This is to certify that the thesis entitled CONSEQUENCES OF ASPARTASE DEFICIENCY IN YERSINIA PESTIS presented by LAWRENCE ALFRED DREYFUS has been accepted towards fulfillment of the requirements for M.S. . MICROBIOLOGY degreeln Major professor : Date 8/1/78 0-7639 CONSEQUENCES OF ASPARTASE DEFICIENCY IN YERSINIA PESTIS BY Lawrence Alfred Dreyfus A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1978 @mw ABSTRACT CONSEQUENCES OF ASPARTASE DEFICIENCY IN YERSINIA PESTIS by Lawrence Alfred Dreyfus Growing cells of Yersinia pseudotuberculosis but not closely related Yersinia pestis rapidly destroyed exogenous L-aspartic and L-glutamic acids thus prompting a comparative study of dicarboxylic amino acid catabolism. Rates of amino acid metabolism by resting cells of both species were deter- mined at pH 5.5, 7.0, and 8.5. Regardless of pH, 3. pseudo- tuberculosis destroyed L-glutamic acid, L-glutamine, L- aspartic acid, and L-asparagine at rates greater than those observed for X. pestis. Proline degradation by X. pestis at pH 8.5 resulted in excretion of glutamic and aspartic acids. Yersinia pestis excreted aspartic acid when incu— bated with L-glutamic acid (pH 8.5) or L-asparagine. Aspartase activity was not detected in extracts of 10 strains of Y; pestis but was present in all of 11 isolates of X. pseudotuberculosis which contained significantly more gluta- minase, asparaginase, and L-glutamate-oxalacetate trans- aminase activity than X. pestis. The observed differences in dicarboxylic amino acid metabolism are traceable to aspartase deficiency in Z. pestis. ACKNOWLEDGMENTS I wish to thank Dr. R. N. Costilow for use of his laboratory space and equipment. Were it not for Dr. Costilow's extreme generosity much of this work would have been extremely difficult if not impossible. I am grateful for the interesting discussions with Dr. R. L. Uffen, Dr. R. N. Costilow, Dr. H. L. Sadoff and my fellow students. I am especially grateful to Dr. R. R. Brubaker for allowing me to work in his laboratory, his help and encouragement over the last three years, and his undying support. ii TABLE OF CONTENTS LIST OF TABLES . . . . LIST OF FIGURES . . . INTRODUCTION . . . . LITERATURE REVIEW . . . MATERIALS AND METHODS . Bacteria . . . . Media and Cultivation Degradation of Amino Acids Cell—free Extracts . Enzymes . . . . . Reagents and Isotopes RESULTS . . . . . . Degradation of L-glutamic and L-proline . . Degradation of L-aspartic L-asparagine . . DISCUSSION . . . . . LIST OF REFERENCES . . iii Acid, Acid L-glutamine Page iv l6 l6 l6 l7 l9 19 20 21 22 23 24 45 LIST OF TABLES 1. Established virulence determinants in Yersinia pestis . . . . . . . . . 2. Some phenotypic differences between wild type Yersinia pestis and Yersinia pseudotuberculosis . . . . . . . . 3. Initial rates of destruction of some amino acids by resting cells of Yersinia pestis EV76 and Yersinia pseudotuberculosis PBl 4. Specific activities of L-glutamate-pyruvate transaminase and L-glutamate-oxalacetate transaminase in dialyzed extracts of cells of Yersinia pestis EV76, Yersinia pseudo- tuberculosIs PBl, and EscheriEhia coII K12 5. Specific activities of aspartase, a-ketoglutarate dehydrogenase, and L- glutamate dehydrogenase in dialyzed extracts of cells of Yersinia pestis and Yersinia pseudotuberculosis . . . 6. Specific activity of aspartase in dialyzed extracts of Yersinia pestis EV76, Yersinia pseudotuberculosis PBl, and Escherichia coli'KlZ prepared after growth in complex synthetic medium (CSM), heart infusion broth containing added 5.0 mM sodium L-aspartate (HIB), and minimal synthetic medium (MSM) . . . . . . . . . . 7. Specific activities of asparaginase in dialyzed extracts of Yersinia pestis, Yersinia pseudotuberculosis, and Escherichia coli . . . . . . . . iv Page 28 29 30 31 32 33 34 Figure 1. LIST OF FIGURES Degradation of L-amino acids by Yersinia pestis EV76 and Yersinia pseudo- tuberculosis chromatogramed at culture optical densities of 0.85 and 0.91, respectively, during aeration at 37°C in preincubation mixture plus 0.01 M D-glucose . . . . . . . . . . Degradation of L-glutamic acid by resting cells of Yersinia pseudotuberculosis PBl and Yersinia pestis EV76 with accumulation of aspartic acid by the latter . . . . Degradation of L-proline by resting cells of Yersinia pseudotuberculosis P81 and Yersinia pestis EV76 with accumulation of glutamic acid and aSpartic acid by the latter . . . . . . . . . . . . Degradation of L-aspartic acid by resting cells of Yersinia pestis EV76 and Yersinia pseudotuberculosis PBl with accumulation by the latter of fumarate, malate, succinate, and an unidentified non- volatile product . . . . . Degradation of L-asparagine by resting cells of Yersinia pestis EV with accumulation of aspartic acid . . . . . . . . Page 35 37 39 41 43 INTRODUCTION Yersinia pestis, the causative agent of bubonic plague, is maintained in nature within a nutritionally enriched, protected, and fixed cycle formed by its mammalian host and insect vector. In contrast, cells of very closely related Yersinia pseudotuberculosis (53, 60) are usually transmitted orally and, like other Entero- bacteriaceae, must exist at least transiently in nutri— tionally depleted environments which often require compe- tition with saprOphytes. Unlike X. pestis, survival of Z. pseudotuberculosis may thus depend upon its maintaining broad biosynthetic and catabolic potential. Cells of I. pestis, however, are unable to express many activities known to exist in Z. pseudotuberculosis including glucose— 6-phosphate dehydrogenase, urease, abilities to synthesize 5 amino acids, and potential to ferment certain carbo- hydrates (11). Many of these determinants can be gained singly by meiotrophic mutation (14, 29, 30) and all are ancillary in the sense that their absence would not be expected to significantly reduce doubling times in nature or in conventional culture media. Nevertheless, typical generation times in enriched media for Z. pseudotuberculo— sis and X. pestis are about 0.5 h and 2 h, respectively. 