'umsl. LIBRARY Michigan State ‘ University ‘ This is to certify that the 7 thesis entitled REPLACEMENT OF Caz” BY EXOGENOUS NUCLEOTIDES AS A GROWTH FACTOR FOR YERSINIA PESTIS AT 37 0 presented by Robert John Zahorchak has been accepted towards fulfillment of the requirements for Ph. D. degree in Microbiology Major professor DateWW/L /,, /f77 0-7 639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. G) 1979 ROBERT JOHN ZAHORCHAK ALL RIGHTS RESERVED REPLACEMENT OF Caz+ BY EXOGENOUS NUCLEOTIDES AS A GROWTH FACTOR FOR YERSINIA PESTIS AT 37 C By Robert John Zahorchak 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 1978 ABSTRACT REPLACEMENT OF Ca2+ BY EXOGENOUS NUCLEOTIDES AS A GROWTH FACTOR FOR YERSINIA PESTIS AT 37 C By Robert John Zahorchak Cells of virulent 1, pestis, a facultative intracellular parasite. require physiological levels of Ca2+ (2.5 mM) for sustained growth jg_vitro at 37 C. This concentration of Ca2+ is not found in mammalian intracellular fluid. In the restrictive environment (no Ca2+) the virulence antigens, V and N, which are essential for success- ful invasion of the host, are expressed. Exogenous ATP allowed for a energy charge in Ca2+-starved and Ca2+-supplemented cells was determined as 0.57-0.67 for the former and 0.75-0.84 for the latter. Exogenous ATP allowed for a growth response equivalent to that of a Ca2+-sufficient control culture. The growth response was dependent upon the concentration of ATP and Mg2+ as well as the pH of the culture medium. Other 5'-ribonucleotides that could replace Ca2+ were ADP, AMP, GTP, GDP, and CTP. The addition of UMP, CMP, or common free bases or ribonucleosides failed to promote growth. An intermediate response was obtained with UTP, UDP, GDP, and GMP. Cells grown in the presence of [8-140] ATP did not incorporate the label into TCA—precipitable material. Cells cultivated in Ca2+- supplemented media failed to express the virulence antigen. Robert John Zahorchak However, the specific activity of V antigen measured in extracts of cells grown in the presence of exogenous ATP was similar to that observed in Ca2+-starved non-growing cells. These findings suggest that certain nucleotides may serve a regulatory role, allowing for both the growth of 1. gggtis and expression of the virulence antigens, in the intracellular environment of the host. ACKNOWLEDGMENTS I am grateful to Dr. R. N. Costilow and Dr. H. L. Sadoff for the use of their laboratory equipment. I wish to express my appreciation for the technical assistance of Janet Fowler. The suggestions openly offered by Dr. R. J. Patterson, Dr. L. R. Snyder, and Dr. N. Band have been very helpful. The advice and guidance of my major professor, Dr. R. R. Brubaker, have been a great asset to my education. For this I wish to extend my sincere appreciation. ii ORGANIZATION This thesis is organized in such a way as to fulfill the departmental requirement for a publishable manuscript as well as to provide the reader with background material helpful in under- standing the implications of the results presented. Section I consists of a review of the literature pertinent to the research discussed. Section II is a manuscript prepared to send to internal review and subsequently to Current Microbiology. The third section consists of data obtained that is related to that presented in Section II. A more complete discussion of the results obtained is also presented in Section III. An appendix consisting of a detailed description of the methods used to determine the adenylate energy charge is also included. iii TABLE OF CONTENTS Page SECTION I Literature Review . . . . . . . . . . . . l SECTION II ARTICLE: REPLACEMENT OF Ca2+ BY EXOGENOUS NUCLEO— TIDES AS A GROWTH FACTOR 0F YERSINIA PESTIS AT 37 C . . . . . . 12 Abstract . . . . . . . . . . . . . . l3 Introduction . . . . . . . . . . . . l5 Materials and Methods . . . . . . . . . . 17 Results . . . . . . . . . . . . . . 21 Discussion . . . . . . . . . . . . 38 Literature Cited . . . . . . . . . . . 43 SECTION III Introduction . . . . . . . . . . . 46 Materials and Methods . . . . . . . . . . 47 Results . . . . . . . . . . . . . . 50 Discussion . . . . . . . . . . . . . . 61 APPENDIX Determination of the Adenylate Energy Charge in _.pestis. . . . . . . . . . . 69 LIST OF REFERENCES . . . . . . . . . . . . . . 76 iv Tables l. LIST OF TABLES Page Inability of Y. pestis sto incorporate exogenous C] ATP. . . . . 31 Effect of various ribonucleotides on the growth of Vwa+ Y. pestis EV76 at 37 C . . . . . . . . . 33 Expression of V antigen . . . . . . . . . . . 37 Phenotypes of Y. pestis . . . . . . . . . . 49 Adenine nucleotide pools in Vwa gestis sstrain EV76 cultivated under various condit1ons . . . 5l Growth response of various strains of Y. estis in the presence and absence of ATP in media .suppe mnted with 20 mM MgClz. . . . . . . 55 Effect of pH on growth of various strains of Y. pestis. at 37 C . . . . . . . . . . . . 60 ‘1 LIST OF FIGURES Figure Page 1. ATP Effect . . . . . . . . . . . . . . . 23 pH Effect . . . . . . . . . . . . . . . 25 Dependence on ATP Concentration . . . . . . . . 28 Inability of 1, pgstj§_to Utilize Exogenous ATP . . 30 ATP-stimulated Growth of I, pgstj§_strain KIM . . . 35 2* Effect . . . . . . . . 53 ATP Antagonism of Ca Effect of MgClz and ATP on the Growth of Y, pestis . 57 Effect of MgCl2 and ATP on the Growth of Y, pestis . 59 tomNOSU'I-hwh) Effect of Mg2+ on the Growth of 1, pestis . . . . 65 vi SECTION I LITERATURE REVIEW Virulence and Yersinia gestis The plague has been of historical significance to man possibly since as early as l320 B.C. (El). Epidemics and pandemics of this disease have been responsible for the deaths of large percentages of the human population. Killing 25 to 40 million people, the Black Death of 1347 A.D. - 1350 A.D. was one of the most disastrous of these (65). In modern times the institution of public health practices and the use of insecticides have been largely responsible for the containment of plague. Even though epidemics are rare, the disease is endemic in the rodent population in many areas of the world (4). The fact that the disease is still prominent, although not overtly manifest, maintains the scientific concern to understand plague. The bacteria that causes the bubonic plague, Yersinia pestis, easily lends itself to scientific investigation of the pathogenic process. The ease with which it is cultivated in the laboratory has helped to establish the organism as a model system to understand infectious disease in general as well as to define specifically the variables of significance in the pathogenesis of plague. The results of investigations into various facets of the