Vol. 28, No. 1INFECTION AND IMMUNITY, Apr. 1980, p. 74-810019-9567/80/04-0074/08$02.00/0

Adenine Nucleotide Degradation by the Obligate IntracellularBacterium Rickettsia typhi

JIM C. WILLIAMS

Rickettsial Diseases Section, Laboratory ofMicrobial Structure and Function, Rocky MountainLaboratories, National Institute ofAllergy and Infectious Diseases, Hamilton, Montana 59840, and Naval

Medical Research Institute, Bethesda, Maryland 20014

Adenosine 5'-triphosphate (ATP) was catabolized by whole cells and cell-freeextracts of Rickettsia typhi to adenosine 5'-diphosphate (ADP) and then toadenosine 5'-monophosphate (AMP), the end product of ATP catabolism underthe experimental conditions used. The only intermediate of the pathway fromATP to AMP which was identified by thin-layer chromatography and quantitatedby the "4C content was ADP, whereas products such as adenine, adenosine,hypoxanthine, inosine, and inosine 5'-monophosphate were not detected. Theenzymes which could be theoretically responsible for the catabolism or theanabolism ofAMP were not detected by standard assay procedures. Most impor-tantly, 5'-nucleotidase or nonspecific phosphatase and AMP nucleosidase activi-ties were undetectable under a variety of experimental conditions. Although thesetwo enzymes remove AMP from the adenylate pool in other cells, they areapparently nonfunctional in R. typhi. The biosynthesis of ATP was initiated byadenylate kinase because no adenine phosphoribosyltransferase or adenosinekinase could be detected. Furthermore, AMP was transported intact withoutprior dephosphorylation. These observations suggest that for R. typhi the in vivoactivity of adenine nucleotide interconversion was limited to the nucleotides, withAMP being the end product of ATP catabolism, and that the salvage of purinebases and nucleosides was not an essential feature of purine metabolism. Theseresults elucidate the findings of a previous study which showed that in the absenceof glutamate as a source of energy, the adenylate energy charge of resting cells ofR. typhi is drastically lowered by the high proportion of AMP.

Previous studies with the typhus group rick-ettsiae have indicated that these obligate intra-cellular bacteria express a limited capacity forenergy metabolism in an environment rich innucleotides (for a review, see reference 20). Re-cent investigations into the mechanisms of nu-cleotide acquisition from the hosts' milieu byRickettsiaprowazekii have indicated that a car-rier-mediated transport system for adenosine 5'-diphosphate (ADP) and adenosine 5'-triphos-phate (ATP) exists and probably accounts forthe accumulation and maintenance of endoge-nous adenine nucleotide pools (26). Furtherstudies of the energy metabolism of Rickettsiatyphi have indicated that an adenylate energycharge (EC) ratio {[ATP + 1/2 ADP]/[ATP +ADP + adenosine 5'-monophosphate (AMP)])of 0.75 can be maintained in an extracellularenvironment (25) during the metabolism of glu-tamate. In the absence of glutamate, the ade-nylate pools are disproportionate, resulting in amarked lowering of the EC via an increasedAMP pool (25). Those studies with whole cellshave shown that adenine nucleotide metabolismis balanced delicately between glutamate avail-

ability and a catabolic pathway which predomi-nates during substrate depletion.Adenine nucleotide degradation to adenine

and adenosine and resynthesis generally are car-ried out by 11 enzymes, as outlined in Fig. 1 (19).The metabolic role of this pathway has beenestablished in autonomously growing bacteria(19) and mammalian tissues (7, 8), whereas thenature of this sequence in R. typhi has beenunclear in view of the disproportionate increasein the AMP pool and the failure to detect ade-nine and adenosine (25). This study wasprompted by the unusual adenylate metabolismof the typhus rickettsiae, which suggests thatsome of these enzymes are not present. Theenzymatic degradation and resynthesis of ATPby whole cells of R. typhi therefore were com-pared with cell-free extracts and cell envelopepreparations to determine how the adenine nu-cleotide content was regulated. The results pro-vide evidence that the salvage of bases and ofnucleosides did not play a role in this bacterialparasitism of eucaryotic cells. However, the ad-enine nucleotides are indispensable for success-ful parasitism, since AMP was not degraded.

