38 Plate II. Phase contrast photomicrographs of (A) megaterium ATCC 19213» (B) megaterium BLC-1, and (G) megaterium BLC-2 in india ink wet mounts. The relative sizes of the capsules are noted for each cell type. Marker "bar represents 5 urn. 39 Plate II. *K> Plate III. Electron photomicrographs of thin sections of (A) E. megaterium ATGG 19213i (®) IL. megaterium BLC-1, and (G) _B;_ megaterium BLG-2. The fibrillar matrix(F) or lack of fibrillar matrix, surrounding each cell type is noted. Marker bar represents 0.5 urn. Plate III. kz Plate IV. Electron photomicrographs of (A) megaterium ATCC 19213» (B) megaterium BLC-1, and (C) 33j_ megaterium BLG-2 which have been critical point dried. The fibrillar matrix(F), or lack of fibrillar matrix, surrounding the cells is noted for each strain. Marker bar represents 0.5 urn. 43 t <■/ \,J r* • * * *t* ^ ft-Li -f / .’ > r tkj‘jkM&: ^Vjrl Plate IV. 44 Table 1 Physiological characteristics and antibiotic of B. megaterium ATCG 19213, B. megaterium BLC-1, and B. megaterium BLG-2. Antibiotic sensitivity was determined by placing a disc containing the antibiotic on nutrient agar seeded with the B. megaterium strain. Physiological test B. megaterium strain or antibiotic Parent BLC-1 I BLG-2 Glucose Aa A A Lactose AA A Maltose A AA Mannitol A AA Sucrose A A A Indole - - - Methyl red --- Voges-Proskauer - - - SIM No H2S No H2S No H2S Bacitracin (2 units) . S* S S Cloromycetin (5 meg) s s S Erythromycin (2 meg) s s S Kanomycin (5 meg) s s S Neomycin (5 meg) s s S Novobiocin (5 meg) s s S Penicillin G (2 units) s s S Tetracycline (5 meg) s s S a - A means acid was produced. b - S means the organism was sensative to the antibiotic. Figure 1 represents a typical growth curve for the parent strain of B. megaterium and the small capsule mutants (BLC-1 and BLC-2) cultured on FMS medium. The rate of growth was very similar for each cell type through the logarithmic phase of the parent strain. Logarithmic growth of both small capsule mutants was extended for a short time,* however, and these cultures reached slightly higher optical densities. Throughout sporulation, the O.D. of the mutant strains did not decrease as much as the parent strain. Phase contrast microscopic observations of wet mounts revealed that a slightly smaller percentage of the small capsule mutants sporulated as compared to the parent strain. The spores of the small capsule mutants were also somewhat more spherical than spores from the normally capsulated parent. Characterization of B. megaterium polysaccharide (BMP) The purified isotonic lysozyme preparation extracts and phenol extracts previously described by Cassity (1976) were found to contain glucose, diaminopimelic acid, alanine, and N-acetyl-glucosamine. Column chromatography of these extracts through Bio-Gel A 1.5m revealed that the polysaccharide portion of these extracts could be isolated free of peptide (Figure 2). The polysaccharide portion eluted as a single peak at the void volume, indicating a mol. wt. of 1.5 x 10^ or greater. The peptide portion was somewhat smaller (mol. wt. approximately 3 x 1 0 - 9 x 10 J and was much less uniform than the polysaccharide portion. k6 Figure 1. Growth curve of B. megaterium ATGG 19213, B. megaterium BLC-1, and BLG-2 cultured on FMS medium. Symbols: (O) B. megaterium ATCG 19213, (Hi) B. megaterium BLC-1, and (^) B. megaterium BLG-2. O D. (Klett units) Figure 1. Figure ie (hours) Time 12 48 Figure 2 . Column gel chromatography of acid purified BMP through Bio-Gel A 1.5m. Symbols: (O) carbohydrate, (O) peptide. iue 2. Figure Carbohydrate(ug/ml) 90i 30- 60- 0 1 2 30 20 10 0 cP'°cr a rcin Number Fraction -30 -60 -90 -120 Peptide(jjg/ml) 50 The pep.tide fractions were pooled, concentrated, hydrolyzed, and the amino acid components analyzed. The results of the amino acid analysis • o f the peptide are shown in Figure 3* Four major amino acid components were identified, those being alanine (ala), glutamic acid (glu), diaminopimelic acid (dap), and isoleucine ( ile). Integration of the peaks indicated that the relative ratio of alas glu: dap: ile was 1 .8 :1 :1 .2 :.5 , respectively. The polysaccharide-containing fractions of the extracts were pooled, concentrated, and called "column purified SIP". Since most gram positive microorganisms contain teichoic acids, which may contain glucose or amino acids, the BMP was analyzed for the presence of phosphate. Phosphate determinations showed the presence of only O.fi^ of organically bound phosphate in the SIP. The SIP isolated in this manner was previously assumed to be capsular material on the basis of its composition and method of isolation (Cassity, 1976). In order to determine if the material was indeed of capsular origin, B. megaterium ATCC 19213 and B. megaterium BLC-1 were analyzed for glucose content throughout growth and sporulation. The results are shown in Table 2. There was a consistently higher concentration of polymeric glucose in BLC-1 cells than the parent strain. This indicated that, if the glucose content of the cells reflected the concentration of BMP in the cells, the BMP was probably not a capsular polymer. In order to further determine if the EMP was of capsular origin or cellular origin, both parent strain and BLC-1 were extracted with hot phenol. A concentration of BMP representing 2.13^5 of the dry wt. of the cells was extracted from the normally capsulated parent strain, while a concentration of HIP representing 51 Figure 3* Amino acid analysis of the peptide-containing fractions of acid purified BMP. The peaks were identified as follows: 1 , alanine; 2 , glutamic acid; 3 , diaminopimelic acid; 4, isoleucinej and 5 » ammonia. Figure 3 . Absorbance 04- 0.8 t Retention time (min) time Retention 40 80 0 2 1 53 Table 2 Glucose content of B. megaterium ATCG 19213 (parent strain) and B. megateritun BLC-1 through growth and sporulation. Time of Glucose Content (ug/mg ce].1 dry wt.) Incubation (hours) B. megaterium ATCC 19213 B. megaterium BLC-1 3.0 NDa 37.15 3-5 13.05 ND 6.0 17.42 28.29 8.0 19.04 ND 9.0 ND 22.41 10.0 20.37 ND 12.0 15.82 21.80 14.0 18.25 ND 15.0 ND 22.82 16.0 19.64 ND 24.5 ND 11.04 26.5 8.33 ND a - ND signified that the glucose content was not determined for that sample. 2.35^ of the cell dry wt. was extracted from BLC-1. This indicated that the BMP most likely was not a capsular polymer, but rather was intracellular or associated with the cell surface in some way. The monosaccharide components of hydrolyzed BMP were identified, and the relative quantities of each determined by gas-liquid chromato graphy of the trimethylsilyl (TMS) ethers of the particular components. The results are shown in Figure 4. Four monosaccharides were evident, those being glucose, galactose, xylose, and arabinose in the ratio of 7.7:1.1:1:0.61, respectively. The BMP isolated from both parent and mutant strains was identical. Table 3 shows the retention times of SIP components and each of the standards used. In order to check these results, standards were injected with the sample. If no new peaks were present, but rather the peak tentatively identified as the standard became much larger, the component was assumed to be identical to the standard. This was done with each component, and again glucose, galactose, xylose, and arabinose were identified. Integration of all peaks showed that 97.3$ of the area under the peaks was within the glucose, galactose, xylose, and arabinose peaks. Glucose anomers accounted for 71* galactose anomers xylose anomers 10.5^, and arabinose anomers accounted for 5>7f° of the BMP. A small amount of ribose was found (0 .