Long-Term Incorporation of Tritiated Adenine into Deoxyribonucleic Acid and Ribonucleic Acid by Treponema pallidum (Nichols Strain) - Infection ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
INFECTION AND IMMUNITY, Sept. 1980, p. 1040-1049 Vol. 29, No. 3
0019-9567/80/09-1040/10$02.00/0
Long-Term Incorporation of Tritiated Adenine into
Deoxyribonucleic Acid and Ribonucleic Acid by Treponema
pallidum (Nichols Strain)
STEVEN J. NORRIS,It* JAMES N. MILLER,' AND JOHN A. SYKES2
Treponemal Research Laboratory, Department of Microbiology and Immunology, University of California-
Los Angeles School ofMedicine, Los Angeles, California 900241; and Research Department, Southern
California Cancer Center, Los Angeles, California 900152
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
Treponema pallidum (Nichols strain), extracted in medium containing Eagle
minimal essential medium, 50% fresh, heat-inactivated normal rabbit serum, and
1.0 mM dithiothreitol, was incubated under 3% oxygen in the presence of tritiated
nucleic acid precursors. [8-3H]adenine was incorporated with high efficiency into
trichloroacetic acid-insoluble material; 2'-deoxyadenosine and uridine were incor-
porated in lower quantities, and thymine and thymidine were not incorporated.
Incorporation of [3H]adenine was inhibited by penicillin G, mitomycin C, acti-
nomycin D, and erythromycin, but was not affected by cycloheximide. Partial
purification of nucleic acids from T. pallidum incubated with [8-3H]adenine for
36 to 72 h and subsequent treatment with ribonuclease and deoxyribonuclease
revealed that 15 to 20% of the trichloroacetic acid-precipitable counts were
resistant to ribonuclease but susceptible to deoxyribonuclease. A simple assay
was developed in which NaOH treatment was used to distinguish incorporation
into ribonucleic acid and deoxyribonucleic acid. Both ribonucleic acid and deoxy-
ribonucleic acid synthesis continued for 6 days of incubation under 3% 02, whereas
incorporation was limited to the first day of incubation in samples incubated
under aerobic or anaerobic conditions. T. pallidum thus appears to be capable of
significant de novo deoxyribonucleic acid and ribonucleic acid synthesis under
microaerobic conditions.
Knowledge of the physiological requirements dithiothreitol (DTT), cysteine, glutathione, so-
and metabolic capabilities of Treponema palli- dium thioglycolate, and dithioerythritol aid in
dum has been rapidly increasing in recent years maintaining the viability of T. pallidum (5, 8-
despite our continued inability to cultivate the 10, 19, 22, 26); their activity appears to involve
bacterium in vitro. T. pallidum has long been the reduction of oxygen toxicity rather than the
considered an obligate anaerobe, but it is now fulfillment of a nutritional requirement (22).
evident that oxygen is utilized in the metabolism Glucose and magnesium salts have been re-
of the organism (6, 15, 16, 25) and that survival ported to bring about slight extensions in the
in vitro can be extended by the presence of small maintenance of motility (13), but the signifi-
amounts of oxygen when compounds capable of cance of the observed differences is open to
reducing its toxic effects are added to the me- question. The difficulty encountered in obtain-
dium (8-10, 22, 24). Two- to fivefold increases in ing continuous multiplication of T. pallidum in
the numbers of T. pallidum have been observed vitro is, in all probability, not the result of a
in tissue culture systems utilizing low O2 tensions single deficiency but rather a reflection of mul-
(9, 24). The nutritional requirements of T. pal- tiple factors, including the delicate balance be-
lidum have yet to be determined. Serum, serum tween oxygen utilization and toxicity, complex
albumin, or tissue extracts promote the reten- nutritional requirements, and the possible ac-
tion of motility and virulence in vitro (19,26), cumulation of toxic components. It is becoming
and the beneficial effect of serum is concentra- increasingly apparent that the delineation of
tion dependent (17). Addition of fatty acids to a conditions for the cultivation of T. pallidum will
prereduced, albumin-containing medium does require the development of sensitive assays for
not extend the retention of motility of T. palli- the metabolism and growth of T. pallidum and
dum (22). Certain sulfhydryl compounds such as the application of these procedures to the care-
t Present address: Department of Pathology M-012, Uni- ful, systematic consideration of the influence of
versity of California-San Diego School of Medicine, La Jolla, incubation parameters on the bacterium.
CA 92093. It is possible that a basic metabolic defect
1040VOL. 129, 1980 DNA AND RNA SYNTHESIS BY T. PALLIDUM 1041
prevents T. pallidum from multiplying outside The present study describes an independent
the host. However, Baseman and co-workers demonstration of DNA synthesis and develop-
have demonstrated that T. pallidum freshly iso- ment of a simple assay for measuring RNA and
lated from infected rabbit tissue is capable of DNA synthesis in small volumes of treponemal
incorporating radiolabeled compounds into pro- suspensions. Results obtained for T. pallidum
tein ribonucleic acid (RNA) and deoxyribonu- incubated under varied environmental condi-
cleic acid (DNA) (1, 3, 21). The amount of triti- tions are presented.