1 Although much is known about the intermediary metabolism of yersiniae, the reason for this difference in rates of growth is unknown. Aerobically grown cells of X. pestis possess an operational Embden-Meyerhof pathway (65), tricarboxylic acid cycle (31, 64), and cytochrome system (31); amino acid transport (52, 68, 69), deamination (41, 49, 63), and incorporation into cellular components (41) have been described. Glycolytic and hexose mono— phOSphate pathways exist in z. pseudotuberculosis (9). This organism can catabolize a number of amino acids (8) presumably by conversion to tricarboxylic acid cycle intermediates (11). In preliminary experiments it was noted that the two organisms could catabolyze common carbohydrates and organic acids at similar rates. However, significant differences were observed in rates of degradation of L-aspartate and L—glutamate. The purpose of this study is to define the basis of the lesion in dicarboxylic amino acid metabolism in Z. pestis and to compare the ability of yersiniae to degrade metabolically related amino acids. LITERATURE REVIEW The genus Yersinia (genus XI of the family Enterobacteriaceae) is currently recognized as having three members all of which are gram negative facultative intracellular parasites of rodents and man (3, 23, 59, 72). Two members of the genus, X. pestis and X. pseudo- tuberculosis, are antigenically and biochemically related to a high degree (11, 12). In fact, X. pestis has been shown to share 85% of the deoxyribonucleic acid (DNA) sequence of X. pseudotuberculosis (53). In this same study X. enterocolitica, the third member of the genus, was demonstrated to possess 20% homology with X. pseudo- tuberculosis (53). When less stringent conditions of hybridization were followed, Brenner et al. (6) found a slightly higher homology between X. pseudotuberculosis and X. enterocolitica than was observed by Moore and Brubaker (53). These data suggest a marginal intrinsic similarity within the genus as a whole and relatedness approaching identity between X. pestis and Z. pseudo— tuberculosis. Yersinia enterocolitica, a recently recognized member of the genus, occurs commonly in nature (33). Clinical isolates are usually avirulent, however, human illness in the form of acute terminal ileitis has been reported (56). Z. pseudotuberculosis, like Z. entero- colitica, causes a relatively mild acute mesenteric lymphadenitis mimicking appendicitis in humans (26). When a comparative study was performed, both X. pseudotubercu- losis and X. enterocolitica successfully produced experi- mental enterocolitis in rabbits resulting in necrobiotic centers in reticuloendothelial tissues of the intestine, mesenteric lymph nodes, liver and spleen (72). The author describes events occurring in infected HeLa cell and infected rabbit peritoneal macrophage model systems. These data correlated well with the pathology observed in the infected whole animal studies (72). The striking similarities observed may reflect the ability of both X. pseudotuberculosis and X. enterocolitica to gain entrance to and multiply within nonprofessional phagocytes and epithelial cells (3, 59, 72). In spite of the marked similarities between X. pseudotuberculosis and X. pestis, the mild mesenteric lymphadenitis caused by the former species bears little resemblance to bubonic plague caused by the latter species. Data concerning the virulence determinants of yersiniae are largely if not solely centered on those determinants elaborated by X. pestis. Much of this work was performed by T. W. Burrows and his colleagues and has been recently reviewed (11, 12). In brief, X. pestis possesses five recognized determinants of virulence. Loss of any one of the five results in varying degrees of avirulence (11, Table 1). It is noteworthy that upon discovery, these same virulence determinants were sought in X. pseudo— tuberculosis. Two of the five determinants of virulence are shared by the two species, namely, the ability to produce V and W antigens (vwa+) and purine independence (pur+) (11). None of the five determinants possessed by X. pestis have been reported to occur in Z. enterocolitica. Fraction 1 (fra+), a glycoprotein capsular antigen, is present in cells of Z. pestis cultivated at 37°C but not at 26°C (32). Fraction 1 antigen is an antiphago- cytic capsule once thought to be obligately associated with virulence in human cases of fleaborne plague (44). Isolation of fra- X. pestis from a fatal case of human plague (76), however, suggests that virulence in man is not selected for by the presence of fraction 1 antigen. Thus, the role of the fra+ phenotype in the pathogenesis of plague is somewhat confusing at this time. Mutation of fra+ isolates to fra- results in only marginal reduc- tion in the virulence of X. pestis for guinea pigs (19) and has no effect on the virulence in mice (11). X. pseudotuberculosis