INFECriON AND IMMUNITY, Mar. 1978, P. 854-860

Vol. 19, No. 3

0019-9567/78/0019-0854$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Ribosomal Ribonucleic Acid Synthesis by Virulent Treponema pallidum J. C. NICHOLS AND J. B. BASEMAN*

Department of Bacteriology and Immunology, University of North Carolina School ofMedicine, Chapel Hill, North Carolina 27514 Received for publication 20 October 1977

Ribosomal ribonucleic acid (rRNA) synthesis by virulent Treponema pallidum monitored by incorporation of [3H]uridine into trichloroacetic acid-precipitable counts and examination of radiolabeled rRNA on polyacrylamide gels. Verification that rRNA synthesis originated with T. pallidum was based upon co-electrophoresis with Escherichia coli rRNA, proportionate reductions in the amount of rRNA synthesized when numbers of treponemes were decreased, and inclusion of appropriate animal cell controls. The rate of treponemal rRNA synthesis was greater at temperatures of 37 and 390C than at 330C; rRNA synthesis was inhibited at 4 and 42°C and was effectively inhibited by actinomycin D. Kinetic experiments indicated that the majority of rRNA synthesis occurred early after extraction of treponemes from infected rabbit testicular tissue. Polyacrylamide gel profiles demonstrated the capacity of virulent T. pallidum to synthesize and process RNA to 23s, 16s, and 4 to 5s classes. Although motility of T. pallidum appeared unaffected during longer periods of incubation, pulselabeling experiments confirmed significant reductions in the rate of rRNA synthesis. When the effect of various environmental conditions upon rRNA synthesis was investigated, optimal synthesis was found to occur in an atmosphere of 20% oxygen whereas virtually no synthesis was observed under anaerobic or lowoxygen conditions. was

Considerable effort has been directed at un- poration of radiolabeled uridine into trichloroaderstanding virulent Treponema pallidum, the cetic acid-precipitable material. This paper deetiological agent of syphilis (18, 23, 29). Despite scribes the resultant data and discusses the apthese investigations, neither the growth require- plicability of this bioassay. ments nor the virulence determinants of the MATERIALS AND METHODS organism have been elucidated. Lack of growth in vitro has proven to be the major obstacle Animals. New Zealand white male rabbits (Pelencountered in treponemal research. We have Freeze, Rogers, Ark.) were injected intratesticularly attempted to develop in vitro metabolic assays with approximately 5 x 107 virulent T. pallidum orfor assessing the anabolic and catabolic capabil- ganisms per testis. Rabbits were housed in isolation ities of T. pallidum (3, 24). Measurements of cubicles kept at 16 to 180C prior to and during treponemal infection. Infected rabbits were injected intraprotein synthesis and carbon degradation have muscularly 25 mg of cortisone acetate (Medwick information about the metabolic yielded poten- Laboratories,with Melrose Park, Ill.) beginning 3 days Inc., tial of T. pallidum and have been helpful in postinfection and continuing until sacrifice. defining favorable in vitro environmental conBacteria. The virulent Nichols strain of T. palliditions (5). However, these assays may only dum used in these studies was kindly provided by the reflect endogenous activity of resting organisms, Center for Disease Control, Atlanta, Ga. Suspensions as no incorporation of carbon from glucose or of treponemes used for intratesticular inoculation were pyruvate into macromolecular material by T. stored in liquid nitrogen. The O111:B4 strain of Eschwas maintained on Trypticase soy agar pallidum can be detected, and no growth has erichiaatcoli room temperature. been observed (2). Consequently, we hoped to plates Chemicals and radioisotopes. [5,6-3H]uridine, 47 develop a metabolic assay which would be more Ci/mmol, and [2-'4C]uridine, 42 mCi/mmol, were pursensitive to optimal nutritive and environmental chased from ICN Radioisotope Division, Irvine, Calif. requirements of T. pallidum. By examining nu- Oxygen (100%) was supplied by Air Products, and cleic acid metabolism of freshly harvested trep- Matheson Gas Products provided a gas mixture of 50% onemes, we could measure ribosomal ribonucleic argon-35% nitrogen-10% hydrogen-5% carbon dioxide. acid (rRNA) synthesis by monitoring the incor- Actinomycin D and N-tris(hydroxymethyl)methyl-2854

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rRNA SYNTHESIS BY VIRULENT T. PALLIDUM

aminomethane-sulfonic acid (TES) were obtained from Sigma Chemical Co. Fetal calf serum and Dulbecco's minimal essential medium (MEM) were purchased from Grand Island Biological Co., Grand Island, N.Y. Proteinase K, a product of E. Merck, Darmstadt, Germany, was provided by Beckman Instruments, Inc. Other chemicals were reagent grade. Assay medium. Dulbecco's MEM was supplemented with 0.35% glucose, 10% tryptose-phosphate broth, 10% fetal calf serum, 10 mM TES, 8 mg of cysteine per ml, and 7 mg of sodium thioglycollate per ml (modified Dulbecco's MEM). This medium optimized anabolic activity of T. pallidum with retained vigorous motility during the performance of the experiment. Although other reported media prolong motility and virulence of T. pallidum beyond 48 h (14, 15, 27), these latter conditions require anaerobiosis or low 02 tensions which, based upon our findings (5; this paper) do not permit or maximize biosynthetic capabilities of the treponemes. Extraction of virulent T. pallidum. Testes were aseptically removed from infected rabbits 1 to 2 days after detection of an orchitis, minced, and shaken in 10 ml of modified Dulbecco's MEM in an air atmosphere. For certain experiments these manipulations were performed under anaerobiosis. The testicular extract was then centrifuged twice at 500 x g for 5 min to sediment tissue debris and the majority of contaminating animal cells. Measurement of RNA synthesis. Fractions of the above supernatant fluid containing treponemes were added to 2-ml glass vaccine vials (Arthur H. Thomas Co.) with 10 liCi of [5,6-3H]uridine (Fig. 1). Vials were stoppered and incubated at 330C unless indicated otherwise for various time intervals. Because a small amount of testicular tissue contamination was present, animal cell controls were established by resuspending in treponeme-free testicular extract the number of animal cells which contaminated the original treponeme fraction (Fig. 1). The number of contaminating cells was determined with a Petroff-Hausser counting chamber. After incubation samples were diluted in 10 ml of cold phosphate-buffered saline (PBS) plus 0.2% unlabeled uridine and were centrifuged at 20,000 x g for 15 min at 40C. Pellets were resuspended in 5 ml of cold PBS plus 0.2% uridine and were applied to a vacuum filter apparatus (Hoefer Scientific Instruments) containing 0.22-am membrane filters (Millipore Corp.). Filters were washed under vacuum with 5 ml of cold 5% trichloroacetic acid, dried, and added to vials containing 5 ml of Omnifluortoluene fluid. The radioactivity in each sample was determined with a liquid scintillation spectrometer (Packard Instrument Co.). Extraction of RNA. Approximately 5 x 108 T. pallidum organisms in 10 ml of modified Dulbecco's MEM were incubated with 250 liCi of [5,6-3H]uridine at 330C for various time periods in an air atmosphere. After incubation treponemes were pelleted by centrifugation at 18,000 x g for 15 min at 41C, washed, and resuspended in 3 ml of cold NTE buffer (100 mM NaCl, 10 mM tris[hydroxymethyl]aminomethane, 1 mM [ethylenedinitrilo]-tetraacetic acid disodium salt), pH 7.2. Appropriate animal cell controls were established in treponeme-free testicular extract as described

