From the Département de Microbiologie et immunologie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7
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ABSTRACT |
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It has been suggested that the essential function
of DNA topoisomerase I in Escherichia coli is to prevent
chromosomal DNA from reaching an unacceptably high level of global
negative supercoiling. However, other in vivo studies have
shown that DNA topoisomerase I is very effective in removing local
negative supercoiling generated during transcription elongation. To
determine whether topoisomerase I is essential for controlling global
or local DNA supercoiling, we have prepared a set of topA
null mutant strains in combination with different plasmid DNAs.
Although we found a correlation between the severity of the growth
defect with both transcription-induced and global supercoiling, near to
complete growth inhibition correlated only with transcription-induced
supercoiling. This result strongly suggests that the major function of
DNA topoisomerase I is to relax local negative supercoiling generated
during transcription elongation.
Based on genetic evidence, it has been postulated that the
maintenance of a global level of chromosomal negative supercoiling within a ±15% range is required for good growth of Escherichia coli cells (1). The global level of DNA supercoiling reflects the
average superhelical density of all supercoiling domains. In this
context, the essential function of DNA topoisomerase I is to prevent
chromosomal DNA from reaching an inappropriate level of negative
supercoiling. This model stems from the observation that
topA null mutants are viable only if they accumulate
compensatory mutations that are very often found in one of the genes
encoding a subunit of DNA gyrase. As a result, global negative
supercoiling of both chromosomal and plasmid DNA were decreased below
the normal level (2, 3). Therefore, this global level of negative
supercoiling is believed to be regulated by the opposing enzymatic
activities of DNA topoisomerase I, encoded by the topA gene,
which specifically relaxes negative supercoiling, and DNA gyrase, with
two different subunits encoded by gyrA and gyrB
that introduces negative supercoiling. However, exactly how a high
level of global negative supercoiling could be detrimental to cell
growth is not known.
In vivo and in vitro studies have shown that DNA
topoisomerase I is highly efficient in removing negative supercoils
produced in the wake of moving transcription complexes (4-6). Such
supercoiling can be generated during transcription elongation because
of the difficulty for a moving transcription complex to rotate around the double helix (the twin-domain model for transcription; see Ref. 7).
In this situation, domains of negative and positive supercoiling are
transiently generated, respectively, behind and ahead of the moving
transcription complex. In the absence of DNA topoisomerase I, the local
negative supercoiled domain can build up, whereas the positive one can
be removed by DNA gyrase. In some cases, especially when the
transcribed genes encode membrane bound proteins, extreme negative
supercoiling is generated by transcription (8, 9). When such genes are
present on a plasmid DNA, transcription in the absence of DNA
topoisomerase I has been shown to generate hypernegatively supercoiled
DNA (5, 8-10). Therefore, in the context of transcription elongation,
the major role of DNA topoisomerase I is to control important local
fluctuations of negative supercoiling, as opposed to simply maintaining
global chromosome supercoiling at a constant level. Given the fact that in all these studies, the experiments were performed with
topA null mutants with compensatory gyrase mutations, one
may conclude that the removal of transcription-induced negative
supercoiling by DNA topoisomerase I is not essential for cell growth.
In the work reported here, we present data suggesting that the
essential function of DNA topoisomerase I is linked to transcription elongation and not to the control of the global level of negative supercoiling. These results were obtained by measuring the linking number deficit of pBR322 derivatives extracted from various
topA mutants having different growth capacities. On these
pBR322 derivatives, the effect of transcription of the tetA
gene that encodes a membrane bound protein is either kept at its
minimum or totally abolished. These plasmid DNAs allowed us to measure
both global negative supercoiling and transcription-induced negative
supercoiling and to make a correlation between these parameters and the
growth of the various topA mutants. Our results indicate
that the essential function of DNA topoisomerase I is linked to
transcription-induced negative supercoiling. Our results presented in
Ref. 11 further suggest that one detrimental consequence of the failure
to relax transcription-induced supercoiling is R-loop formation
(11).
E. coli Strains--
The E. coli strains used are
listed and described in Table I. DM800
derivatives were constructed by P1vir transduction (12). The
RFM475 cold-sensitive strain is well described by Drolet et al. (13).
Plasmids--
pBR322 Media and Growth Conditions--
Unless otherwise indicated,
bacteria were grown in VB Casa or LB medium (13) supplemented with
cysteine (50 µg/ml) at the temperature indicated in the table and
figure legends. When needed, antibiotics were added as follows:
ampicillin at 50 µg/ml, and chloramphenicol at 30 µg/ml. Because of
the acrA13 mutation in the DM800 derivatives, which renders
these cells more permeable to many antibiotics (3), chloramphenicol was
used at 10 µg/ml for these cells.