does not express the fra+ phenotype. Purine independence (pur+) has for some time been recognized to be associated with the virulence of a number of bacterial pathogens including X. pestis and X. pseudo- tuberculosis (ll, 17). This trait is therefore not unique to yersiniae but rather probably reflects the inability of the organism to scavenge purines from a purine deficient environment or inefficient transport of exogenous purines or purine precursors when available, thus overruling growth and therefore virulence. The V and W or virulence antigens of X. pestis, first described by Burrows and Burrows and Bacon (18, 20) have long been recognized to be obligately associated with the virulence of the plague bacillus (11). However, attempts to characterize the nature of their contribution to the pathogenicity of virulent X. pestis have been frustrated by difficulty in their purification (48). Even with good preparations of the antigens their role in plague pathogenesis remains obscure. None-the-less, X. pestis vwa_ cells are avirulent (11). V and W antigens have also been observed in X. pseudotuberculosis (21) and are required for the virulence of this species in mice (11). However, here too the role of the vwa+ gene pro- ducts in regard to virulence is unknown. Virulent (i.e., vwa+) cells of X. pestis possess an unusual temperature dependent requirement for substrate levels (2.5 mM) of Ca2+ (29, 47). Growth at 25°C or loss of the vwa+ phenotype obviates the Ca2+ requirement which is manifest by vwa+ cells at 37°C (12). The correlation between V and W antigen production and calcium dependence was first noted by Brubaker and Surgalla (15). It was found that V and W antigens were produced only when cells pregrown in the absence of calcium at 25°C were shifted to 37°C. This shift resulted in the cessation of cell division and a high production of V and W antigens (15). Brubaker and Surgalla also described a rare phenotype termed "VW+—avirulent" in which the requirement for Ca2+ but not the ability to produce V and W is lost. The finding suggests that the requirement for Ca2+ is directly associated with virulence and not subordinate to the pro— duction of V and W antigens. A redesignation of this virulence determinant from vwa+ to cal+ (representing the calcium dependence) to better describe the phenotype associated with virulence in X. pestis has been proposed by Brubaker (12). The consequences of shifting a growing X. pestis cal+ (virulent) culture from 26°C to 37°C in a medium containing no added Ca2+ and 20 mM Mg2+ (i.e., those con- centrations found within host cell cytoplasm) are mani- fold. The effects described to date of such a temperature shift are turnoff of initiation of new rounds of chromo- somal replication, cessation of stable ribonucleic acid synthesis, expression of the V and W antigens, reduction of nucleoside triphosphate pools, decrease in adenylate energy charge from 0.85 to 0.59 and bacteriostasis (77). Avirulent cal- (vwa- or "VW+—avirulent") isolates grow normally at 37°C in this medium. It was also noted in these studies that at pH 7.8 cal+ cells grew without added calcium in the presence of any one of a number of nucleoside mono-, di-, or triphosphates (10.0 mM) includ— ing adenosine triphosphate (ATP). It should be noted, however, that stimulation was apparently not a result of l4C-ATP bound by cal+ uptake of the nucleotides since cells under these conditions could be dissociated from the cells by the addition of chelating agents (R.J. Zahorchak, personal communication). It is of interest that the V and W antigens and the calcium restriction phenotype are expressed only under conditions which mimic an intracellular environment. For X. pestis, a facultative intracellular parasite, these events which are seemingly required for its virulence may reflect a bioenergetic regulatory mechanism. Conceivably such a mechanism could serve as a signal for expression of intra- vs. extraecellular modes of energy conservation. However, at this time the definitive role of these events in the virulence and success of X. pestis as a facultative intracellular pathogen, remains highly speculative. The fourth recognized virulence determinant of X. pestis concerns the ability of the organism to produce pesticin (pst+), a protein bacteriocin active against serotype I strains of X. pseudotuberculosis, a few isolates of X. enterocolitica, and certain colicin-indicator strains of Escherichia coli (ll). Pesticin production per se may not necessarily promote virulence of the organism since loss of pesticin activity (a means to measure its contri- bution to virulence) is concomitant with the loss of coagulase and fibrinolysin activities (2, l6). Intra- venous injection of pst- cells of otherwise fully virulent X. pestis into lab animals results in death of the animals comensurate with wild type organisms injected subcutaneously (13). Thus pst- cells are avirulent when administered via the natural port of entry (i.e., sub- cutaneous inoculation). These data support the necessity of coagulase and fibrinolytic activities for the expres- sion of virulence in fleaborne plague, presumably by pro- moting dissemination of the organism from the initial foci to deep peripheral tissues (ll, 13). Cells of virulent X. pestis grown on solid media containing either hemin or congo red can absorb the planar chromOphores contained therein and grow as pigmented (pgm+) colonies (42, 71). Avirulent pgm- cells