or“ Ease: RED 3, LYS! MPAI U AND ALT 3.“ LATE T0 SENSITIVEMUTANTS? “T “Hannah ‘RESESTANCE: TBACILLQS. CEREU IN Maury 7701st TEMPERATURE -r..~-.e.xvn:1 nvvxa‘elonriv‘\ -z--V«.-n-o “.- : V V. V. V V . ... .V V ,. VVVV V VV V V V V V V V ‘ V. V V V VV V V . . . V . V V V V .V VV V . . . V V V . V .V . V: ‘ V .V V . V V V . . V Vi 3,52: .2 1, . L EVE. EV?» LIBRARY ‘ MahiganSCaee University T; ._ g": . This is to certify that the thesis entitled 'g; Temperature- Sensitive Mutants of Bacillus cereus T impaired in ability to sporulate ..'and altered in resistance to lysis by bacteriophage. presented by Gerard N. Stelma, Jr. has been accepted towards fulfillment of the requirements for Ph.D. degn¥>h1 Microbiology Major professor 10- 04 - 74 Date 0-7639 (Ix ABSTRACT TEMPERATURE-SENSITIVE MUTANTS OF BACILLUS CEREUS T IMPAIRED IN ABILITY TO SPORULATE AND ALTERED IN RESISTANCE TO LYSIS BY BACTERIOPHAGE BY Gerard Nicholas Stelma Jr. The objectives of this study were to isolate temperature-sensitive mutants of Bacillus cerenus T blocked at an early stage of sporulation and to use these mutants to obtain information on the nature and number of functions which are indispensable to the sporulation pro- cess. Five temperature-sensitive mutants were isolated from cells treated with N-methyl-N-nitro-N-nitrosoguani- dine. Preliminary investigations of these mutants indi- cated that each of them is susceptible to an agent which causes them to lyse in a sporodic manner. This lytic agent was identified as a bacteriophage carried by the parent strain in a latent form but induced at high frequencies in the mutants. Three of the mutants have lost super- infection immunity. Observations of the phage under the electron microscope reveal that they are pleiomorphic. One morphological type has heads 65 to 70nm in diameter and Gerard Nicholas Stelma Jr. tails 200nm in length with triangular base plates. The second type has heads identical to those of the first, but the tails are variable in length; a variable number of plates occur at irregular intervals along the tails and perpendicular to the axis of the tails. One of the mutants, JSZZ, becomes spontaneously cured of the phage genome at a high frequency. Clones are cured if they no longer produce plaque-forming phage and simultaneously become sensitive to the phage. Sedi- mentation of DNA from JSZZ and from the parent strain in CsCl-ethidium bromide density gradients reveals that this spontaneous curing is not due to the occurrence of the phage genome as a plasmid. Strains cured of the lytic phage still carry an apparently defective phage which cannot produce plaques. The lytic phage produced by the mutants was compared with the two g. cereus phages which it most closely resembled. Comparisons in size, chloroform sensitivity, host range, and antigenic components establish that this phage is unique, and hence it has been designated as lytic phage 22 (LP-22). LP-22 is not able to mediate generalized tranduction in E. cereus T. The time course of phage release was measured in broth cultures of JSZZ and the parent strain at both restrictive (37C) and permissive (26C) temperatures. Maximal phage titers in cultures of the mutant grown at Gerard Nicholas Stelma Jr. 37C are 4x109 plaque forming units per milliliter (pfu/ml), while those of the parent strain are 104 pfu/ml. Maximal phage titers in cultures of the mutant and parent strain grown at 26C are 7x108 pfu/ml and 103 pfu/ml respectively. At both temperatures over 99% of the phage produced by the mutant and over 90% of those produced by the parent strain are released after the end of exponential growth. This late release of phage does not appear to be due to a requirement of the phage for a sporulation-specific RNA polymerase or a sporulation-specific protein synthesizing system; infected cells of JSZ9, a mutant which has lost its superinfection immunity, are able to produce high titers of phage during early exponential growth. The only condition in which induction of LP-22 has been observed is mutation to asporogeny. Ultraviolet light, mitomycin C, gig dichlorodiammineplatinum (II) and growth at elevated temperatures all fail to induce the phage in cultures of the parent strain. JSZZ-c, a strain of JSZZ which has been cured of the LP-22 genome, was investigated in some detail in order to determine the nature of the genetic lesion which prevents it from Sporulating at 37C. Growth studies of the mutant and parent strain at 37C reveal that the vegetative growth rate of the mutant is identical to that of the parent strain. Some biochemical events associated with the onset of sporulation, such as production of Gerard Nicholas Stelma Jr. extracellular and intracellular proteases and production of alkaline phosphatase, occur normally at 37C. Failure of the pH to increase after the end of exponential growth in 37C cultures of J822-c indiCates that the mutant is unable to oxidize, via the tricarboxylic acid cycle, the organic acids which accumulate in the medium during growth. Examination by electron microscopy of cells of JSZZ-c incubated at 37C for the time period that allowed sporulation to occur in the parent strain has revealed that the cells are blocked at stage 0 of Sporulation. 3 heat- Cultures of the mutant produce approximately 10 stable spores per ml at 37C, while cultures of the parent strain produce 7x108 heat-stable spores per ml at that temperature. Growth studies of the mutants and parent strain at 26C reveal that functions such as oxidation of organic acids and formation of heat resistant spores, which are blocked at 37C, are somewhat impaired at 26C. The develOpment of a functional tricarboxylic acid cycle and the development of refractile fore-spores is delayed for about three hours in cultures of the mutant. The mutant produces about 60% as many spores as the parent strain at 26C. Cultures of JSZZ-c were shifted from 37C to 26C at various times during growth and Sporulation, and the effect of these shifts on the ability of the mutant to Gerard Nicholas Stelma Jr. form heat-stable spores was determined. Cultures shifted to 26C during exponential growth or during the first hour after the end of exponential growth are able to sporulate at near normal levels, but those shifted later are impaired in ability to sporulate. Complete inhibition of sporulation occurs in cultures shifted at four or more hours after the end of growth. Similar experiments in which cultures of JSZZ-c were shifted from 26C to 37C at various times revealed that cultures shifted during exponential growth or during the first five hours after the end of exponential growth are greatly impaired in their ability to Sporulate. Cultures shifted six hours after the end of exponential growth are able to sporulate at near maximal levels, indicating that the function of the temperature-sensitive gene product is dispensible after this time. Examination of thin sections of JSZZ-c cells removed from 26C cultures six hours after the end of exponential growth reveals that the cells had reached the end of stage II (septum formation) at that time. Although cells shifted from 26C to 37C from one hour after the end of exponential growth through four hours after the end of exponential growth are unable to sporulate, the rise in pH values of these cultures indicates that they are able to form a functional tricarboxylic acid cycle. Rates of oxidative metabolism were followed in cultures of JSZZ-c which had been shifted Gerard Nicholas Stelma Jr. from 26C to 37C by measuring the rate of reduction of 2,6-dichlor0phenolindolphenol. The rates of oxidative metabolism of cultures shifted to 37C three hours after the end of exponential growth increase after the shift, indicating that the temperature-sensitive protein is not one of the enzymes involved in oxidative metabolism. Cultures shifted at the end of exponential growth do not develop detectable levels of oxidative metabolism. These results are consistent with the hypothesis that the lesion affects a gene which has a control function that must be expressed before a functional tricarboxylic acid cycle can be formed. Since cultures shifted three hours after the end of exponential growth have high rates of oxida- tive metabolism but are still unable to sporulate, expression of this gene appears to be necessary for the function of at least one other indispensible event of sporulation. TEMPERATURE-SENSITIVE MUTANTS OF BACILLUS CEREUS T IMPAIRED IN ABILITY TO SPORULATE AND ALTERED IN RESISTANCE TO LYSIS BY BACTERIOPHAGE BY Gerard Nicholas Stelma Jr. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1974 ACKNOWLEDGMENTS I wish to thank my advisor, Dr. Harold L. Sadoff, for his guidance, his support, and his patience throughout the course of this study. I also wish to thank the other members of my guidance committee, Dr. Ralph N. Costilow, Dr. Loren Snyder, Dr. Fritz M. Rottman, and Dr. John Boezi, for their interest and their willing- ness to answer questions. I am grateful to Dr. Robert R. Brubaker for his useful advice on bacteriophage techniques and for serving as a temporary member of my guidance com- mittee during Dr. Costilow's absence. I am also grateful to H. Stuart Pankratz, for assistance with the electron microscopic studies, to Dr. Willard Charnetzky for his advice on transduction procedures, to Ms. Dorothy Okazaki for technical assistance, and to Dr. Reynard Bouknight for his constant encouragement. ii TABLE OF CONTENTS INTRODUCTION 0 O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . Morphological Changes During Sporulation. Biochemical Events Associated With Sporu- lation . . . . . . . . . . . Protease . . . . . . . . . . Peptide Antibiotics. . . . . . Alkaline Phosphatase . . . . Tricarboxylic Acid Cycle Activity . Cortex and Coat Synthesis. . . . Dipicolinic Acid Synthesis . . . Regulation of Sporulation. . . . . . Catabolite Repression . . . . . Changes in Nucleic Acid Synthesis . . Translational and Post-Translational Control . . . . . . . . . . Temperature-Sensitive Asporogenous Mutants Bacteriophage and Sporulation . . . . REFERENCES 0 O O O O O O O O O O 0 ARTICLE 1 PRODUCTION OF BACTERIOPHAGE BY TEMPERATURE- SENSITIVE SPORULATION MUTANTS OF BACILLUS CEREUS T. G. N. Stelma Jr. and H. L. Sadoff. J. Bacteriol. 116:1001-1010. (1973) . iii Page 10 ll 13 15 l6 l6 17 21 22 25 27 37 Page ARTICLE 2 PRELIMINARY INVESTIGATION OF A TEMPERATURE- SENSITIVE SPORULATION MUTANT OF BACILLUS CEREUS T. G. N. Stelma Jr. and H. L. Sadoff. (manuscript in preparation) . . . . . . . 47 APPENDIX SOME FURTHER CHARACTERISTICS OF THE LP-22 - BACILLUS CEREUS T PHAGE-HOST RELATIONSHIP . . 91 iv LIST OF TABLES Table Page ARTICLE 1 l. The extent of sporulation at 26 C of JSZZ in media containing G salts, 0.4% glucose, and varying concentrations of casein hydrolysate . . . . . . . . . . . 40 2. Sensitivities of the parent strain, the ts mutants, and two revertants to phage in the supernatant fluids of broth cultures of the mutants . . . . . . . . . . 42 3. Specificity of antisera for CP-53 and phage from J822. . . . . . . . . . . . 44 ARTICLE 2 1. Oxygen consumption by cells of the parent strain and mutant JSZZ-C after shifting growth temperature from 26 C to 37 C. . . 78 APPENDIX 1. Heat stability of phage carried in spores of JSZZ O O O O O O O O O O O O O 112 LIST OF FIGURES Figure ARTICLE 1 1. Electron micrographs of the phage particles observed in supernatant fluids of JSZZ . . 2. Time course of inactivation of Bacillus cereus phage LP-22 at 60 and 70 C in 1% peptone . . . . . . . . . . . . 3. Inactivation of phages LP-22 and CP-53 by ultraviolet light . . . . . . . . . 4. Growth and phage production of mutant JSZZ and the parent strain at both restrictive (37 C) and permissive (26 C) temperatures . ARTICLE 2 1. Growth of the parent strain and the mutant 2. at the restrictive temperature (37 C) . . Electron micrograph of a longitudinal section of the mutant after growth at 37 C for the time required for the parent strain to sporulate under the same con- ditions . . . . . . . . . . . . Growth of the parent strain and mutant at the permissive temperature (26 C). . . . Sporulation of JSZZ-C cultures shifted-down from 37 C to 26 C at different times during the growth cycle . . . . . . . . . Sporulation of JS22-C cultures shifted-up from 26 C to 37 C at different times during the growth cycle . . . . . . . . . vi Page 41 43 44 45 . 80 82 84 86 88 Figure ARTICLE 2 6. Electron micrOgraph of a longitudinal section of one cell and part of a longitudinal section of a second cell from a sample of JSZZ-C removed at T6 from a culture growing at 26 C . . . . . . . . APPENDIX 1. CsCl-ethidium bromide gradient centrifugation of DNA extracted from Bacillus cereus try? 2. CsCl-ethidium bromide gradient centrifugation of DNA extracted from Bacillus cereus JSZZ 3. Growth and lysis of a broth culture of Bacillus cereus JSZ9 infected with LP-22 at a multiplicity of infection of 1.0 . . 