ated amino acids or uridine incorporated was
maximal in suspensions incubated under 10 or MATERIALS AND METHODS
20% oxygen as compared to higher or lower 02 Reagents. Eagle minimal essential medium with
concentrations (2, 21), but incorporation was glutamine (MEM; GIBCO Laboratories, Grand Island,
limited to the first 24 h of incubation. Utilizing N.Y.) was prepared with double-distilled water and
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
a complex medium with a low redox potential, supplemented with 50% fresh, heat-inactivated normal
Sandok and Jenkin (23) found that uptake of rabbit serum, 20 mM HEPES (N-2-hydroxyethylpi-
'4C-labeled amino acids or uracil was linear over perazine-N'-2-ethanesulfonic acid; Calbiochem), and
a 96-h period, suggesting that the brevity of 1.0 mM DTT (Sigma Chemical Co.) as described pre-
anabolic activity described previously was due viously (22). [8-3H]adenine (16.3 Ci/mmol), 2'-[G-
to unfavorable environmental conditions rather 3H]deoxyadenosine (15.4 Ci/mol), [methyl-3H]thy-
than an inherent deficiency in the organism. mine (14.3 Ci/mmol), and [methyl-3H]thymidine (19.8
Ci/mmol) were purchased from New England Nuclear
Incubation systems developed for the poten- Corp.; [6-3H]uridine (36.7 Ci/mmol) was obtained
tial cultivation of T. pallidum should promote from ICN Radioisotope Division.
both survival and retention of optimal metabolic Extraction and incubation of T. pallidunL The
activity for prolonged periods. Motility and vir- Nichols strain of T. pallidum was maintained by in-
ulence are adequate measures of survival but are tratesticular passage in adult New Zealand white rab-
relatively insensitive to the metabolic state of bits. At 7 to 10 days postinoculation, testes exhibiting
the organism, as exemplified by the ability of T. a firm orchitis were removed aseptically, sliced, and
pallidum incubated in the presence of erythro- extracted for 5 to 15 min in MEM-50% normal rabbit
serum-1.0 mM DTT which had been preequilibrated
mycin to retain motility despite a marked inhi- for >1 h in 95% N2-5% CO2 at 34°C. The extract was
bition of protein synthesis (1, 23). Incorporation removed and centrifuged twice at 250 x g for 7 min to
of radiolabeled precursors into macromolecules remove most of the tissue debris. The resulting sus-
offers a sensitive means of measuring the ana- pensions contained 0.2 x 107 to 3.0 x 107 T. pallidum
bolic activity of the bacterium. Of particular organisms and 90% of the trepo-
DNA synthesis. [3H]uridine incorporation oc- nemes. Contamination of the suspensions by other
curred only during the first 24 h under the bacteria was monitored by inoculation of 0.1 ml into
incubation conditions employed. Although the thioglycolate broth or onto chocolate agar plates, fol-
number of organisms used per assay was not lowed by incubation for 48 h at 34°C. Data from the
few experiments where contamination was detected
stated, it appears that the specific activity ob- were excluded from the results.
tained was quite low. Lysates of T. pallidum Assay of total incorporation by utilizing filtra-
were found to lack detectable thymidine kinase tion. Some of the described experiments utilized a
activity and to exhibit relatively low DNA po- filtration technique to determine the amount of triti-
lymerase activity (3). ated compounds incorporated. Triplicate 300-Ad sam-1042 NORRIS, MILLER, AND SYKES INFECT. IMMUN.
ples of T. pallidum suspensions were added to 2 ml of RESULTS
ice-cold phosphate-buffered saline and drawn by vac-
uum through either Millipore 0.22-ftm nitrocellulose The basal medium used in the described ex-
filters or Whatman GFC glass-fiber filters. Ten ml of periments consisted of Eagle MEM supple-
each of ice-cold phosphate-buffered saline and 5% mented with 50% fresh, heat-inactivated normal
TCA were passed through the filters sequentially. The rabbit serum, 20 mM HEPES, and 1.0 mM DTT.
filters were air-dried before quantitation of acid-insol- Unless otherwise stated, suspensions were incu-
uble radioactivity by liquid scintillation.
Nucleic acid purification and characterization. bated in an atmosphere of 3% 02-92% N2-5%
T. pallidum cells previously incubated with 5 IiCi of C02 at 34°C. These conditions were previously
[8-3H]adenine per ml were centrifuged at 15,000 x g found to allow retention of motility and viru-
at 40C for 30 min, washed three times in 35 ml of lence by T. pallidum for several days (22).
sterile saline-sodium citrate solution (SSC; 0.15 M Comparison of precursors. Tritiated nu-
NaCl with 0.015 M trisodium citrate, pH 7.0), and cleic acid precursors were added to suspensions
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
suspended in 3 ml of SSC. The treponemes were lysed of T. pallidum to determine the efficiency of
by the addition of 25 Ad of a 2-mg/mi solution of their incorporation into TCA-insoluble material.
