Vol. 132, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Dec. 1977, p. 841-846 Copyright ( 1977 American Society for Microbiology

Synthesis of Ribonucleotides and Their Participation in Ribonucleic Acid Synthesis by Coxiella burnetii ROBIN G. CHRISTIAN AND D. PARETSKY* Department of Microbiology, The University of Kansas, Lawrence, Kansas 66045 Received for publication 19 July 1977

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a -P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates. Coxiella burnetii, the rickettsial agent of Q fever, is an obligate intracellular parasite, proliferating in a wide variety of hosts and tissues. In guinea pig liver, numerous rickettsiae may be found inside small vacuoles and, less frequently, as individual cells free in sinusoids (9). When propagated in L cells, C. burnetii is found within cytoplasmic vacuoles, with as many as 103 to 104 rickettsiae per vacuole (4; J. Stueckemann and D. Paretsky, unpublished data). Within these vacuoles, the rickettsiae find the metabolites required for maintenance and proliferation. C. burnetii has several host-independent enzyme systems (14, 17-19, 21, 23, 31), a nucleoid deoxyribonucleic acid (DNA) mass (5, 23), ribosomes (2), and a typically procaryotic cell wail and plasma membrane (1, 5, 13). Efforts to identify the physiological or biochemical lesions responsible for this rickettsial agent's obligate parasitism have been unsuccessful. The central biochemical role of ribonucleic acid (RNA) species stimulated studies on the capabilities of C. burnetii to synthesize RNA. It was found that the rickettsia had a DNA-dependent RNA polymerase (14) and that the organisms could synthesize ureidosuccinate and orotate, uracil precursors (18). It has recently been shown that Rickettsia typhi synthesize uridine 5'-diphosphate (UDP), deoxythymidine diphosphate, and their corresponding nucleoside triphosphates (NTPs) (32). As part of continued efforts to identify host-independent and -dependent biochemical activities of C. burnetii, we now report on the competence of C. burnetii to synthesize the substrates and some of their pre-

cursors required for DNA-dependent RNA polymerase activity. MATERIALS AND METHODS Organisms. The Nine Mile strain of C. burnetii, phase I, was propagated in antibiotic-free embryonated chicken eggs. The organisms were harvested and purified from yolk sacs as previously described (28) and stored at -70°C until use. The phase I condition was verified by R. A. Ormsbee, U.S. Public Health Service, Rocky Mountain National Laboratory, Hamilton, Mont. Preparation of cell-free extracts. Purified C. burnetii was suspended in 0.2 M tris(hydroxymethyl)aminomethane, pH 7.9, to yield a 3% (wt/vol) suspension. The suspension was subjected to sonic treatment five times for 10 s each with 20-s cooling intervals. A Kontes ultrasonic cell disrupter, with a 2-mm probe, 1,200 W/inch2 (ca. was 6.5 x 0-4 Mi2) was used. The sonic extract was kept on ice and used immediately in RNA polymerase assays. For nucleotide phosphorylation, rifampin, and a-amanitin studies, the sonic extract was centrifuged at 10,000 rpm for 5 min in a Beckman Microfuge B, and the supernatant was considered as the enzyme. RNA polymerase assay. DNA-dependent RNA polymerase was measured by the method of Roeder and Rutter (25). A final volume of 64 l1 contained, in nanomoles (unless otherwise stated): guanosine, adenosine, and cytidine 5'-triphosphates (GTP, ATP, and CTP) (or the nucleoside di- or monophosphate [NDP or NMP], as designated), 37.5; UTP or UDP, 0.625; [3H]UTP (14 Ci/mmol; New England Nuclear Corp., Boston Mass.), 0.5 uCi, or [1H]UDP (11 Ci/mmol; New England Nuclear), 0.5 6Ci; NaF, 375; MnCI2, 100;

tris(hydroxymethyl)aminomethane, 3,000, pH 7.9; ,Bmercaptoethanol, 160; calf thymus DNA, 20 Mg; (NH4)2SO4, 100 mM or as stated; and 30 [L of enzyme