^), and the remaining peaks were found to be from the silanizing reagents. Since the BMP was found to be composed of four monosaccharides, the question arose as to whether there was one heteropolysaccharide with four different monosaccharides, or four separate homopolysaccharides. Since column chromatography showed only one polymer, the BMP was electrophoresed through polyacrylamide. A representative scan of the 55 Figure 4. Gas-liquid chromatography analysis of the trimethylsilyl ethers of the monosaccharide components of parent strain (A) and BLC-1 (b ) column purified BMP. The peaks were identified as follows: 1. D-ribose 2. D-arabinose 3. D-xylose 4. IMS reagent 5.' TMS reagent 6. TMS reagent 7. D-glucose 8. D-galactose 9- D-glucose slAM. = 4 ------1 1 5 30 45 52.5 Retention time (min) Figure 4. 57 Table 3 Retention times of BMP components and standards. Sample Retention times (min) BMP component 1 8.2 BMP component 2 9.4 BMP component 3 10.9 BMP component 4 16.4 BMP component 5 19.9 BMP component 6 23.3 BMP component 7 26.3 BMP component 8 28.5 BMP component 9 43.5 N -ac etyl-D-gluco samine 22.9 29.6 D-ribose 8.4 9.0 Glycogen 26.3 43'. 9 D-galactose 28.6 TMS reagents 23.3 16.4 19.9 Arabinose 7.1 9.2 Xylose 7 A ll.l gels is shown in Figure 5* Only one large peak was noted, which migrated only 2 mm from the top of the gel. Some minor peaks were seen "between 22 mm and 51 i™- from the top of the gels; however, these smaller peaks were not reproducable from gel to gel, and they were later conclusively attributed to flaws or nicks in the gels. This data implied, along with column chromatographic data, that the BMP was most likely one large heteropolysaccharide. Even though the BMP was found to be composed of several sugars, the major component was glucose. This necessitated the presence of glucose-glucose linkages. In order to determine the linkages present, microorganisms which produce hydrolases for specific glucose-glucose linkages were employed. Table 4 shows the microorganisms used, linkages they hydrolyze, and the extent to which they degraded BMP. It was noted that essentially none of the BMP was hydrolyzed and used by any of the microorganisms. This would eliminate the presence of large amounts of oC(l->^), o<(l— » 6)f oC(i— > 3), e*(l— >4) in an alternating <*(l— >3) £*(1- ^ ) pattern,^ (1— > 2 ), ^ ( l — >3 ), and 0 ( 1 — > 6) glucose-glucose linkages. All microorganisms grew when BMP was replaced with glucose, even though Sporotrichum pruinosum ATGG 2^782 grew very slowly and only to low cell densities. Since only negative results were obtained for the degradation of BMP by microorganisms with specific known glucan hydrolases, an attempt was made to isolate a microorganism which would hydrolyze the BMP, and then determine which linkages this microorganism would hydrolyze. Attempts to isolate BMP hydrolyzers from 105 samples of soil or decaying organic matter was made. Of these, only the microorganisms in 6 samples displayed ability to utilize BMP for growth. As such cultures were 59 Figure 5 . Spectrophotometeic scan of BMP electrophoresed through 5% polyacrylamide. Figure5- Absorbance Distance (cm) Distance 6 8 10 0 6 61 Table 4 Hydrolysis of IMP by microorganisms which produce specific glucan hydrolases. Microorganism* Linkages hydrolyzed % fflP used Aspergillus fumigatus OSU 156 o(.(l— *>2) 0.0 Aspergillus niger o(,( 1— 5.3) 0.0 o<.( 1-^-4) (l~^4) Bacillus subtilis Marburg c*(l-^6) 0.0 Penicillium brefeldianum ATCC j 9 d - ^ 6) 0.05 10417 Sporotrichum pruinosum ATCC / ( 1 - ^ 3 ) 0.0 24-728 Trichoderma viride ATGG 13631 /3(l— > 3) £*(l— >4) in alternating o(,(i~-^3) * Each microorganism either hydrolyzed control polysaccharides with the specific linkage indicated, or was reported in the literature to hydrolyze polysaccharides containing the linkage indicated. transferred Into new BMP medium, in order to enrich for the micro organism responsible for BMP degradation, the cultures became less efficient. Eventually a microorganism, identified as an Arthrobacter species, was isolated on BMPMS medium solidified with agarose. The utilization of substrates with known linkages and BMP by the Arthrobacter species and mixed soil organism culture from which the Arthrobacter species was isolated is summarized in Table 5* The Arthrobacter species reduced the HIP concentration only ^.0% in IMP medium after 21 days of incubation. However, while in the culture with other soil microorganisms, 63.6^ of the BMP was removed from the culture after 6 days of incubation. When sterile soil extract was added to the BMPMS Arthrobacter culture, the BMP concentration did not decrease further. Since mixed soil cultures utilized polysaccharides with many different types of linkages, as well as HP, no definite conclusions as to what linkages the BMP contained could be drawn. However, from the data from'both BMP utilization experiments, it appeared that several different glucose-glucose linkages were present, as well as bonding of glucose to other constituents of the BMP. In an attempt to discern if BMP contained oflinked terminal glucose residues, and possibly discern the degree of branching, the BMP was tested for its ability to bind Goncanavalin A (ConA). The results are shown in Table 6 . These results showed that BMP bound only a small amount of Con A, indicating that there were few terminally oC-linked glucose residues. Starch and glycogen, with ©<(l— >4), and c<(l— >*!■) and c<(l— > 6 ) linkages, respectively, form colored complexes with iodine solutions which give characteristic absorption curves. In order to 63 Table 5 Utilization of BMP and purified polysaccharides Uy an isolated Arthrobacter species and a mixed culture from soil. % utilization % utilization by Arthrobacter by soil micro spp. (21 days organisms (6 Polysaccharide Linkage of incubation) days of incu bation) BMP 4-.1 63.6 Cellobiose ^(l-»^) 9.7 92.0 Glycogen oc (l— >40, 2.2 95-0 ©<(i-*>6 ) Starch c<(l- » * 0 1.7 97-^ Lichenan ft (l—->3) 0.0 37.0 Gentiobiose 04.