74

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

VOL. 28, 1980

pj_ 41

I~~~~ADP *-PPi

6

Pi 3

8 %, 1P i W., '%' 'R-5-P

,'.1 ,(9* 2) "

,' ATP PRPP " "

AR -{-- - - B-I _1A

P; R-I-P RFIG. 1. Pathways ofadenine nucleotide anabolism

and catabolism. For comprehensive reviews on thebehavior and interrelationships ofthe enzymes in themetabolism of adenine nucleotides, see references 7and 19. A, Adenine; AR, adenosine; R, ribose; R-1-P,ribose-l-phosphate; R-5-P, ribose-5-phosphate; PPi,inorganic pyrophosphate; Pi, inorganic phosphate;PRPP, 5'-phosphoribosyl-a-1-pyrophosphate. En-zymes: 1, AMP nucleosidase; 2, adenine phosphori-bosyl pyrophosphate synthetase; 3, adenylate kinase(AMP kinase); 4, nucleoside diphosphate kinase; 5,ATP phosphatase; 6, ADP phosphatase; 7, ATP py-

rophosphatase; 8, AMP nucleotidase (or phospha-tase); 9, adenosine kinase; 10, adenosinephosphoryl-ase; 11, adenosine nucleosidase. , ---, and * *,Generally encountered enzymes; , in R. typhi;---, not detected in R. typhi; *., not assayed withR. typhi.

MATERIALS AND METHODS

Organisms and growth conditions. The Wil-mington strain of R. typhi was propagated in 6-day-old, embryonated, antibiotic-free hens' eggs (TruslowFarms, Chestertown, Md.) at 35°C, as described pre-viously (22). Salmonella typhimurium LT2 was grownin tris(hydroxymethyl)aminomethane (Tris)-irop me-

dium or brain heart infusion broth at 37°C in a Psy-crotherm incubator shaker (New Brunswick ScientificCo.) (24). Growth was monitored by measuring theincrease in absorbency at 450 nm.

Preparation of cell suspensions and cell-freeextracts. R. typhi organisms were separated fromyolk sac components as described previously (22) on

day 9 postinfection of embryonated hens' eggs at 4°Cwithout prior freezing. The pellets were suspended in100 ml of 50 mM Tris-hydrochloride at pH 7.2 (4°C)

ADENINE NUCLEOTIDE METABOLISM 75

and centrifuged at 7,710 x g (40C) for 10 min. Thecells were resuspended in 6 ml of 50 mM Tris-hydro-chloride and sonicated (Sonifier Cell Disruptor, Bran-son Instruments Co.) at 100 W for 5 min at 40C. Thesuspension was centrifuged at 7,710 x g (40C) for 10min, and one-half of the supernatant fluid was centri-fuged at 80,000 x g for 60 min. The supernatant wasdesignated as the soluble fraction, and the pellet(membrane fraction) was resuspended in 6 ml of 50mM Tris-hydrochloride with a tissue grinder and re-centrifuged. The 7,710 x g and 80,000 x g supernatantsand membrane suspensions were assayed for enzy-matic activity.The S. typhimurium cells grown in 2-liter liquid

cultures were harvested in the late log phase by cen-trifugation at 13,300 x g (40C) for 30 min. The S.typhimurium cell suspensions were processed as de-scribed for R. typhi (see above).

Whole-cell metabolism was carried out on suspen-sions in K36 (23) buffer (pH 7.4) containing the follow-ing components at their respective millimolar concen-trations: K2HPO4, 10; KH2PO4, 5; KCl, 15; NaCl, 15;glycylglycine, 20; MgCl2, 10; and L-glutamate, 20. Pro-tein determinations were carried out by the method ofLowry et al. (12), with bovine serum albumin as thestandard.Enzyme assays. All enzyme assays were carried

out at 370C with radiochemical techniques employinglabeled (3H or '4C) substrates. The reaction productswere resolved by thin-layer chromatography (Poly-gram Cellulose MN300 polyethyleneimine [polyeth-yleneimine-cellulose]-impregnated precoated plasticsheets; J. T. Baker Chemical Co.) (15, 24). Specificactivities of the enzymes were determined under con-ditions in which product formation was proportionalto the amount of cell extract added and to time andare expressed as nanomoles per minute per milligramof protein.

Interconversion enzymes. AMP kinase (or ade-nylate kinase; ATP:AMP phosphotransferase [EC2.7.4.3]) activity was measured by monitoring the con-version of [3H]AMP to [3H]ADP and [3H]ATP. Invar-iably, with crude extracts [3H]ADP was phosphoryl-ated to [3H]ATP, which necessitated the measurementof [3H]ATP. The reaction mixture contained, in a totalvolume of 0.2 ml, the following: 50 mM glycylglycine,pH 7.4; 7.5 mM MgCl2; 1.5 mM ,B-mercaptoethanol;1.25 mM ATP, disodium salt; 1.0 mM AMP, sodiumsalt; 3.0 ttCi of [2-3H]AMP (15 Ci/mmol); and theappropriate dilution of cell-free extract. Enzymaticactivity was monitored by withdrawing three 20-tLIsamples at 2-min intervals and spotting them directlyon polyethyleneimine-cellulose plates, which had beenactivated with 10% NaCl. The plates were washed for10 min in 500 ml of anhydrous methanol and air dried,and the products of the reaction were separated chro-matographically from the substrate with 1.0M sodiumformate (pH 3.4) in rectangular glass tanks. The AMPmigrated about 3 cm above the ADP and ATP, whichremained at the origin. After the plates were dried, theADP-ATP spot was located under ultraviolet light,marked with a soft lead pencil, cut out with scissors,and placed in counting vials with 7.5 ml of scintillationfluid (LSC complete, Yorktown Research); the radio-activity was determined in a Beckman LS335 liquidscintillation spectrometer.