plague comprise a considerable amount of material that has been reviewed extensively (8,l7,l8,20,6l). This literature review is written in order to present those findings of significance in understanding the physiology 0f.!:.2§§£l§ and its relationship to the disease caused by the organism. The etiologic agent of bubonic plague was discovered in l894 by Alexander Yersin (77). The gram-negative bacillus was subse- quently classified as Pasturella pestis (29). Recently, the organism has been reclassified and a new genus, Yersinia, in honor or the discoverer, was defined (62). The genus is composed of three recognized species, 1, pestis, 1,_p§eudotuberculosis, and Y, entercolitica. A fourth member, I. ruckeri, has been nominated (3l). These bacteria have been of scientific interest, mainly to medical bacteriologists, because of their pathogenic properties. Organisms of the three accepted species are all facultative intra- cellular parasites. It is of interest to note that all of these organisms appeared to prefer the fixed macrophages of the mesenteric lymph nodes, liver, and spleen as the host-cell of choice to invade (44,77). The extreme potential for wild-type Y. gestis to infect and disease laboratory animals has been documented (23). The average lethal dose necessary to kill fifty percent of test animals (L.D.50) was observed to be less than ten organisms for both guinea pigs and mice when wild-type 1, p§§31§_was injected by the intraperitoneal route. Three pathologically distinct forms of plague have been defined: pneumonic, bubonic, and septicemic. Each form of the disease is acute and the mortality is very high in untreated cases (29). Pneumonic plague, the most acute, results from infection of the lungs by I, pggtis. This form of plague is transmitted from person to person by aerosol. The most common form of plague, bubonic, is transmitted from rodents to man via an insect vector, the flea. Septicemic plague may be merely a manifestation of either pneumonic or bubonic plague where the bloodstream is invaded before the classic clinical symptoms have surfaced. Much research has been done in order to identify the specific properties of X, 23311; that correlate with the disease producing capabilities of the organism. To date, five phenotypic character- istics have been demonstrated to be directly associated with the pathogenicity of Y. pestis; pigmentation (Pgm), fraction I antigen (Fra), purine independence (Pur), pesticin I (Pst), and the V and W antigens (Vwa). These five virulence determinants have been discussed in detail by Burrows (l7) and Brubaker (8). A brief description of each follows. Wild-type Y, pg§31§.(Pgm+) formed pigmented colonies on the hemin agar of Jackson and Burrows (42) and on the congo red agar of Surgalla and Beesley (69). Mutation resulting in the non-pigmented phenotype (Pgm') occurred at a spontaneous rate of about 10'5 mutations per bacterium per generation (10). An L.D.50 of greater than 107 was observed when mice were experimentally infected with non-pigmented mutants (43). Fra' cells were unable to synthesize the fraction I or capsular antigen and were decreased in virulence for guinea pigs 4 3 to 106) but were fully virulent for mice (23). The (L.D.50 of lo possibility that the fraction I antigen rendered Y, pestig less susceptible to phagocytosis by polymorphonuclear leukocytes, thereby enhancing the invasiveness of the bacteria, has been investigated by Janssen et al. (45). These investigators showed that there was a marked difference in resistance to phagocytosis among the various strains of Y. pestis. They correlated resistance to phagocytosis with the presence of a visible capsule (fraction 1). It has been demonstrated that purine auxotrophs of Y, pestis (Pur') were avirulent (l6). Brubaker (ll) showed that mutation eliminating guanosine monophosphate synthetase activity increased the intraperitoneal lethal dose in mice to greater than l07, whereas mutation affecting the enzymes necessary for the synthesis of inosine monophosphate lowered the virulence of the bacteria only slightly (L.D.50 of approximately 102). Virulent 1, pestig synthesize a bacteriocin that is active against certain strains of Escherchia coli, Y, pseudotuberculosis, and 1. enterocolitica (8). The role of pesticin in disease produc- tion ble, pgstj§_is obscure but it may be significant that the synthesis of the protein (41) is correlated with that of coagulase and fibrinolysin (7). With respect to the latter point it is important to state that, although non-pesticinogenic mutants (Pst') appeared to be avirulent when introduced by the intraperitoneal route, wild-type virulence was observed when the same mutants were injected intravenously into mice (l2,l4). Cells of Y. pgstj§_and Y, pseudotuberculosis must possess the ability to express two antigens, V and W, in order to be fully virulent (15,l9,22). Mutants that did not possess this character- istic exhibited a mouse intraperitoneal L.D.50 of greater than 107 (23). Vwa+ cells also displayed a requirement for relatively high concentrations of calcium ions (2.5 mM) as a growth factor at 37 C but not at 26 C (39). The expression of V and W antigens and the Ca2+ requirement will be discussed in detail in a later section of this review. Y,ng§ti§ rendered avirulent by the mutational loss of any of the first four determinants defined above, have been shown to exhibit wild-type virulence under certain conditions. Pgm- and Pur- mutants exhibited wild-type virulence if the challenge dose was accompanied by an injection of iron or purine, respectively (16, 43). It was suggested that these mutants were avirulent due to their inability to procure sufficient iron or purine for growth inside the host. If, however, the concentrations of these compounds were artificially increased, the invading bacteria would no longer be nutritionally deprived and would cause disease as easily as wild- type 1,‘pg§tj§, As already mentioned, Fra' cells were observed to be fully virulent in mice (23) and wild-type virulence was observed with intravenously injected Pst' Y, pestis (12). To date there is no known manipulation of either the host or the bacteria that results in Vwa- 1, pgstis exhibiting L.D.50 values similar to those observed with wild-type cells. The expression of the virulence antigens appears to be essential for a successful invasion of the host by 1. eggs. Burrows (l5) and Burrows and Bacon (22) discovered two antigens, V and W, in cultures of phagocytosis-resistant virulent Y, pggtjs, The V and W antigens were undetectable in phagocytosis- sensitive avirulent cells. It had already been documented that there was a strong correlation