regain full virulence when injected intravenously with suffi- cient iron to saturate serum transferrin (43). However, whether restoration of virulence was due to the ability of the pgm— strain to successfully acquire iron in the Fe3+ saturated system or to the compromising condition dealt the host upon such action (45) is not distinguishable 10 at this time. Differences in the iron metabolism of pgm+ and pgm- cells have yet to be observed. When finally elucidated, differences between pgm+ and pgm- isolates of X. pestis may reflect a selective advantage of pst+ over pst- cells to bind complexed iron in vivo. Yersiniae possess no unusual growth requirements and grow very well at 26°C in defined media containing a few L—amino acids, organic acids, inorganic salts and a source of energy, usually xylose and gluconate or glucose (10, 14, 22, 38, 40, 61). Prior to the demonstration of a chemically defined medium suitable for the growth of X. pestis, Higuchi and Carlin obtained excellent growth in casein hydrolysate broth with no other additions (37). Subsequent defined medium studies focused on the identi- fication of those factors which would allow growth of X. pestis at 26°C as well as at 38°C while still main- taining the high yield obtained in casein hydrolysate broth (7, 38, 40, 61). Though much discrepancy concerning the require— ment of X. pestis for various amino acids appears in the literature, it is generally accepted that at 26°C most strains require phenylalanine, methionine, glycine, and cysteine for growth (14, 22, 38). The requirement for cysteine may be obviated by the addition of thiosulfate or sulfite but not sulfate (28). The addition of L—threonine but not L-serine will satisfy the requirement 11 for glycine (42, 14). Valine and isoleucine stimulate growth at 26°C (28, 40). A medium containing glucose, inorganic salts, thiosulfate, valine, isoleucine, methionine, and phenylalanine will support excellent growth of X. pestis at 26°C with final yields of %5 x 109 cells per ml (10, and unpublished observation), at 28°C strains of X. pseudotuberculosis and X. enterocolitica have either no requirements for growth (e.g., glucose and inorganic salts will support growth), or require either thiamine or Ca-pantothenate (22). Growth of X. pestis at 38°C is somewhat more restrictive than at 26°C. Brownlow and Wessman reported X. pestis to require thiamine, Ca-pantothenate, biotin, isoleucine, valine, threonine, phenylalanine, cysteine, methionine and hemin at 38°C (7). These authors found that hemin could be replaced in the medium with a- ketoglutarate or high concentrations of glutamic acid. From these results they postulated that the exogenous addition of d-ketoglukarate or glutamic acid was necessary at 38°C for the synthesis of porphoryns by X. pestis (7). However, these findings have not been substantiated. Burrows and Gillett more recently demonstrated reliable growth of X. pestis at 37°C on solid media (glucose and inorganic salts) supplemented with cysteine, methionine, phenylalanine, glycine, valine, isoleucine, glutamic acid and thiamine under C02 enriched air (22). These same 12 authors found that the growth requirements of X. pseudo- tuberculosis and X. enterocolitica at 37°C, like those of X. pestis, are more exacting. Most strains of X. pseudotuberculosis required any three of the four factors, glutamic acid, thiamine, cystine, and pantothenate at 37°C, whereas X. enterocolitica required thiamine and either cysteine or methionine at that same temperature (22). To date the most suitable defined liquid medium for the cultivation of X. pestis at 37°C is that of Higuchi, Kupferberg, and Smith (39) as modified by Brubaker (10). This medium supports high yield growth (’blOlo cells per ml) of X. pestis at 37°C with a doubling time of NZ hours (unpublished observation). When cultured under the same conditions, X. pseudotuberculosis doubles in approximately thirty minutes. Both X. pestis and X. pseudotuberculosis possess an operational Embden-Meyerhoff pathway (65, 9). X. pseudotuberculosis can also oxidize glucose via the hexose monophosphate pathway (9). Claims by Santer and Ajl (66) that X. pestis also contains a functional pentose phosphate shunt could not be substantiated by other investigators, all of whom observed the lack of glucose-6—phosphate dehydrogenase in that organism (54, 55, 5). Gluconate is catabolyzed via the Entner—Douderoff pathway in both X. pestis and X. pseudotuberculosis 13 (54, 9). Pentose synthesis in X. pestis presumably occurs via rearrangement of 3C and 6C fragments by the action of transketolase and transaldolase (ll). Xylose isomerase has been purified from extracts of X. pestis (67). Both X. pestis and X. pseudotuberculosis can oxidize a variety of sugars and organic acids including glucose, gluconate, ribose, xylose, pyruvate and lactate (ll). Anaerobically X. pestis ferments glucose primarily to lactate, ethanol, acetate and formate (31, 65). X. pestis possesses a functional tricarboxylic acid cycle (TCAC) (64, 31). The presence of the TCAC in X. pseudotuberculosis has not been documented yet is assumed to be present (11). The presence of an ADP— dependent phosphoenolpyruvate carboxykinase and an irreversible phosphoenolpyruvate carboxylase serving to catalyze the fixation of CO2 into oxalacetate has been reported in X. pestis (l). X. pestis maintains the ability to transport, catabolyze, deaminate and/or incorporate into cellular material