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

76 WILLIAMS

Guanosine 5'-monophosphate (GMP) kinase (ATP:GMP phosphotransferase [EC 2.7.4.8]) activity wasmeasured by monitoring the conversion of [3H]GMPto [3H]guanosine 5'-diphosphate and [3H]guanosine5'-triphosphate as described for AMP kinase.

Nucleoside diphosphate kinase (NDP kinase; ATP:ADP phosphotransferase [EC 2.7.4.6]) activity wasmeasured by monitoring the conversion of [8-3H]ADPto [8-3H]ATP. The reaction mixture contained, in atotal volume of 0.2 ml, the following: 50mM glycylgly-cine, pH 7.4; 7.5 mM MgCl2; 1.5 mM ,1-mercaptoeth-anol; 1.25 mM ATP, disodium salt; 1.0 mM ADP,sodium salt; 3.0,uCi of [8-3H]ADP (13 Ci/mmol); andthe appropriate dilution of cell-free extract. Enzymaticactivity was monitored as described for AMP kinase(see above) except that the product of the reaction(ATP) was separated chromatographically from ADPwith 4.0 M sodium formate, pH 3.4.ATP phosphohydrolase (ATPase [EC 3.6.1.3]) ac-

tivity was measured by monitoring the dephosphory-lation of [2-3H]ATP to [2-3H]ADP. The reaction mix-ture was that of ADP kinase except that no ADP wasadded and the 3H label was in [2-3H]ATP. The reac-tion products were separated chromatographicallywith 4.0 M sodium formate, pH 3.4.ADP phosphohydrolase (ADPase [EC 3.6.1.6]) ac-

tivity was measured by monitoring the dephosphory-lation of [8-3H]ADP to [8-3H]AMP. The reaction mix-ture was that of ADP kinase except that no ATP wasadded. The reaction products were separated chro-matographically with 1 M sodium formate, pH 3.4.AMP phosphohydrolase (AMPase or 5'-nucleotid-

ase [EC 3.1.3.5]) activity was measured by monitoringthe dephosphorylation of [2-3H]AMP to [2-3H]aden-osine (18) or by the release of 32p, as previously de-scribed (3). The reaction mixture was that of AMPkinase except that no ATP was added. To verify thereaction products, they were separated chromato-graphically with 1 mM ammonium formate. The pol-yethyleneimine-cellulose plates were scored at 2-cmintervals, the reaction mixture was applied to theplates (see above), and the plates were developedtwice with ammonium formate. AMP was located atthe origin and adenosine was located at the top of theplate with ultraviolet light. The radioactivity was de-termined as described above.AMP nucleosidase (EC 3.2.2.4) activity was meas-

ured by monitoring the formation of [8-'4C]adeninefrom [8-'4C]AMP. The reaction mixture for AMP ki-nase was employed in the presence or absence of ATP,and the reaction products were resolved by thin-layerchromatography (see above).

Adenine phosphoribosyltransferase (APRTase [EC2.4.2.7]) activity was measured by monitoring thephosphoribosylation of [8-'4C]adenine to [8-'4C]AMPas described by Hochstadt (9), with a slight modifica-tion. The reaction mixture contained, in a total volumeof 0.2 ml, the following: 100 mM Tris-hydrochloride,pH 7.4; 1.0 mM adenine; 0.3 MCi of [8-'4C]adenine (60,LCi/,umol); 1.0 mM 5'-phosphoribosyl-a-1-pyrophos-phate; 10.0 mM glutathione, reduced; 10.0 mM MgCl2;and the appropriate dilution of cell-free extract. En-zymatic activity was monitored by withdrawing 20-,ulsamples at 5-min intervals and spotting them directlyon polyethyleneimine-cellulose plates (prescored as 1-cm2 areas). The plates were washed with 500 ml of

methanol and then were washed three times with 1mM ammonium formate to remove the adenine. Theplates were dried with methanol, the 1-cm2 areas werecut out and placed in counting vials, and the radioac-tivity was quantitated (see above).