between the disease producing ability of these bacteria and their ability to resist phagocytosis (16,22). It was suggested therefore that the role of the antigens with respect to virulence was to render Y, pestis resistant to phagocytosis. The results obtained by Janssen et al. (45) did not support this hypothesis. Their observations that fixed macrophages phago- cytized virulent cells as readily as avirulent cells led them to suggest that the V and W antigens were important to the survival and multiplication of the bacteria after they had been phagocytized by fixed macrophages. The virulent cells were detected in the blood- stream much earlier than were the avirulent cells (44). Attempts to demonstrate a difference between Vwa+ and Vwa' Y, pggtis with respect to their ability to survive and multiply within macrophages in cell culture have indicated that, in these systems, there is no signifi- cant difference (46). The expression of the antigens is a temperature dependent phenomenon. V and W were detected in cultures of Y. pggtig that were incubated at 37 C but not in those at 26 C (l5,22). Lawton et al. (50) noted that 20 mM Mg2+ enhanced the expression of the antigens and they were able to purify V antigen lOO fold and W antigen 1000 fold. Analysis of these preparations resulted in identifying V as a protein of 90,000 daltons and W as a lipoprotein of 145,000 daltons. The mechanism by which the expression of the antigens is regulated is not known. These same authors demonstrated that antibodies to V antigen could protect mice from experimental plague (50). This observation further indicated the importance of the virulence antigens in the pathogenesis of plague. Vwa+‘1,‘pg§ti§ displayed a requirement for at least 2.5 mM 2+ or An2+) when cultivated in a defined Ca2+ (replaceable by Sr medium at 37 C (38,39). Vwa' mutants did not require Ca2+. Cells of neither phenotype require the cation at 26 C. The mutation from Vwa+ to Vwa- occurred at a spontaneous rate of 10'4 (39) and appeared to be irreversible (21). These observations implied the presence of a plasmid that carries the genetic information necessary for Y, pggtis to express the virulence antigens. No plasmid has been identified by cesium chloride-ethidium bromide density gradient methodology (52). More recently, however, analysis of gently prepared cell extracts by agarose gel electrophoresis revealed the presence of plasmids in Y, pggtis (D. Ferber, personal communica- tion). None of these plasmids as yet have been correlated with the Vwa or any other virulence determinant. In vitro studies performed in order to ascertain the effect on Vwa+ I. gestis cultivated at 37 C revealed that Ca2+- of Ca2+ starved cells were morphologically and physiologically different 2+ from cells cultivated at 26 C or at 37 C in Ca ~supplemented media (35.51.76). Ca2+ starvation resulted in elongated cells with a diffuse nucleoid (35). DNA synthesis and cell growth ceased after a time that allowed two doublings in mass (76). Significantly, it was only under these "restrictive" conditions that V and W were expressed jg_!jxg_(13). Brubaker (9) compared the jg 315:9 Ca2+-deficient environ- ment with that of the intracellular environment of the phagocytic cell and suggested that this ion may regulate the expression of V 2+-starved and W ig_vivo. The conflicting observations that Ca 1, pestis cells cannot multiply lg vitro at 37 C while they pro— liferate in the essentially Ca2+-free intracellular environment 1 vivo have not been resolved. -Bacterial Cell Growth and the Adenylate Energy Charge The ability of a bacterial cell to utilize various compounds as energy sources and/or substrates for the components of the cell is tightly coupled to the growth rate of that cell. Some of the mechanisms by which enzymes involved in macromolecular synthesis are regulated have been elucidated by nutritional shift or growth state transition experiments. When a bacterial cell such as an E, ggflj_is starved for an essential nutrient such as a nitrogen or a carbon source, dramatic effects on macromolecular synthesis are observed. Classically, net RNA and protein synthesis cease. This is followed sequentially by the cessation of DNA synthesis and cell division (55). It has recently been shown that one of the earliest defined events observed in nitrogen-starved E, coli was a drastic change in nucleotide pools (73). The cytoplasmic concentration of all ribo- nucleoside and deoxyribonucleoside triphosphates dropped before net accumulation of RNA and protein stopped. In addition, the concentra- tion of ppGpp or magic spot I rose drastically and reached maximum levels after cell growth had ceased. The nucleotides, ppGpp and pppGpp, accumulate in many bacteria as a result of amino-acid or ammonium ion starvation (24). Only the former nucleotide has been observed in cells starved for a carbon source (34,56). The concentration of the "common" nucleoside triphosphates (ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and TTP) varies directly with the growth rate (58) unlike those of the magic spot compounds which vary inversely with growth rate (24). Based upon experiments with various enzymes, Atkinson (2) proposed that the activities of energy-producing and energy-consuming enzymes in vitro are more influenced by the ratio of adenine nucleo- tides in the cell than by the absolute concentrations of these compounds. The development and definition of the adenylate energy charge theory has been presented (1,3). Briefly, the metabolic energy immediately available to a cell is reflected in the degree of phosphorylation of the adenylate pool. A linear measure of this parameter can be achieved by applying the equation: [ATP] + I [ADP] [ATP] + [ADP] + [AMP] (2). The importance of this parameter in the whole cell has been investigated by Chapman et a1. (27). For instance, it has been 10 shown that the value of the energy charge (EC) is generally above 0.8 for most growing organisms that have been studied (see compiled data in ref. 27). In dividing E, 5911 the EC appears to be stabilized around a value of 0.8 or higher regardless of the growth rate (70). The EC value observed for stationary phase cells slowly decreased from 0.8 to 0.5 over a period of about 60 h (27). During this time the viability of the cells remained constant. However, a decrease in viability was observed when the EC dropped below 0.5. Variations in energy charge have been observed after nutrient deprivation of glucose-limited E. 3911 (27). Adenine limita- tion of an adenine auxotroph of E, Egli_resulted in a drop in EC value from 0.8-0.9 to 0.6-0.7 after growth had ceased (70). In the case of adenine limitation, the total adenylate pool fell to a relatively low level before any significant change in the EC value was observed. Investigations with other organisms, in general, support the concept that Atkinson originally presented. The parameter is generally accepted as a measure of the metabolic state of a cell. Caution should be exercised, however, before applying hard-fast rules with respect to energy charge and bacterial cell growth. For instance, it has been reported that iron limitation of Candida utilis resulted in a low EC value (0.6). These cells, however, continued to grow (71). Furthermore, although it might be inferred from the above discussion that an extremely low EC value corresponds to cell death, it had been shown that freshly isolated Rickettsia typhi exhibit EC values of about 0.2-0.5 (74). 