in Fig. 1 and were processed in a like manner. [2-14C] uridine (5 pCi) was added to 25 ml of E. coli cells (7) grown to early log phase (optical density [OD] of 0.70 at 650 nm) in Trypticase soy broth (TSB) at 370C with agitation in a water bath. Growth was continued to mid-log phase (OD of 1.3 at 650 nm), and subsequently the E. coli organisms were treated in a manner identical to that used for treponemes. Prior to phenol extraction, 109 unlabeled E. coli cells grown to mid-log phase in TSB at 370C were added to all radioactive samples as additional carrier RNA. Sodium dodecyl sulfate was introduced to a final concentration of 0.5% along with Proteinase K at 50 ug per ml (13). Individual samples were kept at room temperature for 15 min, an equal volume of water-saturated redistilled phenol (pH 7.2) was added, and each tube was shaken gently for 20 min (9). The aqueous layer was removed, combined with 0.3 ml of 2 M NaCl and 5 ml of cold absolute ethanol, placed at -20'C overnight, and centrifuged at 3,000 x g for 20 min at 40C. The supernatant was decanted and the pellet was gently washed with a precooled (-200C) 2:1 mixture of absolute ethanol and 0.2 M NaCl. After recentrifugation at 3,000 x g for 20 min at 4VC, the pellet was resuspended in 0.2 ml of NTE buffer with 20 pl of 0.1% bromophenol blue in 40% sucrose (ribonuclease-free sucrose, Schwartz/Mann). Gel electrophoresis. Radiolabeled RNA extracted from treponemes, animal cells, and E. coli was layered on gels consisting of 0.5% agarose and 2% acrylamide (26). Electrophoresis of the gels was performed under a constant current of 200 V and approximately 3 mA per gel for 80 min in Tris-borate buffer [90 mM trs(hydroxymethyl)aminomethane, 2.5 mM ethylenediaminetetraacetic acid, 900 mM boric acid, pH 8.3]. After electrophoresis, 1-mm gel slices were placed in vials containing 0.25 ml of 30% ammonium hydroxide. Samples were incubated overnight, 10 ml of ACS TESTICULAR

EXTRACT

PLEMl |T| AML

ANIMAL CELL | CONTROL

~~~~Smi

SUPERNATANT

18.000

x

9

15

SUPERNATANT

ADD 31-URIDINE INCUBATION

(TREPONEMES)

e ANIMAL CELLS

lu VI

VI

9

I_18000

15 min

lI min

ISOO

X

l

ADD 3H-URIDINE INCUBATION

x g

PRECIPITATE ON _ uJPRECIPITATE ON FILTERS WITH TCA 'ILTERS WITH TCA

CPM

CPM

FIG. 1. Measurement of RNA synthesis by T. pallidum and animal cell controls. Treponemes were extracted from infected rabbit testes by shaking in modified Dulbecco's MEM. Animal cell controls were established by resuspending in treponeme-free testicular extract the number of animal cells which contaminated the original treponeme fraction. Further details are outlined in Materials and Methods.

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(aqueous counting scintillant; Amersham/Searle, Arlington Heights, Ill.) was added to each vial, and the radioactivity was determined. RNA synthesis under various oxygen concentrations. Anaerobic conditions were established as previously described (5). Various oxygen concentrations were obtained by injecting stoppered vials containing treponemes and [5,6-3H]uridine with different amounts of 100% oxygen. Control vials containing redox indicator (5) were utilized to detect any 02 contamination arising during experimental manipulations. After incubation at 330C, the amount of radioactivity incorporated into trichloroacetic acid-precipitable material was determined as described in previous sections. All experiments included animal cell and actinomycin D controls.