(1— ^-6) 3.8 92.0 Trehalose C<(1~ > 2 ) 0.9 90.0 Table 6 Binding of Concanavalin A by BMP, starch, and glycogen. Polysaccharide % Concanavalin A bound DDW 0.0 BMP 2.5 Starch 4-1.7 Glycogen 75.3 determine if BMP was similar to either starch or glycogen, it was mixed with an iodine solution and an absorption spectrum in the visible region obtained. The results are shown in Figure 6. The BMP did not form a colored complex with the iodine solution, even though the BMP is a high mol wt. polymer, indicating that the polymer is not similar to starch or glycogen. In order to determine if the BMP functions as a carbon and energy storage compound, the concentration of BMP in cells and culture supernates was determined throughout growth and sporulation of B. megaterium (Parent strain). The results are shown in Figure 7* It was noted that the concentration of BMP in the cells increased sharply, as did the cell mass, throughout exponential growth. During forespore formation, the cell mass decreased somewhat, as did the cellular concentration of BMP. As phase bright spores and free spores appeared, the concentration of BMP had stabilized. The BMP was gradually released from the cells into the culture medium as forespores began to develop, and then sharply as the cells lysed liberating free spores. Even though BMP was released into the medium, the total concentration of BMP did decrease sharply throughout forespore formation. This strongly suggests that the BMP was used as a carbon and energy storage compound for the synthesis of a spore. Since BMP appeared to function as a carbon and energy source during forespore formation in FMS cultures, the use of BMP by cells placed under conditions of endotrophic sporulation was examined. The total concentration of BMP was determined following removal of the cells from the FMS culture, and the results are shown in Figure 8. The concentration of BMP decreased only slightly in the culture in which 66 Figure 6. Absorption Spectra of BMP, starch, and glycogen complexed with iodine solution. Symbols: ( O ) B M P * (0) starch, and ( A ) glycogen. -90.2 o o (p 460 500 540 580 600 Wavelength (nm) -oc r\ 68 Figure ?• Concentration of BMP in cells, culture supernates, and "both, during growth and sporulation of B. megaterium. Symbols: ( O ) Total BMP, ( g | ) Cellulai* BMP, and ( ® ) BMP in culture supernates. b 20 12 18 24 Time(hours) Figure 7- 70 Figure 8. Concentration of total BMP of cells in which cells had been placed under conditions of endotrophic sporulation. Cells were removed from FMS.cultures after 3 h (O). 6 h (□), and 8 h (A) of incubation. Carbohydrate (jjg/ml) _a no rv> 4^ the cells had been removed from the IMS medium after 3 h of incubation. Observations of wet mount preparations of the cells in the phase contrast microscope revealed that for all three cultures there was little spor ulation under endotrophic conditions. The 3 h culture had a number of forespores after 9.75 h of incubation; however, only a very small percentage when compared with the number of vegetative cells. The 6 and 8 h cultures formed fewer spores under these conditions than did the 3 h culture, even though there were more vegetative cells present. This data, even though not totally conclusive, also suggests that the BMP may be used during spore formation or incorporated into the spore. In order to further determine if the BMP was utilized by B. megaterium during spore formation, the ability of the cells to hydrolyze IMP to reducing sugars was examined. The activity of cell-free extracts and sedimentable cell components prepared from cells collected periodically during growth on FMS was examined, and the results are shown in Table ?• There was not a release of a large quantity of reducing sugars from BMP by either cell-free extracts or sedimentable cell components. The quantity of reducing sugars liberated from sedimentable cell components was much greater than that released by cell-free extracts. Essentially no reducing sugars were liberated from cell-free extracts, while only 1.2-4.0 jig mg of BMP were liberated by sedimentable cell components. Extraction of Glutamyl Polypeptide It was originally thought that the IMP was capsular material; however, when BMP was shown not to be of capsular origin, a search for the true capsular material was implemented. Polyglutamate was isolated Table 7 Release of reducing sugars from M P by cell-free extracts and sedimentable cell components of B. megaterium ATGG 19213 throughout growth and forespore formation. Time of ug/ml reducing ug/ml reducing ug/ml reducing ug/ml reducing Incubation sugars in CFE^a sugars in CPE sugars in SCC“ sugars in SCC (hours) without BMP with BMP without BMP with BMP 5 8.2 8.2 0.0 2.4 7 16.1 0.0 2.4 5.^ 11 8.7 4.0 4.0 7.7 15 11.6 11.1 8.2 16.2 a - CPE represents cell-free extracts, b - SGG represents sedimentable cell components. from the parent strain as shown "by thin layer chromatography of acid hydrolysates of this polymer (Fig. 9)* The polyglutamate was not pure since several spots were evident; however, the glutamic acid spot accounted for most of the amino acids present. Only 4 4 . of the con centration of polyglutamate was extracted from BLC-1 as compared to the normally capsulated parent strain. This polymer was not soluble at pHs required for column chromatography and precipitated in plastic tubing; however, polyglutamate solubilized in dilute HC1 was electro- phoresed through polyacrylamide. A scan of a typical polyacrylamide gel is shown in Figure 10. The material gave one large broad peak from 4 cm from the top to the bottom of the gel (10.4 cm). This indicated that the polyglutamate was quite polydisperse. Accumulation of Metallic ions by Normally Capsulated B. megaterium and Small Capsule Mutants In order to elucidate the possibility that capsule of B. megaterium may accumulate certain metallic ions, the culture medium in which normally capsulated B. megaterium and small capsule mutants were cultured was analyzed by atomic absorption spectrophotometery throughout logarithmic growth. Figure 11 shows the concentrations of sodium (Na) removed from the medium by the parent strain and BLC-2. During lag phase and early log phase, BLC-2 removed Na slightly better than did the parent strain. However, after 4 h of incubation, there was not any reproducable statistical differences between the two cell types, and such analyses showed quite large variations in the concen tration of Na removed from one sample time to the next. 75 Figure 9* Thin layer chromatography of hydrolyzed polyglutamate on microcrystalline cellulose. Symbolss A L-glutamic acid B Hydrolyzed polyglutamate extract from B. megaterium. C hydrolyzed L-glutamic acid 76 ABC Figure 9* Figure 10. Scan of a polyglutamate extract (Cu salt) from B. megaterium electrophoresed through a polyacrylamide. 0 Absorbance 0 Figure10. , — i * 0 2 . 