New topoisomerase essential for chromosome segregation in E. coli

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<.coli B. suht

GyrB PnrE . GyrB

173 171 175

E.coli E.coli B.subt.

GyrB ParE GyrB

231 228 235

E.coli E.coli B.subt.

GyrB ParE GyrB

290 288 294

E.coli E.coli B.subt.

GyrB ParE CyrB

350 347 354

E.coli E.coli B.subt.

GyrB ParE GyrB

409 403 413

E.coli E.coli B.subt.

GyrB ParE GyrB

469 463 473

E.coli E.coli B.subt.

GyrB ParE GyrB

528 520 531

IKDDEAM?~YQISIALDGATLHTNASAPALAGEALEKL X’OQPP~XKVKKEBQEQ~---'IiL i -f AL~EE8;(tQ-tEB~KR’K.-----------------------X~BLPW - . EH Y x* YIAQPPLYKVQQGKRVEYAYNDKELEELLKTL-PQT------------------------

E.coli E.coli

GyrB ParE

584

R.suht.

GyrB

VSEYNATQKMINRMERRYPKAMLKELIYQPTLTEADLSDEQTVTRWVNALVSELNDKEQH --___----_-------------------------------------------------------------------------------------------------------------

E.coli E.coli B.subt.

GyrB ParE GyrB

644

E.col1 E.coli B.subt.

GyrB ParE GyrB

704 556 566

E.?011 E.COll n.subt

GyrB ParE . GyrB

764 588 598

Figure

6. Ammo

Acid Sequence

QVTLEI)A??DADETFEMI,MGDKVEPRRNF

Homology

I EANARl-L’KNLD

of E. coli ParE wrth E. coli GyrB and B. subtills

I

GyrB

The GyrB sequence of E. coli was taken from Adachr et al. (1967) and Yamagishi et al. (1966). The GyrB sequence of B. subtilis was taken from Moriya et al. (1965). Identities are represented by asterisks and conservative substitutions by dots. Homology was identified according to the maximum homology score (Lipman and Pearson, 1965). Of the 57 amino acids conserved among gyrase B subunit of E. coli and B. subtilis and the type II topoisomerases of T4 phage, D. melanogaster, S. cerevisiae, and S. pombe, the 46 amino acids conserved in ParE are underlined.

mined previously. This open reading frame was confirmed to be the parC coding region by the location of the pa&7275 mutation site in it. Although the N-terminal amino acid sequence has not yet been analyzed, the ParC protein was tentatively determined to consist of 730 amino

acids. The deduced amino acid sequence showed homology to several type II topoisomerases. The homology was particularly remarkable in the N-terminal half of the GyrA subunit of gyrase, which interacts with DNA, while there was little homology to the GyrB subunit, which contains

New Topoisomerase 401

of E. Coli

(cell

lysate)

+

DHl /pSY343 DHI /pJK825 Mw

(parC+)

!tN2942/@6 YN2942/pJK2020

:

+

+

+

+ (parE+) ;2;12j

Figure

7. Enhanced

In Vitro Relaxation

Activity

in the Cell Lysates

+

;zj

+ 12;

of ParC and ParE Overproducers

(A) The crude cell lysates prepared as described in Experimental Procedures were examined by SDS-polyacrylamide (12%) gel electrophoresis. The ParC and ParE positions are marked with wedges. Molecular size standards were phospholipase B 97.4 kd. bovine serum albumin 66.2 kd, ovalbumin 42.7 kd, carbonic anhydrase 31.0 kd, and soybean trypsin inhibitor 21.5 kd. (B) Removal of negative supercoils was measured in a standard reaction mixture as described under Experimental Procedures, Reaction mixture contained 0.94 ug (lane l), 0.47 ug (lane 2) and 0.23 ug (lane 3) of each of the crude lysates. Plus signs indicate the presence of the indicated lysate in the reaction mixture and minus signs indicate the absence of the lysate.

