Interconversion of Purine Mononucleotides in Pasteurella Pestis' R

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INFECTION AND IMMUNITY, May 1970, p. 446-454 Vol. 1, No. 5 Copyright © 1970 American Society for Microbiology Printed in U.S.A. Interconversion of Purine Mononucleotides in Pasteurella pestis' R. R. BRUBAKER Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48823 Received for publication 22 December 1969

Included among the five established determinants of virulence in Pasteurella pestis are the abilities of cells to accomplish the de novo biosynthesis of purines and to grow as dark pigmented (P+) colonies on a solid synthetic medium containing hemin. P+ isolates of P. pestis strain KIM-10 (mouse intraperitoneal LD50 < 10 cells) failed to convert exogenous guanine-8-14C to adenine residues of ribonucleic acid (RNA) when cultivated in a minimal medium which favored the pigmentation reaction. This conversion occurred in P+ cells grown in an enriched medium which did not support the pigmentation reaction and was observed in P- mutants cultivated in both types of media. Both P+ and P- isolates converted exogenous adenine-8-14C but not adenine-2-14C at a significant rate to guanosine residues of RNA when grown under a variety of conditions. This difference appeared to reflect a deficiency of adenine deaminase. The mouse intraperitoneal LD60 of purine-auxotrophs was about 102 cells when the metabolic block occurred prior to the de novo formation of inosine monophosphate (IMP). In contrast, the corresponding value for a mutant blocked between IMP and guanine monophosphate was > 107 cells in mice and > 108 cells in guinea pigs.

Purine-dependent mutants of highly patho- tionship between adenine dependence and aviru- genic bacteria such as Pasteurella pestis, Bacillus lence in P. pestis could be established (9). anthracis, Klebsiella pneumoniae, and certain Cells of eight strains of P. pestis studied by species of Salmonella often exhibit dramatic de- Mayskiy (27) were unable to convert exogenous creases in virulence (9, 15, 16, 20). In the case adenine-8-'4C to guanine residues of nucleic of B. anthracis, auxotrophs blocked between acids. This observation indicates that the or- inosine monophosphate (IMP) and adenosine ganisms were unable to convert adenine directly monophosphate (AMP) were avirulent, whereas to IMP via hypoxanthine or indirectly via amino- mutants blocked in the de novo synthesis of imidazole-carboxamide ribotide (AICAR). An IMP or those unable to convert IMP to guano- outline, modified from Magasanik (25), of path- sine monophosphate (GMP) remained fully viru- ways involving the de novo synthesis and inter- lent (20, 21). However, guanine-auxotrophs of conversion of purine nucleotides as reported for some of the gram-negative bacteria were of re- enteric bacteria is shown in Fig. 1. However, a duced virulence. The nature of the mutations block in the latter pathway would necessarily promoting avirulence in P. pestis has not yet prevent the biosynthesis of histidine (25), an been clarified. One avirulent purine-auxotroph amino acid which is not normally required by of this species (strain MI) exhibited an unusual wild-type P. pestis (11, 18). All strains of P. requirement for hypoxanthine, xanthine, or pestis could convert guanine-8-14C to adenine guanine; adenine failed to support growth, sug- residues of nucleic acids (27). gesting that cells of strain MI were unable to One purpose of this study was to resolve the convert adenine to IMP. A second purine-auxo- evident discrepancy between the interconversion troph (strain T3) was able to grow on adenine of purine ribotides and the nutritional require- but not hypoxanthine, xanthine, or guanine and ments of P. pestis. A second objective was to de- thus resembled the avirulent mutants of B. termine which of the relevant enzymes are neces- anthracis. Unlike strain Ml, strain T3 was iso- sary for the expression of virulence. lated from an avirulent prototroph; thus no rela- MATERIALS AND METHODS I Presented in part at the 69th Annual Meeting of the American Bacteria. Mutants obtained from wild-type P. Society for Microbiology, Miami Beach, Fla., May 1969. pestis strain KIM-10 were used in most experiments. 446 VOL. I, 1970 PURINE METABOLISM 447