Isopropyl-
For the extraction of plasmid DNAs for supercoiling analysis, bacterial
cells carrying various pBR322 derivatives were grown overnight in VB
Casa medium at 37 °C and then diluted 1/75 in prewarmed LB medium.
The cells were grown to an A600 of 0.4 at 37 °C at which time they were transferred to the desired
temperature. The plasmid DNAs were extracted when the
A600 reached about 0.7 or after an exposition of
about 2 h at the respective temperature when an
A600 of 0.7 could not be reached. We found that
when hypernegatively supercoiled plasmid DNAs were produced at
21 °C, the proportion of such topoisomers reached a maximum after
about 1 h at this temperature and did not change for at least
another hour. Growth was stopped by transferring the cells in a tube
filled with ice. By this procedure, the temperature of the cultures
immediately dropped to 0 °C. Plasmid DNAs were extracted by an
alkaline lysis procedure (15).
Current Molecular Biology Techniques--
The current molecular
biology techniques were performed essentially as described (16).
CaCl2 transformations were carried out as described by
Drolet et al. (13).
Electrophoresis in the Presence of
Chloroquine--
One-dimensional and two-dimensional agarose gel
electrophoresis in the presence of chloroquine in 0.5 × TBE were
performed essentially as described (16). Chloroquine was used at the
concentration indicated in the figure legends. After electrophoresis,
agarose gels were dried and prepared for in situ
hybridization as described (16).
A Genetic System to Correlate Local and Global Negative
Supercoiling Fluctuations with Cell Growth in the Absence of DNA
Topoisomerase I--
Most of the previous studies related to the
effects of transcription on DNA supercoiling were designed to support
the twin-domain model for transcription (7) but not to test if DNA
topoisomerase I activity during transcription is crucial for cell
growth. In these studies, transcription of genes encoding membrane
bound proteins allowed the extraction of hypernegatively supercoiled plasmid DNAs from topA null mutants (5, 8, 9). Although the
results of these studies supported very well the twin-domain model for
transcription, the question of DNA topoisomerase I activity being
essential during transcription was not addressed or could not even be
addressed, because the topA null mutants used in these studies grew very well. Indeed, the fact that these strains grew very
well may suggest that DNA topoisomerase I activity during transcription
is not normally essential.
Therefore, to better address the role of DNA topoisomerase I in
transcription elongation, we had to design a genetic system in which at
least two requirements needed to be met: 1) the plasmid DNAs used
should not carry genes encoding for membrane bound products; 2) the
topA mutants used should not benefit from compensatory mutations in order for the topA phenotypes to be fully
expressed. Because most of the results supporting the twin-domain model
for transcription were obtained by using pBR322 DNA, we decided to use
several of its derivatives in our studies. In the early studies, the
extraction of hypernegatively supercoiled pBR322 DNA from topA null mutants carrying compensatory mutations, was
clearly shown to be dependent on tetA gene expression, which
encodes a membrane-bound protein (9). One derivative used in one study, pBR322
The relative growth capacity of the various topA null
strains used in our studies is shown in Table
III. It can be seen that the strain
showing the most severe growth defect and therefore, in our view, the
strain that most closely resembles a true topA null mutant
without compensatory mutations is RFM475. Low temperatures are more
restrictive for this strain because of the gyrB(Ts) allele that regains a more wild-type level of activity at these temperatures (13). The other strain that grows poorly is a gyrB+
derivative of the widely used DM800 strain ( The Formation of Transcription-induced Hypernegatively Supercoiled
Plasmid DNA Correlates with the Severity of the Growth Defect in the
Absence of DNA Topoisomerase I--
The various Our results show that severe growth inhibition of Escherchia
coli in the absence of DNA topoisomerase I correlates with
transcription-induced negative supercoiling, but not with global
negative supercoiling. Therefore, it suggests that topA
mutants fail to grow when DNA gyrase is too active during the process
of transcription elongation. Under these conditions, DNA gyrase
activity is efficient whether or not genes encode for membrane bound
proteins. In fact, under these conditions, we have also detected
hypernegatively supercoiled topoisomers for several others plasmid DNAs
that do not carry genes encoding for membrane bound proteins. For the
moment, we cannot exclude the possibility that the apparent increase in
DNA gyrase activity at low temperatures is not, for unknown reasons, only linked to the gyrB(Ts) allele, but to the low
temperature itself. Indeed, our preliminary data with DM800 derivatives
suggest that this might be the
case.1 Either way, the result
of this activity, if not counteracted by DNA topoisomerase I, is
inhibitory to cell growth. The results presented in the accompanying
manuscript (11) show that extensive R-loop formation can occur under
these conditions (11). This is most likely inhibitory to cell growth,
because the cellular level of RNase H activity must be properly
increased to support growth under conditions where topA
mutations are fully expressed (13).