an ATPase domain. Consistent with these homology features, the ParC protein showed DNA binding ability (unpublished data). Among the three topoisomerases identified in E. coli, topo III is nearly the same as the ParC protein in relative molecular mass (Dean et al., 1982; Srivenugopal et al., 1984; DiGate and Marians, 1988). Topo Ill belongs to the type I group represented by topo I (Dean et al., 1982) and its catalytic properties seem to be basically similar to those of topo I. Recently, the structural gene of topo III (top@ was cloned and its DNA sequence was determined (DiGate and Marians, 1989). The map position for the topo III gene is 38.7 min, and it has been clearly shown that the ParC protein is not top0 Ill. We identified another gene, named pa&, in the upstream region of the pafC locus during analysis of the region that caused instability of the plasmid. It is not clear why transformants generated by introducing the high-copy plasmid carrying both theparC7275 mutated gene and the Pstl(2)-BamHl(2) region encoding the 3’-terminal half of the par/E gene hardly grow. A possible explanation is that complexes of the alternated ParC protein encoded by the par0275 gene and the carboxy-terminal part of ParE protein inhibit host cell growth, or, alternatively, change the topology of the plasmids to be unstable. A whole region of theparEgene was needed to complement the par/i mutations, while only the distal part of the parE gene causes the plasmid instability. Topo I deletion mutants (fopA) are greatly impaired in growth rate, and normal growth is generally recovered by

the occurrence of a suppressor mutation in either gyrA or gyff3 (DiNardo et al., 1982; Pruss et al., 1982) and sometimes in the third locus, tot (Raji et al., 1985). The foe mutations have been mapped around 66 min close to the pafC and par,5 locus. The tot mutations might not be point mutations and were always accompanied with DNA amplification and rearrangement of the region including the to/C gene located at 66 min (Dorman et al., 1989). In this work it was found that simultaneous increase in parC and par,!! gene dosage was able to compensate the defect in the topA gene. Since both the pafC and parE plasmids are necessary for the compensation, both ParC and ParE proteins may constitute topoisomerase IV Thus the top4 defect is compensated by either the gyrase mutation that decreases superhelical density or amplification of topo IV genes. This is consistent with the conclusion that one of the topo IV functions is relaxation of supercoiled DNA, like topo I, opposed to gyrase. The enhanced relaxation activity was found in the crude cell lysates containing both overproduced ParC and ParE proteins, as was expected. We are now purifying both ParC and ParE proteins. The activity to relax negative supercoiled DNA, but not supercoiling activity, has been detected so far when both partially purified fractions are combined (unpublished data). These results suggest that top0 IV is a topoisomerase correspondence to the eukaryotic type II topoisomerase. Gyrase is also involved in chromosome segregation, perhaps through its decatenating activity. This study suggests that, in addition to gyrase, topo IV is required for chromosome partitioning. Dumbbell-shaped doublet nu-

Cell 402

cleoids have been isolated from a thermosensitive gyrS mutant incubated at the restrictive temperature and resolved into singlets in vitro by addition of purified gyrase (Steck and Drlica, 1984). Bacterial chromosomes are folded in multilooped structures and organized into nucleoids in which the chromosome is segmented into topological units independent of one another in superhelical density (for review see Drlica, 1987). Such organization might impose a local structural constraint on daughter chromosome separation in each looped segment. Although bacterial chromosomes can be formally segregated by decatenating once at or after replication termination, the local constraint would give rise to a situation similar to that found in eukaryotic chromosomes, which are linear topologically and yet appear to require the action of topoisomerase II to resolve intertwined pairs of daughter chromosomes (DiNardo et al., 1984; Uemura and Yanagida, 1984). (In this context, small independent replicons like plasmids would be exempt from such a situation. In fact, we could not detect even major catenane bands when the plasmids in the pa0275 mutant were examined after incubation at the restrictive temperature for 2 hr or overnight.) A larger supply of to’poisomerases might be required to resolve intertwining in each looped segment. There may be some kinds of intertwined structure, and gyrase and topo IV may have different substrate specificities or may be concerned in different reactions. It is necessary to study the biochemical property of topo IV with purified ParC and ParE proteins for understanding the function of topo IV. Besides the catalytic activities, structural functions have been suggested for gyrase, which binds to the par region of pSC101 (Wahle and Kornberg, 1988). Topo IV may similarly have a dual function, since the ParC protein can associate with both DNA and the membrane (Kato et al., 1988; unpublished data). The basic mechanism of chromosome segregation might be illuminated by studies on topo IV. Experimental

