Vol. 122, No. 2
JOURNAL OF BACrERIOLOGY, May 1975, p. 393-400 Copyright @ 1975 American Society for Microbiology
Printed in U.S.A.
Utilization of L-Cell Nucleoside Triphosphates by Chlamydia psittaci for Ribonucleic Acid Synthesis THOMAS P. HATCH Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Received for publication 4 February 1975
Long-term, 32P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3H ]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3H to 32P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.
Members of the genus Chlamydia are intracellular parasitic bacteria which have not been propagated outside eukaryotic host cells. Although fragments of the tricarboxylic acid cycle and the pentose phosphate and glycolytic pathways have been demonstrated in host-free chlamydiae, mechanisms by which these organisms synthesize adenosine triphosphate (ATP) have not been detected (for reviews, 12, 13). Moulder (11) has suggested that chlamydiae are energy parasites which depend on their hosts for the energy intermediates they require for biosynthesis of macromolecules. The hypothesis is strengthened by the observation that antimycin, an inhibitor of aerobic respiration at the cytochrome level, reduced chlamydial growth (4). However, the action of this inhibitor on chlamydial cytochromes, although none can be detected (25), cannot be ruled out. Tribby and Moulder (24) demonstrated that most exogenously supplied bases and ribonucleosides enter the ribonucleic acid (RNA) of Chlamydia psittaci multiplying within the cytoplasm of L cells; the energy parasite concept
implies that it is the host nucleoside triphosphate pool that is exploited by the parasite. Radioactively labeled uridine can enter the RNA of chlamydiae by one or both of the two pathways outlined in Fig. 1. After transport into the host cell, uridine may be elevated to the nucleotide level by either host or parasite nucleoside kinases. In either case, the uridine label will equilibrate between uridine triphosphate (UTP) and cytidine triphosphate (CTP) in the soluble pool because UTP is the immediate precursor of CTP (7). The rate at which the label equilibrates between the two nucleotides varies from one organism to the next because it is dependent upon several factors, including pool size and rates of synthesis and degradation of RNA (18). If chlamydiae directly utilize the host ribonucleotide pool for RNA synthesis as outlined in pathway 1 of Fig. 1, the proportion of label distributed between the cytidine monophosphate (CMP) and uridine monophosphate (UMP) of chlamydial RNA should be a function of the approach to equilibrium labeling of UTP
393
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HATCH
J. BACTERIOL.
density of 106 cells/ml in medium 199 containing [32P]HSPO4 (approximately 5 ACi/ml, New England Nuclear Corp.) for 72 h. Cells (4.0 x 10') were then infected with C. psittaci in a volume of 5 ml of medium containing "p (5 pCi/ml) and incubated as monolayers in 25-cm' plastic tissue culture flasks. At UR 20 h postinfection, the medium supernatant was decanted and 2.0 ml of fresh medium was added. Depending on the experiment, it contained "2Pi (5 pCi/ml) plus [5-'HJuridine (60 pCi/ml, New England Nuclear Corp.) and carrier uridine to a final concentration of 7.5 pM, [8-_4C]adenine (6.0 pCi/ml; 54.7 mCi/mmol; New England Nuclear Corp.), or [8'Hlguanosine (60 pCi/ml; 15.8 mCi/mmol, Amersham/Searle). At intervals the medium supernatant was detanted, the cells in monolayer were rapidly rinsed two times with 5.0 ml of Dulbecco phosphate-buffered saline, and the acid-soluble nucleotide pool and RNA were extracted. Extraction of the soluble pool and analysis of FIG. 1. Alternative pathways for the entry of nucleotides. Soluble pools were extracted from exogenous uridine into the RNA of C. psittaci. washed monolayers with 2.0 ml of 1.0 N formic acid for 2 h at 4 C. Cellular debris was removed by centrifugation and 100 pl of the formic acid extract and CTP in the host pool. If the parasite was applied directly onto thin layers of poly(ethyleneindependently synthesizes nucleotides from uri-
imine)-cellulose (Brinkmann Instruments, Inc.) together with 10 nmol of carrier nucleoside triphosphates. Nucleotides were separated by the method of Randerath and Randerath (17) and detected by ultraviolet absorption and autoradiography with medical X-ray film (Eastman Kodak Co.). Radioactivity was measured by cutting out areas yielding darkening of the film, eluting the nucleotides with 1.0 N LiCl, and suspending in a toluene-Triton X-100 (Rohm & Haas) scintillation mixture. Isolation and analysis of RNA. Washed monolayers were lysed with a nonionic detergent, 0.5% Nonidet-P40 (a gift from Shell Chemical Co., Ltd.), in buffer containing 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.4), 1.50 mM MgCl,, 0.14 N NaCl, and 500 pg of heparin per ml. Nuclei were removed by centrifugation, sodium dodecyl sulfate was added to the supernatant to a final concentration of 0.5%, and RNA was extracted by the phenol method of Penman (15). Chlamydial 16S RNA and L-cell 18S RNA were separated from other species of rRNA present in infected cells by electrophoresis in buffer E (8) on polyacrylamide gels consisting of 2.6% MATERIALS AND METHODS acrylamide cross-linked with bisacrylamide. Gels Growth of organisms. Suspension cultures of the were then dried on sheets of Whatman GF 83 chroma-
dine as in pathway 2, the distribution of label between CMP and UMP in parasite RNA should be independent of the composition of the host pool. These alternatives are kinetically distinguishable because the approach to equilibrium labeling of CTP and UTP in the soluble pool of a prokaryotic cell such as C. psittaci should not be identical to that of the eukaryotic L cell. The approach to equilibrium labeling of nucleotides method was first utilized by Wu and Soeiro (27) to demonstrate that heterogeneous sedimenting nuclear RNA and ribosomal (r) RNA precursors are supplied from the same pyrimidine nucleoside triphosphate precursor pool. In this investigation, the method has been used to demonstrate that RNA of C. psittaci is synthesized from the purine and pyrimidine nucleoside triphosphate pools of L cells.