proteinase K or protease (Sigma Chemical Co.) and In these experiments, MEM lacking bases and
0.2% (wt/vol) sodium dodecyl sulfate. The lysed sus- nucleosides was used. The compounds listed in
pension was extracted with agitation at ambient tem-
perature with 2 volumes of chloroform-isoamyl alcohol Table 1 were added at 5 ,uCi/ml to 1-ml samples
(24:1, vol/vol) for 1 h. After centrifugation at 2,000 x of the same T. pallidum suspension in each of
g at 41C for 15 min, the aqueous phase was removed, the three experiments. After 0 and 24 h of incu-
and the nucleic acids were precipitated by the addition bation, triplicate 300-pl samples were collected
of 2 volumes of ice-cold absolute ethanol. The precip- onto glass fiber filters, washed with phosphate-
itate was collected by centrifugation at 10,000 x g at buffered saline, and treated with ice-cold 5%
4VC for 30 min and dissolved in 1 ml of 0.085% (wt/ TCA. Significant incorporation of adenine, 2'-
vol) NaCl. Recovery of TCA-precipitable radioactivity deoxyadenosine, and uridine was obtained in all
by this method ranged from 50 to 909%o.
The composition of the nucleic acid preparations three experiments (Table 1). Adenine incorpo-
was determined by their susceptibility to degradation ration was particularly efficient, with increases
by bovine pancreatic RNase A (Sigma, X-A grade) or over background ranging from 1.7 x 105 to 3.0
bovine pancreatic DNase (Sigma, DN-CL grade). x l05 cpm per 107 T. pallidum organisms. Vari-
Heating the RNase preparation at 80°C for 10 min ation in incorporation among experiments has
had no effect on the results obtained. Triplicate 10- or been noted by other investigators (21) and is
20-l samples of the nucleic acid preparations were most likely a reflection of the conditions of the
incubated for 1 h at 37°C with 50 ,ug of RNase per ml, treponemes when harvested from the rabbit tes-
50 jig of DNase per ml + 1 mM MgCl2, or both. ticular tissue. Radioactivity retained on the filter
Nondegraded nucleic acids were precipitated by the was not significantly above background for T.
addition of 4 ml of ice-cold 5% TCA and collected by
filtration onto Millipore 0.22-,tm nitrocellulose filters; pallidum suspensions incubated with tritiated
the filters were washed with an additional 10 ml of 5% thymine or thymidine (Table 1). Autoradiogra-
TCA to remove acid-soluble material. The amount of phy of T. pallidum suspensions incubated for 24
radioactivity retained was determined by liquid scin- to 48 h with 5 ,ui of [8-3H]adenine per ml
tillation. demonstrated that tritium was associated with
Batch technique for assaying incorporation >90% of the treponemes. Because of the high
into DNA and RNA. To quantitate total incorpora- activities obtained, adenine incorporation was
tion, triplicate 100-pl samples of radiolabeled T. pal- studied in detail.
lidum suspensions were applied to 1.5-cm squares of Effects of inhibitors upon adenine incor-
Whatman 3MM filter paper. Measurement of incor-
poration into DNA was accomplished by pretreating poration. Suspensions of T. pallidum were
0.4 ml of the suspensions with 40 pI of 3.3 M NaOH preincubated for 4 h in the presence of 10-,Ag/ml
(final concentration, 0.3 M) for 1 h at 25°C before concentrations of various inhibitors of procar-
application of triplicate 110-pl samples to filter paper yotic and eucaryotic synthetic processes. [3H]-
squares. The air-dried filter paper squares were adenine (5 ,ACi/ml) was then added, and the
treated collectively with 200 to 300 ml of 5% TCA at suspensions were incubated for an additional 24
ambient temperature for 20 min, followed by two h before being subjected to filtration and TCA
additional washes with 5% TCA, two washes with 100
ml of 95% ethanol, and a final dehydration with ether. treatment. Penicillin G was the most effective of
Quantitation of radioactivity. Dried filters were the compounds tested in decreasing incorpora-
placed sample side up in 20-ml vials with 5 ml of a tion, followed by mitomycin C, actinomycin D,
toluene-based liquid scintillation cocktail. A Beckman and erythromycin (Table 2). Rifampin, strepto-
model LS 3133T liquid scintillation spectrophotometer mycin, and cycloheximide did not significantly
with a 3H counting efficiency of 57% was used to affect [3H]adenine incorporation under these
quantitate beta emission. conditions. The adenine incorporation resultsVOL. 29, 1980 DNA AND RNA SYNTHESIS BY T. PALLIDUM 1043
correlated in most cases with treponemal motil- of nucleic acid synthesis and procaryotic metab-
ity at the end of the incubation period. However, olism.
