Cell, Vol. 63, 393-404,

October

19, 1990, Copyright

0 1990 by Cell Press

New Topoisomerase Essential for Chromosome Segregation in E. coli Jun-ichi Kate,’ Yukinobu Nishimura,t Ryu Imamura,* Hironori Niki,* Sota Hiraga,* and Hideho Suzuki5 Department of Bacteriology National Institute of Health of Japan Kamiosaki Shinagawa-ku Tokyo 141 Japan t Department of Microbiology National Institute of Genetics Mishima Shizuoka-ken 411 Japan f Department of Molecular Genetics Institute for Medical Genetics Kumamoto University Medical School Kumamoto 862 Japan 5 Laboratory of Genetics Department of Biology Faculty of Science University of Tokyo Hongo Tokyo 113 Japan l

Summary The nucleotide sequence of the parC gene essential for chromosome partition in E. coli was determined. The deduced amino acid sequence was homologous to that of the A subunit of gyrase. We found another new gene coding for about 70 kd protein. The gene was sequenced, and the deduced amino acid sequence revealed that the gene product was homologous to the gyrase B subunit. Mutants of this gene were isolated and showed the typical Par phenotype at nonpermissive temperature; thus the gene was named parE. Enhanced relaxation activity of supercoiled plasmid molecules was detected in the combined crude cell lysates prepared from the ParC and ParE overproducers. A topA mutation defective in topoisomerase I could be compensated by increasing both the parC and the parE gene dosage. It is suggested that the pad and parE genes code for the subunits of a new topoisomerase, named topo IV. Introduction Escherichia coli temperature-sensitive mutants are classified into several groups, for example, dna mutants defective in DNA replication, fts mutants defective in septum formation, and min mutants defective in determination of the septation sites (Hirota et al., 1968). The par mutant is one of them, and the par mutation has been so termed

because of the nucleoid morphology that would result if chromosomes have been replicated but not partitioned, resulting in large nucleoids in the midcell. Some of the Par phenotypes may arise from defective DNA replication, and, in fact, the parS mutation has been found to be an allele of the dnaG gene (Norris et al., 1986). The par mutations are expected to include, apparently, two categories of partition defects, inasmuch as chromosome partitioning involves topological resolution (decatenation of replicated chromosomes) and topographical segregation (positioning) of daughter chromosomes. Two daughter chromosomes produced by one round of replication are likely to be linked topologically with each other in a catenane, as demonstrated with small replicons (Sakakibara et al., 1976). The primary event in chromosome partitioning would involve topological resolution of the catenated chromosomes through the decatenase activity of topoisomerase. Gyrase participates in resolution of catenated chromosomes in E. coli (Steck and Drlica, 1984). Two par mutant phenotypes described as ParA and ParD were eventually ascribed to gyr6 and gyrA, respectively (Kato et al., 1989; Hussain et al., 1987). The requirement for topological resolution seems not to be confined to resolution of simple catenanes that arise from the circularity of replicons. Topologically linear replicons in eukaryotes also require the action of a type II topoisomerase for resolution of intertwined progeny replicons, as shown by the failure of chromosomes to segregate at the restrictive temperature in thermosensitive type II topoisomerase mutants of yeasts (DiNardo et al., 1984; Holm et al., 1985; Uemura and Yanagida, 1984, 1988). The topographical segregation (positioning) of chromosomes in cell division is the other facet of chromosome partitioning (Jacob et al., 1963; Donachie and Begg, 1989; Hiraga, 1990). The stable inheritance of prokaryotic replicons may be ensured by the association of the replicons with the cell membrane, which segregates the attached replicons as the cell surface grows (Jacob et al., 1963). Low-copy-number plasmids are known to have a certain partition system that ensures the tight inheritance of a plasmid copy by each daughter cell. The partition system includes a cis-acting par region that contains repeating sequences and provides a site for binding of protein(s) required for proper partitioning of the plasmid replicons (for review see Austin, 1988). The par region complexed with the partition-specific protein(s) may constitute a prokaryotic analog of a centromere in eukaryotes and may interact with the membrane to be assembled into a putative mitotic apparatus. The partition proteins may be specified by transacting par genes of the same plasmid or some genes on the host chromosome, or both. In Pl and F plasmids, for example, the protein product of one of two trans-acting par and sop genes is bound to the par and sop region, respectively, while pSClO1 has no Vans-acting par gene of its own. Specific binding of gyrase to the pSC101 par region has been demonstrated, and some structural func-