841

842

CHRISTIAN AND PARETSKY

preparation. Rifampin (Sigma Chemical Co., St. Louis,

Mo.) was 25, 50, or 100 [ig/rnl (final concentration), preincubated at 0°C with the enzyme for 15 min before assay. a-Amanitin (Boehringer Mannheim Chemicals, Indianapolis, Ind.) was used at 100 yg/ml; actinomycin D (Calbiochem, La Jolla, Calif.) was 31 tig/ml, final concentration. Nucleotide phosphorylation assay. The presence of nucleoside phosphate kinase-like activity was assayed by the method of Ginther and Ingraham (7), using [y--2P]ATP and _7-2P]GTP (New England Nuclear). All reagents were prepared in 100 mM tris(hydroxymethyl)aminomethane, pH 8.0-10 mM MgCl2-2 mM 8-mercaptoethanol buffer. In 40 1il of assay mixture were contained the appropriate nucleoside phosphate, 1 mM; ATP or GTP, 2 mM, and 20 ul of enzyme. With ATP was added 50 8Ci of [y_32p]_ ATP, 23.8 Ci/mmol. With GTP was added 50 8Ci of [y-_2P]GTP, 25 Ci/mmol. Reactions were initiated by the addition of enzyme and were continued for 30 min at 20°C. Two controls were included to account for nonspecific phosphate exchange: one contained enzyme, which was inactivated by H202 (30%), and one lacked enzyme. Carrier product NTP was added at the end of the reaction, and the mixture was quickly frozen. Three microliters of the subsequently thawed mixture was spotted on polyethyleneimine-cellulose F-precoated plastic thin-layer chromatography sheet (Brinkmann Instruments Inc., Des Plaines, Ill.), prewashed in absolute methanol. The plates were developed in either 2 M LiCl-2 M HCOOH (1:1), pH 3.4 (24), or 0.85 M KH2PO4, pH 3.4 (7), until the solvent front was approximately 15 cm from the origin. The chromatograms were dried, and the nucleotides were located with ultraviolet light. Autoradiography was carried out by placing the plates against X-ray film (Kodak Royal Blue) for 8 h. The product nucleotide spots were then scraped from the plate and counted in 5 ml of Packard II LS cocktail (Packard Instrument Co., Inc., La Grange, Ill.) in a Beckman liquid scintillation spectrometer. Because protein in the sample resulted in a slightly slower migration rate, enzyme was added to chromatographed standard nucleotides, which were run on each plate to insure positive identification of the product nucleotides. Luciferin-luciferase ATP assay. The ATP content of C. burnetii was measured by spectrophotometry (27). ATP was extracted from C. burnetii with cold 0.6 N perchloric acid and then neutralized with KHCO.3. A Beckman scintillation spectrometer model LSC 300 was used with the coincidence switch turned "off' and set at "sample repeat" for 0.1-min counts. To a 7-rnl vial containing the buffers (27) and ATP was added 25 1Ld of the firefly extract. The vial was capped, swirled gently, and counted exactly 15 s after addition of the enzyme. Four 0.1-min counts were taken for each sample. The first count was discarded due to high chemoluminescence, and the next three counts, usually quite reproducible, were averaged. Background luminescence was subtracted and the net counts for a specific concentration of ATP were plotted to yield a standard curve from which the concentration of ATP in C. burnetii was calculated. Protein. Protein was measured by the method of Lowry et al. (16).

J. BACSTERIOL.

RESULTS The DNA-dependent RNA polymerase of C. burnetii (14) was further investigated. The requirement for all four NTPs for RNA synthesis is shown in Table 1. To confirm that the observed polymerase activity was of rickettsial (procaryotic) and not host (eucaryotic) origin, rifampin and a-amanitin were used. Rifampin inhibits only procaryotic RNA polymerase (30), whereas a-amanitin at 100 fig/ml inhibits eucaryotic polymerases IL and III (12), although not polymerase I. Table 2 shows that 82 to 86% of the observed polymerase activity was sensitive to rifampin, whereas only 1 to 3% of the activity was inhibited with a-amanitin. Although the data do not completely rule out the possibility of contaminating eucaryotic RNA polymerase I, they indicate that at least most of the activity was of rickettsial origin. Actinomycin D, which inhibits transcription by the polymerase (8), inhibited RNA synthesis by the rickettsial enzyme preparation (Table 2). The synthesis of RNA was fairly stable between 26 and 36°C (Fig. la), and the time course of the reaction was linear for 50 min, with a change in slope to 120 min (Fig. lb). The reaction was stimulated four- to fivefold by 100 mM ammonium sulfate. (data not shown). The presence of RNA polymerase activity in C. burnetii (Tables 1 and 2) raises questions on the origin and availability of the substrate NTPs. Experiments were designed to determine whether C. burnetii could phosphorylate NDP to the corresponding NTP and, if so, whether the NTP would participate in RNA synthesis. When one NDP was incubated with the other three NTPs in the presence of C. burnetii enzyme, RNA polymerase activity was demonstrated (Table 3). The level of [3H]UMP incorporation was of the same magnitude regardless of which NDP was used and when all four NTPs were used. When only the four NDPs were the substrate, the level of [3H]UMP incorporation TABLE 1. RNA polymerase activity of C. burnetii Expt no.

