NOTES
amino acid is not the only deterministic factor for protein
folding, besides hydrophobicity, volume and charge of
molecular, contactable area and so on, are all factors that
govern the folding of protein. Had all the factors be included, we are sure of a higher capability for prediction.
Acknowledgements We thank Dr. Huang Yanzhao, Dr. Zhang Linsen
for useful discussion. This work was supported by the National Natural
Science Foundation of China (Grant No. 39870171).
LIU Danxu & HUANG Li
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Induction of the Sulfolobus
shibatae virus SSV1 DNA
replication by mitomycin C
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(Received January 28, 2002)
Chinese Science Bulletin Vol. 47 No. 11 June 2002
State Key Laboratory of Microbial Resources, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100080, China
Correspondence should be addressed to Huang Li (e-mail: huangl@
sum.im.ac.cn)
Abstract The temperate virus SSV1 from the hyperthermophilic archaeon Sulfolobus shibatae provides a useful
model system for the study of archaeal DNA replication.
Southern hybridization showed that SSV1 existed primarily
as a provirus in its host that was grown without shaking.
Upon UV or mitomycin C induction, the cellular level of free
SSV1 DNA increased drastically whereas that of integrated
viral DNA remained unchanged. The results of mitomycin C
induction were more reproducible than those of UV induction. We found that, when the cells that had been grown
without shaking were shaken, the replication of SSV1 DNA
was also induced. Based on our results, we developed a
method for the induction of SSV1 DNA replication by mitomycin C. When the S. shibatae virus production was induced
using this method, the cellular level of free SSV1 DNA started to increase 10 h after induction, and peaked after 12
15 h. A fully induced S. shibatae cell contained ~ 50 molecules
of free SSV1 DNA. The development of this induction
method and the description of the process of SSV1 DNA
replication following induction are valuable to the analysis of
the origin and mode of replication of the virus.
Keywords: hyperthermophilic archaea, Sulfolobus shibatae, SSV1,
DNA replication, induction.
According to Woese et al. [1, 2] , there exists in the
living world, in addition to Bacteria and Eukarya, Archaea,
the third form of life. The uniqueness of Archaea has been
largely confirmed by comparative biochemical and genomic studies[3,4]. Notably, Archaea are similar to Eukarya
in DNA replication, transcription, translation and genetic
recombination despite their morphological and structural
resemblance to Bacteria[ 3,5]. It appears, therefore, that
Archaea are a better prokaryotic model system than Bacteria for the study of eukaryotic genetic mechanisms. Our
laboratory studies DNA replication in the hyperthermophilic archaeon S. shibatae. This organism is used because
it harbors SSV1, a well-studied temperate virus containing
a 15.5-kb double-stranded DNA genome [6,7]. In the host
cell, SSV1 DNA may exist as free extrachromosomal
molecules or become integrated into the host genome
through site-specific recombination. Since viral DNA
replication depends on the replication machinery of its
host, SSV1 may provide a window on DNA replication in
923
NOTES
S. shibatae. The general features of a replicon, e.g. the
locations of the origin and termination site of replication,
are often examined through the analysis of replication
intermediates. However, the low copy number of the free
SSV1 DNA in the host cell[6] makes it difficult to apply
this approach to the study of SSV1 DNA replication. It
was reported that SSV1 DNA replication could be induced by UV irradiation[6]. But, this induction method
suffers from poor reproducibility and is not suitable for
handling large samples. In this report, we describe a
highly reproducible method for the induction of SSV1
DNA replication by mitomycin C. We have also studied
the kinetics of viral replication following induction, and
determined the copy number of free SSV1 DNA molecules in fully induced S. shibatae cells. The availability of
this induction method will be useful for understanding the
process of SSV1 DNA replication.
1 Materials and methods
( ) Growth of S. shibatae. S. shibatae ATCC
51178, purchased from the American Type Culture Collection, was grown in 250-mL serum bottles at 80 with
or without shaking in Brock’s basal medium[8] supplemented with 0.2% tryptone and 0.1% yeast extract. Unless otherwise indicated, each bottle contained 30 50
mL culture.