Cell 394

tion other than the catalytic activity of introducing superhelicity has been suggested for the bound gyrase (Wahle and Kornberg, 1988). In E. coli three topoisomerases are known so far: topoisomerase I (topo I), II (gyrase), and Ill (topo Ill) (Dean et al., 1982). Topo I and topo III are classified as type I topoisomerases that relax DNA supercoils through a transient single-strand break, while gyrase belongs to type II, which can introduce negative supertwists via transient double-strand scission (for review see Wang, 1985). Decatenase activity has been observed in all the three enzymes, while type I topoisomerases can only decatenate if one of the catenated molecules contains a nick (Wang, 1985). Gyrase and topo I, through their opposing action on the superhelical DNA, maintain superhelical tension of cellular DNA at a level appropriate for the action of DNA in transcription and replication (Wang, 1985). Topo III could conceivably participate in regulating chromosome superhelicity by analogy to topo I in catalytic property. The potent activity exhibited by topo Ill for resolution of plasmid catenanes in vitro suggests that topo Ill may exercise the decatenation function rather than the relaxation function (DiGate and Marians, 1988), although the role of topo Ill in vivo still remains to be elucidated. Among four par mutants (parA, -13,-C, and -D) described to date (Hirota et al., 1968, 1971; Hussain et al., 1987; Kato et al., 1988) only the pa& mutation, causing the Par phenotype similar to ParA (or GyrB), appeared not to be related to gyror dna: the mutation was located at map position 65 min; the pa& gene product was identified as a 75 kd protein on SDS-electrophoretic gels; and the association of the ParC protein with the membrane was found (Kato et al., 1988). In this paper, we describe further characterization of the parC gene and identification of another par gene, parE, which is located in the upstream region of pa&. The nucleotide sequence of the parC and parE genes showed that the ParC and ParE proteins are homologous to the A and B subunits of gyrase, respectively. We detected the high level of topoisomerase activity, relaxation activity of supercoiled plasmid molecules, in the crude cell lysates that contained the overproduced ParC and ParE proteins. It was suggested that the new topoisomerase encoded by the pa& and parE genes had relaxation activity, in contrast to gyrase, which caused increase of superhelicity. Results Nucleotide Sequence of the pa& Gene The parC gene has been located on the Hpal-Smal fragment of the chromosome region carried by pLC4-14 (Kato et al., 1988). To confirm and delimit the pa&-coding region more precisely, the Hpal-Smal fragment was subcloned into pUC18 to construct pJK935 and pJK937, and various deletion derivatives were constructed by exonuclease III digestion as shown in Figure 1. Among the deletion derivatives, pJK940, pJK975, and pJK974 complemented the parC7275 mutation, but the others did not, as examined by correction of thermosensitive growth of EJ812 @arC7275), although the complementation by pJK974 ap-



+

+ -

'( \ I 1 -

pJK935 pJK940 pJK941 pJK952

+

pJK953 pJK937 + pJK975 +* pJK974

-

pJK973

parC parC1215

ts mutation Figure

1. Chromosomal

Inserts

O%Tb in Plasmids

The upper line shows the physical map of the parC chromosome region Solid bars indicate chromosomal inserts in pJK935, pJK937, and their deletion derivatives. The ParC coding region is shown by an open arrow, below which the region containing the par0275 mutation is indicated. Pius and minus signs denote complementation positive and negative, respectively. The plus sign with an asterisk indicates incomplete complementation (see text).

peared to be rather incomplete, as judged from small colonies at the restrictive temperature. The above results indicate that the parC gene is contained in the chromosome region shared by pJK940 and pJK975. The nucleotide sequence was determined for this region, in which one open reading frame was found to code for a protein with a maximum of 730 amino acids corresponding to 81,243 in molecular weight (Figure 2). This open reading frame is long enough to accommodate the size of the ParC protein (75 kd) and its orientation agreed with that of the parC gene determined previously (Figure 1; Kato et al., 1988). Three possible initiation codons were found within the region carried by pJK940 (complementation positive) but not by pJK941 (complementation negative): ATG (nucleotides 92-94), GTG (98-loo), and ATG (104-106). The amino acid sequence was deduced on the assumption that the first ATG (92-94) was the initiation codon as shown in Figure 2. The use of the other possible initiation codons yields a product shorter by two or four amino acids without affecting the pl value. Since theparC gene was expressed in pJK940 with insertion in the opposite orientation to that of the lac promoter, sequences of nucleotides 31-36 and 54-59 may serve as a promoter for parC transcription (Figure 2). Determination of the pa&7215 Mutation Site Among the plasmids that did not complement the parC7275 mutation (Figure l), pJK941 and pJK952 caused development of a few thermoresistant clones from the pafC mutant cells transformed with these plasmids, whereas pJK953 and pJK973 did not. The rare appearance of ther-