Adenosine kinase (EC 2.7.1.20) activity was meas-ured by monitoring the phosphorylation of [8-'4C]-adenosine to [8-'4C]AMP. The reaction mixture con-tained, in a total volume of 0.2 ml, the following: 1.0mM adenosine; 0.3 ,uCi of [8-`C]adenosine (53 mCi/mmol); 10 mM ATP; 10 mM MgCl2; 100 mM Tris-hydrochloride, pH 7.4; 5 mM f)-mercaptoethanol; andthe appropriate dilution of cell-free extract. Enzymaticactivity was monitored as described for APRTase (seeabove).Measurement of ATP breakdown products.

The degradation of ATP to smaller metabolites by R.typhi was monitored upon addition of [8-'4C]ATP towhole cells, cell-free extracts, and cell envelope prep-arations in reaction mixtures containing 2.0 mMMgCl2, 50 mM Tris-hydrochloride (pH 7.4), 2.0 mM,8-mercaptoethanol, 2.5mM ATP, and 100 ,ul of sampleat 37°C. Breakdown ofATP was monitored by remov-ing samples of the reaction mixture at appropriatetime intervals. Analysis was carried out by the methoddescribed by Schramm and Lazorik (16), which doesnot detect cyclic AMP. Hence, the levels of cyclicAMP were not measured.Measurement of AMP breakdown products

employing permeabilized cells. R. typhi cells werepurified by the standard procedure (22), suspended inK36 buffer, and washed one time with the same buffer.The cell pellet was suspended in 1.0 ml of ice-coldbuffer (50 mM Tris-hydrochloride plus 5 mM /3-mer-captoethanol) containing concentrations of Triton X-100 ranging from 0 to 1.0%. The samples were mixedwith a Vortex mixer and frozen at -70°C for 48 hbefore assaying for the catabolism of AMP. S. typhi-murium cells were grown to mid-log phase in brainheart infusion broth and harvested by centrifugation,and the cell pellet was washed once with K36 bufferand treated with Triton X-100 as described above.Transport of AMP. Cells were purified and sus-

pended in K36 buffer containing 20 mM glutamateand 5 mM MgCl2 at 4°C. The transport of [2-3H]AMPand [32P]AMP was carried out at 36°C and pH 7.4.The cell suspensions (0.45 ml) were preincubated for3 min at 36°C before the addition of 50,ul of reactionmixture. The final concentration of nucleotide was15.16 ,uM (7.58,M [2-3H]AMP and 7.58 MM [32p]-AMP). Transport was monitored by withdrawing 100M1 at the appropriate time intervals and by filteringthe sample with HAWP-HA 0.45-,um filter pads (Mil-lipore Corp.). The cells were washed with 30 ml of K36buffer containing 2 mM AMP at 36°C.

Chemicals. Bases, nucleosides, and nucleotideswere purchased from Sigma Chemical Co. or Calbi-ochem. All other chemicals were reagent grade. Ra-diochemicals were obtained from Schwarz/Mann andAmersham Corp.

RESULTSDegradation of [8-'4CJATP by R. typhL

The pathways by which ATP was degraded tosmaller metabolites were monitored by remov-

INFECT. IMMUN.

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ADENINE NUCLEOTIDE METABOLISM 77

ing samples of the reaction mixture at appropri-ate time intervals as described above. At zerotime and 40C, 89, 10, and 1% of the total radio-activity were present as ATP, ADP, and AMP,respectively. Incubation of [8-'4C]ATP at 370Cin the presence of whole cells and the 7,710 x gsupernatant fluid caused a rapid degradation ofATP to ADP and AMP (Fig. 2A and B). By 120min, the ratio had shifted to 3% ATP, 67% ADP,and 30% AMP in the whole cells and to 10%ATP, 59% ADP, and 31% AMP in the 7,710 x gsupernatant fluid. The 80,000 x g supernatantfluid and pellet showed qualitative differences inthe pattern ofATP degradation (Fig. 2C and D).The ratios of ATP, ADP, and AMP generatedby the 80,000 X g supernatant fluid were 61, 29,and 10%, respectively, whereas with the cellenvelope preparation the ratios were 44, 51, and4%, respectively. There was no radioactivity as-sociated with adenine, adenosine, hypoxanthine,inosine, or inosine 5'-monophosphate duringthese experiments, and all of the radioactivitywas recovered in the adenine nucleotides. Fur-thermore, the data suggested that ATPase wasa soluble and membrane-bound enzyme andADPase was a soluble enzyme, whereas 5'-nucle-otidase or nonspecific phosphatase activity wasundetectable.

Since AMP phosphatase and nucleosidase ac-

100 A WHOLE CELLS

ATP80 / ADP

60

40 AMP

2020

a0 SUPERNATANT.80.000 x gPLUS DIALY!

