Relaxation of Transcription-induced Negative Supercoiling Is an Essential Function of Escherichia coli DNA Topoisomerase I*

Eric Massé and Marc DroletDagger

From the Département de Microbiologie et immunologie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table I
E. coli strains used in this study

Plasmids-- pBR322Delta 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). pBR322Delta 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.

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-beta -D-thiogalactoside and 5-bromo-4-chloro-3-indolyl beta -D-glucoside were purchased from Sigma.

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, pBR322Delta 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, pBR322Delta 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 sigma 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 pBR322Delta Ptet and pBR322Delta tet5' the promoter sequence that was reconstituted due to the deletion is shown. The presence of promoter activity for pBR322Delta 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.

                              
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Table II
Growth of RFM475 carrying pBR322 derivatives on LB plates with various amounts of tetracycline
RFM475 cells carrying the indicated plasmid were grown overnight in liquid VB Casa medium with cysteine and ampicillin (except for the strain carrying no plasmid) at 37 °C, and 2 µl were streaked on LB plates with the indicated concentration of tetracycline. The plates were incubated for 20 hrs at 37 °C. +, reflects colony size; -, absence of growth.

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 (Delta 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 Delta topA strains used in our studies carry the same topA deletion [Delta (topA cysB)204].

                              
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Table III
Relative colony size on LB medium of the various Delta topA strains used in this study
Bacterial cells were grown overnight in liquid VB Casa medium with cysteine at 37 °C, and 2 µl were streaked on LB plates that were incubated at 37, 28, and 21 °C, respectively, for 20, 42, and 72 h (EM176 and RFM475 were incubated for 6 days at 21 °C). +, reflects colony size; -, absence of growth.

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 Delta topA mutants carrying pBR322Delta 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 pBR322Delta 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 Delta topA mutant completely fails to grow (RFM475 at 21 °C), hypernegatively supercoiled pBR322Delta 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 pBR322Delta 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 pBR322Delta tet5' (Fig. 3). A larger proportion of hypernegatively supercoiled topoisomers was detected when pBR322Delta 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 [Delta (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 pBR322Delta Ptet DNA in various topA null mutant. The various topA null mutants carrying pBR322Delta 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 pBR322Delta tet5' DNA in various topA null mutant. The various topA null mutants carrying pBR322Delta 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

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.

    ACKNOWLEDGEMENTS

We thank Pauline Phoenix for technical assistance and Sonia Broccoli for careful reading of the manuscript.

    FOOTNOTES

* 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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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