Mr

Topoisomerase

of

E. Coli

The parC nucleotide sequence is shown along with the deduced primary structure of its peptide product. Probable -35 and -10 regions of the promoter, as underlined, were identified by comparison with generally accepted consensus sequences. An asterisk indicates the translation stop codon. Three possible initiation codons are shown by broken lines above them. The Dral, Pstl, and Pvull restriction sites are indicated, and the ends of chromosomal regions in pJK940, pJK941, pJK973, pJK952, and pJK974 are shown by arrows. An arrowhead marks the mutation site of parC1275 at nucleotide coordinate 2199.

moresistant clones was regarded as a result of recombination to generate a wild-type allele of pa&. It was thus presumed that the pan3275 mutation was located in the region common to pJK952 and pJK974 (Figure 1). The nucleotide sequence containing the mutation site was determined with the cloned par0275 segment (Kato et al., 1988) by using two synthetic primers. G at nucleotide 2199 was changed to A in parC7275 within the span of nucleotides 1987-2235, which is common to pJK925 and pJK974 (Figure 2). This base change of transition will bring about

the replacement of Asp for Gly at residue 703. The location of the mutation site is consistent with the presumption that the open reading frame in Figure 2 represents the parkoding region. Homology of Par-C to GyrA The deduced amino acid sequence of the ParC protein revealed a high homology to the sequences of type II topoisomerases. In regions that gave the maximum score by the method of Lipman and Pearson (1985) the homol-

Cell 396

E.coli E.coli B.subt..

GyrA PnrC GyrA

1 1 1

E.coli E.coli B.subt.

GyrA ParC GyrA

60 35 61

E.coli E.coli B.subt.

GyrA PnrC GyrA

119 95 120

F.coli E.coli J\.sllbt.

GyrA ParC GyrA

I79 155 180

E.coli E.coli

GyrA ParC

238 215

B.subt.

GyrA

239

E.coli fl. subt

GyrA PnrC . GyrA

293 264 293

E.soii E.coli B.subt.

GyrA PnrC GyrA

350 324 350

E.coli E.coli B.subt.

GyrA ParC GyrA

407 381 410

E.coli E.coli B.subt.

GyrA ParC GyrA

467 404 433

E.coli E.coli R.subt.

GyrA ParC GyrA

527 464 492

E.coli E.coli B.subt.

GyrA ParC GyrA

577 474 510

E.coli E.coli B.subt.

CyrA ParC GyrA

637 531 570

E.coli E.coli B.subt.

GyrA ParC GyrA

697 623 665

E.coli E.coli B. subt

GyrA ParC . GyrA

756 682 723

D.subt.

GyrA GyrA

816 783

I?.COll

Gyr;r

876

E.COIl

E.COll

Figure

3. Amino

Acid Sequence

Homology

of E. coli ParC with E. coli GyrA and B. subtilts

GyrA

The GyrA sequence of E. coli was taken from Swanberg and Wang (1987) and Hussain et al. (1987). The GyrA sequence of B. subtilis was taken from Moriya et al. (1985). Identities are represented by asterisks and conservative substitutions by dots. Homology was identified according to the maximum homology score (Lipman and Pearson, 1985). Active site Tyr of E. coli gyrase is boxed. Of the 33 amino acids conserved among gyrase A subunit of E. coli and B. subtilis and the type II topoisomerases of T4 phage, D. melanogaster, S. cerevisiae, and S. pombe, the 30 amino acids conserved in ParC are underlined.