4. Growth and lysis of a broth culture of Bacillus cereus JSZZ-c infected with LP-22 at a muIti- plicity of infection of 1.0 . . . . . vii Page 90 114 116 118 120 INTRODUCTION Endospores formed by bacteria of the genus Bacillus differ from vegetative cells in their morphology, their dormant physiological state, and their greater resistance to a number of adverse environmental con- ditions. The formation of Bacillus Spores is initiated by conditions in which a nutrient essential for the growth of the organism has become limiting and then occurs as a precisely ordered sequence of biochemical and morphological events. The sporulation process is considered to be a primitive form of cellular differenti- ation. Since bacteria are susceptible to analysis by genetic and biochemical techniques which cannot presently be applied to differentiating cells of higher organisms, bacterial sporulation is being studied extensively as a model to promote better understanding of the process of differentiation in more complex organisms. The nature and the number of indispensible steps involved in the formation of bacterial spores can be analyzed by studying large numbers of asporogenous mutants. Temperature-sensitive mutants are particularly valuable for these kinds of analyses; because they make it easier for one to determine whether the mutation has affected a specific enzyme or a control gene, and they make it possible to determine the time period in which the altered gene normally functions. The initial purpose of this project was to study the function of a sporulation-specific protease of Bacillus cereus through the use of temperature-sensitive mutants. Although the desired mutant was not obtained, five temperature-sensitive mutants were found which were blocked at an early stage of sporulation when grown at 37C. These mutants were also altered in their resistance to lysis by a previously undiscovered bacteriOphage. This dissertation describes the isolation and preliminary characterization of the five mutants, the partial char- acterization of the newly discovered bacteriophage, and a more thorough investigation of one of the mutants. LITERATURE REVIEW Morphological Changes During Sporulation The first photographic evidence demonstrating the correct sequence of the gross morphological changes that occur during sporulation was presented by Bayne- Jones and Petrilli in 1933 (8). Their observations were that one cell gives rise to one spore, that the Spore arises within the cytoplasm at one end of the cell, that the spore area increases in size during the later stages of Spore development, and that the terminal processes of sporulation are the development of refractility followed by release of the spore from the disintegrating cell. Recently, the development of techniques for achieving more synchronous sporulation in batch cultures and the refinement of electron microsc0pic techniques have made it possible to study these changes in much greater detail and to resolve the morphological development of spores into seven distinct stages (18, 20, 37, 50, 64). During stage I the final round of DNA repli- cation is completed, and the two nuclei condense, coalesce, and form an axial thread of chromatin which extends almost to the full length of the vegetative cell (50). The formation of the axial filament is the first major structural change that takes place during sporulation; mutants blocked prior to this change are identical to exponentially growing cells in their appearance and are said to be blocked at stage 0 (30, 37). During stage II a septum is formed near one pole of the cell (37). This results in the segregation of the DNA into two compartments, the mother cell and the fore- spore. Although the forespore is much smaller than the mother cell, both are believed to contain the same amount of DNA (64). No peptidoglycan material is deposited between the invaginating layers of membrane (37, 64). The asymetric nature of the cyt0plasmic division and the absence of cross wall formation are the only major differences between forespore septum formation and binary fission (37). After completion of the forespore septum, the forespore is engulfed or enveloped by the mother cell (stage III). When the forespore has become completely enveloped, the outer sides of the plasma membrane face inward toward each other, and the mechanism by which the cell wall material of the cortex is built or excreted is doubly provided for (64). Cells that have attained stage III continue their development to mature spores upon transfer into fresh medium and are said to be "committed" to sporulation. Prior to this stage, cells transferred into fresh medium resume vegetative growth (20). Cell wall material is deposited between the outer forespore membrane and the inner forespore membrane during stage IV. This cell wall material consists of two layers, an inner thin shell; the germ cell wall, and an outer thick shell; the cortex (37). During stage IV the forespore must remain associated with the mother cell in order to be capable of maturation. It is not until this stage is completed that the forespore can be separated (61). Synthesis of the protein coat and the exosporium occur during stages IV and V. The end of stage V is characterized by a completed coat structure (37). At the end of stage V and during stage VI the spore under- goes a number of changes which have been described as ripening. The mesosomes become compact and barely visible, the refractility of the spore increases, and the spore becomes heat-resistant and impermeable to basic strains (64). Stage VII is marked by the lysis of the Spor- angium and release of the mature spore into the medium (37). Biochemical Events Associated With Sporulation A number of enzymes and products have been associated with Sporulation because they appear for the first time or in much greater quantities after the onset of sporulation (37, 51). It is not certain how many of these enzyme and products are really involved in sporu- lation and how many appear at this time merely because they are repressed by conditions promoting vegetative growth (37). This review will not consider all of the biochemical events which occur during sporulation but will be limited to those events for which a sporulation- specific function is known and those events which, when absent, result in a reduced frequency of sporulation. Protease The correlation of the time course of ex0pro- tease synthesis in B. licheniformis (9), B. cereus (58), and B. subtilis (14, 62) with the onset of sporulation; the absence of one or more proteolytic enzymes from a large number of mutants blocked at stage 0 of sporulation (7, 37, 90); and high rate of protein turn-over normally observed in Sporulating cells (51, 87); suggested that proteases were involved in the Sporulation process. The metal protease which is inhibited by EDTA accounts for nearly all of the extra-cellular proteolytic activity of B. cereus and B. megaterium (1) and approximately 20% of the extra-cellular activity of B. subtilis (62). The function of this enzyme, to supply nutrients in assimilable form to the Sporulating cell (7), may enhance sporulation; but the existence of mutants of B. cereus (l, 2), B, megaterium (l), and B. subtilis (33) which do not produce an active metal protease but are able to sporulate normally indicates that this enzyme is not essential to sporulation. The serine protease, which is inhibited by phenyl methyl sulfonyl floride, appears to be the protease which is essential to sporulation. Sadoff et a1. (75) have shown that the serine protease of B. cereus, in a limited proteolysis, converts vegetative aldolase to spore aldolase. More direct evidence that a functional serine protease is required for the initiation and sub— sequent stages of sporulation was obtained by Leighten et a1. (56, 57, 76) who isolated a temperature-sensitive mutant of B. subtilis with a lesion in the structural gene coding for the serine protease. Studies of this mutant revealed that sporulation is blocked at stage 0, and the mutant is unable to produce antibiotic or sporulation-specific RNA polymerase at the restrictive temperature. The serine protease is not responsible for general protein turnover in B. subtilis (56). Although the results Leighton et a1. (56, 57) indicate that the serine protease of B. subtilis has both intra and extracellular functions, recent studies have shown that the major intracellular serine protease of E. subtillis is different from the extracellular serine protease. Reysset and Millet (70) found that the intra- cellular serine protease had an absolute requirement for Ca++, not found in the extracellular serine protease, and a narrower substrate specificity than the extracellular protease. Hageman and Carlton (33) found that the two serine proteases had different electrophoretic mobilities. Although much evidence has been obtained which supports the supposition that intracellular proteolytic activity is required for sporulation, one report by Slapikoff et al. (86) appears to contradict that evidence. They reported that a strain of g. brevis which lacks detectable intra or extracellular proteolytic activity (using 14C denatured protein as substrate) is able to sporulate normally without undergoing protein turnover. Aronson (1) suggested that the apparent lack of protein turnover observed may be due to the insensitivity of the method used by Slapikoff (86) for detecting small changes in turnover rates. Measurements of labled amino acid release from the metabolic pool of cells would probably be a more sensitive method to determine protein turnover (1). Peptide Antibiotics Several lines of evidence suggest a relationship between the production of peptide antibiotics and sporu- lation: 1. Specific sporulation inhibitors also inhibit antibiotic synthesis (10, 67). 2. Many pleiotrophic stage 0 mutants of B. subtilis are antibiotic negative (79). 3. A few mutants selected for their inability to produce antibiotics are asporogenous (81, 82). 4. Restoration of antibiotic production by reversion, transduction, or transformation also restores ability to sporulate (43). The antibiotics produced by sporulation bacilli have been classified into three groups (73). Group 1 consists of edeines, basic linear peptides which inhibit DNA synthesis (52); group 2 consists of bacitracins, cyclic peptides containing amino acid condensation pro- duct which inhibit cell wall synthesis (85); and group 3 consists of gramacidins, polymixins, and tryocidins, linear (78) or cyclic peptides (72, 88) which modify membrane structure or function. The edeines (53), gramicidins (100), tyrocidins (71), and polymixins (68) are not the products of normal protein synthesis but are synthesized enzymatically. Bacitracins appear to be the products 10 of the proteolytic cleavage of a protein formed earlier in the growth cycle (74). Sadoff (73) has proposed that edienes function in the inhibition of DNA synthesis that occurs during stage I, that gramicidins, tyrocidins, and polymixins each have a role in the membrane alterations that occur during stage II and stage III, and that bac- itracin prevents cell wall synthesis during forespore septum formation and engulfment. It is possible that the quantities of antibiotic required in sporulation may amount to only a few molecules per cell and that the known antibiotic producing strains are mutants which overproduce products that occur in much smaller amounts in all aerobic Sporulating bacilli (73). If these suggestions are correct, the high frequencies of sporulation which have been observed in mutants which produce low levels of antibiotic (82) and the apparent absence of antibiotic in some strains of Bacillus would be eXplained. Alkaline Phosphatase The alkaline phOSphatase which appears during sporulation is much less susceptible to repression by phosphate than that produced by vegetative cells (47, 96). Although production of this alkaline phosphatase, like sporulation, is delayed by addition of glucose to the medium (47), and alkaline phosphatase activity is absent in some Es mutants under conditions in which ll sporulation does not occur (55, 57); it is still not known whether alkaline phosphate activity is necessary for spore development or merely a by—product (47). Tricarboxylic Acid Cycle Activity Hanson et a1. (38) observed that growing cultures of B. cereus are unable to oxidize the acetate that accumulates in the medium while Sporulating cells are able to oxidize acetate at a high rate. Further study of this phenomenon revealed that several of the enzymes of the tricarboxylic acid cycle are absent from vegetative cells and that the appearance of these enzymes corresponds to the onset of Sporulation (39). The appearance of high rates of acetate oxidation in cultures of B. subtilis also corresponds to the onset of Sporulation although low levels of activity can be detected in vegetative cells of that strain (35). The synthesis of the tri- carboxylic acid cycle enzymes, like the initiation of sporogenesis, is subject to catabolite repression (36, 91). In B. cereus, the amount of cytochromes associated with the membranes increase concomitant with the increase in activity of the tricarboxylic acid cycle (19, 54). The results of several early studies suggested that the synthesis of a functional tricarboxylic acid cycle is an essential step in the sporulation process. Hanson et a1. (38, 39) observed that the addition of 12 a picolinic acid to cultures of B. cereus simultaneously inhibited the synthesis of aconitase and the initiation of sporulation. When the inhibition of aconitase syn- thesis was reversed by addition of metal ions the medium, the inhibition of sporulation was also reversed. Szul- majster and Hanson (35, 91) isolated an asporogenic mutant which was aconitase negative and transfer of this defective gene to wild-type strains by transformation caused these strains to become a3porogenic. Freese et al. have observed that a number of mutants, isolated for their inability to sporulate, have lesions in various structural genes of the tricarboxylic acid cycle (23, 24, 25). The method of isolating aSporogenic mutants and screening them for lesions in the tricarboxylic acid cycle precludes the selection of sporogenic mutants which lack a functional tricarboxylic acid cycle. To overcome this experimental limitation, Carls and Hanson (12) developed a technique for the direct isolation of mutants lacking a functional tricarboxylic acid cycle. Clones isolated by this technique were selected for their inability to oxidize organic acids rather than their inability to sporulate. However, mutants isolated by Carls and Hanson were all impaired in their ability to sporulate although three isolates were able to form Spores in l to 10% of the viable cells. Some of these l3 mutants Sporulated poorly while others with the same lesion Sporulated well. Their observations cannot be explained at the present time. Freese and co-workers (24) believed that the poor sporulation of tricarboxylic acid cycle mutants resulted from a deficiency of ATP during Sporogenesis. However, recent studies (26, 99) have shown that the arrest of these mutants at stage I is not due entirely to an insufficient energy supply. If mutants blocked in the first half of the tricarboxylic acid cycle are supplied with a metabolizable carbon source they are able to main- tain high levels of ATP but are not able to sporulate unless they are resuspended in culture fluids from post- exponential phase wild-type E.