11 These cells were presumably viable based on results of metabolic activity and infectivity (74). SECTION II ARTICLE REPLACEMENT OF Ca2+ BY EXOGENOUS NUCLEOTIDES AS A GROWTH FACTOR FOR YERSINIA PESTIS AT 37 C By R. J. Zahorchak and R. R. Brubaker (Manuscript to be published) 12 ABSTRACT REPLACEMENT OF Ca2+ BY EXOGENOUS NUCLEOTIDES AS A GROWTH FACTOR FOR YERSINIA PESTIS AT 37 C By Robert John Zahorchak Cells of virulent 1, gestis, a facultative intracellular parasite, require physiological levels of Ca2+ (2.5 mM) for sustained growth jg_vitro at 37 C. This concentration of Ca2+ is not found in mammalian intracellular fluid. In this restrictive environment the virulence antigens, V and W, which are essential for successful invasion of the host, are expressed. Exogenous ATP allowed for a growth response equivalent to that of a Ca2+-sufficient control culture. The growth response was dependent upon the concentration of ATP and M92+ as well as the pH of the culture medium. Other 5'-ribonucleotides that could replace Ca2+ were ADP, AMP, GTP, and CTP. The addition of UMP, CMP, or common free bases or ribonucleo- sides failed to promote growth. An intermediate response was obtained with UTP, UDP, GDP, and GMP. Cells grown in the presence of [8-14C] ATP did not incorporate the label into TCA-precipitable material. Cells cultivated in Ca2+-supplemented media failed to express the virulence antigen. However, the specific activity of V antigen measured in extracts of cells grown in the presence of 13 l4 exogenous ATP was similar to that observed in Ca2+-starved non-growing cells. These findings suggest that certain nucleotides may serve a regulatory role, allowing for both the growth of 1, ggggig and expression of the virulence antigens, in the intracellular environ- ment of the host. INTRODUCTION Yersinia pestis, the causative agent of bubonic plague. is a facultative intracellular parasite. Wild-type cells (Vwa+) express two antigens, V and W, at 37 C [4,5]. These antigens were not detected in Vwa+ cell cultures incubated at 26 C or in avirulent Vwa' cell cultures. It has been suggested that these antigens are important to the survival and multiplication of 1,.gg§31§_within fixed macrophages [16]. Vwa+ cells required at least 2.5 mM Ca2+ for sustained growth in a defined medium at 37 C whereas Vwa' mutants did not 2+ inhibited the expression require the cation [13]. Furthermore, Ca of the V and W antigens [3]. The response of growth and virulence antigen expression jg_ 2+, led Brubaker .yiggg, with respect to the presence or absence of Ca [2] to postulate that the ion may serve as a signal indicating to the yersinial cell that it is in either an extracellular or an intra- cellular environment. The observation that 1, pg§31§_did divide in the Ca2+-limited intracellular environment of the phagocytic cell [16] indicates that some other intracellular molecule(s) may replace Ca2+ in allowing the sustained growth of these bacteria after phagocytosis. Investigations defining the morphological and physiological consequences of Ca2+ starvation on Vwa+ _Y_. M i_n litr_o have 15 16 provided information helpful in understanding this phenomenon. Cells cultivated in a Ca2+-free defined medium are elongated with a diffuse nucleoid [10]. Ca2+-starved cells were unable to synthesize signifi- cant amounts of DNA [28]. Stable RNA and protein synthesis were also depressed (submitted for publication). Analysis of nucleotide pools in these cells revealed that the ribonucleoside but not the deoxyri- bonucleoside triphosphate pools were significantly lower than those observed in Vwa+‘1, pggEj§.growing cells (submitted for publication). The adenylate energy charge in Ca2+-starved Vwa+ 1, pgggjg cells was significantly lower than in growing cells (submitted for publication). These latter results suggested that Ca2+-starved.1,.pg§31§ may be unable to generate sufficient energy to maintain cell growth. The possibility that exogenous ATP may promote growth in a Ca2+- deficient environment was therefore investigated. In this communica- tion we present results indicating that exogenous ATP and other ribonucleotides promote both cell growth and the expression of V and W antigens in Ca2+-deficient medium at 37 C. The significance of these results with respect to the survival and multiplication of 1, gestis in the intracellular environment of the fixed macrophage is discussed. MATERIALS AND METHODS Bacteria. The laboratory strains of 1, ggggig, EV76 and KIM, were employed in the experiments presented. The organisms are both non-pigmented mutants and, therefore, are avirulent but still retain the potential to express the V and W antigens [15]. Stock suspensions of the bacteria were maintained at -20 C in glycerol-phosphate buffer as previously described [1]. “Eggig. Bacteria were cultivated in the liquid synthetic medium described by Higuchi et al. [13] with the following modifica- tions. It was observed that ammonium acetate and xylose were neither stimulatory nor necessary for growth at 37 C. Therefore, these com- pounds were not included. The L-isomers of the following amino acids were included: phenylalanine, 4.8 mM; tyrosine, 2.2 mM; threonine, 2.8 mM; methionine, 3.2 mM; isoleucine, 7.6 mM; valine, 13.6 mM; leucine, 4.0 mM; glutamic acid, 162.0 mM; proline, 14.0 mM; arginine, 2.0 mM; glycine 5.2 mM. D-L alanine was included at the final con- centration of 9.0 mM. The final KZHPO4 concentration was 2.5 mM and buffering capacity was provided by the inclusion of N-2-Hydroxyethyl- piperazine-N'~2-ethanesulfonic acid (HEPES) at a final concentration of 25 mM. The medium was adjusted to the desired pH with 5.5 N NaOH. The desired MgCl2 and CaCl2 concentrations were achieved by adding the appropriate amount of a sterile 100X stock solution of either of these salts. Nucleotides were prepared as 10X stock solutions in 17 18 25 