6 Top - 0 - Distancefcm) bottom 78 79 Figure 11. Removal of sodium from M S by normally capsulated B. metaterium (parent strain) and the small capsule mutant BI£-2. Symbols: ( O ) Parent strain (A) BLC-2 LOS jug jug Na removed/ml FMS Time (hours) 81 The concentration of potassium (K) removed by the parent strain of B. megaterium was consistently greater than the concentration of K removed by BLC-2 throughout both lag and log phases (Fig. 12). As was the case with Na, the K concentration of the medium varied greatly from one sample time to the next. The concentrations of magnesium (Mg) removed by the parent strain, BLC-1, and BLC-2 are shown in Figure 13• The concentrations of Mg removed by both small capsule mutants was consistently lower than that removed by the parent strain throughout both the lag and log phases. The removal of iron (Fe) from FMS media by normally capsulated B. megaterium and small capsule mutants is shown in Figure 1^. The parent strain removed an increasing concentration of Fe from the medium as logarithmic growth progressed. The mutant BLC-1 removed Fe from the medium equally as well as the parent strain. However, BLC-2 removed only small amounts of Fe throughout logarithmic growth. There was a small difference in the concentrations of zinc (Zn) removed from the medium by the parent strain as compared to BLC-1 and BLC-2, as is seen in Figure 15. The parent strain removed a relatively large concentration of Zn during lag phase, and very little during log phase. Both mutants behaved as the parent strain, except that a marked egression of Zn from the small capsule mutants into the medium was noted at mid-log phase. The results of the concentrations of calcium (Ca) removed from EMS medium by the parent strain, BLC-1, and BLC-2 are shown in Figure 16. The parent strain removed Ca from the medium throughout lag phase and to three-quarter log phase, following which there was an egression back into the medium. This also occurred with BLC-2, except that the egression 82 Figure 12. Removal of potassium from M S medium by normally capsulated B. megaterium (parent strain) and the small capsule mutant BLC-2. Symbols: (O) Parent strain ( A ) BLC-2 CD VjJ fo oi jjg K jjg K removed/ml FMS o o» O )- 0 Time (hours) H* n CD TO 8^ Figure 1 3 . Removal of magnesium from FMS medium "by normally capsulated B. megaterium (parent strain) and the small capsule mutants, BLC-1 and BLC-2. Symbols: (O) Parent strain (0) BLC-1 ( A) BLC-2 85 1 .2-1 -0.6H 3 6 Time (hours) Figure 13* 86 Figure l*k Removal of iron from M S medium by normally capsulated B. megaterium (parent strain) and the small capsule mutants BLC-1 and BLC-2. Symbols: ( O ) Parent strain (□) BLC-1 (A) BLC-2 o ° fu o o (Ji di jjgFe removed/ml FMS Figure lA. 88 Figure 15. Removal of zinc from M S medium by normally capsulated B, megaterium (parent strain) and the small capsulated mutants, BLC-1 and BLG-2. Symbols; ( O ) Parent strain (Q) BLC-1 (A) BLG-2 jug Zn Zn removed/mljug FMS i i i o k) ^ o *-* ro (ji O O O O O W Figure 15* 90 Figure 16. Removal of calcium from FT4S medium by normally capsulated B. megaterium (parent strain) and the small capsule mutants BLC-1 and BLC-2. Symbols! (O) Parent strain (0) BLC-1 ( A ) blc-2 4 VO jjg Ca removed/ml FMS CD-p Figure 16. 92 began at mid-log phase rather than at three-quarter log phase. BLC-1, following an initial period of egression during lag phase, removed only a small amount of Ca from IMS between the beginning of log phase and three-quarter log phase, at which time another egression from the cells occurred. The concentrations of manganese (Mn) removed from IMS medium by the parent strain was consistently greater than those removed by either of the small capsule mutants (Fig. 17). The removal of the Mn by the parent strain was rapid until slightly past mid-log phase, at which time an egression of Mn from the cells was noted. The small capsule mutants behaved similar to the parent strain, except they removed much less Mn, and the egression from BLC-1 began 1 h earlier than the parent strain. Analysis of the culture medium in which the parent strain and small capsule mutants strains were cultured indicated that the capsule of B. megaterium accumulated K, Mg, and Mn. In order to substantiate these results, the metallic content of cells of the parent strain, BLC-1, and BLC-2 were determined. These results are summarized in Table 8 . Calcium, Magnesium, and Manganese were accumulated in much lower concentrations by BLC-1 and BLC-2, as compared to the parent strain. Cell analyses showed no consistent differences in the concentrations of Na, K, Fe, or Zn between normally capsulated cells and small cap sule mutants. The inhibition of respiration by toxic metals provided a sensitive method for determining if these metallic ions reached the cytoplasmic membrane, or were retained by the capsule. The rate of 0^ consumption by the normally capsulated parent strain and small capsule Figure 17. Removal of manganese from M S medium by normally capsulated B. megaterium (parent strain) and the small capsule mutants, BLC-1 and BLC-2. Symbols: (O) Parent strain (Q) BLC-1 (A) BLC-2 i i jjg Mn removed/ml FMS "(Ji "(Ji O (ji o O O O ^ Figure 17* Table 8 Percentage of metallic ions accumulated by small capsule mutants (BLC-1 and BLG-2) relative to the normally capsulated parent strain of B. megaterium, which was taken as 100%. Time of Element incubation Na K Mg Fe Zn Ca Mn (hours) BLG-2 BLC-2 BLC-1 BLG-2 BLC-1 BLG-2 BLC-1 BLG-2 BLC-1 BLG-2 BLC-1 BLC-2 3 138.6 70.4 58.1 21.5 120.0 100.0 142.4 76.5 100.0 83.O 59.6 21.5 6 62.0 114.9 57.8 58.6 114.3 97.5 78.4 I63.8 96.4 37-0 32.9 67.9 9 83.2 69.5 83.7 74.1 92.3 80.8 93.3 92.4 72.5 58.1 22.7 83.3 vo Kjx mutants in the presence of small amounts of copper (Cu), mercury (Hg), and silver (Ag) was determined. Typical curves of the rate of 0^ consumption versus time which were generated from such experiments are shown in Figures 18, 19, and 20. After the addition of the toxic metal the cells did not respire at a constant rate. After addition of each of the toxic metals, it was noted that the small capsule mutants were inhibited much more rapidly than the normally capsulated cells. The time required to totally inhibit respiration was determined and the coefficient of inhibition calculated for each cell type and heavy metal, and the results are shown in Table 9. In each case the coeffi cient of inhibition was smaller for the mutant cells, signifying that the mutant cells were inhibited more rapidly than the parent strain, Characterization of B. megaterium Bacteriophage CK-1 A B. megaterium bacteriophage was isolated from the soil. The isolate described here was named CK-1. An electron photomicrograph of a negatively stained preparation of CK-1 is seen in Plate The phages had icosahedral heads averaging 52 nm in diameter and long tails averaging 280 nm in length. The density of CK-1 was 1.49-1.50 g/cm^ in CsCl gradients. The host range of CK-1 is shown in Table 10- Plaques were produced on all strains of B. megaterium tested; however, no plaques were produced on any other Bacillus' species. The influence of pH on the stability of CK-1 at 37 C was • studied, and the results are shown in Table 11. The phage was most stable at pHs between 8-9. Titers decreased rapidly below a pH of 5.2 and above a pH of 9*0. Figure 18. Inhibition of respiration of normally capsulated B. megaterium (parent strain) and the small capsule mutant, BLG-2, by 19.6 n mole of silver. Figure18. o O 0 8 Saturation(OJ - 0 6 i 0 4 ie Cmin) Time (D BLC-2 Parent 99 Figure 19. Inhibition of respiration of normally capsulated B. megaterium (parent strain) and the small capsule mutant, BLG-2, hy 115 m mole of copper. Figure19* - 0 8 % Saturation(Cu) - 0 6 i O 0 Time (min) Time C 9-0 0 2 l ) BLC-2 Parent 4 6 101 Figure 20. Inhibition of respiration of normally capsulated B. megaterium. (parent strain) and the small capsule mutant, BLG-2, by 3*07 n mole of mercury. 