5b clone of L cells (mouse fibroblasts) were grown by procedures previously described (23) in medium 199 (Grand Ibland Biological Co.) containing 0.1% sodium bicarbonate, 200 pg of streptomycin sulfate per ml, and 5% heat-inactivated fetal calf serum (International Scientific Industries, Cary, Ill.). No mycoplasma was isolated from cultures tested before the initiation and after the completion of these experiments (Flow Laboratories). All experiments were done with the 6BC strain of C. psittaci. L-cell cultures were infected with 10 times the amount of C. psittaci required to infect 50% of the cells in the culture, a multiplicity sufficient to infect more than 95% of the host cells. Labeling experiments. L cells were grown to a
tography paper, RNA bands were excised with a razor blade, and the RNA was recovered by the method of Young and Young (28) and hydrolyzed in 0.30 M KOH at 37 C for 18 h. The hydrolysate was neutralized with 35% perchloric acid, the potassium salt was removed by centrifugation, and the supernatant was applied to Whatman 3MM chromatography paper together with 40 nmol of carrier nucleotides. Nucleoside monophosphates were separated by high-voltage paper electrophoresis with 67 mM sodium citratecitric acid buffer (pH 3.5). Radioactive areas were detected by ultraviolet absorption, eluted in 1.0 N LiCl, and counted in a toluene-Triton X-100 scintillation mixture. Definitions. The specific activity of a nucleotide is
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USE OF L-CELL NUCLEOTIDES BY C. PSITTACI
VOL. 122, 1975
defined as the ratio of the short-term 3H or 14C counts to the long-term 82P counts in that nucleotide. The 32P counts per uninfected cell in ATP, guanosine triphosphate (GTP), CTP, and UTP were constant between 48 and 72 h post-82P label. The ratio of the specific activities of two different nucleotides is designated the specific ratio following the terminology of Wu and Soeiro (27). Calculations. A theoretical calculation of the specific CMP:UMP and specific guanosine monophosphate (GMP):adenosine monophosphate (AMP) of chlamydial 16S rRNA has been made by assuming the parasite draws on the total acid-soluble nucleoside triphosphate pool. The calculation is based on the average specific CTP:UTP or GTP:ATP in the pool over a given time interval. For example, if the average specific CTP:UTP in the pool is 0.33 during the interval 15 to 30 min post- [3H ]uridine label, the average mole of UTP incorporated into 16S RNA during that interval will contain three times as many atoms of tritium as each mole of CTP incorporated. The cumulative 30-min specific CMP:UMP is obtained by adding the 15- to 30-min interval ratio to the cumulative ratio obtained from previous intervals after adjusting for the contribution of this interval to the total incorporation of tritium into 16S RNA. RESULTS
Nucleoside triphosphate pools in uninfected and infected celis. The effect of infection on the amount of [32P]HSPO4 incorporated into the nucleoside triphosphate pools of L cells is shown in Table 1. There was significantly less label in the pyrimidine nucleoside triphosphate pools of infected cells. The decreased incorporation of 3 Pi into purine pools was less obvious, but it was observed consistently. Gill and Stewart (3) have also reported a slight decrease in the ATP pool of L cells infected with C. psittaci
for 24 h. Although the drop in the nucleoside triphosphate pools in infected cells may be a nonspecific effect of infection, evidence will be presented in later sections that indicates the drop may be the result of increased drainage of host pools by the parasite for its own biosynthetic purposes. Nonspecific leaking of nucleotides is unlikely since the percentage drop in the pyrimidine pool is considerably greater than in the purine pool. Entry of [3H]uridine into the soluble pool and 16S RNA. L cells prelabeled for 72 h with [32P ]HSPO4 were infected with C. psittaci in the presence of 32Pi. At 20 h postinfection, [3H Juridine was added and the cells were sampled at intervals for -H and 32P counts in CTP and UTP in the acid-soluble pool and in CMP and UMP in chlamydial 16S RNA (Table 2) as described in Materials and Methods. The long-term 32P TABLE 1. Nucleoside triphosphate pools in infected and uninfected L cells after labeling with 32pa GTP
ATP
UTP
CTP
1,123 858 265
12,217 11,439 778
552 231 321
602 231 371
Sample
Uninfected ..... Infected .... Difference .....
.
aL cells were grown in suspension culture to a density of 106/ml in the presence of [32P]H,PO4 (5 ACi/ml) for 72 h. Cells (40 x 106) were then infected with C. psittaci and incubated along with an equal portion of uninfected cells as monolayers in 5 ml of medium containing 32Pi (5 uCi/ml). At 24 h, the monolayers were washed, the soluble pools were extracted, and the components were separated as described in Materials and Methods. Data are expressed as counts per minute per 105 L cells.
TABLE 2. [3Hluridine label; nucleotide analysis of the soluble pool and 16S ribosomal RNA in L cells infected with C. psittaci 16S rRNA
Soluble pool
Time of____
(min)
Sp
Sp
labeling
Sp act UTPb
Sp act CTPa
CTP:
Sp act CMPd
Sp act UMPe
310/639 = 0.485 = 1.58 2,641/894 = 2.95
4.55 4,100/901 7,482/972 = 7.70
UTPC 5 15
30 45
7,011/1,166 12,101/1,377 = 14,498/1,489 = 9.74 43,306/2,121 = 20.4 0.477 =
6.01 27,396/1,468 8.79 38,812/1,764
=
=
18.7 0.322 22.0 0.399
1,398/886
12,830/1,289 = 9.95 33,689/1,966 = 17.1 0.581 5,372/1,041 = 5.16
aSpecific activity of CTP is calculated from counts per minute of
[32PJCTP in the soluble pool.
b Specific activity of UTP is [32P]UTP in the soluble pool.
calculated from counts per minute of
1,175/673
= =
CMP: UMP/
1.75 0.278 0.347
0.384
10,641/950 = 11.2 0.461
[3H]CTP
per counts per minute of
[3H]UTP
per counts per minute of
c Specific CTP:UTP equals specific activity of CTP per specific activity of UTP in the soluble pool. dSpecific activity of CMP is calculated from counts per minute of [3H]CMP per counts per minute of [32P]CMP in 16S RNA. 'Specific activity of UMP is calculated from counts per minute of [3H]UMP per counts per minute of [82P]UMP in 16S RNA. t Specific CMP:UMP equals specific activity of CMP per specific activity of UMP in 16S RNA.
396
J. BACTERIOL.
HATCH
label permits the calculation of the specific activity of the various nucleotides with respect to the 3H label. The use of specific activities in turn corrects for differences in the pool size of UTP and CTP, differential loss of nucleotides during extraction and separation, and differential expansion of pools owing to the addition of exogenous uridine. The specific activity of UTP in the acid-soluble pool at first rose and then dropped (Table 2). This was probably due to the depletion of the exogenous uridine by its rapid conversion by an L-cell nucleosidase to uracil, which accumulates in the medium and is not transported into L cells (Hatch, unpublished observation). The rate of approach to equilibrium of the 3H label between UTP and CTP was obtained from the ratio of the specific activities of CTP and UTP and is designated specific CTP:UTP in Table 2. In long-term experiments not reported here, an equilibrium value of unity was approached between 2 and 3 h post-3H label. If the parasite draws directly on the total acid-soluble pyrimidine nucleoside triphosphate pool, the specific CMP:UMP of chlamydial 16S RNA can be predicted from the average specific CTP:UTP of the pool and the rate of incorporation of total 3H counts into 16S RNA (see Materials, and Methods). Theoretical ratios are compared with the observed specific CMP:UMP obtained from the hydrolysate of 16S RNA in Fig. 2. The observed ratios follow closely the predicted ones. These data suggest that parasite RNA is supplied from a pyrimidine nucleoside triphosphate precursor pool that is labeled with the same kinetics as the total pool extracted from infected L cells. Entry of [14C]adenine and [3H]guanosine into the soluble pool and 16S and 18S RNA. The entry of ["4C]adenine into the pool and RNA of infected cells is reported in Table 3. The adenine label approached equilibrium between GTP and ATP at a much slower rate than did the uridine label between CTP and UTP, possibly because of the much larger size of the purine pool. As was done for the pyrimidines, a theoretical calculation was made for the specific GMP:AMP in 16S chlamydial RNA if the parasite utilizes the total acid-soluble nucleoside triphosphate pool for RNA synthesis (Fig. 3). The observed specific ratios obtained from the hydrolysates of 16S RNA are shown for only the 2- and 4-h samples because so few counts entered the RNA during the first hour of labeling (Table 3). The close correlation of the observed values with the theoretical ratios (Fig. 3) suggests that chlamydial 16S RNA is derived from the host purine nucleoside triphosphate
0.60
0
0.40 L U
0
0. (0
0.20 _.