RESULTS

Uridine incorporation by T. pallidum. When the rate of uptake of [5,6-3H]uridine by T. pallidum was examined (Fig. 2), accumulation of radioactivity based upon trichloroacetic acid-precipitable counts was linear for a limited time period (3 h). Further net accumulation occurred between 4 and 24 h of incubation, but in the interval between 24 and 48 h a slight decrease in total counts per minute was observed. This decrease in net accumulation of radioactivity does not appear to be the result of cell death, as the majority of treponemes remained actively motile over the entire incuba161

1

s~~~~~~~~~~~~1

tion period. The radioactivity measured in animal cell controls indicated that the major portion of RNA synthesis was the consequence of incorporation of [5,6-3H]uridine into macromolecular material by T. pallidum. At a concentration of 5 ,ug per ml, actinomycin D, a specific inhibitor of RNA synthesis, reduced net treponeme counts per minute by 90 to 95%. When the kinetics of RNA synthesis were investigated by monitoring utilization of [3H]uracil by T. pallidum, a similar pattern was obtained (data not shown). Incorporation of [3H]uridine into macromolecular material by various concentrations of treponemes diluted in treponeme-free testicular extract was measured to further confirm that T. pallidum was the predominant source of RNA synthesis (Table 1). Proportionately smaller amounts of radioactivity were utilized by decreasing numbers of treponemes, demonstrating that T. pallidum was the agent responsible for synthesis. Effect of temperature variation on RNA synthesis by treponemes. Previous investigations suggested that an incubation temperature below 370C was advantageous for maintenance of treponemal viability (16, 23, 29). Also, when incorporation of amino acids was monitored, maximal protein synthesis occurred at 34°C as compared to 32 and 36°C (3). When the effect of temperature variation on uridine utilization by treponemes was assayed over a 24-h incubation period, little synthesis was observed at 40C (Fig. 3). At 420C some synthesis was evident during the first 2 h of incubation, little further accumulation resulted by 8 h, and a TABLE 1. Incorporation of f3H]uridine into trichloroacetic acid-precipitable material by various concentrations of virulent T. pallidum

0.

0

Counts/mina 6 22

6

12

24

O

No. of treponemes

48

1.0 5.0 2.5 1.3 6.5 3.3

INCUBATION TIME (h)

FIG. 2. Rate of incorporation of J;HJuridine into trichloroacetic acid-precipitable material by virulent T. pallidum. Samples containing treponemes or animal cells and 10 1Ci of [5,6-3Hjuridine were incubated at 33°C in air for various time intervals. Radioactivity was determined as specified in Fig. I and Materials and Methods. Each point represents the average value of triplicate samples from three separate experiments. Within each experiment, individual samples varied less than 10% from the mean value. Treponemes were actively motile at all time points as determined by dark-field microscopy. Thepresence of 5 jig of actinomycin D per ml reduced radioactivity to background levels. Symbols: treponeme samples; 0 .

animal cell controls.

a

x x x x x x

107 106 106 106 105 105

Treponeme samples 6,760 4,574

2,804 1,695 982 595

Animal cell controls

442 294 286 253 320 314

Treponemes and animal cells were diluted with

treponeme-free testicular extract prepared by centri-

fuging treponemes extracted from testicular tissue at 18,000 x g for 15 min at 4VC. Quadruplicate samples were incubated at 330C for 8 h with 10 jtCi of [5,6-3H] uridine and then processed for radioactivity determination as outlined in Materials and Methods and Fig. 1. Actinomycin D (5 ,ug/ml) inhibited incorporation of uridine by 90%. Individual samples did not vary more than 15% from the mean value.

rRNA SYNTHESIS BY VIRULENT T. PALLIDUM

VOL. 19, 1978

857

9 -

30

-

30

11-M

On 6 x

I-,

x 20

k-*

31

20

0-0 -

20 f A.-M

I

10

h-.AI:11.

:0

0

a

0-2

3

I

0-8

0-24

INCUBATION TIME (h)

3. Effects of temperature variation on the incorporation of f3Hjuridine into macromolecular material by virulent T. pallidum. Each sample was incubated in air with 10,iCi of[5,6-3H~uridine for the indicated time intervals. Values have been corrected for animal cell contribution. Actinomycin D (5 pg/ml) reduced utilization of uridine by 90%. Counts per minute (CPM) are average values of three trials each with triplicate samples which did not deviate more than 13% from the mean. Symbols within open bars: U. 40C; O. 25 C; 0, 330C; 0, 370C. Open bar, 390C; FIG.