Procedures

Bacterial Strains, Plasmids, and Culture Media The E. co11 strains used were DHI (F-, recA7 endA gyrA96 thi-7 hsdR77supE44) (Maniatis et al., 1982), JMl09 (recA7 A (lacpro) endA gyrA96 W-7 hsdR77 supE44 re/Al/F freD36 pro/@ /ac/%ZAMl5) (Maniatis et al., 1982). EJ812 @a&7275; a derivative of C600) (Kato et al , 1988), TH1219 (F- miflB rpsL tsrfar recA) (Harayama and Hazelbauer, 1982), CGOOrecA (F- fhi fhr leu6 lacy tonA supf recA), BR83 (fopA57,, argA s~pD74’~ rpsL A lac-574) (Dorman et al.. 1989). and YN2942 (TAPlOG) ([A (~nf-cl/l)BAM N,:Kan cl857 A (cro-NoA)]) (Inada et al., 1989). Plasmlds used were pUC4K (Vieira and Messing, 1982), pSY343 (Yasuda and Takagl. 1983), pJL6 (Inada et al., 1989). pJL3-4779, pJK800, pJK803, pJK811, pJK820 (Kato et al., 1988). and pLC4-14 (Clarke and Carbon, 1975). Bacteria were grown routinely in LB broth and antIbiotIc medium 3 (Difco) broth, or on antibiotic medium 3 plates. Where relevant, antiblotlcswere added to 50 vglml (amplclllm and kanamycin) and 15 pglml (chloramphenicol). Plasmid Construction The plasmids shown In Figure 1 were constructed as follows. The Hpal fragment containing the parC gene was excised from pJK803 (Kato et al., 1988) and Inserted into the Smal site on pUC8 to produce pJK871. Plasmld pJK871 was subjected to replacement of the Pstl fragment containing the distal parC region wtth the Pstl fragment containing the