additional studies showed that as little as 0.1 ,ug Characterization of the products of ade-
of mitomycin C per ml could reduce [3H]adenine nine incorporation. Suspensions (40 ml) con-
incorporation >60% without affecting the reten- taining 3 x 107 T. pallidum per ml were incu-
tion of motility (data not shown). The pattern bated for 36 or 72 h (Table 3) in the presence of
of sensitivity demonstrated, as expected, that 5 ,uCi of [8-3H]adenine per ml. The treponemes
adenine incorporation is decreased by inhibitors were then harvested by centrifugation, washed,
TABLE 1. Incorporation of nucleic acid precursors by T. palliduma
TCA-insoluble activity (cpm per 300-1LI sample ± SD)'
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
Tritiated com-
pound Expt 1 Expt 2 Expt 3
(5 tCi/ml)
Oh 24h Oh 24h Oh 24h
Adenine 1,291 ± 344 61,816 ± 11,229 1,356 ± 388 41,783 ± 8,953 1,293 ± 67 33,705 ± 2,528
2'-Deoxyadeno- 844 ± 756 18,255 ± 670 958 ± 76 4,406 ± 1,696 522 ± 56 7,027 ± 185
sine
Uridine 495 ± 157 2,242 ± 502 462 ± 129 1,509 ± 394 241 ± 31 1,436 ± 435
Thymine 935 ± 245 2,054 ± 573 1,211 + 258 1,276 ± 1,209 378 ± 153 245 ± 94
Thymidine 766 ± 261 1,432 ± 644 1,074 ± 499 1,160 ± 1,062 250 ± 29 269 ± 156
a
Concentration of T. pallidum = 1.5 x 107, 2.0 x 107, and 1.6 x 107 per ml in experiments 1, 2, and 3,
respectively.
b SD, Standard deviation.
TABLE 2. Effect of selective inhibitors on the incorporation of [8-3Hladenine by T. palliduma
Adenine incorporationb (% of control) Motility (% motile)
Inhibitor (1 ILg/ml) Expt Expt 3
Expt 1 Expt 2 Expt 3 Mean Expt 1 2P Mean
None (100) (100) (100) (100) 96 88 100 94.7
Penicillin G 0 0 1.1 0.4 0 0 0 0
Mitomycin C 4 5 7 5.3 4 2 10 5.3
Actinomycin D 22 13 3 12.7 68 26 10 34.7
Erythromycin 66 54 42 54.0 100 92 84 92.0
Rifampin 95 73 81 83.0 96 96 98 96.7
Cycloheximide 97 90 86 91.0 98 84 96 92.7
Streptomycin 100 96 93 96.3 96 86 92 91.3
a T. pallidum concentration was 1 x 107 to 2 x 107 per ml. Inhibitors were added at 0 h, and 5 ,tCi of [8-
3H]adenine was added per ml at 4 h. Samples (300 pA) were harvested by filtration at 28 h.
b Corrected for background (0.5, 0.9, and 1.0 kcpm per sample for experiments 1, 2, and 3, respectively).
Control values (100%) were 59.2, 105.1, and 104.8 kcpm per sample, respectively.
TABLE 3. Susceptibility of nucleic acids isolated by [3Hladenine-labeled T. pallidum to degradation by
RNase and DNase
TCA-precipitable cpm ± SD' Percent of control ± SD
Treatment
Expt 1 Expt 2 Expt 1 Expt 2
None 11,195 ± 1,418 26,093 ± 990 (100 ± 12.7) (100 ± 3.8)
RNase 1,858 ± 121 5,907 ± 145 16.6 ± 1.1 22.6 ± 0.6
DNase 8,216 ± 983 15,580+ 1,675 73.4 ± 8.7 59.7 ± 6.4
RNase + DNase 175 ± 31 722 ± 143 1.6 ± 0.2 2.8 ± 0.5
Percent recovery 91.6 85.1
jtg
a 50 of each enzyme was used per ml; 1 mM MgCl2 was added to DNase-treated
'Based on three triplicate determinations. SD, Standard deviation.
samples.1044 NORRIS, MILLER, AND SYKES INFECT. IMMUN.
and subjected to the nucleic acid purification lectively in batches of up to 100 samples with
procedure as described. The resulting prepara- 5% TCA, ethanol, and ether. The reproducibility
tions were tested for their suscepta bility to pu- and counting efficiency achieved with this pro-
rifled bovine pancreatic RNase ancd DNase. As cedure were equal to or better than those ob-
shown in Table 3, TCA-precipitalble material tained by the filtration method, and the time
remained after treatment with either RNase or required to process each sample was reduced
DNase but was virtually abolished Iby treatment substantially. Direct treatment of 0.4-ml sam-
with both enzymes. Furthermore, ti ie sum of the ples of T. pallidum suspensions previously in-
TCA-precipitable activities for the RNase- and cubated with [3H]adenine with 40 ,Al of 3.3 M
DNase-treated samples was nearly equal to the NaOH (0.3 M final concentration) resulted in a
control value in each experiment. IRoughly 20% reduction in TCA-insoluble radioactivity corre-
of the TCA-precipitable radioactiveity exhibited sponding to the hydrolysis of RNA (Table 4).
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
the enzymatic susceptibility of DNiA, with the The decrease in counts occurred within the first
remaining activity residing in RNA,. 30 min of treatment at each of the temperatures
Centrifugation of purified nuclei c acids from tested; the NaOH-resistant activity, represent-
T. pallidum labeled with [3H]adeniine in a CsCl ing incorporation into DNA, was stable for at
gradient resulted in the formation c A a shoulder least 24 h at either 25 or 370C. The standard
of 3H-labeled TCA-precipitable matterial at the NaOH treatment used in the assay was 1 h at
bottom of the gradient and a well-cdefined peak 250C. Incorporation of [3H]adenine into DNA
at a lower density (Fig. 1). Pretrealtment of the was quantitated as the mean counts per minute
nucleic acids with RNase abolish ad the high- for the NaOH-treated samples minus the mean
density shoulder without affectingg the DNA counts per minute for samples collected at 0 h
peak. and treated with NaOH. Incorporation into
Simplified assay for DNA andI RNA syn- RNA was calculated as the mean counts per
thesis. A batch method was developped whereby minute for the untreated samples, minus the
triplicate 100-gil samples of T. par ilidum were DNA-associated counts and the mean counts
applied to 1.5-cm squares of WhaLtman 3MM per minute for the untreated 0-h samples.