‘subtilis or E, 2211. Mutants blocked in the second half of the tricarboxylic acid cycle are not able to sporulate even under these conditions. These observations suggest that tricar- boxylic acid cycle mutants are unable to remove a suppressor(s) of Sporulation from the growth medium. Cortex and Coat Synthesis Two of the events that occur only during sporu- lation for which a sporulation-specific function is known are the synthesis of the cortical peptidoglycan and the synthesis of the spore coat protein. Although the cortex is made up of peptidoglycan (97, 98) there is evidence that it differs in chemical structure from 14 the vegetative cell wall and the germ cell wall. The cortex is hydrolysed during germination, while the germ cell wall persists and becomes the cell wall of the germ- inated spore (64). The cortex, which is sensitive to lysozyme, differs from the germ cell wall and vegetative cell wall which are not (97, 98). The cortex is synthe- sized immediately after the closure of the forespore septum. It is synthesized £2.2222I not from cell wall turnover (94). The number and nature of unique enzymes involved in the synthesis of the cortex are not known, due to the difficulties in recognizing and enriching for mutants devoid of these enzymes (37). The spore coat of B. cereus T contains primarily protein (6) and consists of two morphological layers (5). Recent studies have shown that the proteins in both coat layers are identical and that there may be only a single major species of polypeptide in the spore coat (5). There appear to be two major biochemical events associated with coat formation in B. cereus T (6). The first is the initiation of coat polypeptide precursor synthesis shortly after the termination of exponential growth. The second is the incorporation of cystine into the spore coat at the time of the formation of well- defined coat layers. The cystine appears to be involved in the formation of intermolecular disulphide bonds (5). Studies of exoprotease regulatory mutants which over or 15 underproduce coat protein suggest that coat synthesis is subject to regulation by catabolic repressors which are separate from but metabolically related to the catabolic repressors for ex0protease synthesis (4). Dipicolinic Acid Synthesis The synthesis of dipicolinic acid (DPA) like the synthesis of the spore cortex and the spore coat is con- sidered to be a Sporulation-specific event. DPA is absent from vegetative cells, but it is present in high concentration in all bacterial endospores, presumably as a calcium chelate (64, 69). Calcium and DPA are con- sidered to play important roles in the heat-resistance of spores. Mutants which contain low levels of DPA or no DPA produce spores with reduced heat-resistance (3, 21, 28, 34). Starvation of cells for calcium also results in the production of heat-sensitive spores (21). DPA synthesis occurs during cortex formation and matur- ation of the sporangia (stages IV to VI) (40, 93) and is accompanied by incorporation of calcium into the endospores (ll, 40). Analysis of mutants has shown that some of the enzymes of the lysine biosynthetic pathway participate in DPA synthesis (21, 28). The first enzyme of that pathway, aspartakinase, loses its sensitivity to feedback inhibition by lysine about two hours before DPA synthesis begins. This change appears to be due to an alteration of the pre-existing vegetative enzyme (21), 16 but other enzymes in the pathway do not appear to be altered (13, 21). DPA synthase, which catalyzes the conversion of dihydrodipicolinic acid to DPA, can only be detected in sporulating cultures just prior to the onset of DPA accumulation (13). Regulation of Sporulation Catabolite Repression Massive sporulation is suppressed as long as the growth medium contains an excess of rapidly metabolizable carbon and nitrogen sources (79). Depletion of these energy and nitrogen sources appears to be a factor involved in the initiation of sporulation (64). With E. subtilis, the probability that sporulation will occur depends on the nature and concentration of the carbon source and on the nature of the available nitrogen source. With E, megaterium sporulation occurs whether either the carbon or the nitrogen source is removed or depleted (81). These observations strongly suggest that sporulation is under the control of catobolite repression (81). Freese and co-workers (24) have identified at least three compounds, not interconvertible in certain mutants, which are involved in the repression of sporu- lation. They are D-glucose-6-phosphate, L-a-glycerol- phosphate, and some compound(s) derived from L-malate. The mechanism of catabolite repression in Bacillus Species is not understood. Cyclic 3', 5'-monophosphate l7 (cAMP) is the chemical messenger that signals low energy levels in E. coli and it is required for the transcription of catabolite-repressible genes in that strain (66). Attempts to detect cAMP in Bacillus species have not been successful (48, 84), and therefore it is likely that an alternative chemical is used in Bacillus. Although the nature of the signal molecule in Bacillus has not been determined, its presence or absence is dependent on the energy charge of the cells (46). Changes in Nucleic Acid Synthesis Replication of DNA is not irreversibly arrested during stage I or stage II in either B. subtillis or B. cereus. Transfer of these strains into fresh medium during stage I or stage II results in renewed vegetative growth. However, cells that have attained stage III have become committed to sporulate and cannot initiate DNA synthesis (20, 37). Although the reason for this irre— versible cessation of DNA replication is not known, it is probably not due to the state of the DNA in the axial chromatin filament but is more likely due to the bio- chemical changes associated with the membrane rearrange- ment that take place during engulfment (37). A number of studies have shown that gene tran— scription is altered at the onset of sporulation. Hussey et a1. (44) observed that the incorporation of radioactive 18 uracil into B. subtilis 30 s and 50 s ribosomal subunits stops during early sporulation, indicating that ribosomal RNA is not synthesized during sporulation. Doi and Igarashi (16) used DNA—RNA hybrid competition studies to demonstrate that genes are transcribed during sporu- lation which are not expressed during growth or germi- nation. They observed that a 15-30% fraction of labled sporulation mRNA from B. subtilis was able to hybridize. with DNA in the presence of an 150 fold excess of unabled exponential phase or germination phase mRNA. Using the ,same technique, Di Cioccio and Strauss (15) found that unique mRNA species occur at specific times during the sporulation process, that significant transcription occurs on the light DNA strand, and that only about 20% of the vegetative RNA Species are turned off during sporulation. Sumida-Yasumoto and Doi (89) have established that dif- ferential transcription occurs during sporulation in a sequential manner and that spore—specific genes are present in both complementary strands of DNA. Early in sporulation (stage 0 to I) only the light strand of DNA is specifically transcribed for sporulation genes but in stages III and IV both strands are transcribed. The first evidence that the changes in tran- scription that occur during sporulation are due, at least in part, to a change in the RNA polymerase was presented by Losick and Sonenshein (60). They compared 19 the ability of RNA polymerase obtained from vegetative cells of B. subtilis with that of RNA polymerase obtained from Sporulating B. subtilis to transcribe the DNA of ¢e, a phage which only replicates in exponetially growing cells. The results of this study showed that the RNA polymerase from exponentially growing cells actively transcribes ¢e DNA while that from Sporulating cells is inactive with the same DNA. Leighton et a1. (56, 57) have proposed that the change in the template specificity of B. subtilis RNA polymerase was due to a specific cleavage of the 8 subunit of the polymerase by a serine protease. Their evidence was derived from observations on their temperature-sensitive serine protease mutant which failed to modify the polymerase at the restrictive temperature but was able to modify the polymerase at the permissive temperature. Their strongest evidence was the observation that the template Specificity of RNA poly- merase was altered when the mutant was shifted to the permissive temperature in the presence of inhibitors of protein synthesis (56). Millet et a1. (63) have observed a similar modification of the 8 subunit upon treatment of the B. subtilis RNA polymerase with intracellular serine protease from B. megaterium. Recent evidence obtained by Linn et al. (59) indicates that the modification of the 8 subunit by the serine protease is not responsible for the observed changes 20 in template specificity but is an artifact of the pro- cedures used by Leighton et a1. (56, 57) and Millet et a1. (63) to obtain and purify the polymerase. Linn et al. (59) observed that no modification takes place when the cells are harvested rapidly at 0C, immediately washed with a salts medium containing the serine protease inhibitor phenyl methyl sulfonyl floride (PMSF), and if the entire RNA polymerase purification procedure is performed in the presence of PMSF. Although no proteolytic modifi— cation occurs by this procedure the change in template specificity is still evident. This change is due, par- tially, to a loss in activity of the 6 subunit which occurs during the first two hours of sporulation (59). The core enzyme which contains no 6 activity not only loses the ability to transcribe ¢e mRNA, but it also loses the ability to transcribe rRNA in_zi£rg (45). Greenleaf et a1. (30, 31) have purified a polypeptide with a molecular weight of 70,000 daltons which binds to the core polymerase. This polypeptide, which first appears during the third hour of sporulation, appears to be sporulation-specific. Thus, the most recent evidence suggests that the change in template specificity of the RNA polymerase during sporulation is due to loss of 6 activity and the acquisition of a new polypeptide sub- unit during sporulation. 