same region plus a Cm’ (chloramphenicol resistance) marker to construct pJK872. This Pstl fragment was derived from pJK860, which was constructed by inserting the BamHl fragment containing a Cm’ marker excised from pJL3-4779 into one of the BamHl sites of pLC4-14 (BamHI(2) site in the previous work; Kato et al., 1988). The EcoRISmal fragment containing the parC gene was excised from pJK872, converted to a blunt-end fragment by using Klenow fragment, and Inserted into the Smal site of pUCl8 to produce pJK935. To construct pJK937, the same EcoRI-Smal fragment of pJK872 was ligated with the EcoRI-Smal fragment of pUCl8. The deletion derivatives of pJK935 (pJK940, pJK941, pJK952, and pJK953) and those of pJK937 (pJK975, pJK974, and pJK973) were obtained by using a deletion kit (Takara Shuzo Corp., Kyoto, Japan). To isolate these deletion derivatives, pJK935 and pJK937 were digested with both Sphl and Sall, treated with exonuclease Ill, mung bean nuclease, and Klenow fragment, and then ligated. The ligated DNA was digested with Sall before transformation. The plasmids shown in Figure 4A were constructed as follows. To clone the parE gene, the DNA of the h phage clone 1782. which covers the parE region of the E. coli W3110 genomic library (Kohara et al.. 1987), was purified and the EcoRl fragment (EcoRI(l)-EcoRI(2); Figure 4A) containing the parE gene was cloned into pUC118 to produce pJK2002. The BamHl fragment containing the Km’ marker was excised from pUC4K and inserted into the Bglll site downstream the parE gene of pJK2002 to produce pJK2001, pJK2000 was constructed by inserting the EcoRl fragment of pJK2001 into the EcoRl site of ColEl. To obtain pJK200OT and pJK2001T, the translational terminator (Kato et al., 1988) was inserted into the BamHI(2) site of pJK2000 and pJK2001, respectively. To construct pJK914, the BamHl fragment (BamHI(l)-BamHI(2); Figure 4A) containing the parC7275 gene was excised from pJK820 (Kato et al., 1988) and inserted into the BamHl site of pBR322. The Hpal fragments were deleted from pJK914, to produce pJK2027. The BamHI(l)-Hpal(1) fragment containing the par-C1275 gene was excised from pJK820 and ligated with the BamHI-Smal fragment of pUC8 to construct pJK822 and the BamHIEcoRV fragment of pACYCl84 to construct pJK2030. The EcoRIBamHl fragment of pJK822 was ligated with the EcoRI-BamHI fragment of pBR322 to obtain pJK902. The EcoRI-BamHI fragment containing the parC gene of pJK87l was excised and ligated with the EcoRIBamHl fragment of pBR322 to construct pJK901. pJK2029 was obtained by ligating the BamHI(l)-Hpal(1) fragment of pLC4-14 with the BamHI-EcoRV fragment of pACYCl84. pJK2028 was constructed by cloning the Pstl(2)-BamHl(2) fragment of pJK914 into pUCl8. pJK887 and pJK885 were constructed by inserting the BamHl fragment of pJK820 (parC7275) into pUC18 in opposite orientation to each other. A Sall fragment was deleted from pJK887 to obtain pJK894. pJK2010 and pJK2011 wereobtained by digesting with exonuclease from the Hpal(2) site and constructing the deletion plasmid. To construct pJK888, an EcoRl fragment was deleted from pJK885. pJK2014, a deletion denvative of pJK888, was obtained by digesting wtth exonuclease from the BamHl(2) site and constructing the deletion plasmid. pJKl8Tc, pJK2OlOTc, and pJK2OllTc were constructed by excising the Pvull fragment containing a Tc’ marker from pBR325 and inserting it into the Seal site in the Ap’ marker of pUCl8, pJK2010, and pJK2011. respectively. For overproductlon of the ParC and the ParE proteins, pJK825 and pJK2020 were obtained, respectively. To construct pJK825. the BamHIEcoRl fragment of pSY343 was ligated with the BamHI-EcoRI fragment containing the parC gene of pJK871. To construct pJK2020, the Hindlll-BamHI fragment of pJL6 was ligated with the HIndIll-Bglll fragment containing the parf gene of pJK2002. Preparation, Manipulation, and Sequencing of DNA The techniques for preparation of plasmld DNA, manipulation of DNA, and transformation were as described (Kato et al., 1984). Nucleotlde sequences were determined by the dideoxy chain termination method (Sanger et al., 1977) using double-stranded DNA as templates according to the manual of the sequencing kit (Takara Shuzo Corp., Kyoto, Japan) and Sequenase version 2.0 (US Biochemical Corp.). The nucleotide sequence of the both strands of the parC region was determined using the deletion derivatives of pJK935 and pJK937, and the one of a strand of the parC7275 region was determmed using pJK887 and the specific primers. The nucleotide sequence of both strands of the part