New Topoisomerase 397

of E. Coli

pJK2002 pJK2000,

pJK2001

pJK2000T, pJK2001T pJK914 pJK2027 pJK902, pJK2030 pJK2029 pJK901, pJK2028 pJK888 pJK2014 pJK894 pJK20 10 pJK2011

MWl

23

45 6

7

8 ParC

Figure

4. Identification

of the par.E Gene

(A) The upper physical map of the parC and pafE chromosome region was drawn from the one made by Kohara et al. (1987). Solid bars and hatched bars indicate chromosomal DNA cloned from W3110 @err?) and EJ612 (parC7215), respectively. Open bars with Km indicate the inserts of the Km’ marker. The insertion of the translational terminator is indicated by an inverted triangle. The parC and par/Z genes are shown by open arrows. (6) In lanes 1 through 6, proteins were synthesized in vitro. Lane 1, pLJC18Tc; lane 2, pJK2010; lane 3, pJK2011; lanes 4 and 6, Pstl digest of pJK2001; lane 5, Pstl-BamHI double digest of pJK2001. Lane 7, molecular weight markers. Lane 8, proteins were synthesized in minicells with pJK87l @WC+). The positions of ParE (70 kd protein) and ParC are shown by wedges in lanes 6 and 8, respectively. The labeled products were processed as described in Experimental Procedures.

ogy accounted for 35.9% of the span of 679 residues to the E. coli GyrA subunit (Swanberg and Wang, 1987; Hussain et al., 1987) and 35.3% of the span of 888 residues for the Bacillus subtilis GyrA subunit (Moriya et al., 1985) (Figure 3). A region with high homology was found in the N-terminal half of ParC and GyrA of E. coli and 6. subtilis. The

homology was more remarkable around the Tyr residue of the gyrase active center. The amino acid sequence (amino acids 410-443) of E. coli GyrA is unique, when the GyrA amino acid sequence is compared with the other type II topoisomerase sequences (Wyckoff et al., 1989). The amino acid sequence homologous to this E. coli GyrA unique sequence does not exist in ParC as other type II topoisomerases. The amino acid sequence of ParC was fairly homologous to other topoisomerases: homology was 21.2% with human topoisomerase II over 443 residues (Tsai-Pflugfelder et al., 1988) 22.3% with Saccharomyces cerevisiae topoisomerase II over 417 residues (Giaever et al., 1986) 19.5% with Schizosaccharomyces pombe topoisomerase II over 481 residues (Uemura et al., 1986) and 30.8% with the bacteriophage T4 gene 52 product over 224 residues (Huang, 1986a). Thirty of the 33 amino acids conserved among the gyrase A subunits of E. coli and B. subtilis and the type II topoisomerases of T4 phage, S. cerevisiae, S. pombe, and Drosophila melanogaster are conserved in ParC protein. Identification of a New Gene Relating to ParC Protein The BamHI(l)-BamHI(2) fragment containing the parC7275 mutation has been cloned from the mutant chromosome into a mini-F vector (Figure 4A; Kato et al., 1988). When this BamHl fragment was inserted into the BamHl site of a high-copy vector, pBR322, the resulting plasmid pJK914 was found to become very unstable: more than 99% of cells lost the plasmids after overnight cultivation under nonselective conditions, although the mini-F plasmid pJK820 that carried the same BamHl segment of pa70275 was stably inherited (Table 1). Since plasmid pJK902 carrying the BamHI(l)-Hpal(1) segment that contained the mutated pafC7275 gene was not as unstable, the upstream region of the pa& locus in the BamHl fragment seems to be responsible for this phenomenon (Table 1; Figure 4A). The region that gave instability to plasmids was located in the Hpal(2)-BamHI(2) region, since pJK2027 lacking the Hpal(l)-Hpal(2) segment of pJK914 was as unstable as pJK914 (Figure 4A). When pJK2030 (Cm3 carrying the BamHI(l)-Hpal(1) segment of the parC7275 allele was introduced into cells carrying pJK2028 (APr), which carried the Pstl(P)-BamHl(2) segment, Ap’ Cm’ transformants formed minute colonies at 30°C on LB agar containing ampicillin and chloramphenicol. However, the transformants hardly grew at 30°C in LB broth containing both antibiotics. This indicates that the plasmid instability is caused by the plasmid-encoding transacting products and that the Pstl(2)-BamHI(2) region was thus enough to confer instability on plasmids. The deletion analysis of the Pstl(P)-BamHI(2) segment showed that almost all of the ml kb Pstl-BamHI region was essential for giving instability, because pJK2014 with a ~100 bp deletion from the BamHI(2) site and pJK2011 with a ~150 bp deletion from the Pstl(2) site were stably maintained (Table 1; Figure 4A). Nucleotide sequencing revealed the occurrence of an open reading frame coding for a ~70 kd protein and covering the BamHI(2) site as described later. The Pstl(2)-