102 Parent (Q Q. 40- Q. Q. 60- 8 0 2 6 8 Time (min) Figure 20. Table 9 Inhibition of respiration of normally capsulated B. megaterium (parent strain) and the small capsule mutants, BLC-1 and BLC-2, by silver, copper, and mercury. Coefficient of inhibition (min2 Sat."1) Metallic Concentration ion (n moles) Parent BLC-1 BLC-2 „ 2+ Hg M i 0.505 NDa u 2+ Hg 3.0? 0.435 ND 0.348 Hgu 2 + 3.68 o.?54 ND 0.218 u 2 + Hg 2.30 0.431 ND 0.195 Ag+ 19.6 0.118 0.112 ND Ag+ 39-2 0.216 0.160 ND Cu2+ 115 0.044 ND 0.034 Cu2+ 57.5 0.059 ND 0.036 ^ signifies that the experiment was not performed with that strain at that concentration of metal. H o 104 Plate V. Electron, photomicrograph of a negatively stained preparation of megaterium bacteriophage CK-1. Marker tar represents 0.1 um. 105 Plate V. 1 0 6 Table 10 Host range of B. megaterium bacteriophage CK-1 Microorganism Plaque Formation Bacillus brevis OSU 142 Bacillus cereus OSU 123 Bacillus circulans OSU 38 Bacillus globigii OSU 556 Bacillus M Bacillus megaterium ATCC 19213 + Bacillus megaterium NRRL-B 369^ + Bacillus megaterium NRRL*B 3^95 + Bacillus megaterium BLC-1 + Bacillus megaterium OSU 23 + Bacillus megaterium KM + Bacillus megaterium QM + Bacillus megaterium V + Bacillus subtilis OSU 782 10? Table 11 Influence of pH on the stability of B. megaterium bacteriophage CK-1 pH CK-1 titer (PFU/ml) 3.4 0.0 4.5 3.8 X 103 5.2 1.6 x 10-5 6.3 4.0 x 103 7.2 4.6 x 103 8.0 6.6 x 103 8.8 6.3 x 103 9.7 1.8 x 10-5 V 108 The influence of temperature on the stability of CK-1 was studied, and the results are shown in Table 12. CK-1 was appreciably inactivated at 60 C, with only 9*9?£ and 0.071$ of the original number of phages remaining active after 5 rain and ^5 rain of exposure, respec tively, Inactivation of CK-1 was complete at 70 C. Bacteriophage CK-1 was also 81.9^ inactivated by treatment with chloroform for 30 min at ambient temperature. The requirement of metallic ions for replication of CK-1 was studied, by plating CK-1 with spores on medium containing varying coneen- 2+ 2+ 24* 4* trations of Ca , Mg , Mn , and Na . ' The results are shown in Table 13. CK-1 was found to require divalent cations for replication. * 2*i* 2+ 2+ Medium containing Ca , Mg , on Mn stimulated the production of plaques. Medium without cations, or Na , did not permit the formation 2+ 2+ 2+ of plaques. The optimal concentrations of Ca , Mg , and Mn were found to be 2.5 raM, 5*0 raM, and 2.5 raM, respectively Replication of CK-1 in Different Media In devising a medium and growth system for the optimal production of CK-1, it was noted that when phage was added to the culture prior to, or at the time of, germination of the spores, that the phage would replicate and lyse the cells. However, when CK-1 was added to cultures in the early log phase, no lysis of the cells occurred (Fig. 2l). In attempting to explain this phenomenon, the replication of CK-1 added to cells cultured on various types of media, some of which permitted the formation of large capsule, and some only small capsules, was studied. The results are shown in Table 1^. CK-1 did not replicate in FMS medium, even though CK-1 replicated in nutrient broth supplemented 109 Table 12 Inactivation of B. megaterium "bacteriophage CK-1 by heat Length of Temperature Exposure 60 C 70 C (Min) PFU/ml PFU/ml 0 b.2 x 10^ 6.7 x 10^ 5 ^.1 x 103 0.0 45 3.0 x 101 0.0 110 Table 13 Effect of metallic ions on plaque formation by CK-1 Cation „ 1+ Concentration Ca2+ Mgmt 2+ Mnv 2+ Na (mM) PFU/ml PFU/ml PFU/ml PFU/ml 0 0 0 0 0 0.625 12 112 88 0 1.25 13^ 182 17^ 0 2.5 kz6 600 6l6 0 5-0 6 650 392 0 10.0 0 600 305 0 Table Ik Replication of B. megaterium bacteriophage CK-1 in media which promotes the formation of various sized capsules State of cell Capsule PFU/ml CK-1 PFU/ml CK-1 when inoculated size at at time of at 7 h post Medium with CK-1 early log inoculation infection phase ON MS spore moderate 7.0 X 106 X o 3,1 M S germinating spore small 6.2 X 106 9.0 X 103 MS early log moderate 3.8 X 106- 2.7 X 106 Aa germinating spore small 1.7 X !06 2.0 X 1010 A germinating spore small 2.0 X 106 8.3 X 1010 A early log small 2.9 X 106 5.8 X 109 Bb spore large 1.7 X 106 1.5 X 106 C° spore small 1.7 X 106 1.5 X i o 6 PA spore small 4.8 X i o 6 1.0 X i o 1 1 PA germinating spore small 5.0 X 103 2.6 X i o 10 PA early log small 9.0 X 10^ 1.7 X i o 1 1 111 a - mediumA is nutrient broth with M S trace salts. b - medium B is PA medium supplemented with 0.2$ sucrose. c - medium C is nutrient broth, FMS trace salts, and FMS phosphate buffer, 112 Figure 21. Lysis of normally capsulated B. megaterium by CK-1 added with the spore inoculum (0 h of incubation), at the time of germination of the spores (after 3 h of incubation), and at early log phase (after 5 h of incubation). Symbols: ( O ) megaterium with no CK-1 added. (©) B. megaterium with CK-1 inoculated with the spore inoculum. (@) B. megaterium with CK-1 inoculated at the time of germination of the spores. ( A ) B. megaterium with CK-1 inoculated at early log phase. Figure21. O D (Klett units) 3 ie (hours) Time 6 113 Ilk with JMS trace salts# The buffer could have been responsible for this, however, in that CK-1 did not replicate in nutrient broth supplemented with EMS trace salts and FMS buffer components. CK-1 also did not replicate in PA medium supplemented with 0.2% sucrose. It was noted that large capsules were produced by B. megaterium in this medium. Prom.these data it appeared that the capsule may block attachment of the bacteriophage to the cell, and thus replication. In order to test this theory, the adsorption rate constants for the adsorption of CK-1 to cells with differing amounts of capsule were determined. A summary of the results is shown in Table 15• It was noted that as the capsule size increased, the adsorption of CK-1 to the cells decreased. The relationship between adsorption rate constants and average capsule size is shown in Figure 22. A direct relationship was observed; however, this relationship was neither arithmatic nor logarithmic. Effect of Drying on the Viability of Normally Capsulated B. megaterium and Small Capsule Mutants The effect which drying in soil had upon the viability of normally capsulated B, megaterium and BLC-1, was determined. The results are shown in Figure 23. The rate of loss of viability of both cell types was similar. This indicated that the capsule did not provide protection to the cell when it was dried. 