80 /

tivities were not detected with cell-free extractsor cell envelope preparations, whole cells wereincubated with [8-"C]AMP (2.5 mM) in thepresence and absence of ATP (1 mM) and in thepresence of 5 mM glutamate. In these experi-ments, no adenine, adenosine, hypoxanthine, orinosine 5'-monophosphate could be detected.However, '4C from [8-'4C]AMP was accumu-lated by whole cells (data not shown).Distribution of adenine nucleotide en-

zymes of R typhi The enzymes responsiblefor the formation and catabolism of ATP wereexamined in cell-free extracts of R. typhi (Table1). Because AMP accounted for most of theaccumulation of adenine nucleotides in gluta-mate starvation (25) and during the breakdownof [4C]ATP (Fig. 2A and B), the activities of thecatabolic and the anabolic enzymes were inves-tigated. AMP kinase was distributed in the cy-toplasm (93%) and the membrane (7%), whereasADP kinase was located only in the cytoplasm(Table 1). No adenosine kinase or APRTaseactivity was detected. GMP kinase activity wasreadily separated from AMP kinase by molecu-lar sieve chromatography and via sedimentationin sucrose density gradients (data not shown).Also, no inosine 5'-monophosphate kinase activ-ity was demonstrated. ATPase activity was ap-proximately equally distributed between the cy-

20 ; _11=- i~~~~fMP AMP

0 20 40 60 80 100 120 0 20 40 60 80 100 120

TIME (min) TIME (min)FIG. 2. Degradation ofATP by R. typhi. Metabolism of [8-'4CJATP was followed as described in the text.

(A) Whole cell (5.75 mg of protein per ml of reaction mixture); (B) supernatant fluid (7,710 x g) dialyzedovernight against the reaction buffer (3.8 mg of protein per ml of reaction mixture); (C) supernatant fluid(80,000 X g) dialyzed overnight against the reaction buffer (1.45 mg ofprotein per ml of reaction mixture); (D)cell envelope preparation from the 80,000 x g pellet (1.4 mg ofprotein per ml of reaction mixture).

VOL. 28, 1980

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

78 WILLIAMS

TABLE 1. Distribution of adenine nucleotideenzymes of R. typhi

Sp act'Enzyme Mem-

Cytoplasmb brane'APRTase None NoneAdenosine kinase None NoneAMP kinase 197 (93)d 14 (7)ADP kinase 2,488 (100) NoneAMPase None NoneADPase 3 (100) NoneATPase 62 (42) 86 (58)

a Nanomoles per minute per milligram of protein.b Supernatant fluid after centrifugation at 80,000 x

g for 60 min.'Membrane preparation of 80,000 x g supernatant

fluid.d Percentage in cytoplasm or membrane given

within parentheses.

toplasm and membrane, whereas ADPase activ-ity was located only in the cytoplasm.

Degradation of AMP was not detected undera variety of conditions employing two differentassay procedures. The highly sensitive radiomi-croassay of Torrance et al. (18) was employedfor the detection of enzyme activity by R. typhiand S. typhimurium whole cells and extracts.The assay detects adenine and adenosine re-maining in the solution after either dephosphor-ylation of AMP to adenosine or the hydrolysisof AMP in the presence of ATP and inorganicphosphate to adenine plus ribose-5-phosphateor both. Hence, the detection of adenosine oradenine in the solution would be a measure ofAMP phosphatase or nucleosidase activity. An-other highly sensitive assay for AMP phospha-tase which measures the release of 3P fromAMP was carried out as described by Burgerand Lowenstein (3). Both AMP phosphataseand nucleosidase activities were undetectableunder the following conditions. (i) Heat inacti-vation at 45°C for 20 min suggested that a pro-tein inhibitor (6) was not causing the lack ofactivity, whereas heat treatment of S. typhimu-rium preparations resulted in an average of 1.9-fold increased enzyme activity (data not shown).(ii) No inhibition of S. typhimurium enzymeactivity was observed with extracts of R. typhi.(iii) Metal ion requirement for enzyme activitywas tested in 50 mM Tris-hydrochloride bufferat pH 7.4 for both R. typhi and S. typhimurium.No activity was detected with R. typhi extracts(7,710 x g supernatant fluid) in the presence ofZnSO4, FeCl2, CaCl2, MgCl2, MnCl2, CoCl2,PbCl2, or all of the above, whereas AMP phos-phatase activity of S. typhimurium was metalion dependent (10). The addition of ethylenedi-