Cell 398

Table

1. Stability

of Transformants

Plasmid

and Used

pJK820 pJK914 pJK2027 pJK902 pJK2028 pJK2028 pJK2028 pJK888 pJK2014 pJK894 pJK2010 pJK2011

(Km’) (Ap’) (Ap’) (Ap’) (Ap’) (Ap’) + pJK2029 (Cmr) (Ap’) + pJK2030 (Cmr) (Ap’) + pJK2030 (Cm’) (Ap’) + pJK2030 (Cm’) (Ap’) + pJK2030 (Cm’) (Ap’) + pJK2030 (Cm’) (Ap’) + pJK2030 (Cm’)

Host was strain a See the text.

Marker

Stabilitya Stable Unstable Unstable Stable Stable Stable Unstable Unstable Stable Unstable Unstable Stable

CGOOrecA.

BamHl(2) segment carried the distal part of the open reading frame, although the N-terminal part of the open reading frame was deleted in this segment. These results sugPyuI

-35

gested that the distal part of the polypeptide encoded by the Pstl(2)-BamHl(2) segment was related to the altered ParC protein encoded by the parC7275 allele, causing the instability of plasmids by an unknown mechanism. In vitro protein synthesis using various plasmids as templates revealed that pJK2001, which carried the EcoRI(l)EcoRI(2) segment (Figure l), directed synthesis of a 70 kd protein. The 70 kd protein was synthesized with the Pstl digest of pJK2001 (Figure 48, lane 4) but not with the Pstl and BamHl double digest of pJK2001 (Figure 48, lane 5). The 70 kd protein was not synthesized from pUC18Tc (lane l), pJK2010Tc (a Tcf derivative of pJK2010) (lane 2), and pJK2011Tc (a Tcr derivative of pJK2011) (lane 3). These results suggested that the BamHI(2) site existed within the coding region for the 70 kd protein. In addition, pJK2001T, which was a pJK2001 derivative carrying a translational terminator sequence at the BamHI(2) site (see Figure 4A), did not direct the 70 kd protein (data not shown). As shown in Figure 5, if the ATG at nucleotides 64-66 is assumed to be the initiation codon, the open reading

-10

The nucleotide sequence of the parf coding region is shown along with the deduced primary structure of Its peptide product. Probable -35 and -10 regions of the promoter, as underlined, were identified by comparison with generally accepted consensus sequences. An asterisk indicates the translation stop codon. The Pvul and BamHl restriction sites are indicated and the ends of chromosomal regions in pJK888, pJK2014, and pJK2011 are shown by arrows.

New Topoisomerase 399

of E. Coli

frame codes for a protein composed of 601 amino acids corresponding to 66,772 in molecular weight. A possible promoter sequence is located at nucleotides 16-21 and 39-43. Isolation of the Mutants of the New Gene To obtain the mutants of this gene, 40 unidentified temperature-sensitive par mutants in the E. coli mutant bank (Suzuki et al., 1976) were surveyed by complementation with pJK2000, which carried the EcoRI(l)-EcoRl(2) DNA segment of the wild-type strain. Four mutants were isolated, the thermosensitivity of which was corrected by pJK2000, but not by pJK2000T, which had the translational terminator inserted at the BamHI(2) site. All four mutations were genetically mapped with transduction with phage Pl at the expected loci; the cotransduction frequency of the mutations with to/C (tolerance to colicin El) was about 90%, while the one of parC with to/C was about 60%. The parE mutations were transferred into W3110. All of the four mutants with W3110 genetic background showed the typical Par phenotype at the nonpermissive temperature, and the Par phenotype was corrected by introducing pJK2000. Thus, we named the gene parf. Homology of ParE to GyrB By searching for other proteins homologous to the deduced amino acid sequence of ParE protein, it was found that the ParE protein was homologous to type II topoisomerases, especially the B subunit of gyrase. Homology was found almost over the whole polypeptide. In regions that gave the maximum score by the method of Lipman and Pearson (1965) the homology accounted for 40.1% of the span of 604 residues to E. coli GyrB subunit (Adachi et al., 1967; Yamagishi et al., 1966) and 39.7% of the span of 605 residues for the B. subtilis GyrB subunit (Moriya et al., 1965) (Figure 6). E. coli GyrB protein has a unique amino acid sequence (amino acids 561-731) as well as E. coli GyrA protein, but the amino acid sequence homologous to this E. coli GyrB unique sequence does not exist in ParE protein as the other type II topoisomerases (Wyckoff et al., 1989). The amino acid sequence of ParE was fairly homologous to other topoisomerases: homology was 21.8% with human topoisomerase II over 579 residues, 22.8% with S. cerevisiae topoisomerase II over 584 residues (Giaever et al., 1986) 21.8% with S. pombe topoisomerase II over 574 residues (Uemura et al., 1986) and 26.7% with the bacteriophage T4 gene 39 product over 445 residues (Huang, 1986b). Forty-eight of the 57 amino acids conserved among the gyrase B subunits of E. coli and B. subtilis and the type II topoisomerases of T4 phage, S. cerevisiae, S. pombe, and D. melanogaster are conserved in ParE protein.