Reaction

constituents'

[r H]UMP

incorporated (pmol/mg of protein) 6.13 ± 0.48

Enzyme plus ATP, GTP, CTP, UTP As expt 1, minus ATP 1.13 ± 0.11 As expt 1, minus CTP 0.72 ± 0.22 As expt 1, minus GTP 0.86 ± 0.25 a Reaction mixture, as described in text, included (in a final volume of 64 ,l): 0.56 pM [3H]UTP, 14 Ci/mmol; (NH4)2S04, 0.1 M; C. burnetii protein, 73 to 100 jg. It was incubated at 30°C for 30 miin. 'Average of two experiments ± standard error of the mean. 1

2 3 4

C. BURNETII RIBONUCLEOTIDE AND RNA SYNTHESIS

VOL. 132, 1977

843

TABLE 2. Inhibition of C. burnetii RNA polymerase Inhibitor (Qg/ml)

No. of expts

[IH]UMP of protein)' (pmol/mg incorporated

Inhibition (%)

35.2 + 5.6 4 2 ± 1.2 2 34.7 ± 12.4 (100) 82 ± 1.0 6.3 ± 0.4 2 (25) Rifampin 84.5 ± 0.5 5.4 ± 0.5 2 (50) 86 ± 2 4.8 ± 0.1 2 (100) 85 0.5 1 Actinomycin D (31) M; protein, 18 0.1 64 volume, jd): with (final text, in described as (NH4)2SO4, mixture Polymerase reaction to 24 [Lg; inhibitor concentration as shown. Rifampin was preincubated with enzyme for 15 min at 0°C, before reaction. Reaction was conducted for 30 min at 30°C. 'Average ± standard error of the mean. None

a-Amanitin

a

a

4

i

I

*

*

35

33

*TEM

I,

,

31

29

27

25

I

*

T E MP It b

25

o

a20

C 2

0

a:0

/

50

30

0

FIG. of C.

SO

70 TIME

110

MINI"

1. DNA-dependent RNA polymerase activity burnetii.

Conditions as described in text.

Effect of temperature;

mmn

incubation time, 30

(a)

(b)

Time course of the reaction; incubation temperature, 300 C.

was about

10%

that

of the

complete

system,

which contained all four NTPs. When ATP was incubated

with

the

GDP,

UDP,

and

CDP,

[3H]UMP incorporation was about 40% that of the complete system (Table 3). The data indi-

cate that C. burnetii has NDP kinase-like activity and that the NTP product can participate in an RNA polymerase reaction.

Direct evidence was sought for nucleotide kinase-like activities. [-y-32P]GTP was incubated with an NDP together with C. burnetii enzyme,