21 Iganslational and Post- Translational Control Fortnagel and Bergman (22) have demonstrated by gel electrophoresis that there are significant quantita- tive differences between the ribsomal proteins of vege- tative cells and those of Sporulating cells and that at least one new protein is present in ribosomes of Sporu- lating cells. They also observed that vegetative cells and ribosomes from vegetative cells are sensitive to the antibiotic fusidic acid, while Sporulating cells and ribosomes from Sporulating cells are resistant. Graham and Bott (Bacteriol. Proc. P. 39, 1974) studied mutants resistant to antibiotics that act at different ribosomal sites and found that some of the mutants grow normally in the presence or absence of the antibiotic but are not able to Sporulate; others grow normally but are only able to Sporulate in the absence of the antibiotic. The differential effects of these antibiotics on growth and sporulation strongly suggests that there are changes in the translational mechanisms of the ribosomes during sporulation. The appearance of some unique Species of tRNA in spores (95) suggests that these may also be involved in translational control during sporulation. The best evidence for post-translational control is the demonstration by Sadoff et a1. (75) that the serine protease converts vegetative aldolase of B. cereus to the Spore form in vitro, and the demonstration by Sadoff 22 and Celikkol (74) that antibiotic is formed by the cleavage of a pre-existing cellular protein by the serine protease. Temperature-Sensitive Asporogenous Mutants A valuable tool for studying gene function during sporulation, which has only recently been utilized, is the temperature-sensitive (Es) asporogenous mutant. Temperature-sensitive mutants can be used to obtain information which totally negative mutants are unable to provide. Point mutants which are ts enable one to determine whether the mutation resides in a structural gene or a control gene; they also permit one to control the phenotypic expression of a Single gene product by shifting the temperature of the cultures. From these temperature-shift experiments, the time period during sporulation in which the function of the mutated gene is indispensible can be determined (17, 42). Szulmajster and co-workers (32, 65, 90) have isolated a Es mutant of B, subtilis (ts-4) which grows normally at both permissive (30C) and restrictive (42C) temperature but is blocked at stage 0 at 42C. The mutant does not produce antibiotic, has low rates of RNA and protein turnover, and is defective in the late enzymes involved in DPA synthesis. Experiments in which the capacity of the mutant to sporulate in cultures shifted 23 from 42C to 30C or from 30C to 42C at various times during growth and sporulation revealed that the tem~ perature-sensitive event occurs in an early stage of spore develOpment and that the loss in sporulation capacity at 42C is irreversible if the shift is made more than one hour after the onset of sporulation. Although the nature of the genetic lesion in the ts-4 mutant is not known, it has been found that stationary phase cells have a low capacity for protein synthesis at 42C, due to the presence of large amounts of an mondialyzable inhibitor which affects amino acylation of phenylalanyl tRNA. The inhibitor is not specific to stationary ts-4 cells. It is also found in exponentially growing cells of the wild-type. The critical factor appears to be in the large quantities of inhibitor found in the mutant during stationary phase. Hitchins and Sadoff (41, 42) have isolated a Es mutant (TH-14) of Bacillus megaterium which has a lesion in a gene essential to the formation of both cell division septa and forespore septa. DNA synthesis continues when the cells are shifted to the restrictive temperature, and the mutant forms multinucleate aseptate filaments. The Sporulation frequency of TH-l4 at the restrictive temperature is approximately 10-5. Experi- ments in which cultures were shifted from permissive to restrictive temperature during sporulation have shown 24 that the septation and engulfment stages are particularly sensitive to the temperature shift. Although there is no qualitative change in extractable membrane proteins when the mutant is grown at the restrictive temperature, there appears to be a partial derepression of a membrane protein(s) with a molecular weight of approximately 80,000, and there is a reduced content of a small molecular weight protein(s). Numerous spherical inclusions, recently identified as glycogen, are present in the cytoplasm of TH-l4 at the restrictive temperature. These are concentrated at only one pole of each filament. Leighton and co-workers have isolated ts mutants of B. subtilis which are blocked in specific functions believed to be involved in the sporulation process (55, 56, 57, 76, 77). One of these mutants (ts-5) has a genetic lesion in the structural gene for a serine pro- tease. When grown at the restrictive temperature, this mutant is blocked at stage 0. Results of temperature- Shift experiments with ts-5 indicated that the serine protease activity is required throughout the sporulation sequence. The evidence from these studies indicated that the serine protease is not responsible for the general intracellular protein turnover. Other mutants isolated by Leighton include ts rifampin—resistant which are also ts RNA polymerase 25 mutants which appear to block sporulation at uniquely different time periods including stages 0, II, and IV. Some of these mutants have very long temperature-sensitive periods while others have very short and discrete tem- perature-sensitive periods (55). One of these mutants (ts-l4) has been studied in detail. The mutation in ts-l4 is expressed during the middle third of the Sporu- lation sequence. Early events such as protease and anti- biotic synthesis are not affected by growth at the restrictive temperature while alkaline phosphatase syn- thesis is blocked (55). Morphological studies of ts-l4 cells grown at the restrictive temperature have revealed that abnormal synthesis of cell wall occurs along the forespore membrane; this prevents the engulfment process and causes abortive Sporulation to occur (77). Bacteriophage and Sporulation The expression of the lytic cycles of several bacteriOphage of Bacillus Species appears to be related in some way to the sporulation properties of the host strains. While studies of this particular phenomenon are not extensive, in at least two instances phage pro- duction has been associated with an impairment in the ability of the host strain to Sporulate. Thorne (92) observed that lysogenic cultures of Bacillus subtilis W-23-Sr produce very low titers of phage SP-10 when they are grown in a medium that produces high yields of 26 spores, and they produce high titers of phage when they are grown in a medium that produce low yields of spores. Ito and Spizizen (49) found that bacteriophage HZ is not able to replicate in mutants of that strain blocked in early sporulation functions. They have suggested that an alteration in the ribonuceic acid polymerase in the mutants or an alteration in the membranes involved in phage restrictions may be responsible for this phenomenon. Goldberg and Gollakota (29) have isolated a bac- teriOphage from B. cereus T which may be related to Sporulation in an entirely different way. This phage does not produce mass lysis of the bacterial culture until after the onset of sporulation, and the production of this phage is inhibited by agents that inhibit Sporu- lation. These observations suggest that replication of this phage may be dependent on changes that occur in the cells during sporulation. REFERENCES REFERENCES Aronson, A. I. 1973. The function of various pro- teases in bacterial sporulation. Colloques Internationoux C.N.R.S. No. 227-Regulation de la sporulation microbienne. Aronson, A. 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Bacteriol. 83:106-111. Vinter, V. 1960. Spores of microorganisms. VIII. The synthesis of Specific calcium and cystine containing structure in Sporulating bacilli. Folia Microbiol. 5:217-230. Vinter, V. 1963. Spores of microorganisms. Non participation of the preexisting sporangial cell wall in the formation of spore envelopes and the gradual synthesis of DAP-containing structures during sporogenesis of Bacilli. Folia Microbiol. 8:147-155. Vold, B. S. and S. Minatogawa. 1972. Characteri- zation of changes in transfer ribonucleic acid during sporulation of Bacillus subtilis. pp. 254- 263. In H. O. Halvorson, R. S. Hanson, and L. L. CampBell (ed.), Spores V. American Society for Microbiology, Washington, D.C. Warren, S. C. 1968. Sporulation in Bacillus sub- tilis. Biochemical Changes. Biochem. J. 109: 811-818. Warth, A. D., D. F. Ohye, and W. G. Murrell. 1963. The composition and structure of bacterial spores. Jo cello BiOlo 16:570—592. Warth, A. D., D. F. Ohye, and W. G. Murrell. 1963. Location and composition of spore mucopeptide in Bacillus species. J. Cell. Biol. 16:592- Yousten, A. A. and R. 8. Hanson. 1972. Sporulation of tricarboxylic acid cycle mutants in Bacillus subtilis. J. Bacteriol. 109:886-894. Yukioka, M., Y. Tsukamoto, Y. Saito, T. Tsuji, S. Otani and S. Otani. 1965. Biosynthesis of gramicidin S by a cell—free system of Bacillus brevis. Biochem. Biophys. Res. Commun. : 204-258. ARTICLE 1 PRODUCTION OF BACTERIOPHAGE BY TEMPERATURE-SENSITIVE SPORULATION MUTANTS OF BACILLUS CEREUS T BY G. N. Stelma Jr. and H. L. Sadoff (Reprinted from the Journal of Bacteriology, Volume 116, pp. 1001- 1010, November 1973, American Society for Microbiology, Washington, D. C. ) JOURNAL or BACTEaioLom', Nov. 1973, p. 1001-1010 Copyright © 1973 American Society for Microbiology til 116' No' 2 V . Prin ed in U.S.A. Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T1 G. N. STELMA, JR., AND H. L. SADOFF Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48823 Received for publication 17 August 1973 ' Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permrssrve temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction ls accompanied, in some ofthe mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage w1th those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 103 plaqueiorming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 X 109 PFU/ml at 37 C and 7 x 108 PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase. / phan auxotroph of B. cereus T, isolated in our laboratory. The tryplophan requirement was a con- venient marker for verifying the origin of the sporula- tion mutants. B. cereus NRRL569 and B. cereus ATCC6464 UM4, which are sensitive to bacteriophage Preliminary investigations of five tempera- turesensitive (ts) sporulation mutants of Bacil< lus cereus T indicated that a sporadically in- duced lytic agent was present in the culture supernatant fluids of all five mutants. Through its ability to form discrete plaques on lawns of senSItive bacteria and through observation of the particles under the electron microscope, we WBre able to identify the lytic agent as a bac— teriophage. The present paper describes the isolation and Preliminary characterization of the ts mu- tants, some physical and biological properties 0f the phage which distinguish it from similar phages previously studied (1, 6, 16, 17: Gold- berg and Gollakata, Bacteriol. Proc. p. 47, 1960', Gollakata and Halvorson. Bacteriol. Proc. p. 86. 1950i, and some aspects of the phagehost cell :lationship in the mutants and in the parent rain. MATERIALS AND METHODS f BaCterial strains and cultivation. The strain mm which our mutants were derived was a trypto- 'Journal Article no. 6l84 trom the Michigan Agricultural Experiment Station. CP-53, and B. cereus ATCC6464. which carries phage CP-53. were obtained from Curtis B. Thorne (Univer— sity of Massachusetts, Amherst). Stocks of all bacter- ial strains were stored in 60% glycerol buffered with 0.1 M sodium phosphate (pH 7.0) at «20 C. These stocks were transferred at 6-month intervals. The stock spore suspensions, from which inoculations were made routinely, were grown either in G medium (8) or CH medium at 26 C, washed three times with distilled water, and stored in distilled water at 4 C. Chemicals. The N-methyl-N-nitro-N-nitrosoguan» idine (NTG) was purchased from the Aldrich Chemi- cal Co., Milwaukee, Wis. The acridine orange was purchased from Allied Chemicals, New York. The 2,3,5-triphenyltetrazolium chloride was obtained from General Biochemicals Inc., Chagrin Falls. Ohio. Pancreatic ribonuclease (RNase) (grade A) was a product of Sigma, St. Louis, Mo. Pancreatic deoxy- ribonuclease (DNase) was obtained from the Cali- fornia Corporation for Biochemical Research. Los Angeles, Calif. Media. Skim milk agar was used for detection oi asporogenic mutants, which form translucent colonies on this medium. whereas sporulating B. cereus form llllll 1002 white colonies. The exoprotease activity of the mu- tants was simulataneously scored on this medium, which consisted of 0.3% skim milk (Carnation), 0.02% yeast extract (BBL), and 1.5% agar (Difco). The skim milk was sterilized separately and added just before the plates were poured. Modified G medium was prepared by the method of Hashimoto et al. (8). CH medium was the same composition as modified G medium except 0.6% vitamin-free casein hydrolysate (NBC) was substituted for 0.2% yeast extract. Bromo- cresol purple medium had the same salt and yeast extract concentrations as modified G medium but contained only 0.2% glucose instead of the usual 0.4%, and 0.015 g of bromocresol purple (Difco) per liter. NBY medium consisted of 0.8% nutrient broth (Difco) and 0.3% yeast extract (15). The soft agar used in overlays contained 0.5% agar. All media except the NBY medium were supplemented with ‘20 pg of L~tryptophan per ml. Isolation of mutants. An effort was made to isolate temperature—sensitive mutants which were protease negative and asporogenic at the restrictive tempera- tures. Nutrient broth cultures, started from spores heat-shocked at 70C for 30 min were harvested in mid-exponential growth phase by centrifugation at 12,000 x g for 10 min at 4 C. The cells were washed twice with an equal volume of 0.05 M tris(hydroxy- methyl)aminomethane—maleic acid buffer, pH 6.0. sus- pended in the same buffer containing 200 pg of NTG/ml, and shaken for 30 min at 30 C. Normally 80 to 90% of the cells were killed by this procedure. The surviving cells were washed with sterile G medium, resuspended in G medium, and grown at 37C on a New Brunswick model G76 water bath-shaker at 240 rpm. The culture was observed periodically under the phase microscope until most cells reached the fore- spore stage and became irreversibly committed to sporulate (5). At this time. glucose and yeast extract were added to final concentrations of 0.4 and 0.2"}. respectively. This supplement permitted the growth of asporogenous mutants while most of the cells in the Culture were completing sporulation. When free spores were observed, the culture was harvested. washed, and resuspended in sterile G medium. A .1 ml amount of these cells was used to inoculate .50 ml of fresh G medium, and the enrichment tor asporogenic mutants was repeated. On completion of the semnd enrichment, the cells and spores were harvested by centrifugation. washed with sterile 0.1 M phosphate buffer (pH 7.0), and suspended in 6091' glycerol. Samples of cells in glycerol were stored at 20 C. Dilutions of these samples which produced .50 to 100 colonies per plate were spread on skim milk agar, and the plates were incubated at :17 (‘. After 12 h of growth at .37 C the colonies were examined for pro» tease activity, and those having no apparent activity were restreaked on fresh skim milk medium The plates were then placed in the 37C incubator for another 12 h, and after '24 h total incubation replicate plates were made. These were exposed to chloroform for 5 min and incubated at 1.7 C for 12 h. Colonies which produced Spores alter 24 ii were not sensitive to the chloroform treatment and were able to grow on the replicate plates, whereas asporogenous mutants failed STELMA AND SADOFF J. BACTERIOL. to grow on the replicate plates. All mutants which were asporogenic at 37 C were restreaked and grown at 26 C to test their ability to sporulate at lower temper atures. Isolation of revertants. Cultures of the ts mutants were grown in modified G medium at .37 C. A sample of early stationary- phase cells was diluted and spread on nutrient agar plates for determination of the total viable count. Forty- eight hours after inoculation. a second sample was removed and placed in a screw-cap vial which was submerged in a 70 C water bath for :10 min. The surviving spores were spread on skim milk medium and incubated at 37 C. Revertant colonies could be distinguished after 24 h of growth by their white appearance. The reversion frequency was deter mined by dividing the number of revertants per milli- liter by the total viable count per milliliter. Electron microscopy. Phage were produced on plates in soft agar overlays. They were prepared for electron microscopy by scraping the son agar layer from the plates. mixing in a small volume or 0.1 .