115 Table 15 Adsorption of CK-1 to B. megaterium surrounded by different sized capsules Medium in which Average Adsorption Strain of cells were cul capsule rate con- ^ B, megaterium tured. radius stant (cell (pm) min-1) Parent PA 0.6 1.04 x 10-10 BLC-1 PA 0.1 1.67 x 10~7 Parent PA +0.2$ sucrose 2.0 0.0 Parent M S 1.2 1.9 x 10-11 BLC-1 MS 0.3 3.51 x 10"9 1 1 6 Figure 22. Relationship of CK-1 adsorption rate constants to size of capsule of B, megaterium. Figure22. Adsorption rate constant (cell min~ 1 1 10 0 0 -7 0 Capsule radius (um)radius Capsule 0.9 117 118 Figure 23* Viability of normally capsulated. B. megaterium (Parent strain) and the small capsule mutant BLC-1 dried in soil. Symbols! ( O ) Parent strain (0) BLC-1 10 10 8 6 ro 10 5 10 10A 0 8 16 24 32 Time (days) Figure 23. DISCUSSION Characterization of Small Capsule Mutants The small capsule mutants of B. megaterium ATCC 19213» BLC-1 and BLC-2 gave the same physiological reactions, were sensitive to the same antibiotics, were prototrophic, and grew at the same rate as the parent strain, indicating that the mutants were strains of B. megaterium rather than chance contaminants. The bacteriophage CK-1 also infected both mutants, and it was shown that this bacteriophage only infected strains of B. megaterium. The only differences that could be discerned between the two mutants and the parent strain were that the capsules of the mutants were smaller, their colonies were less mucoid, and that both mutants grew to slightly higher cell densities, with a slightly smaller percentage of the cells sporulating, as compared to the parent strain. The differences in cell densities could have been due to the availability of more substrate for the production of cells, since substrate was not needed for capsule synthesis. Characterization of BMP Column chromatography of acid purified BMP through Bio-Gel A 1 .5m revealed that the carbohydrate components could be separated from the peptide components. Analysis of the amino acid components of the peptide-containing fractions revealed the presence of alanine, glutamic acid, diaminopimelic acid, and isoleucine in the ratio of 1.8:1:1.2:0.5, respectively. With the exception of the isoleucine, 120 121 these were the same amino acids in the same relative ratios as those previously reported from B. megaterium peptidoglycan, (Ghuysen, 1968). The isoleucine appeared to be a part of the peptide, but its presence in cell wall polymers is quite unusual. It may link another polymer to the cell wall, however. The polysaccharide fraction, or column purified BMP, was shown not to be a teichoic acid since there was very little phosphate associated with the polymer, and that which was present was most likely from a small amount of nucleic acid. The BMP was also not a teichuronic acid, since no uronic acid components were present in the polymer. Glucose was found to be the primary monosaccharide component of BMP. By thin layer chromatography, no other components could be identified (Cassity, 1976); however, by gas-liquid chromatography, galactose, xylose, and arabinose were also found to be present. This is an unusual composition for a polysaccharide from a Gram positive microorganism. The BMP was shown to be one polymer since only one peak could be discerned in polyacrylamide gels. However, it may be noted that the BMP migrated only a short distance through the gel. It is possible that there could have been more than one very large polymer which also did not migrate very far. Another possibility is that the BMP is a glucan, and that the other components arose during hydrolysis. However, this would seem unlikely, since' glycogen and cellobiose were hydrolyzed and handled in an identical manner as the BMP, without the appearance of any component other than glucose. The polymer was also shown to be different from other polysaccharides isolated from sporeformers in that it did not form colored complexes with iodine. The BMP also bound very little Con A. This indicated 122 that the polymer either does not contain ©<-linked terminal glucopyranosyl residues, or is a very long chained, unhranched polymer (Goldstein, 1972). The EMP was extremely difficult to enzymatically hydrolyze. Microorganisms which could hydrolyze the most common polysaccharide linkages could not attack the BMP. The presence of large amounts of ct(l— > 6), *(l— >3 ). o((l-»4)f c<(l- » * 0 in alternating oC(l— >3)^ °C(l— j3{ 1— > 2 ), p (l— > 3)» pi. 1— and p i x — > 6) glucose- glucose linkages were eliminated. This implies that the glucose- glucose linkages may he in an alternating sequence, there may be a small number of several different types of glucose-glucose linkages, or the galactose, xylose, and arabinose may be linked in such a way as to protect the polymer from hydrolysis by these enzymes. The Arthrobacter species, which degraded the BMP slightly, was much more efficient when it was cultured with the other microorganisms with which it was associated in the soil. This may indicate that several types of enzymes from several different microorganisms are required for the complete hydrolysis of the polymer, thus favoring the hypothesis that several types of linkages are present in the polymer. It is also possible, however, that the Arthrobacter species required a substance elaborated by some of the other soil microorganisms, or from the soil itself, to grow well and degrade IMP. This would not seem likely, however, since soil extract failed to enhance the degradation of BMP. The BMP, previously believed to be a capsular polymer (Cassity, 1976), was shown not to be of capsular origin, since BLC-1 contained more of the polymer than the normally capsulated strain. A polymer, 123 similar to BMP, was described by Aubert (l95l)* He also assumed this polymer to be of capsular origin, since it was isolated from a boiling water extract of B, megaterium KM, The exact location of the BMP was not determined. It is possible that it could have been the remaining capsular polymer on the small capsule mutants. This is not likely, however, since small capsule mutants also contained some polyglutamate. The BMP is most likely an intracellular polymer, even though such a polymer could be associated with the cell wall. The most likely function of a polymer with the composition of BMP would be as a carbon and energy storage compound, since it possesses a large amount of potential chemical energy and organic car bon. It was shown that some of the BMP was liberated into the medium when the cells lysed, but during the formation of forespores and phase bright spores, the total concentration of polymeric glucose in the culture decreased sharply. This indicated that the BMP was probably used as a carbon and energy storage compound for sporulation. Glycogen-like compounds which behave similar to BMP have been isolated from a number of bacteria (Slock and Staley, 197^» Levene et al., 1953» Aubert, 1951* and Strasdine, 19^8). Bacillus megaterium did not sporu- late well endotrophically; hence, it was not possible to conclusively determine if the BMP, along with PHB, provided a sufficient quantity of energy and carbon for sporulation. It is not known why this microorganism did not sporulate endotrophically, but it seems likely that, since the BMP was not utilized under conditions of endotrophic sporulation, some nutrient which the microorganisms may synthesize earlier and secrete into the medium is necessary for spore formation. 