The prototroph is a typical virulent strain of the Isolation of purine auxotrophs. Mutations were in- variety mediaevalis of Devignat (14). duced in P. pestis by irradiation with ultraviolet light Media. The composition of the minimal medium (UV), and auxotrophs were selected by a modification used for the selection and characterization of purine of the penicillin technique (24); all operations were auxotrophs is shown in Table 1. An enriched medium, performed at 26 C. After incubation on slopes of en- containing 1% yeast extract (Difco) and 2% Bacto riched medium for 24 hr, the cells were removed and Casitone (Difco) in place of the nitrogenous compo- diluted in sterile 0.033 M potassium phosphate buffer, nents ofthe minimal medium, was used fortheisolation pH 7.2 (phosphate buffer), to yield a concentration of and routine cultivation of auxotrophs. The complex 5 X 107 viable organisms per ml. About 6 ml of this synthetic medium of Higuchi, Kupferberg, and Smith suspension received sufficient irradiation with UV to (19), modified as shown in Table 2, and the minimal reduce viability by 50%. To 5 ml ofliquid enriched me- medium described previously were used to prepare dium (prepared at double strength) in a screw-cap tube cells containing radioactive ribonucleic acid (RNA). (20 by 125 mm) was added 5 ml of irradiated cells. The tube was then slanted and, after incubation for 24 hr, the cells were collected by centrifugation at ADENOSINE 10,000 X g for 15 min, washed twice with phosphate ADENINE buffer, and transferred in 5 ml of phosphate buffer to a { / \~~* INOSINE sterile 50-ml Erlenmeyer flask fitted with a cotton x;HYPOXAN-, INTHINE XANTHINE stopper. The flask was aerated on a shaker for 2 hr, AMP \ f-A and the organisms were centrifuged and resuspended /,, XMP ATP (\ in phosphate buffer at a concentration of 5 X 107 cells I per ml. To 4.5 ml of minimal medium in a screw-cap IMP tube (20 by 125 mm) was added 0.5 ml of cell suspen- / w'* .GMPP k25 sion. The tube was slanted and, after incubation for 8 IMIDAZOLE y"< 45 hr, 0.05 ml of a fresh sterile 0.7% solution of po- GLYCEROL ,- AICAR GUANINE> tassium penicillin G (E. R. Squibb and Sons, New PHOSPHATE York, N.Y.) was added aseptically to yield a final I GUANOSINE I I1 . concentration of approximately 100 units per ml. After AICA further incubation for 2 to 3 days, samples of 0.1 ml were plated directly onto the surface of solid enriched THIAMINE AIR medium. Colonies arising after incubation for 2 days were replicated onto solid minimal medium. The latter 46 step, however, was not essential, because about 50% PHOSPHATE of the surviving cells were auxotrophs and the ma- FIG. 1. Schema illustrating the enzymatic pathlways jority of these exhibited a nutritional requirement for (broken lines) or individual reactions (solid lines) of the hypoxanthine. de novo biosynthesis and interconversions of purine Characterization of purine auxotrophs. The nutri- nucleotides as reported for enteric bacteria. Abbrevia- tional response to purines was determined by layering tions are defined in the text; nwnerals refer to auxo- 5 ml of minimal agar, inoculated with 105 cells per ml, trophic strains possessing corresponding metabolic onto plates of solid minimal medium. After overnight blocks. incubation at 26 C, drops of 0.001 M or saturated solu-

TABLE 1. Composition of minimal synthetic mediuma Component Concn (mm) Component Concn (mm)

Salt solutionsb Amino acids' K2HPO4 25 L-Isoleucine 0.5 Citric acid 10 L-Methionine 0.5 Ammonium lactate 10 L-Phenylalanine 0.5 MgCl2 2.5 L-Valine 0.5 FeCI2 0.1 Glycine 10 MnCl2 0.01 L-Threonine 0.5 Miscellaneous solutions L-Tyrosine 0.5 D-Glucosec 10 L-Arginine 0.5 Na2S203c 2.5 L-Aspartic acid 10 CaCl2d 2.5 a Neutralized with 5 N NaOH. b Prepared as XIO stock; sterilized by autoclaving. c Prepared as X100 stock; sterilized by filtration. d Prepared as X100 stock; sterilized by autoclaving. e Sterilized by autoclaving. 448 BRUBAKER INFEC. IMMUN.