0
15
30
I 45
MINUTES
FIG. 2. The rate of approach to equilibrium C:U labeling of the acid-soluble nucleoside triphosphate pool of infected and uninfected L cells and the 16S RNA of C. psittaci. The specific C:U ratios are plotted as a function of time of labeling with [3H]uridine. *, CTP:UTP in uninfected pool; 0, CTP:UTP in infected pool; A, CMP:UMP in 16S RNA, observed; 0, CMP: UMP in 16S RNA, theoretical.
pool. Similar results were obtained from the hydrolysates of chlamydial 23S RNA (data not shown). The specific GMP:AMP from the hydrolysate of L-cell 18S rRNA in the infected cells also are shown in Table 3. These ratios are similar to the observed and theoretical 16S RNA ratios. A direct comparison of 16S to 18S RNA in this type of experiment is difficult because of a presumed significant difference in the time required by L cells and chlamydiae for the processing of the rRNA precursor. However, the relatively long labeling periods minimize this problem. Consequently, the data of Fig. 3 are consistent with the conclusion that host and parasite RNA are derived from the same purine nucleoside triphosphate precursor pool and that this pool is accurately represented by the pool extracted with formic acid. When [3H Iguanosine was supplied to infected cells, label entered only GTP in the nucleoside triphosphate pool (Table 4). Similarly, the label entered only GMP in both 16S and 18S RNA (data not shown). Failure of labeled guanosine
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USE OF L-CELL NUCLEOTIDES BY C. PSITTACI
397
TABLE 3. ["4CJadenine label; nucleotide analysis of the soluble pool, 16S ribosomal RNA, and 18S ribosomal RNA in L cells infected with C. psittaci
laeln
he)Sp act GTPa (h)
0.5
Sp act ATP'
18S rRNA
16S rRNA
Soluble pool
Time
Sp
GTP: Sp act GMPd
~~~~ATPc
Sp act AMPe
Sp
Sp
GMP: Sp act GMP' Sp act AMPh GMP: AMP' AMP'
5/315 NC- 10/260 NC NC 21/681 NC 15/364 NC NC 0.120 = 0.527 436/1,914 18,621/22,102 0.270 14/307 NC 42/251 NC NC 11/720 NC 28/372 NC NC = 0.228 = 0.843 154/391 0.328 92/711 276/310 0.274 97/398 783/2,430 20,729/21,523 0.335 = 0.322 = 0.243 = 0.890 = 0.394 = 0.963 = 0.129 312/451 0.364 202/801 973/496 0.370 422/582 1,952/2,758 35,454/23,970 0.478 = 0.692 = 0.252 = 0.725 = 1.96 = 0.708 = 1.48
267/2,218 12,060/22,865 0.228 =
1.0 2.0 4.0
aSpecific activity of GTP is calculated from counts per minute of [14C]GTP per counts per minute of
[32PJGTP in the soluble pool.
Specific activity of ATP is calculated from counts per minute of [14C ]ATP per counts per minute of
['2P]ATP in the soluble pool.
Specific GTP:ATP equals specific activity of GTP per specific activity of ATP in the soluble pool. Specific activity of GMP is calculated from counts per minute of ["C]GMP per counts per minute [3P]GMP in 16S RNA. eSpecific activity of AMP is calculated from counts per minute of [14C]AMP per counts per minute [32P]AMP in 16S RNA. t Specific GMP:AMP equals specific activity of GMP per specific activity of AMP in 16S RNA. 'Specific activity of GMP is calculated from counts per minute of [14C]GMP per counts per minute [32P]GMP in 18S RNA. hSpecific activity of AMP is calculated from counts per minute of [14C ]AMP per counts per minute [32P ]AMP in 18S RNA. I Specific GMP:AMP equals specific activity of GMP per specific activity of AMP in 18S RNA. J NC, Not calculated due to insufficient counts. c
to enter adenine nucleotides has also been reported for sea urchins (6). However, labeled guanine can enter the RNA of Enterobacteriaceae as GMP and AMP (10). Moreover, a pathway leading from GMP to AMP in mammalian cells exists in theory (5). The failure of infected L cells to incorporate [3H ]guanosine into adenine nucleotides may be due to the presence of adenine (50 MM) in medium 199. Exogenous adenine has been used to prevent purine interconversion in Bacillus subtilis (18). Entry of [3H lguanosine into only GMP of both host and parasite RNA is consistent with both host and parasite drawing on the same precursor pool. However, the action of separate feedback mechanisms for the control of
purine nucleotide interconversion pathways cannot be excluded with certainty. DISCUSSION Moulder (11) coined the term "energy parasite" to describe the probable dependency of C. psittaci on its host for undefined energy intermediates. The energy parasite concept has been generated by negative evidence: the failure to detect mechanisms by which host-free chlamydiae might generate ATP. The purpose of
of of
of of
these investigations was to make a positive test of the energy parasite hypothesis. The most straightforward approach, isolation of chlamydiae and determination of their energy requirements in vitro, was not feasible because these organisms do not multiply or synthesize significant amounts of macromolecules outside of their host cells (26). Therefore, the approach to equilibrium labeling method of Wu and Soeiro (27) was used to identify in intact, infected host cells the source of the nucleoside triphosphate precursors of chlamydial RNA. It was found that the 6BC strain of C. psittaci draws on the total pyrimidine and purine nucleoside triphosphate pools of infected L cells for biosynthesis of its own RNA. The approach to equilibrium method requires the extraction with formic acid of the total nucleotide pool of infected L cells, which is the sum of the host and parasite pools. However, the total nucleotide pool is probably almost exclusively host pool because (i) the mass of the parasite represents not more than 5% of the total mass of the infected cell at 20 h postinfection (Hatch, unpublished observation), and (ii) the approach to equilibrium of an adenine label between GTP and ATP in infected cells is
398
HATCH
IF
I
0.40
-.
0
L.)
CD
w
0
0.20k
a.
CO)
I
I 0
2
3
HOURS FIG. 3. The rate of approach of equilibrium G:A labeling of the acid-soluble nucleoside triphosphate pool of infected and uninfected L cells and the 16S RNA of C. psittaci and the 18S RNA of L cells. The specific G:A ratios are plotted as a function of time of labeling with [14C]adenine. 0, GTP:ATP in uninfected pool; 0, GTP:ATP in infected pool; A, GMP:AMP in 16S RNA, observed; V, GMP:AMP in 18S RNA, observed: 0, GMP:AMP in 16S RNA, theoretical.
TABLE 4. [3HJguanosine label; nucleotide analysis of the soluble pool in L cells infected with C. psittaci Time of
labeling (h)
GTPa
ATPb
0.5 1.0 2.0 4.0
18,734/2,553 17,642/2,396 10,236/2,286 5,559/2,809
43/25,039 12/25,380 22/25,608 10/28,273
aExpressed as counts per minute of [3HJGTP per counts per minute of [32P]GTP. b Expressed as counts per minute of [3H ]ATP per counts per minute of [32P ]ATP.
identical to that of uninfected cells (Fig. 3), which suggests a negligible parasite contribution. Although the initial rate of conversion of UTP to CTP in infected cells is greater than in uninfected cells, the rate at which the uridine label is equilibrated between UTP and CTP in infected cells parallels that of uninfected cells at later time samplings (Fig. 2). It is not known why the initial rates differ; however, the smaller