124 Cell-free extracts and sedimentable cell components of B. megaterium prepared during forespore formation did not possess appre ciable hydrolytic activity towards the BMP with the release of reducing sugars. The hydrolysis of this material may be directly dependent upon its utilization. Extraction of Polyglutamate Since the BMP was shown not to be the capsular material, isolation of another substance which could be shown to be capsular in nature was attempted. Previous reports in the literature showed that the capsular material of several Bacillus species, including B. megaterium, was, at least in part, polyglutamate (Tomcsik, 1956; and Hanby and Rydon, 1946). Much less (55*4^) polyglutamate was isolated from BLC-1 compared to the parent strain. This indicated this was a capsular polymer. It had been shown previously, by immunofluorescent staining with anti-poly-D-glutamic acid antiserum prepared against B, anthracis poly-D-glutamic acid, that B. megaterium possessed capsu lar poly-D-glutamate when cultured on sodium bicarbonate agar under a high CO^ concentration, even though cells cultured on FMS did not stain with this antiserum (Cassity, 1976), The polyglutamate isolated from M S cultured cells most likely contained both the D and L isomers, which would not stain with fluorescein-labeled anti- poly-D-glutamic acid antiserum. Leonard et al. (1958) demonstrated that the ratio of D-glutamic acid to L glutamic acid in the glutamyl polypeptide produced by B. licheniformis ATCC 9945 A was dependent 2+ upon the concentration of Mn in the medium. This could account for the failure of B. megaterium cultured on M S to stain with anti- 225 poly-D-glutamic acid antiserum, since M S contains a larger quantity 2+ of Mn than sodium bicarbonate agar. The synthesis of polyglutamate by cells cultured on medium which did not contain L-glutamic acid is also unusual (Housewright, 1962). Polyacrylamide gel electrophoresis of B. megaterium polyglutamate indicated that the polymer was of a relatively low mol. wt; and was nonuniform. Accumulation of Metallic Ions by Normally Capsulated B. megaterium and Small Capsule Mutants There were no significant differences in the accumulation of Na, Fe, or Zn between the normally capsulated cells and small capsule mutants. From analyses of the culture medium, it was shown that Mg, Mn, and K accumulated in the parent strain to a much higher concentra tion than in the small capsule mutants. From analysis of digested cells, Ca, Mg, and Mn were shown to accumulate in higher concentrations in the parent strain than in small capsule mutants also. The reason for the discrepancy between Ca and K accumulation between the two types of analysis is not understood. This- indicated that the capsule may function as a polymer which can bind metallic ions. A rapid uptake of Mg, Ca, and Mn was observed during lag phase and early log phase, with an egression of these metals from the cells taking place 1-2 h prior to the beginning of stationary phase. These results are similar to those observed by Kreuger (1972). He hypothesized that the initial uptake of these metals was due to capsular binding, and that the decrease in capsular material around the cell was responsible for the egression of the metals from the cells. This does not appear to be the case, however, since the small capsule mutants, which have 126 small capsules throughout all stages of growth, also showed the rapid uptake of Mg, Ca, and Mn during early logarithmic growth, and an egression of these minerals 1-2 h prior to stationary phase. It is likely that a polymer composed of glutamic acid would hind certain cations, since amino acids are capable of forming stable five-membered rings with metallic ions (Gurd and Wilcox, 1956). Magnesium and manganese, in particular, tended to combine with carboxyl groups forming a metal-oxygen ionically bonded complex (Gurd and Wilcox, 1956). A polyglutamate would have one free carboxyl group per each glutamic acid residue, giving the polymer a large capacity for the binding of metallic ions. Measurement of the inhibition of respiration by toxic metals, such as Hg^+ , Cu^+ , and Ag+ , provided a very sensative method for determining if these metallic ions reached the cytoplasmic membrane, where the respiratory enzymes are located. However, one must be certain that, each type of cell suspension contains the same biomass, that each cell type is the same age, and that each culture is respiring at the same rate. The results from these experiments revealed that toxic metals required a much longer time to reach the cytoplasmic membrane of normally capsulated cells compared to small capsule mutants, since the small capsule mutants were inhibited much more rapidly than the normally capsulated cells. The differences in the inhibition of respiration between the two cell types due to Hg was the greatest, followed by Ag, then Gu. Mercury has the largest atomic radius, followed by Ag, then Gu. It is possible that the larger the atomic radius of the metal, the more efficiently it is retained by the capsule. From these studies it is apparent that 127 certain metallic ions, such as Mg, Mn, K, Hg, Ag, and Cu are bound by the capsular polymer of B. megaterium. These results are similar to those reported by Rorem (l955)» who showed that capsulated Streptococcus salivarius, and Leuconostoc mesenteriodes accumulated up to twenty times more phosphate and rubidium when they were cultured under conditions which permitted the formation of large quantities of dextran and levan. Ghiorse and Hirsh (1978) reported that iron and manganese oxides combined with the extracellular polymer from a Pediomicrobium-1ike budding bacterium. This would provide an advantage to encapsulated organisms, since they would be able to tolerate small amounts of toxic metals in their environments better than nonencapsu lated cells. They would also be able to retain usable metallic ions near the cell surface if they were in an environment where there were few metallic ions. Characterization of Bacteriophage CK-1 The bacteriophage, CK-1, was similar to the B. megaterium CS-1 bacteriophage described by Cooney et al. (1975) with respect to density, physical dimensions, chloroform sensitivity, and host range. However, several non-overlapping strains of B. megaterium were tested in each of these reports. The CS-1 phage was most stable at a pH of 7-8, while CK-1 was most stable at a pH of 8-9. Cooney et al. (1975) noted that CS-1 did not require metallic ions for replication, even though the efficiency of plating was increased by the addition of 0.8 mM CaCl2*2H20, and 1.8 mM MgC^^H^O, while Mn had no effect. In p |_ O | constrast, CK-1 was shown to require divalent cations (Mg , Ca , or 2+ \ Mn ), with no plaques produced at all without these metallic ions or 128 with Na+ . CS-1 was not inactivated at 60 C for 4-5 min (Cooney et al,, 1975)» while CK-1 was 99*929^ inactivated at 60 C for 45 min. Cooney et al. (1975) reported that CS-1 had a hexagonal head, while CK-1 had an icosahederal head, CS-1 also lysed logarithmically growing cultures of B. megaterium ATCC 19213 in nutrient troth, while CK-1 would not lyse such cultures. These results clearly showed that, even though there are some