aminetetraacetate (7.5 mM) to the reaction mix-ture without MgCl2 indicated that a divalentcation was required for S. typhimurium enzymeactivity, whereas no activity was observed for R.typhi (data not shown). (iv) The addition ofRenografin, used for the purification of R. typhi,to the reaction mixture did not cause inhibitionof enzyme activity of S. typhimurium extracts.In fact, this combination stimulated the enzymeactivity by 13.4-fold. (v) No enzyme activity wasdetected when cellular extracts were prepared inbuffers with and without dithiothreitol orf?-mer-captoethanol (1 to 5 mM) or when the buffer(pH 7.4) was 50 mM glycylglycine, 50 mM Tris-hydrochloride, or K36-phosphate buffer. Nei-ther dialysis of a 48,200 x g supernatant fluidnor 1% streptomycin sulfate treatment followedby dialysis resulted in detectable enzyme activ-ity. (vi) No enzyme activity could be detected ina 205,000 x g membrane fraction of R. typhi,whereas the S. typhimurium enzyme activitywas easily demonstrated with [3H]AMP and[32P]AMP as substrates (data not shown). (vii)Triton permeabilization (13) of R. typhi wholecells did not result in enzyme activity, whereasactivity with S. typhimurium cells was clearlydemonstrable (data not shown). In contrast,counts from [3H]- or ['4C]AMP were accumu-lated when R. typhi cells were incubated in K36buffer. The simultaneous transport of [2-3H]-AMP and [3P]AMP indicated that AMP wastransported without prior dephosphorylation(Fig. 3). The ratio of3P to 3H remained constantthroughout the transport process. Under thesame conditions described for AMP transport,adenine and adenosine were not accumulatedintracellularly. Preliminary studies showed thatthe AMP transport process was energy depend-ent. Complete details of this transport systemwill be published elsewhere. (viii) No enzymeactivity was detected with R. typhi extractswhen a pH range of 5.9 to 8.7 was tested.

DISCUSSIONEnergy metabolism of procaryotes and eu-

caryotes generally centers around anabolic path-ways, which generate ATP for macromolecularsynthesis, and catabolic sequences, which de-grade ATP and macromolecules to smaller me-tabolites. The maintenance of a molar balancebetween ATP, ADP, and AMP has been consid-ered a prerequisite for a cell to be able to adjustto different environmental and regulatory needs(1). The environmental factors which regulatethe receptor activity and the permeability prop-erties of cells have been investigated extensivelyfor autonomously growing bacteria and eucary-

INFECT. IMMUN.

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ADENINE NUCLEOTIDE METABOLISM 79

zu

u

tl

Z' 200

0

10

0 3 6 9 12

TIME (min)

FIG. 3. Transport ofAMP by R. typhi cells. Trans-port activity was expressed as the percentage of thefirst time point. One hundred percent represents 0.28pmol for [3H]AMP and 0.32pmol for [32PJAMP. Thebuffer solution (K36) contained 15mM P04, 15.16 WMAMP, and 625 nM 32PO4, which was a contaminantof the [32PJAMP. The ratio of [32PAMP to [3H]-AMP averaged 1.13 throughout the transport interval(x).

otes. However, only limited studies have beenconducted with procaryotes which grow in thecytoplasm of eucaryotes. A revealing feature ofthe obligate intracellular bacteria is that theseorganisms must express certain bacterial func-tions while preserving key host functions. There-fore, obligate intracellular organisms must relyon the host to supply unstable intermediatesand, perhaps, macromolecules (21, 25).The present investigation centered around the

enzymatic components detectable in whole cellsor cell-free extracts which are responsible for themetabolic fate of the endogenous and exogenousadenine nucleotides of R. typhi. S. typhimuriumLT2 was employed as a positive control for thoseenzyme activities which were negative with R.typhi fractions. During these studies, severalfeatures of rickettsial adenine nucleotide metab-olism were discovered. Whole cells and cell-freeextracts ofR. typhi were unable to degrade AMPto nucleoside and inorganic phosphate or baseand ribose-5-phosphate, whereas ATP was de-phosphorylated to ADP and AMP, the end prod-uct of ATP degradation. The only intermediateduring ATP degradation was ADP, and AMP

was not converted to hypoxanthine, inosine, orinosine 5'-monophosphate. Hence, the catabolicpathway apparently was limited to the produc-tion of mononucleotide via either ATPase orATP pyrophosphatase (not examined in thisstudy) or both and ADPase. Since AMP ap-peared to be the end product of ATP degrada-tion, several parameters which theoreticallycould affect AMP breakdown were investigated.There was no readily apparent (i) protein inhib-itor (6), (ii) metal ion requirement for activation(19), (iii) inhibition by the components of Ren-ografin, (iv) reducing agent requirement, (v) in-hibition by nucleic acids or other nucleotides, or(vi) loss of enzyme activity due to mechanicalbreakage of the cells. The possibility that puri-fication resulted in osmotic extrusion of the en-zyme was examined by ultrastructural cyto-chemistry. This technique allowed direct visu-alization of ATPase and ADPase activity in R.typhi grown in tissue culture cells, whereas noAMP phosphatase activity was detected (T. F.McCaul and J. C. Williams, unpublished obser-vations).The anabolic sequence of adenine nucleotide