TABLE 2. Composition of complex synthetic mediulna

Component Concn (mm) Component Concn (mM)

Salt solutionb Amino acids- K2HPO4 25 DL-Alanine 9.0 Citric acid 10 L-Isoleucine 7.6 Potassium gluconate 10 L-Leucine 4.0 Ammonium acetate 10 L-Methionine 3.2 MgCl2 2.5 L-Phenylalanine 4.8 FeCl2 0.1 L-Threonine 2.8 MnCl2 0.01 L-Valine 13.6 Vitamin solutionc L-Arginine 2.0 Thiamine 0.01 L-Proline 14.0 Calcium pantothenate 0.01 L-Glutamic acid 162 Biotin 0.005 L-Lysine 2.2 Miscellaneous solutions Glycine 5.2 D-Xylosec 61 L-Tyrosine 2.2 L-Cysteined 1 .0 L-Tryptophanc 0.1 a Neutralized with 5 N NaOH. b Prepared as X1O stock; sterilized by autoclaving. c Prepared as X10 stock; sterilized by filtration. d Prepared as XIOO stock; sterilized by filtration. e Sterilized by autoclaving. tions of purines or their derivatives were spotted on the of cells. The interrelationship between these isolates is agar surface; zones of growth were recorded after shown in Fig. 2. further incubation for 3 to 4 days. Purine nucleotide interconversions. A modification The resulting response facilitated the identification of the Schmidt-Thannhauser technique (30) was used by enzymatic assay of blocks in the de novo pathway to isolate mononucleotides from cells cultivated with of purine biosynthesis. Mutant cells were incubated radioactive purines. To 50 ml of liquid minimal or overnight at 26 C on a model R25 shaker (New Bruns- complex synthetic medium per 500-ml flask was wick Scientific Co., Inc., New Brunswick, N.J.) in 100 added 5 X 104 M radioactive purine (specific ac- ml of liquid enriched medium per 1-liter flask, washed tivity of 0.13 ,Ac/,umole). After inoculation with 107 twice in phosphate buffer, and then aerated under the cells per ml, the cultures were aerated at 26 C for 24 to same cultural conditions for 24 hr in liquid minimal 36 hr. The cells were harvested by centrifugation, medium. The organisms were then washed in phos- washed twice with phosphate buffer, and extracted for phate buffer, suspended at a concentration of about 90 min with 10 ml of cold 5% HCI04. The resulting 1011 cells per ml of cold 0.1 M tris(hydroxymethyl)- precipitate was again centrifuged and washed with 10 aminomethane-hydrochloride (pH 7.4) plus 0.001 M ml of cold 5% HCl04 prior to extraction in a closed mercaptoethanol, and disrupted by treatment for 1 vessel for 15 min at 50 C with 10 ml of ether:ethanol min with an ultrasonic probe (Instrumentation Asso- (1:1, v/v). The suspension was centrifuged and the ciates, New York, N.Y.). After removal of cellular precipitate was dissolved in 10 ml of 0.5 M KOH; after debris by centrifugation, quantitative determinations hydrolysis for 14 hr at 37 C, the solution was chilled of enzymes were performed by methods outlined by toO C and incubated for 1 hr with 10 ml of added cold McElroy (28) and Magasanik (26); qualitative pro- 20% HC104. The resulting precipitate was removed cedures are described under Results. In some cases the by centrifugation and re-extracted with 1 ml of cold supernatant fluids from the cultures were assayed by 20% HC104, and the combined supernatant fluids the Bratton-Marshall test (15). were neutralized with 5 N KOH. After KCl04 was re- Virulence determinants. The ability of cells to ab- moved by centrifugation, the sample was brought to sorb hemin and thus grow as dark or pigmented (P+) dryness on a model 3-2100 flash evaporator (Buchler colonies was determined with the hemin agar of Jack- Instruments, Inc., Ft. Lee, N.J.), reconstituted with son and Burrows (22). The modified magnesium oxa- distilled water to 10 ml and centrifuged, and the super- late agar described previously (4) was used as an assay natant fraction was again brought to dryness. The for calcium dependence. The expression of this prop- sample was dissolved in 5 ml of distilled water and re- erty closely correlates with the ability to produce the frigerated for 1 hr to permit precipitation of remaining virulence or V and W antigens (VW+) (7, 8). Remain- KC104. The