and the reacted mixture was chromatographed

on polyethyleneimine-cellulose F plates. The position of UTP was established with an authentic sample, and the presence of [32P]UTP was confirmed by autoradiography; [-y-32P]UTP was scraped from the chromatogram, and its radioactivity was counted. In a similar manner, [y"2P]GTP was reacted with CDP and ADP, and the corresponding NTPs were identified and the radioactivities were measured (Table 4). When UMP was coupled with [-y-32P]GTP, [32P]UDP and [32P]UTP were identified (Table 4). Parallel experiments were performed with [y32P]ATP as the -P donor, coupled with UDP, GDP, and CDP and with CMP and UMP (Table 5). The corresponding 32P-labeled NTP and 32Plabeled NDP were isolated. It is clear from these data that not only does C. burnetii have nucleoside phosphate kinase capability, but that the NTP products can participate in an RNA polymerase reaction. NTP synthesis in vivo by the type of transphosphorylation demonstrated in vitro should require an ATP pool in the organisms. The ATP content of C. burnetii, as measured by the luciferin-luciferase method (27), was 1.96 ± 0.63 nmol/mg of protein. This is comparable to the ATP levels reported for Rickettsia prowazeki of 2.9 ± 0.6 nmol/mg of protein (33) and 1.5 to 1.9 nmol/mg of Madrid E typhus rickettsial protein (3). DISCUSSION C. burnetii is an example of an obligate intracellular procaryote. Efforts to cultivate C. burnetii in axenic media have proven unsuccessful. Among the factors considered responsible for the obligate parasitism may be biochemical and physiological defects. "Energy parasitism," membrane defects, or lesions in pathways of intermediary metabolism have been suggested for rickettsiae and the chlamydia (10, 11, 20, 31). The genomic size of C. burnetii has been estimated as 1.8 x 107 daltons (26), and 109 daltons may be calculated for the genome of Rickettsia rickettsii (15). Although those data

844

J. BACTE'REIOL

CHRISTIAN AND PARETSKY

TABLE 3. Cooperative (nucleoside phosphate kinase-RNA polymerase) activity in C. burnetti [:'H]UJMP incorporation Reaction constituents" Expt no. (pmol/mg of protein) 6.1 ± 0.5 Enzyme + (ATP, GTP, CTP, [3H]UTP) 5.9 ± 0.4 Enzyme + (GTP, CTP, [3H]UTP) + ADP 6.6 ± 0.8 Enzyme + (ATP, GTP, [3H]UTP) + CDP 6.8 ± 0.8 Enzyme + (ATP, CTP, [3H]UTP) + GDP 7.7 ± 0.7 Enzyme + (ATP, GTP, CTP) + [,H]UDP 0.56 ± 0.18 Enzyme + (ADP, GDP, CDP, [:3H]UDP) 2.5 ± 0.4 Enzyme + ATP + (GDP, CDP, [3H]UDP) 0 Boiled enzyme + (ATP, GTP, CTP, [3H]UTP) a 14 pM 0.56 64 included Ci/mmol, in described pl): as volume, ['H]UTP, (final text, Reaction mixture, 0.71 pM [3H]UDP, 11 Ci/mmol, where indicated; protein, 73 to 100 pg. It was incubated for 30 min at 300C. h Average of two experiments ± standard error of the mean.

1 2 3 4 5 6 7 8

or

TABI,E 4. GTP-linked nucleotide synthesis by C. burnetii

[32P]GTP + NDP - ;32P-labeled NTP

Avg ±

no.

[32P]CTP

[2P]ATP

[2P] JTP

1 2 3

418 937

1,694 2,062

2,600 286 1,471

678 ± 260

1,878 ± 184

1,452 + 668

SEM'

[02P]GTP + NMP 32P-labeled NTP

;32P-labeled NDP +

-

Product (nmol/mg of protein)

Expt

Reaction'

[r-P]UDP

315 154 384

1,010 1,725 1,007

1 2 3

Avg ± SEM 2 mM GTP; 0.5 aReaction mixture contained (final volume, 40 of C. burnetii protein. It was incubated for 30 min at 20°C. SEM, Standard error of the mean.

pul):

284 ± 68 25 Ci/mmol; 15 to 20

1,247 + 239

pM [y-32P]GTP,

pg

TABILE 5. ATP-linked nucleotide synthesis by C. burnetii Reaction (t

[I2P]ATP

+ NDP

-

Expt no.