metabolism in R. typhi begins with AMP sincethe salvage of either adenine or adenosine orboth via APRTase and adenosine kinase activi-ties was undetectable. However, the transport ofAMP without dephosphorylation was demon-strated clearly by cells which were otherwiserefractile to bases and nucleosides. The anabolicsequence to ATP was carried out byAMP kinaseand ADP kinase. Theoretically, the interconver-sions outlined in Fig. 1 could be carried out byR. typhi, resulting in EC regulation similar toautonomously growing bacteria. The apparentpathway, at least for R. typhi, is illustrated inFig. 1 by the solid lines. This pathway wouldexplain the observed increases in the cellularadenylate pool, with the greater proportion re-siding in the AMP pool (25). Clearly, the meta-bolic role of these interconversions is of primaryimportance. Although it is desirable for autono-mously growing bacteria to regulate their AMPlevels, it may not be so critical in the case of R.typhi.The adenylate EC has been proven an essen-

tial parameter of metabolic capability for bothautonomously growing procaryotes and eucary-otes (1). However, the mechanisms of maintain-ing the EC vary greatly in autonomously grow-ing bacteria in which AMP is degraded by AMPnucleosidase (17), adenine deaminase (11), andAMP deaminase (14). AMP nucleotidase ap-pears to be an extracellular enzyme primarilyresponsible for uptake of AMP degradationproducts (27). Eucaryotes do not appear to have

VOL. 28, 1980

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

80 WILLIAMS

AMP nucleosidase (17), whereas AMP deami-nase (4), AMP nucleotidase (2), and adenosinedeaminase (6) are primarily responsible for thedegradation capacity. The function of these en-zymes is to remove AMP from the total adeninenucleotide pool so that the EC can be main-tained. Since AMP nucleotidase is a membrane-bound enzyme, it functions as a mediator innucleotide transport and thus provides the cellwith adenosine and inorganic phosphate. Hence,the intracellular concentration of nucleotides isgreater than the extracellular concentration.For those organisms residing intracellularly,

the size of the exogenous (cytoplasmic) nucleo-tide pool may be the critical factor. The salvageof bases and nucleosides, which are low in rela-tive concentration to nucleotides, should not bean essential feature of obligate intracellular par-asites (21, 24). Hence, the metabolic cooperation(5) between host and parasite may be a mecha-nism of cell interaction in which the phenotypeof the enzyme-deficient parasite is alleviated byspecified host functions. The nature of this co-operation (i.e., a receptor, informational macro-molecule, or metabolic function) may ultimatelygauge the degree of pathogenicity of a givenorganism for its host. At least for R. typhi orga-nisms, which are incapable of regulating theirendogenous adenylate pools, the rapid degrada-tion of ATP to AMP with a subsequent accu-mulation of AMP appears to represent a mech-anism of nucleotide conservation. Since AMPappears to be the end product of ATP degrada-tion, this mononucleotide (and perhaps others)would act as an energy reservoir when a sub-strate such as glutamate is available for oxida-tion via the tricarboxylic acid cycle (25). Alter-natively, and perhaps in addition, the transportof either ADP or ATP or both (26) in a nucleo-tide-rich environment would allow adenylate ki-nase and nucleoside diphosphate kinase to phos-phorylate the endogenous AMP, thereby greatlyincreasing the available ADP for exchange orphosphorylation and, in turn, markedly enhanc-ing the EC. Under these conditions, the orga-nism could maintain an adequate EC for growth,provided other nutritional requirements arepresent in the host cytoplasm.

ACKNOWLEDGMENTSThis work was supported in part by Research Task MRO

410501.0030 from the Naval Medical Research and Develop-ment Command, Department of the Navy.

I am indebted to E. Weiss for discussion of the manuscript.

LITERATURE CITED1. Atkinson, D. E. 1975. Biochemical function and homeo-

stasis: the payoff of the genetic program, p. 193-211. InR. H. Meints and E. Davis (ed.), Control mechanisms

in development, activation, differentiation and modu-lation in biological systems. Plenum Publishing Corp.,New York.

2. Baer, H. P., and G. L. Drummond. 1968. Catabolism ofadenine nucleotides by the isolated perfused rat heart.Proc. Soc. Exp. Biol. Med. 127:33-36.

3. Burger, R. M., and J. M. Lowenstein. 1970. Preparationand properties of 5'-nucleotidase from smooth muscleof small intestine. J. Biol. Chem. 245:6274-6280.