mM HEPES buffer, adjusted to pH 7.8, and filter sterilized before addition to the growth medium. Cultivation of bacteria. Cells were preliminarily cultivated on slopes of blood agar base (BBL, Cockeyville, MD) at 26 C for 48 h. The growth was then washed from the slope with freshly prepared sterile medium. This cell suspension was used directly to inoculate the liquid growth medium. Prior to all experiments, bacteria were cultivated in the growth medium for at least 12 h in order to eliminate possible nutritional shift complications. This primary culture was used directly as an inoculum for experimental cultures. Experimental cultures, consisting of medium in an Erlenmeyer flask (10% vol/vol), were inoculated at a cell density of approximately 1x108 cells per ml (0.0.620 = 0.1). The cultures were aerated at the appropriate temperature by shaking at 200 rpm on a model G76 gyrotory water bath shaker (New Brunswick Scientific Co., Inc., New Brunswick, NJ). Bacterial growth in the cultures was monitored by assessing the optical density of culture aliquots, appropriately diluted, with a spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, 0H) at a wavelength of 620 nm. Utilization of exggenous ATP. Cells were cultivated in the defined liquid medium supplemented with 20 mM ATP. At designated times aliquots were removed and filtered through a millipore filter (0.22 um pore size). The filtrate was diluted appropriately and ATP was determined spectrophotometrically with hexokinase and glucose- 6-phosphate dehydrogenase as previously described [21]. 19 Incorporation of radioactive ATP. The rationale behind these experiments is based on the fact that if [8—]4C] ATP was transported into the cytoplasm of the bacterial cells, it would become available for RNA synthesis. Therefore, an increase in radioactivity in trichloroacetic acid (TCA) precipitable material over time would indicate uptake. [8-]4C] ATP (0.1 uCi/mmol) was added to a growing culture of 1, pggEig EV76. Aliquots of 0.1 ml were removed at the designated times and immediately added to 10 ml of cold 5% TCA. The TCA precipitable material was collected on millipore filters (0.22 pm pore size) after incubation on ice for 30 minutes. After drying, the filters were placed in scintillation vials containing 10 m1 of 0.4% 2,5-diphenyloxazole (PPO) and 0.005% 1,4—di-2(5-phenyloxazolyl)- benzene (dimethyl POPOP) in toluene. Radioactivity was measured on a Model 3320 Packard Tri-Carb Liquid Scintillation Spectrophotometer. Immunological Methods. Anti-V serum was obtained essentially as previously described [22]. The quantitation of V antigen has also been described [22]. Protein determination. Protein was determined by the method of Lowry et a1. [23] with bovine serum albumin as a standard. Electrgphoresis of[8-]4C] ATP. [8-]4C] ATP purchased from Amersham Corporation was further purified by paper electrophoresis at 1500 volts for 1.5 h in 0.5 M acetate buffer, pH 3.5, prior to the incorporation studies presented herein. This procedure was necessary in order to remove a minor unidentified contaminant that resulted in artifactual results. 20 Chemicals. All chemicals were of the highest purity avail- able from commercial sources. Enzymes were purchased from Sigma Chemical Company, St. Louis, MO. RESULTS Replacement of Ca2+ by ATP as a growth factor at 37 C. Because Ca2+-starved cells had decreased ATP pools and energy charge value, the possibility that exogenous ATP could sustain cell growth at 37 C in the absence of Ca2+ was investigated. Figure 1 shows that the addition of 20 mM ATP to Ca2+-deficient medium resulted in cell growth equivalent to that observed for a Ca2+-sufficient control culture. pH dependence. Preliminary experiments indicated that the growth of EV76 in the presence of exogenous ATP was strongly dependent on the pH of the medium at the time of inoculation. The effect of this variable was therefore investigated in order to define conditions for further experimentation. The growth curves presented in Figure 2 indicate that slightly alkaline conditions favored growth in the presence of the nucleotide. The medium pH did not significantly affect the growth response in medium containing Ca2+. At pH 8.0 the growth yield in Ca2+-deficient medium was increased but was not equal to that observed in either Ca2+- or ATP-supplemented cultures. Cultivation at a pH higher than 8.0 resulted in reduced growth yields with exogenous ATP compared to those obtained at pH 7.8 (data not shown). In all future experiments the medium was adjusted to pH 7.8. This allowed for a 21 22 FIGURE l.--ATP effect. Growth of X:.E§§£i§ strain EV76 at 37 C in the defined medium (see Materials and Methods), pH 7.8, containing 2.5 mM MgCl2 and either 2.5 mM CaCl2 (Cl), 20 mM ATP (0). or no supplement (0). DENSITY OPTICAL 10.0 8.0 4.0 2.0 1.0 0.8 0.4 0.2 0.1 23 _ L— .— E J .— . .—1 - 4 »- -4 _ "l — -—l — ' —1 _ 1'. _ 30000.00... 1. I _. Q /“ ”1 1 J 1 1 J 1 1.1 o 2 4 6 8 101214 HOURS Figure l.--ATP Effect. 24 FIGURE 2.--pH effect. The effect of pH on the growth of X:.E§§£i§ strain EV76 in an unsupplemented medium (A) or medium containing either 20 mM ATP (8) or 2.5 mM CaCl2 (C) as a supplement. Bacterial cells, previously cultured at pH 7.0, were used to inoculate media adjusted to either pH 7.0 (ID), 7.3 (CD), 7.5 (II). 7.8 (C3). or 8.0 (I)). The cultures were then incubated with aeration at 37 C and the optical density was monitored. Cultures contained 2.5 mM MgC12. 25 0. o. N. .eddeem =a--.~ deemed mane: ammo BEN. TTllll [— l l 111] ILL l TTTTTN . _ 1a _ . .r 4O ..0 «.0 V0 0.0 0.. 0.N 0.? 0.0 "IVOlldO ALISNBO 26 reproducible system in which to study the effect of ATP on Ca2+- starved cells. Concentration dependence. The growth response of 1, pestis was dependent upon the concentration of ATP present in the medium (Figure 3). At 10 and 20 mM ATP the cell growth was equal to or better than that observed in a Ca2+-supplemented culture. Utilization of exgggnous ATP. As a direct method to determine whether the ATP added to the medium was utilized by the cells, the extracellular concentration of the nucleotide was enzymatically determined in a growing culture (see Materials and Methods). The results indicated that there was no measurable decrease in the extracellular concentration of ATP over a period of 10 h (Figure 4). During this time the cell mass in culture, determined as a function of optical density, doubled four times. Uptake and incorporation of ATP. Additional evidence support- ing the conclusion that exogenous ATP was not utilized by these cells was obtained by investigating the ability of X:.E§§Ei§ to incorporate exogenous [8-14C] ATP into TCA-precipitable material. The results of this experiment are presented in Table 1. No increase in the radioactivity in the TCA-precipitable material was observed over an 8 h period. During this time, cell mass increased almost ten-fold. Specificity. It is known that Ca2+ could be replaced by 2+ Sr or Zn2+ as a growth factor for Vwa+.1, pestis [13]. In order to ascertain how specific the phenomenon was with respect to 27 FIGURE 3.