( ) Induction of SSV1 DNA replication. S. shibatae was grown without shaking to an A600 of ~ 0.7. For
UV induction, the culture was poured into a flat dish to
form a ~2-mm-thick layer and placed for indicated times
under a 15 W UV light (Philip) at a distance of 23 cm. For
mitomycin C induction, the antibiotic was added to the
culture to the indicated final concentrations. The sample
was incubated in the dark for 30 min at 22 . After induction, the culture was shaken at 150 r/min or incubated
without shaking at 80 .
( ) Isolation of the total cellular DNA from S. shibatae. Cells of S. shibatae were harvested by centrifugation (4000 r/min, 4 , 10 min) and resuspended in 10
mmol/L Tris-HCl, pH 8.0, 0.1 mmol/L EDTA (TE). SDS
and proteinase K were added to final concentrations of 1%
and 1 mg/mL, respectively. After incubation for 3 h at
50 , the sample was extracted successively with phenol,
phenol/chloroform/isoamyl alcohol (25/24/1) and chloroform/isoamyl alcohol (24/1). DNA was precipitated with
ethanol and resuspended in TE containing RNase at 20
µg/mL.
( ) Detection of SSV1 DNA in S. shibatae cells.
The S. shibatae DNA was digested with EcoR .
The restriction fragments were resolved by electrophoresis in agarose and electrotransferred to a Hybond-N+ nylon membrane (Amersham-Pharmacia Biotech). The
membrane was processed for Southern hybridization as
described in ref. [9]. An SSV1-specific probe (P SSV1 ) was
924
used in hybridization experiments. When indicated, an
additional probe (P Ssh7) specific for the host genomic DNA
was also included in the hybridization reactions. The sequences of the two probes were as follows.
PSSV1: 5 -ATTCGTATCCGCTTCGATTGATCAGCTCAATGATCAGCTT-3 [10];
PSsh7: 5 -GTAAGCGAGAAAGACGCTCCAAAAGA-3 .
The sequence of P Ssh7 was derived from a conserved
region in ssh7a (GenBank accession number: ABO13922)
and ssh7b (ABO13923) genes encoding the Ssh7 proteins
of S. shibatae. This probe was used to provide a control
for the copy number of the host genome. Both probes
were labeled with Digoxigenin (Roche) at the 3 -end according to the manufacturer’s instruction. Chemiluminescence detection was carried out as described by the
manufacturer. The resultant X-ray film was scanned for
quantitation using the Shimadzu scanner.
( ) Isolation of SSV1 DNA from S. shibatae. The
total cellular DNA from S. shibatae was digested with
and Avr , and electrophoresed in low
Xba , Kas
gelling point agarose. A gel slice containing SSV1 DNA
was excised under a long wavelength UV light. The DNA
was recovered from the gel as described in ref. [9].
( ) Extraction of SSV1 DNA from the extracellular
fluid of the S. shibatae culture. The growing culture was
centrifuged at 5000 r/min for 10 min at 4 . SSV1 particles in the supernatant were conce ntrated using a 30-ku
Microsep concentrator (Pall), digested for 2 h at 50
with 1 mg/mL proteinase K in the presence of 1% SDS
and extracted successively with phenol, phenol/chloroform and chloroform. DNA was precipitated with ethanol
and resuspended in TE.
2 Results
( ) Induction of SSV1 DNA replication by UV or
mitomycin C. In order to detect SSV1 DNA in the S.
shibatae cells by Southern hybridization, we designed an
SSV1 DNA-specific oligonucleotide probe (PSSV1), which
recognized a 7.9-kb EcoR
fragment of free SSV1 DNA
and a 12-kb EcoR
fragment of the S. shibatae genomic
DNA containing the sequence of integrated SSV1
DNA[ 6,10]. In our induction experiments, the S. shibatae
culture was grown to the late log phase and then exposed
to UV or treated with mitomycin C. In the uninduced cells,
only integrated SSV1 DNA was found and little free viral
DNA was detected (fig. 1 (a)). However, the amount of
free SSV1 DNA increased drastically upon UV induction.