15 9 INCUBATION

21 TIME (h)

27

45

FIG. 4. Oxygen effects on RNA synthesis by T. pallidum. Anaerobic conditions were initially established with the use of an anaerobic glove box (Coy Manufacturing, Ann Arbor, Mich.). Varying amounts of 100% oxygen were injected into vials containing treponemes and 10,Ci of [5,6-3Hluridine. Incubation temperature was 33C. Values have been corrected for animal cell contribution and represent quadruplicate samples from each of two experiments. Individual samples varied less than 15% from the mean value in each experiment. Actinomycin D (5 pg/ml) reduced counts per minute to background levels. Treponemes were motile at all time points. Symbols: V anaerobic; 0, 5% 02; *, 10% 02; *, 15% 02;'E

solid bar, 420C.

20% 02-

decrease in net accumulation was seen by 24 h. Maximal stimulation of incorporation at 2 and 8 h occurred at 390C, but between 8 and 24 h RNA synthesis was reduced at this temperature while continual net increases of counts per minute were obtained at 24, 33, and 370C. When motility was examined, organisms placed at 40C for 24 h were noticeably sluggish, and 60 to 70% of the total population of treponemes was nonmotile by 24 h at 420C, correlating with the net decrease in radioactivity observed at this temperature. All other 24-h samples contained actively motile treponemes representing greater than 90% of the total population. Oxygen effects on RNA synthesis by T. pallidum. A functional role for oxygen in T. pallidum metabolism has been suggested by several studies (5, 8, 20). It was of interest to examine the effect of various oxygen tensions on RNA synthesis since incorporation of [3H]uridine into macromolecular material suggested a kinetic pattern (Fig. 2) different from that observed for utilization of radiolabeled amino acids (3). Previously described procedures were used to establish initial anaerobic conditions (5). A response similar to that observed for protein synthesis resulted: maximal incorporation of [5,6-3H]uridine occurred in an atmosphere of 20% oxygen (Fig. 4), and a profound reduction in RNA synthesis was measured under anaerobic and low 02 concentrations, providing further

evidence that oxygen plays a critical role in T. pallidum metabolism. Concentrations of 02 beyond the 20% level were avoided since we had earlier observed decreased macromolecular synthesis and loss of motility in T. pallidum at higher 02 tensions (5). Treponemes were actively motile under the oxygen concentrations and time periods presently assayed, demonstrating the inadequacy of motility as an exclusive indicator of T. pallidum metabolism. In addition, treponemes incubated anaerobically in modified Dulbecco's MEM retained motility, in contrast to a previous report in which treponemes were maintained in a less enriched medium (5). Comparison of the data in Fig. 2 and 4 demonstrates a phenomenon encountered frequently: significant differences in total counts per minute between treponemal preparations from separate rabbits are obtained, although general patterns of RNA synthesis are consistent. Therefore, data are expressed in average counts per minute from all experiments with the percentage of deviation from the mean computed in each experiment rather than the standard deviation. This variation in T. pallidum metabolic capability is probably the result of a complex interaction of factors, including stage of orchitic development, the immune response mounted by the infected rabbit, and the metabolic state of treponemes at the time of harvest.

INFECT. IMMUN.