--Dependence on ATP concentration. Growth of 1,.pgggig strain EV76 at 37 C in a defined medium (see Materials and Methods), pH 7.8, containing exogenous ATP at a concentra- tion of 20 mM (0), 10 mM (0), 5 mM (D), 2.5 mM (I), 1.25 mM (A), or 0.0 mM (A). Cultures contained 2.5 mM MgClz. DENSITY OPTICAL .03 T 1 l T 1 1 1 1 1 1 1 l_ 8.0 ~— — L'— O 6.0 ~ 4 L. , _l 4.0 - - 2.0 — 4 I 1.0 : I 0.8 r- "l 0.6 ~— .._. A 0.4 -- — 0.2 —- - O .1 ° ' [’1 1 1 1 1 J 1 L 1 1 1 1 o 2 4 s 8 IO 12 HOURS Figure 3.--Dependence on ATP Concentration. 29 FIGURE 4.--Inabi1ity of X, 22531; strain EV76 to utilize exogenous ATP. l Bacterial cells previously cultivated at 26 C in medium, pH 7.8, were used to inoculate the experimental cultures consisting of the same medium supplemented with 20 mM ATP. At the times indicated, samples were removed, filtered, and assayed for ATP as described in Materials and Methods (0). The optical density of the culture was monitored at 620 nm ((3). Cultures contained 2.5 mM MgC12. OPTICAL DENSITY 6.0 4.0 2.0 1.0 0.8 0.6 0.4 0.2 0.1 Figure 4.--Inability of _Y, utilize exogenous . 30 I lllll VI bj l---1 C) HOURS estis strain EV76 to 31 TABLE l.--Inability of 1. pestis to incorporate exogenous [8-14C] ATP. 1..) Cultgggliggtical “Alissa-":55: (cpm) l 0.23 73 2 0.32 79 3 0.44 70 4 0.57 62 5 0.75 49 6 1.12 79 7 1.37 65 8 1.69 65 aDetermined as described in Materials and Methods. 32 ribonucleotides, the ability of other ribonucleotides to replace Caz+ was assessed. The results of these experiments are presented in Table 2. It was obvious that a number of ribonucleotides could replace Ca2+ as a growth factor for 1:.EEEEIE EV76 at 37 C. There appeared to be no preference for purine or pyrimidine nucleotides nor was a "high-energy" phosphate a necessary moiety. Pyrophosphate was also observed to enhance the growth yield over that of a Ca2+- deficient control culture. However, this effect was not observed consistently and the growth rate observed in pyrophosphate- supplemented cultures was much slower than that in other growing cultures. Ethylenediaminetetraacetic acid did not support the growth of I, pg§31§_EV76 at the concentrations used. None of the respective ribosides or free bases supported growth in Ca2+-deficient medium at 37 C. Expression of V antigen by ATP-supplementedll,Aggggig. The expression of the V antigen appears to be essential for the successful invasion of the mammalian host by 1,Igg§Ej§_[4,5]. It was, therefore, of interest to determine whether this antigen was expressed by cells cultivated at 37 C in Ca2+-deficient media that was supplemented with ATP. For these experiments the KIM strain of .1, EEEEiE was chosen because it exhibited an enhanced growth response in the presence of 20 mM "92+, previously shown to poten- tiate the expression of the V and W antigens [22]. The growth of these cells under various conditions is shown in Figure 5. The cells were harvested at the end of the experiment, disrupted by sonication, 33 TABLE 2.--Effect of various ribonucleotides on the growth of Vwa+ I, pestis EV76 at 37 C. Supplementa Fina1b 0.0.620 None 0.37 Ca2+ 4.36 ATP 5.87 GTP 3.81 CTP 3.51 UTP 0.94 ADP 4.83 GDP 4.01 CDP 1.16 UDP 1.00 AMP 4.39 GMP 0.75 CMP 0.40 UMP 0.40 a2.5 mM Ca2+, all nucleotides were 20 mM. bThe optical densities presented are those measured after 13 h of incubation (ATP, ADP, AMP) or when growth ceased (all other cultures). 34 FIGURE 5.--ATP-stimulated growth of I, £2351; strain KIM. Growth of I, pggEig strain KIM at 37 C in the defined medium (see Materials and Methods), pH 7.8, containing 2.5 mM MgCl2 (A) or 20 mM MgCl2 (B). The medium was either supplemented with 3.0 mM CaCl2 (1D), 20 mM ATP ([3), or remained unsupplemented (CD). 35 DENSITY OPTICAL 02468101202468 HOURS Figure 5.--ATP-stimulated growth of _Y_. pestis strain KIM. 10 12 36 and the resultant extracts were assayed for V antigen. It was observed that the cells cultivated either in the presence or the 2 absence of the nucleotide expressed the antigen as long as Ca + was absent and sufficient Mg2+ was available (Table 3). 37 TABLE 3.--Expression of V antigen. V antigen (sp. act.)a Addition 2.5 mM M92+ 20 mM Mg2+ 3.0 mM Ca2+ < 0.05 < 0.04 20 mM ATP < 0.08 1.30 None 1.00 1.30 aAfter cultivation for 12 h at 37 C in medium containing the designated supplements, the cells were harvested by centrifugation at 27,000 X G for 15 min, washed in 0.033 M potassium phosphate buffer (pH 7.0), resuspended in buffer and disrupted by sonication. The resultant extract was assayed for V antigen and protein as described in Materials and Methods. The results are expressed as units of V antigen per mg of protein. DISCUSSION The expression of the V and W antigens appears to be essential for a successful invasion of the host by X, pestis [4,5]. Vwa+ cells cultivated in vitro in a simulated intracellular environment with 7 M Caz+ and 20 mM Mgz+ respect to Ca2+ and Mg2+ concentration (10' [18]) express V and W [3]. These same cells fail to multiply at 37 C unless 2.5 mM Ca2+ is present [13]. This latter environment mimics the high extracellular Ca2+ concentration in the mammalian host [19]. However, under these permissive conditions, V and W are not expressed [3]. It has been suggested that the inability of Vwa+‘1, gggEig to grow in vitro in the absence of Ca2+ at 37 C reflects a physio- logical change that makes the bacterium dependent upon the host cell [2]. The observations that both the ATP pool and the energy charge (submitted for publication) were decreased in Ca2+-starved Vwa+.1..gg§31§ prompted investigations to see if exogenous ATP would stimulate the growth of these cells. Two obligate intracellular parasites, the rickettsiae and the chlamydiae, have been shown to transport host-cell derived nucleo- tides. Rickettsia prowazeki possesses an ADP-ATP exchange system and thus has access to the host ATP pool [27]. Chlamydia psittaci incorporated host-cell derived UTP and CTP into 16S RNA [11]. 