Zillig’s laboratory reported earlier that UV irradiation
induced the replication of SSV1 DNA in S. shibatae, and
the induction was independent of the UV dosage within a
certain range [ 6 ] . Treatment with mit omycin C also
resulted in the induction of SSV1 DNA re plication.
We found that mitomycin C induction was reproducibly
Chinese Science Bulletin Vol. 47 No. 11 June 2002
NOTES
Fig. 1. Induction of the SSV1 DNA replication by UV or mitomycin C. (a) Lane 1, molecular weight markers with sizes (in kb) indicated; lane 2, uninduced control; lanes 3 and 4, UV induction: irradiation times were 40 and 60 s, respectively; lanes 5 7, mitomycin C
induction: mitomycin C concentrations were 5, 10 and 15 µg/mL, respectively. The treated samples were incubated without shaking. The
total cellular DNA (2 µg) from each sample was loaded. Probe: PSSV1. (b) Lane 1, an uninduced control; lane 2, 15 µg/mL mitomycin C.
The treated sample was incubated without shaking. The total cellular DNA (4 µg) from each sample was loaded. Probes: PSSV1 and PSsh7.
, an EcoR fragment from integrated SSV1 DNA (I);
, an EcoR fragment from free SSV1 DNA (F);
, an ssh7b-containing
fragment; , an ssh7a-containing fragment. SSV1 DNA (F)/SSV1 DNA (I), the ratio of free SSV1 DNA to integrated SSV1 DNA.
more effective than UV induction. In addition, similar
levels of induction were achieved with mitomycin C at
5 15 µg/mL. In order to determine if the level of integrated SSV1 DNA changed upon induction, we included
in a hybridization experiment two probes, PSSV1 and PSsh7.
The latter probe detected the ssh7a and ssh7b genes encoding the two isoforms, respectively, of the DNA binding
protein Ssh7. Since both ssh7a and ssh7b are single copy
genes (to be published elsewhere), they were used as a
control for the genomic copy number. As shown in fig.
1(b), the scanning intensity of the band of integrated viral
DNA from uninduced cells (0.84) was similar to that from
induced cells (0.73). It is possible that SSV1 DNA in a
fraction of cells in the induced culture replicated, leading
to the degradation of the host genome, while no viral
DNA replication occurred in the remaining cells. So the
hybridization signals of the ssh7 genes and integrated
SSV1 DNA were from the uninduced cells in the treated
culture. However, it was shown previously that S. shibatae
could survive induction and be reinduced [11]. In this study,
no degradation of genomic DNA was found in the induced
cells. Both of these findings argue against the above possibility. Therefore, we conclude that integration of SSV1
DNA into the host genome is unaffected by induction. Our
data also suggest that the ratio of free viral DNA to integrated one can be used for quantification of the induction.
It is worth noting that the two tested induction procedures
differed significantly in reproducibility. While each of the
13 mitomycin C induction trials in this study was successful, 5 out of 12 UV induction experiments failed to produce a significant amount of free viral DNA (free SSV1
DNA/integrated SSV1 DNA < 0.1). This discre- pancy
remains to be investigated.