;12P_labeled

NTP

Avg ± SEM r PIATP + NMP 2P-labeled NDP + O2p labeled NTP

1 2 3 4

of protein) [

[32P]CTP

Product (nmol/mg [-2P]GTP [;2P]UTP

574 488 542 461 516 ± 26

387 403 312 434 384 ± 26

2I4]CDP

[

2P]UDP

536 450 551 514 513 ± 22

50 30 132 252 31 56 141 242 140 115 478 480 133 151 230 346 ± 24 ± ± 93 ± 30 84 81 245 ± 330 55 SEM Avg a Reaction mixture contained (final volume, 40 p1): 2 mM ATP; 0.53 pM [Y-_2PIATP, 23.8 Ci/mmol; 15 to 20 pLg of C. burnetii protein. It was incubated for 30 min at 20°C. -

1 2 3 4

may not be exact, it is clear that the rickettsial genome is significantly smaller than the 2 x 109dalton genome of the free-living Escherichia coli. This implies fewer coding capacities for C. burnetii and a likelihood of enzyme protein deficiencies. Studies of the enzyme systems of C. burnetii have uncovered numerous host-independent anabolic and catabolic activities, includ-

ing glycolysis and glucose-6-phosphate oxidation (6, 17) as well as amino acid, pyrimidine, and DNA-dependent RNA synthesis (14, 18, 19, 23). Because intact organisms were frequently apparently inactive, the use of cell-free preparations from the rickettsiae were required in most cases to mediate the reactions. DNA-dependent RNA polymerase requires

VOL. 132, 1977

C. BURNETII RIBONUCLEOTIDE AND RNA SYNTHESIS

the four NTPs, UTP, ATP, GTP, and CTP, so that it became of interest to learn whether C. burnetii has the competence to synthesize NTP from the corresponding NDP or NMP. This communication confirms earlier observations (14) of C. burnetii RNA polymerase dependence on the four NTPs (Table 1). The differential response to polymerase inhibitors (Table 2) indicates little or no eucaryotic polymerase contamination. When one NTP was omitted from the reaction mixture and replaced with its corresponding NDP, full polymerase activity was restored (Table 3), indicating phosphorylation of the NDP to NTP. That the synthesized RNA was not the product of an Ochoa-like polyribonucleotide phosphorylase reaction (22) is shown by the virtual lack of RNA synthesis in the presence of the four NDPs (Table 3, experiment 6). When ATP replaced ADP (Table 3, experiment 7), polymerase activity was partially restored, again suggesting phosphorylation of the NDP to the NTP. Direct evidence of this reaction was obtained by reacting [32P]GTP or [32P]ATP with an NDP and isolating the 32p_ labeled NTP product (Tables 4 and 5). Nucleotide monophosphates as well were phosphorylated to the NDP and NTP products. In the present paper, we show that C. burnetii contains 2 nmol of ATP per mg of protein, which would support the likelihood of in vivo synthesis of NTP from NDP via an ATP-linked reaction. Cell-free preparations of R. typhi have recently been shown to possess several nucleotide kinase activities (32) with no capability to phosphorylate uridine and deoxyribosylthymidine, but the fate of the synthesized products was not determined. On the other hand, Chiamydia psittaci utilized host L-cell NTP for RNA synthesis (11). The meningopneumonitis agent could utilize most host L-cell nucleic acid precursors, but not host deoxycytidine, thymidine, or thymidine nucleotides (29). It was proposed that this parasite synthesizes its own thymidine and deoxycytidine 5'-triphosphates from host nucleic acid pools. Winkler has shown R. prowazeki could use exogenous ATP and that, whereas the rickettsiae had a carrier-mediated ADP-ATP transport system, AMP could not be taken in (33). The present communication demonstrates that UDP, GDP, and CDP, as well as UMP and CMP, can be phosphorylated to the NTPs in the presence of exogenous ATP. GTP may also serve as a -P donor for UDP, ADP, CDP, and UMP (Tables 4 and 5). The presence of nucleotide kinases, but no uridine or thymidine kinase, in R. typhi has led Williams and Peterson to propose that these rickettsiae rely on host nucleoside monophosphates or that they are synthesized de novo

(32).