38 39 Although the nucleotide sustained the growth of Vwa+'1,.pg§31§ under normally restrictive conditions, it does not seem likely that X,.pg§Ei§_depends upon host cell ATP as a substrate for growth as in the previously mentioned systems. The effect is not specific to ATP or even to triphosphorylated ribonucleosides. Furthermore, ATP was not utilized by l, gg§51§ even though growth proceeded normally. The observation that ATP was not transported into 1, pgggig cells was not totally surprising. Bacterial cell membranes are usually impermeable to these highly charged compounds and their transport has been demonstrated in very few cases [11,27]. However, exogenous nucleotides have been observed to affect other bacterial systems [8,9,17,26,29]. Transport of the nucleotides in these systems was not evaluated. The mechanism by which the various ribonucleotides promote the growth of Vwa+ X, pgggi§_remains as obscure as does that for the effect of Ca2+ [3,13]. Any model presented to explain the observed results must account for the observations that either specific 2+, or Zn2+) or specific anions (see Table 4) cations (Ca2+, Sr produce similar effects. It may be that Ca2+ competes with another cation in the medium that inhibits the growth of Vwa+ X, EEEEiE. The effect observed with the various ribonucleotides may then be due to the chelation of the same cation, thereby preventing growth inhibition. It has been demonstrated that increasing concentrations of Mg2+ hastened the onset of restriction of Vwa+ X, pgggig in Ca2+- deficient medium at 37 C (submitted for publication). ATP and other 40 nucleotides form complexes with Mg2+ although the association con- stants vary over a large range [25]. The observations that ATP, ADP, and AMP were able to equally substitute for Ca2+, even though their affinities for Mg2+ are enormously different, suggests that Mg2+ complexing is probably not of sole importance to the effect observed. Another possibility surfaces upon examination of the litera- ture with respect to the effects of Ca2+ and ATP on biological mem- branes. It has been shown that Ca2+ can affect the activity of membrane-bound enzymes by its interaction with the membrane [14]. The fluidity of artificial membranes is affected by the cation [24]. Of particular interest to this study was the observation that ATP, 2+ in the stabilization of neural ADP, or AMP could substitute for Ca membranes [20]. It is possible that the specific interactions of either C32+ or a ribonucleotide with the yersinial membrane results in the stabilization of some membrane state that is necessary for growth. Obviously, there is at this time not enough data to support directly either the above or any alternative possibility. The most significant observation presented here is that ATP- supplemented X: pgggig expressed the V antigen. The ability to express this antigen significantly affects the virulence of 1. BETA—s [4,5,6]. If it is true that the virulence antigens are necessary to the survival and multiplication of the bacterial cells after phago- cytosis by fixed macrophages, as previously suggested [16], then they should be expressed in a simulated intracellular environment. Pre- vious studies indicated that the virulence antigens were expressed jg_vitro only by restricted cells [3]. However, the results 41 obtained here indicate that growing X, ggggj§_cells do express V antigen in nucleotide-supplemented media. The observation presented in this stUdy may mean that certain ribonucleotides replace Ca2+ as a growth factor intracellularly. In the essentially Ca2+-free environment of the cytoplasm, the expression of the V and W antigens would not be inhibited and the cells would still be able to multiply due to the presence of the effective ribonucleotide. Intracellular concentrations of adenine nucleotides have been measured [12] and they approach the concentrations that were found to be effective jg_vitro. Since the most abundant nucleotide in a growing cell is ATP [7,12] it is quite possible that this nucleotide is the biological effector. ACKNOWLEDGMENTS This work was supported by Public Health Service research grants AI 13590 from the National Institute of Allergy and Infectious Diseases and GM 01911 from the Department of Health, Education, and Welfare. The excellent technical assistance of Janet Fowler is gratefully appreciated. 42 10. LITERATURE CITED Beesley, E. 0., Brubaker, R. R., Janssen, W. A., Surgalla, M. J. 1967. Pesticins. III. Expression of coagulase and the mechanism of fibrinolysin. Journal of Bacteriology. .25: 19-26. Brubaker, R. R. 1967. Growth of Pasteurella pseudotuberculosis in simulated intracellular and extracellular environments. The Journal of Infectious Diseases. .llZF403‘4‘7- Brubaker, R. R., Surgalla, M. J. 1963. The effect of Ca2+ and M92+ on lysis, growth, and production of virulence antigens by Pasteurella pestis. The Journal of Infectious Diseases. 114:13-25. Burrows, T. W. 1956. An antigen determining virulence in Pasteurella pestis. Nature. .lZZ:426-427. Burrows, T. W., Bacon, G. A. 1956. The basis of virulence in Pasteurella pestis: an antigen determining virulence. Briti§h Journal of Experimental Pathology. 31:481-493. Burrows, T. W., Bacon, 0. A. 1958. The effect of the loss of different virulence determinants on the virulence and immunogenicity of strains of Pasteurella pestis. British Journal of Experimental Pathology. Eg;278-291. Chapman, A. 6., Fall, L., Atkinson, 0. E. 1971. Adenylate energy charge in Escherichia coli during growth and starvation. Journal of Bacteriology. 19§:1072-1086. Fanica-Gaignier, M., Clement-Metral, J., Kamen, M. D. 1971. Adenine nucleotide levels and photopigment synthesis in a growing photosynthetic bacterium. Biochimica et Biophysica Acta. EEE:135-143. Gajdos, A., Gajdos-Torok. 1969. The quantitative regulation of the biosynthesis of porphyrins by intracellular ATP concentration. Biochemical Medicine. 3:372-388. Hall, Yang+G. ,Little, R. V. Brubaker, R. R. 1974. Effect of Ca on morphology and division of Yersinia pestis. Infection and Immunity. .2: 1105- 1113. 43 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 44 Hatch, T. P. 1975. Utilization of L-cell nucleoside tri- phosphates by Chlamidia psittaci for ribonucleic acid synthesis. Journal of Bacteriology. .1ggza93-400. Hauschka, P. V. 1973. Analysis of nucleotide pools in animal cells, pp. 361-462. In: Prescott, D. M. (ed.), Methods in cell biology, vol. 7. New York: Academic Press. Higuchi, K., Kupferberg, L. L., Smith, J. L. 1959. Studies on the nutrition and physiology of Pasteurella pestis. III. Effects of calcium ions on the growth of virfilent and aviru- lent strains of Pasteurella pestis. Journal of Bacteriology. 