\1 sodium phosphate buffer (pH 7.0). and sedimenting at 12,000 x g to separate the cells and the agar from the phage. Alternatively. the supernatant fluids of broth cultures were spun at 125,000 x g for 2 h. and the resultant phage pellet was suspended in phos- phate buffer. The phage were stained with 05'? phosphotungstic acid (PTA) and were observed and photographed under a Philips EM<300 elecrron micro- scope. Propagation and assay of phage. Broth cultures of the ts B. cereus mutant JSZ'Z Iyse spontaneously due to the induction of a lysogenic hacieriophage (LP-‘22). The initial propagation of LB?) was as follows. Mutant JS22 was grown from a spore inocu- lum in 100 ml of CH medium at 37 C for 10 11 into the stationary phase. The cells were pelleted by centrilu» gation at 12,000 \ g for 10 min in a Sorvull Rt‘LZ-B centrifuge. The supernatant fluids were 1hen sulr jected to 125,000 \ g in an International preparative ultracentril‘uge (model BBO) for 2 h at 4 C to pellet the phage particles. The phage were washed with 1’1 peptone (Difco). resuspended in 10 ml of 1H peptone. and stored at 4 C over chloroform. Phage CP-Sil was induced in B. cereus A'I‘t‘t‘tflttl by the method of Yelton and Thorne (16) and was stored in l“; peptone at 4 C. Spontaneously cured strains of mutant .1522 were isolated in the following manner. Flasks (.500 ml) containing .50 ml of nutrient broth were moculnted with 3 \ 10" heat»shocked spores per ml and incu- bated at 37 C. After approximately four generations. samples were removed. diluted, and spread on nutri- ent agar plates. The plates were incubated at :17 C for 12 h, after which time smooth colonies were selected and tested for sensitivity to the phage and for phage activity in their culture supernatants. Phage isolates were assayed by standard techniques using soft nutrient agar overlays seeded with cells from a cured strain of .1522 (.JS22-C). Phage (‘I’a'm was assayed by the method of Yelton and Thorne I 161 using lawns of B. cereus A'I‘CC6464 UM4. Preparation of antisera. Antisera to the newly isolated phage. 1.1122, and to CP—53 were prepared in VOL. 116, 1973 the same manner. A 2»ml amount of phage suspended in sterile saline at a concentration of 10W plaque- forming units (PFU)/ml was mixed with an equal volume of Freund complete adjuvant and injected subcutaneously into rabbits. Booster injections of 10m PFU/ml in Freund incomplete adjuvant were given at 3. to 4-day intervals. A total of six injections was given. The rabbits were bled from the ear vein 1 week after the last injection. The antisera and the preimmune sera taken from each rabbit before the first injections were sterilized by filtration through membrane filters (pore size 0.45 pm; Millipore Corp), heated at 56C for 30 min to destroy the complement, and stored at 7‘20 C. Nucleic acid staining. Suspensions of phage LP-22 (10“ PFU/ml) in 0.1 M sodium phosphate buffer (pH 7.0) were treated with 0.1% pancreatic RNase and 0.2% pancreatic DNase plus 10.: M Mg“ at 37 C for 2 h. The phage were then pelleted by centrifugation at 125,000 x g, washed with 0.1 M sodium phosphate buffer, and resuspended in buffer at a concentration of 10” PFU/ml. Four drops of the phage suspension were added to each of several slides. The slides were treated with Carnoy fixative (2), then with RNase, DNase, or buffers containing no nucleases. and stained with acridine orange as described by Bradley (2). The stained preparations were observed under a Blak-Ray long-wave ultraviolet lamp (Ultra-Violet Products Inc., San Gabriel, Calif). Ultraviolet irradiation. The phage were sus- pended in 10 ml of 0.1 M sodium phosphate buffer (pH 7.0) in a glass petri dish 9 cm in diameter and irradiated with a 30-W General Electric germicidal ultraviolet lamp at a distance of 40 cm. During exposure, the phage suspension was stirred constantly on a magnetic stirrer. Time course of phage production. The time course of phage production and maximal PFU per milliliter were measured in cultures of both JS22 and the parent strain at 26 and at 37 C. Flasks (500 ml) containing 100 ml of CH medium were inoculated With 3 x 106 spores per ml (heat-shocked at 70 C for 30 min) and incubated on the water bath-shaker at 240 rpm. Growth was followed by measuring absorbance at 620 nm on a Bausch and Lomb Spectronic 20 Spectrophotometer. At various intervals, samples were removed and subjected to centrifugation at 12,000 X g for 15 min to remove the cells. The Supernatant fluids were treated with a few drops of chloroform to kill any residual cells, diluted, and tltrated by the agar overlay technique with strain JS22-C as the indicator strain. RESULTS Isolation of mutants. The original screening was for temperature-sensitive protease mutants with impaired ability to sporulate at 37 C, the restrictive temperature. However, of 25,000 col- onies screened for protease activity, only 20 protease mutants were observed. None of these mutants was completely blocked in exoprotease secretion, and all but one sporulated normally. In addition to the protease mutants, 924 muA PHAGE INDUCTION IN 8. CEREUS 1003 tants were obtained which had translucent colonies on skim milk agar, rather than the normal white colonies. Since early~--_....z 7 ..n - A "mm." M... .- was observed in cultures of the parent strain immediately after To' but in cultures of the mutant this increase was not seen until T3 (Figure 3). Later events in sporulation also appeared to be delayed with respect to the time of expression. The develOpment of refractility occurred between T and T in cultures of the parent strain 10 16 and between T13 and T19 in cultures of the mutant (Figure 3). Cultures of the parent strain produced approximately 6 x 108 heat-stable (70 C, 30 min.) Spores per m1, while those of the mutant produced 3.5 x 108 heat-stable spores per m1. Temperature-sensitive period. Temperature shift experiments.were performed with the mutant strain JSZZ-C to determine the time period in which the ts gene func- tioned and to determine whether the loss in sporulation capacity at 37 C was reversible. A similar experiment was.performed using the parent strain to determine whether the temperature shift had any effect on its capacity to sporulate. The results of two typical shift down- eXperiments with mutant J822-C are shown in Figure 4. The pH changes were identical in both experiments. Sub- cultures shifted from 37 C to 26 C at T1 or earlier were 61 able to recover completely and produce approximately the same number of heat-stable Spores per ml as cultures maintained at 26 C. If subcultures were shifted after T0.5 the sporulation capacity of the mutant decreased progressively. If the shift was made at T less than 3! one percent of the cells were able to Sporulate and, if it was made at T4 or later, the cells were unable to sporulate. Measurements of the final pH of the sub- cultures revealed that the cells were unable to oxidize organic acids in the medium if the shifts were made at T4 or later. In similar experiments subcultures of the ts mutant were shifted up from 26 C to 37 C (Figure 5). Those shifted at T4 or earlier Sporulated at very low frequencies. Sporulation capacity increased signifi- cantly if the shift was made at T and reached near 5 maximal values if the shift was made at T suggesting 6 that the ts gene product was required until T6‘ Thin sections of J822-C cells removed from 26 C cultures at T6 were examined under the electron microscope. All cells had formed completed or nearly completed forespore septa, but none had begun the engulfment process. These observations indicate that the cells were at the end of Stage II (forespore septum formation) when the function of the ts gene was completed. The appearance of a typical cell at T is shown in Figure 6. 6 62 Measurements of the pH in the culture fluids at T revealed that all subcultures shifted after T had 30 1 gained the capacity to derepress the enzymes of the tri- carboxylic acid cycle; while those shifted before Tl never gained that capacity. These data suggest that the expression of that gene was necessary before the tri- carboxylic acid cycle could become functional. Although expression of the ts gene until T was sufficient to l derepress the tricarboxylic acid cycle enzymes, continued function of this gene until T was necessary before 6 sporulation could occur at near normal frequencies (Figure 5). Effect of temperature shift on tricarboxylic acid cycle activity. Since mutants of Bacillus subtilis blocked in the inability to produce a functional tri- carboxylic acid cycle have been observed to be impaired in their ability to sporulate (3, 6, 9), the possibility that the genetic lesion in J822-C resided in a structural gene for one of the tricarboxylic cycle enzymes was investigated. The extent of oxidative metabolism and thus, indirectly, the activity of the tricarboxylic acid cycle was determined by dye reduction studies. Rates of oxidation determined in this manner for the parent strain were similar to those previously reported by Gollakota and Halvorson for B. cereus (7). Cultures of the parent strain and mutant were shifted from 26 to 37 C at the 63 times when the rise in pH was first detected in the culture medium (Figure 3). Activities of the tricarboxylic acid cycle were eXpected to the highest at these times. When J822-C cultures were shifted at T3, the rate of oxygen consumption 30 min. after the shift had increased sig- nificantly over the initial rate, indicating that the activity of the tricarboxylic acid cycle had increased (Table 1). One hour after the shift the rate of oxygen consumption had decreased somewhat but was still higher than the initial values. The maximal rate found at T3.5 was almost as high as the maximal rate attained by 1. If the temperature-sensitive protein of the mutant were one of the tricarboxylic acid the parent strain at T cycle enzymes a significant decrease in the activity of 'the tricarboxylic acid cycle would have occurred rather than the increase that was observed. Cultures of the mutant shifted from 26 C to 37 C at T and shifted back 3: to 26 C at T4, produced 40% fewer heat-stable spores than control cultures of JSZZ-C which were left at 26 C. Sporulation was thus much more sensitive to the temper- ature shift during this interval than was the activity of the tricarboxylic acid cycle. Cultures of the mutant shifted to 37 C at T1 had no detectable oxidative capacity at the time of the shift (Table 1). At T significant activity was detected 2 although this activity was low compared to the activities 64 observed in the parent strain. This activity remained about the same at T and declined sharply at T4. The 3 initial increase in oxidative metabolism, coupled with the slow decrease after the shift to 37 C, is inconsistent with the supposition that the temperature—sensitive protein was one of the tricarboxylic acid cycle enzymes. DISCUSSION Growth studies of mutant J822—C at 37 C have shown that the function of the ts gene is not essential for normal vegetative growth or for the production of proteases and alkaline phosphatase at the onset of sporu- lation. However, the function of the ts gene appears to be essential for the expression of a functional tricarbo- xylic acid cycle and for the cells to undergo any of the morphological changes associated with sporulation. Cells of JSZZ-C maintained at 37 C throughout growth were blocked at stage 0 of sporulation. Growth studies of the ts mutant at 26 C revealed that it was slightly impaired in its ability to sporu- late at that temperature. The mutant at 26 C produced only 60% as many spores as cultures of the parent strain. At the permissive temperature the number of heat-resistant spores per ml produced was 105 fold greater than the number produced at 37 C, the restrictive temperature. At the permissive temperature, the