filter paper, allowed to air dry, and treated col- Long-term DNA and RNA synthesis. The
kinetics of [3H]adenine (10 JLCi/ml) incorpora-
tion for T. pallidum incubated under 21% 02,3%
30 02, and anaerobic conditions were examined uti-
lizing the described assay technique. As dem-
onstrated in a representative experiment (Fig.
20
2), nucleic acid synthesis was limited to the first
0y 12 h of incubation when DTT was not included
10
in the medium, with incorporation under 3%-02
'I exceeding that obtained under aerobic and an-
aerobic conditions. In the presence of 1.0 mM
I-
ka&maaadu DTT, net incorporation of [3H]adenine into both
10 20 30 40 50 DNA and RNA occurred in samples incubated
FRACTION NUMBER under 3% 02 for a period of 24 to 48 h, at which
FIG. 1. CsCl gradient density profile e of 3H-nucleic
acids isolated from T. pallidum incubaited for 48 h in TABLE 4. Kinetics of degradation of TCA-insoluble
medium containing 10 fiCi of [8-3Hia6ienine per ml. material from a [8-3Hiadenine-labeled T. pallidum
A purified nucleic acid preparation rejPresenting 2 x suspension by 0.3 M NaOH0
10°
0
T. palidum
n8 Mn _s _ _
was divided
_ _
into two portions; one
_ -I,- -I AF _
.
TCA-insoluble cpm ± SDh
was left untreated, and the other was treated with 50
pg of RNase per ml at 370C for 30 min. The prepa-
Treatment time
rations were subjected to centrifugation at 160,000 x 250C 370C 650C
g for 40 h in a CsCl gradient with an average density 0 (untreated) 64,478 ± 934
of 1.55 g/ml. Fractions (250 Ml) were taken, and TCA- 30 min 11,869 ± 253 10,795 ± 378 9,009 ± 216
insoluble radioactivity was determined by applying 60 min 10,210 ± 454 9,532 ± 381 8,566 ± 906
100-,.l samples to filter paper squares and processing 120 min 10,054 ± 429 9,403 ± 1,357 9,767 ± 1,506
24 h 9,262 ± 431 10,122 ± 277 ND
as described in the text. (0) Untreated; (0) treated
0 T.
with RNase. Although the gradient was not of suffi- pallidum organisms (2 x 107 per ml) were incubated
cient density to resolve the RNA- and DNA-associ- with 10 ACi of [8- 'H]adenine per ml for 60 h at 340C under 3%
ated radioactivity, the DNA peak was clearly demon- 02-92% N2-5% C02. Suspension was treated directly with 0.3
strable in the RNase-treated preparation. Average M NaOH by addition of 40 ;d of 3.3 NaOH to 0.4 ml of
suspension.
CsCl density of the DNA peak in three experiments h 100-1d samples for untreated control, 110 pl for NaOH-
was 1. 7026 g/ml (range, 1. 7010 to 1. 7040). treated samples. SD, Standard deviation. ND, Not done.VOL. 29, 1980 DNA AND RNA SYNTHESIS BY T. PALLIDUM 1045
A RNA
60
50
AA 21% 0 --WITHOUT DTT
40
0)3%Ot
OE0 AN
_
WITH 10 M DT a
0
o30
20
.0- .,I-
10
ol
-P o-
Ilp -Ho0,,,
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
_ is GodkEAA
B DNA
7 7
6 6
0.
5 N5
0.
0 4 04
Bf
3 ~~-/ o.........-
2
I/ O/°-.°O --°-_ -_
A/
0,- 0- -°----=8
C MOTILITY
goo
" _
't \' w
F:
0
z-s50-
La
\\ z
w
50
0
0x \ -".
A
..Im Ia~ ~ ~ ~i
24 48 72 HOURS
FIG. 2. Incorporation of [8-3H~adenine (10 ILCi/ml) into RNA (A) and DNA (B) and retention of motility
(C) by T. pallidum incubated for 72 h under 21% 02, 3% 02, and anaerobic (AN) atmospheres in the presence
or absence of 1.0 mM DTT. A suspension containing 1.5 x 107 T. pallidum per ml in MEM plus 50% normal
rabbit serum was divided into 1-ml samples after the addition of [3Hladenine and, in half the suspension, 1.0
mM DTT. Samples were incubated at 340C under either 21% 02-74% N2-5% CO2, 3% 02-92% N2-5% CO2, or
95% N2-5% C02, removed at the designated intervals, and subjected to the NaOH assay procedure as described
in the text. Standard deviations were less than 15% of the mean in all cases. Background values were 2,977
cpm (without DMT) and 2,543 cpm (with DTT) for untreated samples and 1,003 cpm (without DTT) and 960
cpm (with DTT) for NaOH-treated samples.