4. Chapman, A. G., and D. E. Atkinson. 1973. Stabiliza-tion of adenylate energy charge by the adenylate de-aminase reaction. J. Biol. Chem. 248:8309-8312.

5. Cox, R. P., M. R. Krauss, M. E. Balis, and J. Dancis.1974. Metabolic cooperation in cell culture: studies ofthe mechanisms of cell interaction. J. Cell. Physiol. 84:237-252.

6. Drummond, G. I., and M. Yamamoto. 1971. Nucleotidephosphomonoesterases, p. 337-354. In P. D. Boyer (ed.),The enzymes, vol. 4, 3rd ed. Academic Press Inc., Lon-don.

7. Fox, I. H. 1974. Purine ribonucleotide catabolism: clinicaland biochemical significance. Nutr. Metab. 16:65-78.

8. Fox, I. H., and W. N. Kelley. 1978. The role of adenosineand 2'-deoxyadenosine in mammalian cells. Annu. Rev.Biochem. 47:655-686.

9. Hochstadt, J. 1978. Adenine phosphoribosyltransferasefrom Escherichia coli. Methods Enzymol. 51:558-568.

10. Kier, L. D., R. Weppelman, and B. N. Ames. 1977.Resolution and purification of three periplasmic phos-phatases ofSalmonella typhimurium. J. Bacteriol. 130:399-410.

11. Koch, A. L., and G. Vallee. 1959. The properties ofadenosine deaminase and adenosine nucleoside phos-phorylase in extracts of Escherichia coli. J. Biol. Chem.234:1213-1218.

12. Lowry, 0. H., N. J. Rosebrough, A. L Farr., and H.J. Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

13. Miozzari, G. F., P. Niederberger, and R. Hutter. 1978.Permeabilization of microorganisms by Triton X-100.Anal. Biochem. 90:220-223.

14. Niven, D. F., P. A. Collins, and C. J. Knowles. 1977.Catabolism of adenosine 5'-monophosphate by extractsof the murine bacterium Beneckea natriegens. J. Gen.Microbiol. 100:5-13.

15. Randerath, K., and E. Randerath. 1967. Thin-layerseparation methods for nucleic acid derivatives. Meth-ods Enzymol. 12:323-348.

16. Schramm, V. L., and F. C. Lazorik. 1975. The pathwayof adenylate catabolism in Azotobacter vinelandii. Ev-idence for adenosine monophosphate nucleosidase asthe regulatory enzyme. J. Biol. Chem. 250:1801-1808.

17. Schramm, V. L., and H. Leung. 1973. Regulation ofadenosine monophosphate levels as a function of aden-osine triphosphate and inorganic phosphate. J. Biol.Chem. 248:8313-8315.

18. Torrance, J., C. West, and E. Beutler. 1977. A simplerapid radiometric assay for pyrimidine 5'-nucleotidase.J. Lab. Clin. Med. 90:563-568.

19. Vogels, G. D., and C. Van der Drift. 1976. Degradationof purines and pyrimidines by microorganisms. Bacte-riol. Rev. 40:403-468.

20. Weiss, E. 1973. Growth and physiology of rickettsiae.Bacteriol. Rev. 37:259-283.

21. Weiss, E. 1979. Metabolism of Rickettsia typhi: specula-tions on regulatory mechanisms, p. 144-149. In D.Schlessinger (ed.), Microbiology-1979. American So-ciety for Microbiology, Washington, D.C.

22. Weiss, E., J. C. Coolbaugh, and J. C. Williams. 1975.Separation of viable Rickettsia typhi from yolk sac andL cell host components by Renografin density gradientcentrifugation. Appl. Microbiol. 30:456-463.

INFECT. IMMUN.

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ADENINE NUCLEOTIDE METABOLISM 81

23. Weiss, E., H. B. Rees, Jr., and J. R. Hayes. 1967.Metabolic activity of purified suspensions of Rickettsiarickettsii. Nature (London) 213:1020-1022.

24. Williams, J. C., and J. C. Peterson. 1976. Enzymaticactivities leading to pyrimidine nucleotide biosynthesisfrom cell-free extracts of Rickettsia typhi. Infect. Im-mun. 14:439-448.

25. Williams, J. C., and E. Weiss. 1978. Energy metabolism

of Rickettsia typhi: pools of adenine nucleotides andenergy charge in the presence and absence of glutamate.J. Bacteriol. 134:884-892.

26. Winkler, H. H. 1975. Rickettsial permeability and ADP-ATP transport system. J. Biol. Chem. 251:389-396.

27. Yagil, E., and I. R. Beacham. 1975. Uptake of adenosine5'-monophosphate by Escherichia colt. J. Bacteriol.121:401-405.

VOL. 28, 1980

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from