NICHOLS AND BASEMAN

858

Pulse labeling of virulent T. pallidum. Kinetic data suggested that incorporation of [3H]uridine into RNA by T. pallidum decreased after a short incubation period (Fig. 2), although some net accumulation of radioactivity occurred during 24 h of in vitro incubation and active motility was observed up to 48 h. Treponemes were pulsed with [5,6-3H]uridine for 3-h intervals over 48 h of incubation. A significant portion (66%) of the total RNA synthesis occurred during the first 3 h of incubation (Fig. 5). By 23 h the amount of synthesis observed over a 3-h pulse was approximately 12% of the value obtained in the initial 3-h intervals. Virtually no net accumulation resulted thereafter. Thus, although motility of treponemes was unaffected for up to 48 h of incubation, pulse labeling confirmed that incorporation of [3H]uridine into macromolecular material was significantly curtailed after a much shorter incubation time. Gel electrophoresis of radiolabeled RNA. When isolation of RNA from T. pallidum was attempted by use of normal phenol extraction techniques (30), intact molecules of rRNA could not be obtained. However, utilization of a more gentle technique employing Proteinase K, an endoprotease made from the culture filtrate of the fungus Tritirachium album (11), permitted recovery of intact 23s, 16s, and 4 to 5s RNA molecules which could be demonstrated on polyacrylamide gels (Fig. 6). Animal cell controls 12

C,,

x

0.8

() 4 FLm Fl. 23-26 45-48 0-3 9-12 INCUBATION TIME (h)

were responsible for a negligible portion of the measurable radioactivity. E. coli and treponeme rRNA comigrated (Fig. 6), furnishing additional evidence that T. pallidum is the source of measured RNA synthesis. Also, when T. pallidum [3H]RNA (23s, 16s, 4 to 5s) was subjected to coelectrophoresis with ['4C]RNA from normal rabbit testicular cells (28s, 18s, 4 to 5s), the majority of RNA from treponemes and eucaryotic cells migrated at different rates, as expected (data not shown). Gel analysis of pulse-labeled rRNA further confirmed that synthesis of 23s and 16s molecules by T. pallidum was markedly reduced by 8 to 11 h of incubation at 33°C, although motility was unaffected up to 24 h (Table 2).

GEL SLICE FIG. 6. Migration of pHluridine RNA from T. pallidum on polyacrylamide gels. Treponemes and animal cell controls were incubated for 8 h at 330C with 25 ,uCi of [5,6-3Hjuridine per ml. RNA was phenol extracted and subjected to electrophoresis with [214C]uridine E. coli RNA on 2% polyacrylamide, 0.5% agarose gels. Gels were sliced and the radioactivity was determined as outlined in Materials and Methods. Symbols: * * *3Hluridine T. pallidum RNA; -_- -, /'4C]uridine E. coli RNA; * 0, 13H]uridine animal cell control * RNA. TABLE 2. Percentages of 23s and 16s rRNA synthesized at various time intervals by virulent T. palliduma Expt 1 Incubation time (h)

23s

16s 100 33 27

Expt 2

23s

16s

FIG. 5. Pulse labeling of virulent T. pallidum with pHluridine. At the indicated incubation times, 10 jiCi of [5,6-3Hluridine was added to treponeme samples and animal cell controls for 3-h intervals. Samples were incubated at 33°C in an air atmosphere. Radioactivity was measured as outlined in Fig. I and Materials and Methods. Treponemes were motile at all incubation times. Actinomycin D (5 pg/ml) inhibited uridine incorporation by 90%. The experiment was performed four times in triplicate. Samples in each experiment did not vary more than 10% from the mean value. Symbols: O. treponeme samples; U, an-

phoresis of virulent T. pallidum rRNA. Percentages were determined as (counts per minute under 16s or 23s peaks at various time intervals)/(counts per minute under 16s or 23s peaks at 0 to 3 h) x 100. Greater than 90% of the population of treponemes was vigorously motile at all time periods as determined by dark-

imal cell controls.

field microscopy.