various amino acids (52, 68, 69, 57, 63, 41). Levine et al. (49) demonstrated the presence of serine dehydratase in X. pestis which is apparently responsible for the rapid degradation of L-serine observed in other studies (57, 41). Other amino acids which have been reported to undergo degradation by X. pestis are L-alanine and L—glutamate (41). X. pseudotuberculosis degrades l4 L—serine and L-aspartate very rapidly followed more slowly by L—glutamate, L-proline, L-threonine and glycine (8). The inability of X. pestis to convert cysteine to cystathionine resulting in the nutritional requirement for methionine was documented by Englesberg (28). No such block exists in X. pseudotuberculosis which can utilize methionine as its sole source of sulfur (11). No other unusual biochemical features have been reported for either of these yersiniae. The central anabolic pathways of these two organisms are presumably identical to those in other enteric bacteria (11). Some of the phenotypic differences between wild type X. pestis and X. pseudotuberculosis have already been mentioned. These characteristics and others are listed in Table 2. Special emphasis should be given to those traits in X. pestis which have been shown to become positive via mutation. Englesberg first used the expres- sion meiotrophic mutation to describe the occurrence of rhamnose positive X. pestis isolates which occurred 11 (29). Like- naturally at the low frequency of 2.6 x 10‘ wise, wild type X. pestis which normally cannot ferment melibiose gives rise to melibiose meiotrophs when plated on selective media (11). Additional meiotrOphic con- versions recognized to date are the ability to synthesize glycine and assimilate low levels of ammonium salts (l4) and the ability to synthesize phenylalanine and methionine 15 (30, 7). Judicious employment of novel selective methods will undoubtedly lead to the generation of additional meiotrophic markers in X. pestis. The ability of X. pestis to undergo such meiotrophic conversions suggests a deficiency or lack of a specific mechanism for the excision of genetic informa- tion no longer in normal metabolic use. Further investi- gation into the mechanisms involved in the retrieval of these gene products may explain how the gene functions were initially rendered inactive and what role environ- mental factors play in this evolutionary pattern. MATERIALS AND METHODS Bacteria. X. pestis EV76, X. pseudotuberculosis PBl and Escherichia coli K-12 were used in most experi- ments. All organisms were preserved in buffered glycerol as previously described (2). Media and Cultivation. Upon removal from storage, cells were incubated at 26°C for 36 to 48 h on slopes of blood agar base (Difco). Organisms were removed in 0.033 M potassium phosphate buffer, pH 7.0 (phosphate buffer), washed by centrifugation at 48,000 x g for 10 min at 5°C, and inoculated at a density of about 108 cells/ml into 25 ml (per 250 ml Erlenmeyer flask) of the complex synthetic medium of Higuchi, Kupferberg, and Smith (39) as modified by Brubaker (10). After aeration at 26°C for 16 h at 170 rpm on a model G76 gyrotory shaker (New Brunswick Scientific Co., New Brunswick, NJ), the culture served as inoculum for 200 ml of the same medium contained in a 2 liter flask. The latter was similarly aerated at 37°C and, upon reaching late logo- rithmic growth, the bacteria were collected by centrifuga- tion and suspended in 200 ml (per 2 liter flask) of preincubation mixture consisting of the inorganic salts 16 l7 and vitamin components of the complex synthetic medium plus 5.0 mM concentrations of the 20 naturally occurring amino acids. This step was performed to insure induction of pertinent catabolic enzymes. After aeration for 3 h at 37°C the organisms were collected by centrifugation, and immediately used for determination of rates of amino acid degradation. The same procedure was used to prepare cells for sources of enzymes except that the organisms were washed in phosphate buffer. Preincubation mixture supplemented with D-glucose (0.01 M) was also used to monitor by paper chromatography the degradation of amino acids by growing cells. For determination of aspartase activity the organisms were also grown in a minimal synthetic medium (10) and heart infusion broth (Difco) supplemented with L—aspartic acid (5.0 mM). Degradation of Amino Acids. Destruction of amino acids by growing cells was assayed by sterilizing samples of culture by passage through 0.45 pm pore membrane filters (Millipore Corp., Bedfore, MA), exchange and elution with Dowex 50 W (H+ form), and paper chromatography (14). Rates of degradation were determined in a reaction system con- taining distilled water-washed cells (%5 mg), 33 umoles of buffer, 1 pmole of MgCl and 10 umoles of L-[U-14C] 2’ amino acid (0.1 uCi/pmole) in a volume of 1.0 ml contained 18 in a 25 ml Erlenmeyer flask. Buffers used were sodium citrate (pH 5.5), sodium morpholinopropane sulfonate (pH 7.0), or tris(hydroxymethyl)aminomethane-HCI (Tris- HCl buffer) (pH 8.5). Reactions were initiated by addi- tion of radioactive amino acid and the flasks were aerated at 37°C as previously described. At appropriate intervals samples of 0.1 ml were removed, added to 0.1 ml of cold 10% trichloroacetic acid, stored for 30 min in an icebath, centrifuged, and 10 ul of the supernatant was applied to Whatman no. 1 paper. After chromatography in one dimension with either n-butanol:acetic acid:water (100:22:50 