Our results also strongly suggest that gyr mutations arose
in topA mutants to reduce gyrase activity during
transcription elongation. We can also conclude that the increase in the
level of global negative supercoiling in some topA mutants
(see Ref. 1 and this work), is only a secondary consequence of the
absence of DNA topoisomerase I and is not linked to the essential
function of this enzyme. This notion is further supported by our recent observations that the overproduction of both RNase H (11) and topoisomerase III (TopB)2 can
very well correct the growth defect of topA null mutants without, however, altering the global supercoiling level.
In conclusion, although the previous studies with various
topA null mutants have been very useful to reveal the
regulatory potential of negative supercoiling on DNA functions, they
did not reveal the essential function of DNA topoisomerase I. In our view, this enzyme should be considered, at least in part, as a transcription factor and not as a regulator of the global supercoiling level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E. coli strains used in this study
Ptet is a pBR322 derivative
with a small deletion within the tetA promoter region (5).
This deletion was further characterized in the present study by
sequencing the appropriate region of the plasmid (Fig. 1).
pBR322
tet5' was obtained by deleting the
HindIII-EcoRV DNA fragment from pBR322. The
HindIII-EcoRV-digested pBR322 vector was treated
with the Klenow enzyme to fill in the HindIII site, before
being treated with DNA ligase. Transformants were selected on LB medium
with ampicillin and were screened for sensitivity to tetracycline. The
plasmid DNA of some Tets transformants was analyzed by
using appropriate restriction enzymes to confirm the
HindIII-EcoRV deletion.
-D-thiogalactoside and
5-bromo-4-chloro-3-indolyl
-D-glucoside were purchased
from Sigma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ptet, has a deletion within the promoter
responsible for tetA expression (Fig.
1A), which was shown to
abolish the production of hypernegatively supercoiled pBR322 DNA (5).
We therefore decided to use this pBR322 derivative in our studies.
However, as can be seen in Table II, this
plasmid confers residual tetracycline resistance, because bacterial
cells carrying it can grow in the presence of 4 µg/ml tetracycline,
whereas cells carrying no plasmid DNAs can only grow when tetracycline
concentrations do not exceed 1 µg/ml. This residual tetA
expression can be explained by the fact that a weak
10 promoter
region, according to the consensus, was created during plasmid
construction (Fig. 1B). We constructed an additional pBR322
derivative, pBR322
tet5' (Fig. 1A), from which
tetA gene expression is not detectable (Table II). This plasmid has an HindIII-EcoRV deletion, removing
the 5' part of the gene including the original
10 promoter region,
the Shine-Dalgarno sequence, the ATG initiator codon, and one
transmembrane domain responsible for the anchorage of the TetA protein
to the membrane (Fig. 1A). Interestingly, by performing such
a deletion, a
10 region that restored a promoter sequence was
produced (Fig. 1B). Indeed, promoter activity was detected
when the lacZ gene was cloned downstream in the appropriate
orientation (Fig. 1B).
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Fig. 1.
The tetA promoter
region of the various pBR322 derivatives used in this study. In
panel A, a schematic drawing of the 5'
tetA region of the various pBR322 derivatives used in this
study is shown. 35 and
10 represent the respective consensus
sequences for
70 E. coli promoters (17), +1 shows the
transcriptional start site of tetA, and ATG is the initiator
codon of the TetA protein. One transmembrane domain responsible for the
anchorage of the TetA protein to the membrane and hence responsible for
generating hypernegatively supercoiled pBR322 DNA in topA
mutants (9) is also indicated. The deleted region within the various
pBR322 derivatives is indicated by thin lines. In
panel B, the nucleotide sequence of the tetA
promoter region of the various pBR322 derivatives used in this study is
shown. For both pBR322
Ptet and pBR322
tet5'
the promoter sequence that was reconstituted due to the deletion is
shown. The presence of promoter activity for pBR322
tet5'
was detected when the lacZ gene was cloned in the
appropriate orientation within the BamHI cloning site of
this plasmid (data not shown). Letters in bold type reflect
the most frequently found nucleotides at these positions (17). 16 to 19 indicates the number of nucleotides separating the
35 and
10
regions, with 17 being the most frequently found number.
Growth of RFM475 carrying pBR322 derivatives on LB plates with various
amounts of tetracycline
,
absence of growth.
topA and
gyrB225). The fact that it is possible to introduce a
wild-type gyrB allele in DM800 indicates a possible presence
of additional compensatory mutation(s) as previously considered when a
similar transduction experiment was performed (3). Moreover a
gyrB(Ts) derivative of DM800, a strain that should almost be
identical to RFM475, grows significantly better than RFM475 (Table
III), again suggesting that additional compensatory mutation(s) might
be present. It is also important to note that all the
topA strains used in our studies carry the same
topA deletion [
(topA
cysB)204].