Chinese Science Bulletin Vol. 47 No. 11 June 2002
( ) Factors affecting the mitomycin C induction of
SSV1 DNA replication. Since mitomycin C induction
worked consistently with S. shibatae, we further tested the
induction conditions. We found that more effective induction could be achieved if cells were shaken following the
mitomycin C treatment, as compared to a non-shaking
control (fig. 2(a)). In fact, when an uninduced culture that
had been grown without shaking was transferred to a
shaker and incubated with shaking, the cellular SSV1
DNA level went up. Furthermore, it appears that the effects of mitomycin C treatment and a change from nonshaking to shaking incubation on the induced replication
of SSV1 DNA were additive. How could a change in
cultivation conditions for S. shibatae affect the replication
of SSV1 DNA? In an attempt to address this question, we
grew an S. shibatae culture without shaking to the late log
phase (A600 = ~ 0.7). The culture was then transferred to
three 250-mL serum bottles to final volumes of 50, 100
and 200 mL, respectively. The three bottles were shaken
for 19 h. Although the three cultures reached similar cell
densities (A600 = ~1.0), the ratios of free SSV1 DNA to
integrated SSV1 DNA in cells from the 50, 100 and 200mL cultures were 8.1, 2.5 and 1.6, respectively (fig. 2(b)),
suggesting that shaking incubation may induce viral DNA
replication by changing the level of dissolved O2 in the
culture. In order to analyze the effect of the physiological
state of the host cell on the inducibility of SSV1 production, cells grown without shaking to the late log, stationary and death phases (A600 = 0.6, 1.0 and 0.8, respectively)
were treated with mitomycin C. Induction of viral DNA
replication was observed in all the tested samples. Therefore, the inducibility of SSV1 appears to be independent
of the growth phase of the host cell. Based on
925
NOTES
Fig. 2. Effects of growth conditions on the induction of
SSV1 DNA replication by mitomycin C. (a) Lane 1,
molecular weight markers with sizes (in kb) indicated;
lane 2, an uninduced control; lane 3, mitomycin C (5
µg/mL) treatment followed by incubation with shaking;
lane 4, mitomycin C (5 µg/mL) treatment followed by
incubation without shaking; lane 5, an uninduced shaking
culture control. (b) An S. shibatae culture was grown
without shaking to the late log phase and aliquoted. The
aliquoted cultures were incubated with shaking for 19 h.
Lane 1, 50 mL aliquoted culture in a 250-mL serum
bottle; lane 2, 200 mL aliquoted culture in a 250-mL
,
,
and
, the same as in fig.
serum bottle.
1(b).
our data, we have designed a protocol for the induction of
SSV1 DNA replication which entails the addition of
mitomycin C to the S. shibatae culture grown without
shaking to the late log phase and subsequent incubation of
the treated cells in a shaking bath.
( ) Time course of the induction of SSV1 DNA
replication. The cellular level of SSV1 DNA started to
increase ~10 h after mitomycin C induction, peaked after
12 15 h and declined gradually thereafter (fig. 3(a)). In a
Fig. 3. Time courses of the change in cellular free SSV1 DNA (a) and
the release of SSV1 (b) following the induction of the host by mitomycin
C. , SSV1 DNA concentration;　, A600 .
926
parallel experiment, the same time course was obtained
for the change of SSV1 DNA in cells following a switch
from non-shaking to shaking incubation. It appears that
the responses of S. shibatae to mitomycin C and to the
change in cultivation conditions involve similar molecular
steps. Previous studies suggest that S. shibatae releases
progeny viral particles without cell lysis[11]. In the present
study, we also observed the appearance of SSV1 DNA in
the extracellular fluid of the S. shibatae culture following
induction. The DNA was presumably released from the
induced host without cell lysis since little integrated SSV1
DNA, an indicator of the presence of genomic DNA, was
detected in the extracellular fluid during this period of
incubation. Both intracellular and extracellular viral DNA
molecules appeared to increase in the same pattern following induction.