J. BACTERIOL.
size of the pyrimidine nucleoside triphosphate pool in infected cells (Table 1) and the expansion of the UTP pool in the presence of exogenous uridine (as indicated by the rise in 32p counts in UTP from 1468 to 1966 in the experiment reported in Table 2) may be contributing factors. Although the kinetics of CMP:UMP and GMP:AMP labeling of chlamydial 16S RNA is consistent with the parasite drawing on preformed host nucleoside triphosphate pools, it is possible that chlamydiae independently synthesize nucleoside triphosphates from either uridine or adenine but with the same kinetics of labeling as the host cell. However, one would expect the parasite pool to reach a steady state much sooner than the host pool since chlamydiae are prokaryotes with a generation time of about 2 h (1), and L cells divide about once every 24 h. The few published C:U labeling studies suggest the generalization that prokaryote pyrimidine pools equilibrate rapidly as compared to the pools of mammalian cells. McCarthy and Britten (9) found a uracil label entered Escherichia coli RNA (generation time of 51 min) with equilibrium kinetics between 15 and 20 min postadministration of label. Nierlich and Vielmetter (14) had difficulty distinguishing between the labeling kinetics of uridine and cytidine nucleotides from a uracil label administered to B. subtilis (generation of 112 min). The results presented in Fig. 2 favor the entry of uridine into chlamydial RNA by pathway 1 and not pathway 2 as outlined in Fig. 1, but they do not distinguish between direct utilization of host-synthesized nucleoside triphosphates and uptake of dephosphorylated derivatives of these nucleotides by the parasite. The latter possibility requires the simultaneous presence of chlamydial nucleoside kinase and the absence of a chlamydial cytidine synthetase for pyrimidine interconversion. A similar argument would have to be made for the entry of adenine into chlamydial RNA. The direct utilization of host nucleoside triphosphates by the parasite seems more likely. In discussing the model presented in Fig. 1, it has been tacitly assumed that the exogenously supplied labeled nucleosides enter the L-cell host and thence into the chlamydial parasite. However, there is a remote possibility that the nucleosides supplied in the medium never reach the parasite in that form. For example, they may be phosphorylated during transport into the host. If this is true, then the kinetic studies must be interpreted as follows. First, pathway 2 (Fig. 1) does not operate, and all of
VOL. 122, 1975
USE OF L-CELL NUCLEOTIDES BY C. PSITTACI
the 'H label in the chlamydial 16S RNA must have been supplied by the nucleoside triphosphate pools of the host. Consequently, the observed specific CMP:UMP and GMP:AMP in 16S RNA would have to coincide with the corresponding theoretical specific ratios at all times of sampling. However, if the chlamydiae synthesize precursor nucleotides from endogenous sources, the 16S RNA synthesized from these precursors would be unlabeled and undetected. Therefore, if labeled nucleosides supplied in the medium do not enter the chlamydial cells, the data of Fig. 2 and 3 unambiguously demonstrate that the L cell supplies nucleotide precursors for synthesis of chlamydial 16S RNA, although the chlamydiae may also partially supply independently synthesized nucleotide precursors. On the other hand, if exogenous nucleotides freely enter the chlamydial cell, then the close agreement between observed and theoretical specific ratios (Fig. 2 and 3) strongly suggest that t-he L-cell pools are the exclusive precursors for chlamydial 16S RNA. The present findings complement investigations with host-free chlamydiae. Weiss and Wilson (26) demonstrated a requirement for exogenous ATP for lipid biosynthesis by the meningopneumonitis strain of C. psittaci, and Tamura (22) observed polymerization of exogenous nucleotides by reticulate bodies of this agent. Sarov and Becker (19) have reported that otherwise metabolically inert elementary bodies of C. trachomatis will synthesize minute amounts of RNA if treated with mercaptoethanol and supplied with all four nucleoside triphosphates. These experiments also indicate that the 18S RNA of L cells is derived from a purine precursor pool which is equilibrated with the same kinetics as the total acid-soluble purine nucleoside triphosphate pool (Fig. 3). In contrast, Plagemann (16) and Fields and Brox (2) have suggested that the total acid-soluble pyrimidine pool of Novikoff rat hepatoma cells and human lymphocytes is not available for RNA synthesis. In particular, Plagemann postulates the existence of at least two pools in Navikoff cells: an expandable cytoplasmic pool which only slowly equilibrates with a second, smaller and nonexpandable pool in the nucleus. Plagemann concluded the RNA of mengovirus reproducing in the cytoplasm is derived from the expandable pool, whereas cell nuclear RNA is derived from the nuclear pool. On the other hand, Soeiro and Ehrenfeld (20), using the approach to equilibrium labeling method, obtained results which are consistent with the present findings. They concluded that poliovirus and HeLa-cell nu-
399
clear RNA derive from the same precursor pyrimidine nucleoside triphosphate pool and that the total acid-soluble cellular pool always reflects the ratio of uridine and cytidine nucleotides in both cytoplasmic viral and nuclear RNA. These investigations suggest that C. psittaci draws on the total acid-soluble pyrimidine and purine nucleoside triphosphate pools of its Lcell host for biosynthesis of its own RNA. Both host and parasite RNA appear to be fed from the same purine nucleoside triphosphate pool. This first demonstration of the use of an energyrich product of host metabolism by the obligately intracellular chlamydiae reinforces the concept that chlamydiae are energy parasites. The requirement for energy intermediates alone would be enough to restrict them to an intracellular habitat, although other unrecognized hostproduced factors may also be required. Since chlamydiae grow inside cytoplasmic inclusion vacuoles bounded by membranes of host origin (21), the conclusion that chlamydiae depend on the nucleoside triphosphates of their hosts implies the existence of unusual mechanisms for the natural transport of nucleoside triphosphates across both the eukaryotic inclusion vacuole membrane and the prokaryotic cytoplasmic membrane of the parasite itself. ACKNOWLEDGMENTS I thank James W. Moulder for his advice throughout this study. This investigation was supported by Public Health Service research grant Al-1594 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service and postdoctoral fellowships from the National Science Foundation and the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Alexander, J. J. 1969. Effect of infection with the meningopneumonitis agent on deoxyribonucleic acid and protein synthesis by its L-cell host. J. Bacteriol.
97:653-657. 2. Fields, T., and L. Brox. 1973.
Specific activities of the
UTP pools of human lymphocytes after uridine-'Hlabeling. Can. J. Biochem. 51:954-957. 3. Gill, S. D., and R. B. Stewart. 1970. Respiration of L cells infected with Chiamydia psittaci. Can. J. Microbiol. 16:1033-1039. 4. Gill, S. D., and R. B. Stewart. 1970. Effect of metabolic inhibitors on the production of Chlamydia psittaci by
infected L cells. Can. J. Microbiol. 16:1079-1085. 5. Kelley, W. N. 1972. Purine and pyrimidine metabolism of cells in culture, p. 211-256. In G. H. Rothblat and V. J. Cristofalo (ed.), Growth, nutrition, and metabolism of cells in culture, vol. 1. Academic Press Inc., New York. 6. Kijima, S., and F. H. Wilt. 1969. Rate of nuclear ribonucleic acid turnover in sea urchin embryos. J. Mol. Biol. 40:235-246. 7. Koerner, J. F. 1970. Enzymes of nucleic acid metabolism,
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HATCH
p. 291-322. In E. E. Snell, P. D. Boyer, A. Meister, and R L. Sinsheimer (ed.), Annual review of biochemistry, vol 39. Annual Reviews Inc., Palo Alto. 8. Loening, U. E. 1969. The determination of the molecular weight of ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 113:131-138. 9. McCarthy, B. J., and R. J. Britten. 1962. The synthesis of ribosomes in E. coli. I. The incorporation of C14-uracil into the metabolic pool and RNA. Biophys. J. 2:35-47. 10. Mager, J., and B. Magasanik. 1960. (iuanosine 5'-phosphate reductase and its role in the conversion of purine nucleotides. J. Biol. Chem. 235:1474-1478. 11. Moulder, J. W. 1962. The biochemisyry of intracellular parasitism, p. 123. The University of Chicago Press, Chicago. 12. Moulder, J. W. 1969. A model for studying the biology of parasitism. Chiamydia psittaci and mouse fibroblasts (L cells). BioScience 19:875-881. 13. Moulder, J. W. 1971. The contribution of model systems to the^understanding of infectious diseases. Perspect. Biol. Med. 14:486-502. 14. Nierlich, D. P., and W. Vielmetter. 1968. Kinetic studies on the relationship of ribonucleotide precursor pools and ribonucleic acid synthesis. J. Mol. Biol. 32:135-147. 15. Penman, S. 1966. RNA metabolism in the HeLa cell nucleus. J. Mol. Biol. 17:117-130. 16. Plagemann, P. G. W. 1971. Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent nucleotide pools for nucleic acid synthesis. J. Cell. Physiol. 77:241-258. 17. Randerath, E., and K. Randerath. 1964. Resolution of complex nucleotide mixtures by two-dimensional anion-exchange thin-layer chromatography. J. Chromatogr. 16:126-129. 18. Salser, W., J. Janin, and C. Levinthal. 1968. Measure-