100 100 100 21 70 73 42 51 49 a Values are based on polyacrylamide gel electro-

0-3 8-11 21-24

VOL. 19, 1978

rRNA SYNTHESIS BY VIRULENT T. PALLIDUM

DISCUSSION We have continued our efforts to delineate general metabolic characteristics of T. pallidum, attempting to develop an assay which would more readily reflect favorable conditions for in vitro growth of virulent treponemes. Measurement of RNA synthesis by T. pallidum provides a sensitive indicator of treponeme metabolic activity and has contributed additional information about the biosynthetic competence of T. pallidum in vitro. RNA synthesis was examined by monitoring the incorporation of [5,6-3H]uridine into macromolecular material by freshly harvested suspensions of virulent T. pallidum. Evidence that T. pallidum was the specific agent responsible for RNA synthesis was the result of comigration with E. coli RNA on polyacrylamide gels, proportionate reductions in the amount of RNA synthesized when treponemes were diluted in treponeme-free testicular extract, and the small amount of radioactivity contributed to the total counts per minute by animal cell controls. Actinomycin D, a specific inhibitor of RNA synthesis, reduced [3H]uridine incorporation into trichloroacetic acid-precipitable material by 90 to 95%. Temperature effects on RNA synthesis by T. pallidum were different from those exerted on protein synthesis. The optimal temperature for protein synthesis in vitro was 340C, whereas the maximal level of continual RNA synthesis during a 24-h incubation occurred at 37CC. The highest levels of uridine .incorporation were obtained at 390C, but the reduction in total counts per minute seen between 8 and 24 h (Fig. 3) suggests that 390C is not a physiologically favorable incubation temperature. Utilization of [3H]uridine at 25 and 330C was not markedly different from that observed at 370C, but small temperature variations exerted a profound influence on protein synthesis (3). Another difference exhibited in the two biosynthetic activities was the linearity of protein synthesis over a 24-h period versus linear RNA synthesis for an initial 2- to 4-h interval. This considerable disparity in the kinetic patterns of the two processes may be the product of unequal turnover rates which could influence intracellular precursor pools. RNA synthesis is maximal under a concentration of 20% 02 as compared to lower oxygen concentrations and anaerobiosis. These data are consistent with earlier observations concerning the stimulatory effect of 20% oxygen on protein synthesis and carbon degradation (5). These results, taken together with the report by Lysko and Cox that cytochrome o is the terminal oxidase in T. pallidum (20), provide strong support

859

for a functional role of 02 in T. pallidum metabolism, although final substantiation awaits in vitro growth. However, it should be emphasized that no data exist concerning the relationship between in vitro metabolic data and the in vivo capabilities of treponemes. Certain abnormalities in RNA synthesis appear to arise shortly after in vitro incubation. When utilization of [5,6-3H]uridine by T. pallidum was measured over a 48-h interval, a marked decrease in the incorporation of [3H]uridine into RNA was observed after a brief incubation at 330C (Fig. 2). Pulse-labeling experiments confirmed that the uptake of [3H] uridine was significantly reduced (Fig. 5). This reduction was apparently not the consequence of death of the organisms, as motility was unaffected up to 48 h. Also, in numerous experiments, a decline in net radiolabeled RNA accumulation was detected after 24 h of incubation. Several possibilities exist for the net loss in radioactivity in addition to an accelerated turnover rate. The instability of phenol-extracted RNA and the favorable effect on the isolation of intact RNA molecules by Proteinase K, an inhibitor of ribonucleases (31), indicate that ribonuclease activity may be stimulated in treponemes shortly after isolation from peak orchitis. The 1:1 ratio of 23s to 16s rRNA extracted from T. pallidum (Fig. 6) as compared to the normal 2:1 ratio obtained with E. coli on polyacrylamide gels also might reflect increased ribonuclease activity since 23s rRNA molecules are more labile than 16s molecules and consequently are more susceptible to ribonuclease degradation (22). It is possible that treponemes are exhibiting a phenomenon seen in Myxococcus xanthus organisms undergoing morphogenesis: no net accumulation of RNA is evident, but RNA synthesis is occurring and is masked by extensive turnover and/or increased nuclease activity (1). Also, net rRNA synthesis is markedly reduced in sporulating Bacillus subtilis (6, 17). Furthermore, when rapidly proliferating E. coli is placed in a "step-down" medium, as much as a 75% decrease in the rate of rRNA synthesis can be immediately observed (12, 19). However, this effect is temporary in E. coli whereas the reduction in rRNA synthesis by T. pallidum appears to be permanent under our experimental conditions. Perhaps T. pallidum is entering a similar dormant or resting phase and is capable of growth only if placed under more appropriate environmental conditions. This suggestion is consistent with the report that T. pallidum can remain virulent for 2 to 3 weeks in vitro without detectable growth (14). A picture of T. pallidum metabolism is begin-

860

INFECT. IMMUN.