time a plateau was reached. Under 21% 02 in the tween 12 and 24 h. In suspensions incubated
presence of DTT, incorporation was equivalent under anaerobic conditions, survival in terms of
to that seen under 3% 02 for the first 6 to 12 h motility was extremely limited, with 50% motil-
(Fig. 2). The TCA-insoluble counts then de- ity endpoints of 14 and 17 h with and without
creased, corresponding to a loss of motility be- DTT, respectively. Incorporation in these sam-1046 NORRIS, MILLER, AND SYKES INFECT. IMMUN.
ples was only slightly above background levels 24-h periods revealed that RNA and DNA syn-
and was limited to the first few hours of incu- thesis was continuous during the first 144 h (6
bation. days) of incubation under 3% 02 in medium
Labeling of 1-ml samples of a T. pallidum containing 1.0 mM DTT (Fig. 3), corresponding
suspension with 10 ,uCi of [3H]adenine per ml for to the retention of motility under these condi-
so
A
4
RNA
IL 0
3%02
O AN
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
E2 CELL
iei I i I
42-72 7- 9-012-4 14- 0 19
LABELING PERIOD20120-(O4
RS14 M-1"*
LABELING PERIOD (HOURS)
P
o
~
\ ~ t~ < MOTILITY
* * 3% 2
I
I oo. \ O0--OAN
5-
0. N
24 46 7 120 6
144 42 I,
HOURS
FIG. 3. Incorporation of [8_3Hjadenine into RNA (A) and DNA (B) by T. pallidum and rabbit testicular
cell controls during 24-hour labeling periods. T. pallidum (8 x 106 per ml) was extracted and incubated in
MEM-50% normal rabbit serum-1.0 mM DTT at 34C under either 3% 02-92% N2-5% CO2 (3% 02) or 95% N2-
5% CO2 (AN). Animal cell controls (CELL) containing 5 x 10 tissue cells and -5 x 10Os T. pallidum, per ml
were incubated under the 3% 02 atmosphere. [8_3HJadenine at 10 ,iCi/ml was added to samples at 24-h
intervals; motility (C) and incorporation were determined as described in the text. Standard deviations were
less than 15% of the mean in all cases. RNA and DNA were synthesized throughout the 6-day period that
motility was retained under 3% 02.VOL. 29, 19 DNA AND RNA SYNTHESIS BY T. PALLIDUM 1047
tions. This long-term incorporation occurred de- at lower levels, and incorporation of thymine
spite the lack of significant increases in cell and thymidine was not detected. It is likely that
numbers. RNA and DNA synthesis was consid- T. pallidum, like other bacteria (4, 11), possesses
erably reduced under anaerobic conditions, even transport mechanisms and enzymes which aid in
during the first day of incubation when the the efficient uptake and incorporation of exoge-
percentage of motile organisms was still high. nous adenine, although specific assays for these
Tissue cell controls containing rabbit testicular activities in T. pallidum have not been per-
cells but reduced numbers of T. pallidum did formed. Adenine is also the compound of choice
not incorporate significant quantities of [3H]ad- for labeling nucleic acids in Neisseria sp. be-
enine. Similar results were obtained in two ad- cause of its high efficiency of incorporation (14).
ditional experiments. Thymine and thymidine are metabolized to
The reason for the cessation of incorporation other compounds by wild-type Escherichia coli
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
within 2 days of incubation in continuously la- and Neisseria meningitidis, so that radiolabel-
beled T. pallidum suspensions is not presently ing with these precursors is relatively inefficient
known. Most of the tritium remained in the and not specific for DNA (7, 12). It is interesting
medium after incubation, indicating that the that incorporation of radiolabel initially associ-
cessation does not result from depletion of label. ated with thymine or thymidine does occur in
It is possible that the [3H]adenine breaks down N. meningitidis despite the lack of detectable
or is degraded during incubation into com- thymidine kinase, thymidine phosphorylase, or
pounds which are not incorporated into nucleic nucleoside deoxyribosyltransferase activity (12);
acids by T. pallidum (7, 14). Turnover of RNA this is presumably due to the incorporation of
species was found to occur: addition of 50 ,ug of breakdown products through other pathways.
unlabeled adenine per ml to T. pallidum suspen- The assay technique developed for the quan-
sions preincubated with 10 jCi of [8-3H]adenine titation of RNA and DNA synthesis offers sev-
per ml resulted in a 20% reduction in RNA- eral advantages over previously described meth-
associated counts within 2 h and a 40% reduction ods (3, 21, 23). The high efficiency of adenine
by 24 h. However, a steady-state phenomenon incorporation allows the use of smaller volumes
could not explain the cessation of net incorpo- (0.1 ml as compared to 2.0 ml), increasing the
ration into DNA, because the level of radioactiv- possible number of samples per unit volume by
ity in DNA did not change dramatically after 20-fold. Despite the reduction in volume and use
the addition of unlabeled adenine (data not of similar quantities of radiolabeled precursors,
shown). maximal counts per minute per sample were
actually greater than in previous reports. Use of
DISCUSSION batch techniques and direct treatment of the
T. pallidum appears to be capable of de novo treponemal suspensions with NaOH avoid the
synthesis of RNA and DNA in vitro as demon- need for filtration or centrifugation. These fea-
strated by incorporation of nucleic acid precur- tures will permit the use of this assay for the
sors. Under optimal incubation conditions, sus- detailed analysis of nutritional requirements and
pensions of T. pallidum were found to incorpo- other environmental conditions conducive to nu-
rate [3H]adenine into both RNA and DNA for cleic acid synthesis by T. pallidum.