expression of the tricarboxylic acid cycle enzymes and the development of refractility did not occur in the mutant cultures 65 66 until about three hours after they had occurred in the parent strain. However, the kinetics of exoprotease production were the same in both mutant and parent strain. These observations are in accord with the data obtained in studies at the restrictive temperature show— ing that protease production is not impaired by the mutation. Those cellular activities leading to Sporu- lation which are blocked at 37 C appear to be impaired in JSZZ-C at 26 C. Those not blocked at the restrictive temperature are normal at the permissive temperature. ‘ Experiments in which cultures of the mutant were shifted from 37 C to,g61C (Figure 4) or from 26 Cito 37 C (Figure 5) at various times during the growth cycle have shown that the temperature-sensitive event occurs very early in the Sporulation sequence, those cells of JSZZ-C maintained at 37 C for four or more hours after the end of exponential growth cannot recover the ability to pro- duce spores if they are shifted to 26 C. Morphological studies have shown that the expression of the ts gene begins at the sporulation stage preceding the formation of the axial filament (Figure 2) and ends when the fore- spore septum has been completed (Figure 6). Since the tricarboxylic acid cycle is not functional in the mutant at 37 C, and a block in any one of the enzymes of that cycle has been shown to impair the ability of mutants to Sporulate (3, 6), the 67 temperature-sensitivity of the tricarboxylic acid cycle of JSZZ-C was investigated. The results of these studies (Table 1) indicated that the temperature-sensitive lesion had not occurred in any of the structural genes which code for the tricarboxylic acid cycle enzymes or for any of the electron transport components ultimately reacting with the dye. The initial increase followed by a slow decrease in oxidative metabolism after the mutant cultures were shifted from 26 C to 37 C would not be expected if one of the many proteins involved were subject to decay at the higher temperature. If any one of the enzymes involved in oxidation of organic acids were subject to decay at a very slow rate at 37 C, some oxidative metabolism should have been detectable in cultures of the mutant maintained at 37 C throughout growth. However, cultures maintained at 37 C and assayed several times between T0 and T5 did not produce detectable dye reduction (unpublished results). The delay in expres- sion of oxidative metabolism (TCA cycle activity) in cultures grown at 26 C is not consistent with the sup— position that the ts mutation affects the activity of one of the individual enzymes of that cycle. The hypothesis that the lesion has affected a control gene which must be eXpressed before derepression of the tri- carboxylic acid cycle can occur is more consistent with our data. 68 Cultures of the mutant growing at 26 C have under- gone derepression of the tricarboxylic acid cycle at T4 (Figure 3, Table 1); but these cultures are not able to produce a significant number of spores if they are shifted to 37 C. Thin sections of cells removed from 26 C cultures and fixed at T4 have been compared with thin sections of cells removed from cultures which were shifted from 26 C to 37 C at T4. The cells which were shifted up were observed to be arrested at the stage of morphological develOpment they had attained at the time of the shift (unpublished observations). These obser- vations imply that the function of the ts gene is neces- sary for the expression of at least one sporulation- specific event that occurs after the tricarboxylic acid cycle has been derepressed. The ts mutation which blocks the ability of this mutant to sporulate at 37 C also appears to be responsible for the induction of LP22, a bacteriophage carried by the parent strain in a latent form. Revertants of mutant J822 selected for their ability to sporulate at 37 C concurrently lost the ability to induce the phage at high frequencies (34). Revertants of J822-C which were able to sporulate at 37 C at high frequencies did not support plaque formation as well as JSZZ-C or as partial rever- tants which sporulated at frequencies of 1% or less. This inverse relationship between the ability of cured 69 strains to sporulate and their ability to support plaque formation further supports the hypothesis that the ts mutation somehow affects the stability of the phage-host relationship between LP-22 and B. cereus (34). Studies of the P22-Sa1monella typhimurium system have shown that the establishment of lysogeny or repli- cation depends upon the level of cyclic adenosine 3', 5' - monophosphate (cAMP) in the host cells. Under con- ditions of strong catabolite repression, when the supply of energy and biosynthetic components is abundant and the concentration of cAMP is low, the phage multiplies and lyses the cell. When the supply of energy is deficient and the concentration of cAMP is high the phage lysogenizes the cell (16). Although cAMP is apparently absent from cells of Bacillus (18, 33), some alternative signal must be used to indicate a depletion in energy levels since sporulation has been shown to be subject to catabolite repression (32) and dependent on the energy charge of the cell (17). If the lytic cycle of LP22, like that of P22, is dependent on the energy level of the host cell, a mutation which produces a state of strong catabolite repression could cause induction of phage to occur at an increased fre- quency and concomitantly prevent derepression of sporu- lation. 70 If the ts mutation in J822 has affected the ability of the cells to overcome catabolite repression, it has affected a specific mechanism for release of catabolite repression rather than a generalized mechanism. This is evident from the ability of the mutant to synthe- size normal levels of exoprotease at the restrictive temperature. Aronson et al. (1) have demonstrated the existence of closely related but distinct catabolic con- trols for extracellular protease production and for spore formation in Bacillus cereus. Furthermore, the parent and mutant strains undergo diauxic growth at 37 C in modified G-medium containing low levels of glucose supplemented with maltose or sucrose. Thus normal cata— bolite repression seems to function in the mutant J822-C but the release of special catabolite repression associated with certain sporulation events seems to be blocked. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant Al-01863 from the National Insti- tute of Allergy and Infectious Diseases. G. N. Stelma, Jr., is a Public Health Service Trainee on grant GM-019ll from the National Institute of General Medical Sciences. We thank H. Stuart Pankratz for assistance with the electron microscopic studies. 71 LITERATURE CITED Aronson, A. I., N. Angelo, and S. C. Holt. 1971. Regulation of extracellular protease production in Bacillus cereus T. J. Bacteriol. 106:1016-1025. Beaman, T. C., H. S. Pankratz, and P. Gerhardt. 1972. Ultrastructure of the exosporium and underlying inclusions in spores of Bacillus megaterium strains. J. Bacteriol. 109:1198-1209. Carls, R. A., and R. S. Hanson. 1971. Isolation and Characterization of tricarboxylic acid cycle mutants of Bacillus subtilis. J. Bacteriol. 106:848-855. Doi, R. H., and T. J. Leighton. 1972. Regulation during initiation and subsequent stages of bac- terial sporulation. pp. 225-232. In H. O. Halvorson, R. Hanson and L. L. Campbell (ed.). Spores V. American Society for Microbiology, Washington, D.C. 72 73 5. Fahmy, A. 1967. An extemporaneous lead citrate stain for electron microsc0py, pp. 148-149. In C. J. Arceneaux (ed.) 25th Proc. electron microscopy soc. Amer. 1967. Claitor's Book Store, Baton Rouge, La. 6. Fortnagel, P., and E. Freese. 1968. Analysis of sporulation mutants II. Mutants blocked in the citric acid cycle. J. Bacteriol. 25:1431-1438. 7. Gollakota, K. G., and H. O. Halvorson. 1961. Bio- chemical changes occurring in Bacillus cereus during sporulation. pp. 113-119. I2_H. Orin Halvorson (ed.). Spores II. Burgess Publishing Co., Minneapolis, Minn. 8. Guha, S. 1973. Studies on in zitrg protein syn- thesis in a thermosensitive sporulation mutant (ts-4) of Bacillus subtilis. 9. Hanson, R. S., J. Blicharska, and J. Szulmajster. 1964. Relationship between the tricarboxylic acid cycle enzymes and sporulation of B. subtilis. Biochem.-Biophys. Res. Commun. 11:1-7. 10. Hanson, R. S., J. A. Peterson, and A. A. Yousten. 1970. Unique biochemical events in bacterial sporulation. Ann. Rev. Microbiol. 24:53-90. (11. Hanson, R. S., V. R. Srinivason, and H. O. Halvorson. 1963. Biochemistry of Sporulation.l II Enzymatic changes during sporulation of Bacillus cereus. J. Bacteriol. 86:45-50. 12. l3. 14. 15. l6. 17. 74 Hageman, J. H. and B. C. Carlton. 1973. Effects of mutational loss of Specific intracellular pro- teases on the sporulation of Bacillus subtilis. J. Bacteriol. 114:612-617. Hashimoto, T., S. H. Black, and P. Gerhardt. 1960. Development of fine structure, thermostability, and dipicolinate during sporogenesis in a Bacillus. Can. J. Microbiol. 6:203-212. Hitchins, A. D., and H. L. Sadoff. 1972. Cell division and sporulation septation in Bacillus megaterium. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.). Spores V. American Society for Microbiology, Washington, D.C. Hitchins, A. D., and H. L. Sadoff. 1974. Proper- ties of a thermosensitive asporagenous filamentous mutant of Bacillus megaterium. J. Bacteriol. 118:1167-1175. Hong, J. S., G. R. Smith, and B. N. Ames. 1971. Adenosine 3':5'-cyclic mon0phosphate concentration in the bacterial host regulates the viral decision between lysogeny and lysis. Proc. Nat. Acad. Sci. U.S. 68:2258-2262. Hutchinson, K. W., and R. S. Hanson. 1974. Adenine nucleotide changes associated with the initiation of Sporulation in Bacillus subtilis. J. Bacteriol. 119:70-75. 75 18. Ide, M. 1971. Adenyl cyclase of Bacteria Arch. Biochem. Biophys. I44;262-268. 19. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Electron microscopic study of DNA-containing plasma. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in dif- ferent physiological states. J. Biophys. Biochem. Cytol. 4:671-678. 20. Kornberg, A., J. A. Spudich, D. L. Nelson, and M. G. Deutscher. 1969. Origin of proteins in sporu- lation. Ann. Rev. Biochem. 31:51-78. 21. Leighton, T. J. 1973. An RNA polymerase mutant causing temperature-sensitive sporulation in Bacillus subtilis. Proc. Nat. Acad. Sci. U.S. 10:1179-1183. 22. Leighton, T. J., R. H. Doi, R. A. J. Warren, and R. A. Kelln. 1973. Relationship of serine pro- tease activity to RNA polymerase modification and sporulation in Bacillus subtilis. J. Mol. Biol. 16:103-122. 23. Leighton, T. J., P. K. Freese, R. H. Doi, R. A. J. Warren, and R. A. Kelln. 1972. Initiation of sporulation in Bacillus subtilis: Requirement for serine protease activity and ribonucleic acid polymerase modification. pp. 238-246. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.). Spores V. American Society for Microbiology, Washington, D.C. 24. 25. 26. 27. ‘28. 29. 30. 76 Levisohn, S., and A. I. Aronson. 1967. Regulation of extracellular protease production in Bacillus cereus. J. Bacteriol. _I:1023-1030. Luft, J. H. 1961. Improvements in epoxy resin methods. J. Biophys. Biochem. Cytol. 2:407-414. Millet, J. 1970. Characterization of proteinases excreted by Bacillus subtilis Marburg strain during sporulation. J. Appl. Bact. 32:207-219. Murrell, G. W. 1967. The biochemistry of the bac— terial Spore. In A. H. Rose, and J. F. Wilkinson (ed.). Advances in Microbial Physiology Vol. I. Sabatini, D. D., K. Bensch, and R. J. Barnett. 1963. Cytochemistry and electron microscopy. The pre- servation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell. Biol. Ilzl9-58. Santo, L., T. J. Leighton, and R. H. Doi. 1972. Ultrastructural analysis of sporulation in a con- ditional serine protease mutant of Bacillus sub- EIIIg. J. Bacteriol. III:248-253. Santo, L., T. J. Leighton, and R. H. Doi. 1973. Ultrastructural studies of sporulation in a con- ditionally temperature-sensitive ribonucleic and polymerase mutant of Bacillus subtilis. J. Bacteriol. 115:703-706. 31. 32. 33. 34. 35. 36. 37. 77 Schaeffer, P. 1969. Sporulation and the production of antibiotics, exoenzymes, and exotoxins. Bact. Rev. 23:48-71. Schaeffer, P., J. Millet, and J. Aubert. 1965. Catabolite repression of bacterial sporulation. Proc. Nat. Acad. Sci. U.S.A. Eiz704-7ll. Setlow, P. 1973. Inability to detect cyclic AMP in vegetative or Sporulating cells or dormant spores of Bacillus megaterium. Biochem. Biophys. Res. Commun. 52:365-372. Stelma, G. N., Jr., and H. L. Sadoff. 1973. Pro- duction of bacteriophage by temperature-sensitive sporulation mutants of Bacillus cereus T. J. Bacteriol. 116:1001-1010. Szulmajster, J., C. Bonamy, and J. Laporte. 1970. Isolation and properties of a temperature- sensitive sporulation mutant of Bacillus sub- tilis. J. Bacteriol. 101:1027-1037. Warren, S. C. 1968. Sporulation in Bacillus sub- EIIIg. Biochemical Changes. Biochem. J. I92: 811—818. Watson, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. BiOphys. Biochem. Cytol. 4:475-478. 