vol/vol), phenol:water (4:1 g/ml), or pyridinezacetic acid:water (50:35:15 vol/vol) the papers were cut into strips and examined with a model 7201 radiochromatogram scanner (Packard Instrument Co., Downers Grover, IL). Radioactive areas corresponding to those of ninhydrin-positive controls and of non-volatile products were removed, cut into squares of about 1 mm, and placed into scintillation vials with 10 ml of ACS counting fluid (Amersham-Searle Co., Arlington Heights, IL). Radioactivity was determined with a model 3320 Packard Tricarb liquid scintillation spectrometer. Appropriate controls were constructed to monitor nonSpecific binding of radioactive amino acids to glassware and bacteria; such losses were never observed. l9 Cell-free Extracts. Organisms previously washed in phosphate buffer were suspended in 0.1 M Tris-HCl buffer (pH 7.8) containing 0.001 M 2-mercaptoethanol. After dis- ruption at 20,000 psi in a French pressure cell or by treatment for l min with a sonic probe (MSE Ltd., London, England), debris was removed by centrifugation and the supernatant was dialyzed overnight at 5°C against 0.005 M TriS'HCl buffer (pH 7.8) containing 0.001 M 2—mercapto— ethanol and 0.001 M MgCl Protein was determined by the 2. method of Lowry et al. (50) using bovine serum albumin as a standard. Enzymes. The method of Reed and Willms (58) was used to determine d-ketoglutarate dehydrogenase where a unit of activity is defined as that amount of enzyme required to reduce 2 umoles of ferricyanide per h. Specific activities of other enzymes are defined in terms of number of umoles of substrate converted to product per min per mg of protein. L-Glutamate dehydrogenase was measured as described by Coolbaugh, Proger, and Weiss (25). L—Glutamate—oxalacetate transaminase and L- glutamate—pyruvate transaminase activities were determined by the methods of Hebeler and Morse (36). Aspartase was assayed by measuring the release of ammonia from L—aspartate. The reaction mixture consisted of 50 umoles of Tris-HCl buffer (pH 7.0), 50 umoles of 20 sodium L—aspartate, l umole of MgSO 0.1 umoles of 4, ethylenediaminetetracetate, and bacterial extract in a volume of 1.0 ml. After starting the reaction by addition of aspartate, samples of 0.1 ml were added at intervals to 0.1 ml of cold 10% trichloroacetic acid. The resulting precipitate was immediately removed by centrifugation and 0.1 m1 of the supernatant was analyzed with Nessler's reagent. Glutaminase activity was determined by radio— assay in a reaction mixture containing 20 umoles of potassium phosphate (pH 7.2), 20 umoles of L—[U-14C] glutamine (0.1 uCi/umole), and bacterial extract in a volume of 1.0 ml. Samples of 0.1 ml of reaction mixture were added to 0.1 ml of 10% cold trichloroacetic acid at intervals and immediately clarified by centrifugation. The supernatant was chromatographed with phenol:water (4:1 g/ml) and radioactive glutamine and glutamic acid were determined as previously described. Asparaginase activity was measured by an identical procedure except that L-[U-14C] asparagine was substituted for radioactive glutamine. Reagents and Isotopes. All chemicals were com- mercial products of highest available purity. Radioactive compounds were purchased from Amersham—Searle. RESULTS Cells of X. pseudotuberculosis and X. pestis exhibited typical growth in preincubation mixtures plus glucose (see Materials and Methods) yielding maximum optical densities of 1.8, and 1.5, respectively. Prior to the termination of log phase growth essentially all of the L-aspartic acid and much of the L—asparagine and L- glutamic acid had disappeared from the culture medium of X. pseudotuberculosis (Figure 1). Significant concentra- tions of these amino acids were detectable in comparable cultures of X. pestis; at this time both organisms had eliminated L-serine from the medium. Chromatograms of stationary phase cultures showed that cells of both species could also degrade L-proline, glycine, L-alanine, and L—threonine. Having identified in these preliminary experiments those amino acids which underwent significant metabolism, individual rates of degradation were determined by radioassay. These results, shown in Table 3, suggested the existence of marked differences between the two species in abilities to catabolize dicarboxylic amino acids. 21 22 Degradation of L-glutamic Acid, L-glutamine, and L—proline. Cells of X. pseudotuberculosis degraded L- glutamic acid at pH 5.5 and 7.0 about 5 times more rapidly than did preparations of X. pestis. Metabolism of L—glutamic acid was markedly stimulated in both organisms at pH 8.5 where X. pestis but not X. pseudo- tuberculosis excreted aspartic acid (Figure 2). Further- more, at pH 8.5 but not pH 5.5 or 7.0 both glutamic and aspartic acids were excreted by X. pestis incubated with L—proline (Figure 3). Similar degradation products were not detected with X. pseudotuberculosis. L—Glutamine was not significantly degraded by X. pestis and only slow destruction occurred with X. pseudotuberculosis; neither organism catabolized L-arginine. Specific activities for L—glutamate-pyruvate transaminase and L—glutamate-oxalacetate transaminase for yersiniae and E. coli control are shown in Table 4. Cells of X. pseudotuberculosis were somewhat deficient in the former enzyme whereas those of X. pestis possessed reduced levels of the latter. The specific activities of L—glutamate dehydrogenase and d-ketoglutarate dehydrogenase in both yersiniae were comparable (Table 5). L-glutaminase activity was present in X. pestis and X. pseudotuberculosis at a specific activity of 0.003 and 0.012, respectively. 