845

C. burnetii proliferates in host cell vacuoles (4; Stueckemann and Paretsky, unpublished data). The vacuoles contain lysosomal enzymes (4), presenting a possible nucleoside or nucleotide source that could be used for rickettsial RNA synthesis. The level of C. burnetii polymerase activity in vitro (Table 1) is of sufficient magnitude to account for much of the rickettsial RNA synthesized in one generation time, of about 16 h. Whereas this is based on an assumption of undiminished polymerase activity and unrestricted nucleoside phosphate transport, the demonstration of the enzyme systems in C. burnetii is suggestive of their role in vivo. The present report extends infornation on the anabolic competence of the obligate intracellular parasite C. burnetii. Coupled with earlier observations on its host-independent ability to synthesize orotate (18) and RNA (14) and the presence of typically procaryotic RNA and ribosomes (2, 28) and an endogenous ATP pool (this paper), a picture of host-independent RNA anabolism is presented. Other critical RNA species, such as transfer RNA, have not as yet been studied, nor has DNA synthesis been examined. Defects or deficiencies in any of these species could be responsible for the strict host-dependence of C. burnetii. ACKNOWLEDGMENTS We express appreciation to M. L. Anthes for valuable technical assistance and discussion. Grants from the National Science Foundation (PCM 7611705) and the University of Kansas General Research Fund (3623-x038) are gratefully acknowledged. LITERATURE CITED 1. Allison, A. C., and H. R. Perkins. 1960. Presence of cell walls like those of bacteria in rickettsiae. Nature (London) 188:796-798. 2. Baca, 0. G., R. T. Hersh, and D. Paretsky. 1973. Ribosomes and ribonucleic acids of Coxiella burnetii. J. Bacteriol. 116:441-446. 3. Bovarnick, M. R., and E. G. Allen. 1957. Reversible inactivation of the toxicity and hemolytic activity of typhusrickettsiaebystarvation. J. Bacteriol. 74:637-645. 4. Burton, P. R., N. Kordova, and D. Paretsky. 1970. Electron microscopic studies of the rickettsia Coxiella burneti: entry, lysosomal response, and fate of rickettsial DNA in L-cells. Can. J. Microbiol. 17:143-150. 5. Burton, P. R., J. Stueckemann, and D. Paret8ky. 1975. Electron microscopy studies on the limiting layers of the rickettsia Coxiella burneti. J. Bacteriol.

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6. Consigli, R. A., and D. Pareteky. 1962. Oxidation of glucose 6-phosphate and isocitrate by Coxiella burnetii. J. Bacteriol. 83:206-207. 7. Ginther, C. L., and J. L. Ingraham. 1974. Nucleoside diphosphokinase of Salmonella typhimurium. J. Biol. Chem. 249:3406-3411. 8. Goldberg, L H., M. Rabinowitz, and E. Reich. 1962. Basis of actinomycin action. I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc. Natl. Acad. Sci. U.S.A. 48:2094-2101. 9. Handley, J., D. Paretsky, and J. Stueckemann. 1967.

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J. BACTER{IOLJ. 22. Ochoa, S. 1956. Enzymatic synthesis of ribonucleic acidlike polynucleotides. Fed. Proc. Fed. Am. Soc. Exp. Biol. 15:832-840. 23. Paretsky, D. 1968. Biochemistry of rickettsiae and their infected hosts, with special reference to Coxiella burnetii. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 206:284-291. 24. Randerath, K., and E. Randerath. 1964. Ion-exchange chromatography of nucleotides on poly(ethyleneimine) cellulose thin layers. J. Chromatogr. 16:111-125. 25. Roeder, R. G., and W. J. Rutter. 1969. Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature (London) 224:234-237. 26. Schramek, S. 1968. Isolation and characterization of deoxyribonucleic acid from Coxiella burneti. Acta Virol. (Engl. Ed.) 12:18-22. 27. Stanley, P. E., and S. G. Williams. 1969. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal. Biochem. 29:381-392. 28. Thompson, H. A., 0. G. Baca, and D. Paretsky. 1971. Presence of ribosomal ribonucleic acid in the rickettsia Coxiella burneti. Biochem. J. 125:365-366. 29. Tribby, I. I. E., and J. W. Moulder. 1966. Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis. J. Bacteriol. 91:2362-2367. 30. Wehrli, W., and M. Staehelin. 1971. Actions of the rifamycins. Bacteriol. Rev. 35:290-309. .31. Weiss, E. 1973. Growth and physiology of rickettsiae. Bacteriol. Rev. 37:259-283. 32. Williams, J. C., and J. C. Peterson. 1976. Enzymatic activities leading to pyrimidine nucleotide biosynthesis for cell-free extracts of Rickettsia typhi. Infect. Immun. 14:439-448. 33. Winkler, H. H. 1976. Rickettsial permeability. An ADPATP transport system. J. Biol. Chem. 251:389-396.