J. BACTERIOL.
ment of the unstable RNA in exponentially growing cultures of Bacillus subtilis and Escherichia coli. J. Mol. Biol. 31:237-266. 19. Sarov, I., and Y. Becker. 1971. Deoxyribonucleic aciddependent ribonucleic acid polymerase activity in purified trachoma elementary bodies: effect of sodium chloride on ribonucleic acid transcription. J. Bacteriol. 107:593-598. 20. Soeiro, R., and E. Ehrenfeld. 1973. Cytoplasmic and nuclear pyrimidine ribonucleotide pools in HeLa cells. J. Mol. Biol. 77:177-187. 21. Stokes, G. V. 1973. Formation and destruction of internal membranes in L cells infected with Chlamydia psittaci. Infect. Immun. 7:173-177. 22. Tamura, A. 1967. Studies on RNA synthetic enzymes associated with meningopneumonitis microorganism. Ann. Rep. Inst. Virus Res. Kyoto Univ. 10:26-36. 23. Tribby, I. I. E. 1970. Cell wall synthesis by Chiamydia psittaci growing in L cells. J. Bacteriol. 104:1176-1188. 24. 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. 25. Weiss, E., and L. A. Kiesow. 1966. Incomplete citric acid cycle in agents of the psittacosis-trachoma group (Chlamydia). Bacteriol. Proc. Biol. Med. p. 85-86. 26. Weiss, E., and N. N. Wilson. 1969. Role of exogenous adenosine triphosphate in catabolic and synthetic activities of Chlamydia psittaci. J. Bacteriol. 97:719-724. 27. Wu, R. S., and R. Soeiro. 1971. Turnover of nuclear RNA in HeLa cells: evidence for a single ribonucleotide pool. J. Mol. Biol. 58:481-487. 28. Young, Y. P. L., and R. J. Young. 1974. A method for the recovery of nucleic acids from polyacrylamide gels. Anal. Biochem. 58:286-293.
JOURNAL OF BACrERIOLOGY, May 1975, p. 393-400 Copyright @ 1975 American Society for Microbiology
Printed in U.S.A.
Utilization of L-Cell Nucleoside Triphosphates by Chlamydia psittaci for Ribonucleic Acid Synthesis THOMAS P. HATCH Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Received for publication 4 February 1975
Long-term, 32P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3H ]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3H to 32P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.
Members of the genus Chlamydia are intracellular parasitic bacteria which have not been propagated outside eukaryotic host cells. Although fragments of the tricarboxylic acid cycle and the pentose phosphate and glycolytic pathways have been demonstrated in host-free chlamydiae, mechanisms by which these organisms synthesize adenosine triphosphate (ATP) have not been detected (for reviews, 12, 13). Moulder (11) has suggested that chlamydiae are energy parasites which depend on their hosts for the energy intermediates they require for biosynthesis of macromolecules. The hypothesis is strengthened by the observation that antimycin, an inhibitor of aerobic respiration at the cytochrome level, reduced chlamydial growth (4). However, the action of this inhibitor on chlamydial cytochromes, although none can be detected (25), cannot be ruled out. Tribby and Moulder (24) demonstrated that most exogenously supplied bases and ribonucleosides enter the ribonucleic acid (RNA) of Chlamydia psittaci multiplying within the cytoplasm of L cells; the energy parasite concept
implies that it is the host nucleoside triphosphate pool that is exploited by the parasite. Radioactively labeled uridine can enter the RNA of chlamydiae by one or both of the two pathways outlined in Fig. 1. After transport into the host cell, uridine may be elevated to the nucleotide level by either host or parasite nucleoside kinases. In either case, the uridine label will equilibrate between uridine triphosphate (UTP) and cytidine triphosphate (CTP) in the soluble pool because UTP is the immediate precursor of CTP (7). The rate at which the label equilibrates between the two nucleotides varies from one organism to the next because it is dependent upon several factors, including pool size and rates of synthesis and degradation of RNA (18). If chlamydiae directly utilize the host ribonucleotide pool for RNA synthesis as outlined in pathway 1 of Fig. 1, the proportion of label distributed between the cytidine monophosphate (CMP) and uridine monophosphate (UMP) of chlamydial RNA should be a function of the approach to equilibrium labeling of UTP
393
394
HATCH
J. BACTERIOL.
density of 106 cells/ml in medium 199 containing [32P]HSPO4 (approximately 5 ACi/ml, New England Nuclear Corp.) for 72 h. Cells (4.0 x 10') were then infected with C. psittaci in a volume of 5 ml of medium containing "p (5 pCi/ml) and incubated as monolayers in 25-cm' plastic tissue culture flasks. At UR 20 h postinfection, the medium supernatant was decanted and 2.0 ml of fresh medium was added. Depending on the experiment, it contained "2Pi (5 pCi/ml) plus [5-'HJuridine (60 pCi/ml, New England Nuclear Corp.) and carrier uridine to a final concentration of 7.5 pM, [8-_4C]adenine (6.0 pCi/ml; 54.7 mCi/mmol; New England Nuclear Corp.), or [8'Hlguanosine (60 pCi/ml; 15.8 mCi/mmol, Amersham/Searle). At intervals the medium supernatant was detanted, the cells in monolayer were rapidly rinsed two times with 5.0 ml of Dulbecco phosphate-buffered saline, and the acid-soluble nucleotide pool and RNA were extracted. Extraction of the soluble pool and analysis of FIG. 1. Alternative pathways for the entry of nucleotides. Soluble pools were extracted from exogenous uridine into the RNA of C. psittaci. washed monolayers with 2.0 ml of 1.0 N formic acid for 2 h at 4 C. Cellular debris was removed by centrifugation and 100 pl of the formic acid extract and CTP in the host pool. If the parasite was applied directly onto thin layers of poly(ethyleneindependently synthesizes nucleotides from uri-
imine)-cellulose (Brinkmann Instruments, Inc.) together with 10 nmol of carrier nucleoside triphosphates. Nucleotides were separated by the method of Randerath and Randerath (17) and detected by ultraviolet absorption and autoradiography with medical X-ray film (Eastman Kodak Co.). Radioactivity was measured by cutting out areas yielding darkening of the film, eluting the nucleotides with 1.0 N LiCl, and suspending in a toluene-Triton X-100 (Rohm & Haas) scintillation mixture. Isolation and analysis of RNA. Washed monolayers were lysed with a nonionic detergent, 0.5% Nonidet-P40 (a gift from Shell Chemical Co., Ltd.), in buffer containing 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.4), 1.50 mM MgCl,, 0.14 N NaCl, and 500 pg of heparin per ml. Nuclei were removed by centrifugation, sodium dodecyl sulfate was added to the supernatant to a final concentration of 0.5%, and RNA was extracted by the phenol method of Penman (15). Chlamydial 16S RNA and L-cell 18S RNA were separated from other species of rRNA present in infected cells by electrophoresis in buffer E (8) on polyacrylamide gels consisting of 2.6% MATERIALS AND METHODS acrylamide cross-linked with bisacrylamide. Gels Growth of organisms. Suspension cultures of the were then dried on sheets of Whatman GF 83 chroma-
dine as in pathway 2, the distribution of label between CMP and UMP in parasite RNA should be independent of the composition of the host pool. These alternatives are kinetically distinguishable because the approach to equilibrium labeling of CTP and UTP in the soluble pool of a prokaryotic cell such as C. psittaci should not be identical to that of the eukaryotic L cell. The approach to equilibrium labeling of nucleotides method was first utilized by Wu and Soeiro (27) to demonstrate that heterogeneous sedimenting nuclear RNA and ribosomal (r) RNA precursors are supplied from the same pyrimidine nucleoside triphosphate precursor pool. In this investigation, the method has been used to demonstrate that RNA of C. psittaci is synthesized from the purine and pyrimidine nucleoside triphosphate pools of L cells.