6 days, i.e., the period for which active motility The effects of oxygen tension and DTT upon
was retained. Similarly, Sandok and Jenkin (23) RNA and DNA synthesis by T. pallidum par-
have reported that 14C-labeled uracil is assimi- allel those previously observed by Norris et al.
lated by T. pallidum for at least 96 h. In contrast, (22) for the retention of motility and virulence.
Nichols and Baseman (21) and Baseman et al. In the absence of DTT, [3H]adenine incorpora-
(3) found that incorporation of [3H]uridine into tion was limited to the first 24 h of incubation,
ribosomal RNA and DNA occurred only during with that occurring under 3% 02 far exceeding
the first 6 to 24 h of incubation, suggesting that that obtained under either aerobic or anaerobic
the conditions of incubation used were detrimen- conditions (Fig. 2). When 1.0 mM DTT was
tal to the maintenance of anabolic activity. The added to the medium, RNA and DNA syntheses
lack of significant multiplication by T. pallidum were essentially equivalent for the first 12 h in
in vitro is apparently not due to a deficiency in samples incubated under either 3% 02 or 21% 02.
nucleic acid synthesis, which continues despite After this initial period, incorporation ceased in
the absence of significant increases in cell num- the samples exposed to 21% 02, corresponding to
bers. a concurrent decrease in motility. In contrast,
[3H]adenine was incorporated with extremely samples incubated under 3% 02 continued to
high efficiency under 3% 02 in MEM-50% nor- incorporate [3H]adenine into RNA and DNA for
mal rabbit serum-1.0 mM DTT. Tritiated 2'- 6 days (Fig. 3). Both RNA and DNA syntheses
deoxyadenosine and uridine were incorporated were minimal under anaerobic conditions, even1048 NORRIS, MILLER, AND SYKES INFECT. IMMUN.
during the period when the treponemes re- LITERATURE CrIED
mained motile. 1. Baseman, J. B., and N. S. Hayes. 1974. Protein synthe-
It has been shown previously that T. pallidum sis by Treponema pallidum extracted from infected
retains its motility and virulence for longer pe- rabbit tissue. Infect. Immun. 10:1350-1355.
2. Baseman, J. B., J. C. Nichols, and N. S. Hayes. 1976.
riods under conditions of limited oxygen expo- Virulent Treponema pallidum: aerobe or anaerobe. In-
sure than under aerobic or anaerobic conditions fect. Immun. 13:704-711.
(8, 10, 22, 24). Norris et al. (22) reported that, in 3. Baseman, J. B., J. C. Nichols, and S. Mogerley. 1979.
the absence of reducing agents, suspensions of Capacity of virulent Treponema paUidum (Nichols) for
deoxyribonucleic acid synthesis. Infect. Immun. 23:392-
T. pallidum extracted in air survived longer 397.
under anaerobiosis than under an atmosphere 4. Burton, K. 1977. Transport of adenine, hypoxanthine and
containing 3% 02. However, T. pallidum ex- uracil into Escherichia coli. Biochem. J. 168:195-204.
tracted in an anaerobic chamber lost its motility 5. Chalmers, W. S. K., and D. Taylor-Robinson. 1979.
Downloaded from http://iai.asm.org/ on January 26, 2021 by guest
The effects of reducing and other agents on the motility
and ability to synthesize nucleic acids within 24 of Treponema pallidum in an acellular culture medium.
to 48 h when incubated anaerobically (Fig. 2 and J. Gen. Microbiol. 114:443-447.
3), suggesting that the amount of dissolved ox- 6. Cox, C. D., and M. K. Barber. 1974. Oxygen uptake by
ygen obtained during aerobic extraction in the Treponema pallidum. Infect. Immun. 10:123-127.
7. Fangman, W. L. 1969. Specificity and efficiency of thy-
earlier study was sufficient to allow extended midine incorporation in Escherichia coli lacking thy-
survival of the anaerobic samples. Addition of midine phosphorylase. J. Bacteriol. 99:681-687.
DTT to the medium appreciably extends the 8. Fieldsteel, A. H., F. A. Becker, and J. G. Stout. 1977.
retention of motility, virulence, and the capacity Prolonged survival of virulent Treponema pallidum
for nucleic acid synthesis in T. pallidum suspen- (Nichols strain) in cell-free and tissue culture systems.
Infect. Immun. 18:173-182.
sions incubated under 3% 02, apparently due to 9. Fieldsteel, A. H., J. G. Stout, and F. A. Becker. 1979.
the ability of this reducing agent to counteract Comparative behavior of virulent strains of Treponema
oxygen toxicity without affecting oxygen utili- pallidum and Treponema pertenue in gradient cultures
of various mammalian cells. Infect. Immun. 24:337-345.
zation (5, 22). 10. Fitzgerald, T. J., R. C. Johnson, J. A. Sykes, and J.