NICHOLS AND BASEMAN

ning to emerge. When treponemes are extracted directly from infected rabbit testes, at least part of their metabolic machinery appears to be intact (3, 5, 20, 24, 28). Also, treponemes synthesize a broad spectrum of high- and low-molecularweight proteins which can be demonstrated on polyacrylamide gels (4). The banding pattern obtained is similar to that of the avirulent and cultivable Reiter treponeme. To date all procaryotes examined synthesize rRNA molecules approximately 23s, 16s, and 5s in size (25), although 23s rRNA exists only transiently in the photosynthetic organism Rhodopseudomonas spheroides and in the blue-green alga Anacystis nidulans (10, 21). Electrophoretic analysis of radiolabeled RNA from T. pallidum on polyacrylamide gels has been a useful probe of treponeme metabolic activity. Gel analysis has provided conclusive proof that T. pallidum is the source of measured RNA synthesis and has established that the virulent organism can synthesize and process RNA to 23s, 16s, and 4 to 5s classes for at least a short period of time in vitro. Concomitantly, however, treponemes seem to have been profoundly affected in some manner after extraction from the infected host. Decreased uptake of radiolabeled uridine into RNA occurs during early stages of in vitro incubation. Perhaps this alteration is another manifestation of inherent biological inadequacies of T. pallidum when subjected to unfavorable environments (2, 5, 24). ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI-11283 and Research Career Development Award 1-K04AI-00178 from the National Institute of Allergy and Infectious Diseases to J.B.B. LITERATURE CITED 1. Bacon, K. and E. Rosenberg. 1967. Ribonucleic acid synthesis during morphogenesis in Myxococcus xanthus. J. Bacteriol. 94:1883-1889. 2. Baseman, J. B. 1977. Report of a workshop: the biology of Treponema pallidum. J. Infect. Dis. 136:308-311. 3. Baseman, J. B., and N. S. Hayes. 1974. Protein synthesis by Treponema pallidum extracted from infected rabbit tissue. Infect. Immun. 10:1350-1355. 4. Baseman, J. B., and N. S. Hayes. 1977. Anabolic potential of virulent Treponema pallidum. Infect. Immun. 18:857-859. 5. Baseman, J. B., J. C. Nichols, and N. S. Hayes. 1976. Virulent Treponema pallidum: aerobe or anaerobe. Infect. Immun. 13:704-711. 6. Bonamy, C., L. Hirschbein, and J. Snulmajster. 1973. Synthesis of ribosomal ribonucleic acid during sporulation of Bacillus subtilis. J. Bacteriol. 113:1296-1306. 7. Bremer, H., and D. Yuan. 1968. Uridine transport and incorporation into nucleic acids in Escherichia coli. Biochim. Biophys. Acta 169:21-34. 8. Cox, C. D., and M. K. Barber. 1974. Oxygen uptake by Treponema pallidum. Infect. Immun. 10:123-127. 9. Dahlberg, A. E., and A. C. Peacock. 1971. Studies of 16 and 23s ribosomal RNA of Escherichia coli using composite gel electrophoresis. J. Mol. Biol. 55:61-74. 10. Doolittle, W. F. 1973. Postmaturational cleavage of 23s

11. 12. 13.

14.

15.

16. 17. 18. 19.

20.

21. 22.

23. 24. 25. 26.

27.

28.

29. 30.

31.

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