23 Degradation of L-aspartic acid and L-asparaginine. L-Aspartic acid was degraded slowly by cells of X. pestis at pH 5.5; no detectable destruction occurred at pH 7.0 or pH 8.5. In contrast, L-aspartic acid was rapidly destroyed by X. pseudotuberculosis over the pH range of 5.5 to 8.5 yielding fumarate, malate, succinate, and an unidentified product (Figure 4). Accumulation of these tricarboxylic acid cycle intermediates would be expected if the amino acid had undergone deamination via aspartase. L—Asparagine was degraded more rapidly by cells of X. pseudotuberculosis than by those of X. pestis (Table 3). The latter accumulated stoichiometric amounts of aspartic acid while catabolyzing L-asparagine under the 3 conditions of pH; results obtained at pH 8.5 are shown in Figure 5. Aspartase activity was present in extracts of all of 11 isolates of X. pseudotuberculosis but was not detected in similar preparations of 10 strains of X. pestis (Table 5). Similarly, aspartase was not observed in X. pestis grown in minimal synthetic medium (see Materials and Methods) or heart infusion broth supplemented with 5.0 mM L-aspartic acid. Extracts of X. pseudotuberculosis and E. 921$ contained aSpartase activity after growth in these media (Table 6). Significantly less L-asparaginase was present in X. pestis than in comparable extracts of X. pseudotuberculosis (Table 7). DISCUSSION Cells of X. pestis are capable of actively trans- porting some (52, 68, 69) if not all naturally occurring amino acids. This process of accumulation probably had little influence on the rates of destruction reported here where favorable equilibria caused by catabolic enzymes were sufficient to have promoted entrance by passive mechanisms. In fact, in the low-potassium and carbohydrate—deficient environment that was chosen, an internal negative potential (created by alkaline pH) would have hindered active transport of neutral and acidic amino acids (24, 35). Since alkaline or neutral pH enhanced the destruction of L-glutamic acid and all tested neutral amino acids, degradation was not limited by the process of uptake. The role of alkaline pH in stimulating amino acid destruction was not resolved in this study. Elevated pH may promote turnover of critical catabolic enzymes including L—glutamate dehydrogenase (74) and can favor exit of acidic degradation products (75), especially carbonate and acetate ions. Aspartic acid was another anionic degradation product that accumulated in suspensions of X. pestis 24 ‘ 25 containing added L-proline, L-glutamate or L-asparagine. Cells of X. pseudotuberculosis, however, rapidly destroyed L—aspartic acid in a reaction yielding fumarate. Further work showed that extracts of 10 isolates of X. pestis from diverse geographical sources lacked detectable aspartase activity when tested under conditions that yielded positive results for X. pseudotuberculosis and an E. 221$ control. This finding was not anticipated because Korobeinik and Domaradskii (46) had reported the presence of aspartase in X. pestis of Russian origin. A recent search of the literature, however, revealed that Domaradskii (27) sub- sequently found 19 of 24 X. pestis isolates to lack aspartase. Accordingly, this property may be variable among Russian strains, many of which resemble X. pseudo— tuberculosis with respect to other properties (51, 62, 70). The results reported here indicate that aspartase is absent in typical X. pestis. As a consequence of this deficiency, significant conversion of L-glutamic acid to d-ketoglutarate can only occur in X. pestis via the action of L-glutamic dehydrog- enase. In addition to this mechanism, X. pseudotubercu- losis can form d-ketoglutarate by transamination of L- glutamic acid with a catalytic amount of oxalacetate to yield L-aspartate which can then be catabolized via aspartase, fumarase, and malic dehydrogenase, ‘- 26 consecutively, to regenerate oxalacetate. This second method of L-glutamic acid oxidation was originally pro- posed to account for the inability of L-glutamate decarboxylase-negative mutants of E. 2211 to utilize L- glutamate as a sole source of carbon (73). The importance of this pathway in normal metabolism was later underscored by isolation of aspartase-negative mutants of E, ggXX by selection for inability to utilize L-glutamic acid for growth (34). Some consequences of aspartase-deficiency in X. pestis are that the organisms are unable to catabolize exogenous L-aspartic acid or aspartic acid arising from L-asparagine. Likewise, oxidation of exogenous L-glutamic acid (or glutamic acid arising from L-glutamine or L- proline) via the tricarboxylic acid cycle is limited by the activity of L-glutamate dehydrogenase. This primary dehydrogenation, unlike that of its counterpart in X. pseudotuberculosis (malic dehydrogenase), yields NADPH which must undergo transhydrogenation with NAD+ before initiating oxidative phosphorylation (14). Furthermore, oxalacetate can undergo transamination with glutamic acid in X. pestis yielding catabolically inert aSpartate thus preventing the generation of citrate via the condensing enzyme. A block at this level of the tricarboxylic acid cycle resulting in the accumulation of acetate has been reported in X. pestis (31). The occurrence of these 27 events in yersiniae result in marked differences in dicarboxylic amino acid metabolism and may contribute to the longer generation time of X. pestis relative to that of X. pseudotuberculosis. Aspartase deficiency is uncommon in bacteria. Francisella tularensis is Said to lack the enzyme (27) and Rickettsia prowaseki and Rickettsia mooseri excrete aspartic acid as a function of L-glutamate metabolism (4); the Specific activity of aspartase in rickettsiae has not yet been reported. It may be significant that Yersinia, Francisella, and Rickettsia are mammalian intra- cellular parasites. 