78 TABLE 1. Oxygen consumption by cells of thenparent strain and mutant J822—C after shifting growth temperature from 26 C to 37 C Oxygen consumption Sample To T0.5 T1 T2 T3 T3.5 T4 parent strain 1.8 7.8 8.8 8.3 - - - shifted at T0 shifted at T3 mutant J822-C - - 0 3.0 2.7 - 1.0 shifted at T1 Cultures of the parent strain and mutant JSZZ-C were grown in G medium at 26 C. At the times indicated, above the cultures were shifted to 37 C. Oxygen con- sumption was measured immediately and several times after the shift. Oxygen consumption was measured by measuring the rate of reduction of the dye 2, 6 dichlorophenol- indolphenol at 540 nm by whole cells. In the presence of KCN, the dye becomes the terminal electron acceptor. Two moles of the dye reduced are equivalent to a mole of oxygen consumed. Oxygen consumption is expressed in ul 02 consumed per 108 cells per h at STP. 79 .cflmnum usmnmm muflawpomnmmu o “Ulmmmb mpfl>fiuom mmmmpoumoxm < “samuum usmumm mufiflpom mmmmuoumoxm 4 “Ulmmmb mm _U “aflmnpm vacuum mm I “Ulmmmb ommao o “:Hmuum ucmnmm ommoo O "maonemm OB mo mfinwu CH Ummmmumxm ma mEHH .Um:HMon mosam> HmEmeE mnu mo pamoumm mo mason ca cmmmmumxm mum muonu .pmHSmmmE Omam mumB MDHHfluomnmmu mo ucmEmon>mU map can mpfl>fluom mmmmuoumoxm Hmuoe .Umnsmmme Oman mums modam> mm .COHUMHsHomm mo pomso tam susoum Hwflpsmcomxm mo cam may .08 msflfiumump on Hmpno CH .ommoo CH mwmcmco mcflusmmme an Umuouficoa mmB susouo .AO nmv chapmummsmp w>HuoHHpmmH map #8 unmusfi asp tam samuum ucmumm mzu mo fiuaouo .H mmeHm .smfifimfi.. 80 PERCENT OF MAXIMUM 9.- on: OCT ow ., ad 1. o5 ., m6 1. ON ., ms : mm _ — F - h _ _ _ 81 FIGURE 2. Electron micrograph of a longitudinal section of the mutant after growth at 37 C for the time required for the parent strain to sporulate under the same conditions. The bar in the lower left-hand corner represents one micron. 82 83 .Ulmmmb muflafluomummu 0 “Gasman ucmumm muflaflpomuwmu o “Ulmmmb mufl>fluom mmmmpoumoxm < ucflmnum pcmnmm MuH>Huom mmmououmoxm 4 “Olmmmb man. “GHMUBm usmumm mm I “Olmmmb ommao o 250qu ucmumm ommoo o umHOQEMm .susoum Hwfiucmcomxm mo tam msu .09 mo memo» Ga pmmmmnmxm ma mafia .pmsflmuno mosam> HmEmeE may wo uswoumm mo mfiumu ca pmmmmumxm mum hpflafluomummu paw mafi>flpom mmmmuoumoxm .cmndmmme omam mumz muflaflp locummu mo usmfimoam>op map tam >ua>fluom mmmmuoumoxm Hopou .mm as momsmsu .omwao CH mmmcmso mcflusmmme mg pwuouflcoe mwz cusouw .AU mmv musumummBmu m>HmmHEHmm mnu um pcmusa Ugo sflmuum ucmumm map mo cuzouu .m MMDon 84 PERCENT OF MAXIMUM 18 20 16 TIME 85 .mm 4 “N quEHHmmxm monomm quumHmmHIummC o “a UCmEHHmmxm .mmuomm quumHmmulummC . "maonsmm .quEHummxm mCu mo mmusoo mCu UCOCmCOHCu 0 um um meHmqumE Ulmmmh mo mmuspaso CH pamflw muomm may mUCmmmHmmn 0 mm meMCmHmmU 0 Hon mCB . B um 0 mm cu UODMHCm mumB COHCB Camuum qunmm map mo mmusuaso CH Gama» macaw map mqummummH 3 meMConmU non 0C8 .4NB um consumme u . 4m mos mm mCB CHE om How O on um mCflummC Hmumm 9 pm mCOHmCmmwsm mnomm map mCflumam an pmuCCoo mum? mmuomm quumHmmHIummm .Cuzoum HMHquComxm mo 6C0 map mmUMCmflmmp OB .maomo Cuzoum mCu OCHHCU mmEHu pCmHmmmap um O mm 0p 0 am scum czooumeMACm mausuaso onmmmm mo conumHsnomm .4 mmoon 86 HRH—Am RC @529 o _ a 1 — _ _ _. ~b 'IW HHd SEIHOcIS 90'] 60 l l N l V- 1 l 87 .mm 4 “N quEHHmmxm .mmuomm qupmHmmulummC o “a UCmEHHmmxm .mmuomm pCmpmHmmulummC o "maonshm .quEHHmmxm me mo mmusoo map uCOCmCOHCu 0 mm um UmCHmuCHmE Olmmmb mo mwusuaso Ca mpamflm whomm may mqummummu 0 mm meMCmHmmU Hon OCH .09 pm 0 mm on UOMMHCm muo3 CUHC3 Cflmuum “Comma me mo monsuaso CH pamflh muomm map mUCmmmummu DB pauMCmflme Hon mCB .omB um Consumme mos mm 0C9 .Cfla om How O on an mCHummC Hmpmm ome um mCOHmCmm Imsm whomm mCflumam ha UmuCCoo mum3 mmuomm quumHmmulummm .Cuzonm amapCmC Iomxm mo UCm 0C» wmmemHmmp OB .maoho Cuzonm map mCHHCC mmaflp quHmMMHU no 0 mm on 0 mm scum msuemuCHnm mmusnaso cummmn Co coanmasuomm .m mmaon 88 l I I k . 'IW 3:5ch I l I 4 l I I I I I to m — SHHOdS DO'I TIME OF SHIFT 89 FIGURE 6. Electron micrograph of a longitudinal section of one cell and part of a longitudinal section of a second cell from a sample of JS22—C removed at T6 from a culture growing at 26 C. The bar in the lower left-hand corner represents one micron. 90 APPENDIX APPENDIX SOME FURTHER CHARACTERISTICS OF THE LP-22 - BACILLUS CEREUS T PHAGE-HOST RELATIONSHIP 91 (ABSTRACT) The high frequency of induction of bacteriophage LP-22 by cells of temperature-sensitive sporulation mutant J822 is accompanied by a high frequency of spon- taneous curing in the mutant strain. This curing is not due to the occurrence of the phage genome as a non- integrating plasmid. Although the phage is induced in the mutant at high frequencies, it appears to be unin- ducible in the wild-type. Treatments with ultraviolet light, mitomycin C, and nIs dichlorodiammineplatinum (II) and growth at elevated temperatures all fail to cause phage induction in the wild-type. Although 99% of the phage induced by mutant JS22 do not appear in the culture fluid until after the end of exponential growth, the phage does not appear to require sporulation specific RNA polymerase or protein synthesizing machinery for replication. The appearance of the phage at that time may be due to the fact that conditions for induction are less favorable during exponential growth. LP-22 does not appear to be capable of mediating generalized transduction. 92 93 LP-22, a bacteriophage of unusual morphology, was discovered in lysates of temperature sensitive (En) mutants of Bacillus cereus T which were blocked in an early function of sporulation and which had concurrently become sensitive to lysis by the phage. Preliminary studies suggested a relationship between the inability of the mutants to Sporulate and the increased sensitivity of the mutants to lysis by the phage. The mutants pro- duced titers of phage as high as 3 X 109 plaque forming units (pfu)/ml; while the parent strain produced titers of 104 pfu/ml or less. Single step revertants of one of the mutants, JS 22, produced titers approximately the same as those produced by the parent strain. Over 99% of the phage released by the mutants did not appear until after the end of exponential growth. Many of the phage particles released by the mutants were morphologi- cal variants having tails of different lengths with unusual plate-like structures placed at irregular intervals along the tails and perpendicular to the length of the tails (8). A better understanding of factors which influence the stability of virus-host relationships could be achieved through knowledge of the reasons for the dis- ruption of the normally stable LP—22-B. cereus T phage- host relationship by mutation to asporogeny. This investigation was therefore undertaken to gain further 94 information on the nature of the LP-ZZ—B. cereus phage- host relationship in both wild-type and mutant strains and to determine whether LP-22 could be used in genetic studies as mediator of generalized transduction. MATERIALS AND METHODS Bacterial Strains and Phage. The bacterial strains used in this study were a tryptOphan auxotrOph of Bacillus cereus T and two In Sporulation mutants, JSZZ and J829, which were derived from the try-auxotroph. The auxotrOphs used in the transduction experiments, J83 nnn’:§n:, JSll E15 :Sn:, and JS32 SEE :Sni, were all derived from wild-type B. cereus T. The procedures for cultivation and maintenance of these strains and for the propagation and assay of the phage (LP-22) were described in an earlier publication (8). Screening for Clear-Plaque Mutants. In attempts to find spontaneous clear-plaque mutants, 109 to 2.5 X 109 pfu of LP-22 were added to 3.0 ml of soft nutrient agar (0.5% agar) which had been seeded with B. cereus try-; the seeded agar was layered over nutrient agar, and the plates were incubated at 37 C. After 12 to 15h incu- bation the plates were examined for clear plaques. Mutagenesis by nitrous acid was accomplished by mixing 2.0 m1 of LP-22 (1.5 x 1010 pfu/ml) with 1.0 m1 60mM sodium nitrate and 1.0 m1 1.0M sodium acetate buffer 95 96 (pH 4.2) and allowing the mixture to stand at room tem— perature. At 30-minute intervals samples were removed, diluted 1:10 in nutrient broth and added to soft agar seeded with B. cereus try-. The last sample was removed at 3h. Hydroxylamine treatments consisted of mixing 10 0.5 ml LP-22 (1.5 X 10 pfu/m1) with 1.0 ml 4M hydroxyla- mine, l.0 ml 1.5M NaOH, and 2.5 ml 0.M NaHPO buffer 4 (pH 6.0) and incubating the mixture at 37 C. In the period 6h to 10h after incubation, samples were removed at 2h intervals, diluted 1:10 in nutrient broth and added to soft agar seeded with B. cereus try“. Detecting cured strains. Individual colonies of mutant J822 and of the parent strain were tested for their capacity to produce LP-22 by three different methods. The first method consisted of replicating plates containing colonies derived from single spores unto plates which had been overlayed with soft nutrient agar (0.5% agar) seeded with JSZZ-C, incubating at 37 C and looking for lysis of the lawns. The second method consisted of overlaying the colonies with soft nutrient agar which had been seeded with JSZZ-C, incubating at 37 C and looking for zones of lysis around the colonies. The third, and most effective method, consisted of inoculating 5.0 m1 nutrient broth with cells from single isolated colonies, growing the cultures overnight at 97 37 C and testing the supernatant fluids for phage by spotting 0.2 ml onto plates overlayed with soft agar which had been seeded with J322—C. Cesium chloride-ethidium bromide density gradients. Twenty ml of modified G medium (1) supplemented with casein hydrolysate (0.2%) in place of yeast extract, deoxyadeno- sine (250 g/ml), and 3H thymidine (0.1 m Ci, New England Nuclear) were inoculated with 2 X 108 spores of B, cereus try-'or J822 and were grown at 37 C until early stationary phase. The cells were pelleted by spinning at 10,000 rpm for 10 min (4 C) and were washed with cold TES buffer [0.02m tris (hydroxymethyl) aminomethane (Tris), 0.01 MCaCl, 0.005 M ethylenedromine-tetraacetic acid (EDTA), pH 7.5]. Lysates were prepared by the method of Lovett (4), with one modification: immediately after pronase treatment the cells were frozen and thawed six times to achieve complete lysis. The final volume of the lysate was adjusted to 5.0 ml with TES buffer, and 6.9 grams of CsCl (General Biochemicals) was added. A 7.0 ml amount of the resulting solution was mixed with 3.0 ml ethidium bromide (EB; Sigma, 4 mg/ml in 0.1 M phosphate buffer pH 7.0) and distributed equally between two polyallomer tubes. The tubes were topped with mineral oil, and Spun for 42 h at 36,000 rpm in a Ti 50 fixed angle rotor at 15 C in a Beckman model L3-50 preperative ultracentrifuge (4). Seven drop fractions (approximately 0.15 ml) were 98 collected through holes pierced in the bottoms of the tubes, and 10 pl portions from each were spotted on squares (1.0 cm) of Whatman no. 1 filter paper. The squares were dried, washed in cold 10% trichloraacetic acid (TCA), rinsed in 95% ethanol, and dried. After drying, the squares were placed in scintillation vial containing 5.0 ml toluene scintillation fluid [toluene containing 0.4% 2, 5 diphenyloxazole (PPO) and 0.005% 1, 4 -di- 2 — (5 phenyloxozolyl) - benzine (dimethyl POPOP)] and counted on a Packard Tricarb liquid scintillation counter. Induction of LP-22. Several procedures were used in attempts to induce LP-22 from wild-type B. cereus. In the first procedure, approximately 108 cells were sus- pended in 6.0 ml sterile saline in a glass petri dish and were exposed to ultraviolet light (G.E. 30 watt ger- micidal lamp) at a distance of 45 cm for 30 sec. The cells were agitated constantly during the exposure. The surviving cells (approximately 37% of the initial number) were used to inoculate 500 ml flasks containing 50 ml nutrient broth. These cultures were grown at 37 C for periods ranging from 2h to 5h and the number of pfu of LP-22 in the supernatant fluids determined by plaque assay on lawns of J822-C (8). Both exponentially growing cells and sporulating cells were subjected to this treat- ment. 99 In the second procedure, 5.0 ml subcultures were removed from a 100 m1 culture of wild-type B, cereus which was growing in modified G medium at 37 C and were treated with 2 mg/ml mitomycin C for 10 min. The cells were spun down at 10,000 rpm for 10 min., washed once with 0.1 M phosphate buffer (pH 7.0) and resuspended in modified G medium with no glucose. After 5h incubation at 37 C the cultures were assayed for the number of phage in the supernatant fluids. The subcultures were taken 2h before the end of exponential growth; at the end of exponential growth, and 1h, 2h, and 3h after the end of exponential growth. In the third method, cultures of wild-type E. cereus growing in nutrient broth at 37 C were treated with gInfdichlorodiammineplatinum (II) at concentrations ranging from 0.1 umolar for a period of 2.5h (7); and supernatant fluids were assayed for phage. The final method consisted of growing wild-type B. oereus in nutrient broth at 45 C and assaying supernatant fluids of exponentially growing cultures and of stationary cultures for phage. Production of phnge during exponential growth. Early exponential cultures of JSZZ-C and JS29 were infected with LP-22 at a multiplicity of infection of 1.0. Growth of infected cultures and uninfected control cultures was followed by measuring OD at 30 min. 620 100 intervals. The number of phage in the culture super- natant fluids were assayed on lawns of JSZZ-C. Isolation of auxotrophs. Nutrient broth cultures of wild-type B. cereus were harvested during mid-exponen- tial growth by spinning at 10,000 rpm for 15 min. The cells were washed with an equal volume of tris-maleic acid buffer (pH 6.0), resuspended in 50.0 ml of the same buffer containing 100 pg of N-methyl-N-nitro-N-nitro- soguanidine (NTG, Aldrich Chemical Co.), and