5b clone of L cells (mouse fibroblasts) were grown by procedures previously described (23) in medium 199 (Grand Ibland Biological Co.) containing 0.1% sodium bicarbonate, 200 pg of streptomycin sulfate per ml, and 5% heat-inactivated fetal calf serum (International Scientific Industries, Cary, Ill.). No mycoplasma was isolated from cultures tested before the initiation and after the completion of these experiments (Flow Laboratories). All experiments were done with the 6BC strain of C. psittaci. L-cell cultures were infected with 10 times the amount of C. psittaci required to infect 50% of the cells in the culture, a multiplicity sufficient to infect more than 95% of the host cells. Labeling experiments. L cells were grown to a
tography paper, RNA bands were excised with a razor blade, and the RNA was recovered by the method of Young and Young (28) and hydrolyzed in 0.30 M KOH at 37 C for 18 h. The hydrolysate was neutralized with 35% perchloric acid, the potassium salt was removed by centrifugation, and the supernatant was applied to Whatman 3MM chromatography paper together with 40 nmol of carrier nucleotides. Nucleoside monophosphates were separated by high-voltage paper electrophoresis with 67 mM sodium citratecitric acid buffer (pH 3.5). Radioactive areas were detected by ultraviolet absorption, eluted in 1.0 N LiCl, and counted in a toluene-Triton X-100 scintillation mixture. Definitions. The specific activity of a nucleotide is
395
USE OF L-CELL NUCLEOTIDES BY C. PSITTACI
VOL. 122, 1975
defined as the ratio of the short-term 3H or 14C counts to the long-term 82P counts in that nucleotide. The 32P counts per uninfected cell in ATP, guanosine triphosphate (GTP), CTP, and UTP were constant between 48 and 72 h post-82P label. The ratio of the specific activities of two different nucleotides is designated the specific ratio following the terminology of Wu and Soeiro (27). Calculations. A theoretical calculation of the specific CMP:UMP and specific guanosine monophosphate (GMP):adenosine monophosphate (AMP) of chlamydial 16S rRNA has been made by assuming the parasite draws on the total acid-soluble nucleoside triphosphate pool. The calculation is based on the average specific CTP:UTP or GTP:ATP in the pool over a given time interval. For example, if the average specific CTP:UTP in the pool is 0.33 during the interval 15 to 30 min post- [3H ]uridine label, the average mole of UTP incorporated into 16S RNA during that interval will contain three times as many atoms of tritium as each mole of CTP incorporated. The cumulative 30-min specific CMP:UMP is obtained by adding the 15- to 30-min interval ratio to the cumulative ratio obtained from previous intervals after adjusting for the contribution of this interval to the total incorporation of tritium into 16S RNA. RESULTS
Nucleoside triphosphate pools in uninfected and infected celis. The effect of infection on the amount of [32P]HSPO4 incorporated into the nucleoside triphosphate pools of L cells is shown in Table 1. There was significantly less label in the pyrimidine nucleoside triphosphate pools of infected cells. The decreased incorporation of 3 Pi into purine pools was less obvious, but it was observed consistently. Gill and Stewart (3) have also reported a slight decrease in the ATP pool of L cells infected with C. psittaci
for 24 h. Although the drop in the nucleoside triphosphate pools in infected cells may be a nonspecific effect of infection, evidence will be presented in later sections that indicates the drop may be the result of increased drainage of host pools by the parasite for its own biosynthetic purposes. Nonspecific leaking of nucleotides is unlikely since the percentage drop in the pyrimidine pool is considerably greater than in the purine pool. Entry of [3H]uridine into the soluble pool and 16S RNA. L cells prelabeled for 72 h with [32P ]HSPO4 were infected with C. psittaci in the presence of 32Pi. At 20 h postinfection, [3H Juridine was added and the cells were sampled at intervals for -H and 32P counts in CTP and UTP in the acid-soluble pool and in CMP and UMP in chlamydial 16S RNA (Table 2) as described in Materials and Methods. The long-term 32P TABLE 1. Nucleoside triphosphate pools in infected and uninfected L cells after labeling with 32pa GTP
ATP
UTP
CTP
1,123 858 265
12,217 11,439 778
552 231 321
602 231 371
Sample
Uninfected ..... Infected .... Difference .....
.
aL cells were grown in suspension culture to a density of 106/ml in the presence of [32P]H,PO4 (5 ACi/ml) for 72 h. Cells (40 x 106) were then infected with C. psittaci and incubated along with an equal portion of uninfected cells as monolayers in 5 ml of medium containing 32Pi (5 uCi/ml). At 24 h, the monolayers were washed, the soluble pools were extracted, and the components were separated as described in Materials and Methods. Data are expressed as counts per minute per 105 L cells.
TABLE 2. [3Hluridine label; nucleotide analysis of the soluble pool and 16S ribosomal RNA in L cells infected with C. psittaci 16S rRNA
Soluble pool
Time of____
(min)
Sp
Sp
labeling
Sp act UTPb
Sp act CTPa
CTP:
Sp act CMPd
Sp act UMPe
310/639 = 0.485 = 1.58 2,641/894 = 2.95
4.55 4,100/901 7,482/972 = 7.70
UTPC 5 15
30 45
7,011/1,166 12,101/1,377 = 14,498/1,489 = 9.74 43,306/2,121 = 20.4 0.477 =
6.01 27,396/1,468 8.79 38,812/1,764
=
=
18.7 0.322 22.0 0.399
1,398/886
12,830/1,289 = 9.95 33,689/1,966 = 17.1 0.581 5,372/1,041 = 5.16
aSpecific activity of CTP is calculated from counts per minute of
[32PJCTP in the soluble pool.
b Specific activity of UTP is [32P]UTP in the soluble pool.
calculated from counts per minute of
1,175/673
= =
CMP: UMP/
1.75 0.278 0.347
0.384
10,641/950 = 11.2 0.461
[3H]CTP
per counts per minute of
[3H]UTP
per counts per minute of
c Specific CTP:UTP equals specific activity of CTP per specific activity of UTP in the soluble pool. dSpecific activity of CMP is calculated from counts per minute of [3H]CMP per counts per minute of [32P]CMP in 16S RNA. 'Specific activity of UMP is calculated from counts per minute of [3H]UMP per counts per minute of [82P]UMP in 16S RNA. t Specific CMP:UMP equals specific activity of CMP per specific activity of UMP in 16S RNA.
396
J. BACTERIOL.
HATCH
label permits the calculation of the specific activity of the various nucleotides with respect to the 3H label. The use of specific activities in turn corrects for differences in the pool size of UTP and CTP, differential loss of nucleotides during extraction and separation, and differential expansion of pools owing to the addition of exogenous uridine. The specific activity of UTP in the acid-soluble pool at first rose and then dropped (Table 2). This was probably due to the depletion of the exogenous uridine by its rapid conversion by an L-cell nucleosidase to uracil, which accumulates in the medium and is not transported into L cells (Hatch, unpublished observation). The rate of approach to equilibrium of the 3H label between UTP and CTP was obtained from the ratio of the specific activities of CTP and UTP and is designated specific CTP:UTP in Table 2. In long-term experiments not reported here, an equilibrium value of unity was approached between 2 and 3 h post-3H label. If the parasite draws directly on the total acid-soluble pyrimidine nucleoside triphosphate pool, the specific CMP:UMP of chlamydial 16S RNA can be predicted from the average specific CTP:UTP of the pool and the rate of incorporation of total 3H counts into 16S RNA (see Materials, and Methods). Theoretical ratios are compared with the observed specific CMP:UMP obtained from the hydrolysate of 16S RNA in Fig. 2. The observed ratios follow closely the predicted ones. These data suggest that parasite RNA is supplied from a pyrimidine nucleoside triphosphate precursor pool that is labeled with the same kinetics as the total pool extracted from infected L cells. Entry of [14C]adenine and [3H]guanosine into the soluble pool and 16S and 18S RNA. The entry of ["4C]adenine into the pool and RNA of infected cells is reported in Table 3. The adenine label approached equilibrium between GTP and ATP at a much slower rate than did the uridine label between CTP and UTP, possibly because of the much larger size of the purine pool. As was done for the pyrimidines, a theoretical calculation was made for the specific GMP:AMP in 16S chlamydial RNA if the parasite utilizes the total acid-soluble nucleoside triphosphate pool for RNA synthesis (Fig. 3). The observed specific ratios obtained from the hydrolysates of 16S RNA are shown for only the 2- and 4-h samples because so few counts entered the RNA during the first hour of labeling (Table 3). The close correlation of the observed values with the theoretical ratios (Fig. 3) suggests that chlamydial 16S RNA is derived from the host purine nucleoside triphosphate
0.60
0
0.40 L U
0
0. (0
0.20 _.