28 .cumcmmosmocoa mnemoafl oHOmoQ xoon .owmnmmOSQOCOE mcflmocfi Hmpmw xoono n .cOADHGHmmp mom uxmu comm HOH mOHA hoae + Hoe mafia moae + ROHA moaA ROHA + II wOHA N.OHA DO I- see NOH no II voa oav + oav oav oav + fiauelxrompan one mad 8502 mmcflsw Munoz Emm pmm AEV HMO Hum mum omoq Amocouflummmuch mucmcflsumuoo .AHHV mflnmom mHCHmHow CH mucmcflaumump moccasufl> UmanHQmeMII.H mqm m>fluflmom mEooon :mom .0 -+ + -+ + -+ + -+ + -+ + -+ + c: o c: 0 .0.0.0.0 OOOOOOOOOOOO++++ (defoUrUrU f6 f0 mononmoumaouno msocmmoxo mo COHDQHOmQ4 Caxou ocean: :Hmwaocflnnflm cam .wmwasmmoo .cfl0flgmmm somflucm Ammasmmmov H cofluocnm comm um mueafluoz mpfluwaoommwaomomfla Ga monoxmnmxoocflplm.m mmmcmmoucwnmp mpmsmmonmlonmmoodaw mammnpcmwofln mafiam>\mcaosmHOmH mammnucmmofln mcflcmamamcmam mHmQSDCMmofin mcflaoflnumz mflmonucmmofln wqflomam\mcflcomnna mmmmno mmz mo mao>oa 30H 00 coflumaweflmm< mmmuummmd :owpmucmenmm mmocEmam cofluwucmfiumm wmoanflamz mHmoHDOHmnsuocsmmm mflcflmuow maummm macflmwow pcmcflahoumo .AHHV mflmoasoumnsuocsmmm macflmnmw cam mflpmmm mecflmumw omwu UHHB cmm3pmn mmocmummmap 0Hmmuocw£m mEomI|.m mqmfle 3O .meHEuoumw poz .cflom oawummmm mo COHDMHSEDUOM me Umpasmmu was“ coauflpcouo n .uanmB Hamo who mom CHE Hod powoupmmc cfiom OCHSM mo mmaoac mm commoumxm ohm measmwmw ODCZ n.0H m.HHH H.mH h.h OQOZ m.NH m.mvm mzflomaw wcflcomnnelq mcflmmummmdlq oawuummmmlq mcflaoumlq mcHEmuuslq mumfimusaouq mcflmpm%UIq maficmaalq ocflummlq m.m mm o.s mm mflmoasoumbsuocsmmm macflmuow mflpmmm aflcflmhmw caom ocflsfi m.Hmm memoHsoumnspOUsmmm MHGHmumw can wn>m mflummm macamumw wo maaoo mcflummu ha mpflom ocHEm OEOm mo coawosuwmmp mo mmumu HmwpflaHll.m mqmde 31 Hopcs pmcHMOUV .CHououm mo 08 mom Amconumz can mamflumumz means 00 mayo» CH cm>Hm mum mmflyfl>flpom camaoommm mao.o moo.o Haoo manuancsomm HHo.o Hoo.o mamOasoumospopsmmm mflaflmuow Hoo.o noo.o mflummm macflmucw omMGHEMmcmup ommGHEmmcmup m0m¢momamxo mpm>suwm Emflcmmuo ummemnsaouq umumsmusaouq m.NHM Haoo mmaomnmnomm pom .Hmm mHmoHDouwnspOUsmmm macfimumw .mh>m mflummm macflmnmw mo mHHmo mo mpomupxm commamflp ca mmm:HEwmcmuu mpmumomameImmemvsHmlq can mmmGHEMmcmup mum>summlmmem05Hmlq mo mmflufl>flgom oameommmll.v mqmflm me mufl>fluom cemaoommm 33 mo.o ma.o ma.o ohm fiaoo weaveumnomm Ho.o .o.z .Q.z 0mm no.0 Hm.o mm.o ohm mHmOHSOHmnsuocDowm macflmumw mo.o .Q.z .o.z 0mm Hoo.ov Hoo.ov Hoo.ov ohm mflummm aflcflmnmw Hoo.ov Hoo.ov Q.o.z 0mm Ems mHE Emu on npsoum no musumummame Ewflcmmuo EDHUGE 8.xzmzv Seneca oeumnncSm Hegeces cam .AmHmv mumaummmMIq EDHUOm SE o.m cocoa mcflcfimucoo auoun coflmswcfl “Ham: .Azmuv asfipoe oapmnpcmm memEoo ca npzonm Houmm Umummcum mam flHoo manoaumnomm com .Hmm mfimoasoumnsuocsomm wflcflmno» .oh>m mflpmmm macflmuow mo muomnuxm GONSHMH© CH mmmvummmm mo wufl>fipom oawfloommll.w mamme 34 TABLE 7.-—Specific activities of aSparaginase in dialyzed extracts of Yersinia pestis, Yersinia pseudo— tuberculosis, and Escherichia coli. Organism Asparaginasea Yersinia pestis EV76 0.004 Yersinia pestis KUMA 0.009 Yersinia pestis KIM 0.005 Yersinia pestis A1122 0.007 Yersinia pseudotuberculosis PBl 0.024 Escherichia coli K12 0.180 aSpecific activity is given in terms of units (defined in Materials and Methods) per mg of protein. 35 Figure 1.——Degradation of L-amino acids by Yersinia pestis EV 76 (middle) and Yersinia pseudo- tuberculosis (bottom) chromatogramed at culture optical densities of 0.85 and 0.91, respectively, during aeration at 37°C in preincubation mixture plus 0.01 M D—glucose. Position of amino acids (top) correspond to: lysine (l), histidine (2), arginine (3), asparagine (4), glutamine (5), methione sulfoxide (6), proline (7), threonine (8), alanine (9), glycine (10), serine (11), glutamic acid (12), aspartic acid (13), tyrosine (14), valine, tryptophan and methionine (15, 16, 17), phenylalanine (18), leucine and isoleucine (19, 20); solvent 1 was phenol:water (4:1 g/ml) and solvent 2 was n—butanolzacetic acid:water (100:22; 50 V/V). 37 Figure 2.——Degradation of L-glutamic acid by resting cells of Yersinia pseudotuberculosis PBl (<’) and Yersinia pestis EV76 (.) with accumulation of aspartic acid ((3) by the latter. 38 .=>_\mm_._05_0m0:>_ HOURS 39 Figure 3.——Degradation of L—proline by resting cells of Yersinia pseudotuberculosis PBl (.) and Yersinia pestis EV76 (‘>) with accumulation of glutamic acid (C,) and aspartic acid ((3) by the latter. 40 L _ lQO 80+ 0. o. 6 4 ..s_\mm._o_20mo_ —| D 2 2 L0 L5 20 HOURS 05 41 Figure 4.——Degradation of L—aspartic acid by resting cells of Yersinia pestis EV76 (C,) and Yersinia pseudotuberculosis PBl (.) with accumulation by the latter of fumarate (CD), malate (GD), succinate (C5), and an unidentified non—volatile product (‘)). 42 8.0 *- .=>_ \mMAOEOm—Ozz HOURS 43 Figure 5.——Degradation of L—asparagine (CD) by resting cells of Yersinia pestis EV76 with accumulation of aspartic acid (.) . 44 O... 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