Relative colony size on LB medium of the various topA strains used
in this study
, absence of growth.
topA
mutants carrying pBR322
Ptet were grown in LB medium at
37 °C and exposed to the indicated temperatures as described under
"Experimental Procedures." All the strains were exposed to various
temperatures to discriminate between true temperature effects and
allele specific effects [gyrB(Ts)] on DNA supercoiling.
The extracted plasmid DNAs were subjected to electrophoresis in agarose
gel in the presence of chloroquine at 7.5 µg/ml, as described under
"Experimental Procedures." Under these conditions the more
negatively supercoiled topoisomers migrate slowly except for the
fastest migrating band pointed to by an arrow (Fig.
2A, lane 12 [-
-]), which represents hypernegatively supercoiled plasmid DNAs. It can
be seen that the global DNA supercoiling level in the various strains,
represented by the topoisomers distributions of
pBR322
Ptet DNA without considering hypernegatively
supercoiled DNA, reflects very well the level of gyrase activity within
these strains (Fig. 2A). Indeed, plasmid DNAs extracted from
the DM800 strain that carries the gyrB225 mutation are less
negatively supercoiled than plasmid DNAs extracted from the DM800
gyrB+ derivative (EM176) or the gyrB(Ts) strains
(EM169 and RFM475) exposed to temperatures of 28 °C and below (Fig.
2A). It is also obvious that the global negative
supercoiling level correlates with the growth defects (Table III).
However, this correlation is not complete because the global negative
supercoiling level eventually reaches a maximum value even though some
bacterial strains are still growing, albeit slowly (EM169 at 28 °C
and 21 °C, EM176 at 28 °C, and very slowly at 21 °C), whereas
others are not (RFM475 at 28 °C (almost undetectable growth) and
21 °C). Under more restrictive conditions, when the
topA mutant completely fails to grow (RFM475 at
21 °C), hypernegatively supercoiled pBR322
Ptet DNA is
found (Fig. 2, A, lane 12; B,
bottom right panel). The formation of such
topoisomers is completely dependent on transcription, because it is
abolished by rifampicin treatment (data not shown). Such topoisomers
are not found when pBR322
Ptet DNA is either extracted
from DM800 carrying the gyrB+ allele (Fig. 2, A,
lanes 4-6; B, top
middle panel), or DM800 carrying the gyrB(Ts)
allele and grown at 21 °C (Fig. 2, A, lane 9;
B, top right panel), again supporting our
conclusion about the presence of additional compensatory mutation(s)
within DM800. Similar results are also obtained when the same set of
experiments are performed with pBR322
tet5' (Fig.
3). A larger proportion of
hypernegatively supercoiled topoisomers was detected when
pBR322
tet5' DNA was extracted from RFM480 grown at
21 °C (and a small amount from cells grown at 28 °C), a strain
identical to RFM475, but carrying a
topA::Tn10 allele instead of the
[
(topA cysB)204] found in RFM475 (11). This
is possibly due to the fact that RFM480 is genetically more stable than
RFM475.
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Fig. 2.
Transcription-induced hypernegative
supercoiling of pBR322 Ptet
DNA in various topA null mutant. The
various topA null mutants carrying pBR322
Ptet
were grown, and the plasmid DNAs were extracted as described under
"Experimental Procedures." In panel A, the samples were
analyzed by electrophoresis in an agarose gel containing 7.5 µg/ml of
chloroquine. [- -] indicates hypernegatively supercoiled topoisomers.
In panel B, some samples were also analyzed by
two-dimensional agarose gel electrophoresis. The chloroquine
concentrations used were 7.5 µg/ml and 30 µg/ml, respectively, in
the first and second dimension. Under the chloroquine concentrations
used, hypernegatively supercoiled plasmid DNAs migrate at the end of
the left part of the curve.
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Fig. 3.
Transcription-induced hypernegative
supercoiling of pBR322 tet5' DNA in
various topA null mutant. The various
topA null mutants carrying pBR322
tet5' were
grown, and the plasmid DNAs were extracted and analyzed as described in
the legend to Fig. 2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Pauline Phoenix for technical assistance and Sonia Broccoli for careful reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Grant MT-12667 from the Medical Research Council of Canada (to M. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a scholarship from les Fonds de la recherche en
santé du Québec. To whom correspondence should be
addressed: tel.: 514-343-5796; Fax: 514-343-5701; E-mail:
Marc.Drolet{at}umontreal.ca
1 E. Massé and M. Drolet, unpublished results.
2 S. Broccoli, P. Phoenix, E. Massé, and M. Drolet, manuscript in preparation.
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REFERENCES |
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