named rot. DNA rearrangement has been described occurring in the 65-66 min region of the tot mutants, leading to the amplification of the to/C gene region (Dorman et al., 1989). To examine the possibility that the compensation of the topA amber mutation is due to the amplification of the pa& and parf genes, the topA amber mutant was transformed with the parC and/or the par/Z plasmids. Strain BR83 (fopA,, supDis) can grow at both 42% and 30% in media of low osmolarity in the absence of any compensatory mutation, while the plating efficiency decreases drastically at 42% in media of high osmolarity. BR83 carrying pJK831 @arC+) and/or pJK2000 (par/f+) grew at 42% on LB agar containing no NaCI. However, only BR83 that carried both pJK831 and pJK2000 grew at 42% on LB agar containing 1% NaCI. This means that the compensation of the topA defect was caused by increasing both parC and parE gene dosage. Enhanced Relaxation Activity in the Cell Lysates of the ParC and the ParE Overproducers Relaxation of negative supercoils was assayed using crude cell lysates prepared from the ParC and the ParE overproducing strains. To overproduce the ParC protein, pJK825 was constructed by cloning the parC gene into the runaway plasmid vector pSY343 (Yasuda and Takagi, 1983). To construct the overproducing strain of the ParE protein, pJK2020 was obtained by cloning the parE gene downstream the PL promoter of h phage in the plasmid vector pJL6 (Inada et al., 1989). Crude cell lysates were prepared from each of the overproducers and their control strains as described in Experimental Procedures. When these overproducers were grown at the restrictive temperature, ParC and ParE proteins were synthesized to the extent that these proteins could be observed as protein bands in the electrophoretic profiles of the crude lysates (Figure 7A). The crude cell lysates were examined for the topoisomerase activity in vitro. Most of the supercoiled pBR322 DNA was relaxed when the cell lysate prepared from the ParC overproducer (DHl/pJK825) was combined with the one prepared from the ParE overproducing strain (YN2942/pKJ2020) as shown in Figure 78. The enhanced relaxation activity could not be detected when the cell lysate prepared from each of the control strains was used. The result, along with the homology in amino acid sequence found among ParC, ParE, and type II topoisomerases, strongly suggests that subunits of a new topoisomerase, topo IV, is encoded by the parC and pa& gene. In addition, it was suggested that the complex of ParC and ParE proteins causes decrease of superhelicity as well as eukaryotic type II topoisomerases, in contrast to gyrase, which caused increase of superhelicity. Discussion

Compensation of the fopA Mutation by Increasing Both ParC and parE Gene Dosage Suppressor mutations that compensate a top4 amber mutation defective in topo I have been mapped at three loci: gyrA (48 min), gyrB (83 min), and the 65-86 min region of the E. coli genetic map (Raji et al., 1985) where parC and par/Z are located. The mutation of the third locus was

The pafC gene product, which is essential for chromosome partition in E. coli, has been identified as a 75 kd protein by SDS-gel electrophoresis (Kato et al., 1988). Sequencing the pa& gene region revealed one open reading frame large enough to code for this size of protein and consistent with the orientation of the parC gene deter-

Cell 400

F.coli I:.coli R,subt..

GyrB PnrE cyrn

1 1 1

11.col1 1; . 00 1 i B.subt.

CyrB FnrE GyrB

55 51 57

E.coli E.coli

GyrB ParE

115 111

B. auht,.

GyrB

117

E.coli I

New topoisomerase essential for chromosome segregation in E. coli.

The nucleotide sequence of the parC gene essential for chromosome partition in E. coli was determined. The deduced amino acid sequence was homologous ...
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