similarities between CS-1 and CK-1, the two are distinctly different B. megaterium bacteriophages. CK-1 was also much different from the other B, megaterium bacteriophages reported in the literature (Friedman and Cowles, 1953; Murphy, 1957; Erlich and Pfau, 1957; and Carvalho and Vary, 1977)• Replication and Adsorption of CK-1 to B. megaterium with Varying Sized Capsules CK-1 appeared to adsorb to the cell wall when observed in the electron microscope. The replication of CK-1 on cells which, either by mutation or manipulation of growth medium, produced small capsules, and the failure of CK-1 to replicate on B. megaterium which produced large capsules, indicated that the capsule was possibly blocking the receptor site of the phage on the cell wall. This also explained why CK-1 would not lyse logarithmically growing cultures, since cells in early log phase contained a large amount of capsular material, while there was essentially no capsule surrounding germinating spores. The adsorption rate constants of cells with varying sized capsules substantiated the previous conclusion that the larger the capsule, the less likely that CK-1 adsorbed to the cells. The relationship between capsule size and adsorption rate constant was neither 129 arithmatic nor logarithmic; however, adsorption of phage decreased rapidly as the capsule size increased. This demonstrated that capsules can protect a bacterium from bacteriophage infection. McCloy (1951)» Maxted (1952), Bernheimer and Tiraby (l9?6), and Burt et al. (1978) using different microorganisms and different techniques, also showed that capsules tend to protect bacteria from infection by bacteriophages. Drying of Normally Capsulated B. megaterium and BLC-1 Both the normally capsulated parent strain and the small capsule mutant BLC-1 lost viability at similar rates when dried in soil. This indicated that the capsule did not protect the capsulated cells from the lethal effects of desiccation. Similar results have been obtained by Bitton et al. (1976) with Klebsiella aerogenes, and were in contrast to the results of Morgan and Beckwith (1939)• CONCLUSIONS 1. A high mol wt polysaccharide (<1.5 x 10^) was isolated from Bacillus megaterium ATCC 19213. This polymer was shown not to be of capsular origin, since comparable quantities of this material were found in both normally capsulated cells and small capsule mutants. This polysaccharide was composed of glucose, galactose, xylose, andarabinose in a ratio of 7*7!l*lsl*0.6l, respectively. The polysaccharide neither bound Concanavalin A nor formed a colored complex with iodine. Microorganisms which hydrolyzed specific linkages c<(l— ^2), o4(l— >3)» c^(l— >6), ^ ( l — >6), j £ ( l — 5>3), and p (l— >2) did not hydrolyze the B. megaterium polysaccharide, indicating the polymer may contain more than one type of linkage. The polysaccharide appeared to be utilized by the cells as a storage compound, which provided a source of carbon and energy for ohe cell during sporulation. 2. Polyglutamic acid was shown to be the primary capsular polymer of B, megaterium, since a much higher concentration was extracted from normally capsulated cells as compared to small capsule mutants. 3 . Capsulated B, megaterium was found to accumulate more Ca, Mg, and Mn, than small capsule mutants, but not more Fe, K, Na, or Zn. 130 Respiration of small capsule mutants was inhibited much more rapidly by Ag, Gu, and Hg as compared to normally capsulated cells, indicating that the capsule may bind these metals and prevent them from reaching the cytoplasmic membrane. A B. megaterium bacteriophage was isolated and characterized. Adsorption rate constants of the bacteriophage were shown to decrease as the size of the capsule surrounding the cell increased, indicating that the capsule inhibits adsorption of the phage to the cell wall. The capsule was shown to provide no advantage for survival of capsulated cells when normally capsulated B, megaterium and BLC-1 were dried in soil. APPENDIX 133 Figure 2 l4-. Standard curve for the determination of total carbohydrate by the anthrone method of Ashwell (1957)* Figure24. Absorbance 0.6 j/l Glucose jjg/ml 20 0 4 135 Figure 25. Standard curve for the determination of protein by the method of Lowry et al. (l95l)* 0 .2 - mg/ml BSA Figure 25. 13? Figure 26. Standard curve for the determination of protein by the method of Kalb and Bernlohr (197?)• Figure26. [P] Value - 0 4 - 0 8 gm BSA 4g/ml 0 4 138 Figure 27. Standard curve for the determination of nucleic acid by the method of Kalb and Bernlohr (1977)* 5 0 4 0 20 jjg /m l DNA 20 5 0 30 4 0 snieA IN] Figure 27. Figure Figure 28. Standard curve for the determination of amino acids by the method of Moore and Stein (19^8). 142 0.6- jjg/ml Glutamic Acic Figure 28. 143 Figure 29. Standard curve for the determination of phosphate according to the method of Ames (1966). 0.6-r j a < 0 .2- 0 10 20 25 jjg/m l P04 Figure 29. 145 Figure 30. Standard curve for the determination of glucose "by the glucose oxidase-peroxidase method. Figure30. Absorbance j/ Glucose l jjg/m 0 4 146 120 147 Figure 31. Standard curve for the determination of reducing sugars according to the method of Somoygi (1945)- 148 5 0 jjg/ml Glucose Figure 31. 1^9 Figure 32. Standard curve for the determination of calcium by atomic absorption spectrophotometery. 150 0 0 0 • 0) O 0 Absorbs nee IV) 0 0 O O Figure 32. Figure 33* Standard curve for the determination of iron by atomic absorption spectrophotometery. I ^ Absorbance VjJ • o O o o b O o o o ro -few 0) 00 t (Q \ 3 ■n CD OJ - 153 Figure 3^* Standard curve for the determination of potassium "by atomic absorption spectrophotometery. T -CO U) OJ *• O GDueqjosqv § 155 Figure 35* Standard curve for the determination of magnesium "by atomic absorption spectrophotometery. 0.3n r o 0 . 2 H JQ L. o _Q 1 H ' < 0 c 0.25 jjg/ml Mg Figure 35* 157 Figure 36. Standard curve for the determination of manganese by atomic absorption spectrophotometery. 0.121 0.16- (D §0^2- X3 £_ 1 0.08- < 0-04- 0 — i 0 4 jjg/ml Mn Figure j6. V_n CD 159 Figure 37. Standard curve for the determination of sodium by atomic absorption spectrophotometery. l6o - OJ CO \ O) LO c n C\J o O O O O O 0 Dueqjosq\/ Figure 37. Figure Figure J8. Standard curve for the determination of zinc by atomic absorption spectrophotometery. *=d H* Absorbance p o o p O ^ K) u) t (Q \ 3 N D H CN ro LITERATURE CITED Adams, M. H. 1959 a-. Bacteriophages, pp. 450-451. Interscience, London. Adams, M. H. 1959 !>• Bacteriophages, pp. 466-473* Interscience, London. Adams, M. H., and B. H. Park. 1956. An enzyme produced "by a phage- host cell system. II. The properties of the polysaccharide depolymerase. Virology 2: 719“736* Ames, B. N. 1966. Assay of inorganic phosphate, total phosphate, and phosphatases, pp. 115-118. In E. F. Neufeld and V. Ginsburg (ed.) Methods in enzymology Vol. VIII. Academic Press, Inc., New York. Ashwell, G. 1957* Colorimetric analysis of sugars, pp. 73“1°5* In S.P. Colowick and N. 0. Kaplan (ed.) Methods in enzymology Vol. III. 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