The occurrence of long-term RNA and DNA N. Miller. 1977. Interaction of Treponema pallidum
synthesis under 3% oxygen, in contrast to the with cultured mammalian cells: effects of oxygen, re-
minimal synthesis observed under aerobic and ducing agents, serum supplements, and different cell
anaerobic conditions, provides further evidence types. Infect. Immun. 15:444-452.
11. Hochstadt-Ozer, J., and E. R. Stadtman. 1971. The
for the microaerophilic nature of T. pallidum regulation of purine utilization in bacteria. II. Adenine
(22). It has been reported that incorporation of phosphoribosyltransferase in isolated membrane prep-
tritiated precursors into protein and RNA by T. arations and its role in transport of adenine across the
pallidum was maximal at 10 to 20% 02 when membrane. J. Biol. Chem. 246:5304-5311.
12. Jyssum, S. 1972. Labelling of DNA and RNA from thy-
compared to that obtained at lower 02 tensions mine and thymidine in Neiweria menmgitidis. Acta
(2, 21), but incorporation in these studies was Pathol. Microbiol. Scand. Sect. B 80:325-334.
limited to the first 24 h of incubation. Whereas 13. Kimm, G. E., R. Allen, H. J. Morton, and J. F. Mor-
T. pallidum may be able to maintain its meta- gan. 1960. Enhancement of survival in vitro of Trepo-
nema pallidum by addition of glucose and magnesium.
bolic functions for brief periods in an aerobic J. Bacteriol. 80:726-727.
atmosphere, it is apparent from the results of 14. Kingsbury, D. T., and J. F. Duncan. 1967. Use of
this investigation that reduced oxygen tensions exogenous adenine to label the nucleic acids of wild-
are more conducive to survival and extended type Neisseria meningitidis. J. Bacteriol. 94:1262-1263.
15. Lysko, P. G., and C. D. Cox. 1977. Terminal electron
maintenance of biosynthetic activity. Biosyn- transport in Treponema pallidum. Infect. Immun. 16:
thetic assays may aid in defining conditions al- 885-890.
lowing growth of T. pallidum by revealing those 16. Lysko, P. G., and C. D. Cox. 1978. Respiration and
whose implementation stimulates or deletion re- oxidative phosphorylation by Treponema pallidum. In-
fect. Immun. 21:462-473.
duces anabolic activity. The long-term retention 17. Matthews, H. M., H. M. Jenkin, K. Crilly, and P. L
of DNA and RNA synthetic activity observed in Sandok. 1978. Effects of fatty acids on motility reten-
the present study could serve as a basis for such tion by Treponema pallidum in vitro. Infect. Immun.
an approach. 19:814-821.
18. Miao, R., and A. H. Fieldsteel. 1978. Genetics of Tre-
ponema pallidum and five cultivatable treponemes. J.
ACKNOWLEDGMENTS Bacteriol. 133:101-107.
19. Nelson, R. A. 1948. Factors affecting the survival of
We thank Raymond Miao, Felix 0. Wettstein, and Edmund Treponema pallidum in vitro. Am. J. Hyg. 48:120-131.
Choi for their advice, Frank Fazzan and Wayne Ichikawa for 20. Nichols, J. C., and J. B. Baseman. 1975. Carbon sources
their technical assistance, and Elizabeth Janes and Renee utilized by Treponema pallidum. Infect. Immun. 12:
Norris for their secretarial help. 1044-1050.
This work was supported in part by World Health Organi- 21. Nichols, J. C., and J. B. Baseman. 1978. Ribosomal
zation agreement V3/181/26. S.J.N. is the recipient of predoc- ribonucleic acid synthesis by virulent Treponema pal-
toral Public Health Service National Research Service Award lidum. Infect. Immun. 19:854-860.
GM-07185 in Cellular and Molecular Biology from the Na- 22. Norris, S. J., J. N. Miller, J. A. Sykes, and T. J.
tional Institute of General Medical Sciences. Fitzgerald. 1978. Influence of oxygen tension, sulflhy-VOL. 29, 1980 DNA AND RNA SYNTHESIS BY T. PALLIDUM 1049
dryl compounds, and serum on the motility and viru- S. Roberts. 1978. Unsustained multiplication of Tre-
lence of Treponema pallidum (Nichols strain) in a cell- ponema pallidum (Nichols virulent strain) in vitro in
free system. Infect. Immun. 22:689-697. the presence of oxygen. Infect. Immun. 19:421-429.
23. Sandok, P. L, and H. M. Jenkin. 1978. Radiolabeling 25. Schiller, N. L, and C. D. Cox. 1977. Catabolism of
of Treponema pallidum (Nichols virulent strain) in glucose and fatty acids by virulent Treponema palli-
vitro with precursors for protein and RNA biosynthesis. dum. Infect. Immun. 16:60-68.
Infect. Immun. 22:22-28. 26. Weber, M. D. 1960. Factors influencing the in vitro sur-
24. Sandok, P. L, H. M. Jenkin, H. M. Matthews, and M. vival of Treponema pallidum. Am. J. Hyg. 71:401-417.
Downloaded from http://iai.asm.org/ on January 26, 2021 by guestYou can also read