clear solution was then decanted, dried, ing determinants of virulence and the LD5o were as- and dissolved in 2 ml of distilled water. sayed by methods described previously (2, 7). Samples of 20 to 50 uliters were subjected to electro- Known avirulent P- and VW- mutants were iso- phoretic analysis on Whatman no. 1 paper in 0.05 M lated for use in experiments requiring large numbers ammonium acetate buffer (pH 3.5) at 65 v per cm for VOL. 1, 1970 PURINE METABOLISM 449 60 min (12). Nucleotides were located with a model R51 minerolight (Ultra-Violet Products, Inc., San KIM-1O(VW+,P+) C Gabiel, Calif.), and the distribution of radioactivity B (WILD-TYPE) was determined with a model 7201 radiochromato- KIM-O I(VW+K1M-O l gram scanner (Packard Instrument Co., Downers Grove, Ill.). In certain experiments, the purine nucleo- , ~~~~B tides were eluted in distilled water and determined quantitatively with the use of established extinction 45(VW,P+) 25(VW+,P+) KIM-IO(VW-,P-) coefficients, and radioactivity was estimated with a 46(VW-,P+) B 45(VW ,P+) Mark 1 liquid scintillation counter (Nuclear-Chicago 61(VW-,P+) 46(VW+,P+) C Corp., Des Plaines, Ill.) using Bray's scintillation 161 (VW+,P+) fluid (3). 45(VW-,P-) B 45(VW+,P-) Reagents. All organic constituents of synthetic 46(VW-,P-) 46(VW+,P-) media were obtained from Calbiochem, Los Angeles, - Calif. Purine nucleotides and cofactors used in enzy- 61(VW P-) 61(VW+,P-)I matic determinations were products of the Sigma FIG. 2. Schema illustrating the origini of substrains Chemical Co., St. Louis, Mo. Radioactive purines used in this investigationt. A, intductiont by ultraviolet were purchased from the Volk Radiochemical Co., light and selection with penicillini; B, selection ont mag- Skokie, Ill. nesium oxalate agar; C, selection on hemint agar plius 0.0005 M hypoxanthine. RESULTS By use of the penicillin technique, 100 auxo- intermediate of divergent pathways leading to trophs of strain KIM-10 (VW+, P+) were iso- IMP and the pyrimidine moiety of thiamine (29). lated which grew in the presence of hypoxan- The nutritional response of mutant 61 (VW+, thine. These mutants were separated into three P+), an example of the second class of purine classes on the basis of their nutritional response auxotrophs, was similar to that of isolate 46 to other purines and purine ribosides (Table 3). (VW+, P+) except that a nutritional requirement A representative isolate of each class was then for thiamine was not detected. The fact that cells preserved for determinations of virulence; VW- of mutant 61 (VW+, P+) were thiamine-inde- and P- mutants of these isolates were also se- pendent and able to grow in the presence of lected (Fig. 2). AICA suggested that the metabolic block was The first class of mutants, exemplified by iso- located before the biosynthesis of AICAR but late 46 (VW+, P+), exhibited a dual nutritional after the formation of AIR. Subsequent study requirement for thiamine plus hypoxanthine, showed that cells of strain 61 (VW-, P+) excreted adenine, or aminoimidazole carboxamide a compound yielding an orange color in the Brat- (AICA). Isolates of this phenotype were assumed ton-Marshall test (emax = 500 nm) which closely be blocked prior to the formation of amino- matched the spectrum of aminoimidazole ribo- imidazole ribotide (AIR), the last common side. This accumulation is consistent with the absence of phosphoribosyl-aminoimidazole car- TABLE 3. Growth of purinie-auxotrophs ont solid boxylase (EC 4.1.1.21), although it could also minimal medium supplemented with various result from the loss of phosphoribosyl-amino- purines and derivatives imidazole-succinocarboxamide synthetase (EC 6.3.2.6), because the substrate of the latter spon- Mutant strain taneously decarboxylates to AIR (30). Added source of of the third class, such as isolate 45 purine 46 (VW+, 61 (V\\, 45 (VWV, 25 (V\\+, Mutants p+)a p+) b p+)b P+)b (VW+, P+) expressed a specific requirement for hypoxanthine (or inosine). Single-step mutants AICAc. + + 0 0 of this phenotype cannot be isolated from species Adenine ...... + + 0 0 of bacteria which possess the direct (deaminating) Adenosine ...