0
15
30
I 45
MINUTES
FIG. 2. The rate of approach to equilibrium C:U labeling of the acid-soluble nucleoside triphosphate pool of infected and uninfected L cells and the 16S RNA of C. psittaci. The specific C:U ratios are plotted as a function of time of labeling with [3H]uridine. *, CTP:UTP in uninfected pool; 0, CTP:UTP in infected pool; A, CMP:UMP in 16S RNA, observed; 0, CMP: UMP in 16S RNA, theoretical.
pool. Similar results were obtained from the hydrolysates of chlamydial 23S RNA (data not shown). The specific GMP:AMP from the hydrolysate of L-cell 18S rRNA in the infected cells also are shown in Table 3. These ratios are similar to the observed and theoretical 16S RNA ratios. A direct comparison of 16S to 18S RNA in this type of experiment is difficult because of a presumed significant difference in the time required by L cells and chlamydiae for the processing of the rRNA precursor. However, the relatively long labeling periods minimize this problem. Consequently, the data of Fig. 3 are consistent with the conclusion that host and parasite RNA are derived from the same purine nucleoside triphosphate precursor pool and that this pool is accurately represented by the pool extracted with formic acid. When [3H Iguanosine was supplied to infected cells, label entered only GTP in the nucleoside triphosphate pool (Table 4). Similarly, the label entered only GMP in both 16S and 18S RNA (data not shown). Failure of labeled guanosine
VOL. 122, 1975
USE OF L-CELL NUCLEOTIDES BY C. PSITTACI
397
TABLE 3. ["4CJadenine label; nucleotide analysis of the soluble pool, 16S ribosomal RNA, and 18S ribosomal RNA in L cells infected with C. psittaci
laeln
he)Sp act GTPa (h)
0.5
Sp act ATP'
18S rRNA
16S rRNA
Soluble pool
Time
Sp
GTP: Sp act GMPd
~~~~ATPc
Sp act AMPe
Sp
Sp
GMP: Sp act GMP' Sp act AMPh GMP: AMP' AMP'
5/315 NC- 10/260 NC NC 21/681 NC 15/364 NC NC 0.120 = 0.527 436/1,914 18,621/22,102 0.270 14/307 NC 42/251 NC NC 11/720 NC 28/372 NC NC = 0.228 = 0.843 154/391 0.328 92/711 276/310 0.274 97/398 783/2,430 20,729/21,523 0.335 = 0.322 = 0.243 = 0.890 = 0.394 = 0.963 = 0.129 312/451 0.364 202/801 973/496 0.370 422/582 1,952/2,758 35,454/23,970 0.478 = 0.692 = 0.252 = 0.725 = 1.96 = 0.708 = 1.48
267/2,218 12,060/22,865 0.228 =
1.0 2.0 4.0
aSpecific activity of GTP is calculated from counts per minute of [14C]GTP per counts per minute of
[32PJGTP in the soluble pool.
Specific activity of ATP is calculated from counts per minute of [14C ]ATP per counts per minute of
['2P]ATP in the soluble pool.
Specific GTP:ATP equals specific activity of GTP per specific activity of ATP in the soluble pool. Specific activity of GMP is calculated from counts per minute of ["C]GMP per counts per minute [3P]GMP in 16S RNA. eSpecific activity of AMP is calculated from counts per minute of [14C]AMP per counts per minute [32P]AMP in 16S RNA. t Specific GMP:AMP equals specific activity of GMP per specific activity of AMP in 16S RNA. 'Specific activity of GMP is calculated from counts per minute of [14C]GMP per counts per minute [32P]GMP in 18S RNA. hSpecific activity of AMP is calculated from counts per minute of [14C ]AMP per counts per minute [32P ]AMP in 18S RNA. I Specific GMP:AMP equals specific activity of GMP per specific activity of AMP in 18S RNA. J NC, Not calculated due to insufficient counts. c
to enter adenine nucleotides has also been reported for sea urchins (6). However, labeled guanine can enter the RNA of Enterobacteriaceae as GMP and AMP (10). Moreover, a pathway leading from GMP to AMP in mammalian cells exists in theory (5). The failure of infected L cells to incorporate [3H ]guanosine into adenine nucleotides may be due to the presence of adenine (50 MM) in medium 199. Exogenous adenine has been used to prevent purine interconversion in Bacillus subtilis (18). Entry of [3H lguanosine into only GMP of both host and parasite RNA is consistent with both host and parasite drawing on the same precursor pool. However, the action of separate feedback mechanisms for the control of
purine nucleotide interconversion pathways cannot be excluded with certainty. DISCUSSION Moulder (11) coined the term "energy parasite" to describe the probable dependency of C. psittaci on its host for undefined energy intermediates. The energy parasite concept has been generated by negative evidence: the failure to detect mechanisms by which host-free chlamydiae might generate ATP. The purpose of
of of
of of
these investigations was to make a positive test of the energy parasite hypothesis. The most straightforward approach, isolation of chlamydiae and determination of their energy requirements in vitro, was not feasible because these organisms do not multiply or synthesize significant amounts of macromolecules outside of their host cells (26). Therefore, the approach to equilibrium labeling method of Wu and Soeiro (27) was used to identify in intact, infected host cells the source of the nucleoside triphosphate precursors of chlamydial RNA. It was found that the 6BC strain of C. psittaci draws on the total pyrimidine and purine nucleoside triphosphate pools of infected L cells for biosynthesis of its own RNA. The approach to equilibrium method requires the extraction with formic acid of the total nucleotide pool of infected L cells, which is the sum of the host and parasite pools. However, the total nucleotide pool is probably almost exclusively host pool because (i) the mass of the parasite represents not more than 5% of the total mass of the infected cell at 20 h postinfection (Hatch, unpublished observation), and (ii) the approach to equilibrium of an adenine label between GTP and ATP in infected cells is
398
HATCH
IF
I
0.40
-.
0
L.)
CD
w
0
0.20k
a.
CO)
I
I 0
2
3
HOURS FIG. 3. The rate of approach of equilibrium G:A labeling of the acid-soluble nucleoside triphosphate pool of infected and uninfected L cells and the 16S RNA of C. psittaci and the 18S RNA of L cells. The specific G:A ratios are plotted as a function of time of labeling with [14C]adenine. 0, GTP:ATP in uninfected pool; 0, GTP:ATP in infected pool; A, GMP:AMP in 16S RNA, observed; V, GMP:AMP in 18S RNA, observed: 0, GMP:AMP in 16S RNA, theoretical.
TABLE 4. [3HJguanosine label; nucleotide analysis of the soluble pool in L cells infected with C. psittaci Time of
labeling (h)
GTPa
ATPb
0.5 1.0 2.0 4.0
18,734/2,553 17,642/2,396 10,236/2,286 5,559/2,809
43/25,039 12/25,380 22/25,608 10/28,273
aExpressed as counts per minute of [3HJGTP per counts per minute of [32P]GTP. b Expressed as counts per minute of [3H ]ATP per counts per minute of [32P ]ATP.