incubated with shaking at 30 C and 200 rpm for 30 min. Normally 80 to 90% of the cells were killed during this treatment. The cells were washed with 50.0 ml double strength nutrient broth, resuspended in 50.0 ml fresh double strength nutrient broth, and incubated on a reciprocal shaker at 30 C and 200 rpm for 3h, to allow for segre- gation of the mutant genomes and dilution of the wild-type enzymes. The cells were again spun down at 10,000 rpm for 15 min., washed with minimal medium (5), resuspended in minimal medium containing 200 ug D-cycloserine/ml (Nutritional Biochemicals) and incubated on the shaker for 5h at 30 C. During this treatment approximately 99% of the cells were killed. The cells were again spun down, washed with double strength nutrient broth, to remove the D-cycloserine, and grown in double strength nutrient broth for 6h at 30 C and 200 rpm. 101 Samples were removed from the enriched culture, diluted to yield about 100 colonies per plate, and spread on nutrient agar plates. After the plates had been incubated for 12h at 30 C, replicate plates were made on minimal agar. Colonies which failed to grow on the minimal medium after 8h incubation at 30 C were restreaked on minimal medium and incubated overnight. All strains which failed to grow were screened for specific growth requirements on plates of minimal agar supplemented with pools of various amino acids, vitamins, and nitrogen bases. Streptomycin resistant strains were derived from each stable auxotrOph for use in transduction experiments. Transduction. The phage used in the transduction experiments were obtained from infected cultures of J829. Optimal numbers of pfu/ml were obtained from nutrient broth cultures infected during early exponential growth at a multiplicity of infection of 0.1. The phage sus- pensions were passed through a 0.45 filter (Gelman) before their use in the experiments. The procedures used in the transduction experiments were the same as those of Yelton and Thorne (10) except each set of plates was done in triplicate rather than in duplicate, and the experiments were repeated with cells which had reached the end of exponential growth. Multiplicities of infection varied from 1.0 to 3.0. RESULTS Clear-plague mutants. Although large numbers of phage have been treated with nitrous acid and hydroxyla- mine, no clear plaque mutants have been isolated. The lack of clear-plaque mutants makes it impossible to study phage replication in the wild-type at this time. High frequency of spontaneous curing. The first indications that mutant J822 became cured of the LP-22 genome at a high frequency were the observations that serial subculturing of single colonies produced cured strains (8) and from data concerning the stability of phage in heated and unheated Spores (Table l). Colonies derived either from heat-shocked spores or from unheated spores were overlayed with soft agar seeded with J822-C, and the plates were incubated at 37 C for 12h.. The colonies which produced phage caused a localized clearing of the indicator strain. The number of colonies producing detectable titers of phage increased with the age of the colonies. After 24h the entire lawns underwent lysis; so it was not possible to determine the percentage of colonies capable of producing phage after longer periods 102 103 of incubation. Only a little over one-third of the colonies derived from single spores produced detectable phage. This was true of colonies derived from heat- shocked spores and from unheated spores (Table 1). These data indicated that nearly two-thirds of the spores had either become cured or were not induced until they were very old. Since the agar overlay method did not appear to be a highly sensitive method for measuring the capacity of an individual colony to produce phage, the experiment was repeated using a more sensitive technique. Indi- vidual colonies, which had been derived from spores, were used to inoculate tubes containing 5.0 ml nutrient broth. The cultures were incubated for 24h, spun down at 10,000 rpm for 10 min. and 0.2 ml samples from each supernatant fluid were spotted on sensitive lawns. After 12 h incubation at 37 C, the plates were examined for lysis. Only 11 of 20 cultures produced phage. The colonies which did not produce phage had become sensi- tive to plaque formation by the phage; therefore they appeared to be cured. Attempts to repeat this experiment with the parent strain were not successful. Cultures of the parent strain sometimes produced as few as 5 to 10 pfu/ml, and these were not always detected during the first screening. In order to obtain higher phage yields 104 from the parent strain so that cured strains could be readily observed, a means of inducing the phage was sought. Attempts to induce the phage by treatment with ultraviolet light, mitomycin C, and cis-dichlorodiam- mineplatinum (II) all failed both in exponentially grow- ing cells and in sporulating cells. Attempts to induce the phage by raising the temperature of incubation to 45 C also failed. Cesium chloride - ethidium bromide density gradient centifugation. The higher frequency at which cured strains of J822 were found would not be expected to occur in cells carrying an integrated prophage; therefore the possibility that LP-22 was carried as a nonintegrating plasmid was investigated. Cleared lysates obtained from wild-type B. cereus, which had been labeled with 3 H thymidine, were layered on a cesium chloride-ethidium bromide solution and subjected to centrifugation until equilibrium was reached. Figures 1 and 2 show that there was only one peak of radioactivity in the gradients. If plasmid DNA had been present a second, smaller, peak composed of covalently-closed circular DNA should have been present in the denser fractions nearer to the bottom of the tube (6). A control experiment was performed using E. coli JC 411 (col. E.), and the results indicated that plasmid DNA was present (data not shown). 105 Although the LP-22 genome did not appear to occur as a nonintegrating plasmid in the wild-type cells, it was possible that it was unable to integrate properly, and did occur as a plasmid in JS22. To determine whether J822 carried the phage as a plasmid, cleared lysates of that strain were subjected to the same procedure used for the wild-type. The results (Figure 2) indicated that JSZZ did not carry plasmid DNA. Infection of exponentially growing cells. The observed release of 99% of the total pfu of LP-22 by mutant JSZZ after the end of exponential growth (8) could have been due to infection of the cured cells by the few phage which were released during exponential growth com- bined with an unusually long latent period. Alterna— tively it could have been due to the fact that conditions during exponential growth were less favorable either for phage induction or for phage replication. It was of particular interest to determine whether exponentially growing cells were able to replicate the phage since failure to do so could indicate a requirement by the phage for a sporulation-specific RNA polymerase or Sporulation-specific protein synthesizing machinery. The ideal way to determine whether the phage could be replicated in vegetative cells would be to infect the wild-type cells with a clear-plaque mutant and to look for massive lysis accompanied by a large increase in 106 the number phage in the supernatant fluid. Since no clear-plaque mutants have been found, the experiment was performed by infecting J829, a mutant that has lost its superinfection immunity and forms clear plaques (8). A nutrient broth culture of J829 was divided into two portions; one was infected during the early stages of exponential growth at a multiplicity of infection of 1.0; the other was not infected. The growth of both cultures was followed by measuring the OD on a B. 620 and L. Spectronic 20 spectrophotometer. The final number of pfu/m1 was measured at 2.5h by standard plaque assay methods. Figure 3 shows that lysis did occur during exponential growth. The phage titers 2.5h after infection had risen from an initial 107 pfu/ml to 9 x 109 pfu/m1 indicating that the drOp in OD was 620 not due to lysis from without but to the production of bursts of phage by the infected cells. When the same experiment was performed with J822-C very little lysis was observed, but phage titers did increase from an initial 3.5 x lo6 pfu/ml by the end of exponential growth. The number of phage present in the supernatant fluid at the end of exponential growth was approximately 35% of the total number found 5.5h later. These data indicate that the phage could be replicated and assembled by vegetative cells. The data from the experiment in which J822-C cells were infected imply that most of the 107 phage enter into a stable lysogenic or pseudolysogenic relationship with that strain, rather than producing a burst. Transduction. It was of great interest to deter- mine whether LP-22 was capable of mediating generalized transduction; since only two phage have been shown to mediate generalized transduction in B. cereus, and only one of these is effective with n. cereus T (9, 10, 11). Preliminary experiments to determine whether LP-22 could mediate generalized transduction were per- formed using three auxotrophs: J83 (pur_8mr), JSll (try-Smr), and JS32 (arg-Smr) as recipients. Although a few spontaneous revertants of strains J83 and J832 were observed, no transductants were observed in any experiments. DISCUSSION The loss in the ability of 45% of the strains derived from single spores of mutant J822 to produce plaque-forming particles of phage LP-22, accompanied by the acquisition of sensitivity of these strains to plaque formation by LP-22, strongly suggests that these strains have been "cured" of the phage genome. This high frequency of curing is not due to the occurrence of the LP-22 genome as nonintegrating plasmid in the host cells. This high frequency of curing has not been seen in the parent strain, and the parent strain appears to carry the phage as a stable lysogen (8). Therefore, it is probable that the genetic lesion that occurred in J822 has altered that mutant in such a way that it is no longer able to carry the phage as a lysogen but carries it as a pseudolysogen. The pseudolysogenic relationship of phage SP-lO and Bacillus subtilis (3) shows some similarity to the relationship between LP-22 and J822. Cultures of B, subtilis W-23-Sr produce high titers of SP-lO and, simultaneously, a high frequency of cured cells. The cured cells are believed to result 108 109 from a lack of coordination in the replication of the phage genome and the bacterial genome which causes the phage genome to be unavailable for distribution to both daughter cells at division time. The observed ability of LP-22 to replicate in exponentially growing cells of J829, a mutant which has lost superinfection immunity, indicates that the phage probably does not require a sporulation-specific ribo- nucleic acid polymerase or Sporulation-specific protein synthesis in order to replicate. It is unlikely that J829 vegetative cells possess a sporulation-specific RNA polymerase or a modified protein synthesizing system since they grow normally at the restrictive temperature and are blocked at stage 0 of sporulation. It would be desirable to verify this result by infecting the parent strain with a clear-plaque mutant of LP-22, but no clear-plaque mutants have been isolated at this time. Determination of the latent period and burst size also depend on the isolation of a clear-plaque mutant. Single step growth studies using J829 as the host have not been successful due to a high "background" caused by the spon— taneous induction of phage by the cells. The inability of LP-22 to transduce three auxo- trophic mutants which have lesions in three unrelated biosynthetic pathways indicates that it is unlikely that LP-22 is capable of mediating generalized transduction in Bacillus cereus T. LITERATURE CITED Hashimoto, T., 8. H. Black and P. Gerhardt. 1960. Development of fine structure, thermostability, and dipicolinate during sporogenesis is a Bacillus. Can. J. Microbiol. 6:203-212. Ikeda, H. and J. Tomizawa. 1968. Prophage Pl, an extrachromosomal replication unit. Cold Spring Harbor Symp. Quant. Biol. 33:791-798. Kawakami, M. and O. E. Landman. 1968. Nature of the carrier state of bacteriophage 8p-10 in Bacillus subtilis. J. Bacteriol. 95:1804-1812. Lovett, P. S. 1973. Plasmid in Bacillus pumilus and the enhanced sporulation of pIasmid-negative variants. J. Bacteriol. 115:291-298. Nakata, H. M. 1964. Organic Nutrients required for growth and sporulation of Bacillus cereus. J. Bacteriol. 88:1522-1524. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dye-bouyant-density method for the detection and isolation of closed circular duplex DNA in Hela cells. Proc. Nat. Acad. Sci. 57:1514-1521. Reslova, 8. 1971/1972. The induction of lySOgenic strains of Escherichia coli by cis dichloro- diammineplatlnum (II). Chem.-BloI. Interactions 4:66-70. Stelma, G. N. Jr. and H. L. Sadoff. 1973. Pro- duction of bacteriOphage by temperature-sensitive sporulation mutants of Bacillus cereus. T. J. Bacteriol. 116:1001-1010. Thorne, C. B. 1968. Transducing bacteriophage for Bacillus cereus. J. Virol. 2:657-662. 110 111 10. Yelton, D. B. and C. B. Thorne. 1970. Transduction in Bacillus cereus by each of two bacteriophage. J. Bacteriol. 102:573-579. ll. Yelton, D. B. and C. B. Thorne. 1971. Comparison of Bacillus cereus bacteriophage CP-51 and CP-53. J. 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