( ) The copy number of free SSV1 DNA in an induced cell. In order to obtain a reliable estimate of the
copy number of SSV1 DNA in an S. shibatae cell following mitomycin C induction, we digested the total DNA
isolated from induced cells with three restriction enzymes
(Xba , Kas and Avr ), which had no cleavage sites
on SSV1 DNA. Restriction fragments were resolved by
electrophoresis in agarose. The viral DNA, well separated
from the genomic DNA fragments, was recovered. An
digest of a known amount of the total DNA isoEcoR
lated from the S. shibatae cells 15 h after induction along
with dilutions of an EcoR digest of the purified SSV1
DNA were run on the same gel. The gel was processed for
hybridization (fig. 4). Comparison of the intensities of the
hybridization signals of the 7.9 kb bands in the pure SSV1
DNA and total cellular DNA samples revealed that SSV1
DNA amounted to ~10% of the total DNA in the induced
cells. Considering the size difference between the viral
DNA and the host genome, we estimated that the ratio of
free SSV1 DNA to integrated SSV1 DNA was ~20. This
value is consistent with the result obtained by comparing
the amount of free SSV1 DNA with that of integrated
Chinese Science Bulletin Vol. 47 No. 11 June 2002
NOTES
SSV1 DNA in the induced cells (fig. 2(a), lane 3). Based
on our estimate of the amount of DNA per cell, an induced S. shibatae cell contained ~50 viral DNA molecules.
The estimates of the ratio of free SSV1 DNA to integrated
SSV1 DNA and the copy number of free viral DNA in an
induced cell are not contradictory in view of the previous
finding that an S. shibatae cell had ~2 copies of chromosomes in the stationary phase[12].
DNA increased significantly after the host cells grown
without shaking were incubated in a shaker. This phenomenon has not yet to be explained. Our preliminary
results suggest a possible link between the observed
shaking-mediated induction of viral DNA replication and
the change in dissolved O 2 level in the S. shibatae culture.
S. shibatae is an aerobe but shows low demand for O2[16].
The possibility exists that the shaking-mediated induction
of the viral DNA replication is part of a general physiological response of S. shibatae to the change in the availability of O2.
Acknowledgements This work was supported by the National Natural
Science Foundation of China (Grant Nos. 39770009 and 39925001) and
the fund for Knowledge Innovation Program of the Chinese Academy of
Sciences (Grant No. KSCX2-3-01-02).
References
Fig. 4. Quantitation of free SSV1 DNA in induced cells by hybridization. The total sample DNA was extracted from the S. shibatae cells that
had been shaken for 15 h following mitomycin C induction. The purified
SSV1 DNA was prepared by cleaving the total sample DNA with Xba ,
Kas and Avr , and gel purification. , see the legend to fig. 1.
1.
2.
3 Discussion
SSV1 is a temperate virus found in the hyperthermophilic archaeon S. shibatae. In this report, we present the
first detailed description of the induction of the viral DNA
replication by mitomycin C, an inhibitor of DNA synthesis. Previous studies have shown that SSV1 exists in the
host cell both as a free extrachromosomal element and as
a specifically integrated provirus in the host genome[6].
Our data revealed that, SSV1 existed almost exclusively
as a provirus in an uninduced S. shibatae cell grown
without shaking. Interestingly, the level of free viral DNA
increased significantly whereas that of integrated viral
DNA remained unchanged upon induction. SSV1 appears to resemble bacteriophage λ in several aspects of
integration: both SSV1 and λ genomes are integrated into
a tRNA gene in their host genomes; and the integrases
encoded by the two viruses are homologous in structure[10,13]. However, once a λ lysogen is induced, the integrated λ DNA is excised and undergoes active replication,
leading eventually to the lysis of the host cell and the
release of progeny phage particles[14]. How could the level
of integrated SSV1 DNA be unaffected by induction? A
possible explanation is that there is a dynamic equilibrium
between the integration and excision of the viral DNA,
with the former reaction being more efficient, as implied
from a previous in vitro study[15]. Induction may only
affect the replication of free viral DNA. It was also shown
in the present study that, unlike the induction of λ, the
induction of SSV1 was a slow process. We speculate that
the excision of integrated viral DNA is probably a ratelimiting step.
We observed in this study that the level of free SSV1
Chinese Science Bulletin Vol. 47 No. 11 June 2002
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(Received December 24, 2001)
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