77:317-321. Horvath, C., Sovak, M. 1973. Membrane coarctation by calcium as a regulator for bound enzymes. Biochimica et Biophysica Acta. IEg§:850-860. Jackson, S., Burrows, T. W. 1956. The virulence enhancing effect of iron on non-pigmented mutants of virulent strains of Pasteurella pestis. British Journal of Experimental Pathology. E15577-583. Janssen, W. A., Surgalla, M. J. 1969. Plague bacillus: survival within host phagocytes. Science. ‘1§§:950-952. Kight-Olliff, L. C., Fitzgerald, J. W. 1978. Inhibition of induction if Pseudomonas aeruginosa by exogenous nucleotides. Canadian Journdl’of Microbiology. 25:811-817. Kretsinger, R. H. 1977. Evolution of the informational role of calcium in eukarotes. pp. 63-72. Inc: Wasserman, R. H., et al. (eds.), Calcium binding proteins and calcium function. New York: North Holland. Kugelmass, N. I. 1959. Biochemistry of blood in health and disease. Springfield, Illinois: Ch. C. Thomas, Inc. Kuperman, A. S., Okamoto, M., Gallin, E. 1967. Nucleotide action on spontaneous electrical activity of calcium deficient nerve. Journal of Cellular Physiology. 29,257- 264. Lamprecht, W., Trautschold, I. 1963. Adenosine-S'-triphosphate: determination with hexokinase and glucose-6-phosphate dehydrogenase. Pp. 543- . In: Bergmeyer, H. U. (ed.), Methods of enzymatic analysis. New York: Academic Press. Lawton, W. 0., Erdman, R. L., Surgalla, M. J. 1963. Bio- synthesis and purification of V and W antigen in Pasteurella pestis. Journal of Immunology. .gl:179-184. 23. 24. 25. 26. 27. 28. 29. 45 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 1g§:265-275. Papahadjopoulos, 0., Portis, A. 1978. Calcium-induced lipid phase transitions and membrane fusion. Annals of the New York Academy of Science. ‘§Q§:50-66. Rose, I. A. 1968. The state of magnesium in cells as estimated from the adenylate kinase equilibrium. Proceed- ings of the National Academy of Sciences. El;1079-1086. Schmidt, G. L., Kamen, M. D. 1971. Control of chlorophyll synthesis in Chromatium vinosum. Archives of Microbiology. 76:61-64. Winkler, H. H. 1976. Rickettsial permeability: an ADP-ATP transport system. Journal of Biological Chemistry. 'EE1:389-396. Yang, G. C. H., Brubaker, R. R. 1971. Effect of Ca2+ on the synthesis of deoxyribonucleic acid in virulent and avirulent Yersinia. Infection and Immunity. .§:59-65. Zilinsky, J. W., Sojka, G. A., Gest, H. 1971. Energy charge regulation in photosynthetic bacteria. Biochemical and Biophysical Research Communications. 5E5955-961. SECTION III INTRODUCTION The data presented in Section II of this thesis indicate that exogenous ATP present jg_vitro can replace Ca2+ in sustaining growth of Vwa+ 1, pgggj§_at 37 C. The growth response observed was dependent upon the pH of the medium as well as the concentration of ATP present. Growing cells did not appear to either hydrolyze significant amounts of exogenous ATP nor did they incorporate the nucleotide into TCA precipitable material. Various other ribonucleotides could serve equally well to sustain the growth of these bacteria. Unlike cells cultivated in the presence of 2.5 mM Ca2+ , those grown in 20 mM ATP expressed the V antigen. These results are consistent with the hypothesis that a ribonucleotide, possibly ATP, may serve a regulatory role in sustaining the intracellular growth of X, pgggjg, This section is included in this thesis in order to present the data relating to energy charge in I, p§§31§.which will appear in a separate communication. In addition, experiments that further investigated the phenomenon of nucleotide-stimulated growth of I, pestis at 37 C are presented. 46 MATERIALS AND METHODS Bacteria. The following strains of I, gestis were employed in these studies: EV76, KIM, MP6, 625, G32. The phenotypes of these strains with respect to the virulence determinants are presented in Table 4. Adegylate energy charge. Adenine nucleotides were extracted by the addition of a 2.0 ml aliquot of the bacterial culture to 0.4 m1 of 35% HC104 (70) containing 67 mM EDTA (54). The resulting precipitate was removed by centrifugation and 2.0 ml of the super- natant was neutralized by the addition of 0.7 m1 of 2.5 M KOH, 0.58 M KHCO3 (70). This extract was either assayed directly for ATP or was assayed after enzymatic conversion of AMP and ADP to ATP (27). ATP was determined by the luciferase assay in a scintillation vial containing 1.8 ml of the assay buffer (40 mM glycylglycine, 3 mM MgSO pH 7.4) and 0.2 ml of the extract. A 75 pl volume of partially 4. purified luciferase and luciferin (57) was added and after 15 sec of incubation, the light emitted over a period of 6 sec was measured with a Model 3320 Packard Tri-Carb scintillation spectrophotometer as previously described (66). Quintuplicate assays were performed on each sample and the mean value was used to determine the adenylate energy charge. All values fell in the linear portion of an ATP standard curve. Analysis of external standards which were treated 47 48 identically to culture aliquots resulted in recoveries of 101, 107, and 91% for ATP, ADP, and AMP, respectively. Less than 2% cross- reactivity was observed with GTP and ADP. Other methods. In the presentation of the energy charge data, optical density was converted to dry weight with a standard curve comparing the two parameters in the appropriate environment. All other methods used have been described in Section II of this thesis. 49 TABLE 4.--Phenotypes of X, pestis. Phenotypesa Strain Vwa Pgm Pst Fra EV76 + o + + KIM + 0 + + MP6 + 0 + + G32 + O o + 625 + 0 o + M23 + 0 + 0 ana, virulence antigen production; Pgm, pigmentation, Pst, pesticin production; Fra, Fraction I antigen; +, expressed; -, not expressed. RESULTS Adenylate energy charge. The adenylate energy charge was determined for cells of 1, pestis cultivated under various conditions. The results of these experiments are presented in Table 5. The 2+-deficient media was energy charge in cells cultivated at 37 C in Ca significantly lower than that observed in cells cultivated at 37 C in Ca2+-supplemented media or at 26 C. The decrease in energy charge observed appeared to be due to a decrease in intracellular ATP and a concommitant increase in intracellular AMP. ATP antagonism of Ca2+-sustained growth. The effect of ATP on the growth of EV76 cells at 37 C in medium containing 2.5 mM Ca2+ was investigated. It was observed that exogenous ATP resulted 2+ on these cells (Figure 6). The in antagonizing the effect of Ca degree of the antagonism was dependent upon the concentration of ATP added to the medium. Effect of ATP on other strains of 1, pestis. In order to assure the fact that the phenomenon observed with exogenous ribo- nucleotides was not unique to EV76, other strains were tested for their response to exogenous ATP. Various strains of I, pg§31§_were 2+-deficient medium at 37 C in the presence of 2+ or 20 mM not able to grow in Ca 20 mM M92+. 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