+ ..+ 0 0 and indirect (histidine cycle) pathways leading Hypoxanthine + + + 0 from adenine to IMP (24). The fact that cells of Inosine ...... + + + 0 strain 45 (VW+, P+) did not exhibit a nutritional Xanthine . ..0. ..0 0 0 requirement for histidine indicated that this Guanine 0 0 0 + mutant was capable of forming AICAR during Guanosine.... 0 0 0 + the biosynthesis of imidazole-glycerol phosphate, a precursor of histidine (25). It seemed probable, a Growth occurred only in the presence of block in thiamine (10-5 M). therefore, that the induced metabolic b Growth occurred in the absence of thiamine. strain 45 (VW+, P+) was located after the bio- c Aminoimidazole carboxamide. synthesis of AICAR but before the formation of 450 BRUBAKER INFEC. IMMUN. IMP. An excretion product from cells of isolate from strain 45 (VW-, P+) had undergone the 45 (VW-, P+) was noted which yielded a purple mutation from P+ to P-. A similar relationship color in the Bratton-Marshall test; the spectrum between expression of the P- phenotype and abil- of this derivative closely matched that of AICA ity to grow in the presence of guanine was also riboside (emax = 540 nm). Attempts to demon- noted in the case of strains 46 (VW", P) and strate the conversion of IMP to AICAR in cell- 61 (VW-, P-). In addition, all of 25 P- clones free extracts of isolate 45 (VW-, P+) containing obtained from strain 45 (VW-, P+), after selec- added tetrahydrofolic acid were not successful, tion on hemin agar plus hypoxanthine, were able whereas AICAR was demonstrated qualitatively to grow in the presence of guanine. in similar experiments utilizing strain KIM-10 Addition of 0.01% hemin to the miniimal agar (VW", P+). These findings indicate that isolate described in Table 1 resulted in a medium which 45 (VW+, P+) lacks aminoimidazole carboxamide supported an intense pigmentation reaction simi- ribotide formyltransferase (EC 2.1.2.3). lar to that of the hemin agar of Jackson and Further study showed that adenine-8-14C was Burrows (22). In contrast, the pigmentation reac- efficiently converted to GMP by strains KIM-10 tion was not expressed by cells of the P+ pheno- (VW+, P-) and KIM-10 (VW-, P+) in minimal type on complex synthetic medium (Table 2) and complex synthetic media (Table 4). In con- supplemented with the same concentration of trast, results obtained in identical experiments hemin. It was of interest, therefore, to determine with adenine-2-'4C varied from essentially no whether P+ cells blocked in the de novo synthesis conversion to values approaching 15% of those of IMP could grow in complex synthetic medium obtained with adenine-8-14C. This finding sug- supplemented with guanine. P+ cells of strain gested that the primary mechanism for the con- 45 grew almost as rapidly on complex synthetic version of adenine to GMP in the prototroph medium containing added 0.0005 M guanine as was via the histidine cycle rather than by deam- did cells of the P- phenotype (Table 5). ination. Subsequent attempts to detect adenine An additional attempt was made to charac- deaminase (EC 3.5.4.2) in five derivatives of terize the metabolic block between exogenous strain KIM-10 were not successful. guanine and IMP in P+ cells grown in minimal Attention was next directed towards deter- medium. Ribotides were isolated from cells of mining why mutants blocked in the de novo syn- strains KIM-10 (VW-, P+) and KIM-10 (VW+, thesis of IMP were unable to grow in the P-) after cultivation in minimal and complex presence ofexogenous guanine, guanosine, or xan- synthetic media containing guanine-8-'4C. The thine. In preliminary experiments, a large num- specific activity of recovered GMP was essentially ber of apparent revertants of strain 45 (VW-, identical to that of the exogenous guanine re- P+) were observed to form colonies on solid gardless of the medium or the strain employed