New Topoisomerase 403

of E. Coli

of the parE gene lower than the BamHI(2) site was determined using the deletion derivatives of pJK888 and pJK894. It was confirmed that the nucleotide sequence of this region cloned from strain EJ812 (parC1215) was the same as the one from strain W3110 by determining the nucleotide sequence of a strand of the region of pJK2002. The nucleotide sequence of the part of the parE gene higher than the BamHI(2) site was determined for both strands using the deletion derivatives of phHl3, which carried the chromosomal region of strain W3110 (Hiraga et al., 1989). The nucleotide sequence around the BamHl(2) site was confirmed by that of pJK2002. Computer analysis of the pa& and pafE sequences was performed using GENETYX programs (Software Development Co. Ltd., Tokyo, Japan). Analysis of Gene Products Minicells were prepared from strain TH1219 carrying appropriate plasmids and labeled with [%]methionine (1200 Cilmmol; New England Nuclear, Boston) at 37% for 30 min. In vitro synthesis of protein was performed by a coupled transcription-translation system according to the instructions of the manufacturer of the DNA expression system (NEK038; New England Nuclear, Boston). The labeled gene products were analyzed by SDS-polyacrylamide gel electrophoresis, as described (Kato et al., 1984). Preparation of Crude Cell Lysates and Assay of Relaxation Activity To prepare the crude cell lysates from the ParC overproducing strain and its control strain, DHl/pJK825 @afC+) and DHllpSY343 (vector) were grown at 27oC for 1 hr in 400 ml of antibiotic medium 3, shifted to 37% for amplification of the plasmid, and harvested at 5 hr after the temperature shift. Cells were washed, suspended in 15 ml of TE, buffer (10 mM Tris-HCI [pH 7.51, 10 mM EDTA, 10 mM P-mercaptoethanol), and disrupted by two passages at 20,000 psi through a prechilled French pressure cell. The lysate was centrifuged at 39,000 rpm for 1 hr with a Beckman 70.1 Ti rotor. The supernatant was made 0.6 M in NaCl and 0.3% in polymin P by dropwise addition of 20% poly min P solution (pH 7.5) with stirring in an ice bath. The precipitate was removed by centrifugation at 10,000 rpm for 30 min. Solid ammonium sulfate was added to the supernatant to 60% saturation. The suspension was stirred for 30 min in an ice bath and centrifuged at 10,000 rpm for 30 min. The pellet was suspended in 2 ml of 10 mM Tris-HCI (pH 8.0), 1 mM EDTA, 0.3 M NaCI. 1 mM 2-mercaptoethanol (lE,N3 buffer) and dialyzed against buffer TE,NS. The dialysate was centrifuged at 15,000 rpm for 15 min and the supernatant was saved. To prepare the crude cell lysates from the ParE overproducing strain and its control strain, YN2942/pJK2020 @afE+) and YN29421pJL6 (vector) were grown at 30% for 2 hr in 400 ml of LB broth, shifted to 42% for induction of PL promoter, and harvested at 2 hr after the temperature shift. Cells were washed, suspended in 14 ml of 10 mM Tris-HCI (pH 8.0), 5 mM EDTA, 10% sucrose, and 0.6 M NaCI, incubated at 0% for 1 hr after addition of 0.7 ml of 10 mglml lysozyme, and lysed by four cycles of freezing and thawing. The lysate was centrifuged at 10,000 rpm for 30 min. The supernatant was treated with polymin P and the proteins were precipitated by addition of ammonium sulfate to 60% saturation as described above. Relaxation of negative supercoils was assayed in a reaction mixture (20 ~1) containing 39 mM Tris-HCI (pH 7.5), 50 pg/ml bovine serum albumin, 5.8 mM MgCI*, 30 mM KCI, 1 mM dithiothreitol, 0.5 mM ATP, 60 mM NaCI, and 0.55 pg of negatively supercoiled pBR322 DNA. Reaction mixtures were incubated for 1 hr at 37% The reaction was stopped with 2 ~1 of a solution containing 16.7% SDS and 0.017% bromophenol blue. Five microliters of the samples was electrophoresed through a 1.3% agarose gel for 15 hr at 1.5 V/cm using 50 mM Tris-phosphate (pH 7.5) and 1 mM EDTA. The gel was stained with ethidium bromide and photographed under UV illumination. Acknowledgments We thank C. J. Dorman and Y. Nakamura for kindly providing us their strains and plasmid. We thank Drs. Yoshimasa Sakakibara, Akihiko Kikuchi, Seiich Yasuda, and Mitsuhiro Yanagida for helpful suggestions and discussions and Dr. Haruo Watanabe for support of this study. This work was supported by a Grant-In-Aid for Scientific Research on Priority Areas 63615005 from the Ministry of Education, Science and Culture of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

April 18, 1990; revised

July 6, 1990.

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Numbers

The accession numbers for the sequences M37832 (pafC) and M37833 (par.E).

reported

in this paper

are