identical to that of uninfected cells (Fig. 3), which suggests a negligible parasite contribution. Although the initial rate of conversion of UTP to CTP in infected cells is greater than in uninfected cells, the rate at which the uridine label is equilibrated between UTP and CTP in infected cells parallels that of uninfected cells at later time samplings (Fig. 2). It is not known why the initial rates differ; however, the smaller
J. BACTERIOL.
size of the pyrimidine nucleoside triphosphate pool in infected cells (Table 1) and the expansion of the UTP pool in the presence of exogenous uridine (as indicated by the rise in 32p counts in UTP from 1468 to 1966 in the experiment reported in Table 2) may be contributing factors. Although the kinetics of CMP:UMP and GMP:AMP labeling of chlamydial 16S RNA is consistent with the parasite drawing on preformed host nucleoside triphosphate pools, it is possible that chlamydiae independently synthesize nucleoside triphosphates from either uridine or adenine but with the same kinetics of labeling as the host cell. However, one would expect the parasite pool to reach a steady state much sooner than the host pool since chlamydiae are prokaryotes with a generation time of about 2 h (1), and L cells divide about once every 24 h. The few published C:U labeling studies suggest the generalization that prokaryote pyrimidine pools equilibrate rapidly as compared to the pools of mammalian cells. McCarthy and Britten (9) found a uracil label entered Escherichia coli RNA (generation time of 51 min) with equilibrium kinetics between 15 and 20 min postadministration of label. Nierlich and Vielmetter (14) had difficulty distinguishing between the labeling kinetics of uridine and cytidine nucleotides from a uracil label administered to B. subtilis (generation of 112 min). The results presented in Fig. 2 favor the entry of uridine into chlamydial RNA by pathway 1 and not pathway 2 as outlined in Fig. 1, but they do not distinguish between direct utilization of host-synthesized nucleoside triphosphates and uptake of dephosphorylated derivatives of these nucleotides by the parasite. The latter possibility requires the simultaneous presence of chlamydial nucleoside kinase and the absence of a chlamydial cytidine synthetase for pyrimidine interconversion. A similar argument would have to be made for the entry of adenine into chlamydial RNA. The direct utilization of host nucleoside triphosphates by the parasite seems more likely. In discussing the model presented in Fig. 1, it has been tacitly assumed that the exogenously supplied labeled nucleosides enter the L-cell host and thence into the chlamydial parasite. However, there is a remote possibility that the nucleosides supplied in the medium never reach the parasite in that form. For example, they may be phosphorylated during transport into the host. If this is true, then the kinetic studies must be interpreted as follows. First, pathway 2 (Fig. 1) does not operate, and all of
VOL. 122, 1975
USE OF L-CELL NUCLEOTIDES BY C. PSITTACI
the 'H label in the chlamydial 16S RNA must have been supplied by the nucleoside triphosphate pools of the host. Consequently, the observed specific CMP:UMP and GMP:AMP in 16S RNA would have to coincide with the corresponding theoretical specific ratios at all times of sampling. However, if the chlamydiae synthesize precursor nucleotides from endogenous sources, the 16S RNA synthesized from these precursors would be unlabeled and undetected. Therefore, if labeled nucleosides supplied in the medium do not enter the chlamydial cells, the data of Fig. 2 and 3 unambiguously demonstrate that the L cell supplies nucleotide precursors for synthesis of chlamydial 16S RNA, although the chlamydiae may also partially supply independently synthesized nucleotide precursors. On the other hand, if exogenous nucleotides freely enter the chlamydial cell, then the close agreement between observed and theoretical specific ratios (Fig. 2 and 3) strongly suggest that t-he L-cell pools are the exclusive precursors for chlamydial 16S RNA. The present findings complement investigations with host-free chlamydiae. Weiss and Wilson (26) demonstrated a requirement for exogenous ATP for lipid biosynthesis by the meningopneumonitis strain of C. psittaci, and Tamura (22) observed polymerization of exogenous nucleotides by reticulate bodies of this agent. Sarov and Becker (19) have reported that otherwise metabolically inert elementary bodies of C. trachomatis will synthesize minute amounts of RNA if treated with mercaptoethanol and supplied with all four nucleoside triphosphates. These experiments also indicate that the 18S RNA of L cells is derived from a purine precursor pool which is equilibrated with the same kinetics as the total acid-soluble purine nucleoside triphosphate pool (Fig. 3). In contrast, Plagemann (16) and Fields and Brox (2) have suggested that the total acid-soluble pyrimidine pool of Novikoff rat hepatoma cells and human lymphocytes is not available for RNA synthesis. In particular, Plagemann postulates the existence of at least two pools in Navikoff cells: an expandable cytoplasmic pool which only slowly equilibrates with a second, smaller and nonexpandable pool in the nucleus. Plagemann concluded the RNA of mengovirus reproducing in the cytoplasm is derived from the expandable pool, whereas cell nuclear RNA is derived from the nuclear pool. On the other hand, Soeiro and Ehrenfeld (20), using the approach to equilibrium labeling method, obtained results which are consistent with the present findings. They concluded that poliovirus and HeLa-cell nu-
399
clear RNA derive from the same precursor pyrimidine nucleoside triphosphate pool and that the total acid-soluble cellular pool always reflects the ratio of uridine and cytidine nucleotides in both cytoplasmic viral and nuclear RNA. These investigations suggest that C. psittaci draws on the total acid-soluble pyrimidine and purine nucleoside triphosphate pools of its Lcell host for biosynthesis of its own RNA. Both host and parasite RNA appear to be fed from the same purine nucleoside triphosphate pool. This first demonstration of the use of an energyrich product of host metabolism by the obligately intracellular chlamydiae reinforces the concept that chlamydiae are energy parasites. The requirement for energy intermediates alone would be enough to restrict them to an intracellular habitat, although other unrecognized hostproduced factors may also be required. Since chlamydiae grow inside cytoplasmic inclusion vacuoles bounded by membranes of host origin (21), the conclusion that chlamydiae depend on the nucleoside triphosphates of their hosts implies the existence of unusual mechanisms for the natural transport of nucleoside triphosphates across both the eukaryotic inclusion vacuole membrane and the prokaryotic cytoplasmic membrane of the parasite itself. ACKNOWLEDGMENTS I thank James W. Moulder for his advice throughout this study. This investigation was supported by Public Health Service research grant Al-1594 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service and postdoctoral fellowships from the National Science Foundation and the National Institute of Allergy and Infectious Diseases.
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UTP pools of human lymphocytes after uridine-'Hlabeling. Can. J. Biochem. 51:954-957. 3. Gill, S. D., and R. B. Stewart. 1970. Respiration of L cells infected with Chiamydia psittaci. Can. J. Microbiol. 16:1033-1039. 4. Gill, S. D., and R. B. Stewart. 1970. Effect of metabolic inhibitors on the production of Chlamydia psittaci by
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p. 291-322. In E. E. Snell, P. D. Boyer, A. Meister, and R L. Sinsheimer (ed.), Annual review of biochemistry, vol 39. Annual Reviews Inc., Palo Alto. 8. Loening, U. E. 1969. The determination of the molecular weight of ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 113:131-138. 9. McCarthy, B. J., and R. J. Britten. 1962. The synthesis of ribosomes in E. coli. I. The incorporation of C14-uracil into the metabolic pool and RNA. Biophys. J. 2:35-47. 10. Mager, J., and B. Magasanik. 1960. (iuanosine 5'-phosphate reductase and its role in the conversion of purine nucleotides. J. Biol. Chem. 235:1474-1478. 11. Moulder, J. W. 1962. The biochemisyry of intracellular parasitism, p. 123. The University of Chicago Press, Chicago. 12. Moulder, J. W. 1969. A model for studying the biology of parasitism. Chiamydia psittaci and mouse fibroblasts (L cells). BioScience 19:875-881. 13. Moulder, J. W. 1971. The contribution of model systems to the^understanding of infectious diseases. Perspect. Biol. Med. 14:486-502. 14. Nierlich, D. P., and W. Vielmetter. 1968. Kinetic studies on the relationship of ribonucleotide precursor pools and ribonucleic acid synthesis. J. Mol. Biol. 32:135-147. 15. Penman, S. 1966. RNA metabolism in the HeLa cell nucleus. J. Mol. Biol. 17:117-130. 16. Plagemann, P. G. W. 1971. Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent nucleotide pools for nucleic acid synthesis. J. Cell. Physiol. 77:241-258. 17. Randerath, E., and K. Randerath. 1964. Resolution of complex nucleotide mixtures by two-dimensional anion-exchange thin-layer chromatography. J. Chromatogr. 16:126-129. 18. Salser, W., J. Janin, and C. Levinthal. 1968. Measure-
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