minimal medium supplemented with guanine. (Table 6). In contrast, no radioactivity was de- The fact that these organisms failed to grow in tected in AMP recovered from P+ cells after the absence of guanine indicated that they had not growth in the minimal medium although both P+ recovered the ability to convert AICAR to IMP but rather had acquired the ability to convert guanine to IMP. Further study showed that all TABLE 5. Doubling time ofP+ and P cells ofstrain 45 in minimal and complex synthetic medium of 50 suppressor mutants of this type obtained supplemented with purine Added purine TABLE 4. Conversion of adenine-8-14C to adenine resi- Strain (0.0005 Mt) Doubling time dues (isolated as adenosine monophosphate) and guanine residues (isolated as guanosine monophosphate) ofribonucleic acid by growing cellsa Minimal synthetic medium 45 (VW-, P+) None >24 hr Specific activity 45 (VW-, P+) Hypoxanthine 4 hr 10 min of isolated 45 (VW-, P+) Guanine >24 hr Strain Medium mononucleotides 45 (VW-, P-) None >24 hr 45 (VW-, P-) Hypoxanthine 2 hr 45 min AMP GMP 45 (VW-, P-) Guanine 2 hr 40 min Complex synthetic medium KIM-10 (VW+, P-) Minimal synthetic 0.14 0.10 45 (VW-, P+) None >24 hr KIM-10 (VW-, P+) Minimal svnthetic 0.12 0.09 45 (VW-, P+) Hypoxanthine 1 hr 35 min KIM-10 (VW+, P1) Complex synthetic 0.11 0.08 45 (VW-, P+) Guanine 1 hr 45 min KIM-10 (VW+, P+) Complex synthetic 0.10 0.09 45 (VW-, P-) None >24 hr 45 (VW-, P-) Hypoxanthine 1 hr 20 min a Specific activity of exogenous adenine-8-14C was 0.13 45 (VW-, P-) Guanine 1 hr 10 min pc/IAm. VOL. I1, 1970 PURINE METABOLISM 451 and P- organisms produced labeled adenine resi- A search for single-step purine-auxotrophs dues of RNA during cultivation in the complex capable of fulfilling their nutritional require- synthetic medium. Typical separations of mono- ment with compounds other than hypoxanthine nucleotides and determinations of radioactivity or inosine resulted in the isolation of strain 25 in hydrolysates of ribonucleic acid (RNA) (VW+, P+). Cells of this mutant grew only in the from P+ cells grown in minimal and complex presence of guanine or guanosine (Table 3) and synthetic media containing guanine-8-'4C are lacked detectable guanosine monophosphate syn- shown in Fig. 3. thetase (EC 6.3.4.1) when grown under conditions which yielded a specific activity of 0.004 in TABLE 6. Conversion ofguanine-8-14C to adenine residues extracts of strain KIM-10 (VW-, P+). (isolated as adenine monophosphate) and guanine resi- Mutants 46 (VW+, P+), 61 (VW+, P+), 45 dues (isolated as guaniosine monophosphate) ofribonu- (VW+, P+), and 25 (VW+, P+) were able to syn- cleic acid by growing cellsa thesize capsular antigen or fraction I and were Specific activity pesticinogenic. Accordingly, they possessed all of isolated of the established determinants of virulence in Strain Medium mononucleotides P. pestis (6, 11) except purine independence. AMP GMP The intraperitoneal LD50 in mice of these isolates and their prototroph is listed in Table 7. It is KIM-10 (VW+, P- Minimal synthetic 0.05 0.12 evident that mutations preventing the de novo KIM-10 (VW-, P+) Minimal synthetic 0.0 0.10 biosynthesis of IMP promoted only a slight de- KIM-10 (VW+, P-) Complex synthetic 0.04 0.11 crease in virulence in mice (LD5o 102 cells), KIM-10 (VW-, P+) Complex synthetic 00.04 0.12 whereas a metabolic block which prevented the a Specific activity of exogenous guanine-8-14C was 0.13 conversion of IMP to GMP resulted in outright jucIAm. avirulence (LD5o > 107 cells). The intraperitoneal

FIG. 3. Radioautographs of hydrolysates of ribonucleic acid isolated from cells of strain KIM-1O (VW-, P4) after growth with guanine-8-_4C and paper electrophoresis; the time constant, counting range, andchart speed were 10 sec, 13 counts per min, and I cm per min, respectively. A, cells grown in complex synthetic medium; B, cells grown in minimal synthetic medium; C, location of mononucleotides as observed under ultraviolet light. 452 BRUBAKER INFEC. IMMUN.

TABLE 7. Virulence ofpurine-auxotrophs of transferase, which is necessary for both the de Pasteurella pestis obtained from novo biosynthesis of IMP and the conversion of strain KIM-10 AMP to IMP via AICAR, might directly account Intraperitoneal for the nutritional response of strain 45 (VW+, Strain Metabolic block LD6o in white P+). This possibility was tested by determining mice the rates of incorporation of adenine-2-14C and adenine-8-'4C into guanine residues of RNA. KIM-10 (Prototroph) <10 cells Both carbon 8 and carbon 2 would be expected 46 (VW+, P+) Early block in de 102 cells to novo synthesis remain in the purine moiety during deamina- 61 (VW+, P+) Formation of 102 cells tion. However, carbon 2 but not carbon 8 would AICARa be transferred to imidazoleglycerol phosphate 45 (VW+, P+) Conversion ofAICAR _102 cells after metabolism via the histidine cycle (24). to inosine mono- The fact that incorporation from adenine-2- phosphate 14C was no more than 15% of that obtained with 25 (VW+, P+) Conversion of xan- > 107 cells adenine-8-14C indicates that the histidine cycle thine monophos- represents the major route in P. pestis for the con- phate to guanosine version of adenine to IMP. This interpretation monophosphate was strengthened by the inability to detect ade- a Aminoimidazolecarboxamide ribonucleotide. nine deaminase in cell-free extracts of the organisms. This result does not, of course, prove that P. LD50 of strain 25 (VW+, P+) in guinea-pigs was pestis lacks the genetic potential to synthesize >108 cells. adenine deaminase; further study of extracts of organisms grown and prepared under different DISCUSSION conditions will be necessary to clarify this situa- Mutational loss of AICAR formyltransferase tion. The significant point is that these experi- in E. coli merely results in a general nutritional ments, in contrast to those of Mayskiy (27), requirement for purine which can be satisfied demonstrate that cells of P. pestis can convert by adenine, hypoxanthine, xanthine, or guanine. exogenous adenine to guanine residues of RNA. It is also necessary to prevent the deamination of A possible explanation for this discrepancy adenine to hypoxanthine and the conversion of would be the presence of histidine in the me- xanthine and guanine to IMP in this organism dium used by Mayskiy. This amino acid is known to produce a mutant which exhibits a mono- to repress the initial enzyme of the histidine specific requirement for hypoxanthine (25). cycle (25) and might thus prevent the conversion Nevertheless, this phenotype was expressed by of AMP to IMP via AICAR in P. pestis. How- P. pestis after the loss of AICAR formyltrans- ever, preliminary attempts to demonstrate such ferase; mutants blocked prior to the formation an effect of histidine in strain KIM-10 (VW+, of AICAR could utilize adenine, AICA, or hypo- P-) have been unsuccessful (unpublished observa- xanthine as a source ofpurine. Xanthine and guan- tions). ine were never observed to support the growth of Of considerable interest was the finding that single-step mutants blocked in the de novo bio- P+ mutants blocked in the de novo pathway of synthesis of IMP. purine biosynthesis were unable to grow in the The possibility was considered that the pres- presence of xanthine or guanine under conditions ence of a metabolic block which prevented the which favored the phenotypic expression of the de novo biosynthesis of IMP might indirectly pigmentation reaction. Although this property promote stasis in the presence of exogenous is an important determinant of virulence in P. adenine. For example, under certain conditions pestis, little is known about the mechanism of adenine is known to repress formyltetrahydro- hemin absorption or its biological significance. folate synthetase (EC 6.3.4.3) in Streptococcus Pesticin I (5) and certain artificial dyes (22, E. faecalis (1). Similarly, toxic concentrations of D. Beesley, personal communication) are also intermediates can accumulate in mutants lacking absorbed by P+ cells, and various modifications enzymes which catalyze the interconversion of of the environment such as elevated temperature purinre nucleotides (13, 25). These or related phe- or use of complex media can inhibit the absorp- nomona might account for the observed inabil- tion ofhemin (10, 22). Burrows and his colleagues ity of cells of strain 45 (VW+, P+) to utilize ade- have suggested that expression of the P+ pheno- nine as a source of purine. However, it seemed type in vivo is associated with the ability of P. equally probable that loss of AICAR formyl- pestis to derive iron for growth (11, 23). This VOL. 1, 1970 PURINE METABOLISM 453 hypothesis is supported by the fact that the lethal- progress. The inability of Mayskiy (27) to detect ity of P- isolates which have retained the remain- a metabolic block between guanine and AMP ing determinants of virulence is restored in mice could reflect use of P strains or a medium which by concomitant injection with iron or hemin (23). would not support the pigmentation reaction. However, the injection of iron with cells which It is evident from these studies that the viru- are avirulent due to the loss of the pesticin I lence of purine-auxotrophs of P. pestis varies determinant also results in the restoration of considerably depending upon the location of the virulence (6). Accordingly, the possibility exists metabolic block. Mutants which were unable to that some common mechanism which defends synthesize IMP via the de novo pathway retained the host against P- and nonpesticinogenic cells a considerable degree of virulence in mice, is inactivated by iron and that this defense mech- whereas strain 25 (VW+, P+), blocked between anism is in turn overcome by pesticinogenic cells IMP and GMP, was completely avirulent (Table of the P+ phenotype. 6). Attempts to isolate VW+, P+ adenine auxo- A correlation between P+ and sensitivity to trophs, blocked between IMP and AMP, have pesticin I has been described (5). However, the so far been unsuccessful. Such mutants, how- discovery that P+ but not P- purine-auxotrophs ever, might also be expected to be avirulent as blocked in the de novo biosynthesis of IMP fail to grow on exogenous guanine is the first indi- judged by the results reported for B. anthracis cation that metabolic differences exist between (21). It is also of interest that guanine auxo- the two types of cell. Of additional interest is the trophs of the latter species, blocked between finding that the presumptive block between IMP and GMP, were fully virulent. Presumably guanine and AMP in P+ cells was only expressed this difference reflects a greater ability of B. under conditions which favor the pigmentation anthracis to obtain guanine in vivo. reaction. The fact that strain 25 (VW+, P+), blocked between XMP and GMP, was able to ACKNOWLEDGMENTS grow in the presence of guanine indicated that The technical assistance of Roselyn V. Little is gratefully this purine was able to enter P+ cells. This sup- acknowledged. Preliminary aspects of this study were undertaken position was verified by the results of at the U.S. Army Biological Laboratories, Frederick, Md. isotope- This investigation was supported by the Michigan Agricultural dilution experiments which showed that exoge- Experiment Station (Article no. 4911) and Public Health Service nous guanine was efficiently incorporated into grant Al 08468-01 from the National Institute of Allergy and guanine residues of RNA under all sets of ex- Infectious Diseases. perimental conditions. Obviously the block could not occur between IMP and AMP; thus atten- LITERATURE CITED tion has become focused on the of GMP 1. Albrecht, A. M. and D. J. Hutchison. 1964. Repression by activity adenine of the formyltetrahydrofolate synthetase in an reductase, an enzyme which catalyzes the conver- antifolic-resistant mutant of Streptococcus faecalis. J. sion of GMP to IMP. Bacteriol. 87:792-798. A deficiency of GMP reductase in P+ cells 2. Beesley, E. D., R. R. 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